Nitric oxide synthase gene diagnostic polymorphisms

Disclosed is a method for determining a genetic predisposition to hypertension, end stage renal disease due to hypertension, non-insulin dependent diabetes mellitus, end stage renal disease due to non-insulin dependent diabetes mellitus, breast cancer, lung cancer or prostate cancer by detecting the presence or absence of single nucleotide polymorphisms in the nitric oxide synthase gene. Also disclosed are kits for detecting the presence or absence of the single nucleotide polymorphisms, methods for the treatment and/or prophylaxis of diseases, conditions, or disorders associated with the single nucleotide polymorphisms.

EXAMPLES 
 Example 1 
 G to A Transition at Position 2548 Amplification of eNOS promoter genomic DNA Leukocytes were obtained from human whole blood collected with EDTA. Genomic DNA was purified from the collected leukocytes using standard protocols well known to those of ordinary skill in the art of molecular biology (Ausubel et al., Short Protocols in Molecular Biology, 3 rd ed, John Wiley & Sons, 1995; Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989; and Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, 1986). DNA comprising the eNOS promoter region was amplified by the polymerase chain reaction (PCR). Twenty-five ng of leukocyte genomic DNA was used as template for each PCR amplification. Twenty-five microliters of an aqueous solution of genomic DNA (1 ng/ul) was dispensed to the wells of a 96-well plate, and dried down at 70° C. for 15 minutes. The DNA was rehydrated with 7 ul of ultra-pure but not autoclaved water (Milli-Q, Millipore Corp., Bedford Mass.). PCR conditions were as follows: 5 minutes at 94° C., followed by 45 cycles, where each cycle consisted of 94° C. for 45 seconds to denature the double-stranded DNA, then 64° C. for 45 seconds for specific annealing of primers to the single-stranded DNA, then 72° C. for 45 seconds for extension. After the 45th cycle, the reaction mixture was held at 72° C. for 10 minutes for a final extension reaction. The PCR reaction contained a total volume of 20 microliters (ul), and consisted of 10 ul of a pre-made PCR reaction mix (Sigma “JumpStart Ready Mix with RED Taq Polymerase” Sigma Chemical, St. Louis, Mo.). Primers at 10 &mgr;M were diluted to a final concentration of 0.3 &mgr;M in the PCR reaction mix. The forward primer was 5′ gagtctggccaacacaaatcc 3′ (SEQ ID NO: 3) and the reverse primer was 5′ ctctagggtcatgcaggttct c 3′ (SEQ ID NO: 4). The primers amplified the region spanning nucleotides 2356 to 2559, inclusive of SEQ ID NO: 1. Post-PCR clean-up was performed prior to submission of PCR product to pyrosequencing. 
 Sequencing of PCR Product Pyrosequencing is a method of sequencing DNA by synthesis, where the addition of one of the four dNTPs that correctly matches the complementary base on the template strand is detected. Detection occurs via utilization of the pyrophosphate molecules liberated upon base addition to the elongating synthetic strand. The pyrophosphate molecules are used to make ATP, which in turn drives the emission of photons in a luciferin/luciferase reaction, and these photons are detected by the pyrosequencer. A Luc96 Pyrosequencer (Pyrosequencing AB, Uppsala Sweden) was used under default operating conditions supplied by the manufacturer. Sequencing primers were designed to anneal within 5 bases of the polymorphism. Patient genomic DNA was subject to PCR using amplifying primers that amplify an approximately 200 base pair amplicon containing the polymorphisms of interest as described in Example 1. One of the amplifying primers, whose orientation is opposite to that of the sequencing primer, was biotinylated. This allowed selection of single stranded template for pyrosequencing, whose orientation was complementary to the sequencing primer. Amplicons prepared from genomic DNA were isolated by binding to streptavidin-coated magnetic beads. After denaturation in NaOH, the biotinylated strands were separated from their complementary strands using magnetic beads (DYNAL, Olso, Norway). After washing the magnetic beads, the biotinylated template strands still bound to the beads were transferred into 96-well plates. The sequencing primers were added, annealing was carried out at 95° C. for 2 minutes, and plates placed in the pyrosequencer. The enzymes, substrates and dNTPs used for synthesis and pyrophosphate detection were added to the instrument immediately prior to sequencing. The Luc96 software requires definition of a program of adding the four dNTPs that is specific for the location of the sequencing primer, the DNA composition flanking the SNP, and the two possible alleles at the polymorphic locus. The order of adding bases generates theoretical outcomes of light intensity patterns for each of the two possible homozygous states and the single heterozygous state. The Luc96 software then compares the actual outcome to the theoretical outcome and calls a genotype for each well. Each sample is also assigned one of three confidence scores: pass, uncertain, or fail. The results for each plate were output as a text file and processed in Excel using a Visual Basic program to generate a report of genotype and allele frequencies for the various disease and population cell groupings represented on the 96 well plate. 
