Patent Publication Number: US-2020299771-A1

Title: Machine assay and analysis for selecting antihypertensive drugs

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of U.S. patent application Ser. No. 16/789,383, filed Feb. 12, 2020, which is a continuation of U.S. patent application Ser. No. 15/314,641, filed Nov. 29, 2016, which is a U.S. National Stage Filing under 35 U.S.C. § 371 from International Application No. PCT/US2015/032651, filed May 27, 2015, and published as WO 2015/183938 on Dec. 3, 2015, which application claims benefit of the priority filing date of United States Provisional Application Ser. No. 62/004,460, filed May 29, 2014, the contents of which applications are specifically incorporated herein by reference in their entireties. 
    
    
     BACKGROUND 
     Hypertension (high blood pressure) is one of the most significant preventable contributors to disease and death in the world and represents the most common condition seen in the primary care setting (Kearney et al.,  Lancet  365:217-223 (2005)). According to the American Heart Association, approximately 78 million adults (1 in 3) living in the United States have hypertension with more than 5 million new diagnoses made each year. Of these individuals, 82% are aware they have it, 75% are currently being treated for it, but only 52% have their blood pressure under control (thus, ˜48% do not have adequate blood pressure control). 
     Hypertension can lead to myocardial infarction (heart attack), stroke, renal failure, and death if not detected early and treated appropriately. In 2009, high blood pressure was listed as a primary or contributing cause of death in about 350.000 of the approximate 2.4 million U.S. deaths (14% of all deaths). From 1999-2009 the number of deaths attributable to hypertension increased by 44%. 
     Refractory (or resistant) hypertension is defined as blood pressure that remains above clinical guideline goals in spite of concurrent use of three antihypertensive agents of different classes. Critically, refractory hypertension is noted in approximately 25-30% of all individuals being treated for hypertension. Refractory hypertension is a common clinical problem which contributes to the high levels of morbidity and mortality. In 2009, the direct and indirect economic burden on the United States health care system associated with hypertension was estimated at $51 billion. 
     Globally, nearly 1 billion individuals have been diagnosed with hypertension, with an estimate of an additional 400 million living with undiagnosed hypertension. Hypertension is the leading cause of premature death and the leading cause of cardiovascular disease worldwide. Similar to the continued upward trend in prevalence as seen in the United States, it is estimated that in 2025 about 1.56 billion adults will be living with hypertension. Because nearly two-thirds of the people living with hypertension worldwide reside in developing countries, providing optimal treatment at the lowest cost is critically important. 
     Unfortunately, despite a significant impulse in the medical community to move towards an “individualized medicine” approach to patient centered treatment, the current clinical treatment strategy is based on a set algorithm which does not take into account individual patient differences. Rather, physicians are guided to choose a drug (one out of many options) in a given class of drugs and use that specific drug as a “first line therapy” (typically initiating with the diuretic class) and titrate that specific drug of choice to therapeutic dosage regardless of efficacy. It is only after a prolonged course of treatment with that specific class of drug that clinical efficacy is determined (typically three months). At this stage, if clinical guideline goals for blood pressure have not been met, it is often recommended that the patient remain on the “first line therapy” whilst an additional drug from a different class of drugs (typically an Angiotensin converting enzyme inhibitor (ACE inhibitor) or Angiotensin II receptor blocker (ARB)) is added to the pharmacologic regimen. Again, this drug is titrated to recommended therapeutic dosage and another prolonged course of treatment is initiated before clinical efficacy is determined (an additional three months—six months since initiation of treatment). If at this point, clinical guideline goals for blood pressure have not been met, a third drug from a third class of drugs (typically a beta-blocker) is added and the process is repeated (another three months—nine months from initiation of treatment). Further, if clinical guideline goals have continued to be elusive, the diagnosis of refractory hypertension is added and the process is reinitiated with a different combination of drugs, different classes of drugs, different drug options within a given class of drugs, different dosages, or all of the above. Thus, from the time of initial diagnosis and the start of treatment to the point in which blood pressure is adequately controlled may take anywhere from three months to well over one year. This trial-and-error standard of care is clearly not optimal. 
     SUMMARY 
     The invention relates to improved methods, devices, and kits for identifying and implementing an appropriate treatment regimen for subjects suffering from hypertension. The methods, devices, and kits comprehensively assess common genetic variants in the cardiac, vascular, and renal systems in an effort to improve therapeutic guidance for high blood pressure treatment. Detection of an individual&#39;s genetic variants permits selection appropriate drug classes for that individual. Clinicians can then guide blood pressure therapy using knowledge that is specific to their individual patient, rather than the currently employed “trial-and-error” procedures that are based on population data and use of drugs with the least initial side effects. 
     One aspect of the invention is a method that includes:
         (a) administering a loop diuretic to a subject as a first line therapy, without a beta blocker and without a vasodilator, if the subject&#39;s genome comprises a WNK1 nucleic acid with a cytosine at the variable position of rs1159744 or rs2107614;   (b) administering a hydrochlorothiazide to a subject as a first line therapy, without a beta blocker and without a vasodilator, if the subject&#39;s genome comprises an ADD1 nucleic acid with a thymine at the variable position of rs4961, or if the test sample comprises a SLC12A3 nucleic acid with a thymine at the variable position of rs1529927; or   (c) administering a hydrochlorothiazide to a subject as a first line therapy, without a beta blocker and without a vasodilator, if the subject&#39;s genome comprises an ADD1 nucleic acid with a thymine at the variable position of rs4961, or if the test sample comprises a SLC12A3 nucleic acid with a thymine at the variable position of rs1529927.       

     Another aspect of the invention is a method that includes: administering a beta-blocker drug to a subject as a first line therapy, without a diuretic and without a hydrochlorothiazide, if the subject&#39;s genome does not comprise:
         (a) a WNK1 nucleic acid with a cytosine at the variable position of rs1159744;   (b) a WNK1 nucleic acid with a cytosine at the variable position of rs2107614;   (c) an ADD1 nucleic acid with a thymine at the variable position of rs4961; or   (d) a SLC12A3 nucleic acid with a thymine at the variable position of rs1529927       

     but the subject&#39;s genome does comprise:
         1. a CYP2D6 nucleic acid with an adenine at the variable position of Rs3892097;   2. an ADRB1 nucleic acid with a cytosine at the variable position of rs1801253;   3. an ADRB1 nucleic acid with an adenine at the variable position of rs1801252;   4. an ADRB2 nucleic acid with a guanine at the variable position of rs1042714; or   5. an ADRB2 nucleic acid with a guanine at the variable position of rs1042713.       

     Another aspect of the invention is a method that includes: administering an angiotensin II receptor blocker to a subject as a first line therapy, without a diuretic, without a hydrochlorothiazide, and without a beta-blocker, if the subject&#39;s genome does not comprise:
         (a) a WNK1 nucleic acid with a cytosine at the variable position of rs1159744;   (b) a WNK1 nucleic acid with a cytosine at the variable position of rs2107614;   (c) an ADD1 nucleic acid with a thymine at the variable position of rs4961; or   (d) a SLC12A3 nucleic acid with a thymine at the variable position of rs1529927       

     but the subject&#39;s genome does comprise:
         1. a renin nucleic acid with a cytosine at the variable position of rs12750834; or   2. an AGT1R nucleic acid with a cytosine at the variable position of rs5186.       

     Another aspect of the invention is a method that includes: administering an ACE inhibitor to a subject without an angiotensin II receptor blocker as a first line therapy, without a diuretic, without a hydrochlorothiazide, and without a beta-blocker, if the subject&#39;s genome does not comprise:
         (a) WNK1 nucleic acid with a cytosine at the variable position of rs1159744;   (b) a WNK1 nucleic acid with a cytosine at the variable position of rs2107614;   (c) an ADD1 nucleic acid with a thymine at the variable position of rs4961; or   (d) a SLC12A3 nucleic acid with a thymine at the variable position of rs1529927       

     but the subject&#39;s genome does comprise:
         1. an ACE nucleic acid with a deletion in rs1799752; or   2. an AGT nucleic acid with a cytosine at the variable position of rs699.       

     Another aspect of the invention is a method that includes: administering an amiloride as a first line therapy to a subject without an ACE inhibitor, without an angiotensin II receptor blocker, without a diuretic, without a hydrochlorothiazide, and without a beta-blocker, if the subject&#39;s genome does not comprise:
         1. a WNK1 nucleic acid with a cytosine at the variable position of rs1159744;   2. a WNK1 nucleic acid with a cytosine at the variable position of rs2107614;   3. an ADD1 nucleic acid with a thymine at the variable position of rs4961; or   4. a SLC12A3 nucleic acid with a thymine at the variable position of rs1529927.   but if the subject&#39;s genome does comprise a SCNN1A nucleic acid with an adenine at the variable position of rs2228576.       

     The methods can also include administering a second line therapy drug after administration of the first line therapy for at least 1 month, wherein the second line therapy drug is selected from the group consisting of diuretic, a beta-blocker, an ACE inhibitor, a vasodilator, and a combination thereof. 
     The methods can include providing a therapy recommendation according to a mathematical algorithm that includes: 1.) computing a weighted score for each genetic variant identified in an individual subject; 2.) calculating values for variables pertaining to each organ or system (e.g., cardiac, renal and vascular system) based on the weighted scores for each genetic variant; and 3.) determining recommendations based on the values for each of the variables pertaining to each organ or system. 
     Devices, compositions, methods, and kits are also described herein for identifying and implementing an appropriate treatment regimen for subjects suffering from hypertension. 
    
    
     
       DESCRIPTION OF THE FIGURES 
         FIG. 1  is a schematic diagram illustrating the interplay between the heart, blood vessels, and kidney in blood pressure regulation. 
         FIG. 2  is a schematic diagram illustrating of the types of genes useful for evaluating hypertension, and representative single nucleotide polymorphisms that are correlated with blood pressure drug responses. 
         FIG. 3A-3B  are schematic diagrams illustrating processing of test samples. For example, each subject can collect two swabs. The A swab can collect cell material from the inside of the right cheek, while the B swab can collect cell material from the left cheek. For  FIG. 3A , the A swab can be the initial swab entered into the process (from DNA Extraction to Reporting). If the A swab fails, during DNA Yield and Purity Analysis. Genetic analysis, or the PCR QA Assay then the B swab can be entered into the system as illustrated.  FIG. 3B  shows the same process but without the steps of DNA Stocks Storage and Future Testing if the sample Passes Yield and Purity Assays. 
         FIG. 4  is a schematic diagram illustrating handling of DNA samples. 
         FIG. 5  is a schematic diagram illustrating processing of the sample after PCR amplification. 
         FIG. 6  illustrates alignment of sample results to a human reference sequence using the CLC DNA workbench program for creating an alignment file from which the allele call is made and added to the final SNP call report (SEQ ID NOs:82-85). 
         FIG. 7  is an example of 2% agarose gel used to score the presence or absence of a 288 bp ALU by visually examining the gel for either the higher molecular weight band (indicating the presence of the 288 bp ALU), the lower molecular weight band (indicating the absence of the 288 bp ALU), or both (indicating a heterozygous state). 
         FIG. 8  is a bar graph of urinary sodium output as a function of genetic variation of SCNN1A. 
         FIG. 9  is a bar graph of mean arterial blood pressure as a function of genetic variation of SCNN1A. 
         FIG. 10  is a flowchart of a method for providing a hypertension treatment recommendation in accordance with some embodiments. 
         FIG. 11  is a flowchart of an algorithm for providing a hypertension treatment recommendation based on a gene assay in accordance with some embodiments. 
         FIG. 12  illustrates a block diagram of an example machine upon which any one or more of the techniques (e.g., methodologies) discussed herein may perform. In alternative aspects, the machine may operate as a standalone device or may be connected (e.g., networked) to other machines. 
     
    
    
     DETAILED DESCRIPTION 
     Methods, devices, and kits are described herein for selecting individualized hypertension treatment regimens that are more effective than currently available regimens. The methods, devices, and kits include assays for identifying genetic variants in individual subjects that make the individual more or less responsive to specific medications. When the appropriate medication is administered, the subject&#39;s blood pressure is appropriately controlled, and side effects are avoided. Genetic variants present in genes such as ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptor, renin. Na +  channels (such as SCNN1A), adducin, sodium (Na + ) chloride (Cl − ) co-transporters (such as SLC12A3), and/or WNK1 genes are correlated with heightened or reduced responsiveness to various blood pressure medications. Although there are a number of hypertension drugs available on the market, subjects react differently to such drugs. The kits, methods, and devices described herein are useful for detecting which subjects benefit from which therapeutic regimen. 
     High Blood Pressure (Hypertension) 
     The development of high blood pressure in humans is the result of one or more of three physiologic maladaptations: 1) elevated cardiac output (liters of blood ejected from the heart per minute) that increases the amount of blood pressing against the vessels, 2) relatively narrow blood vessels that results in increased pressure towards the lumen of the blood vessel due to Poiseuille&#39;s Law (which describes the inverse relationship between the diameter of a tube (vessel) and the pressure against the walls of the tube (vessel), all else being equal), or 3) increased sodium (Na + ) absorption in the kidney which results in increased blood volume (and the amount of blood pumped per minute, cardiac output) and subsequently increased outward pressure against the tubes (vessels). 
     Blood pressure is highly regulated and tightly controlled in humans, such that in the event of a drop in cardiac output, the heart sends a signal to the kidneys (via the proteins atrial natriuretic peptide and brain-type natriuretic peptide, among others) and vessels with the ultimate function of increasing Na +  reabsorption to increase plasma volume and vasoconstriction to increase blood pressure. Similarly, if there is a drop in blood pressure, there is an increase in cardiac output (primarily via an immediate increase in heart rate through inhibition of the parasympathetic nervous system) to compensate and an increase in the renin-angiotensin aldosterone system which results in both constriction of blood vessels (which increases blood pressure) and an increase in Na +  and, therefore, fluid retention in the kidney, which increases plasma volume and can increase blood pressure. Hence, the human body provides redundant functions to maintain blood pressure both in the short-term and in the long-term, by regulating the interplay between the heart, blood vessels, and kidney.  FIG. 1  is a schematic diagram illustrating the interplay between the heart, blood vessels, and kidneys involved in regulating blood pressure. 
     Blood pressure therapy following diagnosis traditionally follows a regimented algorithm based on therapies effective across the general population. Currently, clinicians start a patient who has been diagnosed with high blood pressure on a diuretic (to reduce renal Na +  reabsorption). If such a diuretic does not work within a period of time, the clinician next tries a vasodilator, and if this is not effective, then a clinician will lastly prescribe a beta-blocker. This trial-and-error process to lower a patient&#39;s blood pressure can take several months, is costly, and threatens the health of the patient because the patient&#39;s hypertension is frequently not adequately controlled in a timely manner. 
     Such currently available methods are in stark contrast to the methods, devices, and kits described herein that involve specifically testing an individual&#39;s genetic profile and, as illustrated herein, basing therapeutic treatment on the results of such testing. Hence, the methods, devices, and kits described herein for evaluating a blood pressure genetic panel to improve treatment of hypertensive subjects, by quickly identifying more effective medications, thereby avoiding side effects and delays in treatment. Applicants&#39; methods are therefore an improvement over the currently available trial-and-error procedures that frequently result in side effects and delays in effective treatment. 
     Ranking of Genotypes that Affect Therapy 
     The genotype of a subject can significantly affect the response of the subject to blood pressure medications because certain functional polymorphisms have greater effects on the physiology of a subject than others. The following is a summary of polymorphisms in order of their impact on blood pressure and which drugs should be employed by subjects with such genetic variations.
         1. Subjects with the WNK1 polymorphism defined by rs1159744 (SEQ ID NO:34, with cytosine at the variable position), benefit more from loop diuretics.   2. Subjects with the WNK1 polymorphism defined by rs2107614 (SEQ ID NO:33, with cytosine at the variable position), benefit more from loop diuretics.   3. Subjects with the ADD1 polymorphism defined by rs4961 (SEQ ID NO:27, with thymine at the variable position), benefit more from hydrochlorothiazides.   4. Subjects with the SLC12A3 polymorphism defined by rs1529927 (SEQ ID NO:30, with thymine at the variable position), benefit more from hydrochlorothiazides.
 
Any homozygous combination at one or more of the WNK1 Rs1159744, WNK1 Rs2107614, ADD1 Rs4961, and SLC12A3 rs1529927 polymorphisms means that diuretics should be the first-line therapy, especially if the patient is heterozygous for polymorphisms in genes responsive to beta-blockers or vasodilators.
   5. Subjects with the CYP2D6 polymorphism defined by Rs3892097 (SEQ ID NO: 10, with adenine at the variable position), plus the ADRB1 polymorphism defined by rs1801253 (SEQ ID NO:3, with cytosine as the variable nucleotide), plus the ADRB1 polymorphism defined by rs1801252 (SEQ ID NO:2, with adenine as the variable nucleotide) benefit from beta-blockade classes of drugs.   6. Subjects with the renin polymorphism defined by rs12750834 (SEQ ID NO:20, with cytosine as the variable nucleotide) plus the AGT1R polymorphism defined by rs5186 (SEQ ID NO:16, with cytosine as the variable nucleotide), which affect responses to angiotensin II receptor blockers.   7. Subjects with the ACE deletion defined by Rs1799752 (SEQ ID NO:35) and the AGT polymorphism defined by rs699 (SEQ ID NO:14, with cytosine as the variable nucleotide) can benefit from the combination of an angiotensin II receptor blocker and an ACE inhibitor.   8. Subjects with the SCNN1A polymorphism defined by rs2228576 (SEQ ID NO:22, with adenine as the variable position) can benefit from administration of amiloride.   9. Subjects with the ADRB2 polymorphism defined by rs1042714 (SEQ ID NO:7, with guanine as the variable nucleotide) can benefit from administration of a non-selective beta-blockade.   10. Subjects with the ADRB2 polymorphism defined by s1042713 (SEQ ID NO:6, with guanine as the variable nucleotide) can benefit from administration of a non-selective beta-blockade.
 
All patients do not respond to same. Some subjects have genotypes that can significantly affect their response to medications. When clinicians employ currently available procedures (diuretic first, then vasodilator, then beta blocker), some patients will benefit but others will not respond or will respond negatively. Hence, some patients would benefit from initial administration of a vasodilator or a beta-blocker, rather than a diuretic.
       

     Beta-Blocker Responsive Polypeptides and Nucleic Acids 
     There are two primary receptors within the heart that influence both heart rate (chronotropic effect) and heart contractility (inotrpic effect) (Brodde.  Am J Cardiol  62:24C-29C (1988), the beta-1 adrenergic receptors (β 1 AR, encoded by the ADRB1 gene) and the beta-2 adrenergic receptors (β 2 AR, encoded by the ADRB2 gene). 
     The heart is primary comprised of beta-1 adrenergic receptors, which are located on 80% of the ventricular wall surface, 70% of the atrial wall surface, and 95% of the sino-atrial (SA) node. The atria of the heart receive blood that returns from the body (right atria) of lungs (left atria) whereas the ventricles pump blood to the lungs (right ventricle) and body (left ventricle). The sino-atrial node primarily controls heart rate. 
     Although heart rate and cardiac contractility are primarily regulated by β 1 AR, the β 2 AR also play a role, primarily in cardiac contractility. Stimulation of either β 1 AR or β 2 AR can influence heart rate and cardiac contractility through increases in intracellular c-AMP and protein kinase A (PKA) which, ultimately, alter Ca + -channel sensitivity and reduce the threshold needed for an action potential. Therefore, cardiac output (and blood pressure) can be increased through increases in β 1 AR and/or β 2 AR activities. If a variant β 1 AR or β 2 AR gene encodes a more functional receptor, cardiac output is increased. 
     β 1 AR and β 2 AR activities can be modulated through the use of selective (e.g., atenolol and metoprolol) and non-selective (e.g., propranolol and carvedilol) beta-blockers. The selective beta-blockers are selective for inhibiting the β 1 AR. The non-selective beta-blockers inhibit both β 1 AR and β 2 AR. Both types of beta-blockers tend to decrease blood pressure through a decrease in heart rate and cardiac contractility, which ultimately results in a decrease in cardiac output. Similarly, the administration of a β 2 AR-agonist (e.g., albuterol sulfate) tends to increase cardiac output and heart rate (Snyder et al.,  Pharmacotherapy  31:748-756 (2011)). Thus, both β 1 AR and β 2 AR are important in the regulation of cardiac output. 
     Just as stimulation of these receptors can elevate cardiac output and increase blood pressure, so too can genetic variation of the genes that encode β 1 AR and β 2 AR (ADRB1 and ADRB2) elevate receptor activity and increase blood pressure. Conversely, some ADRB1 and ADRB2 genetic variants encode receptors with reduced activity. In addition, some ADRB1 and ADRB2 genetic variants exhibit reduced, or enhanced, responsiveness to blood pressure medications such as beta-blockers. Not all individuals respond similarly to beta-blockade, despite similar clinical and environmental conditions. As described herein, the effectiveness of beta-blockers is dependent to some extent upon the genetic make-up of the subjects to which the beta-blockers are administered. 
     Sequences for various adrenergic receptors are available, for example, from the National Center for Biotechnology Information (see website at ncbi.nlm.nih.gov). 
     For example, a full length human ADRB1 cDNA nucleotide sequence is available from the database maintained by the National Center for Biotechnology Information (see website at ncbi.nlm.nih.gov), which has accession number NM_00064 (GI:110349783), and is shown below as SEQ ID NO: 1. 
     
       
         
           
               
               
            
               
                    1 
                 GCACCACGCC GCCCGGGCTT CTGGGGTGTT CCCCAACCAC 
               
               
                   
               
               
                   41 
                 GGCCCAGCCC TGCCACACCC CCCGCCCCCG GCCTCCGCAG 
               
               
                   
               
               
                   81 
                 CTCGGCATGG GCGCGGGGGT GCTCGTCCTG GGCGCCTCCG 
               
               
                   
               
               
                  121 
                 AGCCCGGTAA CCTGTCGTCG GCCGCACCGC TCCCCGACGG 
               
               
                   
               
               
                  161 
                 CGCGGCCACC GCGGCGCGGC TGCTGGTGCC CGCGTCGCCG 
               
               
                   
               
               
                  201 
                 CCCGCCTCGT TGCTGCCTCC CGCCAGCGAA    A   GCCCCGAGC 
               
               
                   
               
               
                  241 
                 CGCTGTCTCA GCAGTGGACA GCGGGCATGG GTCTGCTGAT 
               
               
                   
               
               
                  281 
                 GGCGCTCATC GTGCTGCTCA TCGTGGCGGG CAATGTGCTG 
               
               
                   
               
               
                  321 
                 GTGATCGTGG CCATCGCCAA GACGCCGCGG CTGCAGACGC 
               
               
                   
               
               
                  361 
                 TCACCAACCT CTTCATCATG TCCCTGGCCA GCGCCGACCT 
               
               
                   
               
               
                  401 
                 GGTCATGGGG CTGCTGGTGG TGCCGTTCGG GGCCACCATC 
               
               
                   
               
               
                  441 
                 GTGGTGTGGG GCCGCTGGGA GTACGGCTCC TTCTTCTGCG 
               
               
                   
               
               
                  481 
                 AGCTGTGGAC CTCAGTGGAC GTGCTGTGCG TGACGGCCAG 
               
               
                   
               
               
                  521 
                 CATCGAGACC CTGTGTGTCA TTGCCCTGGA CCGCTACCTC 
               
               
                   
               
               
                  561 
                 GCCATCACCT CGCCCTTCCG CTACCAGAGC CTGCTGACGC 
               
               
                   
               
               
                  601 
                 GCGCGCGGGC GCGGGGCCTC GTGTGCACCG TGTGGGCCAT 
               
               
                   
               
               
                  641 
                 CTCGGCCCTG GTGTCCTTCC TGCCCATCCT CATGCACTGG 
               
               
                   
               
               
                  681 
                 TGGCGGGCGG AGAGCGACGA GGCGCGCCGC TGCTACAACG 
               
               
                   
               
               
                  721 
                 ACCCCAAGTG CTGCGACTTC GTCACCAACC GGGCCTACGC 
               
               
                   
               
               
                  761 
                 CATCGCCTCG TCCGTAGTCT CCTTCTACGT GCCCCTGTGC 
               
               
                   
               
               
                  801 
                 ATCATGGCCT TCGTGTACCT GCGGGTGTTC CGCGAGGCCC 
               
               
                   
               
               
                  841 
                 AGAAGCAGGT GAAGAAGATC GACAGCTGCG AGCGCCGTTI 
               
               
                   
               
               
                  881 
                 CCTCGGCGGC CCAGCGCGGC CGCCCTCGCC CTCGCCCTCG 
               
               
                   
               
               
                  921 
                 CCCGTCCCCG CGCCCGCGCC GCCGCCCGGA CCCCCGCGCC 
               
               
                   
               
               
                  961 
                 CCGCCGCCGC CGCCGCCACC GCCCCGCTGG CCAACGGGCG 
               
               
                   
               
               
                 1001 
                 TGCGGGTAAG CGGCGGCCCT CGCGCCTCGT GGCCCTGCGC 
               
               
                   
               
               
                 1041 
                 GAGCAGAAGG CGCTCAAGAC GCTGGGCATC ATCATGGGCG 
               
               
                   
               
               
                 1081 
                 TCTTCACGCT CTGCTGGCTG CCCTTCTTCC TGGCCAACGT 
               
               
                   
               
               
                 1121 
                 GGTGAAGGCC TTCCACCGCG AGCTGGTGCC CGACCGCCTC 
               
               
                   
               
               
                 1161 
                 TTCGTCTTCT TCAACTGGCT GGGCTACGCC AACTCGGCCT 
               
               
                   
               
               
                 1201 
                 TCAACCCCAT CATCTACTGC CGCAGCCCCG ACTTCCGCAA 
               
               
                   
               
               
                 1241 
                 GGCCTTCCAG    G   GACTGCTCT GCTGCGCGCG CAGGGCTGCC 
               
               
                   
               
               
                 1281 
                 CGCCGGCGCC ACGCGACCCA CGGAGACCGG CCGCGCGCCT 
               
               
                   
               
               
                 1321 
                 CGGGCTGTCT GGCCCGGCCC GGACCCCCGC CATCGCCCGG 
               
               
                   
               
               
                 1361 
                 GGCCGCCTCG GACGACGACG ACGACGATGT CGTCGGGGCC 
               
               
                   
               
               
                 1401 
                 ACGCCGCCCG CGCGCCTGCT GGAGCCCTGG GCCGGCTGCA 
               
               
                   
               
               
                 1441 
                 ACGGCGGGGC GGCGGCGGAC AGCGACTCGA GCCTGGACGA 
               
               
                   
               
               
                 1481 
                 GCCGTGCCGC CCCGGCTTCG CCTCGGAATC CAAGGTGTAG 
               
               
                   
               
               
                 1521 
                 GGCCCGGCGC GGGGCGCGGA CTCCGGGCAC GGCTTCCCAG 
               
               
                   
               
               
                 1561 
                 GGGAACGAGG AGATCTGTGT TTACTTAAGA CCGATAGCAG 
               
               
                   
               
               
                 1601 
                 GTGAACTCGA AGCCCACAAT CCTCGTCTGA ATCATCCGAG 
               
               
                   
               
               
                 1641 
                 GCAAAGAGAA AAGCCACGGA CCGTTGCACA AAAAGGAAAG 
               
               
                   
               
               
                 1681 
                 TTTGGGAAGG GATGGGAGAG TGGCTTGCTG ATGTTCCTTG 
               
               
                   
               
               
                 1721 
                 TTGTTTTTTT TTTCTTTTCT TTTCTTTCTT CTTCTTTTTT 
               
               
                   
               
               
                 1741 
                 TTTTTTTTTT TTTTTTCTGT TTGTGGTCCG GCCTTCTTTT 
               
               
                   
               
               
                 1801 
                 GTGTGTGCGT GTGATGCATC TTTAGATTTT TTTCCCCCAC 
               
               
                   
               
               
                 1841 
                 CAGGTGGTTT TTGACACTCT CTGAGAGGAC CGGAGTGGAA 
               
               
                   
               
               
                 1881 
                 GATGGGTGGG TTAGGGGAAG GGAGAAGCAT TAGGAGGGGA 
               
               
                   
               
               
                 1921 
                 TTAAAATCGA TCATCGTGGC TCCCATCCCT TTCCCGGGAA 
               
               
                   
               
               
                 1961 
                 CAGGAACACA CTACCAGCCA GAGAGAGGAG AATGACAGTT 
               
               
                   
               
               
                 2001 
                 TGTCAAGACA TATTTCCTTT TGCTTTCCAG AGAAATTTCA 
               
               
                   
               
               
                 2041 
                 TTTTAATTTC TAAGTAATGA TTTCTGCTGT TATGAAAGCA 
               
               
                   
               
               
                 2081 
                 AAGAGAAAGG ATGGAGGCAA AATAAAAAAA AATCACGTTT 
               
               
                   
               
               
                 2121 
                 CAAGAAATGT TAAGCTCTTC TTGGAACAAG CCCCACCTTG 
               
               
                   
               
               
                 2161 
                 CTTTCCTTGT GTAGGGCAAA CCCGCTGTCC CCCGCGCGCC 
               
               
                   
               
               
                 2201 
                 TGGGTGGTCA GGCTGAGGGA TTTCTACCTC ACACTGTGCA 
               
               
                   
               
               
                 2241 
                 TTTGCACAGC AGATAGAAAG ACTTGTTTAT ATTAAACAGC 
               
               
                   
               
               
                 2281 
                 TTATTTATGT ATCAATATTA GTTGGAAGGA CCAGGCGCAG 
               
               
                   
               
               
                 2321 
                 AGCCTCTCTC TGTGACATGT GACTCTGTCA ATTGAAGACA 
               
               
                   
               
               
                 2361 
                 GGACATTAAA AGAGAGCGAG AGAGAGAAAC AGTTCAGATT 
               
               
                   
               
               
                 2401 
                 ACTGCACATG TGGATAAAAA CAAAAACAAA AAAAAGGAGT 
               
               
                   
               
               
                 2441 
                 GGTTCAAAAT GCCATTTTTG CACAGTGTTA GGAATTACAA 
               
               
                   
               
               
                 2481 
                 AATCCACAGA AGATGTTACT TGCACAAAAA GAAATTAAAT 
               
               
                   
               
               
                 2521 
                 ATTTTTTAAA GGGAGAGGGG CTGGGCAGAT CTTAAATAAA 
               
               
                   
               
               
                 2561 
                 ATTCAAACTC TACTTCTGTT GTCTAGTATG TTATTGAGCT 
               
               
                   
               
               
                 2601 
                 AATGATTCAT TGGGAAAATA CCTTTTTATA CTCCTTTATC 
               
               
                   
               
               
                 2641 
                 ATGGTACTGT AACTGTATCC ATATTATAAA TATAATTATC 
               
               
                   
               
               
                 2681 
                 TTAAGGATTT TTTATTTTTT TTTATGTCCA AGTGCCCACG 
               
               
                   
               
               
                 2721 
                 TGAATTTGCT GGTGAAAGTT AGCACTTGTG TGTAAATTCT 
               
               
                   
               
               
                 2761 
                 ACTTCCTCTT GTGTGTTTTA CCAAGTATTT ATACTCTGGT 
               
               
                   
               
               
                 2801 
                 GCAACTAACT ACTGTGTGAG GAATTGGTCC ATGTGCAATA 
               
               
                   
               
               
                 2841 
                 AATACCAATG AAGCACAATC AA 
               
            
           
         
       
     
     The rs1801252 single nucleotide polymorphism (SNP) is present in the ADRB1 gene, where the variable nucleotide at about position 231 (underlined) can be adenine in some individuals and guanine in others. The rs1801252 sequence (SEQ ID NO:2) is shown below, where the underlined A/G is the SNP. 
     
       
         
           
               
            
               
                 CTCGTTGCTGCCTCCCGCCAGCGAA   [   A/G   ]   GCCCCGAGCCGCTGTCTCAG 
               
               
                   
               
               
                 CAGTG. 
               
            
           
         
       
     
     The rs1801253 single nucleotide polymorphism (SNP) is also present in the ADRB1 gene, where the variable nucleotide at about position 1251 (underlined) can be guanine in some individuals and cytosine in others. The rs1801253 sequence (SEQ ID NO:3) is shown below, where the underlined C/G is the SNP. 
     
       
         
           
               
            
               
                 CCCCGACTTCCGCAAGGCCTTCCAG   [   C/G   ]   GACTGCTCTGCTGCGCGCGC 
               
               
                   
               
               
                 AGGGC. 
               
            
           
         
       
     
     The β 1 AR polypeptide encoded by the ADRB1 cDNA with SEQ ID NO:1 has the following sequence (SEQ ID NO:4). 
     
       
         
           
               
               
            
               
                   1 
                 MGAGVLVLGA SEPGNLSSAA PLPDGAATAA RLLVPASPPA 
               
               
                   
               
               
                  41 
                 SLLPPASE   S   P EPLSQQWTAG MGLLMALIVL LIVAGNVLVI 
               
               
                   
               
               
                  81 
                 VAIAKTPRLQ TLTNLFIMSL ASADLVMGLL VVPFGATIVV 
               
               
                   
               
               
                 121 
                 WGRWEYGSFF CELWTSVDVL CVTASIETLC VIALDRYLAI 
               
               
                   
               
               
                 161 
                 TSPFRYQSLL TRARARGLVC TVWAISALVS FLPILMHWWR 
               
               
                   
               
               
                 201 
                 AESDEARRCY NDPKCCDFVT NRAYAIASSV VSFYVPLCIM 
               
               
                   
               
               
                 241 
                 AFVYLRVFRE AQKQVKKIDS CERRFLGGPA RPPSPSPSPV 
               
               
                   
               
               
                 281 
                 PAPAPPPGPP RPAAAAATAP LANGRAGKRR PSRLVALREQ 
               
               
                   
               
               
                 321 
                 KALKTLGIIM GVFTLCWLPF FLANVVKAFH RELVPDRLFV 
               
               
                   
               
               
                 361 
                 FFNWLGYANS AFNPIIYCRS PDFRKAFQ   G   L LCCARRAARR 
               
               
                   
               
               
                 401 
                 RHATHGDRPR ASGCLARPGP PPSPGAASDD DDDDVVGATP 
               
               
                   
               
               
                 441 
                 PARLLEPWAG CNGGAAADSD SSLDEPCRPG FASESKV 
               
            
           
         
       
     
     Note that the underlined amino acid at position 49 is serine because some individuals have SEQ ID NO:1 or 2, where the variable nucleotide at about position 231 of SEQ ID NO: 1 is adenine. However, position 49 of SEQ ID NO:4 can be glycine in some individuals because those individual have guanine at nucleotide position 231 in SEQ ID NO:1. 
     Note also that the glycine at position 389 is an arginine (instead of glycine) as shown for SEQ ID NO:4 when position 1251 of SEQ ID NO:1 is a cytosine. 
     Individuals with serine at β 1 AR amino acid position 49 and/or arginine at position 389 are more responsive to beta-blockers than those with glycines at these positions. Hence, for example, an individual who expresses the β 1 AR polypeptide with SEQ ID NO:4, will be more responsive to beta-blockers than an individual who expresses the β 1 AR polypeptide with glycines at both positions 49 and 389. 
     A full length human ADRB2 cDNA nucleotide sequence is available from the database maintained by the National Center for Biotechnology Information (see website at ncbi.nlm.nih.gov), which has accession number NM_000024 (GI:283483994), and is shown below as SEQ ID NO:5. 
     