 Bioinformatics Prediction of potential transcription binding factor sites was performed using a commercially available software program &lsqb;GENOMATIX MatInspector Professional release 4.2, February, 2000; http://genomatix.qsf.de/cqi-bin/matinspector/matinspector.pl;. Quandt K et al., Nucleic Acids Res 23: 4878-4884 (1995)&rsqb;. 
 DNA Samples Cases consisted of patients with essential hypertension or non-insulin dependent diabetes mellitus (NIDDM) (type II diabetes mellitus), but without evidence of renal disease (<2&plus;proteinuria on random urinalysis; serum creatinine less than or equal to 1.5 mg/dl). Samples were obtained from indigent-care St. Louis-area hospitals between 1994 and 1996. Patients with end-stage renal disease (ESRD) due to hypertension (ESRD/HTN) or due to NIDDM (ESRD/NIDDM) were hemodialysis patients with either hypertension only, or NIDDM (with or without hypertension), being treated in approximately 40 dialysis units in the southeastern US. Their samples were obtained in 1995. Disease-free controls were healthy plasma donors from cities in the central and eastern United States, with normal serum creatinine (less than or equal to 1.5 mg/dl). Controls were screened routinely to ensure the absence of any infectious diseases. Control plasma donors could not be taking insulin or other medication, except for a single anti-hypertensive at a low dose. Thus, controls could have mild essential hypertension, but no renal disease, and no NIDDM. Cases and controls were matched for ethnicity, gender, and sex, but not age. 
 Statistics Allele and genotype frequencies were stratified on the combination of race and gender (hereinafter referred to as a ‘cell’) and then matched to controls for an association study. Three statistics, a point estimate, 95% confidence interval, and a likelihood (p-value), were calculated for each combination of cell and disease. A simple odds ratio was used as the point estimate of association. In the case where a cell count was 0, the Haldane correction was used. This consists of adding 0.5 to each cell prior to calculations. The 95% confidence intervals were calculated using the asymptotic method. P-values for differences in allele or genotype frequencies were calculated using Fisher's exact test, using a two-sided alternative to the null hypothesis. All calculations were done using the SAS suite of statistical software, version 8.1 (SAS Institute, Cary, N.C.) 
 Results Using the methods described above, a substitution mutation (transition) was found in which the G found in the reference sequence (SEQ ID NO: 1) was replaced with an A. Data analysis produced the following results. 1 TABLE 1 ALLELE FREQUENCES Disease Cell CHROMOSOMES G % A % Controls Black men 1340 280 21% 1060 79% Black women 1380 159 12% 1221 88% White men 1412 402 28% 1010 72% White women 1482 532 36% 950 64% Hypertension Black men 568 139 24% 429 76% Black women 348 86 25% 262 75% White men 562 150 27% 412 73% White women 130 46 35% 84 65% ESRD due to HTN Black men 568 261 46% 307 54% Black women 440 196 45% 244 55% White men 306 108 35% 198 65% White women 284 126 44% 158 56% NIDDM Black men 530 161 30% 369 70% Black women 368 135 37% 233 63% White men 472 136 29% 336 71% White women 86 26 30% 60 70% ESRD due to NIDDM Black men 512 48 9.4% 464 91% Black women 496 78 16% 418 84% White men 426 174 41% 252 59% White women 392 115 29% 277 71% 2 TABLE 2 GENOTYPE FREQUENCIES Total Disease Cell ‘n’ G/G % G/A % A/A % Controls Black men 670 35 5.2% 210 31.3% 425 63.4% Black women 690 6 0.9% 147 21.3% 537 77.8% White men 706 62 8.8% 278 39.4% 366 51.8% White women 741 103 13.9% 326 44.0% 312 42.1% Hypertension Black men 284 3 1.1% 133 46.8% 148 52.1% Black women 174 0 0.0% 86 49.4% 88 50.6% White men 281 12 4.3% 126 44.8% 143 50.9% White women 65 5 7.7% 36 55.4% 24 36.9% ESRD due to HTN Black men 284 0 0.0% 261 91.9% 23 8.1% Black women 220 3 1.4% 190 86.4% 27 12.3% White men 153 0 0.0% 108 70.6% 