       
         
           
               
               
            
               
                    1 
                 GCACATAACG GGCAGAACGC ACTGCGAAGC GGCTTCTTCA 
               
               
                   
               
               
                   41 
                 GAGCACGGGC TGGAACTGGC AGGCACCGCG AGCCCCTAGC 
               
               
                   
               
               
                   81 
                 ACCCGACAAG CTGAGTGTGC AGGACGAGTC CCCACCACAC 
               
               
                   
               
               
                  121 
                 CCACACCACA GCCGCTGAAT GAGGCTTCCA GGCGTCCGCT 
               
               
                   
               
               
                  161 
                 CGCGGCCCGC AGAGCCCCGC CGTGGGTCCG CCCGCTGAGG 
               
               
                   
               
               
                  201 
                 CGCCCCCAGC CAGTGCGCTC ACCTGCCAGA CTGCGCGCCA 
               
               
                   
               
               
                  241 
                 TGGGGCAACC CGGGAACGGC AGCGCCTTCT TGCTGGCACC 
               
               
                   
               
               
                  281 
                 CAAT   A   GAAGC CATGCGCCGG ACCACGACGT CACGCAG   C   AA 
               
               
                   
               
               
                  321 
                 AGGGACGAGG TGTGGGTGGT GGGCATGGGC ATCGTCATGT 
               
               
                   
               
               
                  361 
                 CTCTCATCGT CCTGGCCATC GTGTTTGGCA ATGTGCTGGT 
               
               
                   
               
               
                  401 
                 CATCACAGCC ATTGCCAAGT TCGAGCGTCT GCAGACGGTC 
               
               
                   
               
               
                  441 
                 ACCAACTACT TCATCACTTC ACTGGCCTGT GCTGATCTGG 
               
               
                   
               
               
                  481 
                 TCATGGGCCT GGCAGTGGTG CCCTTTGGGG CCGCCCATAT 
               
               
                   
               
               
                  521 
                 TCTTATGAAA ATGTGGACTT TTGGCAACTT CTGGTGCGAG 
               
               
                   
               
               
                  561 
                 TTTTGGACTT CCATTGATGT GCTGTGCGTC ACGGCCAGCA 
               
               
                   
               
               
                  601 
                 TTGAGACCCT GTGCGTGATC GCAGTGGATC GCTACTTTGC 
               
               
                   
               
               
                  641 
                 CATTACTTCA CCTTTCAAGT ACCAGAGCCT GCTGACCAAG 
               
               
                   
               
               
                  681 
                 AATAAGGCCC GGGTGATCAT TCTGATGGTG TGGATTGTGT 
               
               
                   
               
               
                  721 
                 CAGGCCTTAC CTCCTTCTTG CCCATTCAGA TGCACTGGTA 
               
               
                   
               
               
                  761 
                 CCGGGCCACC CACCAGGAAG CCATCAACTG CTATGCCAAT 
               
               
                   
               
               
                  801 
                 GAGACCTGCT GTGACTTCTT CACGAACCAA GCCTATGCCA 
               
               
                   
               
               
                  841 
                 TTGCCTCTTC CATCGTGTCC TTCTACGTTC CCCTGGTGAT 
               
               
                   
               
               
                  881 
                 CATGGTCTTC GTCTACTCCA GGGTCTTTCA GGAGGCCAAA 
               
               
                   
               
               
                  921 
                 AGGCAGCTCC AGAAGATTGA CAAATCTGAG GGCCGCTTCC 
               
               
                   
               
               
                  961 
                 ATGTCCAGAA CCTTAGCCAG GTGGAGCAGG ATGGGCGGAC 
               
               
                   
               
               
                 1001 
                 GGGGCATGGA CTCCGCAGAT CTTCCAAGTT CTGCTTGAAG 
               
               
                   
               
               
                 1041 
                 GAGCACAAAG CCCTCAAGAC GTTAGGCATC ATCATGGGCA 
               
               
                   
               
               
                 1081 
                 CTTTCACCCT CTGCTGGCTG CCCTTCTTCA TCGTTAACAT 
               
               
                   
               
               
                 1121 
                 TGTGCATGTG ATCCAGGATA ACCTCATCCG TAACGAAGTT 
               
               
                   
               
               
                 1161 
                 TACATCCTCC TAAATTGGAT AGGCTATGTC AATTCTGGTT 
               
               
                   
               
               
                 1201 
                 TCAATCCCCT TATCTACTGC CGGAGCCCAG ATTTCAGGAT 
               
               
                   
               
               
                 1241 
                 TGCCTTCCAG GAGCTTCTGT GCCTGCGCAG GTCTTCTTTG 
               
               
                   
               
               
                 1281 
                 AAGGCCTATG GGAATGGCTA CTCCAGCAAC GGCAACACAG 
               
               
                   
               
               
                 1321 
                 GGGAGCAGAG TGGATATCAC GTGGAACAGG AGAAAGAAAA 
               
               
                   
               
               
                 1361 
                 TAAACTGCTG TGTGAAGACC TCCCAGGCAC GGAAGACTTT 
               
               
                   
               
               
                 1401 
                 GTGGGCCATC AAGGTACTGT GCCTAGCGAT AACATTGATT 
               
               
                   
               
               
                 1441 
                 CACAAGGGAG GAATTGTAGT ACAAATGACT CACTGCTGTA 
               
               
                   
               
               
                 1481 
                 AAGCAGTTTT TCTACTTTTA AAGACCCCCC CCCCCAACAG 
               
               
                   
               
               
                 1521 
                 AACACTAAAC AGACTATTTA ACTTGAGGGT AATAAACTTA 
               
               
                   
               
               
                 1561 
                 GAATAAAATT GTAAAATTGT ATAGAGATAT GCAGAAGGAA 
               
               
                   
               
               
                 1601 
                 GGGCATCCTT CTGCCTTTTT TATTTTTTTA AGCTGTAAAA 
               
               
                   
               
               
                 1641 
                 AGAGAGAAAA CTTATTTGAG TGATTATTTG TTATTTGTAC 
               
               
                   
               
               
                 1681 
                 AGTTCAGTTC CTCTTTGCAT GGAATTTGTA AGTTTATGTC 
               
               
                   
               
               
                 1721 
                 TAAAGAGCTT TAGTCCTAGA GGACCTGAGT CTGCTATATT 
               
               
                   
               
               
                 1761 
                 TTCATGACTT TTCCATGTAT CTACCTCACT ATTCAAGTAT 
               
               
                   
               
               
                 1801 
                 TAGGGGTAAT ATATTGCTGC TGGTAATTTG TATCTGAAGG 
               
               
                   
               
               
                 1841 
                 AGATTTTCCT TCCTACACCC TTGGACTTGA GGATTTTGAG 
               
               
                   
               
               
                 1881 
                 TATCTCGGAC CTTTCAGGTG TGAACATGGA CTCTTCCCCC 
               
               
                   
               
               
                 1921 
                 ACTCCTCTTA TTTGCTCACA CGGGGTATTT TAGGCAGGGA 
               
               
                   
               
               
                 1961 
                 TTTGAGGAGC AGCTTCAGTT GTTTTCCCGA GCAAAGTCTA 
               
               
                   
               
               
                 2001 
                 AAGTTTACAG TAAATAAATT GTTTGACCAT GCCTTCATTG 
               
               
                   
               
               
                 2041 
                 CAAAAAAAAA AAAAAAAA 
               
            
           
         
       
     
     The rs1042713 single nucleotide polymorphism (SNP) is present in the ADRB2 gene, where the variable nucleotide at about position 285 (underlined) can be in adenine some individuals and guanine in others. The rs1042713 sequence (SEQ ID NO:6) is shown below, where the underlined A/G is the SNP. 
     
       
         
           
               
            
               
                 CAGCGCCTTCTTGCTGGCACCCAAT   [   A/G   ]   GAAGCCATGCGCCGGACCAC 
               
               
                 CACGT. 
               
            
           
         
       
     
     The rs1042714 single nucleotide polymorphism (SNP) is also present in the ADRB2 gene, where the variable nucleotide at about position 318 (underlined) can be cytosine in some individuals and guanine in others. The rs1042714 sequence (SEQ ID NO:7) is shown below, where the underlined C/G is the SNP. 
     
       
         
           
               
            
               
                 TGCGCCGGACCACGACGTCACGCAG   [   C/G   ]   AAAGGGACGAGGTGTGGGTG 
               
               
                   
               
               
                 GTGGG. 
               
            
           
         
       
     
     The β 2 AR polypeptide encoded by the ADRB2 cDNA with SEQ ID NO:5 has the following sequence (SEQ ID NO:8). 
     
       
         
           
               
               
            
               
                   1 
                 MGQPGNGSAF LLAPN   R   SHAP DHDVTQ   Q   RDE VWVVGMGIVM 
               
               
                   
               
               
                  41 
                 SLIVLAIVFG NVLVITAIAK FERLQTVTNY FITSLACADL 
               
               
                   
               
               
                  81 
                 VMGLAVVPFG AAHILMKMWT FGNFWCEFWT SIDVLCVTAS 
               
               
                   
               
               
                 121 
                 IETLCVIAVD RYFAITSPFK YQSLLTKNKA RVIILMVWIV 
               
               
                   
               
               
                 161 
                 SGLTSFLPIQ MHWYRATHQE AINCYANETC CDFFTNQAYA 
               
               
                   
               
               
                 201 
                 IASSIVSFYV PLVIMVFVYS RVFQEAKRQL QKIDKSEGRF 
               
               
                   
               
               
                 241 
                 HVQNLSQVEQ DGRTGHGLRR SSKFCLKEHK ALKTLGIIMG 
               
               
                   
               
               
                 281 
                 TFTLCWLPFF IVNIVHVIQD NLIRKEVYIL LNWIGYVNSG 
               
               
                   
               
               
                 321 
                 FNPLIYCRSP DFRIAFQELL CLRRSSLKAY GNGYSSNGNT 
               
               
                   
               
               
                 361 
                 GEQSGYHVEQ EKENKLLCED LPGTEDFVGH QGTVPSDNID 
               
               
                   
               
               
                 401 
                 SQGRNCSTND SLL 
               
            
           
         
       
     
     Note that the underlined arginine at position 16 of SEQ ID NO:8 is arginine because some individuals have nucleotide sequence SEQ ID NO:5, where the nucleotide at about position 285 is adenine. However, position 16 of SEQ ID NO:8 can be glycine in some individuals because those individuals have guanine at nucleotide position 285 in SEQ ID NO:5. 
     Note also that the glutamine at position 27 of SEQ ID NO:8 is a glutamic acid when position 318 of nucleotide sequence SEQ ID NO:5 is a guanine. 
     Individuals with glycine at position 16 and/or glutamic acid at β 2 AR position 27 are more responsive to beta-blockers than those with arginine and glutamine, respectively, at these positions. Hence, for example, an individual who expresses the β 2 AR polypeptide with SEQ ID NO:5, will be more responsive to beta-blockers than an individual who expresses the β 2 AR polypeptide with arginine and glutamine at positions 16 and 27. 
     The gene that encodes cytochrome P450 2D6 (CYP2D6) has been shown to alter the metabolism of the drugs in the beta-blocker class. This alteration in drug metabolism can alter the amount of bioavailable drug. Poor drug metabolizers tend to have more drugs available in the body for longer and will, therefore, have a greater response to therapy. In contrast, active metabolizers of a drug will have less of the drug available in their system and will respond poorly to therapy. 
     Because of the importance of CYP2D6 on beta-blocker metabolism, this gene is a useful marker of responsive to beta-blocker therapy. 
     A full length human CYP2D6 cDNA nucleotide sequence is available from the database maintained by the National Center for Biotechnology Information (see website at ncbi.nlm.nih.gov), which has accession number NM_000106.5 GI:392513720), and is shown below as SEQ ID NO:9. 
     
       
         
           
               
               
            
               
                    1 
                 GTGCTGAGAG TGTCCTGCCT GGTCCTCTGT GCCTGGTGGG 
               
               
                   
               
               
                   41 
                 GTGGGGGTGC CAGGTGTGTC CAGAGGAGCC CATTTGGTAG 
               
               
                   
               
               
                   81 
                 TGAGGCAGGT ATGGGGCTAG AAGCACTGGT GCCCCTGGCC 
               
               
                   
               
               
                  121 
                 GTGATAGTGG CCATCTTCCT GCTCCTGGTG GACCTGATGC 
               
               
                   
               
               
                  161 
                 ACCGGCGCCA ACGCTGGGCT GCACGCTACC CACCAGGCCC 
               
               
                   
               
               
                  201 
                 CCTGCCACTG CCCGGGCTGG GCAACCTGCT GCATGTGGAC 
               
               
                   
               
               
                  241 
                 TTCCAGAACA CACCATACTG CTTCGACCAG TTGCGGCGCC 
               
               
                   
               
               
                  281 
                 GCTTCGGGGA CGTGTTCAGC CTGCAGCTGG CCTGGACGCC 
               
               
                   
               
               
                  321 
                 GGTGGTCGTG CTCAATGGGC TGGCGGCCGT GCGCGAGGCG 
               
               
                   
               
               
                  361 
                 CTGGTGACCC ACGGCGAGGA CACCGCCGAC CGCCCGCCTG 
               
               
                   
               
               
                  401 
                 TGCCCATCAC CCAGATCCTG GGTTTCGGGC CGCGTTCCCA 
               
               
                   
               
               
                  441 
                 AGGGGTGTTC CTGGCGCGCT ATGGGCCCGC GTGGCGCGAG 
               
               
                   
               
               
                  481 
                 CAGAGGCGCT TCTCCGTGTC CACCTTGCGC AACTTGGGCC 
               
               
                   
               
               
                  521 
                 TGGGCAAGAA GTCGCTGGAG CAGTGGGTGA CCGAGGAGGC 
               
               
                   
               
               
                  561 
                 CGCCTGCCTT TGTGCCGCCT TCGCCAACCA CTCC   G   GACGC 
               
               
                   
               
               
                  601 
                 CCCTTTCGCC CCAACGGTCT CTTGGACAAA GCCGTGAGCA 
               
               
                   
               
               
                  641 
                 ACGTGATCGC CTCCCTCACC TGCGGGCGCC GCTTCGAGTA 
               
               
                   
               
               
                  681 
                 CGACGACCCT CGCTTCCTCA GGCTGCTGGA CCTAGCTCAG 
               
               
                   
               
               
                  721 
                 GAGGGACTGA AGGAGGAGTC GGGCTTTCTG CGCGAGGTGC 
               
               
                   
               
               
                  761 
                 TGAATGCTGT CCCCGTCCTC CTGCATATCC CAGCGCTGGC 
               
               
                   
               
               
                  801 
                 TGGCAAGGTC CTACGCTTCC AAAAGGCTTT CCTGACCCAG 
               
               
                   
               
               
                  841 
                 CTGGATGAGC TGCTAACTGA GCACAGGATG ACCTGGGACC 
               
               
                   
               
               
                  881 
                 CAGCCCAGCC CCCCCGAGAC CTGACTGAGG CCTTCCTGGC 
               
               
                   
               
               
                  921 
                 AGAGATGGAG AAGGCCAAGG GGAACCCTGA GAGCAGCTTC 
               
               
                   
               
               
                  961 
                 AATGATGAGA ACCTGCGCAT AGTGGTGGCT GACCTGTTCT 
               
               
                   
               
               
                 1001 
                 CTGCCGGGAT GGTGACCACC TCGACCACGC TGGCCTGGGG 
               
               
                   
               
               
                 1041 
                 CCTCCTGCTC ATGATCCTAC ATCCGGATGT GCAGCGCCGT 
               
               
                   
               
               
                 1081 
                 GTCCAACAGG AGATCGACGA CGTGATAGGG CAGGTGCGGC 
               
               
                   
               
               
                 1121 
                 GACCAGAGAT GGGTGACCAG GCTCACATGC CCTACACCAC 
               
               
                   
               
               
                 1161 
                 TGCCGTGATT CATGAGGTGC AGCGCTTTGG GGACATCGTC 
               
               
                   
               
               
                 1201 
                 CCCCTGGGTG TGACCCATAT GACATCCCGT GACATCGAAG 
               
               
                   
               
               
                 1241 
                 TACACGGCTT CCGCATCCCT AAGGGAACGA CACTCATCAC 
               
               
                   
               
               
                 1281 
                 CAACCTGTCA TCGGTGCTGA AGGATGAGGC CGTCTGGGAG 
               
               
                   
               
               
                 1321 
                 AAGCCCTTCC GCTTCCACCC CGAACACTTC CTGGATGCCC 
               
               
                   
               
               
                 1361 
                 AGGGCCACTT TGTGAAGCCG GAGGCCTTCC TGCCTTTCTC 
               
               
                   
               
               
                 1401 
                 AGCAGGCCGC CGTGCATGCC TCGGGGAGCC CCTGGCCCGC 
               
               
                   
               
               
                 1441 
                 ATGGAGCTCT TCCTCTTCTT CACCTCCCTG CTGCAGCACT 
               
               
                   
               
               
                 1481 
                 TCAGCTTCTC GGTGCCCACT GGACAGCCCC GGCCCAGCCA 
               
               
                   
               
               
                 1521 
                 CCATGGTGTC TTTGCTTTCC TGGTGAGCCC ATCCCCCTAT 
               
               
                   
               
               
                 1561 
                 GAGCTTTGTG CTGTGCCCCG CTAGAATGGG GTACCTAGTC 
               
               
                   
               
               
                 1601 
                 CCCAGCCTGC TCCCTAGCCA GAGGCTCTAA TGTACAATAA 
               
               
                   
               
               
                 1641 
                 AGCAATGTGG TAGTTCCAAA AAAAAAAAAA AAA 
               
            
           
         
       
     
     The rs3892097 single nucleotide polymorphism (SNP) is present in the CYP2D6 gene, where the variable nucleotide at a splice site at about position 595 in SEQ ID NO:9 (underlined), which can be in adenine some individuals and guanine in others. 
     The rs3892097 sequence (SEQ ID NO: 10) of the CYP2D6 gene is shown below, where the underlined A/G is the SNP. 
                    CCCTTACCCGCATCTCCCACCCCCA   [A/G]   GACGCCCCTTTCGCCCCAA               CGGTCT.            
Because the SNP occurs near a splice site, the sequences to the left of the SNP site in SEQ ID NO: 10 do not appear in the SEQ ID NO:9 nucleotide CYP2D6 cDNA sequence.
 
     The cytochrome P450 2D6 polypeptide encoded by the CYP2D6 cDNA with SEQ ID NO:9 has the following sequence (SEQ ID NO: 11). 
     
       
         
           
               
               
            
               
                   1 
                 MGLEALVPLA VIVAIFILLV DLMHRRQRWA ARYPPGPLPL 
               
               
                   
               
               
                  41 
                 PGLGNLLHVD FQNTPYCFDQ LRRRFGDVFS LQLAWTPVVV 
               
               
                   
               
               
                  81 
                 LNGLAAVREA LVTHGEDTAD RPPVPITQIL GFGPRSQGVF 
               
               
                   
               
               
                 121 
                 LARYGPAWRE QRRFSVSTLR NLGLGKKSLE QWVTEEAACL 
               
               
                   
               
               
                 161 
                 CAAFANHS   G   R PFRPNGLLDK AVSNVIASLT CGRRFEYDDP 
               
               
                   
               
               
                 201 
                 RFLRLLDLAQ EGLKEESGFL REVLNAVPVL LHIPALAGKV 
               
               
                   
               
               
                 241 
                 LRFQKAFLTQ LDELLTEHRM TWDPAQPPRD LTEAFLAEME 
               
               
                   
               
               
                 281 
                 KAKGNPESSF NDENLRIVVA DLFSAGMVTT STTLAWGLLL 
               
               
                   
               
               
                 321 
                 MILHPDVQRR VQQEIDDVIG QVRRPEMGDQ AHMPYTTAVI 
               
               
                   
               
               
                 361 
                 HEVQRFGDIV PLGVTHMTSR DIEVQGFRIP KGTTLITNLS 
               
               
                   
               
               
                 401 
                 SVLKDEAVWE KPFRFHPEHF LDAQGHFVKP EAFLPFSAGR 
               
               
                   
               
               
                 441 
                 RACLGEPLAR MELFLFFTSL LQHFSFSVPT GQPRPSHHGV 
               
               
                   
               
               
                 481 
                 FAFLVSPSPY ELCAVPR 
               
            
           
         
       
     
     Note that the underlined glycine at position 169 of SEQ ID NO: 11 is glycine because some individuals have nucleotide sequence SEQ ID NO:9, where the nucleotide at about position 595 is guanine. However, position 169 of SEQ ID NO: 11 can be arginine in some individuals because those individuals have adenine at nucleotide position 295 in SEQ ID NO:9. 
     A patient with that is homozygous for adenine (AA) at the rs3892097 variable site will express CYP2D6 with arginine at position 169 and will not metabolize metoprolol and propranolol as quickly as those with guanine (glycine). Hence, homozygous individuals with adenine (AA) at the rs3892097 variable site have higher plasma levels of metoprolol and propranolol after taking these drugs than subjects that are not homozygous for adenine (AA) at the rs3892097 variable site. Homozygous individuals with adenine (AA) at the rs3892097 variable site would respond more normally to atenolol and carvedilol, which do not require CYP2D6 for their metabolism. 
     Vasodilation Therapy 
     Dilation of blood vessels results in decreases in blood pressure, whereas constriction of blood vessels results in increases in blood pressure. The blood vessels are controlled through local neural signaling that is largely under parasympathetic control, and circulating hormones that are largely under sympathetic control, as well as other circulating proteins. Blood pressure increases following stimulation of the angiotensin receptors, which results in vasoconstriction. Angiotensin receptors are stimulated by angiotensin II, which is converted from angiotensin I through the angiotensin converting enzyme (ACE). Angiotensin II is a potent vasoconstrictor and actively inhibits bradykinin, which is a potent vasodilator. 
     Therefore, angiotensin converting enzyme is a common target of blood pressure therapy. ACE inhibitors such as lisinopril promote vasodilation which, ultimately, reduces the bioavailability of angiotensin-II. Similarly, angiotensin II receptor antagonists such as losartan act as competitive inhibitors, which decrease the number of receptors that are available to bind to angiotensin-II. Despite the method used for promoting vasodilation (through reductions in ACE or receptor inhibition) the end result, on average in the population, is vasodilation which results in a drop in blood pressure due to the inverse relationship between the size of the vessel and the pressure exerted on the vessel all else being equal. Despite this benefit there is a “bell-curve” response to these therapies in humans. Not all individuals are responsive to vasodilator therapy. 
     Several functional polymorphisms of the genes that encode for ACE and angiotensin-II receptors exist, which can affect how a subject responds to vasodilation. 
     Examples of functional ACE polymorphisms include the insertion or deletion polymorphisms such as a 287 base pair fragment (Ulgen et al.,  Coron Arlery Dis  18:153-157 (2007)). The rs1799752 polymorphism is an insertion/deletion in an intron of the ACE gene, and with the sequence (SEQ ID NO:12) shown below, where sequences in the bracket are the insertion/deletion. 
                    TCCCATTTCTCTAGACCTGCTGCCT[-/ATACAGTCACTTTTTTTTTTT               TTTTGAGACGGAGTCTCGCTCTGTCGCCC]ATACAGTCACTTTTATGTG               GTTTCG.            
The deletion removes the bracketed nucleic acid segment so that the rs1799752 polymorphism will have the following sequence (SEQ ID NO:35).
 
     
       
         
           
               
            
               
                 TCCCATTTCTCTAGACCTGCTGCCTATACAGTCACTTTTATGTGGTTTCG. 
               
            
           
         
       
     
     Research indicates that such an ACE deletion polymorphism results in higher ACE plasma levels and greater reduction in ejection fraction in patients following myocardial infraction (likely from elevations in blood pressure) (McNamara et al.,  J Am Coll Cardiol  44:2019-2026 (2004), Pilati et al.,  Congest Heart Fail  10:87-93 (2004). In addition, patients with the deletion polymorphism are more likely to have left-ventricular hypertrophy when compared to patients with the insertion polymorphism (left-ventricular hypertrophy results secondary to prolonged exposure to high blood pressure). Subjects with the deletion polymorphism would therefore be most responsive to ACE-inhibition or angiotensin-II receptor inhibition. 
     At least one functional variant of angiotensin has been found in humans: a cytosine to threonine substitution at nucleotide 4072 (Pilbrow et al.,  Hypertension  49:322-327 (2007); Tang et al.,  Am Heart J  143:854-860 (2002)). Human angiotensinogen is expressed from the AGT gene. A cDNA nucleotide sequence for human angiotensinogen is provided below as SEQ ID NO: 13 (accession number NM_000029.3 GI:188595658, from the NCBI database). 
     
       
         
           
               
               
            
               
                    1 
                 ATCCCATGAG CGGGCAGCAG GGTCAGAAGT GGCCCCCGTG 
               
               
                   
               
               
                   41 
                 TTGCCTAAGC AAGACTCTCC CCTGCCCTCT GCCCTCTGCA 
               
               
                   
               
               
                   81 
                 CCTCCGGCCT GCATGTCCCT GTGGCCTCTT GGGGGTACAT 
               
               
                   
               
               
                  121 
                 CTCCCGGGGC TGGGTCAGAA GGCCTGGGTG GTTGGCCTCA 
               
               
                   
               
               
                  161 
                 GGCTGTCACA CACCTAGGGA GATGCTCCCG TTTCTGGGAA 
               
               
                   
               
               
                  201 
                 CCTTGGCCCC GACTCCTGCA AACTTCGGTA AATGTGTAAC 
               
               
                   
               
               
                  241 
                 TCGACCCTGC ACCGGCTCAC TCTGTTCAGC AGTGAAACTC 
               
               
                   
               
               
                  281 
                 TGCATCGATC ACTAAGACTT CCTGGAAGAG GTCCCAGCGT 
               
               
                   
               
               
                  321 
                 GAGTGTCGCT TCTGGCATCT GTCCTTCTGG CCAGCCTGTG 
               
               
                   
               
               
                  361 
                 GTCTGGCCAA GTGATGTAAC CCTCCTCTCC AGCCTGTGCA 
               
               
                   
               
               
                  401 
                 CAGGCAGCCT GGGAACAGCT CCATCCCCAC CCCTCAGCTA 
               
               
                   
               
               
                  441 
                 TAAATAGGGC ATCGTGACCC GGCCGGGGGA AGAAGCTGCC 
               
               
                   
               
               
                  481 
                 GTTGTTCTGG GTACTACAGC AGAAGGGTAT GCGGAAGCGA 
               
               
                   
               
               
                  521 
                 GCACCCCAGT CTGAGATGGC TCCTGCCGGT GTGAGCCTGA 
               
               
                   
               
               
                  561 
                 GGGCCACCAT CCTCTGCCTC CTGGCCTGGG CTGGCCTGGC 
               
               
                   
               
               
                  601 
                 TGCAGGTGAC CCGGTGTACA TACACCCCTT CCACCTCGTC 
               
               
                   
               
               
                  641 
                 ATCCACAATG ACAGTACCTG TGAGCAGCTG GCAAAGGCCA 
               
               
                   
               
               
                  681 
                 ATGCCGGGAA GCCCAAAGAC CCCACCTTCA TACCTGCTCC 
               
               
                   
               
               
                  721 
                 AATTCAGGCC AAGACATCCC CTGTGGATGA AAAGGCCCTA 
               
               
                   
               
               
                  761 
                 CAGGACCAGC TGGTGCTAGT CGCTGCAAAA CTTGACACCG 
               
               
                   
               
               
                  801 
                 AAGACAAGTT GAGGGCCGCA ATGGTCGGGA TGCTGGCCAA 
               
               
                   
               
               
                  841 
                 CTTCTTGGGC TTCCGTATAT ATGGCATGCA CAGTGAGCTA 
               
               
                   
               
               
                  881 
                 TGGGGCGTGG TCCATGGGGC CACCGTCCTC TCCCCAACGG 
               
               
                   
               
               
                  921 
                 CTGTCTTTGG CACCCTGGCC TCTCTCTATC TGGGAGCCTT 
               
               
                   
               
               
                  961 
                 GGACCACACA GCTGACAGGC TACAGGCAAT CCTGGGTGTT 
               
               
                   
               
               
                 1001 
                 CCTTGGAAGG ACAAGAACTG CACCTCCCGG CTGGATGCGC 
               
               
                   
               
               
                 1041 
                 ACAAGGTCCT GTCTGCCCTG CAGGCTGTAC AGGGCCTGCT 
               
               
                   
               
               
                 1081 
                 AGTGGCCCAG GGCAGGGCTG ATAGCCAGGC CCAGCTGCTG 
               
               
                   
               
               
                 1121 
                 CTGTCCACGG TGGTGGGCGT GTTCACAGCC CCAGGCCTGC 
               
               
                   
               
               
                 1161 
                 ACCTGAAGCA GCCGTTTGTG CAGGGCCTGG CTCTCTATAC 
               
               
                   
               
               
                 1201 
                 CCCTGTGGTC CTCCCACGCT CTCTGGACTT CACAGAACTG 
               
               
                   
               
               
                 1241 
                 GATGTTGCTG CTGAGAAGAT TGACAGGTTC ATGCAGGCTG 
               
               
                   
               
               
                 1281 
                 TGACAGGATG GAAGACTGGC TGCTCCCTGA    T   GGGAGCCAG 
               
               
                   
               
               
                 1321 
                 TGTGGACAGC ACCCTGGCTT TCAACACCTA CGTCCACTTC 
               
               
                   
               
               
                 1361 
                 CAAGGGAAGA TGAAGGGCTT CTCCCTGCTG GCCGAGCCCC 
               
               
                   
               
               
                 1401 
                 AGGAGTTCTG GGTGGACAAC AGCACCTCAG TGTCTGTTCC 
               
               
                   
               
               
                 1441 
                 CATGCTCTCT GGCATGGGCA CCTTCCAGCA CTGGAGTGAC 
               
               
                   
               
               
                 1481 
                 ATCCAGGACA ACTTCTCGGT GACTCAAGTG CCCTTCACTG 
               
               
                   
               
               
                 1521 
                 AGAGCGCCTG CCTGCTGCTG ATCCAGCCTC ACTATGCCTC 
               
               
                   
               
               
                 1561 
                 TGACCTGGAC AAGGTGGAGG GTCTCACTTT CCAGCAAAAC 
               
               
                   
               
               
                 1601 
                 TCCCTCAACT GGATGAAGAA ACTATCTCCC CGGACCATCC 
               
               
                   
               
               
                 1641 
                 ACCTGACCAT GCCCCAACTG GTGCTGCAAG GATCTTATGA 
               
               
                   
               
               
                 1681 
                 CCTGCAGGAC CTGCTCGCCC AGGCTGAGCT GCCCGCCATT 
               
               
                   
               
               
                 1721 
                 CTGCACACCG AGCTGAACCT GCAAAAATTG AGCAATGACC 
               
               
                   
               
               
                 1761 
                 GCATCAGGGT GGGGGAGGTG CTGAACAGCA TTTTTTTTGA 
               
               
                   
               
               
                 1801 
                 GCTTGAAGCG GATGAGAGAG AGCCCACAGA GTCTACCCAA 
               
               
                   
               
               
                 1841 
                 CAGCTTAACA AGCCTGAGGT CTTGGAGGTG ACCCTGAACC 
               
               
                   
               
               
                 1881 
                 GCCCATTCCT GTTTGCTGTG TATGATCAAA GCGCCACTGC 
               
               
                   
               
               
                 1921 
                 CCTGCACTTC CTGGGCCGCG TGGCCAACCC GCTGAGCACA 
               
               
                   
               
               
                 1961 
                 GCATGAGGCC AGGGCCCCAG AACACAGTGC CTGGCAAGGC 
               
               
                   
               
               
                 2001 
                 CTCTGCCCCT GGCCTTTGAG GCAAAGGCCA GCAGCAGATA 
               
               
                   
               
               
                 2041 
                 ACAACCCCGG ACAAATCAGC GATGTGTCAC CCCCAGTCTC 
               
               
                   
               
               
                 2081 
                 CCACCTTTTC TTCTAATGAG TCGACTTTGA GCTGGAAAGC 
               
               
                   
               
               
                 2121 
                 AGCCGTTTCT CCTTGGTCTA AGTGTGCTGC ATGGAGTGAG 
               
               
                   
               
               
                 2161 
                 CAGTAGAAGC CTGCAGCGGC ACAAATGCAC CTCCCAGTTT 
               
               
                   
               
               
                 2201 
                 GCTGGGTTTA TTTTAGAGAA TGGGGGTGGG GAGGCAAGAA 
               
               
                   
               
               
                 2241 
                 CCAGTGTTTA GCGCGGGACT ACTGTTCCAA AAAGAATTCC 
               
               
                   
               
               
                 2281 
                 AACCGACCAG CTTGTTTGTG AAACAAAAAA GTGTTCCCTT 
               
               
                   
               
               
                 2321 
                 TTCAAGTTGA GAACAAAAAT TGGGTTTTAA AATTAAAGIA 
               
               
                   
               
               
                 2361 
                 TACATTTTTG CATTGCCTTC GGTTTGTATT TAGTGTCTTG 
               
               
                   
               
               
                 2401 
                 AATGTAAGAA CATGACCTCC GTGTAGTGTC TGTAATACCT 
               
               
                   
               
               
                 2441 
                 TAGTTTTTTC CACAGATGCT TGTGATTTTT GAACAATACG 
               
               
                   
               
               
                 2481 
                 TGAAAGATGC AAGCACCTGA ATTTCTGTTT GAATGCGGAA 
               
               
                   
               
               
                 2521 
                 CCATAGCTGG TTATTTCTCC CTTGTGTTAG TAATAAACGT 
               
               
                   
               
               
                 2561 
                 CTTGCCACAA TAAGCCTCCA  
               
               
                   
               
               
                 2581 
                 AAAAAAA 
               
            
           
         
       
     
     The rs699 single nucleotide polymorphism (SNP) is present in the AGT gene, where the variable nucleotide is at about position 1311 in SEQ ID NO: 13 (underlined), which can be in thymine some individuals and cytosine in others. The rs699 sequence (SEQ ID NO: 14) is shown below, where the underlined C/T is the SNP. 
     
       
         
           
               
            
               
                 GGATGGAAGACTGGCTGCTCCCTGA   [C/T]   GGGAGCCAGTGTGGACAGCA 
               
               
                   
               
               
                 CCCTG. 
               
            
           
         
       
     
     The human angiotensinogen polypeptide encoded by the AGT cDNA with SEQ ID NO:13 has the following sequence (SEQ ID NO:15). 
     
       
         
           
               
               
            
               
                   1 
                 MRKRAPQSEM APAGVSLRAT ILCLLAWAGL AAGDRVYIHP 
               
               
                   
               
               
                  41 
                 FHLVIHNEST CEQLAKANAG KPKDPTFIPA PIQAKTSPVD 
               
               
                   
               
               
                  81 
                 EKALQDQLVL VAAKLDTEDK LRAAMVGMLA NFLGFRIYGM 
               
               
                   
               
               
                 121 
                 HSELWGVVHG ATVLSPTAVF GTLASLYLGA LDHTADRLQA 
               
               
                   
               
               
                 161 
                 ILGVPWKDKN CTSRLDAHKV LSALQAVQGL LVAQGRADSQ 
               
               
                   
               
               
                 201 
                 AQLLLSTVVG VFTAPGLHLK QPFVQGLALY TPVVLPRSLD 
               
               
                   
               
               
                 241 
                 FTELDVAAEK IDRFMQAVTG WKTGCSL   M   GA SVDSTLAFNT 
               
               
                   
               
               
                 281 
                 YVHFQGKMKG FSLLAEPQEF WVDNSTSVSV PMLSGMGTFQ 
               
               
                   
               
               
                 321 
                 HWSDIQDNFS VTQVPFTESA CLLLIQPHYA SDLDKVEGLT 
               
               
                   
               
               
                 361 
                 FQQNSLNWMK KLSPRTIHLT MPQLVLQGSY DLQDLLAQAE 
               
               
                   
               
               
                 401 
                 LPAILHTELN LQKLSNDRIR VGEVLNSIFF ELEADEREPT 
               
               
                   
               
               
                 441 
                 ESTQQLNKPE VLEVTLNRPF LFAVYDQSAT ALHFLGRVAN 
               
               
                   
               
               
                 481 
                 PLSTA 
               
            
           
         
       
     
     Note that the underlined methionine at position 268 of SEQ ID NO:15 is methionine because some individuals have nucleotide sequence SEQ ID NO: 13, where the nucleotide at about position 1311 is thymine. However, position 268 of SEQ ID NO: 15 can be threonine in some individuals because those individuals have cytosine at nucleotide position 1311 in SEQ ID NO: 13. 
     The threonine polymorphism of angiotensin results in higher angiotensin levels and higher resting blood pressure values. Therefore, patients with the threonine genetic variant will benefit primarily from an ACE inhibitor (preventing the conversion of the higher levels of angiotensin I to angiotensin II) or an angiotensin receptor inhibitor. 
     An example of a functional polymorphism of an angiotensin II receptor type-1 involves an adenine to cytosine substitution at nucleotide 1166 (Miller et al.  Kidney Int  56:2173-2180 (1999); Baudin.  Pharmacogenomics  3:65-73 (2002)). Human angiotensin II receptor type-1 is expressed from the AGT1R gene. One example of an AGT1R single nucleotide polymorphism is the so-called A1166-&gt;C polymorphism, which is in the 3′ untranslated region of the AGT1R gene. This A1166-&gt;C polymorphism is also identified as the rs5186 single nucleotide polymorphism (SNP), which has the following sequence (SEQ ID NO: 16) where the underlined A/C is the variable SNP site. 
                    TGCAGCACTTCACTACCAAATGAGC   [A/C]   TTAGCTACTTTTCAGAATTG               AAGGA.            
A portion of a 3′ untranslated region of the AGT1R gene with NCBI accession number NG_008468.1 (GI: 198041751) is shown below (SEQ ID NO:17) that contains the rs5186 SNP with the variant nucleotide (adenine) identified below in bold and with underlining.
 
     
       
         
           
               
               
            
               
                 48961 
                 ATTCAACTAG GCATCATACG TGACTGTAGA ATTGCAGATA 
               
               
                   
               
               
                 49001 
                 TTGTGGACAC GGCCATGCCT ATCACCATTT GTATAGCTTA 
               
               
                   
               
               
                 49041 
                 TTTTAACAAT TGCCTGAATC CTCTTTTTTA TGGCTTTCTG 
               
               
                   
               
               
                 49081 
                 GGGAAAAAAT TTAAAAGATA TTTTCTCCAG CTTCTAAAAT 
               
               
                   
               
               
                 49121 
                 ATATTCCCCC AAAAGCCAAA TCCCACTCAA ACCTTTCAAC 
               
               
                   
               
               
                 49181 
                 AAAAATGAGC ACGCTTTCCT ACCGCCCCTC AGATAATGTA 
               
               
                   
               
               
                 49201 
                 AGCTCATCCA CCAAGAAGCC TGCACCATGT TTTGAGGTTG 
               
               
                   
               
               
                 49241 
                 AGTGACATGT TCGAAACCTG TCCATAAAGT AATTTTGTGA 
               
               
                   
               
               
                 49301 
                 AAGAAGGAGC AAGAGAACAT TCCTCTGCAG CACTTCACTA 
               
               
                   
               
               
                 49321 
                 CCAAATGAGC    A   TTAGCTACT TTTCAGAATT GAAGGAGAAA 
               
               
                   
               
               
                 49361 
                 ATGCATTATG TGGACTGAAC CGACTTTTCT AAAGCTCTGA 
               
               
                   
               
               
                 49401 
                 ACAAAAGCTT TTCTTTCCTT TTGCAACAAG ACAAAGCAAA 
               
               
                   
               
               
                 49441 
                 GCCACATTTT GCATTAGACA GATGACGGCT GCTCGAAGAA 
               
               
                   
               
               
                 49481 
                 CAATGTCAGA AACTCGATGA ATGTGTTGAT TTGAGAAATT 
               
               
                   
               
               
                 49521 
                 TTACTGACAG AAATGCAATC TCCCTAGCCT GCTTTTGTCC 
               
               
                   
               
               
                 49561 
                 TGTTATTTTT TATTTCCACA TAAAGGTATT TAGAATATAT 
               
               
                   
               
               
                 49601 
                 TAAATCGTTA GAGGAGCAAC AGGAGATGAG AGTTCCAGAT 
               
               
                   
               
               
                 49641 
                 TGTTCTGTCC AGTTTCCAAA GGGCAGTAAA GTTTTCGTGC 
               
            
           
         
       
     
     This polymorphism has been shown to influence resting blood pressure values which suggest which patients may benefit more from angiotensin-II receptor inhibition. Specifically, patients with the C variant of the angiotensin receptor type I tend to demonstrate higher resting blood pressure values, have more detrimental cardiovascular events, and have a greater chance of developing high blood pressure during pregnancy, when compared to the A variant. Subjects with the C variant will therefore be more responsive to angiotensin receptor blockers. 
     A cDNA sequence for human angiotensin II receptor is provided in the NCBI database as accession number X65699.1 (GI:510983), which has the following sequence (SEQ ID NO:18). 
     
       
         
           
               
               
            
               
                    1 
                 GGCAGCAGCG AGTGACAGGA CGTCTGGACC GGCGCGCCGC 
               
               
                   
               
               
                   41 
                 TAGCAGCTCT GCCGGGCCGC GGCGGTGATC GATGGGAGCG 
               
               
                   
               
               
                   81 
                 GCTGGAGCGG ACCCAGCGAG TGAGGGCGCA CAGCCGGACG 
               
               
                   
               
               
                  121 
                 CCGAGGCGGC GGGCGGGAGA CCGCACCGCG ACGCCGGCCC 
               
               
                   
               
               
                  161 
                 TCGGCGGACG AGTCGAGCGC CCGGGCGCGG GTGTATTTGA 
               
               
                   
               
               
                  201 
                 TATAGTGTTT GCAACAAATT CGACCCAGGT GATCAAAATG 
               
               
                   
               
               
                  241 
                 ATTCTCAACT CTTCTACTGA AGATGGTATT AAAAGAATCC 
               
               
                   
               
               
                  281 
                 AAGATGATTG TCCCAAAGCT GGAAGGCATA ATTACATATT 
               
               
                   
               
               
                  321 
                 TGTCATGATT CCTACTTTAT ACAGTATCAT CTTTGTGGTG 
               
               
                   
               
               
                  361 
                 GGAATATTTG GAAACAGCTT GGTGGTGATA GTCATTTACT 
               
               
                   
               
               
                  401 
                 TTTATATGAA GCTGAAGACT GTGGCCAGTG TTTTTCTTTT 
               
               
                   
               
               
                  441 
                 GAATTTAGCA CTGGCTGACT TATGCTTTTT ACTGACTTTG 
               
               
                   
               
               
                  481 
                 CCACTATGGG CTGTCTACAC AGCTATGGAA TACCGCTGGC 
               
               
                   
               
               
                  521 
                 CCTTTGGCAA TTACCTATGT AAGATTGCTT CAGCCAGCGT 
               
               
                   
               
               
                  561 
                 CAGTTTCAAC CTGTACGCTA GTGTGTTTCT ACTCACGTGT 
               
               
                   
               
               
                  601 
                 CTCAGCATTG ATCGATACCT GGCTATTGTT CACCCAATGA 
               
               
                   
               
               
                  641 
                 AGTCCCGCCT TCGACGCACA ATGCTTGTAG CCAAAGTCAC 
               
               
                   
               
               
                  681 
                 CTGCATCATC ATTTGGCTGC TGGCAGGCTT GGCCAGTTTG 
               
               
                   
               
               
                  721 
                 CCAGCTATAA TCCATCGAAA TGTATTTTTC ATTGAGAACA 
               
               
                   
               
               
                  761 
                 CCAATATTAC AGTTTGTGCT TTCCATTATG AGTCCCAAAA 
               
               
                   
               
               
                  801 
                 TTCAACCCTC CCGATAGGGC TGGGCCTGAC CAAAAATATA 
               
               
                   
               
               
                  841 
                 CTGGGTTTCC TGTTTCCTTT TCTGATCATT CTTACAAGTT 
               
               
                   
               
               
                  881 
                 ATACTCTTAT TTGGAAGGCC CTAAAGAAGG CTTATGAAAT 
               
               
                   
               
               
                  921 
                 TCAGAAGAAC AAACCAAGAA ATGATGATAT TTTTAAGATA 
               
               
                   
               
               
                  961 
                 ATTATGGCAA TTGTGCTTTT CTTTTTCTTT TCCTGGATTC 
               
               
                   
               
               
                 1001 
                 CCCACCAAAT ATTCACTTTT CTGGATGTAT TGATTCAACT 
               
               
                   
               
               
                 1041 
                 AGGCATCATA CGTGACTGTA GAATTGCAGA TATTGTGGAC 
               
               
                   
               
               
                 1081 
                 ACGGCCATGC CTATCACCAT TTGTATAGCT TATTTTAACA 
               
               
                   
               
               
                 1121 
                 ATTGCCTGAA TCCTCTTTTT TATGGCTTTC TGGGGAAAAA 
               
               
                   
               
               
                 1161 
                 ATTTAAAAGA TATTTTCTCC AGCTTCTAAA ATATATTCCC 
               
               
                   
               
               
                 1201 
                 CCAAAAGCCA AATCCCACTC AAACCTTTCA ACAAAAATGA 
               
               
                   
               
               
                 1241 
                 GCACGCTTTC CTACCGCCCC TCAGATAATG TAAGCTCATC 
               
               
                   
               
               
                 1281 
                 CACCAAGAAG CCTGCACCAT GTTTTGAGGT TGAGTGACAT 
               
               
                   
               
               
                 1321 
                 GTTCGAAACC TGTCCATAAA GTAATTTTGT GAAAGAAGGA 
               
               
                   
               
               
                 1361 
                 GCAAGAGAAC ATTCCTCTCC AGCACTTCAC TACCAAATGA 
               
               
                   
               
               
                 1401 
                 GC   A   TTAGCTA CTTTTCAGAA TTGAAGGAGA AAATGCATTA 
               
               
                   
               
               
                 1441 
                 TGTGGACTGA ACCGACTTTT CTAAAGCTCT GAACAAAAGC 
               
               
                   
               
               
                 1481 
                 TTTTCTTTCC TTTTGCAACA AGACAAAGCA AAGCCACATT 
               
               
                   
               
               
                 1521 
                 TTGCATTAGA CAGATGACGG CTGCTCGAAG AACAATGTCA 
               
               
                   
               
               
                 1561 
                 GAAACTCGAT GAATGTGTTG ATTTGAGAAA TTTTACTGAC 
               
               
                   
               
               
                 1601 
                 AGAAATGCAA TCTCCCTAGC CTGCTTTTGT CCTGTTATTT 
               
               
                   
               
               
                 1641 
                 TTTATTTCCA CATAAAGGTA TTTAGAATAT ATTAACTCGT 
               
               
                   
               
               
                 1681 
                 TAGAGGAGCA ACAGGAGATG AGAGTTCCAG ATTGTTCTGT 
               
               
                   
               
               
                 1721 
                 CCAGTTTCCA AAGGGCAGTA AAGTTTTCGT GCCTGTTTTC 
               
               
                   
               
               
                 1761 
                 AGCTATTAGC AACTGTGCCT ACACTTGCAC CTGGTCTGCA 
               
               
                   
               
               
                 1801 
                 CATTTTGTAC AAAGATATGC TTAAGCAGTA GTCGTCAAGT 
               
               
                   
               
               
                 1841 
                 TGCAGATCTT TGTTGTGAAA TTCAACCTGT GTCTTATAGG 
               
               
                   
               
               
                 1881 
                 TTTACACTGC CAAAACAATG CCCGTAAGAT GGCTTATTTG 
               
               
                   
               
               
                 1921 
                 TATAATGGTG TTACCTAAAG TCACATATAA AAGTTAAACT 
               
               
                   
               
               
                 1961 
                 ACTTGTAAAG GTGCTGCACT GGTCCCAAGT AGTAGTGTCT 
               
               
                   
               
               
                 2001 
                 TCCTAGTATA TTAGTTTGAT TTAATATCTG AGAAGTGTAT 
               
               
                   
               
               
                 2041 
                 ATAGTTTGTG GTAAAAAGAT TATATATCAT AAAGTATGCC 
               
               
                   
               
               
                 2081 
                 TTCCTGTTTA AAAAAAGTAT ATATTCTACA CATATATGTA 
               
               
                   
               
               
                 2121 
                 TATGTATATC TATATCTCTA AACTGCTGTT AATTGATTAA 
               
               
                   
               
               
                 2161 
                 AATCTGGCAA AGTTATATTT ACCCC 
               
            
           
         
       
     
     In addition to angiotensin, angiotensin II receptors and ACE, renin has been shown to be a potent vasoconstrictor that can result in high blood pressure. Renin converts angiotensinogen to angiotensin I which can result in vasoconstriction due to the down-stream effects (angiotensin-I conversion to angiotensin II through ACE). One example of a functional and common renin polymorphism (Vangjeli et al.,  Circulation Cardiovascular genetics  3:53-59 (2010)) can influence the blood pressure response to vasodilator therapy. This renin polymorphism is present in rs12750834. The nucleotide sequence surrounding the renin rs12750834 single nucleotide polymorphism is shown below, where the underlined A/G in the sequence (SEQ ID NO:19) is the SNP. 
                    AGAACACCAAAGCAGGCTTAATCTG   [A/G]   GGGCACTTACAGAGACTGC               TTTAAA.            
The complementary sequence of SEQ ID NO:19 is the following sequence (SEQ ID NO:20).
 
                    TTTAAAGCAGTCTCTGTAAGTGCCC   [C/T]   CAGATTAAGCCTGCTTTGG               TGTTCT.            
Note that the cytosine to thymine substitution is a guanine to adenine substitution in the opposite strand.
 
     The rs12750834 SNP contains a cytosine to thymine substitution, or a guanine to adenine substitution depending upon the DNA strand, at about nucleotide position 5312 upstream of the renin start site. The cytosine (guanine) variant of renin has been shown to correlate with greater reduction in blood pressure upon administration of angiotensin II receptor blockers such as valsartan. 
     Sodium/Diuretic Regulation of Blood Pressure 
     The kidneys are the center of long-term blood pressure regulation. Alterations in Na +  reabsorption in the kidneys result in alterations in fluid retention, which leads to increases or decreases in blood plasma volume as well as to changes in the pressure against the vessels. There are several proteins that are important in renal Na +  handling and in the response to diuretic therapy including the epithelial Na +  channels, alpha-adducin, the Na + Cl −  co-transporter, and lysine deficient protein kinase-1 (WNK). 
     The epithelial sodium (Na + ) channel is responsible for Na +  reabsorption on the apical portion of epithelial cells in the kidneys. The Na +  channel is made up of three different subunits: the alpha, beta, and gamma. The alpha subunit of the epithelial Na +  channel is highly functional and removal of this subunit abolishes channel activity in cell and animal models. The gamma subunit is also extremely important in channel function. Functional gamma genetic variants result in pseudohypoaldosteronism type-I and Liddle&#39;s syndrome, two severe genetic diseases resulting in salt wasting and high salt conservation (salt sensitivity), respectively. Adducin is made up of an alpha, beta, and gamma subunit. The alpha subunit increases sodium (Na + ) reabsorption in the kidneys through the activity of Na + K +  ATPase (which moves Na +  and potassium into and out of cells). The sodium (Na + ) chloride (Cl − ) co-transporter is important in regulating Na +  and Cl −  movement between the kidney and the rest of the body. Active Na + —Cl −  transport results in Na +  reabsorption and can, therefore, result in higher blood pressure. The WNK1 protein is a key regulator of long-term Na +  and chloride Cl −  reabsorption in the kidneys. WNK1 regulates the activity of Na + —Cl −  co-transporters. If a patient has a more active WNK1 genotype, they likely have greater Na +  and Cl −  reabsorption in the kidneys which can increase blood volume and, therefore, pressure on the vessels. 
     A functional and common polymorphism of the gene that encodes the epithelial Na+channel (SCNN1A) has been identified, where the polymorphism is an alanine to threonine substitution at about position 663-722. A cDNA sequence for the human SCNN1A gene is available from the NCBI database as accession number NM_001159576.1 (GI:227430288). This sequence is provided below as SEQ ID NO:21. 
     
       
         
           
               
               
            
               
                    1 
                 AAACAGAAGG CAGATAGAGA GGGAGTGAGA GGCAGGAGCT 
               
               
                   
               
               
                   41 
                 GAGACACAGA TCCTGGAGGA AGAAGACCAA AGGAAGGGGG 
               
               
                   
               
               
                   81 
                 CAGAGACAGA AAGGGAGGTG CTAGGACAAA ACTCGAAAGG 
               
               
                   
               
               
                  121 
                 TGGCCCTATC AGGGAAGCAG AGGAGAGGCC GTTCTAGGGA 
               
               
                   
               
               
                  161 
                 AGCCCAGCTC CGGCACTTTT GGCCCCAACT CCCGCAGGTC 
               
               
                   
               
               
                  201 
                 TGCTGGCTCC AGGAAAGGTG GAGGAGGGAG GGAGGAGTGG 
               
               
                   
               
               
                  241 
                 GAGAATGTGG GCGCAGGGTG GGACATGGGC ATGGCCAGGG 
               
               
                   
               
               
                  281 
                 GCAGCCTCAC TCGGGTTCCA GGGGTGATGG GAGAGGGCAC 
               
               
                   
               
               
                  321 
                 TCAGGGCCCA GAGCTCAGCC TTGACCCTGA CCCTTGCTCT 
               
               
                   
               
               
                  361 
                 CCCCAATCCA CTCCGGGGCT CATGAAGGGG AACAAGCTGG 
               
               
                   
               
               
                  401 
                 AGGAGCAGGA CCCTAGACCT CTGCAGCCCA TACCAGGTCT 
               
               
                   
               
               
                  441 
                 CATGGAGGGG AACAAGCTGG AGGAGCAGGA CTCTAGCCCT 
               
               
                   
               
               
                  481 
                 CCACAGTCCA CTCCAGGGCT CATGAAGGGG AACAAGCGTG 
               
               
                   
               
               
                  521 
                 AGGAGCAGGG GCTGGGCCCC GAACCTGCGG CGCCCCAGCA 
               
               
                   
               
               
                  561 
                 GCCCACGGCG GAGGAGGAGG CCCTGATCGA GTTCCACCGC 
               
               
                   
               
               
                  601 
                 TCCTACCGAG AGCTCTTCGA GTTCTTCTGC AACAACACCA 
               
               
                   
               
               
                  641 
                 CCATCCACGG CGCCATCCGC CTGGTGTGCT CCCAGCACAA 
               
               
                   
               
               
                  681 
                 CCGCATGAAG ACGGCCTTCT GGGCAGTGCT GTGGCTCTGC 
               
               
                   
               
               
                  721 
                 ACCTTTGGCA TGATGTACTG GCAATTCGGC CTGCTTTTCG 
               
               
                   
               
               
                  761 
                 GAGAGTACTT CAGCTACCCC GTCAGCCTCA ACATCAACCT 
               
               
                   
               
               
                  801 
                 CAACTCGGAC AAGCTCGTCT TCCCCGCAGT GACCATCTGC 
               
               
                   
               
               
                  841 
                 ACCCTCAATC CCTACAGGTA CCCGGAAATT AAAGAGGAGC 
               
               
                   
               
               
                  881 
                 TGGAGGAGCT GGACCGCATC ACAGAGCAGA CGCTCTTTGA 
               
               
                   
               
               
                  921 
                 CCTGTACAAA TACAGCTCCT TCACCACTCT CGTGGCCGGC 
               
               
                   
               
               
                  961 
                 TCCCGCAGCC GTCGCGACCT GCGGGGGACT CTGCCGCACC 
               
               
                   
               
               
                 1001 
                 CCTTGCAGCG CCTGAGGGTC CCGCCCCCGC CTCACGGGGC 
               
               
                   
               
               
                 1041 
                 CCGTCGAGCC CGTAGCGTGG CCTCCAGCTT GCGGGACAAC 
               
               
                   
               
               
                 1081 
                 AACCCCCAGG TGGACTGGAA GGACTGGAAG ATCGGCTTCC 
               
               
                   
               
               
                 1121 
                 AGCTGTGCAA CCAGAACAAA TCGGACTGCT TCTACCAGAC 
               
               
                   
               
               
                 1161 
                 ATACTCATCA GGGGTGGATG CGGTGAGGGA GTGGTACCGC 
               
               
                   
               
               
                 1201 
                 TTCCACTACA TCAACATCCT GTCGAGGCTG CGAGAGACTC 
               
               
                   
               
               
                 1241 
                 TGCCATCCCT GGAGGAGGAC ACGCTGGGCA ACTTCATCTT 
               
               
                   
               
               
                 1281 
                 CGCCTGCCGC TTCAACCAGG TCTCCTGCAA CCAGGCGAAT 
               
               
                   
               
               
                 1321 
                 TACTCTCACT TCCACCACCC GATGTATGGA AACTGCTATA 
               
               
                   
               
               
                 1361 
                 CTTTCAATGA CAAGAACAAC TCCAACCTCT GGATGTCTTC 
               
               
                   
               
               
                 1401 
                 CATGCCTGGA ATCAACAACG GTCTCTCCCT GATGCTGCGC 
               
               
                   
               
               
                 1441 
                 GCAGAGCAGA ATGACTTCAT TCCCCTGCTG TCCACAGTGA 
               
               
                   
               
               
                 1481 
                 CTGGGGCCCG GGTAATGGTG CACGGGCAGG ATGAACCTGC 
               
               
                   
               
               
                 1521 
                 CTTTATGGAT GATGGTGGCT TTAACTTGCG GCCTGGCGTG 
               
               
                   
               
               
                 1561 
                 GAGACCTCCA TCAGCATGAG GAAGGAAACC CTGGACAGAC 
               
               
                   
               
               
                 1601 
                 TTGGGGGCGA TTATGGCGAC TGCACCAAGA ATGGCAGTGA 
               
               
                   
               
               
                 1641 
                 TGTTCCTGTT GAGAACCTTT ACCCTTCAAA GTACACACAG 
               
               
                   
               
               
                 1681 
                 CAGGTGTGTA TTCACTCCTG CTTCCAGGAG AGCATGATCA 
               
               
                   
               
               
                 1721 
                 AGGAGTGTGG CTGTGCCTAC ATCTTCTATC CGCGGCCCCA 
               
               
                   
               
               
                 1761 
                 GAACGTGGAG TACTGTGACT ACAGAAAGCA CAGTTCCTGG 
               
               
                   
               
               
                 1801 
                 GGGTACTGCT ACTATAAGCT CCAGGTTGAC TTCTCCTCAG 
               
               
                   
               
               
                 1841 
                 ACCACCTGGG CTGTTTCACC AAGTGCCGGA AGCCATGCAG 
               
               
                   
               
               
                 1881 
                 CGTGACCAGC TACCAGCTCT CTGCTGGTTA CTCACGATGG 
               
               
                   
               
               
                 1921 
                 CCCTCGGTGA CATCCCAGGA ATGGGTCTTC CAGATGCTAT 
               
               
                   
               
               
                 1961 
                 CGCGACAGAA CAATTACACC GTCAACAACA AGAGAAATGG 
               
               
                   
               
               
                 2001 
                 AGTGGCCAAA GTCAACATCT TCTTCAAGGA GCTGAACTAC 
               
               
                   
               
               
                 2041 
                 AAAACCAATT CTGAGTCTCC CTCTGTCACG ATGGTCACCC 
               
               
                   
               
               
                 2081 
                 TCCTGTCCAA CCTGGGCAGC CAGTGGAGCC TGTGGTTCGG 
               
               
                   
               
               
                 2121 
                 CTCCTCGGTG TTGTCTGTGG TGGAGATGGC TGAGCTCGTC 
               
               
                   
               
               
                 2161 
                 TTTGACCTGC TGGTCATCAT GTTCCTCATG CTGCTCCGAA 
               
               
                   
               
               
                 2201 
                 GGTTCCGAAG CCGATACTGG TCTCCAGGCC GAGGGGGCAG 
               
               
                   
               
               
                 2241 
                 GGGTGCTCAG GAGGTAGCCT CCACCCTGGC ATCCTCCCCT 
               
               
                   
               
               
                 2281 
                 CCTTCCCACT TCTGCCCCCA CCCCATGTCT CTGTCCTTGT 
               
               
                   
               
               
                 2321 
                 CCCAGCCAGG CCCTGCTCCC TCTCCAGCCT TGACAGCCCC 
               
               
                   
               
               
                 2361 
                 TCCCCCTGCC TATGCCACCC TGGGCCCCCG CCCATCTCCA 
               
               
                   
               
               
                 2401 
                 GGGGGCTCTG CAGGGGCCAG TTCCTCC   A   CC TGTCCTCTGG 
               
               
                   
               
               
                 2441 
                 GGGGGCCCTG AGAGGGAAGG AGAGGTTTCT CACACCAAGG 
               
               
                   
               
               
                 2481 
                 CAGATGCTCC TCTGGTGGGA GGGTGCTGGC CCTGGCAAGA 
               
               
                   
               
               
                 2521 
                 TTGAAGGATG TGCAGGGCTT CCTCTCAGAG CCGCCCAAAC 
               
               
                   
               
               
                 2561 
                 TGCCGTTGAT GTGTGGAGGG GAAGCAAGAT GGGTAAGGGC 
               
               
                   
               
               
                 2601 
                 TCAGGAAGTT GCTCCAAGAA CAGTAGCTGA TGAAGCTGCC 
               
               
                   
               
               
                 2641 
                 CAGAAGTGCC TTGGCTCCAG CCCTGTACCC CTTGGTACTG 
               
               
                   
               
               
                 2681 
                 CCTCTGAACA CTCTGGTTTC CCCACCCAAC TGCGGCTAAG 
               
               
                   
               
               
                 2721 
                 TCTCTTTTTC CCTTGGATCA GCCAAGCGAA ACTTGGAGCT 
               
               
                   
               
               
                 2761 
                 TTGACAAGGA ACTTTCCTAA GAAACCGCTG ATAACCAGGA 
               
               
                   
               
               
                 2801 
                 CAAAACACAA CCAAGGGTAC ACGCAGGCAT GCACGGGTTT 
               
               
                   
               
               
                 2841 
                 CCTGCCCAGC GACGGCTTAA GCCAGCCCCC GACTGGCCTG 
               
               
                   
               
               
                 2881 
                 GCCACACTGC TCTCCAGTAG CACAGATGTC TGCTCCTCCT 
               
               
                   
               
               
                 2921 
                 CTTGAACTTG GGTGGGAAAC CCCACCCAAA AGCCCCCTTT 
               
               
                   
               
               
                 2961 
                 GTTACTTAGG CAATTCCCCT TCCCTGACTC CCGAGGGCTA 
               
               
                   
               
               
                 3001 
                 GGGCTAGAGC AGACCCGGGT AAGTAAAGGC AGACCCAGGG 
               
               
                   
               
               
                 3041 
                 CTCCTCTAGC CTCATACCCG TGCCCTCACA GAGCCATGCC 
               
               
                   
               
               
                 3081 
                 CCGGCACCTC TGCCCTGTGT CTTTCATACC TCTACATGTC 
               
               
                   
               
               
                 3121 
                 TGCTTGAGAT ATTTCCTCAG CCTGAAAGTT TCCCCAACCA 
               
               
                   
               
               
                 3161 
                 TCTGCCAGAG AACTCCTATG CATCCCTTAG AACCCTGCTC 
               
               
                   
               
               
                 3201 
                 AGACACCATT ACTTTTGTGA ACGCTTCTGC CACATCTTGT 
               
               
                   
               
               
                 3241 
                 CTTCCCCAAA ATTGATCACT CCGCCTTCTC CTGGGCTCCC 
               
               
                   
               
               
                 3281 
                 GTAGCACACT ATAACATCTG CTGGAGTGTT GCTGTTGCAC 
               
               
                   
               
               
                 3321 
                 CATACTTTCT TGTACATTTG TGTCTCCCTT CCCAACTAGA 
               
               
                   
               
               
                 3361 
                 CTGTAAGTGC CTTGCGGTCA GGGACTGAAT CTTGCCCGTT 
               
               
                   
               
               
                 3401 
                 TATGTATGCT CCATGTCTAG CCCATCATCC TGCTTGGAGC 
               
               
                   
               
               
                 3441 
                 AAGTAGGCAG GAGCTCAATA AATGTTTGTT GCATGAAGGA 
               
               
                   
               
               
                 3481 
                 AAAAAAAAAA AAAAAAA 
               
            
           
         
       
     
     The rs2228576 single nucleotide polymorphism (SNP) is present in the SCNN1A gene, where the variable nucleotide is at about position 2428 in SEQ ID NO:21 (underlined), which can be adenine in some individuals and guanine in others. The rs2228576 sequence (SEQ ID NO:22) is shown below, where the underlined A/G is the SNP. 
     
       
         
           
               
            
               
                 GGGCTCTGCAGGGGCCAGTTCCTCC   [A/G]   CCTGTCCTCTGGGGGGGC 
               
               
                   
               
               
                 CCTGAGA 
               
            
           
         
       
     
     The human the epithelial Na+channel encoded by the SCNN1A cDNA with SEQ ID NO:21 has the following sequence (SEQ ID NO:23). 
     
       
         
           
               
               
            
               
                 1 
                 MGMARGSLTR VPGVMGEGTQ GPELSLDPDP CSPQSTPGLM 
               
               
                   
               
               
                 41 
                 KGNKLEEQDP RPLQPIPGLM EGNKLEEQDS SPPQSTPGLM 
               
               
                   
               
               
                 81 
                 KGNKREEQGL GPEPAAPQQP TAEEEALIEF HRSYRELFEF 
               
               
                   
               
               
                 121 
                 FCNNTTIHGA IRLVCSQHNR MKTAFWAVLW LCTFGMMYWQ 
               
               
                   
               
               
                 161 
                 FGLLFGEYFS YPVSLNINLN SDKLVFPAVT ICTLNPYRYP 
               
               
                   
               
               
                 201 
                 EIKEELEELD RITEQTLFDL YKYSSFTTLV AGSRSRRDLR 
               
               
                   
               
               
                 241 
                 GTLPHPLQRL RVPPPPHGAR RARSVASSLR DNNPQVDWKD 
               
               
                   
               
               
                 281 
                 WKIGFQLCNQ NKSDCFYQTY SSGVDAVREW YRFHYINILS 
               
               
                   
               
               
                 321 
                 RLPETLPSLE EDTLGNFIFA CRFNQVSCNQ ANYSHFHHPM 
               
               
                   
               
               
                 361 
                 YGNCYTFNDK NNSNLWMSSM PGINNGLSLM LRAEQNDFIP 
               
               
                   
               
               
                 401 
                 LLSTVTGARV MVHGQDEPAF MDDGGFNLRP GVETSISMRK 
               
               
                   
               
               
                 441 
                 ETLDRLGGDY GDCTKNGSDV PVENLYPSKY TQQVCIHSCF 
               
               
                   
               
               
                 481 
                 QESMIKECGC AYIFYPRPQN VEYCDYRKHS SWGYCYYKLQ 
               
               
                   
               
               
                 521 
                 VDFSSDHLGC FTKCRKPCSV TSYQLSAGYS RWPSVTSQEW 
               
               
                   
               
               
                 561 
                 VFQMLSRQNN YTVNNKRNGV AKVNIFFKEL NYKTNSESPS 
               
               
                   
               
               
                 601 
                 VTMVTLLSNL GSQWSLWFGS SVLSVVEMAE LVFDLLVIMF 
               
               
                   
               
               
                 641 
                 LMLLRRFRSR YWSPGRGGRG AQEVASTLAS SPPSHFCPHP 
               
               
                   
               
               
                 681 
                 MSLSLSQPGP APSPALTAPP PAYATLGPRP SPGGSAGASS 
               
               
                   
               
               
                 721 
                 S   T   CPLGGP 
               
            
           
         
       
     
     Another cDNA sequence for the human SCNN1A gene with the same SNP is available from the NCBI database as accession number NM_001038.5 (GI:227430285). This sequence is provided below as SEQ ID NO:24. 
     
       
         
           
               
               
            
               
                 1 
                 CTTGCCTGTC TGCGTCTAAA GCCCCTGCCC AGAGTCCGCC 
               
               
                   
               
               
                 41 
                 TTCTCAGGTC CAGTACTCCC AGTTCACCTG CCCTCGGGAG 
               
               
                   
               
               
                 81 
                 CCCTCCTTCC TTCGGAAAAC TCCCGGCTCT GACTCCTCCT 
               
               
                   
               
               
                 121 
                 CAGCCCCTCC CCCCGCCCTG CTCACCTTTA ATTGAGATGC 
               
               
                   
               
               
                 161 
                 TAATGAGATT CCTGTCGCTT CCATCCCTGG CCGGCCAGCG 
               
               
                   
               
               
                 201 
                 GGCGGGCTCC CCAGCCAGGC CGCTGCACCT GTCAGGGGAA 
               
               
                   
               
               
                 241 
                 CAAGCTGGAG GAGCAGGACC CTAGACCTCT GCAGCCCATA 
               
               
                   
               
               
                 281 
                 CCAGGTCTCA TGGAGGGGAA CAAGCTGGAG GAGCAGGACT 
               
               
                   
               
               
                 321 
                 CTAGCCCTCC ACAGTCCACT CCAGGGCTCA TGAAGGGGAA 
               
               
                   
               
               
                 361 
                 CAAGCGTGAG GAGCAGGGGC TGGGCCCCGA ACCTGCGGCG 
               
               
                   
               
               
                 401 
                 CCCCAGCAGC CCACGGCGGA GGAGGAGGCC CTGATCGAGT 
               
               
                   
               
               
                 441 
                 TCCACCGCTC CTACCGAGAG CTCTTCGAGT TCTTCTGCAA 
               
               
                   
               
               
                 481 
                 CAACACCACC ATCCACGGCG CCATCCGCCT GGTGTGCTCC 
               
               
                   
               
               
                 521 
                 CAGCACAACC GCATGAAGAC GGCCTTCTGG GCAGTGCTGT 
               
               
                   
               
               
                 561 
                 GGCTCTGCAC CTTTGGCATG ATGTACTGGC AATTCGGCCT 
               
               
                   
               
               
                 601 
                 GCTTTTCGGA GAGTACTTCA GCTACCCCGT CAGCCTCAAC 
               
               
                   
               
               
                 641 
                 ATCAACCTCA ACTCGGACAA GCTCGTCTTC CCCGCAGTGA 
               
               
                   
               
               
                 681 
                 CCATCTGCAC CCTCAATCCC TACAGGTACC CGGAAATTAA 
               
               
                   
               
               
                 721 
                 AGAGGAGCTG GAGGAGCTGG ACCGCATCAC AGAGCAGACG 
               
               
                   
               
               
                 761 
                 CTCTTTGACC TGTACAAATA CAGCTCCTTC ACCACTCTCG 
               
               
                   
               
               
                 801 
                 TGGCCGGCTC CCGCAGCCGT CGCGACCTGC GGGGGACTCT 
               
               
                   
               
               
                 841 
                 GCCGCACCCC TTGCAGCGCC TGAGGGTCCC GCCCCCGCCT 
               
               
                   
               
               
                 881 
                 CACGGGGCCC GTCGAGCCCG TAGCGTGGCC TCCAGCTTGC 
               
               
                   
               
               
                 921 
                 GGGACAACAA CCCCCAGGTG GACTGGAAGG ACTGGAAGAT 
               
               
                   
               
               
                 961 
                 CGGCTTCCAG CTGTGCAACC AGAACAAATC GGACTGCTTC 
               
               
                   
               
               
                 1001 
                 TACCAGACAT ACTCATCAGG GGTGGATGCG GTGAGGGAGT 
               
               
                   
               
               
                 1041 
                 GGTACCGCTT CCACTACATC AACATCCTGT CGAGGCTGCC 
               
               
                   
               
               
                 1081 
                 AGAGACTCTG CCATCCCTGG AGGAGGACAC GCTGGGCAAC 
               
               
                   
               
               
                 1121 
                 TTCATCTTCG CCTGCCGCTT CAACCAGGTC TCCTGCAACC 
               
               
                   
               
               
                 1161 
                 AGGCGAATTA CTCTCACTTC CACCACCCGA TGTATGGAAA 
               
               
                   
               
               
                 1201 
                 CTGCTATACT TTCAATGACA AGAACAACTC CAACCTCTGG 
               
               
                   
               
               
                 1241 
                 ATGTCTTCCA TGCCTGGAAT CAACAACGGT CTGTCCCTGA 
               
               
                   
               
               
                 1281 
                 TGCTGCGCGC AGAGCAGAAT GACTTCATTC CCCTGCTGTC 
               
               
                   
               
               
                 1321 
                 CACAGTGACT GGGGCCCGGG TAATGGTGCA CGGGCAGGAT 
               
               
                   
               
               
                 1361 
                 GAACCTGCCT TTATGGATGA TGGTGGCTTT AACTTGCGGC 
               
               
                   
               
               
                 1401 
                 CTGGCGTGGA GACCTCCATC AGCATGAGGA AGGAAACCCT 
               
               
                   
               
               
                 1441 
                 GGACAGACTT GGGGGCGATT ATGGCGACTG CACCAAGAAT 
               
               
                   
               
               
                 1481 
                 GGCAGTGATG TTCCTGTTGA GAACCTTTAC CCTTCAAAGT 
               
               
                   
               
               
                 1521 
                 ACACACAGCA GGTGTGTATT CACTCCTGCT TCCAGGAGAG 
               
               
                   
               
               
                 1561 
                 CATGATCAAG GAGTGTGGCT GTGCCTACAT CTTCTATCCG 
               
               
                   
               
               
                 1601 
                 CGGCCCCAGA ACGTGGAGTA CTGTGACTAC AGAAAGCACA 
               
               
                   
               
               
                 1641 
                 GTTCCTGGGG GTACTGCTAC TATAAGCTCC AGGTTGACTT 
               
               
                   
               
               
                 1681 
                 CTCCTCAGAC CACCTGGGCT GTTTCACCAA GTGCCGGAAG 
               
               
                   
               
               
                 1721 
                 CCATGCAGCG TGACCAGCTA CCAGCTCTCT GCTGGTTACT 
               
               
                   
               
               
                 1761 
                 CACGATGGCC CTCGGTGACA TCCCAGGAAT GGGTCTTCCA 
               
               
                   
               
               
                 1801 
                 GATGCTATCG CGACAGAACA ATTACACCGT CAACAACAAG 
               
               
                   
               
               
                 1841 
                 AGAAATGGAG TGGCCAAAGT CAACATCTTC TTCAAGGAGC 
               
               
                   
               
               
                 1881 
                 TGAACTACAA AACCAATTCT GAGTCTCCCT CTGTCACGAT 
               
               
                   
               
               
                 1921 
                 GGTCACCCTC CTGTCCAACC TGGGCAGCCA GTGGAGCCTG 
               
               
                   
               
               
                 1961 
                 TGGTTCGGCT CCTCGGTGTT GTCTGTGGTG GAGATGGCTG 
               
               
                   
               
               
                 2001 
                 AGCTCGTCTT TGACCTGCTG GTCATCATGT TCCTCATGCT 
               
               
                   
               
               
                 2041 
                 GCTCCGAAGG TTCCGAAGCC GATACTGGTC TCCAGGCCGA 
               
               
                   
               
               
                 2081 
                 GGGGGCAGGG GTGCTCAGGA GGTAGCCTCC ACCCTGGCAT 
               
               
                   
               
               
                 2121 
                 CCTCCCCTCC TTCCCACTTC TGCCCCCACC CCATGTCTCT 
               
               
                   
               
               
                 2161 
                 GTCCTTGTCC CAGCCAGGCC CTGCTCCCTC TCCAGCCTTG 
               
               
                   
               
               
                 2201 
                 ACAGCCCCTC CCCCTGCCTA TGCCACCCTG GGCCCCCGCC 
               
               
                   
               
               
                 2241 
                 CATCTCCAGG GGGCTCTGCA GGGGCCAGTT CCTCC   A   CCTG 
               
               
                   
               
               
                 2281 
                 TCCTCTGGGG GGGCCCTGAG AGGGAAGGAG AGGTTTCTCA 
               
               
                   
               
               
                 2321 
                 CACCAAGGCA GATGCTCCTC TGGTGGGAGG GTGCTGGCCC 
               
               
                   
               
               
                 2361 
                 TGGCAAGATT GAAGGATGTG CAGGGCTTCC TCTCAGAGCC 
               
               
                   
               
               
                 2401 
                 GCCCAAACTG CCGTTGATGT GTGGAGGGGA AGCAAGATGG 
               
               
                   
               
               
                 2441 
                 GTAAGGGCTC AGGAAGTTGC TCCAAGAACA GTAGCTGATG 
               
               
                   
               
               
                 2481 
                 AAGCTGCCCA GAAGTGCCTT GGCTCCAGCC CTGTACCCCT 
               
               
                   
               
               
                 2521 
                 TGGTACTGCC TCTGAACACT CTGGTTTCCC CACCCAACTG 
               
               
                   
               
               
                 2561 
                 CGGCTAAGTC TCTTTTTCCC TTGGATCAGC CAAGCGAAAC 
               
               
                   
               
               
                 2601 
                 TTGGAGCTTT GACAAGGAAC TTTCCTAAGA AACCGCTGAT 
               
               
                   
               
               
                 2641 
                 AACCAGGACA AAACACAACC AAGGGTACAC GCAGGCATGC 
               
               
                   
               
               
                 2681 
                 ACGGGTTTCC TGCCCAGCGA CGGCTTAAGC CAGCCCCCGA 
               
               
                   
               
               
                 2721 
                 CTGGCCTGGC CACACTGCTC TCCAGTAGCA CAGATGTCTG 
               
               
                   
               
               
                 2761 
                 CTCCTCCTCT TGAACTTGGG TGGGAAACCC CACCCAAAAG 
               
               
                   
               
               
                 2801 
                 CCCCCTTTGT TACTTAGGCA ATTCCCCTTC CCTGACTCCC 
               
               
                   
               
               
                 2841 
                 GAGGGCTAGG GCTAGAGCAG ACCCGGGTAA GTAAAGGCAG 
               
               
                   
               
               
                 2881 
                 ACCCAGGGCT CCTCTAGCCT CATACCCGTG CCCTCACAGA 
               
               
                   
               
               
                 2921 
                 GCCATGCCCC GGCACCTCTG CCCTGTGTCT TTCATACCTC 
               
               
                   
               
               
                 2961 
                 TACATGTCTG CTTGAGATAT TTCCTCAGCC TGAAAGTTTC 
               
               
                   
               
               
                 3001 
                 CCCAACCATC TGCCAGAGAA CTCCTATGCA TCCCTTAGAA 
               
               
                   
               
               
                 3041 
                 CCCTGCTCAG ACACCATTAC TTTTGTGAAC GCTTCTGCCA 
               
               
                   
               
               
                 3081 
                 CATCTTGTCT TCCCCAAAAT TGATCACTCC GCCTTCTCCT 
               
               
                   
               
               
                 3121 
                 GGGCTCCCGT AGCACACTAT AACATCTGCT GGAGTGTTGC 
               
               
                   
               
               
                 3161 
                 TGTTGCACCA TACTTTCTTG TACATTTGTG TCTCCCTTCC 
               
               
                   
               
               
                 3201 
                 CAACTAGACT GTAAGTGCCT TGCGGTCAGG GACTGAATCT 
               
               
                   
               
               
                 3241 
                 TGCCCGTTTA TGTATGCTCC ATGTCTAGCC CATCATCCTG 
               
               
                   
               
               
                 3281 
                 CTTGGAGCAA GTAGGCAGGA GCTCAATTTA TGTTTGTTGC 
               
               
                   
               
               
                 3321 
                 ATGAAGGAAA AAAAAAAAAA AAAAA 
               
            
           
         
       
     
     The human epithelial Na+channel encoded by the SCNN1A cDNA with SEQ ID NO:24 has the following sequence (SEQ ID NO:25). 
     