45 29.4% White women 142 6 4.2% 114 80.3% 22 15.5% NIDDM Black men 265 2 0.8% 157 59.2% 106 40.0% Black women 184 0 0.0% 135 73.4% 49 26.6% White men 236 7 3.0% 122 51.7% 107 45.3% White women 43 3 7.0% 20 46.5% 20 46.5% ESRD due to Black men 256 6 2.3% 36 14.1% 214 83.6% NIDDM Black women 248 8 3.2% 62 25.0% 178 71.8% White men 213 27 12.7% 120 56.3% 66 31.0% White women 196 16 8.2% 83 42.3% 97 49.5% The susceptibility allele, the odds ratio (OR), 95% confidence interval, and p-value are given in Table 3. An odds ratio of 1.5 was chosen a priori as the threshold of practical significance based on the recommendation of Austin H et al. ( Epidemiol. Rev. 16:65-76, 1994). “ . . . &lsqb;E&rsqb;pidemiology in general and case-control studies in particular are not well suited for detecting weak associations (odds ratios <1.5) &lsqb;p. 66&rsqb;.” This threshold of 1.5 is supported by our data, considering p<0.05 as the level of significance. All odds ratios attaining p<0.05 or better are underlined below. (Scientific notation is used in some entries below, e.g. 2.9E-9&equals;2.9×10 −9 ). An example of an odds ratio calculation is given below: 3 Hypertension: Black Women Cases Controls G 86 159 A 262 1221 In this example, the odds ratio that the G allele is the susceptibility allele for black women with hypertension is (86) (1221)/(262)(159)&equals;2.5. 4 TABLE 3 ALLELE-SPECIFIC ODDS RATIOS Risk Odds P Disease Cell Allele Ratio 95% CI Value Hypertension Black men G 1.2 1.0-1.5 0.09 Black women G 2.5 1.9-3.4 2.9E-9 White men A 1.1 0.9-1.4 0.44 White A 1.0 0.7-1.5 1.0 women ESRD due to HTN † Black men G 2.6 2.0-3.4 4.0E-14 Black women G 2.4 1.8-3.3 7.3E-9 White men G 1.5 1.1-2.0 0.01 White G 1.5 0.9-2.2 0.09 women NIDDM Black men G 1.7 1.3-2.1 0.00002 Black women G 4.4 3.4-5.8 1.5E-26 White men G 1.0 0.8-1.3 0.90 White A 1.3 0.8-2.1 0.30 women ESRD due to Black men A 4.2 3.0-6.0 7.9E-18 NIDDM ‡ Black women A 3.1 2.3-4.3 2.2E-12 White men G 1.7 1.3-2.3 0.00019 White A 1.0 0.6-1.7 0.89 women † ratios calculated using patients with hypertension as controls ‡ Odds ratios calculated using patients with NIDDM as controls The genotype-specific odds ratios are given in Table 4. In Table 4, the susceptibility allele (S) is indicated. The alternative allele at this locus is defined as the protective allele (P). Also presented is the odds ratio (OR) for the SS and SP genotypes. The odds ratio for the PP genotype is 1, since it is the reference group, and is not presented separately. The 95% confidence interval (C.I.) is also given, in parentheses. An odds ratio of 1.5 was chosen as the threshold of significance based on the recommendation of Austin H et al. ( Epidemiol. Rev. 16:65-76, 1994). “ . . . &lsqb;E&rsqb;pidemiology in general and case-control studies in particular are not well suited for detecting weak associations (odds ratios <1.5) &lsqb;p. 66&rsqb;.” Odds ratios attaining 1.5 are high-lighted below. Where Haldane's zero cell correction was used, the odds ratio is so indicated with a superscript “H”. An example is worked below, assuming that G is the susceptibility allele (S), and A is the protective allele (P). 5 Black Women: ESRD due to HTN Cases Controls GG (SS) 3 0 GA (SP) 190 86 AA (PP) 27 88 Applying Haldane's correction only where the denominator of the odds ratio contains a 0, the SS odds ratio is (3.5)(88.5)/(27.5)(0.5)&equals;22.5 while the SP odds ratio is (190)(88)/(27)(86)&equals;7.2 6 TABLE 4 GENOTYPE-SPECIFIC ODDS RATIOS RISK SS SP Disease Cell ALLELE O.R. 95% C.I. O.R. 95% C.I. Hypertension Black men G 0.2 0.1-0.8 1.8 1.4-2.4 Black women G 0.0 3.6 2.5-5.1 White men A 2.0 1.1-3.9 2.3 1.8-3.1 White women A 1.6 0.6-4.3 2.3 1.3-3.9 ESRD due to HTN Black men G 0.0 12.6 7.8-20.5 Black women G 22.5 H 0.4-1325.4 7.2 4.4-11.9 White men G 0.0 2.7 1.8-4.2 White women G 1.3 0.3-4.9 3.5 1.7-6.9 NIDDM Black men G 0.2 0.1-0.8 3.0 2.2-4.0 Black women G 0.0 10.1 6.9-14.6 White men G 0.4 0.2-0.9 1.5 1.1-2.0 