       
         
           
               
               
            
               
                 1 
                 MEGNKLLQED SSPPQSTPGL MKGNKREEQG LGPEPAAPQQ 
               
               
                   
               
               
                 41 
                 PTAEEEALIE FHRSYRELFE FFCNNTTIHG AIRLVCSQHN 
               
               
                   
               
               
                 81 
                 RMKTAFWAVL WLCTFGMMYW QFGLLFGEYF SYPVSLNINL 
               
               
                   
               
               
                 121 
                 NSDKLVFPAV TICTLNPYRY PEIKEELEEL DRITEQTLFD 
               
               
                   
               
               
                 161 
                 LYKYSSFTTL VAGSRSRRDL RGTLPHPLQR LRVPPPPHGA 
               
               
                   
               
               
                 201 
                 RRARSVASSL RDNNPQVDWK DWKIGFQLCN QNKSDCFYQT 
               
               
                   
               
               
                 241 
                 YSSGVDAVRE WYRFHYINIL SRLPETLPSL EEDTLGNFIF 
               
               
                   
               
               
                 281 
                 ACRFNQVSCN QANYSHFHHP MYGNCYTFND KNNSNLWMSS 
               
               
                   
               
               
                 321 
                 MPGINNGLSL MLRAEQNDFI PLLSTVTGAR VMVHGQDEPA 
               
               
                   
               
               
                 361 
                 FMDDGGFNLR PGVETSISMR KETLDRLGGD YGDCTKNGSD 
               
               
                   
               
               
                 401 
                 VPVENLYPSK YTQQVCIHSC FQESMIKECS CAYIFYPRPQ 
               
               
                   
               
               
                 441 
                 NVEYCDYRKH SSWGYCYYKL QVDFSSDHLG CFTKCRKPCS 
               
               
                   
               
               
                 481 
                 VTSYQLSAGY SRWPSVTSQE WVFQMLSRQN NYTVNNKRNG 
               
               
                   
               
               
                 521 
                 VAKVNIFFKE LNYKTNSESP SVTMVTLLSN LGSQWSLWFG 
               
               
                   
               
               
                 561 
                 SSVLSVVEMA ELVFDLLVIM FLMLLRRFRS RYWSPGRGGR 
               
               
                   
               
               
                 601 
                 GAQEVASTLA SSPPSHFCPH PMSLSLSQPG PAPSPALTAP 
               
               
                   
               
               
                 641 
                 PPAYATLGPR PSPGGSAGAS SS   T   CPLGGP 
               
            
           
         
       
     
     Note that the underlined threonine at position 722 of the SEQ ID NO:23 SCNN1A protein, and the underlined threonine at position 663 of the SEQ ID NO:25 SCNN1A protein, is threonine because some individuals have nucleotide sequence SEQ ID NO:22, where the variable nucleotide is adenine. However, position 722 of SEQ ID NO:23 and position 663 of SEQ ID NO:25 can be alanine in some individuals because those individuals have guanine as the variable nucleotide in sequence SEQ ID NO:22. 
     Patients with the threonine substitution in SCNN1A (adenine in rs2228576) have more functional Na +  channels and consequently higher activity higher voltage currents across the cells. Hence, patients with such a threonine at the variable site in SCNNIA are more susceptible to hypertension than SCNNIA proteins with alanine at that position. Patients with the threonine substitution in SCNN1A can benefit from administration of amiloride. 
     Common and functional genetic variation of alpha adducin at amino acid 460 has also been identified where some individuals have glycine and others have tryptophan. A cDNA sequence for the human alpha adducin gene (ADD1) is available from the NCBI database as accession number NM_001119.4 (GI:346644753). This ADD1 sequence is provided below as SEQ ID NO:26. 
     
       
         
           
               
               
            
               
                 1 
                 GCACCCAGGT CGGGCGGTGG GGGCGAGCGG AGGGGCTGAG 
               
               
                   
               
               
                 41 
                 GGGCGGAGAG GCCTGGCGGG CCGCTGCTGC GGGCCAGGGG 
               
               
                   
               
               
                 81 
                 ACGGGGGCGG AGCCGGAGCC GGAGCCGACG GGCGGTGGCC 
               
               
                   
               
               
                 121 
                 GCACTGGGAC CCCGGAATCC CGCGCGCTGC CCACGATTCG 
               
               
                   
               
               
                 161 
                 CTTCTGAGGA ACCTAGAAAG ATTGTACAAT GAATGGTGAT 
               
               
                   
               
               
                 201 
                 TCTCGTGCTG CGGTGGTGAC CTCACCACCC CCGACCACAG 
               
               
                   
               
               
                 241 
                 CCCCTCACAA GGAGAGGTAC TTCGACCGAG TAGATGAGAA 
               
               
                   
               
               
                 281 
                 CAACCCAGAG TACTTGAGGG AGAGGAACAT GGCACCAGAC 
               
               
                   
               
               
                 321 
                 CTTCGCCAGG ACTTCAACAT GATGGAGCAA AAGAAGAGGG 
               
               
                   
               
               
                 361 
                 TGTCCATGAT TCTGCAAAGC CCTGCTTTCT GTGAAGAATT 
               
               
                   
               
               
                 401 
                 GGAATCAATG ATACAGGAGC AATTTAAGAA GGGGAAGAAC 
               
               
                   
               
               
                 441 
                 CCCACAGGCC TATTGGCATT ACAGCAGATT GCAGATTTTA 
               
               
                   
               
               
                 481 
                 TGACCACGAA TGTACCAAAT GTCTACCCAG CAGCTCCGCA 
               
               
                   
               
               
                 521 
                 AGGAGGGATG GCTGCCTTAA ACATGAGTCT TGGTATGGTG 
               
               
                   
               
               
                 561 
                 ACTCCTGTGA ACGATCTTAG AGGATCTGAT TCTATTGCGT 
               
               
                   
               
               
                 601 
                 ATGACAAAGG AGAGAAGTTA TTACGGTGTA AATTGGCAGC 
               
               
                   
               
               
                 641 
                 GTTTTATAGA CTAGCAGATC TCTTTGGGTG GTCTCAGCTT 
               
               
                   
               
               
                 681 
                 ATCTACAATC ATATCACAAC CAGAGTGAAC TTTACAGTGA 
               
               
                   
               
               
                 721 
                 AACACTTCCT CATTGTCCCT TTTGGGCTTC TTTACAGTGA 
               
               
                   
               
               
                 761 
                 AGTGACTGCA TCCAGTTTGG TTAAGATCAA TCTACAAGGA 
               
               
                   
               
               
                 801 
                 GATATAGTAG ATCGTGGAAG CACTAATCTG GGAGTGAATC 
               
               
                   
               
               
                 841 
                 AGGCCGGCTT CACCTTACAC TCTGCAATTT ATGCTGCACG 
               
               
                   
               
               
                 881 
                 CCCGGACGTG AAGTGCGTCG TGCACATTCA CACCCCAGCA 
               
               
                   
               
               
                 921 
                 GGGGCTGCGG TCTCTGCAAT GAAATGTGGC CTCTTGCCAA 
               
               
                   
               
               
                 961 
                 TCTCCCCGGA GGCGCTTTCC CTTGGAGAAG TGGCTTATCA 
               
               
                   
               
               
                 1001 
                 TGACTACCAT GGCATTCTGG TTGATGAAGA GGAAAAAGTT 
               
               
                   
               
               
                 1041 
                 TTGATTCAGA AAAATCTGGG GCCTAAAAGC AAGGTTCTTA 
               
               
                   
               
               
                 1081 
                 TTCTCCGGAA CCATGGGCTC GTGTCAGTTG GAGAGAGCGT 
               
               
                   
               
               
                 1121 
                 TGAGGAGGCC TTCTATTACA TCCATAACCT TGTGGTTGCC 
               
               
                   
               
               
                 1161 
                 TGTGAGATCC AGGTTCGAAC TCTGGCCAGT GCAGGAGGAC 
               
               
                   
               
               
                 1201 
                 CAGACAACTT AGTCCTGCTG AATCCTGAGA AGTACAAAGC 
               
               
                   
               
               
                 1241 
                 CAAGTCCCGT TCCCCAGGGT CTCCGGTAGG GGAAGGCACT 
               
               
                   
               
               
                 1281 
                 GGATCGCCTC CCAAGTGGCA GATTGGTGAG CAGGAATTTG 
               
               
                   
               
               
                 1321 
                 AAGCCCTCAT GCGGATGCTC GATAATCTGG GCTACAGAAC 
               
               
                   
               
               
                 1361 
                 TGGCTACCCT TATCGATACC CTGCTCTGAG AGAGAAGTCT 
               
               
                   
               
               
                 1401 
                 AAAAAATACA GCGATGTGGA GGTTCCTGCT AGTGTCACAG 
               
               
                   
               
               
                 1441 
                 GTTACTCCTT TGCTAGTGAC GGTGATTCGG GCACTTGCTC 
               
               
                   
               
               
                 1481 
                 CCCACTCAGA CACAGTTTTC AGAAGCAGCA GCGGGAGAAG 
               
               
                   
               
               
                 1521 
                 ACAAGATGGC TGAACTCTGG CCGGGGCGAC GAAGCTTCCG 
               
               
                   
               
               
                 1561 
                 AGGAA   G   GGCA GAATGGAAGC AGTCCCAAGT CGAAGACTAA 
               
               
                   
               
               
                 1601 
                 GTGGACTAAA GAGGATGGAC ATAGAACTTC CACCTCTGCT 
               
               
                   
               
               
                 1641 
                 GTCCCTAACC TGTTTGTTCC ATTGAACACT AACCCAAAAG 
               
               
                   
               
               
                 1681 
                 AGGTCCAGGA GATGAGGAAC AAGATCCGAG AGCAGAATTT 
               
               
                   
               
               
                 1721 
                 ACAGGACATT AAGACGGCTG GCCCTCAGTC CCAGGTTTTG 
               
               
                   
               
               
                 1761 
                 TGTGGTGTAG TGATGGACAG GAGCCTCGTC CAGGGAGAGC 
               
               
                   
               
               
                 1801 
                 TGGTGACGGC CTCCAAGGCC ATCATTGAAA AGGAGTACCA 
               
               
                   
               
               
                 1841 
                 GCCCCACGTC ATTGTGAGCA CCACGGGCCC CAACCCCTTC 
               
               
                   
               
               
                 1881 
                 ACCACACTCA CAGACCGTGA GCTGGAGGAG TACCGCAGGG 
               
               
                   
               
               
                 1921 
                 AGGTGGAGAG GAAGCAGAAG GGCTCTGAAG AGAATCTGGA 
               
               
                   
               
               
                 1961 
                 CGAGGCTAGA GAACAGAAAG AAAAGAGTCC TCCAGACCAG 
               
               
                   
               
               
                 2001 
                 CCTGCGGTCC CCCACCCGCC TCCCAGCACT CCCATCAAGC 
               
               
                   
               
               
                 2041 
                 TGGAGGAAGA CCTTGTGCCG GAGCCGACTA CTGGAGATGA 
               
               
                   
               
               
                 2081 
                 CAGTGATGCT GCCACCTTTA AGCCAACTCT CCCCGATCTG 
               
               
                   
               
               
                 2121 
                 TCCCCTGATG AACCTTCAGA AGCACTCGGC TTCCCAATGT 
               
               
                   
               
               
                 2161 
                 TAGAGAAGGA GGAGGAAGCC CATAGACCCC CAAGCCCCAC 
               
               
                   
               
               
                 2201 
                 TGAGGCCCCT ACTGAGGCCA GCCCCGAGCC AGCCCCAGAC 
               
               
                   
               
               
                 2241 
                 CCAGCCCCGG TGGCTGAAGA GGCTGCCCCC TCAGCTGTCG 
               
               
                   
               
               
                 2281 
                 AGGAGGGGGC CGCCGCGGAC CCTGGCAGCG ATGGGTCTCC 
               
               
                   
               
               
                 2321 
                 AGGCAAGTCC CCGTCCAAAA AGAAGAAGAA GTTCCGTACC 
               
               
                   
               
               
                 2361 
                 CCGTCCTTTC TGAAGAAGAG CAAGAAGAAG AGTGACTCCT 
               
               
                   
               
               
                 2401 
                 GAAAGCCCTG CGCTAACACT GTCCTGTCCG GAGCGACCCT 
               
               
                   
               
               
                 2441 
                 GGCTCTGCCA GCGTCCCCGG CCACGTCTGT GCTCTGTCCT 
               
               
                   
               
               
                 2481 
                 TGTGTAATGG AATGCAAAAA AGCCAAGCCC TCCGCCTAGA 
               
               
                   
               
               
                 2521 
                 GGTCCCCTCA CGTGACCAGC CCCGTGTAGC CCCGGGCTGA 
               
               
                   
               
               
                 2561 
                 CCCAGTGTGT GCTCAGCAGC CCCACCCCAC CCTGCCCCTT 
               
               
                   
               
               
                 2601 
                 GTCCTCTCAG AGCCTCAGCT TCTGGGGGAG ACATGCTCTC 
               
               
                   
               
               
                 2641 
                 CCCACAGGGG GGAGGCACTA AGTCATGGTC CTGGCTGGAA 
               
               
                   
               
               
                 2681 
                 GGTACTGAAG GCTTCTGCAG CTTTGGCTGC ACGTCACCCT 
               
               
                   
               
               
                 2721 
                 CCTGAGCCTC ACCTTTCCTG CCGTCCCTCC TGTTGTGAAA 
               
               
                   
               
               
                 2761 
                 TCACCACATT CTGTCTCTGC TTGGCTTCCC CTCCACCCTA 
               
               
                   
               
               
                 2801 
                 AAGTCTCAGG TGACGGACTC AGACTCCTGG CTTCATGTGG 
               
               
                   
               
               
                 2841 
                 CATTCTCTCT GCTCAGTGAT CTCACTTAAA TCTATATACA 
               
               
                   
               
               
                 2881 
                 AAGCCTTGGT CCCGTGAAAA CACTCGTGTG CCCACCAGCG 
               
               
                   
               
               
                 2921 
                 GCCTTGAAGA GGCAGGTCTG GGCCAGATGC TGGGCAGGAA 
               
               
                   
               
               
                 2961 
                 ACCCCAGCGG CAGATGGGCC TGTGTGCACC CAACGTGATG 
               
               
                   
               
               
                 3001 
                 CTATGCATGT CTGACCGACG ATCCCTCGAC CAGAATCAGA 
               
               
                   
               
               
                 3041 
                 TTCAGGAGCT CAGTTTCTTT TTCACTTGGG TCTCTGGATT 
               
               
                   
               
               
                 3081 
                 CCTGTCATAG GGAAGGTATA TCAGGAGGGG AAGAGGCCTT 
               
               
                   
               
               
                 3121 
                 TCTAGAATTT TCTTTGAGCA GGTTTACAAT TTAGCTTACA 
               
               
                   
               
               
                 3161 
                 TTTTTCGACT GTGAACGTGA ATAGGCTGCT TTTTGCTTTC 
               
               
                   
               
               
                 3201 
                 TTCTTTCCAG ACCCCACAGT AGAGCACTTT TCACTTATTT 
               
               
                   
               
               
                 3241 
                 GGGGGAGGCT TCAGGGGACT GTTCTCACCT TAACTCAGCC 
               
               
                   
               
               
                 3281 
                 AGAAAGATGC CCTAGTTGTG ATCAAAGGTA ACTCGAGGTG 
               
               
                   
               
               
                 3321 
                 GAGGGTAGCC CTGGGGCCCC TCGACATCAC CGTCATTGAT 
               
               
                   
               
               
                 3361 
                 GGAGCCTGAA CCGTGTGCTC CTCGGCAGAT GCTGTTGTTG 
               
               
                   
               
               
                 3401 
                 TTACTTCCCT CCAAGAGGCT GGAAAAGGGC TCAGAGCTGC 
               
               
                   
               
               
                 3441 
                 TGAGCAGGAA CCGGAGGGTG ACCCATTTCA GGAGGTGCCG 
               
               
                   
               
               
                 3481 
                 GTACCAGCCT GACTAGGTAC AGGCAAGCTT GTGTGGGCCC 
               
               
                   
               
               
                 3521 
                 AACAGGCCCT TGGTAGAGCT GGTGCCAGAT GTGGGCTCAG 
               
               
                   
               
               
                 3561 
                 ATCCTGGGCA TGATGGGCCG AGCCACCTCG GATCCCACTG 
               
               
                   
               
               
                 3601 
                 ATTGGCCAGC CGAGCGAGAA CCAGGCTGCT GCATGGCACT 
               
               
                   
               
               
                 3641 
                 GACCGCCGCT TCCAGCTTCC TCTGAGCCGC AGGGCCTGCT 
               
               
                   
               
               
                 3681 
                 ACGCGGGCAA GCGTGCTGCC TCTCTTCTGT GTCGTTTTGT 
               
               
                   
               
               
                 3721 
                 TGCCAAGGCA GAATGAAAAG TCCTTAACCG TGGACTCTTC 
               
               
                   
               
               
                 3761 
                 CTTTATCCCC TCCTTTACCC CACATATGCA ATGACTTTTA 
               
               
                   
               
               
                 3801 
                 ATTTTCACTT TTGTAGTTTA ATCCTTTGTA TTACAACATG 
               
               
                   
               
               
                 3841 
                 AAATATAGTT GCATATATGG ACACCGACTT GGGAGGACAG 
               
               
                   
               
               
                 3881 
                 GTCCTGAATG TCCTTTCTCC AGTGTAACAT GTTTTACTCA 
               
               
                   
               
               
                 3921 
                 CAAATAAAAT TCTTTCAGCA AGTTCCTTGT CTAAAAAAAA 
               
               
                   
               
               
                 3961 
                 AAAAAAAAAA 
               
            
           
         
       
     
     The rs4961 single nucleotide polymorphism (SNP) is present in the ADD1 gene, where the variable nucleotide is at about position 1566 in SEQ ID NO:26 (underlined), which can be guanine in some individuals and thymine in others. The rs4961 sequence (SEQ ID NO:27) is shown below, where the underlined G/T is the SNP. 
     
       
         
           
               
            
               
                 CCGGGGCGACGAAGCTTCCGAGGAA   [G/T]   GGCAGAATGGAAGCAGTCCC 
               
               
                   
               
               
                 AAGTC. 
               
            
           
         
       
     
     The human alpha adducin encoded by the ADD1 cDNA with SEQ ID NO:26 has the following sequence (SEQ ID NO:28). 
     
       
         
           
               
               
            
               
                 1 
                 MNGDSRAAVV TSPPPTTAPH KERYFDRVDE NNPEYLRERN 
               
               
                   
               
               
                 41 
                 MAPDLRQDFN MMEQKKRVSM ILQSPAFCEE LESMIQEQFK 
               
               
                   
               
               
                 81 
                 KGKNPTGLLA LQQIADFMTT NVPNVYPAAP QGGMAALNMS 
               
               
                   
               
               
                 121 
                 LGMVTPVNDL RGSDSIAYDK GEKLLRCKLA AFYRLADLFG 
               
               
                   
               
               
                 161 
                 WSQLIYNHIT TRVNSEQEHF LIVPFGLLYS EVTASSLVKI 
               
               
                   
               
               
                 201 
                 NLQGDIVDRG STNLGVNQAG FTLHSAIYAA RPDVKCVVHI 
               
               
                   
               
               
                 241 
                 HTPAGAAVSA MKCGLLPISP EALSLGEVAY HDYHGILVDE  
               
               
                   
               
               
                 281 
                 EEKVLIQKNL GPKSKVLILR NHGLVSVGES VEEAFYYIHN 
               
               
                   
               
               
                 321 
                 LVVACEIQVR TLASAGGPDN LVLLNPEKYK AKSRSPGSPV 
               
               
                   
               
               
                 361 
                 GEGTGSPPKW QIGEQEFEAL MRMLDNLGYR TGYPYRYPAL 
               
               
                   
               
               
                 401 
                 REKSKKYSDV EVPASVTGYS FASDGDSGTC SPLRHSFQKQ 
               
               
                   
               
               
                 441 
                 QREKTRWLNS GRGDEASEE   G    QNGSSPKSKT KWTKEDGHRT 
               
               
                   
               
               
                 481 
                 STSAVPNLFV PLNTNPKEVQ EMRNKIREQN LQDIKTAGPQ 
               
               
                   
               
               
                 521 
                 SQVLCGVVMD RSLVQGELVT AKSAIIEKEY QPHVIVSTTG 
               
               
                   
               
               
                 561 
                 PNPFTTLTDR ELEEYRREVE RKQKGSEENL DEAREQKEKS 
               
               
                   
               
               
                 601 
                 PPDQPAVPHP PPSTPIKLEE DLVPEPTTGD DSDAATFKPT 
               
               
                   
               
               
                 641 
                 LPDLSPDEPS EALGFPMLEK EEEAHRPPSP TEAPTEASPE 
               
               
                   
               
               
                 681 
                 PAPDPAPVAE EAAPSAVEEG AAADPGSDGS PGKSPSKKKK 
               
               
                   
               
               
                 721 
                 KFRTPSFLKK SKKKSDS 
               
            
           
         
       
     
     Note that the underlined glycine at position 460 of the SEQ ID NO:28 alpha adducin protein is glycine because some individuals have nucleotide sequence SEQ ID NO:26, where the variable nucleotide at position 1566 is guanine. However, position 460 of SEQ ID NO:28 can be tryptophan in some individuals because those individuals have thymine as the variable nucleotide at position 1566 in sequence SEQ ID NO:28. 
     Individuals with the tryptophan variant of alpha adducin are more likely to be salt sensitive, more likely to have hypertension and have a greater response to diuretics. 
     Genetic variation of the sodium (Na + ) chloride (Cl − ) co-transporter (SLC12A3) also has blood pressure consequences. A cDNA sequence for the sodium (Na + ) chloride (Cl + ) co-transporter (SLC12A3) is available from the NCBI database as accession number NM_000339.2 (GI:186910314). This SLC12A3 cDNA sequence is provided below as SEQ ID NO:29. 
     
       
         
           
               
               
            
               
                 1 
                 CTGGCCCCTC CCTGGACACC CAGGCGACAA TGGCAGAACT 
               
               
                   
               
               
                 41 
                 GCCCACAACA GAGACGCCTG GGGACGCCAC TTTGTGCAGC 
               
               
                   
               
               
                 81 
                 GGGCGCTTCA CCATCAGCAC ACTGCTGAGC AGTGATGAGC 
               
               
                   
               
               
                 121 
                 CCTCTCCACC AGCTGCCTAT GACAGCAGCC ACCCCAGCCA 
               
               
                   
               
               
                 161 
                 CCTGACCCAC AGCAGCACCT TCTGCATGCG CACCTTTGGC 
               
               
                   
               
               
                 201 
                 TACAACACGA TCGATGTGGT GCCCACATAT GAGCACTATG 
               
               
                   
               
               
                 241 
                 CCAACAGCAC CCAGCCTGGT GAGCCCCGGA AGGTCCGGCC 
               
               
                   
               
               
                 281 
                 CACACTGGCT GACCTGCACT CCTTCCTCAA GCAGGAAGGC 
               
               
                   
               
               
                 321 
                 AGACACCTGC ATGCCCTGGC CTTTGACAGC CGGCCCAGCC 
               
               
                   
               
               
                 361 
                 ACGAGATGAC TGATGGGCTG GTGGAGGGCG AGGCAGGCAC 
               
               
                   
               
               
                 401 
                 CAGCAGCGAG AAGAACCCCG AGGAGCCAGT GCGCTTCGGC 
               
               
                   
               
               
                 441 
                 TGGGTCAAGG GGGTGATGAT TCGTTGCATG CTCAACATTT 
               
               
                   
               
               
                 481 
                 GGGGCGTGAT CCTCTACCTG CGGCTGCCCT GGATTACGGC 
               
               
                   
               
               
                 521 
                 CCAGGCAGGC ATCGTCCTGA CCTGGATCAT CATCCTGCTG 
               
               
                   
               
               
                 561 
                 TCGGTCACGG TGACCTCCAT CACAGGCCTC TCCATCTCAG 
               
               
                   
               
               
                 601 
                 CCATCTCCAC CAATGGCAAG GTCAAGTCAG GTGGCACCTA 
               
               
                   
               
               
                 641 
                 CTTCCTCATC TCCCGGAGTC TGGGCCCAGA GCTTGGGGGC 
               
               
                   
               
               
                 681 
                 TCCATCGGCC TCATTTTCGC TTTCGCCAAT GCCGTGGGTC 
               
               
                   
               
               
                 721 
                 TGGCCATGCA CACGGTGGGC TTTGCAGAGA CCGTGCGGGA 
               
               
                   
               
               
                 761 
                 CCTGCTCCAG GAGTATGGGG CACCCATCGT GGACCCCATT 
               
               
                   
               
               
                 801 
                 AACGACATCC GCATCATTG   G    CGTGGTCTCG GTCACTGTGC 
               
               
                   
               
               
                 841 
                 TGCTGGCCAT CTCCCTGGCT GGCATGGAGT GGGAGTCCAA 
               
               
                   
               
               
                 881 
                 GGCCCAGGTG CTGTTCTTCC TTGTCATCAT GGTCTCCTTT 
               
               
                   
               
               
                 921 
                 GCCAACTATT TAGTGGGGAC GCTGATCCCC CCATCTGAGG 
               
               
                   
               
               
                 961 
                 ACAAGGCCTC CAAAGGCTTC TTCAGCTACC GGGCGGACAT 
               
               
                   
               
               
                 1001 
                 TTTTGTCCAG AACTTGGTGC CTGACTGGCG GGGTCCAGAT 
               
               
                   
               
               
                 1041 
                 GGCACCTTCT TCGGAATGTT CTCCATCTTC TTCCCCTCGG 
               
               
                   
               
               
                 1081 
                 CCACAGGCAT CCTGGCAGGG GCCAACATAT CTGGTGACCT 
               
               
                   
               
               
                 1121 
                 CAAGGACCCT GCTATAGCCA TCCCCAAGGG GACCCTCATG 
               
               
                   
               
               
                 1161 
                 GCCATTTTCT GGACGACCAT TTCCTACCTG GCCATCTCAG 
               
               
                   
               
               
                 1201 
                 CCACCATTGG CTCCTGCGTG GTGCGTGATG CCTCTGGGGT 
               
               
                   
               
               
                 1241 
                 CCTGAATGAC ACAGTGACCC CTGGCTGGGG TGCCTGCGAG 
               
               
                   
               
               
                 1281 
                 GGGCTGGCCT GCAGCTATGG CTGGAACTTC ACCGAGTGCA 
               
               
                   
               
               
                 1321 
                 CCCAGCAGCA CAGCTGCCAC TACGGCCTCA TCAACTATTA 
               
               
                   
               
               
                 1361 
                 CCAGACCATG AGCATGGTGT CAGGCTTCGC GCCCCTGATC 
               
               
                   
               
               
                 1401 
                 ACGGCTGGCA TCTTCGGGGC CACCCTCTCC TCTGCCCTGG 
               
               
                   
               
               
                 1441 
                 CCTGCCTTGT CTCTGCTGCC AAAGTCTTCC AGTGCCTTTG 
               
               
                   
               
               
                 1481 
                 CGAGGACCAG CTGTACCCAC TGATCGGCTT CTTCGGCAAA 
               
               
                   
               
               
                 1521 
                 GGCTATGGCA AGAACAAGGA GCCCGTGCGT GGCTACCTGC 
               
               
                   
               
               
                 1561 
                 TGGCCTACGC CATCGCTGTG GCCTTCATCA TCATCGCTGA 
               
               
                   
               
               
                 1601 
                 GCTCAACACC ATAGCCCCCA TCATTTCCAA CTTCTTCCTC 
               
               
                   
               
               
                 1641 
                 TGCTCCTATG CCCTCATCAA CTTCAGCTGC TTCCACGCCT 
               
               
                   
               
               
                 1681 
                 CCATCACCAA CTCGCCTGGG TGGAGACCTT CATTCCAATA 
               
               
                   
               
               
                 1721 
                 CTACAACAAG TGGGCGGCGC TGTTTGGGGC TATCATCTCC 
               
               
                   
               
               
                 1761 
                 GTGGTCATCA TGTTCCTCCT CACCTGGTGG GCGGCCCTCA 
               
               
                   
               
               
                 1801 
                 TCGCCATTGG CGTGGTGCTC TTCCTCCTGC TCTATGTCAT 
               
               
                   
               
               
                 1841 
                 CTACAAGAAG CCAGAGGTAA ATTGGGGCTC CTCGGTACAG 
               
               
                   
               
               
                 1881 
                 GCTGGCTCCT ACAACCTGGC CCTCAGCTAC TCGGTGGGCC 
               
               
                   
               
               
                 1921 
                 TCAATGAGGT GGAAGACCAC ATCAAGAACT ACCGCCCCCA 
               
               
                   
               
               
                 1961 
                 GTGCCTGGTG CTCACGGGGC CCCCCAACTT CCGCCCGGCC 
               
               
                   
               
               
                 2001 
                 CTGGTGGACT TTGTGGGCAC CTTCACCCGG AACCTCAGCC 
               
               
                   
               
               
                 2041 
                 TGATGATCTG TGGCCACGTG CTCATCGGAC CCCACAAGCA 
               
               
                   
               
               
                 2081 
                 GAGGATGCCT GAGCTCCAGC TCATCGCCAA CGGGCACACC 
               
               
                   
               
               
                 2121 
                 AAGTGGCTGA ACAAGAGGAA GATCAAGGCC TTCTACTCGG 
               
               
                   
               
               
                 2161 
                 ATGTCATTGC CGAGGACCTC CGCAGAGGCG TCCAGATCCT 
               
               
                   
               
               
                 2201 
                 CATGCAGGCC GCAGGTCTCG GGAGAATGAA GCCCAACATT 
               
               
                   
               
               
                 2241 
                 CTGGTGGTTG GGTTCAAGAA GAACTGGCAG TCGGCTCACC 
               
               
                   
               
               
                 2281 
                 CGGCCACAGT GGAAGACTAC ATTGGCATCC TCCATGATGC 
               
               
                   
               
               
                 2321 
                 CTTTGATTTC AACTATGGCG TGTGTGTCAT GAGGATGCGG 
               
               
                   
               
               
                 2361 
                 GAGGGACTCA ACGTGTCCAA GATGATGCAG GCGCACATTA 
               
               
                   
               
               
                 2401 
                 ACCCCGTGTT TGACCCAGCG GAGGACGGGA AGGAAGCCAG 
               
               
                   
               
               
                 2441 
                 CGCCAGAGGT GCCAGGCCAT CAGTCTCTGG CGCTTTGGAC 
               
               
                   
               
               
                 2481 
                 CCCAAGGCCC TGGTGAAGGA GGAGCAGGCC ACCACCATCT 
               
               
                   
               
               
                 2521 
                 TCCAGTCGGA GCAGGGCAAG AAGACCATAG ACATCTACTG 
               
               
                   
               
               
                 2561 
                 GCTCTTTGAC GATGGAGGCC TCACCCTCCT CATTCCCTAT 
               
               
                   
               
               
                 2601 
                 CTCCTTGGCC GCAAGAGGAG GTGGAGCAAA TGCAAGATCC 
               
               
                   
               
               
                 2641 
                 GTGTGTTCGT AGGCGGCCAG ATTAACAGGA TGGACCAGGA 
               
               
                   
               
               
                 2681 
                 GAGAAAGGCG ATCATTTCTC TGCTGAGCAA GTTCCGACTG 
               
               
                   
               
               
                 2721 
                 GGATTCCATG AAGTCCACAT CCTCCCTGAC ATCAACCAGA 
               
               
                   
               
               
                 2761 
                 ACCCTCGGGC TGAGCACACC AAGAGGTTTG AGGACATGAT 
               
               
                   
               
               
                 2801 
                 TGCACCCTTC CGTCTGAATG ATGGCTTCAA GGATGAGGCC 
               
               
                   
               
               
                 2841 
                 ACTGTCAACG AGATGCGGCG GGACTGCCCC TGGAAGATCT 
               
               
                   
               
               
                 2881 
                 CAGATGAGGA GATTACGAAG AACAGAGTCA AGTCCCTTCG 
               
               
                   
               
               
                 2921 
                 GCAGGTGAGG CTGAATGAGA TTGTGCTGGA TTACTCCCGA 
               
               
                   
               
               
                 2961 
                 GACGCTGCTC TCATCGTCAT CACTTTGCCC ATAGGGAGGA 
               
               
                   
               
               
                 3001 
                 AGGGGAAGTG CCCCAGCTCG CTGTACATGG CCTGGCTGGA 
               
               
                   
               
               
                 3041 
                 GACCCTGTCC CAGGACCTCA GACCTCCAGT CATCCTGATC 
               
               
                   
               
               
                 3081 
                 CGAGGAAACC AGGAAAACGT GCTCACCTTT TACTGCCAGT 
               
               
                   
               
               
                 3121 
                 AACTCCAGGC TTTGACATCC CTGTCCACAG CTCTGAGTGT 
               
               
                   
               
               
                 3161 
                 GTGGGATAAG TTGGAACTTG ATTGCCTCTA GTCCACAGGG 
               
               
                   
               
               
                 3201 
                 ATGAGACTCA TGTTCTGTTG CACTTTAAGT GGCAGCATCT 
               
               
                   
               
               
                 3241 
                 GATGATCTCA CCGAAAAAGA TGGTAGATTT CCAAATCTGG 
               
               
                   
               
               
                 3281 
                 CTGGACTCCA CTTCCATGGG ACACATTCCC TGGGTCTTGT 
               
               
                   
               
               
                 3321 
                 GTTTATAGGC TAGAGAAATA GCAGATGGAG CTGCAAGGAA 
               
               
                   
               
               
                 3361 
                 AACTCTCTAA AGCATCCTAT TCCTTTTAAA GGATTTCTTT 
               
               
                   
               
               
                 3401 
                 TGATTTTGAT GACCATTAAT TAAGAGTTCA GTCTTTGATT 
               
               
                   
               
               
                 3441 
                 TGTATGCAAA TTGGAGTCCC AATGCTGGGC GTGAATCTTG 
               
               
                   
               
               
                 3481 
                 ACAGTTTCTA CAGACCTTCC TGGGTGAAAG TTCCTAAATC 
               
               
                   
               
               
                 3521 
                 ATGCCCTGCT TCCTCCAATA GGAGAATGGG AGCCTCACCT 
               
               
                   
               
               
                 3561 
                 GTAGGACCTA CAGGCTCTCT AAGGAATGCA GGTCTCTCTC 
               
               
                   
               
               
                 3601 
                 TGAGCCTCCA CAGCCAGGCA AATACATATA TATATATTTT 
               
               
                   
               
               
                 3641 
                 TTTTTTAGAT GAAGTTTTTT CTCTTGTTGC CCAGGCTAGG 
               
               
                   
               
               
                 3681 
                 GTGTAATGGC ATGATCTCAG GTCACTGCAA CCTCCTCCCG 
               
               
                   
               
               
                 3721 
                 GGTTCAAGCA TTTCTTCTGT CTCAGCCTCC CGAATAGCTG 
               
               
                   
               
               
                 3761 
                 GGATTACAGG CACCTGCCAT CACACGAGCT AATTTTTGTA 
               
               
                   
               
               
                 3801 
                 TTTTTAGTAG AGATGGGGTT TCACCATGTT GACCAGGCTG 
               
               
                   
               
               
                 3841 
                 GTGTTGAGCT CCTGACCTCA GGTGATCCAC CCACCTCGGT 
               
               
                   
               
               
                 3881 
                 CTCCCAAAGT GCTGGGGTTA CAGGCCTGAG CCACTGCGCC 
               
               
                   
               
               
                 3921 
                 CGGCCCAGGC AAATTTCTTG AACCACTTCT CACTCCCGTC 
               
               
                   
               
               
                 3961 
                 ACTTTCAATA AGGGGTCTTT GATGTCTTCA CTGGTTCTTT 
               
               
                   
               
               
                 4001 
                 GGACGAGGGA CTTTTCGAAC TTTTTTGGTT GCAACACACA 
               
               
                   
               
               
                 4041 
                 GTAAGAAATA TACTTCACAC TGAGACTTGC AGCGCACACA 
               
               
                   
               
               
                 4081 
                 CACGGAAACG ACCAAAACAA AAATGTCACA AAACAATACT 
               
               
                   
               
               
                 4121 
                 TACCCTTCCC TGGGGGACGT CCTCCAGTAT GTTCTGTTCT 
               
               
                   
               
               
                 4161 
                 GTTTATTTTT CACTGTTGGT TGCAATCCAA TAAAATGACT 
               
               
                   
               
               
                 4201 
                 TTGGGATCCA CTCATGGGTG GGGACCCACA CATTTGAAAG 
               
               
                   
               
               
                 4241 
                 GCATGGCCAC CTTTCTGTTG TGCCTTGCAT TTGTCCACAC 
               
               
                   
               
               
                 4281 
                 ACAGGGAGTC TGGCTGAGCT GGGGAAAGGC CACGGCTGGG 
               
               
                   
               
               
                 4321 
                 TGTCATTGCC ATTTTCCCAG CTCATCTCAC CGGGAAGAAA 
               
               
                   
               
               
                 4361 
                 AGCAGATTGA CAGAACACGT GAGGAGGGGT ATTGATGGCA 
               
               
                   
               
               
                 4001 
                 GGAGAGTCAA AAAAGAGTTT TAAAGAAGGG GCAAGGTTGA 
               
               
                   
               
               
                 4441 
                 AGGAGTCTAG TGGCAAGGGT AAGATTTCAG GCATGGTTAA 
               
               
                   
               
               
                 4481 
                 GAACAGACGA CAAGGATGTC AGGAATGAAG ATGTGGAGAG 
               
               
                   
               
               
                 4521 
                 GGGTGTAGAG ATGGCAAGGT TGGCAAGGAA CAGATAGGCA 
               
               
                   
               
               
                 4561 
                 GGAGCAGGTC CAAGCCAAGC CTAGCCCAAG ACCAGGTGAA 
               
               
                   
               
               
                 4601 
                 AGGAGAGGGG AGGAGGAGCC ACCTGCAAGA GATGGAAAGA 
               
               
                   
               
               
                 4641 
                 GCAGGCGGCA GAGGGGGCTG GCAGGGAGGG GCTGTTAAGA 
               
               
                   
               
               
                 4681 
                 GTGGGGTTGG AGGTGGGAGA GAAGCTAGGA CAAGGGAGAT 
               
               
                   
               
               
                 4721 
                 GGAGAAAGGA CCTATACCTG GCTCACGGAA GGCCTTCAGG 
               
               
                   
               
               
                 4761 
                 TCACTACACG TTGAACATCC CCAGTGTTTG AGCCCCCAAA 
               
               
                   
               
               
                 4801 
                 GCTAGGGTGC AAGAGCACTG CCATCGAATG CCAGTGGGTG 
               
               
                   
               
               
                 4841 
                 AGGCCAAGTG AGGGTATTTG CAGCTCTAGA CATAACCAAG 
               
               
                   
               
               
                 4881 
                 AAGCGTAAAG GTGAGTTGTT TGGTGGTACG ACTGCCTGTG 
               
               
                   
               
               
                 4921 
                 CCTTCTTCCG ATGGCACTGG GGTGGCTGAA GGAACAGACA 
               
               
                   
               
               
                 4961 
                 TCTTTGGGTT TCATCAGCCT CCTCCAAGAC TGCTGCAGTG 
               
               
                   
               
               
                 5001 
                 CCTACACTTT AGACTTCAGA AGGAGACTAA AGACTTCTAG 
               
               
                   
               
               
                 5041 
                 AATTTAGAAG GAGATCTGAA GTCTCCTTTC TGGAGTTACA 
               
               
                   
               
               
                 5081 
                 ACCCAAAGGA TGTTAGCATT TCTCAGGTCA TCCCACTGCA 
               
               
                   
               
               
                 5121 
                 AAGCCCAGAA GGCTTGGGGC TCCCAGGCTG CTCTGAAGCC 
               
               
                   
               
               
                 5161 
                 CCACTGTCTG ACCGCCTCAG GGCTTGCTAC GAGGGACTGG 
               
               
                   
               
               
                 5201 
                 GGCACGGCCA AGCTGACTAG GAACAGCTCT CGTGCTCCTG 
               
               
                   
               
               
                 5241 
                 AGGGACCTGGAGGATGGGCC TGCCTCCCAG CCATTGAGCT 
               
               
                   
               
               
                 5281 
                 GGATTCTGGG ATAATTCTTA ACTCGAAATA AGGGGAAGCA 
               
               
                   
               
               
                 5321 
                 TCCATCAGGG AATGCTGGCC TTTCTAGAGC CACGTAGAAA 
               
               
                   
               
               
                 5361 
                 ACAATTTTCT GGTTCTTCAA ACCTCAAAGA GTCCTTGGTC 
               
               
                   
               
               
                 5401 
                 ATTTTCACGA TGTCAGAAAT AGTATGTTTT TAACAATAGT 
               
               
                   
               
               
                 5441 
                 ATTTTCACGA TGTCAGAAAT AGTATGTTTT TAACAATAGT 
               
               
                   
               
               
                 5481 
                 AATAGCTTTG TAAAAAAATA AAAAGCTTTA ACAGCGAGGC 
               
               
                   
               
               
                 5521 
                 CATAAACAAT GAAATGAATA AAAACGGTGG TCATTCAGTC 
               
               
                   
               
               
                 5561 
                 AACGGAAAAA AAAAAAAAAA AA 
               
            
           
         
       
     
     The rs1529927 single nucleotide polymorphism (SNP) is present in the SLC12A3 gene, where the variable nucleotide is at about position 820 in SEQ ID NO:29 (underlined), which can be guanine in some individuals and cytosine in others. The rs1529927 sequence (SEQ ID NO:30) is shown below, where the underlined C/G is the SNP. 
     
       
         
           
               
            
               
                 CCCATTAACGACATCCGCATCATTG   [C/G]   CGTGGTCTCGGTCACTGTG 
               
               
                   
               
               
                 CTGCTG. 
               
            
           
         
       
     
     The human the sodium (Na + ) chloride (Cl − ) co-transporter encoded by the SLC12A3 cDNA with SEQ ID NO:29 has the following sequence (SEQ ID NO:31). 
     
       
         
           
               
               
            
               
                 1 
                 MAELPTTETP GDATLCSGRF TISTLLSSDE PSPPAAYDSS 
               
               
                   
               
               
                 41 
                 HPSHLTHSST FCMRTFGYNT IDVVPTYEHY ANSTQPGEPR 
               
               
                   
               
               
                 81 
                 KVRPTLADLH SFLKQEGRHL HALAFDSRPS HEMTDGLVEG 
               
               
                   
               
               
                 121 
                 EAGTSSEKNP EEPVRFWGVK GVMIRCMLNI WGVILYLRLP 
               
               
                   
               
               
                 161 
                 WITAQAGIVL TWIIILLSVT VTSITGLSIS AISTNGKVKS 
               
               
                   
               
               
                 201 
                 GGTYFLISRS LGPELGGSIG LIFAFANAVG VAMHTVGFAE 
               
               
                   
               
               
                 241 
                 TVRDLLQEYG APIVDPINDI RII   G   VVSVTV LLAISLAGME 
               
               
                   
               
               
                 281 
                 WESKAQVLFF LVIMVSFANY LVGTLIPPSE DKASKGFFSY 
               
               
                   
               
               
                 321 
                 RADIFVQNLV PDWRGPDGTF FGMFSIFFPS ATGILAGANI 
               
               
                   
               
               
                 361 
                 SGDLKDPAIA IPKGTLMAIF WTTISYLAIS ATIGSCVVRD 
               
               
                   
               
               
                 401 
                 ASGVLNDTVT PGWGACEGLA CSYGWNFTEC TQQHSCHYGL 
               
               
                   
               
               
                 441 
                 INYYQTMSMV SGFAPLITAG IFGATLSSAL ACLVSAAKVF 
               
               
                   
               
               
                 481 
                 QCLCEDQLYP LIGFFGKGYG KNKEPVRGYL LAYAIAVAFI 
               
               
                   
               
               
                 521 
                 IIAELNTIAP IISNFFLCSY ALINFSCFHA SITNSPGWRP 
               
               
                   
               
               
                 561 
                 SFQYYNKWAA LFGAIISVVI MFLLTWWAAL IAIGVVLFLL 
               
               
                   
               
               
                 601 
                 LYVIYKKPEV NWGSSVQAGS YNLALSYSVG LNEVEDHIKN 
               
               
                   
               
               
                 641 
                 YRPQCLVLTG PPNFRPALVD FVGTFTRNLS LMICGHVLIG 
               
               
                   
               
               
                 681 
                 PHKQRMPELQ LIANGHTKWL NKRKIKAFYS DVIAEDLRRG 
               
               
                   
               
               
                 721 
                 VQILMQAAGL GRMKPNILVV GFKKNWQSAH PATVEDYIGI 
               
               
                   
               
               
                 761 
                 LHDAFDFNYG VCVMRMREGL NVSKMMQAHI NPVFDPAEDG 
               
               
                   
               
               
                 801 
                 KEASARGARP SVSGALDPKA LVKEEQATTI FQSEQGKKTI 
               
               
                   
               
               
                 841 
                 DIYWLFDDGG LTLLIPYLLG RKRRWSKCKQ RVFVGGQINR 
               
               
                   
               
               
                 881 
                 MDQERKAIIS LLSKFRLGFH EVHILPDINQ NPRAEHTKRF 
               
               
                   
               
               
                 921 
                 EDMIAPFRLN DGFKDEATVN EMRRDCPWKI SDEEITKNRV 
               
               
                   
               
               
                 961 
                 KSLRQVRLNE IVLDYSRDAA LIVITLPIGR KGKCPSSLYM 
               
               
                   
               
               
                 1001 
                 AWLETLSQDL RPPVILIRGN QENVLTFYCQ 
               
            
           
         
       
     
     Note that the underlined glycine at position 264 of the SEQ ID NO:31 sodium (Na + ) chloride (Cl − ) co-transporter protein is glycine because some individuals have nucleotide sequence SEQ ID NO:29, where the variable nucleotide at position 820 is guanine. However, position 264 of SEQ ID NO:29 can be alanine in some individuals because those individuals have cytosine as the variable nucleotide at position 820 in sequence SEQ ID NO:29. 
     Patients with the alanine variant of SLC12A3 (encoded by the rs1529927 site (SEQ ID NO:30)) exhibit a stronger diuretic effect to loop diuretics and demonstrate more excretion of Cl −  and K +  in response to therapy. Hence, subject with alanine or guanine at the rs1529927 site are more response to diuretics. 
     The WNK1 gene has functional and common polymorphisms that affect how a subject&#39;s blood pressure responds to drugs. Several cDNA sequences for the WNT1 gene are available from the NCBI database. 
     The rs2107614 single nucleotide polymorphism (SNP) is present in an intron of the WNK1 gene, where the variable nucleotide can be thymine in some individuals and cytosine in others. The rs2107614 sequence (SEQ ID NO:33) is shown below, where the underlined C/T is the SNP. 
     
       
         
           
               
            
               
                 CACTTCCTCCAAAAAAAAAGAAAAC   [C/T]   CCATTTCCCCTCAACTCTT 
               
               
                   
               
               
                 CCAGTT. 
               
            
           
         
       
     
     Another SNP, rs1159744, is present an intron of the WNK1 gene, where the variable nucleotide can be guanine in some individuals and cytosine in others. The rs1159744 sequence (SEQ ID NO:34) is shown below, where the underlined C/G is the SNP. 
     
       
         
           
               
            
               
                 AATGTTAACAGTATAGAAAATTTTA   [C/G]   CTCAACAAATAGAGAATAT 
               
               
                   
               
               
                 CAGTAA. 
               
            
           
         
       
     
     Patients with the cytosine variant of WNK1 at SNP positions rs1159744 and rs2107614 exhibit greater blood pressure reductions in response to loop diuretic therapy when compared to patients with the guanine or thymine variants at these two sites, respectively (Turner et al.,  Hypertension  46:758-765 (2005)). 
     Therapy 
     The methods, reagents, devices, and kits described herein can be used for determining whether a subject may benefit from treatment with a blood pressure medication, and which medication can be more effective for treating high blood pressure. For example, the methods described herein can be employed for determining whether a subject should be treated with a diuretic, an angiotensin converting enzyme (ACE) inhibitor, or a beta-blocker. Such determination is performed by identifying or detecting whether the subject has a genetic variant or single nucleotide polymorphism in his or her ADRB1, ADRB2. CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin. N +  channels (such as SCNN1A), adducin, sodium (Na + ) chloride (Cl − ) co-transporters (such as SLC12A3), and/or WNK1 polypeptides or nucleic acids. If testing of the subject&#39;s tissue sample shows that the subject has a genetic variant or single nucleotide polymorphism in his or her ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na +  channels (such as SCNN1A), adducin, sodium (Na + ) chloride (Cl − ) co-transporters (such as SLC12A3), and/or WNK1 polypeptides or nucleic acids, a suitable therapeutic regimen can be prescribed for the subject. 
     A diuretic promotes the production or urine. Diuretics are sometimes grouped into three categories: thiazides, loop, and potassium-sparing diuretics. Thiazide diuretics include chlorothiazide, hydrochlorothiazide, indapamide, metolazone, and chlorthalidone. Loop diuretics include furosemide, bumetanide, ethacrynic acid, and torsemide. Examples of potassium-sparing diuretics include amiloride, eplerenone, spironolactone, and triamterene. 
     Examples of diuretics that can be employed also include furosemide, thiazides, carbonic anhydrase inhibitors, potassium-sparing diuretics (e.g., aldosterone antagonists, spironolactone, eplerenone, potassium canreonate, amiloride, triamterene, aldactone, and combinations thereof), calcium-sparing diuretics. For example, the diuretic can be acetazolamide, amiloride, bumetanide, chlorothalidone, chlorothiazide, ethacrynic acid, furosemide, glycerin, hydrochlorothiazide, hydroflumthiazide, indapamide, isosorbide, mannitol, methazolamide, methylchlothiazide, metolazone, dichlorphenamide, spironolactone, torsemide, triamterene, urea, and combinations thereof. 
     The angiotensin converting enzyme inhibitor can be selected from enalapril lisinopril, captopril alacipril, benazapril, cilazapril, delapril, fosinopril, perindopril, quinapril, ramipril, moveltipril, spirapril, ceronapril, imidapril, temocapril, trandolopril, utilbapril, zofenopril CV5975, EMD 56855, libenzapril, zalicipril, HOE065, MDL 27088, AB47, DU 1777, MDL 27467A, Equaten™, Prentyl™, Synecor™, and Y23785; and the diuretic is selected from hydrochlorothiazide (HCTZ), furosemide, altizide, trichlormethazide, triflumethazide, bemetizide, cyclothiazide, methylchlothiazide, azosemide, chlorothiazidc, butizide, bendroflumethazide, cyclopenthiazide, benzclortriazide, polythiazide, hydroflumethazide, benzthiazide, ethiazide, penflutazide, and any combination thereof. 
     The angiotensin II receptor antagonists can, for example, be losartan, valsartan, candesartan, irbesartan, olmesartan, or any combination thereof. 
     The renin inhibitors can be urea derivatives of di- and tri-peptides (See U.S. Pat. No. 5,116,835), amino acids and derivatives (U.S. Pat. Nos. 5,095,119 and 5,104,869), amino acid chains linked by non-peptidic bonds (U.S. Pat. No. 5,114,937), di- and tri-peptide derivatives (U.S. Pat. No. 5,106,835), peptidyl amino diols (U.S. Pat. Nos. 5,063,208 and 4,845,079) and peptidyl beta-aminoacyl aminodiol carbamates (U.S. Pat. No. 5,089,471); also, a variety of other peptide analogs as disclosed in the following U.S. Pat. Nos. 5,071,837; 5,064,965; 5,063,207; 5,036,054; 5,036,053; 5,034,512 and 4,894,437, and small molecule renin inhibitors (including diol sulfonamides and sulfinyls (U.S. Pat. No. 5,098,924), N-morpholino derivatives (U.S. Pat. No. 5,055,466), N-heterocyclic alcohols (U.S. Pat. No. 4,885,292) and pyrolimidazolones (U.S. Pat. No. 5,075,451); also, pepstatin derivatives (U.S. Pat. No. 4,980,283) and fluoro- and chloro-derivatives of statone-containing peptides (U.S. Pat. No. 5,066,643), enalkrein, RO 42-5892, A 65317, CP 80794, ES 1005, ES 8891, SQ 34017, aliskiren ((2S,4S,5S,7S)—N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)phenyl]-oetanamid hemifumarate) SPP600, SPP630 and SPP635), or any combination thereof. 
     Other therapeutic agents can also be administered including endothelin receptors antagonists, vasodilators, calcium channel blockers (e.g., amlodipine, nifedipine, veraparmil, diltiazem, gallopamil, nifedipine, nimodipins, nicardipine), potassium channel activators (e.g., nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam), diuretics (e.g., hydrochlorothiazide), sympatholitics, beta-adrenergic blocking drugs (e.g., propranolol, atenolol, bisoprolol, carvedilol, metoprolol, or metoprolol tartrate), alpha adrenergic blocking drugs (e.g., doxazocin, prazocin or alpha methyldopa) central alpha adrenergic agonists, peripheral vasodilators (e.g. hydralazine), lipid lowering agents (e.g., simvastatin, lovastatin, ezetamibe, atorvastatin, pravastatin), metabolic altering agents including insulin sensitizing agents and related compounds (e.g., muraglitazar, glipizide, metformin, rosiglitazone) or with other drugs beneficial for the prevention or the treatment of disease including nitroprusside and diazoxide. 
     The therapeutic protocol can generally be conducted as follows. An assay of all sixteen genotypes (polymorphic sites) can be performed. The therapeutic decision tree of the results can be as follows. 
     To ascertain whether a diuretic should be administered to a subject, the following can be performed.
         If the subject is (a) homozygous for cytosine at the rs1529927 (SEQ ID NO:30) variable site (expressing alanine at position 264 of the SLC12A3 gene product); (b) homozygous for adenine at the rs2228576 (SEQ ID NO:22) variable site (expressing threonine at about 663 or 722 of the SCNN1A protein); and/or (c) homozygous for thymine at the rs4961 (SEQ ID NO:27) variable site (expressing tryptophan at about position 460 of the adducin protein) then the patient should initially start with a diuretic as the first line of therapy. If the patient is heterozygous at these sites, then genetic variation within the polymorphic sites relating to vasodilator and beta-blocker drug class responses should initially be considered.   If the patient does not carry homozygous variants that are known to be functionally important within the vasodilator and beta-blocker classes, but are heterozygous at rs1529927, rs2228576, and rs4961, then diuretic therapy should initially be considered as first-line therapy.   If the subject is homozygous for cytosine at the WNK1 rs1159744 (SEQ ID NO:34) variable site and also homozygous for cytosine at the WNK1 rs2107614 (SEQ ID NO:33) variable site then the patient should start with a loop diuretic as first-line of therapy.   If the patient does not carry homozygous variants within the vasodilator and beta-blocker classes that are known to be functionally important, but are heterozygous at rs1529927, rs2228576, and rs4961, then loop diuretic therapy should initially be considered as first-line therapeutic agent.       

     To ascertain whether a vasodilator should be administered to a subject the following can be performed.
         If the subject is homozygous for cytosine at the rs5186 (SEQ ID NO:16) variable site of AGT1R, and the subject is homozygous cytosine at the rs12750834 (SEQ ID NO:20) variable site of renin, then the patient should use an angiotensin II (AII) receptor blocker as a first line of therapy.   If the patient is heterozygous for cytosine at the rs5186 and rs12750834 variable sites, but does not present with other important functional genotypes within the diuretic and beta-blockade classes, then the patient should also use an angiotensin II receptor blocker as a first line of therapy.   If the patient is homozygous for cytosine at the rs699 (SEQ ID NO:14) variable site of AGT, or for the deletion at the rs1799752 (SEQ ID NO:12; SEQ ID NO:35) of ACE, then the patient will likely benefit most from an angiotensin-converting enzyme (ACE) inhibitor.   Patients who are homozygous for cytosine at the rs699 (SEQ ID NO: 14) will likely benefit most from BOTH ACE inhibition and angiotensin II (AII) receptor blockade.   Patients who are heterozygous for the deletion at the rs1799752 (SEQ ID NO: 12; SEQ ID NO:35) and heterozygous for cytosine at the rs699 (SEQ ID NO: 14) variable site should be administered other drug classes (e.g., diuretic initially followed by beta-blockade). Although homozygosity at other sites has a greater impact on hypertension than heterozygosity at rs1799752 and rs699, this is generally true only if the patient has combined homozygosity at sites indicating that drug classes other than vasodilators should be administered.       

     To ascertain whether a beta-blocker should be administered to a subject the following can be performed.
         Patients homozygous for adenine at the rs3892097 (SEQ ID NO: 10) variable site of the CYP2D6 gene should initially consider the use of atenolol and carevdilol as therapy. This is PARTICULARLY important if the patient is homozygous for cytosine at the rs1801253 (SEQ ID NO:3) variable site (and expresses arginine at position 389 of the β 1 AR polypeptide), or if the patient is homozygous for adenine at the rs1801252 variable position (and expresses serine at position 49 of the β 1 AR polypeptide).   The rs1042713 (SEQ ID NO:6) and rs1042714 (SEQ ID NO:7) variable sites are less important of the other polymorphism sites within the beta-blocker class of drugs and generally indicate patients who will likely respond to non-selective beta-blockade. Thus, subjects who are homozygous for guanine at the rs1042713 variable site (and express glycine at about position 16 of the ADRB2 gene product) as well as subjects who are homozygous for guanine at the rs1042714 position (and express glutamic acid at β 2 AR position 27) are the most responsive to beta-blocker drugs.   If subjects are non-homozygous for polymorphisms in the beta-blockade class of variants, but are homozygous for cytosine at the rs1801253 (SEQ ID NO:3) variable site (and expresses arginine at position 389 of the β 1 AR polypeptide), or if subjects are homozygous for adenine at the rs1801252 variable position (and expresses serine at position 49 of the β 1 AR polypeptide), the beta-blockade class should be considered a possible line of therapy if they do not carry functional mutations within the diuretic and vasodilator classes of drugs.       