White women A 2.2 0.6-7.6 2.1 1.1-4.0 ESRD due to NIDDM Black men A 0.7 0.1-3.4 0.1 0.0-0.1 Black women A 0.0 0.0 White men G 6.3 2.6-15.2 1.6 1.1-2.4 White women A 0.9 0.2-3.4 0.8 0.4-1.5 Odds ratios calculated using patients with hypertension as controls Odds ratios calculated using patients with NIDDM as controls Hardy-Weinberg analysis was conducted on the control samples. Hardy-Weinberg equilibrium is a term used to describe the distribution of genotypes at a bialleleic locus in a stable population without recent genetic admixture, drift, or selection pressure. The equilibrium distribution is the binomial expansion of the two allele frequencies, p and q&equals;1−p, i.e. (p&plus;q) 2 &equals;p 2 &plus;2pq&plus;q 2 &equals;1. The control samples were in good agreement with Hardy-Weinberg equilibrium, as follows: A frequency of 0.12 for the G allele (“p”) and 0.88 for the A allele (“q”) among black female control individuals predicts genotype frequencies of 1.4% G/G, 21.2% G/A, and 77.4% A/A at Hardy-Weinberg equilibrium (p 2 &plus;2pq&plus;q 2 &equals;1). The observed genotype frequencies were 0.9% G/G, 21.3% G/A, and 77.8% A/A, in excellent agreement with those predicted for Hardy-Weinberg equilibrium. The chi-square statistic for a test of disequilibrium was 1.1, which has a p-value of 0.58, with 2 degrees of freedom. Thus, the observed genotype frequencies do not deviate significantly from Hardy-Weinberg equilibrium (HWE). A frequency of 0.21 for the G allele (“p”) and 0.79 for the A allele (“q”) among black male control individuals predicts genotype frequencies of 4.4% G/G, 33.2% G/A, and 62.4% A/A at Hardy-Weinberg equilibrium (p 2 &plus;2pq&plus;q 2 &equals;1). The observed genotype frequencies were 5.2% G/G, 31.3% G/A, and 63.5% A/A, in excellent agreement with those predicted for Hardy-Weinberg equilibrium. The chi-square statistic for a test of disequilibrium was 1.3, which has a p-value of 0.51 with 2 degrees of freedom. Thus, the observed genotype frequencies do not deviate significantly from Hardy-Weinberg equilibrium. A frequency of 0.36 for the G allele (“p”) and 0.64 for the A allele (“q”) among white female control individuals predicts genotype frequencies of 13.0% G/G, 46.1% G/A, and 40.9% A/A at Hardy-Weinberg equilibrium (p 2 &plus;2pq&plus;q 2 &equals;1). The observed genotype frequencies were 13.9% G/G, 44.0% G/A, and 42.1% A/A, in good agreement with those predicted for Hardy-Weinberg equilibrium. The chi-square statistic for a test of disequilibrium was 0.96, which has a p-value of 0.60 with 2 degrees of freedom. Thus, the observed genotype frequencies do not deviate significantly from Hardy-Weinberg equilibrium. A frequency of 0.28 for the G allele (“p”) and 0.72 for the A allele (“q”) among white male control individuals predicts genotype frequencies of 7.8% G/G, 40.3% G/A, and 51.9% A/A at Hardy-Weinberg equilibrium (p 2 &plus;2pq&plus;q 2 &equals;1). The observed genotype frequencies were 8.8% G/G, 39.4% G/A, and 51.8% A/A, in excellent agreement with those predicted for Hardy-Weinberg equilibrium. The chi-square statistic for a test of disequilibrium was 0.7, which has a p-value of 0.7 with 2 degrees of freedom. Thus, the observed genotype frequencies do not deviate significantly from Hardy-Weinberg equilibrium. Hypertension and NIDDM are necessary but not sufficient to develop ESRD. Patients with hypertension are at approximately a 5% lifetime risk of ESRD, while patients with NIDDM are at about a 20% lifetime risk. Therefore hypertension and NIDDM can be considered as intermediate phenotypes; clinically diseased compared to the average population, yet healthier than hypertensive or diabetic patients with ESRD. In order to detect a dosage effect of the G2548→A polymorphism, a progressive disease model for calculating odds ratios was used. The odds ratio for patients with hypertension alone or NIDDM alone relative to normal controls represents a baseline measurement for