     Polymorphism Detection 
     The polymorphism present in genes such as ADRB1, ADRB2, cytochrome P450 2D6 (CYP2D6), angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin. Na +  channels (such as SCNN1A), adducin, sodium (Na + ) chloride (Cl − ) co-transporters (such as SLC12A3), and/or WNK1 can be detected by any available procedure. For example, samples of cDNA, genomic DNA, and/or mRNA can be obtained from a subject and the sequences of polymorphic or variant sites can be evaluated by procedures such as nucleic acid amplification (e.g., PCR), reverse transcription, insertion/deletion analysis, primer extension, probe hybridization, SNP analysis, sequencing, restriction fragment length polymorphism, Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS), Sequenom MassArray genotyping. Sanger sequencing, polyacrylamide gel electrophoresis, agarose gel electrophoresis, probe array hybridization analysis, and combinations thereof. 
     The methods for detecting polymorphisms can therefore involve detecting an alteration in a nucleic acid. As used herein a “nucleic acid” is a DNA or RNA molecule. A nucleic acid can be a segment of genomic DNA (e.g., an entire gene, an intron of a gene, an exon of a gene, a segment that includes regulatory elements, a 5′ non-coding segment, a 3′ non-coding segment, or any combination thereof). The nucleic acid can also be a cDNA (having exons but not introns), an amplicon, an RNA, a primer, or probe. 
     Probes and/or primers can be used that can hybridize to nucleic acid segments flanking or including of any of SNPs, insertions, deletions, polymorphic, or other variant segments of ADRB1. ADRB2, cytochrome P450 2D6 (CYP2D6), angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na +  channels (such as SCNN1A), adducin, sodium (Na + ) chloride (Cl − ) co-transporters (such as SLC12A3), and/or WNK1 genes. For example, probes and/or primers can be employed that hybridize to nucleic acid segments flanking or including any of the following polymorphisms: rs1801252 (ADRB1), rs1801253 (ADRB1), rs1042713 (ADRB2), rs1042714 (ADRB2), rs3892097 (CYP2D6), rs1799752 (ACE), rs699 (AGT), rs5186 (AGT1R), rs12750834 (renin), rs2228576 (SCNN1A), rs4961 (ADD1), rs1529927 (SLC12A3), rs2107614 (WNK1), or rs1159744 (WNK1). For example, the probes and/or primers can separately hybridize to segments of any of SEQ ID NO:2, 3, 6, 7, 10, 12, 14, 16, 19, 20, 22, 27, 30, 32, 33, 34, as well as to the complementary sequences, amplicons, cDNA, cRNA, and genomic sequences thereof. The probes and/or primers can hybridize to genomic, complementary, amplicon, or cDNA sequences that flank up to 50 nucleotides of any of SEQ ID NO:2, 3, 6, 7, 10, 12, 14, 16, 19, 20, 22, 27, 30, 33, or 34, on either or both of the 5′ and 3′ sides of the polymorphism. 
     Methods and devices described herein can detect at least two of these polymorphisms, or at least three of these polymorphisms, or at least of four of these polymorphisms, or at least five of these polymorphisms, or at least of six of these polymorphisms, or at least seven of these polymorphisms, or at least of eight of these polymorphisms, or at least nine of these polymorphisms, or at least of ten of these polymorphisms, or at least eleven of these polymorphisms, or at least of twelve of these polymorphisms, or at least thirteen of these polymorphisms, or at least fourteen of these polymorphisms, or at least fifteen of these polymorphisms or all of these polymorphisms. In some embodiments, the methods and devices described herein detect no other polymorphisms, although such methods and devices can include steps and probes for detecting 1-4 control target nucleic acids. For example, the methods, devices, and kits described herein can detect and evaluate about sixteen polymorphisms. 
     The probes and primers can be of any convenient length selected by one of skill in the art such as at least 12 nucleotides long, or at least 13 nucleotides long, or at least 14 nucleotides long, or at least 15 nucleotides long, or at least 16 nucleotides long, or at least 17 nucleotides long, or at least 18 nucleotides long, or at least 19 nucleotides long, or at least 20 nucleotides long. In some embodiments, the probes and primers can be less than 150 nucleotides in length, or less than 125 nucleotides in length, or less than 100 nucleotides in length, or less than 75 nucleotides in length, or less than 65 nucleotides in length, or less than 60 nucleotides in length, or less than 55 nucleotides in length, or less than 50 nucleotides in length, or less than 45 nucleotides in length, or less than 40 nucleotides in length. 
     To detect hybridization, it may be advantageous to employ probes, primers and other nucleic acids in combination with an appropriate detection means. Labels incorporated into primers, incorporated into the amplified product during amplification, or attached to probes that can hybridize to the target, or its amplified product, are useful in the identification of nucleic acid molecules. A number of different labels may be used for this purpose including, but not limited to, fluorophores, chromophores, radiolabels, enzymatic tags, antibodies, chemiluminescence, electroluminescence, and affinity labels. One of skill in the art will recognize that these and other labels can be used with success in this invention. 
     Examples of affinity labels include, but are not limited to the following: an antibody, an antibody fragment, a receptor protein, a hormone, biotin, dinitrophenyl (DNP), or any polypeptide/protein molecule that binds to an affinity label. Examples of enzyme tags include enzymes such as urease, alkaline phosphatase or peroxidase to mention a few. Colorimetric indicator substrates can be employed to provide a detection means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples. Examples of fluorophores include, but are not limited to, Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy2, Cy3, Cy5, 6-FAM, Fluorescein, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, ROX, TAMRA, TET, Tetramethylrhodamine, and Texas Red. 
     Means of detecting such labels are well known to those of skill in the art. For example, radiolabels may be detected using photographic film or scintillation counters. In other examples, fluorescent markers may be detected using a photodetector to detect emitted light. In still further examples, enzymatic labels are detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label or by use of spectrometer. 
     So called “direct labels” are detectable labels that are directly attached to or incorporated into a probe or primer, or to the target (sample) nucleic acid prior to hybridization to a probe that can, for example, be present on a plate, chip, microtiter plate, or microarray. In contrast, so called “indirect labels” are joined to the hybrid duplex after hybridization. In some embodiments, the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization. Thus, for example, the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin-bearing hybrid duplexes providing a label that is easily detected. For a detailed review of methods of labeling nucleic acids and detecting labeled hybridized nucleic acids see, for example, Peter C, van der Vliet &amp; Shiv Pillai, eds., Laboratory Techniques in Biochemistry and Molecular Biology (1993). 
     Probe arrays, assay plates, and gene chip technology provide a means of rapidly screening a large number of nucleic acid samples for their ability to hybridize to a variety of probes immobilized on a solid substrate that is part of the probe array, assay plate, gene chip or microarray. The technology capitalizes on the complementary binding properties of single stranded nucleic acid probe to screen nucleic acid samples by hybridization (Pease et al., Proc. Natl. Acad. Sci. U.S.A 91: 5022-5026 (1994); U.S. patent to Fodor et al. (1991)). Basically, a nucleic acid probe array or gene chip consists of a solid substrate with an attached array of single-stranded probe molecules. In some embodiments, the probes can fold back on (i.e., hybridize to) themselves to quench a signal from an attached label, but the probes unfold to hybridize to a target nucleic acid, whereupon the signal from the attached label becomes detectable. In other embodiments, the probe can be complementary to the segment of a target nucleic acid but the 3′ end of the probe terminates one nucleotide short of a SNP in the target nucleic acid. The target nucleic acid can be longer than the probe. A signal can be detected upon primer extension of the probe, where the assay mixture contains just one type of labeled nucleotide that can base pair with the variant nucleotide of the SNP. After washing, the presence or absence of the SNP is detectable by incorporation or non-incorporation of the labeled SNP nucleotide into specific probes of the array or plate. 
     For screening, the chip, plate, or array is contacted with a nucleic acid sample (e.g., genomic DNA, cRNA, cDNA, or amplified copies thereof), which is allowed to hybridize under stringent conditions. The chip, plate, or array is then scanned to determine which targets have hybridized to which probes. The probes are arrayed in known locations so a signal detected at a specific location indicates that its target has hybridized thereto. 
     Methods for directly synthesizing on or attaching nucleic acid probes to solid substrates are available in the art. See, e.g., U.S. Pat. Nos. 5,837,832 and 5,837,860, both of which are expressly incorporated by reference herein in their entireties. A variety of methods have been utilized to either permanently or removably attach the probes to the substrate. Exemplary methods include: the immobilization of biotinylated nucleic acid molecules to avidin/streptavidin coated supports (Holmstrom, (Anal. Biochem. 209: 278283 (1993)), the direct covalent attachment of short, 5′-phosphorylated primers to chemically modified polystyrene plates (Rasmussen et al., Anal. Biochem. 198: 138-142 (1991)), or the precoating of the polystyrene or glass solid phases with poly-L-Lys or poly L-Lys. Phe, followed by the covalent attachment of either amino- or sulfhydryl-modified oligonucleotides using bifunctional crosslinking reagents (Running et al., BioTechniques 8: 276 277 (1990); Newton, C. R. et al., Acids Res. 21: 1155-1162 (1993)). When immobilized onto a substrate, the probes are typically stabilized and therefore can be used repeatedly. 
     Hybridization can performed on an immobilized probe that is attached to a solid surface such as silicon, plastic, nitrocellulose, nylon or glass by addition of one or more target molecules. In some embodiments, the target nucleic acid can be attached to a solid surface and the probe can be added to the immobilized target nucleic acids. Numerous substrate and/or matrix materials can be used, including reinforced nitrocellulose membrane, activated quartz, activated glass, polyvinylidene difluoride (PVDF) membranes, polystyrene, polyacrylamide, poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane), photopolymers (which contain photoreactive species such as nitrenes, carbenes and ketyl radicals capable of forming covalent links with target molecules), and combinations thereof. 
     The term “hybridization” includes a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, primer extension, or the enzymatic cleavage of a polynucleotide by a ribozyme. 
     Hybridization reactions can be performed under conditions of different “stringency”. The stringency of a hybridization reaction includes the difficulty with which any two nucleic acid molecules will hybridize to one another. Under low to medium stringent conditions, nucleic acid molecules at least 60%, 65%, 70%, 75% identical to each other remain hybridized to each other, whereas molecules with lower percent identity cannot remain hybridized. For detection of single base polymorphisms, higher stringency conditions can be used. 
     A preferred, non-limiting example of highly stringent hybridization conditions include hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., more preferably at 60° C., and even more preferably at 65° C. 
     When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, the reaction is called “annealing” and those polynucleotides are described as “complementary”. A double-stranded polynucleotide can be “complementary” and/or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. 
     Complementarity” or “homology” (the degree that one polynucleotide is identical or complementary to another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to hydrogen bond with each other, according to generally accepted base-pairing rules. 
     Detection/Identification of Genetic Variants in Expressed Polypeptides 
     Genetic variants present in polypeptides such as ADRB1. ADRB2, cytochrome P450 2D6 (CYP2D6), angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptor, renin, Na +  channels (such as SCNN1A), adducin, sodium (Na + ) chloride (Cl − ) co-transporters (such as SLC12A3), and/or WNK1 can be detected by use of binding entities such as antibodies. Detection of specific differences in these polypeptides can be used to evaluate which blood pressure mediation is more effective. 
     Altered polypeptides can be detected in a selected fluid or tissue sample (e.g., cell scrapings, saliva, hair follicle, blood, skin tissue, or any convenient sample of a subject&#39;s nucleic acids). Any available methods for detecting polypeptides can be employed. Examples of such methods include immunoassay, Western blotting, enzyme-linked immunosorbant assays (ELISAs), radioimmunoassay, immunocytochemistry, immunohistochemistry, flow cytometry, immunoprecipitation, one- and two-dimensional electrophoresis, mass spectroscopy and/or detection of enzymatic activity. 
     Altered polypeptides can be detected by binding entities. 
     Antibodies and other binding entities can be used to detect genetic variants present in ADRB1, ADRB2, cytochrome P450 2D6 (CYP2D6), angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na +  channels (such as SCNN1A), adducin, sodium (Na + ) chloride (Cl − ) co-transporters (such as SLC12A3), and/or WNK1 polypeptides. Such antibodies and binding entities can be prepared by available methods. For example, available amino acid sequences of non-variant and genetic variant ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na +  channels (such as SCNN1A), adducin, sodium (Na + ) chloride (Cl − ) co-transporters (such as SLC12A3), and/or WNK1, including those illustrated herein, can be used to make antibodies and binding entities. Suitable antibodies may include polyclonal monoclonal, fragments (such as Fab fragments), single chain antibodies and other forms of specific binding molecules. Briefly, these polypeptide detection assays can include contacting a test sample with an antibody specific to the genetic variant site in the polypeptide, detecting the presence of a complex between the antibody and the polypeptide. In some embodiments, a signal from the polypeptide-antibody complex is detected. 
     Such antibody-based detection methods can any convenient immuno-detection method such as Western Blot, ELISA, radioimmunoassay, immunocytochemistry, immunohistochemistry, flow cytometry, and immunoprecipitation. 
     Antibodies can be used to detect or identify the presence of genetic variant forms of ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na +  channels (such as SCNN1A), adducin, sodium (Na + ) chloride (Cl − ) co-transporters (such as SLC12A3), and/or WNK1 polypeptides in a sample. The antibodies are specific for sites of genetic variations, and exhibit substantially no (or significantly less) binding to similar polypeptides that do not have the same genetic variation(s). 
     Generally speaking, such antibodies can be employed in any type of immunoassay, so long as the genetic variations in the polypeptides are reliably identified. This includes both the two-site sandwich assay and the single site immunoassay of the non-competitive type, as well as in traditional competitive binding assays. 
     For example, in a typical forward sandwich assay, unlabeled antibody is immobilized on a solid substrate, e.g., within microtiter plate wells, and the sample to be tested is brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex, a second antibody, labeled with a reporter molecule capable of emitting or inducing a detectable signal, is then added and incubation is continued allowing sufficient time for binding with the antigen at a different site and the formation of a ternary complex of antibody-antigen-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal, which may be quantified by comparison with a control sample containing known amounts of antigen. 
     Variations on the forward sandwich assay include the simultaneous assay, in which both sample and antibody are added simultaneously to the bound antibody, or a reverse sandwich assay in which the labeled antibody and sample to be tested are first combined, incubated and added to the unlabeled surface bound antibody. These techniques are well known to those skilled in the art, and the possibility of minor variations will be readily apparent. As used herein, “sandwich assay” is intended to encompass all variations on the basic two-site technique. 
     For the sandwich assays, the only limiting factor is that both antibodies have different binding specificities for the genetic variant polypeptide. Thus, a number of possible combinations are possible. For example, a primary antibody can bind specifically to the variant epitope of one of the variant polypeptides. A secondary antibody can bind to a different site on the genetic variant polypeptide. As a more specific example, in a typical forward sandwich assay, a primary antibody is either covalently or passively bound to a solid support. The solid surface is usually glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinylchloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surfaces suitable for conducting an immunoassay. 
     Conventional antibody binding processes can be employed. Following binding, the solid phase-antibody complex is washed in preparation for the test sample. An aliquot of the test sample is then added to the solid phase complex and incubated at about 25° C. for a period of time sufficient to allow binding of any genetic variant polypeptides present to the antibody. The primary antibody can bind specifically to the site of the genetic variant (e.g., the region of a variant amino acid and/or the structural changes associated therewith), but not to similar polypeptides that have no such genetic variant. After washing off unbound antibodies, the second antibody is then added to the solid phase complex and incubated at 25° C. for an additional period of time sufficient to allow the second antibody to bind to the primary antibody-antigen solid phase complex (e.g., to a different site on the genetic variant polypeptide than is bound by the primary antibody). The second antibody may be linked to a reporter molecule, the visible signal of which is used to indicate the binding of the second antibody to any antigen in the sample. 
     As used herein, a “reporter molecule” or “label” is a molecule that provides an analytically detectable signal, allowing the detection of antigen-bound antibody. In some embodiments, detection is preferably at least relatively quantifiable, to allow determination of the amount of antigen in the sample, this may be calculated in absolute terms, or may be done in comparison with a standard (or series of standards) containing a known normal level of antigen. The term “label” is used interchangeably with “reporter molecule.” 
     Many commonly used reporter molecules in this type of assay are either enzymes or fluorophores. In the case of an enzyme immunoassay an enzyme is conjugated to the second antibody, often by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are well known to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, among others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates: for peroxidase conjugates, 1,2-phenylenediamine or toluidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody-antigen complex and allowed to bind to the complex, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the tertiary complex of antibody-antigen-labeled antibody. The substrate reacts with the enzyme linked to the second antibody, giving a qualitative visual signal which may be further quantified, usually spectrophotometrically, to give an evaluation of the amount of antigen that is present in the serum sample. 
     Additionally, fluorescent compounds, such as fluorescein or rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorophore-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic longer wavelength. The emission appears as a characteristic color visually detectable with a light microscope. As in the enzyme immunoassay (EIA), the fluorescent-labeled antibody is allowed to bind to the first antibody-tagged polypeptide complex. After washing the unbound reagent, the remaining ternary complex is then exposed to light of the appropriate wavelength, and the fluorescence observed indicates the presence of the antigen. 
     Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the required use. 
     In another embodiment, the sample to be tested may be used in a single site immunoassay wherein it is adhered to a solid substrate either covalently or non-covalently. An unlabeled antibody is brought into contact with the sample bound on the solid substrate. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex a second antibody, labeled with a reporter molecule capable of inducing a detectable signal, is then added and incubation is continued allowing sufficient time for the formation of a ternary complex of antigen-antibody-labeled antibody. For the single site immunoassay, the second antibody may be a general antibody (i.e., xenogeneic antibody to immunoglobulin, particularly anti-(IgM and IgG) linked to a reporter molecule) that is capable of binding an antibody that is specific for the genetic variant polypeptide of interest. 
     Kits 
     Another aspect of the invention is one or more kits for evaluating blood pressure from a test sample provided by, or obtained from, a subject. 
     The kits can include any reagents, components and instructions useful for testing, assaying, detecting, identifying, and/or determining whether genetic variations are present in ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na +  channels (such as SCNNIA), adducin, sodium (Na + ) chloride (Cl − ) co-transporters (such as SLC12A3), and/or WNK1 polypeptides or nucleic acids that can be present in the test samples. 
     The kits can include reagents, components and instructions for detecting, identifying, and/or quantifying such polypeptides or nucleic acids. For example, the kits may include primers, probes, labels, enzymes and/or other components for detecting, and/or identifying genetic variations in such polypeptides or nucleic acids. 
     In other embodiments, the kits may include one or more antibody preparations that selectively bind to genetic variant ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na +  channels (such as SCNNIA), adducin, sodium (Na + ) chloride (Cl − ) co-transporters (such as SLC12A3), and/or WNK1 polypeptides, and a detection means for detecting an antibody complex that can form (e.g., a label or reporter molecule that is either bound to an antibody or is capable of binding to the antibody). 
     One type of kit can include components for obtaining a sample from a subject, and instructions for sample collection. For example, such a sample collection kit can include one or more containers for sample collection such as one or more vials, test tubes, or receptacles. The sample collection containers can include a solution for stabilizing samples placed in the containers. Such a stabilizing solution can include protease inhibitors, nuclease inhibitors, DNase inhibitors, RNase inhibitors, chelators, denaturants, salts, salts, and/or buffers. The sample collection kit can also include components for sample collection such as swabs, droppers, syringes, needles, scalpels, and/or catheters. The instructions can include steps for sample collection, storage of the sample, and submission of the sample. 
     The kits can include one or more probes and/or primers each capable of specifically binding to a nucleic acid segment of at least 13, 14, 15, 16, 17, 18, 19, 20, or 25 nucleotides. In some embodiments, probes and/or primers are each capable of specifically binding to a nucleic acid segment of 15-30, 15-40, 15-50 nucleotides, or any number of nucleotides between 13-50 nucleotides, in a target DNA or RNA. The probes may be part of an array, microarray, microchip, assay plate, or nanochip. Alternatively, the probes or primers may be packaged separately and/or individually. In some embodiments, the probes or primers may be detectably labeled. For example, labels can be included on immobilized probes, where the label signals are quenched until hybridization occurs and then, upon hybridization, the label emits a detectable signal. Alternatively, one or more labels can be included in the kit that can bind to a hybridized complex between a probe and a target DNA or RNA. 
     Additional reagents can be included in the kits. For example, the kits may also contain reagents for detecting or identifying a genetic variant in an ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin. Na +  channels (such as SCNN1A), adducin, sodium (Na + ) chloride (Cl − ) co-transporters (such as SLC12A3), and/or WNK1 nucleic acid in a test sample. Such reagents can include reagents for isolating, storing and detecting nucleic acids. For example, the kits can include reagents and enzymes for nucleic acid amplification, primer extension. RNA reverse transcription, sequencing, restriction enzyme cleavage, and/or separation of nucleic acids. The kits may also include reagents such as solutions for stabilizing nucleic acids, solutions for purifying nucleic acids, nucleotide triphosphates, buffers, and/or other reagents that can be used in in a test tissue sample. 
     Preservatives and/or antimicrobial agents can be included to stabilize reagents and prevent contamination, such as, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. 
     It may also be desirable to include agents such as solvents for nucleic acids, reducing agents (e.g., beta-mercaptoethanol), stabilizing reagents (e.g., reagents for inhibiting nucleases, ribonucleases, disrupting tissues, precipitating nucleic acids, and the like). 
     In further embodiments, the kits can include a computer program product for use in conjunction with a computer system and the methods described herein. A computer program mechanism can be embedded in the computer program product. The computer program product can, for example, be a device with a computer program mechanism encoded thereon, where the computer program mechanism may be loaded into the memory of a computer and cause the computer to carry out at least one step of a method for assessing the malignant/benign status of a test thyroid tissue sample. For example, the device can be a computer readable storage medium, a flash memory, a compact disc (CD), a digital versatile disc, digital video disc, or an article of manufacture that tangibly includes one or more computer programs and memory storage. In some embodiments, the computer program product can be a computer readable storage medium. In such kits, the computer program mechanism can include instructions for determining, detecting, and/or identifying a genetic variant in an ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na +  channel (such as SCNNIA), adducin, sodium (Na + ) chloride (Cl − ) co-transporter (such as SLC12A3), and/or WNK1 nucleic acid or polypeptide in a test sample. 
     In other embodiments, the kits can include a system, such as a computer, having a central processing unit and a memory coupled to the central processing unit. The memory may store instructions for determining, detecting, and/or identifying a genetic variant in an ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na +  channel (such as SCNNIA), adducin, sodium (Na + ) chloride (Cl − ) co-transporter (such as SLC12A3), and/or WNK1 nucleic acid or polypeptide in a test sample. The memory can also store therapeutic options for different genotyping results. 
     The kits can also include one or more therapeutic agents, for example, any blood pressure medications described herein. 
     Definitions 
     Some definitions are provided below; other definitions are provided in the other sections of the applications. 
     As used herein. “obtaining a test sample” involves removing a sample of tissue from a patient, receiving a sample of tissue from a patient, receiving a patient&#39;s tissue sample from a physician, receiving a patient&#39;s tissue sample via mail delivery and/or removing a patient&#39;s tissue sample from a storage apparatus (e.g., a refrigerator or freezer) or a facility. Thus, obtaining a test sample can involve removal or receipt of the test sample directly from the patient, but obtaining a test sample can also include receipt of a test sample indirectly from a medical worker, from a storage apparatus/facility, from a mail delivery service after transportation from a medical facility, and any combination thereof. The test sample can therefore originate in one location, and be transported to another location where it is received and tested. Any of these activities or combinations of activities involves “obtaining a test sample.” 
     As used herein a probe refers to a single DNA or RNA molecule (a nucleic acid oligomer) or a collection of nucleic acid molecules (nucleic acid oligomers) where the DNA molecules have at least one segment with a sequence that is complementary to a region of a target nucleic acid. The probe can hybridize with the target nucleic acid under stringent conditions. In some cases, the probe can hybridize with the target nucleic acid under highly stringent conditions. The probe is not identical to naturally available nucleic acids because has additional components such as one or more labels, one or more (engineered) restriction sites, one or more molecular barcodes, one or more tags for identification or retrieval of the probe (e.g., with or without the target hybridized thereto). In some instances the probe is attached to a solid surface such as a chip, an array, a bead, or other surface. 
     As used herein a primer contains a region that is designed to hybridize to a targeted locus (e.g., a targeted polymorphic locus or a nonpolymorphic locus). The primer and may contain a priming sequence designed to allow PCR amplification. The primer can have at least one segment with a sequence that is complementary to a region of a target nucleic acid. The primer can hybridize with the target nucleic acid under stringent conditions. In some cases, the primer can hybridize with the target nucleic acid under highly stringent conditions. The primer is not identical to naturally available nucleic acids because has additional components such as a molecular barcode, a tag, an engineered restriction site, or a combination thereof. A primer may contain a random region that differs for each individual molecule. The terms “test primer” and “candidate primer” are not meant to be limiting and may refer to any of the primers disclosed herein. 
     As used herein a “binding entity” is a molecule or molecular complex that can recognize and bind to selected target molecules. Such binding entities can be antibodies or any molecule that has a binding domain for a target molecule. 
     A number of proteins can serve as protein scaffolds to which binding domains for targets can be attached and thereby form a suitable binding entity. The binding domains bind or interact with the targets of the invention while the protein scaffold merely holds and stabilizes the binding domains so that they can bind. A number of protein scaffolds can be used. For example, phage capsid proteins can be used. See Review in Clackson &amp; Wells, Trends Biotechnol. 12:173-184 (1994). Phage capsid proteins have been used as scaffolds for displaying random peptide sequences, including bovine pancreatic trypsin inhibitor (Roberts et al., PNAS 89:2429-2433 (1992)), human growth hormone (Lowman et al., Biochemistry 30:10832-10838 (1991)), Venturini et al., Protein Peptide Letters 1:70-75 (1994)), and the IgG binding domain of  Streptococcus  (O&#39;Neil et al., Techniques in Protein Chemistry V (Crabb, L., ed.) pp. 517-524. Academic Press. San Diego (1994)). These scaffolds have displayed a single randomized loop or region that can be modified to include binding domains for selected targets. 
     Researchers have also used the small 74 amino acid α-amylase inhibitor Tendamistat as a presentation scaffold on the filamentous phage M13. McConnell, S. J &amp; Hoess, R. H., J. Mol. Biol. 250:460-470 (1995). Tendamistat is a β-sheet protein from  Streptomyces tendae . It has a number of features that make it an attractive scaffold for binding peptides, including its small size, stability, and the availability of high resolution NMR and X-ray structural data. The overall topology of Tendamistat is similar to that of an immunoglobulin domain, with two β-sheets connected by a series of loops. In contrast to immunoglobulin domains, the β-sheets of Tendamistat are held together with two rather than one disulfide bond, accounting for the considerable stability of the protein. The loops of Tendamistat can serve a similar function to the CDR loops found in immunoglobulins and can be easily randomized by in vitro mutagenesis. Tendamistat is derived from  Streptomyces tendae  and may be antigenic in humans. Hence, binding entities that employ Tendamistat are preferably employed in vitro. 
     Fibronectin type III domain has also been used as a protein scaffold to which binding entities can be attached. Fibronectin type III is part of a large subfamily (Fn3 family or s-type Ig family) of the immunoglobulin superfamily. Sequences, vectors and cloning procedures for using such a fibronectin type III domain as a protein scaffold for binding entities (e.g. CDR peptides) are provided, for example, in U.S. Patent Application Publication 20020019517. See also, Bork, P. &amp; Doolittle, R. F. (1992) Proposed acquisition of an animal protein domain by bacteria. Proc. Natl. Acad. Sci. USA 89, 8990-8994; Jones. E. Y. (1993) The immunoglobulin superfamily Curr. Opinion Struct. Biol. 3, 846-852; Bork, P., Hom, L. &amp; Sander, C. (1994) The immunoglobulin fold. Structural classification, sequence patterns and common core. J. Mol. Biol. 242, 309-320; Campbell, I. D. &amp; Spitzfaden, C. (1994) Building proteins with fibronectin type III modules Structure 2, 233-337; Harpez, Y. &amp; Chothia. C. (1994). 
     The following non-limiting examples further illustrate aspects of the invention. 
     Example 1: Sample Processing 
     Each patient is given a collection kit consisting of two buccal swabs and two uniquely barcoded tubes (termed A and B swabs) containing a proprietary lysis buffer consisting of 50 mM Tris pH 8.0, 50 mM EDTA, 25 mM Sucrose, 100 mM NaCl, and 1% SDS. The patient will use the swab to collect buccal cells by scraping the inside of their cheek and place the swab in the provided barcoded tube, one swab for each check. Once the swab has been placed into the lysis buffer the cells are no longer viable and therefore samples are now considered to be nucleic acids and safe to be shipped via standard mail. All samples are checked-in. The barcodes of the samples are scanned and their arrival in the laboratory is confirmed. 
       FIGS. 3A-3B  show schematic diagrams illustrating slight variations in sample processing. In general, two samples (Swab A and Swab B) are taken. The Swab A sample is subjected to the process (DNA Extraction through Reporting) unless the Swab A sample fails either the DNA Yield and Purity Assays. Genomic Analysis, or the PCR QA Assay. If such failure occurs, then the other sample (Swab B) is subjected to the process, as illustrated in  FIGS. 3A and/or 3B . 
     The samples are grouped into sets of 91 and assigned positions in 96 sample grids (12×8 grid layout) for DNA extraction. The remaining five positions in each grid can be extraction controls (four negative controls [H 2 O] and one non-human positive). The five controls can be assigned random positions in each grid, giving each grid/plate a unique “plate fingerprint.” The randomly assigned controls prevent possible plate swaps or 180° rotations as every plate is now identifiable simply by control positions. All samples are then normalized to a volume of 650 ul by addition of the above mentioned lysis buffer. Additionally, 25 ul of proteinase K (ProK) is added and each sample is incubated in a 55° C. oven for a minimum of 4 hours. 
     Following such incubation, the samples are extracted using a BioSprint96 (KingFisher96) Robotic workstation with magnetic-particle DNA purification chemistry to isolate genomic DNA (GenomicDNA) from tissue samples. This protocol utilizes the chemistry from the eVoMagDNA Extraction KF96 Kit (Verde Labs. Marietta, Ga.) and is run to specifications provided by the manufacturer. 
     Following DNA extraction and subsequent desiccation, the DNA is resuspended in HPLC water. Five microliters of each sample is then aliquoted to assay plates for the first pair of QA assays, both a PicoGreen fluorometric quantification and a spectrophotometric purity estimation. The fluorescence and absorbance data is analyzed for all samples in the 96 well plate, including the five controls. The positions of the negative controls is confirmed and accessed for possible plate contamination. The results for the positive control as well as the samples on the plate are analyzed for quality metrics using a systems analysis approach. The outliers are statistically assessed. After the quantification and purity evaluations. QA assay robotic systems are used to transfer the samples into racks of 96 sample septa sealed plates (to ensure there is no evaporative loss) and a fractional volume of each sample is used to create a daughter plate of the samples at a normalized concentration of 5 ng/μl for the PCR QA assays and subsequent genotyping. The creation of the normalized daughter plate serves two purposes. First, it allows the immediate storage of the primary stock of each sample at −80° C. avoiding the need for unnecessary freeze-thaw of samples and the potential contamination risks associated with repeated accessing of the stock. Second, it avoids unnecessary waste of the DNA associated with the use of full concentration stock for the PCR applications (this −80° C. stock DNA can be used at any time or saved for future testing). 
     Any samples that fail any of the QA assays can re-enter the pipeline and be sorted and re-processed from the B-swab, which is the second tube/swab in the kit sent to the customer mentioned above. By always having a backup sample it is not necessary to go back to the customer to ask for a re-swab. If the quantity and purity are still insufficient then whole genome amplification and/or organic re-extraction can be employed. 
     Following the passage of the QA thresholds normalized fractions of the samples are transferred to PCR plates for genotyping. Each sample is analyzed using three different methodologies, the Sequenom MassArray genotyping platform. Sanger sequencing using the ABI 3730x1 genomic analyzer from Applied Biosystems, and classical PCR and gel sizing to determine insertion/deletion status. The Sequenom MassArray genotyping platform is used to analyze the following SNP sites: rs1042713, rs1042714, rs1159744, rs12750834, rs1801252, rs1801253, rs2107614, rs227869, rs4244285, rs4961, and rs699. Sanger sequencing is used to analyze the following SNPs: rs3892097, rs3758581, rs2228586, and rs5186. Finally classical gel sizing is used to determine the insertion/deletion status of the rs1799752 SNP. 
     Example 2: Sequenom MassArray Assay Design and Processing 
     The Sequenom platform is able to perform genotyping as a twelve-plex assay (testing 12 variable sites in one reaction) in a 96 well format using one aliquot of DNA. The AssayDesign software from Sequenom is used to generate both PCR and single base extension primers using the individual rs number of each variable site to create the final assay design. Table 1 shows examples of primers that can be used to detect various single nucleotide polymorphisms. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Primers for Amplification of Nucleic 
               
               
                 Acid Variant Segments 
               
            
           
           
               
               
               
            
               
                 SNP ID 
                   
                 SEQ ID 1 st   
               
               
                 PCRP 
                 2 nd  PCRP 
                 SEQ ID 
               
               
                   
               
               
                 rs1042714 
                 ACGTTGGATGAGACATGACGATGCCCA 
                 NO: 36 
               
               
                   
                 TGC 
                   
               
               
                   
               
               
                   
                 ACGTTGGATGAGCGCCTTCTTGCTGGC 
                 NO: 37 
               
               
                   
                 AC 
                   
               
               
                   
               
               
                 rs699 
                 ACGTTGGATGCTGTGACAGGATGGAAG 
                 NO: 38 
               
               
                   
                 ACT 
                   
               
               
                   
               
               
                   
                 ACGTTGGATGTGGACGTAGGTGTTGAA 
                 NO: 39 
               
               
                   
                 AGC 
                   
               
               
                   
               
               
                 rs4961 
                 ACGTTGGATGTGTTCGTCCACACCTTA 
                 NO: 40 
               
               
                   
                 AGT 
                   
               
               
                   
               
               
                   
                 ACGTTGGATGACAAGATGGCTGAACTC 
                 NO: 41 
               
               
                   
                 TGG 
                   
               
               
                   
               
               
                 rs12750834 
                 ACGTTGGATGGGAATCCAGGAGAATAG 
                 NO: 42 
               
               
                   
                 GTC 
                   
               
               
                   
               
               
                   
                 ACGTTGGATGACAGGCTACCTGGCTTT 
                 NO: 43 
               
               
                   
                 AAC 
                   
               
               
                   
               
               
                 rs1801252 
                 ACGTTGGATGGCCTCGTTGCTGCCTCC 
                 NO: 44 
               
               
                   
                 CG 
                   
               
               
                   
               
               
                   
                 ACGTTGGATGATCAGCAGACCCATGCC 
                 NO: 45 
               
               
                   
                 CG 
                   
               
               
                   
               
               
                 rs1801253 
                 ACGTTGGATGAGCCCTGCGCGCGCAGC 
                 NO: 46 
               
               
                   
                 A 
                   
               
               
                   
               
               
                   
                 ACGTTGGATGTCAACCCCATCATCTAC 
                 NO: 47 
               
               
                   
                 TGC 
                   
               
               
                   
               
               
                 rs227869 
                 ACGTTGGATGCTGACATTGCCAGCTGT 
                 NO: 48 
               
               
                   
                 ATC 
                   
               
               
                   
               
               
                   
                 ACGTTGGATGGTAGTGGCACTGGCATA 
                 NO: 49 
               
               
                   
                 TTC 
                   
               
               
                   
               
               
                 rs2107614 
                 ACGTTGGATGGCAACCATCACAGTACT 
                 NO: 50 
               
               
                   
                 AAG 
                   
               
               
                   
               
               
                   
                 ACGTTGGATGCACAACTGGAAGAGTTG 
                 NO: 51 
               
               
                   
                 AGG 
                   
               
               
                   
               
               
                 rs1529927 
                 ACGTTGGATGTGGACCCCATTAACGAC 
                 NO: 52 
               
               
                   
                 ATC 
                   
               
               
                   
               
               
                   
                 ACGTTGGATGTCACCTTGGACTCCCAC 
                 NO: 53 
               
               
                   
                 TC 
                   
               
               
                   
               
               
                 rs4244285 
                 ACGTTGGATGCACTTTCCATAAAAGCA 
                 NO: 54 
               
               
                   
                 AGG 
                   
               
               
                   
               
               
                   
                 ACGTTGGATGGCAATAATTTTCCCACT 
                 NO: 55 
               
               
                   
                 ATC 
                   
               
               
                   
               
               
                 rs1042713 
                 ACGTTGGATGATGAGAGACATGACGAT 
                 NO: 56 
               
               
                   
                 GCC 
                   
               
               
                   
               
               
                   
                 ACGTTGGATGGAACGGCAGCGCCTTCT 
                 NO: 57 
               
               
                   
                 TG 
                   
               
               
                   
               
               
                 rs1159744 
                 ACGTTGGATGGAAACAGTGACAGCCAA 
                 NO: 58 
               
               
                   
                 ATG 
                   
               
               
                   
               
               
                   
                 ACGTTGGATGGTTTTTCAGTTCCTGAA 
                 NO: 59 
               
               
                   
                 TTG 
               
               
                   
               
            
           
         
       
     
     DNA samples at a concentration of 5 ng/ul undergo a PCR using the above designed PCR primers and the Sequenom iPLEX Gold Reagent kit under the conditions described in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 PCR Reaction Mixture  
               
            
           
           
               
               
               
            
               
                 Reagent 
                 Final Concentration 
                 Vol/rxn (uL) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Water, HPLC 
                 N/A 
                 1.8 
               
               
                 10x PCR Buffer with 20 mM MgCl 2   
                 2mM MgCl 2   
                 0.5 
               
               
                 25 mM MgCl 2   
                 2 mM 
                 0.4 
               
               
                 25 mM dNTP Mix 
                 500 uM 
                 0.1 
               
               
                 0.5 mM Primer Mix 
                 0.1 uM 
                 1 
               
               
                 5 U/uL PCR Enzyme 
                 1 unit 
                 0.2 
               
               
                 Volume 
                   
                 4 
               
               
                 10 ng/uL DNA 
                 10 ng/rxn 
                 1 
               
               
                 Total Volume 
                   
                 5 
               
               
                   
               
            
           
         
       
     
     The PCR reaction cycling conditions can be as illustrated in Table 3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 PCR Reaction Cycling  
               
               
                 Cycler Program iPlex- PCR 
               
            
           
           
               
               
               
               
            
               
                   
                 Temp (° C.) 
                 Time (mm) 
                   
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 95 
                 2:00 
                   
               
               
                   
                 95 
                 0:30 
                 Repeat 
               
               
                   
                 56 
                 0:30 
                 45 
               
               
                   
                 72 
                 1:00 
                 Cycles 
               
               
                   
                 72 
                 5:00 
                   
               
               
                   
                 4 
                 ∞ 
               
               
                   
                   
               
            
           
         
       
     
     Directly following PCR amplification, excess primers and deoxynucleotide triphosphates are removed via a SAP (shrimp alkaline phosphatase) reaction under the conditions described in Table 4. 
                     TABLE 4                  PCR Clean-Up                         Reagent   Final Concentration   Vol/rxn (uL)                                 Water, HPLC   N/A   1.53       SAP Buffer (10x)   0.24x   0.17       5 U/uL PCR Enzyme   1 unit   0.2       Volume       2       PCR product       5       Total Volume       7                    
The Shrimp Alkaline Phosphatase reaction is incubated at 37° C. for 40 min, followed by incubation at 85° C. for 5 min. The samples can be stored at 4° C. indefinitely.
 
     After the SAP reaction is completed the samples can be subjected to single base extension reactions using the primers described in Table 5, and the conditions described in Table 6 and 7. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Single Base Extension Primers 
               
            
           
           
               
               
               
            
               
                 SNP 
                 Sequence 
                 SEQ ID NO: 
               
               
                   
               
               
                 rs1042714 
                 ACACCTCGTCCCTTT 
                 60 
               
               
                   
               
               
                 rs699 
                 CTGGCTGCTCCCTGA 
                 61 
               
               
                   
               
               
                 rs4961 
                 ACTGCTTCCATTCTGCC 
                 62 
               
               
                   
               
               
                 rs12750834 
                 AGTCTCTGTAAGTGCCC 
                 63 
               
               
                   
               
               
                 rs1801252 
                 GTGCCTCCCGCCAGCGAA 
                 64 
               
               
                   
               
               
                 rs1801253 
                 CGCGCGCAGCAGAGCAGT 
                 65 
               
               
                   
               
               
                 rs227869 
                 AGCTGTATCTGCTCCATTCA 
                 66 
               
               
                   
               
               
                 rs2107614 
                 TCCTCCAAAAAAAAAGAAAAC 
                 67 
               
               
                   
               
               
                 rs1529927 
                 GTTACCGACATCCGCATCATTG 
                 68 
               
               
                   
               
               
                 rs4244285 
                 TAAGTAATTTGTTATGGGTTCC 
                 69 
               
               
                   
               
               
                 rs1042713 
                 GGAGGGGTCCGGCGCATGGCTTC 
                 70 
               
               
                   
               
               
                 rs1159744 
                 CAAATGTTAACAGTATAGAAAATTTTA 
                 71 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Single Base Extension Reaction Conditions  
               
            
           
           
               
               
               
               
            
               
                   
                 Reagents 
                 Final Concentration 
                 Vol/rxn (uL) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Water HPLC 
                 N/A 
                 0.619 
               
               
                   
                 iPlex Gold Buffer 
                 0.222x 
                 0.200 
               
               
                   
                 iPlex Termination Mix 
                 1x 
                 0.200 
               
               
                   
                 iPlex Extend Primer Mix 
                 varies 
                 0.940 
               
               
                   
                 iPlex Enzyme 
                 1x 
                 0.041 
               
               
                   
                 Volume 
                   
                 2.000 
               
               
                   
                 PCR product 
                   
                 7 
               
               
                   
                 Total Volume 
                   
                 9 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Single Base Extension Reaction Cycling conditions  
               
            
           
           
               
               
               
               
               
            
               
                   
                 Temp (∞ C.) 
                 Time 
                   
                   
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 94 
                 0:30 
                   
                   
               
               
                   
                 94 
                 0:05 
                   
                 40 cycles 
               
               
                   
                 52 
                 0:05 
                 5 cycles 
                 ↓ 
               
               
                   
                 80 
                 0:05 
                 ↓ 
                   
               
               
                   
                 72 
                 3:00 
                   
                   
               
               
                   
                 4 
                 forever 
               
               
                   
                   
               
            
           
         
       
     
     After completion of all of the above reactions, the samples are run through resin based clean-up to remove excess salts according to standard Sequenom protocols. The samples are then spotted onto the Sequenom provided SpectroChip using the Sequenom Nanodispenser according to manufacturer protocols and subsequently processed on the Sequenom MALDI-TOF platform. 
     A sample results report is provided in Table 7. The two letters for each polymorphism type are for the two alleles present in the subjects, illustrating that the subjects are homozygous for some polymorphisms (e.g., subject GCE0104 is homozygous (G/G) for the variable site in the rs1042713 polymorphism, but subject GCE0120 is heterozygous (GA) for that site). 
                     TABLE 8                  Results                                  SNP   GCE0120   GCE0104                       rs1042713   GA   GG           rs1042714   GC   GG           rs1159744   AA   AT           rs12750834   GA   GG           rs1529927   GG   GG           rs1801252   AA   AA           rs1801253   GG   GG           rs2107614   CT   TT           rs227869   AA   AG           rs4244285   GG   GA           rs4961   GG   GG           rs699   CG   GG                        
Practice of the foregoing methods can be accomplished with a PCR thermocycler instrument such as an MJ Research Tetrad 2 thermocycler for PCR amplification and an instrument for identification of the amplified DNA fragments such as an Agena MassArray® system or a Sequenom MALDI-TOF system which utilize the function of a time-of-flight mass spectrometer. A patient&#39;s DNA is first extracted as described above and the PCR reaction conducted in the MJ thermocycler to amplify only DNA fragments corresponding substantially to the patient&#39;s genomic SNP&#39;s described above. The amplified genomic material is then introduced into the detection and identification instrument such as an Agena or Sequenom automated DNA fragment identification system. This system enables identification of multiples of PCR amplified DNA fragments and provides identification of the patient&#39;s 14 genotype SNP&#39;s in accordance with embodiments of the invention.
 
     Example 3: Sanger Sequencing Primer Design and Workflow 
     All primers for Sanger sequencing were designed using the free, web-based primer design tool Primer3. 500 base pairs of flanking sequence for each SNP (single nucleotide polymorphism) were entered into Primer3. The top primer candidate for each site was chosen and optimized using a buffer panel with varying MgCl concentrations and a temperature gradient to determine the optimal cycling conditions for each primer pair. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Primers for Sequencing of SNPs 
               
            
           
           
               
               
               
               
            
               
                 Primer Name 
                 Sequence 
                 SEQ ID 
                 Purpose 
               
               
                   
               
               
                 rs3892097_F 
                 TTCAGTCCCTCCTGAGCTA 
                 NO: 72 
                 SNP 
               
               
                   
               
               
                 rs3892097_R 
                 AAGGTGGATGCACAAAGAG 
                 NO: 73 
                 SNP 
               
               
                   
               
               
                 rs3758581_F 
                 GTGCATCTGTAGCAGTCCTC 
                 NO: 74 
                 SNP 
               
               
                   
               
               
                 rs3758581_R 
                 CCAAACTGGAATCAACAGAA 
                 NO: 75 
                 SNP 
               
               
                   
               
               
                 rs2228586_F 
                 GAAGTGGTCTCGTCTAGCAA 
                 NO: 76 
                 SNP 
               
               
                   
               
               
                 rs2228586_R 
                 CAGAGAGAGAGGTCCCATTT 
                 NO: 77 
                 SNP 
               
               
                   
               
               
                 rs5186_F 
                 CCACTCAAACCTTTCAACAA 
                 NO: 78 
                 SNP 
               
               
                   
               
               
                 rs5186_R 
                 TGGACAGAACAATCTGGAAC 
                 NO: 79 
                 SNP 
               
               
                   
               
            
           
         
       
     
     The region encompassing the SNP was amplified from sample nucleic acids by PCR using optimized individual cycling conditions for each SNP site. Directly after PCR amplification each sample is cleaned up using a size exclusion micro-filtration plate from Millipore and entered into the Sanger sequencing reaction. Each sample is sequenced in both the forward (3′) and reverse (5′) direction giving double conformation of the allelic state. These forward and reverse sequences from each patient are then aligned to the human reference sequence using the CLC DNA workbench program creating an alignment file from which the allele call is made and added to the final SNP call report. 
       FIG. 6  illustrates the results from one such alignment. 
     Example 4: Gel Sizing Primer Design and Workflow 
     To accurately call the insertion/deletion status for site rs1799752, PCR amplification of sample nucleic acids is performed followed by gel electrophoresis. The PCR primers for this site were also designed and optimized using Primer3 and the above mentioned buffer and temperature gradient. The following primer sequences and PCR conditions were ultimately chosen: 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Primer Sequences for PCR of rs1799752 
               
               
                 Insertion/Deletion 
               
            
           
           
               
               
               
               
            
               
                 Primer Name 
                 Sequence 
                 SEQ ID 
                 Purpose 
               
               
                   
               
               
                 rs1799752_F-2 
                 CCCATTTCTCTAGACCTGCT 
                 NO: 80 
                 INDEL 
               
               
                   
               
               
                 rs1799752_R-2 
                 GGGATGGTGTCTCGTACATA 
                 NO: 81 
                 INDEL 
               
               
                   
               
            
           
         
       
     
     Following PCR amplification, each sample is loaded into its own well of a 2% agarose gel and run at 150 mV for approximately 45 min and stained in a bath of GelRed for 2 hours prior to imaging with UV light. The resulting image is used to score the presence or absence of a 288 bp ALU visually by examining the gel for either the higher molecular weight band (indicating the presence of the 288 bp ALU), the lower molecular weight band (indicating the absence of the 288 bp ALU) or both (indicating a heterozygous state. A sample image of the gel is provided in  FIG. 7 . 
     Example 5: Genotyping Reports 
     Once all tests are performed a report is generated containing all results for each tested patient. One example of a report for subjects GCE0120 and GCE0104, is shown below. The two letters for each polymorphism type are for the two alleles present in the subjects, illustrating that the subjects are homozygous whereas other subjects are heterozygous for the variable site of each polymorphism. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Results from Analysis of Polymorphisms 
               
            
           
           
               
               
               
               
            
               
                   
                 Polymorphism type 
                 GCE0120 
                 GCE0104 
               
               
                   
                   
               
               
                   
                 Sequenom 
                   
                   
               
               
                   
                 Results 
                   
                   
               
               
                   
                 rs1042713 
                 GA 
                 GG 
               
               
                   
                 rs1042714 
                 GC 
                 GG 
               
               
                   
                 rs1159744 
                 CG 
                 CG 
               
               
                   
                 rs12750834 
                 GA 
                 GG 
               
               
                   
                 rs1529927 
                 GG 
                 GG 
               
               
                   
                 rs1801252 
                 AA 
                 AA 
               
               
                   
                 rs1801253 
                 GG 
                 GG 
               
               
                   
                 rs2107614 
                 CT 
                 TT 
               
               
                   
                 rs227869 
                 AA 
                 AG 
               
               
                   
                 rs4244285 
                 GG 
                 GA 
               
               
                   
                 rs4961 
                 GG 
                 GG 
               
               
                   
                 rs699 
                 TT 
                 TT 
               
               
                   
                 Sanger Sequencing 
                   
                   
               
               
                   
                 Results 
                   
                   
               
               
                   
                 rs3892097 
                 CC 
                 CC 
               
               
                   
                 rs3758581 
                 GG 
                 GG 
               
               
                   
                 rs3758580 
                 CC 
                 CT 
               
               
                   
                 rs2228586 
                 TT 
                 TT 
               
               
                   
                 rs5186 
                 AC 
                 AA 
               
               
                   
                 Gel Results 
                   
                   
               
               
                   
                 rs1799752 
                 +/+ 
                 +/− 
               
               
                   
                   
               
            
           
         
       
     
     Example 6: Clinical Study Protocol 
     Clinical Protocol Summary 
       
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 Study Title: 
                 Assessment of the Relationship between Genes that 
               
               
                   
                 Encode Proteins Important in Blood Pressure Regulation 
               
               
                   
                 and Blood Pressure Therapy in Patients with  
               
               
                   
                 Hypertension. 
               
               
                 Study  
                 The Geneticure Pharmacogenetic Testing Kit. The kit 
               
               
                 Device: 
                 contains two buccal swabs with two buffer solution vials  
               
               
                   
                 to stabilize DNA. These buccal swabs are used for DNA 
               
               
                   
                 collection which is then extracted for analysis of genes 
               
               
                   
                 important in high blood pressure. 
               
               
                 Target  
                 The Geneticure Pharmacogenetic Testing Kit is a 
               
               
                 Indication  
                 pharmacogenomic treatment decision support product that 
               
               
                 for Use: 
                 tests for clinically important genetic variants affecting a 
               
               
                   
                 patient&#39;s response to antihypertensive medications. 
               
               
                 Study  
                 This is a post-hoc association study of patients who have 
               
               
                 Design: 
                 been diagnosed with high blood pressure and have been 
               
               
                   
                 stable on medication treatment for at least 6 months. 
               
               
                 Study  
                 To be enrolled in this study, subjects must meet ALL of 
               
               
                 Population: 
                 the inclusion criteria and NONE of the exclusion criteria: 
               
               
                   
                 Inclusion Criteria 
               
            
           
           
               
               
               
            
               
                   
                 1. 
                 Subject is able and willing to provide informed 
               
               
                   
                   
                 consent 
               
               
                   
                 2. 
                 Subject is ≥30 and ≤70 years of age 
               
               
                   
                 3. 
                 Subject with diagnosis of Hypertension for a 
               
               
                   
                   
                 minimum of 1 year 
               
               
                   
                 4. 
                 Subject has been on the same class/classes of blood 
               
               
                   
                   
                 pressure medication for a minimum of 6 months. 
               
               
                   
                   
                 Note: A change in dosage, frequency, or specific 
               
               
                   
                   
                 medication is acceptable as long as there have been 
               
               
                   
                   
                 no changes to the class/classes of medications 
               
               
                   
                   
                 prescribed. 
               
               
                   
                 5. 
                 Subject with a Body Mass Index (BMI) ≥19 and ≤35 
               
               
                   
                 6. 
                 Subject is currently prescribed and taking one of 
               
               
                   
                   
                 the following classes of medications alone or in 
               
               
                   
                   
                 combination with each other or a Ca+ channel 
               
               
                   
                   
                 blocker. 
               
               
                   
                   
                  Diuretics 
               
               
                   
                   
                  ACE Inhibitors 
               
               
                   
                   
                  Angiotensin Receptor Blocker (ARB) 
               
               
                   
                   
                  Beta-blockers 
               
            
           
           
               
               
            
               
                   
                 Exclusion Criteria 
               
            
           
           
               
               
               
            
               
                   
                 1. 
                 Subject has clinically significant kidney disease as 
               
               
                   
                   
                 determined by the investigator. 
               
               
                   
                 2. 
                 Subject has clinically significant cardiac disease as 
               
               
                   
                   
                 determined by the investigator. 
               
               
                   
                 3. 
                 Subject has clinically significant vascular disease as 
               
               
                   
                   
                 determined by the investigator. 
               
               
                   
                 4. 
                 Subject has a diagnosis of secondary hypertension 
               
               
                   
                   
                 or is experiencing a complication of pregnancy. 
               
               
                   
                 5. 
                 Subject is currently prescribed and taking any 
               
               
                   
                   
                 additional class of medication(s) for high blood 
               
               
                   
                   
                 pressure not included in the list above, with the 
               
               
                   
                   
                 exception of a Ca+ channel blocker. 
               
               
                   
                 6. 
                 Subject has Systolic BP &gt; 190 or Diastolic BP &gt; 
               
               
                   
                   
                 120 documented within the six months prior to 
               
               
                   
                   
                 visit. 
               
               
                   
                 7. 
                 Subject has a regular alcohol intake of greater than 
               
               
                   
                   
                 21 units per week in the past 6 months 
               
               
                   
                 8. 
                 Subject has smoked greater than two packs of 
               
               
                   
                   
                 cigarettes (total) or equivalent nicotine intake in the 
               
               
                   
                   
                 past 6 months. 
               
               
                   
                 9. 
                 Subject has an anticipated survival less than 12 
               
               
                   
                   
                 months. 
               
               
                   
                 10. 
                 Any other reason that the subject is inappropriate 
               
               
                   
                   
                 for study enrollment in the opinion of the 
               
               
                   
                   
                 Investigator. 
               
            
           
           
               
               
            
               
                 Primary  
                 To assess the relationship between the drug therapy 
               
               
                 Study 
                 class/combination of therapy classes that resulted in the 
               
               
                 Objective: 
                 best blood pressure control for a patient vs. what the 
               
               
                   
                 Geneticure high blood pressure panel would have 
               
               
                   
                 predicted. 
               
               
                 Secondary  
                 To assess the clinical time to achieve optimal blood 
               
               
                 Study 
                 pressure treatment. 
               
               
                 Objectives: 
                 To assess the number of office visits required to 
               
               
                   
                 achieve optimal blood pressure treatment. 
               
               
                   
               
            
           
         
       
     
     1 Introduction 
     Hypertension (high blood pressure) is one of the most important preventable contributors to disease and death in the United States and represents the most common condition seen in the primary care setting (The sixth report of the Joint National Committee on prevention, detection, evaluation, and treatment of high blood pressure. Arch Intern Med. 157(21):2413-2446 (1997); Chobanian et al. JAMA 289(19):2560-2572 (2003)). According to the American Heart Association, approximately 78 million adults (1 in 3) living in the United States have hypertension with more than 5 million new diagnoses made each year (American Heart Association. Heart and Stroke Statistics—2004 Update. Paper presented at: Dallas: American Heart Association (2004); Roger et al. Circulation. 125(1):e2-e220 (2012)). Of these individuals, 82% are aware they have it, 75% are currently being treated for it, but only 52% have their blood pressure under control. Thus, about 48% of individuals with hypertension do not have adequate blood pressure control. Hypertension is known to lead to myocardial infarction (heart attack), stroke, renal failure, and death if not detected early and treated appropriately. In fact, in 2009, high blood pressure was listed as a primary or contributing cause of death in ˜350,000 of the ˜2.4 million U.S. deaths (14% of all deaths). From 1999-2009 the number of deaths attributable to hypertension increased by 44%. 
     Refractory (or resistant) hypertension is defined as blood pressure that remains above clinical guideline goals in spite of concurrent use of three antihypertensive agents of different classes (Akpunonu et al., Disease-a-month: DM. October 1996; 42(10):609-722). Critically, refractory hypertension is noted in approximately 25-30% of all individuals being treated for hypertension. Refractory hypertension is a common clinical problem which contributes to the high levels of morbidity and mortality. The inability to gain control of blood pressure in these individuals may be related to the pharmacogenetics of the individual coupled with the specific classes of drugs and/or combination of classes chosen for that individual (Calhoun et al. Circulation 117(25):e510-526 (2008); Johnson &amp; Turner, Curr Opin Mol Ther 7(3):218-225 (2005)). In 2009, the direct and indirect economic burden on the United States health care system associated with hypertension was estimated at $51 billion. With the advent of improved diagnostic techniques, increased rates of health care utilization and screening, and the increasing age of the population, a continual upward trend in this expenditure is expected. 
     Globally, nearly 1 billion individuals have been diagnosed with hypertension with an estimate of an additional 400 million living with undiagnosed hypertension. Hypertension is the leading cause of premature death and the leading cause of cardiovascular disease worldwide. Similar to the continued upward trend in prevalence as seen in the United States, it is estimated that in 2025 approximately 1.56 billion adults will be living with hypertension. Because nearly two-thirds of the people living with hypertension worldwide reside in developing countries, providing optimal treatment at the lowest cost is critically important. 
     Unfortunately, despite a significant impulse in the medical community to move towards an “individualized medicine” approach to patient centered treatment, the current clinical treatment strategy is based on a set algorithm which does not take into account individual patient differences. Rather, physicians are guided to choose a drug (one out of many options) in a given class of drugs and use that specific drug as a “first line therapy” (typically initiating with the diuretic class) and titrate that specific drug of choice to therapeutic dosage regardless of efficacy (Chobanian et al. JAMA 289(19):2560-2572 (2003)). It is only after a prolonged course of treatment with that specific class of drug that clinical efficacy is determined (typically three months). At this stage, if clinical guideline goals for blood pressure have not been met, it is often recommended that the patient remain on the “first line therapy” whilst an additional drug from a different class of drugs (typically an Angiotensin converting enzyme inhibitor (ACE inhibitor) or Angiotensin II receptor blocker (ARB)) is added to the pharmacologic regimen. Again, this drug is titrated to recommended therapeutic dosage and another prolonged course of treatment is initiated before clinical efficacy is determined (an additional three months—six months since initiation of treatment). If at this point, clinical guideline goals for blood pressure have not been met, a third drug from a third class of drugs (typically a beta-blocker) is added and the process is repeated (another three months—nine months from initiation of treatment). Further, if clinical guideline goals have continued to be elusive, the diagnosis of refractory hypertension is added and the process is reinitiated with a different combination of drugs, different classes of drugs, different drug options within a given class of drugs, different dosages, or all of the above. Thus, from the time of initial diagnosis and the start of treatment to the point in which blood pressure is adequately controlled may take anywhere from three months to well over one year. This trial-and-error standard of care is clearly not optimal. 
     The blood pressure panel created by Geneticure has been created to comprehensively assess fourteen common genetic variants in the cardiac, vascular, and renal systems that can improve therapeutic guidance for the clinician based on known functional alterations of the protein through these genetic changes, as well as demonstrated effects of certain drug classes on these various genotypes. Based on this information, a clinician can guide therapy with knowledge specific to their patient, rather than “trial-and-error” based on population data and using drugs with least side effects initially. 
     1.1 Investigational Device: Geneticure Pharmacogenetic Testing Kit 
     The Geneticure pharmacogenetic testing kit contains two buccal swabs with two buffer solution vials to stabilize DNA. These buccal swabs are used for DNA collection which is then extracted for analysis of genes important in high blood pressure. 
     The Geneticure Pharmacogenetic Testing Kit is a pharmacogenomic treatment decision support product that tests for clinically important genetic variants affecting a patient&#39;s response to antihypertensive medications. 
     1.2 Genetic Analysis 
     Each sample can be analyzed for fourteen common genetic variants using 3 different methodologies, the Sequenom MassArray genotyping platform, Sanger sequencing using the ABI 3730x1 genomic analyzer from Applied Biosystems, and classical PCR and gel sizing to determine insertion/deletion status (see,  FIGS. 3A-3B ). 
     2 Methodology 
     2.1 Study Design and Protocol Overview 
     This is a post-hoc association study of patients who have been diagnosed with high blood pressure and have been stable on medication treatment for at least 6 months. The purpose of this study is to evaluate the relationship between optimal medication therapy (or the therapy that has resulted in the most stable blood pressure for that particular patient) and the predicted optimal medication therapy based on a patient&#39;s genetic profile. 
     Chart reviews for the patient&#39;s history of antihypertensive therapy can be coupled with buccal swabs and blood pressure readings collected from eligible patients who have provided informed consent. The swab can be analyzed for fourteen genetic variants that are associated with antihypertensive therapy response (efficacy, side-effects). 
     2.2 Study Objective 
     To assess the relationship between the drug therapy class/combination of therapy classes that resulted in the best blood pressure control for a patient vs. what the Geneticure high blood pressure panel would have predicted. 
     2.3 Secondary Objectives 
     The secondary objectives are as follows:
         To assess the clinical time to achieve optimal blood pressure treatment.   To assess the number of office visits required to achieve optimal blood pressure treatment.       

     3 Investigational Study Center 
     This study will be conducted at up to 5 study centers within the United States that have adequate resources for trial responsibilities. 
     4 Study Population 
     To be enrolled in this study, subjects must meet ALL of the inclusion criteria and NONE of the exclusion criteria: 
     4.1 Inclusion Criteria
         1. Subject is able and willing to provide informed consent   2. Subject is ≥30 and ≤70 years of age   3. Subject with diagnosis of Hypertension for a minimum of 1 year   4. Subject has been on the same class/classes of blood pressure medication for a minimum of 6 months. Note: A change in dosage, frequency, or specific medication is acceptable as long as there have been no changes to the class/classes of medications prescribed.   5. Subject with a Body Mass Index (BMI) ≥19 and ≤35   6. Subject is currently prescribed and taking one of the following classes of medications alone or in combination with each other or a Ca +  channel blocker.
           Diuretics   ACE Inhibitors   Angiotensin Receptor Blocker (ARB)   Beta-blockers   
               

     4.2 Exclusion Criteria
         1. Subject has clinically significant kidney disease as determined by the investigator.   2. Subject has clinically significant cardiac disease as determined by the investigator.   3. Subject has clinically significant vascular disease as determined by the investigator.   4. Subject has a diagnosis of secondary hypertension or is experiencing a complication of pregnancy.   5. Subject is currently prescribed and taking any additional class of medication(s) for high blood pressure not included in the list above, with the exception of a Ca+ channel blocker.   6. Subject has Systolic BP&gt;190 or Diastolic BP&gt;120 documented within the six months prior to visit.   7. Subject has a regular alcohol intake of greater than 21 units per week in the past 6 months   8. Subject has smoked greater than two packs of cigarettes (total) or equivalent nicotine intake in the past 6 months.   9. Subject has an anticipated survival less than 12 months.   10. Any other reason that the subject is inappropriate for study enrollment in the opinion of the Investigator.       

     5 Informed Consent 
     The investigator will prepare an informed consent form in accordance with this study protocol and all regulatory requirements (21 CFR Part 50) using the template informed consent form provided by the sponsor. The informed consent form must be submitted to the IRB and a copy of the final IRB-approved consent form must be submitted to the Study Management Center prior to the start of the study at that investigational site. 
     Prior to any study procedures, all subjects must document their consent for study participation and authorization for use and disclosure of health information by signing the IRB-approved Informed Consent Form. As part of the consent process, the subject will have the opportunity to ask questions of, and receive answers from the personnel conducting the study. 
     The investigator will notify the Study Management Center and the IRB within 5 working days if device use occurs without subject informed consent. 
     6 Study Assessments and Data Management 
     6.1 Screening
         Identify Potential Study Subjects. Refer to the Inclusion and Exclusion Criteria sections of this protocol for a complete list of eligibility criteria.   Obtain Written Informed Consent. Each potential study participant must be given time to review the IRB-approved informed consent form, have his/her questions answered to their satisfaction and sign the form prior to any study procedures being performed. A subject will be given a copy of the informed consent form.   Review Inclusion/Exclusion Criteria. The investigator and/or designee will review all criteria to determine if the subject is eligible for enrollment. Eligibility of all subjects must personally be confirmed by the Investigator and will be documented on the CRF.       

     6.2 Enrollment 
     
         
         
           
             Assign Identification Number to Eligible Subjects. See Protocol section 6.3. 
             Record Demographics, Antihypertensive Medical History and current Blood Pressure. Data will be documented in the source document and recorded on the CRF, including but not limited to the following:
           Age   Height   Weight   Race   Ethnicity   Length of Hypertension diagnosis   Previously and currently prescribed antihypertensive medications   Blood pressure measurements   
         
           
         
       
    
     6.3 Specimen Collection
         Collect Buccal Specimen.   Using the collection kit consisting of two buccal swabs and two uniquely barcoded tubes the investigator or designee will remove the first buccal brush and scrape the brush end across a Subject&#39;s right cheek repeatedly (for five seconds). The investigator/designee will place the brush end over the open buffer vial and press the opposite end of the swab stick to release the brush into the buffer and then close the vial. The process can be repeated on the left cheek. Each of the right and left cheek vial numbers must be recorded on the CRF and accountability log as right (R), or left (L).   Adverse Event Recording   Perform Product Accountability       

     6.4 Subject Numbering 
     Subjects meeting the criteria for enrollment (and their specimens) can be identified by unique numbers that can be assigned sequentially by order of enrollment. The pre-assigned investigational site number can be prefixed to the identification number and separated by a hyphen (e.g., site 01 would number their subjects sequentially as 01-001, 01-002, 01-003, etc.). Throughout the descriptions within the protocol the A swab will be referring to the swab that has originated from the right cheek, while the B swab will be that that has originated from the left cheek. To further clarify. Subject 01-001 can be given two barcoded tubes. These barcode numbers can be recorded for each patient. These can also be recorded as originating from the right check (A) or left check (B). 
     At no time should any study paperwork or specimens be marked with the subject&#39;s name or any other traceable identifier except for the informed consent form, which is signed by the subject and kept at the site. At no time should the original (signed) or a copy of this form be collected by the Sponsor or its representative. 
     6.5 Subject Completion and Withdrawal 
     Once subjects undergo the sampling procedure, their study participation is complete. There are no follow-up visits. Subjects will be instructed to notify the Investigator if they experience any symptoms or complications from the sampling procedure. 
     Subjects are free to withdraw consent and discontinue participation in the study at any time. A subject&#39;s participation in the study may be discontinued at any time at the discretion of the Investigator or Geneticure. The following may be justifiable reasons for the Investigator or Geneticure to remove a subject from the study:
         The subject was erroneously included in the study or was found to have an exclusion criterion.   The subject was uncooperative.   The subject experienced an AE/SAE during the sample collection procedure that is considered intolerable by the subject or Investigator.       

     To the extent possible, safety data will be collected on subjects who discontinue participation in the study due to safety reasons. 
     The following may be justifiable reasons for the Investigator or Geneticure to remove a specimen from the study:
         Sample is determined to be of poor or inadequate quality for analysis (e.g., contamination, insufficient material for analysis).   The sample was erroneously included in the study.   The specimen was not collected or processed per protocol procedures.       

     6.6 Concomitant Medications/Treatment/Procedures 
     This study protocol does not require change to any existing treatments or those prescribed during the course of the study by the Investigator or any other provider whom the subject sees for any medical reason. Outside of eligibility screening, there are no clinical evaluations as part of this study. 
     6.7 Data Management 
     The Investigator is responsible to ensure the accuracy, completeness, and timeliness of reported data. 
     All data will be sent to Geneticure who will enter it into the study database using a secure, protected Excel spreadsheet. The database will be validated prior to use in the study. All required data will be recorded on CRFs or paper facsimiles. 
     Data collected within the CRFs will be supported by source documents as appropriate and may be updated to reflect the latest observations on the subjects participating in the study. Corrections to the source documentation can be made in a manner that does not obscure the original entry and will be dated and initialed by the Investigator or assigned designee. It is important for data entry to occur in a timely manner, therefore, data collected on source documents should be transferred into CRFs as soon as possible following study visits. 
     Study subject data can be reviewed at the investigational site by monitors at regular intervals throughout the study. Information on the CRFs can be compared to information originally recorded on source documents related to the study (i.e. professional notes, study-specific worksheets, etc.) 
     7 Genomic Core Laboratory 
     The subjects&#39; cheek vials will be sent to the Geneticure processing center. The vials will then be batched and sent to the Genomic Core laboratory for DNA extraction and genetic analysis. Following analysis, results will be sent to Geneticure for statistical analysis and DNA will be destroyed. 
     A protocol for the extraction and analysis will be followed to ensure consistency and objectivity. 
     8 Adverse Events 
     The procedures outlined in this protocol do not involve significant risk to subject safety. Subjects will be provided the investigator&#39;s contact information and will be instructed to notify the investigator of any adverse events they experience during or secondary to the sample collection procedures. 
     8.1 Definitions 
     8.1.1 Adverse Event
         For the purposes of this study, an adverse event is defined as any undesirable/unusual medical experience that occurs to a subject in conjunction with the use of the product, whether or not considered product related, including (but not limited to) those events that result from the use as stipulated in the protocol.   The following adverse events will not be collected in this study:
           Adverse events which, in the opinion of the Investigator, are unrelated to the swab collection procedure, but rather related to the subject&#39;s underlying medical conditions or status   Adverse events that may be related to the sample collection procedure but result only in local, mild and transient discomforts.   
           The Investigator is responsible for documenting all Adverse Events on the Adverse Event CRF, except for those events noted above.       

     8.1.2 Serious Adverse Event 
     A Serious adverse event is an adverse event that:
         led to death,   led to serious deterioration in the health of a subject that
           resulted in a life-threatening illness or injury,   resulted in permanent impairment of a body structure or body function,   required inpatient hospitalization or prolongation of existing hospitalization.   resulted in medical or surgical intervention to prevent permanent impairment to a body structure or a body function.   
           led to fetal distress, fetal death or a congenital anomaly or birth defect.       

     8.2 Event Reporting 
     Any AE, or SAE experienced by a subject after signing the informed consent until twenty-four (24) hours following study completion or termination will be recorded in the progress notes and on the CRF. The Investigator and/or designee will continue to monitor the subject with additional assessments until the AE is considered resolved, stabilized, or is lost to follow up. 
     A full description of an adverse event, including the nature, date and time of onset and resolution, determination of seriousness, frequency, severity, treatment, outcome, and relationship to the study will be recorded on the Adverse Event CRF. 
     SAEs must be reported to RCRI within 48 hours of the Investigator&#39;s first knowledge of the event. 
     9 Statistical Methods 
     Following is a summary of the Statistical Analysis Plan for the study. The following objectives have been prospectively defined; however, due to the nature of these data, additional analyses may be conducted or additional subsets may be identified that are not listed in this protocol. 
     9.1 Sample Size
         Up to 300 subjects may be enrolled at each site. The minimum number of subjects for meaningful statistical analysis is 100 subjects.       

     9.2 Data Analysis
         All data will be coded for statistical analysis (i.e. drug classes and genotypes will be coded numerically). All data will be analyzed with SPSS v.20. Normality of the data will be assessed using Levene&#39;s test prior to statistical analysis and any correction for non-normal data distribution will be used. Descriptive statistics will be computed (average time for blood pressure control, average number of visits to the clinician for blood pressure control, age, height, weight, BMI, etc.).   Data will be initially analysed following the collection of samples/data from 100 subjects. This will allow for direction for power calculations/etc. for future statistical analysis. Although some of the genes have been analysed individually, no mean or standard deviation data exists to allow for a true a priori power calculation. Data will be analysed again after two months or following 300 subjects for which data has been collected. Statistical tests will be corrected for the number of tests run (preservation of alpha).   Ordinary least squares regression via univariate modelling will be used to estimate the magnitude of linearity between drug class that yielded the best blood pressure control and genetic profile of the subject. Multiple regression analysis will be performed to determine the impact of confounding variables (height, weight, age, race) on blood pressure control. For all statistical analyses an alpha level of 0.05 will be used to determine statistical significance.       

     9.3 Other Statistical Considerations 
     Justification of Pooling Data across Centers 
     There is no need to keep the data from different centers separate for data collection. Primary reasons for not pooling blood pressure data from different centers could include different races (which we are collecting as a demographic and analyzing as a co-variate in a multiple-regression) and different cultures (i.e. southern vs. northern habits of diet, exercise, etc.). The study will take race, height, weight, age into account as co-variates in a multiple regression model, but will not be powered to take into account possible geographic influences on the data. 
     Missing Data 
     All patients with available data will be included in the analyses of primary and secondary objectives. Because some of the data was not recorded as part of a prospective protocol an unknown amount of data will be permanently missing. No patients will be contacted to retrieve missing data, and no sensitivity analyses will be performed on missing data. 
     10 Risk Analysis 
     10.1 Device Description 
     The procedures outlined in this protocol do not involve significant risk to subject safety. Subjects will be provided the investigator&#39;s contact information and will be instructed to notify the investigator of any adverse events they experience during or secondary to the specimen collection procedures. 
     The collection kit includes a small, soft, brush for cheek swabbing and a buffer solution in a small vial, one of each for each cheek, two in total. Once the swab has been placed into the lysis buffer the cells are no longer viable and therefore samples are now considered to be nucleic acids and safe to be shipped via standard mail. 
     11 Study Materials 
     11.1 Handling and Storage
         The Investigator must ensure that the investigational product is stored in a controlled location with limited access.       

     11.2 Product Accountability
         The investigator is responsible for investigational product accountability, reconciliation and record maintenance. The investigator must maintain investigational product accountability records throughout the course of the study.   Upon completion or termination of the study, all unused product, together with a copy of the product accountability form will be returned to Geneticure or its representative.   All supplies are to be used only for this protocol and not for any other purpose.       

     12 Study Administration 
     12.1 Subject Confidentiality 
     All information and data sent to Geneticure, and/or its designees concerning subjects and their participation in this study are considered confidential by Geneticure and it designees (subcontractors or contract research organization). Only authorized Geneticure personnel or approved contracted agents of Geneticure will have access to some portions of these confidential files and will act in accordance with applicable regulations as required by HIPAA. The IRBs and FDA also have the right to inspect and copy all records pertinent to this study. All data used in the reporting of the study will eliminate identifiable reference to the subjects. 
     12.2 Investigational Center Qualification 
     Investigational Center qualification visits or phone calls will be conducted by the Study Management Center prior to acceptance of the site into this study. The site qualification visit will be scheduled to include time with the Principal Investigator and other study personnel as applicable. Areas of discussion include a review of personnel training, investigator qualifications, adequacy of potential subject pool. FDA-regulated study experience, this study&#39;s specific requirements for procedures, and a review of staffing availability and appropriateness. A written report of the qualification visit will be drafted by the Study Management Center. 
     12.3 Site Training 
     Study-specific training of study personnel is the responsibility of the Sponsor or Study Management Center and the Principal Investigator. Study training will occur before the first device use. To ensure protocol and regulatory compliance as well as accurate data collection, site training will include a detailed review of the protocol, CRF completion, study specific procedures, monitoring logistics, and regulatory requirements. 
     12.4 Investigator Responsibilities 
     The investigator is responsible for ensuring that the study is conducted according to the investigational plan and all applicable FDA regulations, including reporting and record-keeping requirements, and controlling the devices undergoing investigation and HIPAA. In addition, the principal investigator is responsible for ensuring that informed consent is obtained from each subject prior to participating in the study, as well as protecting the rights, safety and welfare of participating subjects. Specific responsibilities are listed in this investigational plan. 
     Records and reports must remain on file at the investigational site for a minimum of two years after the later of either the completion/termination of the investigational study or the date it is determined the records are no longer required to support submissions to regulatory authorities. They may be discarded only upon approval from Geneticure. The Principal Investigator must contact Geneticure before destroying any records and reports pertaining to the study to ensure that they no longer need to be retained. In addition, Geneticure must be contacted if the investigator plans to leave the investigational site to ensure that arrangements for a new investigator or records transfer are made prior to investigator departure. 
     12.4.1 Records
         Records to be maintained by the investigator in the designated investigational center&#39;s study file include:
           Investigational plan and all amendments   Signed Financial Disclosure   IRB approval letter including consent and HIPAA authorization form(s)   IRB Membership list or Letter of Assurance   All correspondence relating to the study between the site and Geneticure, and the Study Management Center   CVs and professional licenses for all investigators   Site personnel signature and responsibility list   Clinical monitor sign-in log   Blank set of each version of CRFs   Subject Screening/Enrollment log   Investigational device accountability log including: date, quantity, lot numbers of all devices, identification of all persons the device was used on and final disposition.   
               

     The following records are maintained for each subject enrolled in the study:
         Signed Consent Form and Authorization for the Use and Disclosure of Health Information   Compete, accurate and current CRFs and DCFs   Adverse event reports and any supporting documentation   Protocol deviations   Complete medical records, including procedure reports, lab reports, professional notes, etc.       

     Geneticure reserves the right to secure data clarification and additional medical documentation on subjects enrolled in this study at any time. 
     13 Abbreviations 
     
         
         
           
             AE=Adverse Event CRF=Case Report Form 
             DCF=Data Clarification Form FDA=Food and Drug Administration 
             HIPAA=Health Insurance Portability and Accountability Act of 1996 
             IRB/IEC=Institutional Review Board/Independent Ethics Committee 
             ITT=Intent-to-Treat PP=Per Protocol 
             SAE=Serious Adverse Event 
             UADE=Unanticipated Adverse Device Effect 
           
         
       
    
     Example 7: Results and Summary of Phase I Clinical Study Introduction 
     For this phase-I research study 14 genes within the Geneticure blood pressure (BP) panel were assessed as they relate to time to BP control and absolute BP values in 99 patients with hypertension. The study design utilized a post-hoc patient chart review carried out by two clinical sites through the direction of RCRI (a third-party clinical research firm) exploring genes important in drug metabolism, renal Na +  handling, vascular function, and cardiac output (all of which can result differences in BP and response to BP therapy). Although the primary aim was BP control in response to therapy relative to genetic data, the time on average, it takes patients to achieve BP control without consideration of genetic information was also determined. 
     In summary, the study demonstrated that the genes in the Geneticure panel were predictive of time to BP control in patients with hypertension. In addition, there was an effect of several of the genes being predictive of BP taken within the clinic at the time of the research study. In addition, mechanistic data was gathered for the genes that encode the alpha subunit of the epithelial Na +  channel (SCNN1A, rs #2228576) and found that SCNN1A was predictive of urinary Na +  concentration and mean arterial BP. 
     Methods: 
     The BP history for patients was collected and the current BP levels were measured in patients with controlled hypertension. DNA was collected using a buccal swab and analyzed the genes within the Geneticure panel. The study sought to determine if patients with “functional” genotypes of proteins important in certain drug classes responded better if they were taking the drug that would inhibit the activity of that protein. As an example, the beta-1 adrenergic receptor (ADRB1) is important in heart rate control and patients who are on a beta-blocker can demonstrate a drop in BP because of inhibition of this protein. Therefore, one would hypothesize that if a patient with a functional protein of the ADRB1 is put on a beta-blocker early, they will demonstrate better BP control (because of a greater drop in heart rate and, therefore BP). This was assessed according to 14 genes and 3 major classes of BP drugs (diuretic, vasodilator, beta-blocker) and one drug metabolizing enzyme (CYP2D6). 
     Results: Subject Characteristics 
     Demographics (n=99) 
                                             Variable   mean ± SEM                          Age (yrs)   58 ± 0.8           Sex (% female)   46           Diabetes (% with)   28 ± 4             Weight (kg)   86 ± 1.4           Height (cm)   169 ± 1             BMI (kg/m 2 )   29.9 ± 0.4                          
Results: Blood Pressure Data (n=99)
 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Variable 
                 Mean ± SEM 
               
               
                   
                   
               
             
            
               
                   
                 Initial SBP (mmHg) 
                 151 ± 2 
               
               
                   
                 Initial DBP (mmHg) 
                  91 ± 1 
               
               
                   
                 Initial MAP (mmHg) 
                 111 ± 1 
               
               
                   
                 Lowest SBP in past two years (mmHg) 
                 115 ± 1 
               
               
                   
                 Lowest DBP in past two years (mmHg) 
                  72 ± 1 
               
               
                   
                 Current SBP (mmHg) 
                 134 ± 2 
               
               
                   
                 Current DBP (mmHg) 
                  82 ± 1 
               
               
                   
                 Current MAP (mmHg) 
                  99 ± 1 
               
               
                   
                 Time to BP control (months) 
                  22 ± 10 
               
               
                   
                 Clinic Visits in the Past two years for HTN 
                  3.6 ± 0.3 
               
               
                   
                   
               
            
           
         
       
     
     Results: Current Blood Pressure Therapy Information 
     Drug Class Usage (n=99) 
                                             Variable   mean ± SEM                          Number of Classes of Drugs for HTN    1.8 ± 0.08           Diuretic (% taking)   42 ± 5           ACE Inhibitor (% taking)   62 ± 5           ARB (% taking)   27 ± 5           B-Blocker (% taking)   33 ± 5           Ca +  Channel Blocker (% taking)   16 ± 4                        
These data describe the number of different drug classes that the patients were taking. In addition, we assessed the percent of subjects who were on drugs within the vasodilator class (ACE-inhibitor and ARB), the cardiac class (B-blocker Ca +  channel blocker), and the renal class (diuretic).
 
Time to Control According to Drug Class (n=99)
 
     
       
         
           
               
               
               
            
               
                   
               
               
                   
                 Months For Control 
                 Clinic Visits/2Years 
               
            
           
           
               
               
               
               
               
            
               
                   
                 On the 
                 Not on the 
                 On the 
                 Not on the 
               
               
                 Drug Class 
                 Drug Class 
                 Drug Class 
                 Drug Class 
                 Drug Class 
               
               
                   
               
               
                 Diuretic 
                 39.5 ± 20.4 
                 7.9 ± 4.2 
                 4.5 ± 0.6 
                 3.0 ± 0.4* 
               
               
                 ACE Inhibitor 
                 22.27 ± 11.4  
                 22.5 ± 16.4 
                 3.1 ± 0.4 
                 4.5 ± 0.6* 
               
               
                 Antiotensin  
                 32.8 ± 23.1 
                 17.1 ± 9.1  
                 3.9 ± 0.6 
                 3.5 ± 0.4  
               
               
                 Receptor Blocker 
                   
                   
                   
                   
               
               
                 B-Blocker 
                 24.5 ± 16.9 
                 21.2 ± 12.0 
                 4.9 ± 0.7 
                 3.1 ± 0.4* 
               
               
                 Ca+ Channel  
                 9.9 ± 4.5 
                 25.0 ± 11.7 
                 5.1 ± 0.7 
                 3.3 ± 0.4  
               
               
                 Blocker 
               
               
                   
               
               
                 *p &lt; 0.05 compared to those patients who were on the class of drugs 
               
            
           
         
       
     
     These data describe the time it took for BP control according to which class of drugs the patient was taking. While there are no significant differences in months taken for BP control according to drug class, there was an effect of number of clinic visits (specific to hypertension) within the past 2 years according to drug class. Patients using beta-blockade and diuretic therapy to control their BP had fewer clinic visits, when compared to those patients not on these therapies. Patients on an ACE-inhibitor had significantly more clinic visits per year, when compared to patients not on this therapy. 
     Blood Pressure Control According to Genotypes (n=86)
 
1. Genes Important in Renal Na +  Handling (and Those that are Differentially Responsive to Diuretic Therapy).
 
     
       
         
           
               
            
               
                   
               
               
                 WNK1 (RS# 1159744) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy (Diuretic) 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 GG 
                 C•containing 
                 GG 
                 C•containing 
               
               
                   
               
               
                 n 
                 26 
                 24 
                 19 
                 15 
               
               
                 Systolic Blood  
                 133.7 ± 3.2  
                 133.5 ± 2.3  
                 137.8 ± 4.1  
                 132.1 ± 5.2  
               
               
                 Pressure  
                   
                   
                   
                   
               
               
                 (mmHg) 
                   
                   
                   
                   
               
               
                 Diastolic Blood 
                 79.7 ± 2.3 
                 84.6 ± 2.0 
                 88.7 ± 2.7* 
                 79.5 ± 3.7 
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 Mean Arterial  
                 97.7 ± 2.4 
                 100.9 ± 1.81 
                 105.1 ± 2.7*  
                 97.1 ± 3.6 
               
               
                 Blood Pressure  
                   
                   
                   
                   
               
               
                 (mmHg) 
                   
                   
                   
                   
               
               
                 Months to BP  
                  3.6 ± 1.4 
                  4.8 ± 2.6 
                 8.2 ± 5.6 
                 16.5 ± 6.2 
               
               
                 Control 
               
               
                   
               
               
                 *P &lt; 0.05 compared to same genotype not on target therapy. 
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 SLC12A3 (RS# 1529927) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy (Diuretic) 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 GG 
                 C•containing 
                 GG 
                 C•containing 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 n 
                 45 
                 6 
                 33 
                 2 
               
               
                 Systolic Blood  
                 134.2 ± 2.31  
                 136.8 ± 5.9  
                 136.4 ± 3.4  
                 128.0 ± 8    
               
               
                 Pressure  
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Diastolic Blood  
                 81.8 ± 1.7 
                 86.5 ± 2.3 
                 85.9 ± 2.5 
                 75.0 ± 5.0 
               
               
                 Pressure  
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Mean Arterial  
                 99.3 ± 1.7 
                 103.3 ± 3.1  
                 102.7 ± 2.4  
                 92.3 ± 6.0 
               
               
                 Blood Pressure  
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Months to  
                  2.5 ± 0.7 
                 17.7 ± 7   
                 10.5 ± 3.9 
                 42 
               
               
                 BP Control 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 WNK1 (RS# 2107614) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 GG 
                 C•containing 
                 G 
                 C•containing 
               
               
                   
               
               
                 n 
                 9 
                 41 
                 7 
                 27 
               
               
                 Systolic Blood  
                 130.3 ± 5.6  
                 134.4 ± 2.1  
                 147.7 ± 7.9  
                 132.1 ± 3.3  
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 (mm Hg)  
                   
                   
                   
                   
               
               
                 Diastolic 
                   
                   
                   
                   
               
               
                 Diastolic  
                 80.4 ± 4.7 
                 82.4 ± 1.6 
                 84.6 ± 4   
                 84.7 ± 2.8 
               
               
                 Blood  
                   
                   
                   
                   
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Mean Arterial  
                 97.1 ± 4.6 
                 99.7 ± 1.6 
                 106 ± 4.4 
                 100.5 ± 2.6  
               
               
                 Blood 
                   
                   
                   
                   
               
               
                 Pressure  
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Months to  
                 10.5 ± 7.2 
                  2.6 ± 0.9 
                 10.5 ± 9.5 
                 13.1 ± 4.8 
               
               
                 BP Control 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 Alpha Adducin (RS# 4961) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 GG 
                 T•containing 
                 GG 
                 T•containing 
               
               
                   
               
               
                 n 
                 40 
                 11 
                 24 
                 11 
               
               
                 Systolic  
                 134.2 ± 2.3  
                 135.0 ± 5.3  
                 135.3 ± 3.9  
                 137.4 ± 5.9  
               
               
                 Blood  
                   
                   
                   
                   
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Diastolic  
                 83.1 ± 1.8 
                 79.7 ± 3.3 
                 85.8 ± 2.9 
                 84.2 ± 4.3 
               
               
                 Blood  
                   
                   
                   
                   
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Mean  
                 100.2 ± 1.8  
                 98.2 ± 3.6 
                 102.3 ± 2.7  
                 101.9 ± 4.5  
               
               
                 Arterial  
                   
                   
                   
                   
               
               
                 Blood 
                   
                   
                   
                   
               
               
                 Pressure  
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Months to  
                  4.4 ± 1.6 
                   3.6 ± 15.1 
                 10.5 ± 5.2 
                 15.1 ± 6.7 
               
               
                 BP Control 
               
               
                   
               
            
           
         
       
     
     Of the four genes explored in the clinical study RS #1159744 (the gene that encodes cytoplasmic serine-threonine kinase that is expressed in the kidney, WNK-1) was most predictive of response to therapy. Patients with the C genotype of WNK-1 had the best response to therapy demonstrating 8 mmHg lower DBP, when compared to patients with this genotype who were not on diuretic therapy. Subjects who were homozygous for G for this gene actually had a lower blood pressure if they were not on a diuretic, indicating that they may be benefiting from alternate therapy. Although just a trend (due to small sample size of the minor allele) the C polymorphism of SLC12A3 also may be predictive of response to diuretic therapy with patients demonstrating an 11 mmHg drop in DBP with therapy, compared to the G polymorphism which demonstrated a small increase in DBP with therapy. 
     In addition to this clinical BP data 24-hour urinary and resting BP data were gathered according to genetic variation of the alpha sub-unit of the epithelial Na +  channel (SCNN1A, RS #2228576). It was found that subjects homozygous for the T variant of SCNN1A demonstrated more Na +  excretion from the kidneys and they also demonstrated lower mean arterial blood pressure, when compared to genotype groups containing the A variant (See  FIGS. 8 and 9 ). 
     2. Genes Important in Cardiac Function (and Those that May Respond Differentially to Beta-Blocker Therapy).
 
The beta-1 adrenergic receptor (ADRB1) is important in controlling heart rate and cardiac contractility.
 
     
       
         
           
               
            
               
                   
               
               
                 Beta-1 Adrenergic Receptor 49 (RS# 1801252) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy (Beta-Blocker) 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Ser/  
                   
                 Ser/ 
                   
               
               
                   
                 Ser 
                 Thr•containing 
                 Ser 
                 Thr•containing 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 n 
                 1 
                 55 
                 2 
                 28 
               
               
                 Systolic  
                 137 
                 136.4 ± 2.3   
                 136 ± 6  
                 132.4 ± 3.2   
               
               
                 Blood  
                   
                   
                   
                   
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 (mm Hg)  
                   
                   
                   
                   
               
               
                 Diastolic 
                   
                   
                   
                   
               
               
                 Diastolic  
                 86 
                 85.5 ± 1.7  
                 75 ± 3 
                 80.3 ± 2.1* 
               
               
                 Blood  
                   
                   
                   
                   
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Mean  
                 103 
                 102.4 ± 1.7   
                 95 ± 0 
                 97.7 ± 2.1  
               
               
                 Arterial  
                   
                   
                   
                   
               
               
                 Blood 
                   
                   
                   
                   
               
               
                 Pressure  
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Months to  
                 N/A 
                 7.0 ± 2.4 
                 N/A 
                 9.4 ± 3.8 
               
               
                 BP Control 
               
               
                   
               
               
                 *P &lt; 0.05 compared to same genotype not on target therapy. 
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 Beta-1 Adrenergic Receptor 389 (RS# 1801253) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy (Beta-Blocker) 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 GG 
                 C•containing 
                 GG 
                 C•containing 
               
               
                   
               
               
                 n 
                 56 
                 0 
                 30 
                 0 
               
               
                 Systolic  
                 136.4 ± 2.3  
                   
                 132.6 ± 3.0   
                   
               
               
                 Blood  
                   
                   
                   
                   
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 (mm Hg)  
                   
                   
                   
                   
               
               
                 Diastolic 
                   
                   
                   
                   
               
               
                 Diastolic  
                 85.5 ± 1.7 
                   
                 80.0 ± 2.0* 
                   
               
               
                 Blood  
                   
                   
                   
                   
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Mean  
                 102.4 ± 1.7  
                   
                 97.5 ± 1.98 
                   
               
               
                 Arterial  
                   
                   
                   
                   
               
               
                 Blood 
                   
                   
                   
                   
               
               
                 Pressure  
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Months  
                  7.0 ± 2.3 
                   
                 8.8 ± 3.5 
                   
               
               
                 to BP  
                   
                   
                   
                   
               
               
                 Control 
               
               
                   
               
               
                 *P &lt; 0.05 compared to same genotype not on target therapy. 
               
            
           
         
       
     
     These data indicate a differential BP response to beta-blocker therapy according to genetic variation at position 49 of the ADRB1. Specifically, the inventors found that subjects with the Ser genotype at position 49 of ADRB1 benefit from beta-blocker therapy with an average drop in DBP of 11 mmHg, compared with a drop of 5 mmHg with Thr at this position. Therefore, although patients with the Thr polymorphism also demonstrated a drop in BP with beta-blocker therapy, the effect was most pronounced in patients with the Ser polymorphism. 
     The beta-2 adrenergic receptor (ADRB2) is important in cardiac contractility, which controls stroke volume, and can influence BP through differences in cardiac output. 
     
       
         
           
               
            
               
                   
               
               
                 Beta-2 Adrenergic Receptor 16 (RS# 1042713) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy (B-Blocker) 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Arg/Arg 
                 Gly-containing 
                 Arg/Arg 
                 Gly-containing 
               
               
                   
               
               
                 n 
                 5 
                 51 
                 3 
                 27 
               
               
                 Systolic Blood Pressure (mmHg) 
                 143.6 ± 5.9  
                 135.7 ± 2.4  
                 129 ± 5  
                 133.3 ± 3     
               
               
                 Diastolic Blood Pressure (mmHg) 
                 84.0 ± 7.4 
                 85.6 ± 1.8 
                 80.3 ± 6.6 
                 79.9 ± 2.1* 
               
               
                 Mean Arterial Blood Pressure (mmHg) 
                 104.0 ± 6.82 
                 102.3 ± 1.8  
                 96.6 ± 5.9 
                 97.6 ± 2.1  
               
               
                 Months to BP Control 
                  7 ± 5 
                    7 ± 2.6 
                  4.3 ± 3.8 
                 9.9 ± 4.3 
               
               
                   
               
               
                 *P &lt; 0.05 compared to same genotype not on target therapy. 
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 Beta-2 Adrenergic Receptor 27 (RS# 1042714) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy (B-Blocker) 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Gln/Gln 
                 Glu-containing 
                 Gln/Gln 
                 Glu-containing 
               
               
                   
               
               
                 n 
                 6 
                 48 
                 4 
                 24 
               
               
                 Systolic Blood Pressure (mmHg) 
                 142.0 ± 6.9   
                 134.4 ± 2.2  
                 125.5 ± 5.9   
                 132.7 ± 3.6  
               
               
                 Diastolic Blood Pressure (mmHg) 
                 82.5 ± 2.7  
                 85.1 ± 1.8 
                 82.5 ± 2.7  
                 79.5 ± 2.4 
               
               
                 Mean Arterial Blood Pressure (mmHg) 
                 102.3 ± 4.1   
                 101.5 ± 1.8  
                 102.3 ± 4.1   
                 97.2 ± 2.3 
               
               
                 Months to BP Control 
                 8.5 ± 7.8 
                    7 ± 2.8 
                 5.3 ± 2.3 
                  9.6 ± 4.4 
               
               
                   
               
            
           
         
       
     
     These data demonstrate a generally favorable response to beta-blocker therapy with both genotype groups. However, the Gly16 genotype demonstrated a statistically significant difference in BP control if the patients were on a beta-blocker (drop in DBP of 5 mmHg), when compared to patients with the Arg16 genotype. Generally, there is a similar pattern for a greater drop in BP with subjects who have the most functional gene that encodes the ADRB2 (Gly at position 16 and Glu at position 27). There is strong linkage disequilibrium between these two sites (amino acids 16 and 27), so the similar response between the sites is expected. 
     Observations on Genetic Variation of Cytochrome P450 2D6 (CYP2D6), which is Important in Drug Metabolism, Especially of Particular Beta-Blockers. 
     
       
         
           
               
            
               
                   
               
               
                 CYP 2D6 (RS#) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy (B-Blocker) 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 CC 
                 T-Containing 
                 CC 
                 T-Containing 
               
               
                   
               
               
                 n 
                 35 
                 22 
                 23 
                 7 
               
               
                 Systolic Blood Pressure (mmHg) 
                 140.6 ± 2.7  
                 128.5 ± 3.1   
                 133.4 ± 3.6   
                 130.0 ± 5.7   
               
               
                 Diastolic Blood Pressure (mmHg) 
                 86.0 ± 2.4 
                 83.2 ± 2.4  
                 79.2 ± 2.1* 
                 82.6 ± 5.3  
               
               
                 Mean Arterial Blood Pressure (mmHg) 
                 104.2 ± 2.3  
                 983 ± 2.4  
                 97.3 ± 2.2* 
                 98.4 ± 4.8  
               
               
                 Months to BP Control 
                  7.8 ± 2.9 
                 5.08 ± 3.7  
                 6.25 ± 3.2  
                 16.5 ± 10.8 
               
               
                   
               
               
                 *P &lt; 0.05 compared to same genotype not on target therapy. 
               
            
           
         
       
     
     These data demonstrate that the CC homozygous group of CYP2D6 demonstrates the greatest response to beta-blocker therapy, when compared to the CT and TT groups. Patients with the CC polymorphism had demonstrated a 6 mmHg lower DBP and a 7 mmHd lower MAP when on beta-blocker therapy, compared to this genotype not on beta-blocker therapy. In contrast, patients in the T-containing group (those with the CT and TT genotypes) did not respond to beta-blocker therapy. 
     3. Genes Important in Vascular Function (and Those that May Respond Differentially to Vasodilator Therapy). 
     The following are observations on the genetic variation of the angiotensin gene (encoding a precursor to angiotensin-II, a potent vasoconstrictor, which is converted via angiotensin converting enzyme, ACE) and the responses to various therapies. 
     
       
         
           
               
            
               
                   
               
               
                 Angiotensin (RS# 699) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy (Angiotensin Receptor Blocker)  
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 CC 
                 T-containing 
                 CC 
                 T-containing 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 n 
                 15 
                 50 
                 4 
                 17 
               
               
                 Systolic Blood Pressure (mmHg) 
                 135.3 ± 2.6  
                 135.1 ± 2.6   
                 135.0 ± 7.4  
                 135.1 ± 4.5  
               
               
                 Diastolic Blood Pressure (mmHg) 
                 89.5 ± 2.9 
                 82.6 ± 1.7  
                 77.5 ± 7.8 
                 82.7 ± 3.4 
               
               
                 Mean Arterial Blood Pressure (mmHg) 
                 104.7 ± 2.6  
                 100.1 ± 1.7   
                 96.6 ± 7.5 
                 100.2 ± 3.3  
               
               
                 Months to BP Control 
                  2.7 ± 1.3 
                 6.7 ± 2.7 
                 12 
                 12.4 ± 4.8 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 Angiotensin (RS# 699) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy (ACE-Inhibitor) 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 CC 
                 T-containing 
                 CC 
                 T-containing 
               
               
                   
               
               
                 n 
                 6 
                 24 
                 13 
                 43 
               
               
                 Systolic Blood Pressure (mmHg) 
                 131.7 ± 5.3  
                 133.7 ± 3.5   
                 136.8 ± 2.7   
                 135.8 ± 2.9   
               
               
                 Diastolic Blood Pressure (mmHg) 
                 78.5 ± 5.0 
                 80.4 ± 2.1  
                 90.8 ± 3.2* 
                 83.9 ± 2.0  
               
               
                 Mean Arterial Blood Pressure (mmHg) 
                 96.2 ± 4.7 
                 98.2 ±      
                 106.2 ± 2.8*  
                 101.1 ± 2.0   
               
               
                 Months to BP Control 
                  6.7 ± 3.1 
                 7.7 ± 3.2 
                 2.3 ± 1.7 
                 9.9 ± 3.8 
               
               
                   
               
               
                 *P &lt; 0.05 compared to same genotype not on target therapy 
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 All Receptor Type-1 (RS# 5186) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy (Angiotensin Receptor Blocker) 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 AA 
                 C-containing 
                 AA 
                 C-containing 
               
               
                   
               
               
                 n 
                 39 
                 28 
                 9 
                 11 
               
               
                 Systolic Blood Pressure (mmHg) 
                 137.3 ± 2.6  
                 131.0 ± 3.1  
                 1.363 ± 4.6  
                 134.1 ± 6.5  
               
               
                 Diastolic Blood Pressure (mmHg) 
                 85.4 ± 2.1 
                 80.9 ± 2.0 
                 87.6 ± 3.9 
                 77.9 ± 4.5 
               
               
                 Mean Arterial Blood Pressure (mmHg) 
                 102.7 ± 2.0  
                 97.6 ± 2.1 
                 103.8 ± 2.9  
                 96.6 ± 4.9 
               
               
                 Months to BP Control 
                  7.3 ± 2.9 
                  3.8 ± 2.5 
                 13.4 ± 7.8 
                 11.9 ± 5.7 
               
               
                   
               
            
           
         
       
     
     These data indicate that patients homozygous for the C genotype of angiotensin may benefit from an angiotensin receptor blocker (ARB). When on an ARB, patients with the CC genotype demonstrated a 12 mmHg lower DBP when compared to patients with this genotype who were not on this therapy. In contrast, patients in the T-containing group (those with the CT or TT genotypes) did not show a response to ARB therapy. Furthermore, inhibition of ACE (which converts angiotensin-I to angiotensin-II) results in higher BP levels, possibly due to a “build-up” of angiotensin. Therefore, these data indicate that patients homozygous for C should be given an angiotensin receptor blocker with an ACE inhibitor. 
     Angiotensin Converting Enzyme (ACE) Genotype and ACE-Inhibition 
     
       
         
           
               
            
               
                   
               
               
                 ACE (RS# 1799752) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy (ACE-Inhibition) 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Ins/Ins 
                 Del•Containing 
                 Ins/Ins 
                 Del•Containing 
               
               
                   
               
               
                 n 
                 3 
                 27 
                 12 
                 47 
               
               
                 Systolic  
                 142.7 ± 11.1 
                 132.3 ± 3.1  
                 129.5 ± 3.5   
                 137.1 ± 2.6   
               
               
                 Blood  
                   
                   
                   
                   
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Diastolic  
                 79.6 ± 6.1 
                 80.0 ± 2.1 
                 80.4 ± 14.4 
                 85.7 ± 1.9* 
               
               
                 Blood  
                   
                   
                   
                   
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Mean  
                 100.7 ± 7.7  
                 97.4 ± 2.1 
                 96.8 ± 3.8  
                 102.8 ± 1.8   
               
               
                 Arterial  
                   
                   
                   
                   
               
               
                 Blood 
                   
                   
                   
                   
               
               
                 Pressure  
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Months to  
                  4.7 ± 3.7 
                 8.0 ± 3  
                 7.4 ± 5.8 
                 7.3 ± 3.0 
               
               
                 BP Control 
               
               
                   
               
               
                 *P &lt; 0.05 compared to same genotype not on target therapy. 
               
            
           
         
       
     
     These data indicate that those with the Insertion polymorphism (Ins) of the ACE gene will respond best to ACE-inhibition. Patients with the Del polymorphism actually demonstrated higher DBP with ACE-inhibition, when compared to this patient group not on ACE-inhibitors. 
     Observations on Renin Genotype and Angiotensin Receptor Blocker 
     Renin is a precursor to angiotensin and, therefore, patients with a functional genotype of renin may benefit from Angiotensin Receptor Blocker (ARB) therapy because a more functional genotype can lead to greater angiotensin levels which can result in high BP. 
     
       
         
           
               
            
               
                   
               
               
                 Renin (RS# 12750834) 
               
            
           
           
               
               
            
               
                   
                 On Target Therapy 
               
               
                   
                 (Angiotensin Receptor Blocker) 
               
            
           
           
               
               
               
            
               
                   
                 No 
                 Yes 
               
            
           
           
               
               
            
               
                   
                 Genotype 
               
            
           
           
               
               
               
               
               
            
               
                   
                 G/G 
                 A•containing 
                 G/G 
                 A•containing 
               
               
                   
               
               
                 n 
                 48 
                 17 
                 14 
                 7 
               
               
                 Systolic  
                 134.8 ± 2.3  
                 135.9 ± 4.4  
                 136.4 ± 4.6   
                 132.2 ± 7.3   
               
               
                 Blood  
                   
                   
                   
                   
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Diastolic  
                 83.8 ± 1.7 
                 85.1 ± 2.9 
                 82.0 ± 2.9  
                 81.0 ± 7.4  
               
               
                 Blood  
                   
                   
                   
                   
               
               
                 Pressure 
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Mean  
                 100.8 ± 1.7  
                 102.1 ± 2.9  
                 100.2 ± 3.2   
                 98.1 ± 6.6  
               
               
                 Arterial  
                   
                   
                   
                   
               
               
                 Blood 
                   
                   
                   
                   
               
               
                 Pressure  
                   
                   
                   
                   
               
               
                 (mm Hg) 
                   
                   
                   
                   
               
               
                 Months to  
                 4.75 ± 1.9 
                  8.0 ± 5.8 
                 15.5 ± 5.3* 
                 1.3 ± 0.7 
               
               
                 BP Control 
               
               
                   
               
               
                 *P &lt; 0.05 compared to same genotype not on target therapy. 
               
            
           
         
       
     
     These data indicate that the functional genotype of renin (A) may benefit from ARB therapy. Specifically, the BP response to therapy was not significant, however, the response to therapy time was pronounced. Patients who have the functional genotype of renin (the AG and AA genotype groups) demonstrate a much shorter time to BP control, when compared to patients within this group who do not take this therapy. In contrast, patients in the GG group demonstrate a longer time to control if they take this therapy, possibly due to greater response to another class of drugs. 
       FIG. 10  is a flowchart of a method 1000 for providing a hypertension treatment recommendation in accordance with some embodiments. In operation  1010 , a tissue sample is input into a machine for assay to obtain genotype data. In some examples, patient DNA, primers, reagents and PCR polymerase are loaded into an MJ Tetrad thermoplexer (e.g., a MJ Research Tetrad 2 instrument available from GMI headquartered in Ramsey, Minn.) and the desired genotype SNPs are amplified to yield gene fragment material. This gene fragment material is loaded into, for example an Athena MassArray machine (available from CD Genomics of Shirley, N.Y.), which identifies and quantifies the amplified gene material. This result provides the patient&#39;s genotype profile for the 14 SNP sequences of interest. 
     In operation  1020 , gene sequences can be determined. These can include, for example: 
     1. gene sequences having cardiac effect:
         a. an ADRB2 nucleic acid with a guanine at the variable position of rs1042713 (hereinafter ADRB2_16), which predicts non-selective beta-blocker use;   b. an ADRB2 nucleic acid with a guanine at the variable position of rs1042714 (hereinafter ADRB2_27), which predicts a non-selective beta-blocker use;   c. an ADRB1 nucleic acid with an adenine at the variable position of rs1801252 (hereinafter ADRB1_49), which predicts response to a beta blocker;   d. an ADRB1 nucleic acid with a cytosine at the variable position of rs1801253 (hereinafter ADRB1_389), which predicts response to a beta blocker; and   e. a CYP2D6 nucleic acid with an adenine at the variable position of Rs3892097 (hereinafter CYP2D6).       

     2. gene sequences having renal effect:
         a. a WNK1 nucleic acid with a cytosine at the variable position of rs1159744 (hereinafter WNK1(a), which predicts response to thiazide diuretic;   b. a SLC12A3 nucleic acid with a thymine at the variable position of rs1529927 (hereinafter SLC12A3(2));   c. a WNK1 nucleic acid with a cytosine at the variable position of rs2107614 (hereinafter WNK1(b)), which predicts response to thiazide diuretic;   d. a SCNN1A nucleic acid with an adenine at the variable position of rs2228576, which predicts response to diuretic (hereinafter SCNNIA);   e. WNK1(c) at position rs2277869, which predicts response to a thiazide diuretic (hereinafter WNK1(c)); and   f. an ADD1 nucleic acid with a thymine at the variable position of rs4961 (hereinafter Alpha Adducin), which predicts response to diuretic therapy.       

     3. gene sequences having vascular effect:
         a. a renin nucleic acid with a cytosine at the variable position of rs12750834 (hereinafter Renin), which predicts response to vasidilating agents;   b. a first Angiotensin (rs5051, hereinafter Angiotensin(1));   c. an AGT1R nucleic acid with a cytosine at the variable position of rs5186 (hereinafter A-II Receptor);   d. an AGT nucleic acid with a cytosine at the variable position of rs699 (hereinafter Angiotensin(2));   e. an ACE nucleic acid with a deletion in rs1799752 (hereinafter ACE); and   f. a third Angiotensin (rs7079, hereinafter Angiotensin(3)).       

     In operation  1030  and  1040 , SNP scoring and weighting can be performed, and a treatment recommendation can be provided, using the algorithm described with respect to  FIG. 11 . 
       FIG. 11  is a flowchart of an algorithm  1100  for providing a hypertension treatment recommendation based on a gene assay in accordance with some embodiments. At least some operations of algorithm  1100  can be performed by machine  1200  ( FIG. 12 ) or by a component thereof such as processing circuitry  1202 , or instructions  1224  stored thereon or on main memory  1204 , static memory  1206 , machine-readable medium  1222 , etc. 
     In operation  1110 , each single nucleotide polymorphism (SNP) is assigned a weighted score. It will be appreciated from above that each gene relates to one area or site that corresponds to an organ or system, e.g., cardiac, vascular, and renal. Each single nucleotide polymorphism (SNP) is given a score for functionality (“functional score”, or “recommendation score”). This score is on a scale of 1 (not functional) to 10 (very functional) and values are assigned as 1, 5, and 10, as the level of moderate functional cannot be further fine-tuned beyond a score of 5, given the complexity of what is considered somewhat functional. A functional genotype is one that has demonstrated enhanced receptor function, altered drug metabolism, greater protein expression, or higher channel activity, depending on the genotype. In addition, the functional genotype has, in most cases, been shown to be associated with greater drug responsiveness for a given drug class (i.e. if it is a Na +  channel the enhanced drug response is that of a diuretic). 
     Each gene has a weight that can be based on, for example, the number of scholarly articles related to that gene, the findings of those articles, the quality of those articles (based e.g., on level of review), etc. For example, the inventors have implemented a review that determined that some of the genotypes have considerable scientific alignment with hundreds of studies and almost all studies are in agreement with the degree and direction of functionality for a given genotype. In contrast, some of the genotypes in the panel are more novel and have fewer research studies with some mixed results on degree and direction of functionality. Based on this, the inventors provide a scoring system that aligns the functionality with the number and level of scientific agreement in the previous literature, this is the “weight” score and is on a continuous scale of 1-10. 
     A weighted score assigned therefore equals a recommendation score times the weight, where example data for assigning each of these weights is shown below in Table 12. This data can be stored remotely or on machine  1200 . It will be appreciated that the below is merely example data and embodiments can include additional data (e.g., additional genes, etc.) or different values for the below data (e.g., different weights can be assigned, or other parameters can be provided). It will be noted that “rs” denotes a reference SNP cluster ID (a universal way to identify SNPs), which is the genotype nucleotide fragment with the nucleotide variation that for example generates a trait. 
     “rs1042713”: {“name”: “ADRB2_16”, “varname”: “adrb2_16”, “area”: “cardiac”, “rs”: “rs1042713”. “weight”: 9,}, 
     
       
         
           
               
             
               
                 TABLE 12 
               
               
                   
               
               
                 SNP names and weights. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                  “rs1042714″: {″name″: ″ADRB2_27″, ″varname″: ″adrb2_27″, ″area″: 
               
               
                 ″cardiac″, ″rs″: ″rs1042714″, ″weight″: 8, }, 
               
               
                  ″rs1801252″: {″name″: ″ADRB1_49″, ″varname″: ″adrb1_49″, ″area″: 
               
               
                 ″cardiac″, ″rs″: ″rs1801252″, ″weight″: 9.5,}, 
               
               
                  ″rs1801253″: {″name″: ″ADRB1_389″, ″varname″: ″adrb1_389″, ″area″: 
               
               
                 ″cardiac″, ″rs″: ″rs1801253″, ″weight″: 10, }, 
               
               
                  ″rs3892097″: {″name″: ″CYP2d6″, ″varname″: ″metabolism″, ″area″: 
               
               
                 ″cardiac″, ″rs″: ″rs3892097″, ″weight″: 9,}, 
               
               
                  ″rs4961″: {″name″: ″Alpha Adducin″, ″varname″: ″alpha_adducin″, ″area″: 
               
               
                 ″renal″, ″rs″: ″rs4961″, ″weight″: 9}, 
               
               
                  ″rs1159744″: {″name″: ″WNK1(a)″, ″varname″: ″wnk1_a″, ″area″: 
               
               
                 ″renal″, ″rs″: ″rs1159744″,″weight″: 7,}, 
               
               
                  ″rs1529927″: {″name″: ″SLC12A3(2)″,″varname″: ″slc12a3″,″area″: 
               
               
                 ″renal″, ″rs″: ″rs1529927″,″weight″: 5,}, 
               
               
                  ″rs2107614″: {″name″: ″WNK1(b)″,″varname″: ″wnk1_b″, ″area″: 
               
               
                 ″renal″, ″rs″: ″rs2107614″,″weight″: 7,}, 
               
               
                  ″rs2228576″: {″name″: ″SCNN1A″, ″varname″: ″scnn1a″, ″area″: 
               
               
                 ″renal″,″rs″: ″rs2228576″,″weight″: 5,}, 
               
               
                  ″rs2277869″: {″name″: ″WNK1(c)″,″varname″: ″wnk1_c″, ″area″: 
               
               
                 ″renal″, ″rs″: ″rs2277869″,″weight″: 8,}, 
               
               
                  ″rs699″: {″name″: ″Angiotensin(2)″, ″varname″: ″angiotensin_2″, ″area″: 
               
               
                 ″vascular″,″rs″: ″rs699″,″weight″: 7,}, 
               
               
                  ″rs5051″: {″name″: ″Angiotensin(1)″,″varname″: angiotensin_1″, ″area″: 
               
               
                 ″vascular″,″rs″: ″rs5051″,″weight″: 7,}, 
               
               
                  ″rs5186″: {″name″: ″A:II Receptor″, ″varname″: ″a_ii″,″area″: 
               
               
                 ″vascular″,″rs″: ″rs5186″,″weight″: 8,}, 
               
               
                  ″rs7079″: {″name″: ″Angiotensin(3)″,″varname″: ″angiotensin_3″, ″area″: 
               
               
                 ″vascular″, ″rs″: ″rs7079″,″weight″: 5,}, 
               
               
                  rs1799752″: {″name″: ″ACE″,″varname″: ″ace″,″area″: 
               
               
                 ″vascular″,″rs″: ″rs1799752″, ″weight″: 8,}, 
               
               
                  ″rs12750834″: {″name″: ″Renin″,″varname″: ″renin″,″area″: 
               
               
                 ″vascular″,″rs″: ″rs12750834″,″weight″: 6,} 
               
               
                   
               
            
           
         
       
     
     The recommendation score is calculated based on homozygosity versus heterozygosity: In some examples, individuals who are homozygous for the functional genotype (SNP) are always given a score of 10 for that genotype and individuals who are homozygous for a non-functional genotype are given a score of 1 for that genotype. In some examples, the heterozygous condition is sometimes semi-functional, sometimes non-functional, and sometimes fully-functional. The genotype combinations (at one site, i.e. GC, GG, or CC) may be given a score based on the genetic pair in which non-functional condition (homozygous for the non-functional genotype or heterozygous condition has demonstrated that this is most in-line with the non-functional genotype) is given a score of 1, semi-functional condition (heterozygous and this has demonstrated moderate functionality) is given a score of 5, and the fully-functional condition (homozygous for the functional genotype or heterozygous condition has demonstrated that this is most in-line with the functional genotype) is given a score of 10. 
     In operation  1120 , values are calculated for a number of variables based on the weighted scores generated in operation  1110 , to assess which organ system (cardiac, renal, or vascular) is the most functional, and will therefore likely lead to best drug response, based on the average scoring for that organ system (individual genotypes averaged for a given organ system). In some examples, there is a minimum threshold of 40 for an organ system&#39;s individual genotype averages that must be met in order for that organ system to default to the top drug class choice. If two or more organ systems have a score greater than 40 then the highest number defaults as the top choice. If no organ system meets the threshold of 40, then individual genotypes and haplotypes (genotype combinations that have been deemed particularly functional) are used to determine drug class choices. 
     Once a first drug class choice has been selected then a second drug class can be determined using the same algorithm (genotype average &gt;40 but less than first drug class will determine next choice, if not &gt;40 then SNP scoring and haplotype combinations determine next choice). Once a second-choice drug class has been selected, the scoring of the algorithm occurs again for a third, and so on until all drug classes have been selected. 
     For the cardiac and vascular systems there are two drug class choices (selective vs non-selective 1-blocker for the cardiac, and angiotensin-converting enzyme (ACE) inhibitor vs angiotensin-II receptor blocker for the vascular system). When the cardiac and vascular organ systems are selected as functional, then scoring of the SNPs occurs to determine if a patient will respond to specific drug class within that organ system. 
     For the cardiac system, the algorithm is used to determine if a patient will respond to a selective vs non-selective β-blocker (based on the functional genotypes of the β 1  adrenergic receptor, which directs towards selective, vs β 2  adrenergic receptor, which directs towards non-selective). Because calcium channel blockers physiologically act like beta-blockers (decreasing cardiac output), we have written in the algorithm that if a patient is likely to respond to a β-blocker, but has a co-morbidity that would exclude such treatment, that a clinician should consider a calcium-channel blocker. When the vascular system is determined to be the functional organ system the algorithm is used to determine if they will respond best to an ACE inhibitor vs angiotensin-II receptor blocker based on the functional genotypes of ACE and the angiotensin receptor. Within the cardiac system a patient can only be responsive to a selective or a non-selective b-blocker (meaning if one is selected then the other cannot be selected further down the treatment algorithm). In contrast, a patient can have a choice of ACE-inhibitor followed by a choice of angiotensin-II receptor blocker, or vice versa, because these drug classes are more independent. 
     Some variables include those listed below in Table 13. Variables make reference to SNP varnames provided above in Table 12. 
     
       
         
           
               
             
               
                 TABLE 13 
               
               
                   
               
               
                 variable calculations. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                  {″varname″: ″cardiac_sum″,″eval″: ″$[′adrb2_16′][′wt′] + $[′adrb2_27′][′wt′] 
               
               
                 + $[′adrb1_49′][′wt′] + $[′adrb1_389′][′wt′] + $[′metabolism′][′wt′]″ }, 
               
               
                  {″varname″: ″cardiac_average″,″eval″: ″round($[′cardiac_sum′] / 5)″}, 
               
               
                  {″varname″: ″adrb1_sum″,″eval″: ″$[′adrb1_49′][′wt′] +[′adrb1_389′][′wt′]″}, 
               
               
                  {″varname″: ″adrb2_sum″,″eval″: ″$[′adrb2_16′][′wt′] +$[′adrb2_27′][′wt′]″}, 
               
               
                  {″varname″: ″adrb1_adrb2_ratio″,″eval″: ″($[′adrb1_49′][′score′] + 
               
               
                 $[′adrb1_389′][′score′]) / ($[′adrb2_16′][′score′] + $[′adrb2_27′][′score′])″}, 
               
               
                  {″varname″: ″cardiac_functional_limit″,″eval″: 50}, 
               
               
                  {″varname″: ″cardiac_is_functional″, ″eval″: ″$[′cardiac_average′] &gt; 
               
               
                 [′cardiac_functional_limit′]″}, 
               
               
                  {″varname″: ″vascular_sum″, ″eval″: ″$[′angiotensin_1′][′wt′] + 
               
               
                 [′angiotensin_2′][′wt′] + $[′angiotensin_3′][′wt′] + $[′a_ii′][′wt′] + $[′ace′][′wt′] + 
               
               
                 $[′renin′][′wt′]″}, 
               
               
                  {″varname″: ″vascular_average″,″eval″: ″round($[′vascular_sum′] / 6)″}, 
               
               
                  {″varname″: ″angiotensin_sum″,″eval″: ″$[′angiotensin_1′][′wt′] + 
               
               
                 $[′angiotensin_2′][′wt′] + $[′angiotensin_3′][′wt′],″}, 
               
               
                  {″varname″: ″angiotensin_scores_sum″,″eval″: ″$[′angiotensin_1′][′score′] + 
               
               
                 $[′angiotensin_2′][′score′] + $[′angiotensin_3′][′score′]″}, 
               
               
                  {″varname″: ″vascular_functional_limit″,″eval″: 50}, 
               
               
                  {″varname″: ″vascular_is_functional″,″eval″: ″$[′vascular_average′] &gt; 
               
               
                 $[′vascular_functional_limit′]″}, 
               
               
                  {″varname″: ″renal_sum″,″eval″: ″$[′alpha_adducin′][′wt′] + 
               
               
                 $[′wnk1_a′][′wt′] + $[′slc12a3′][′wt′] + $[′wnk1_b′][′wt′] + $[′scnn1a′][′wt′] + 
               
               
                 $[′wnk1_c′][′wt′]″}, 
               
               
                  {″varname″: ″renal_average″, ″eval″: ″round($[′renal_sum′] / 6)″}, 
               
               
                  {″varname″: ″wnk_sum″,″eval″: ″$[′wnk1_a′][′wt′] + $[′wnk1_b′][′wt′] + 
               
               
                 $[′wnk1_c′][′wt′]″}, 
               
               
                  {″varname″: ″renal_functional_limit″,″eval″: 50}, 
               
               
                  {″varname″: ″renal_is_functional″,″eval″: ″$[renal_average′] &gt; 
               
               
                 $[′renal_functional_limit′]″}, 
               
               
                  {″varname″: ″bb_selective_list″,″condition″: ″$[′metabolism′][′score′] &gt;= 
               
               
                 5″,″eval″: ″′ (metoprolol or nebivolol)′″}, 
               
               
                  {″varname″: ″bb_nonselective_list″, ″condition″: ″$[′metabolism′][′score′] 
               
               
                 &gt;= 5″, ″eval″: ″′ (timolol, alprenolol, carvedilol, or propranolo)″′}, 
               
               
                  {″varname″: ″bb_dose″,″eval″: ″[′normal′, ′moderate′, 
               
               
                 ′low′][round($[′metabolism′][′score′] / 5)]″} 
               
               
                   
               
            
           
         
       
     
     The variables cardiac_average, cardiac_sum, vascular_sum, vascular_average, renal_sum, and renal_average, are calculated so that recommendations can be made based on a descending order of those averages. The variables cardiac_functional_limit, cardiac_is_functional, vascular_functional_limit, vascular_is_functional, renal_functional_limit, and renal_is_functional are calculated to determine whether functional or non-functional recommendations should be provided for that organ. Other variables may be calculated because the inventors have determined that threshold values for those variables indicate that a particular recommendation should be made. 
     In operation  1130 , recommendations are provided. These can be provided on a user display or in a printout, using components of the example machine  1200 . 
       FIG. 12  illustrates a block diagram of an example machine  1200  upon which any one or more of the techniques (e.g., methodologies) discussed herein may perform. In alternative aspects, the machine  1200  may operate as a standalone device or may be connected (e.g., networked) to other machines. In a networked deployment, the machine  1200  may operate in the capacity of a server machine, a client machine, or both in server-client network environments. In an example, the machine  1200  may act as a peer machine in peer-to-peer (P2P) (or other distributed) network environment. The machine  1200  may be, or be a part of, a communications network device, a cloud service, a personal computer (PC), a tablet PC, a personal digital assistant (PDA), a mobile telephone, a smart phone, or any machine capable of executing instructions (sequential or otherwise) that specify actions to be taken by that machine. In some aspects, the machine  1200  may be configured to implement a portion of the methods discussed herein. Further, while only a single machine is illustrated, the term “machine” shall also be taken to include any collection of machines that individually or jointly execute a set (or multiple sets) of instructions to perform any one or more of the methodologies discussed herein, such as cloud computing, software as a service (SaaS), other computer cluster configurations. 
     Examples, as described herein, may include, or may operate on, logic or a number of components, modules, or mechanisms. Modules are tangible entities (e.g., hardware) capable of performing specified operations and may be configured or arranged in a certain manner. In an example, circuits may be arranged (e.g., internally or with respect to external entities such as other circuits) in a specified manner as a module. In an example, the whole or part of one or more computer systems (e.g., a standalone, client or server computer system) or one or more hardware processors may be configured by firmware or software (e.g., instructions, an application portion, or an application) as a module that operates to perform specified operations. In an example, the software may reside on a machine readable medium. In an example, the software, when executed by the underlying hardware of the module, causes the hardware to perform the specified operations. 
     Accordingly, the term “module” or “engine” is understood to encompass a tangible entity, be that an entity that is physically constructed, specifically configured (e.g., hardwired), or temporarily (e.g., transitorily) configured (e.g., programmed) to operate in a specified manner or to perform part, or all of any operation described herein. Considering examples in which modules are temporarily configured, each of the modules need not be instantiated at any one moment in time. 
     For example, where the modules comprise a general-purpose hardware processor configured using software, the general-purpose hardware processor may be configured as respective different modules at different times. Software may accordingly configure a hardware processor, for example, to constitute a module at one instance of time and to constitute a different module at a different instance of time. A module or engine can be implemented using processing circuitry configured to perform the operations thereof. 
     Machine (e.g., computer system)  1200  may include a hardware processing circuitry  1202  (e.g., a central processing unit (CPU), a graphics processing unit (GPU), a hardware processor core, or any combination thereof), a main memory  1204  and a static memory  1206 , some or all of which may communicate with each other via an interlink (e.g., bus)  1208 . The machine  1200  may further include a display unit  1210 , an alphanumeric input device  1212  (e.g., a keyboard), and a user interface (UI) navigation device  1214  (e.g., a mouse). In an example, the display unit  1210 , input device  1212  and UI navigation device  1214  may be a touch screen display. The machine  1200  may additionally include a storage device (e.g., drive unit)  1216 , a signal generation device  1218  (e.g., a speaker), a network interface device  1220 , and one or more sensors  1220 , such as a global positioning system (GPS) sensor, compass, accelerometer, or another sensor, or an input for receiving tissues samples as described earlier herein. The machine  1200  may include an output controller  1228 , such as a serial (e.g., universal serial bus (USB), parallel, or other wired or wireless (e.g., infrared (IR), near field communication (NFC), etc.) connection to communicate or control one or more peripheral devices (e.g., a printer, card reader, etc.). 
     The storage device  1216  may include a machine readable medium  1222  on which is stored one or more sets of data structures or instructions  1224  (e.g., software) embodying or utilized by any one or more of the techniques or functions described herein. The instructions  1224  may also reside, completely or at least partially, within the main memory  1204 , within static memory  1206 , or within the hardware processing circuitry  1202  during execution thereof by the machine  1200 . In an example, one or any combination of the hardware processing circuitry  1202 , the main memory  1204 , the static memory  1206 , or the storage device  1216  may constitute machine readable media. 
     While the machine readable medium  1222  is illustrated as a single medium, the term “machine readable medium” may include a single medium or multiple media (e.g., a centralized or distributed database, and/or associated caches and servers) configured to store the one or more instructions  1224 . 
     The term “machine readable medium” may include any medium that is capable of storing, encoding, or carrying instructions for execution by the machine  1200  and that cause the machine  1200  to perform any one or more of the techniques of the present disclosure, or that is capable of storing, encoding or carrying data structures used by or associated with such instructions. Non-limiting machine-readable medium examples may include solid-state memories, and optical and magnetic media. Specific examples of machine-readable media may include: non-volatile memory, such as semiconductor memory devices (e.g., Electrically Programmable Read-Only Memory (EPROM). Electrically Erasable Programmable Read-Only Memory (EEPROM)) and flash memory devices; magnetic disks, such as internal hard disks and removable disks; magneto-optical disks; Random Access Memory (RAM); Solid State Drives (SSD); and CD-ROM and DVD-ROM disks. In some examples, machine readable media may include non-transitory machine-readable media. In some examples, machine readable media may include machine readable media that is not a transitory propagating signal. 
     The instructions  1224  may further be transmitted or received over a communications network  1226  using a transmission medium via the network interface device  1220 . The machine  1200  may communicate with one or more other machines utilizing any one of a number of transfer protocols (e.g., frame relay, internet protocol (IP), transmission control protocol (TCP), user datagram protocol (UDP), hypertext transfer protocol (HTTP), etc.). Example communication networks may include a local area network (LAN), a wide area network (WAN), a packet data network (e.g., the Internet), mobile telephone networks (e.g., cellular networks), Plain Old Telephone (POTS) networks, and wireless data networks (e.g., Institute of Electrical and Electronics Engineers (IEEE) 802.11 family of standards known as Wi-Fi®. IEEE 802.16 family of standards known as WiMax®). IEEE 802.15.4 family of standards, a Long Term Evolution (LTE) family of standards, a Universal Mobile Telecommunications System (UMTS) family of standards, peer-to-peer (P2P) networks, among others. In an example, the network interface device  1220  may include one or more physical jacks (e.g., Ethernet, coaxial, or phone jacks) or one or more antennas to connect to the communications network  1226 . In an example, the network interface device  1220  may include a plurality of antennas to wirelessly communicate using at least one of single-input multiple-output (SIMO), multiple-input multiple-output (MIMO), or multiple-input single-output (MISO) techniques. In some examples, the network interface device  1220  may wirelessly communicate using Multiple User MIMO techniques. 
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     All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications. 
     The specific methods, devices, and kits described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims. 
     As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a nucleic acid” or “a polypeptide” includes a plurality of such nucleic acids or polypeptides (for example, a solution of nucleic acids or polypeptides or a series of nucleic acid or polypeptide preparations), and so forth. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants. 
     The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention.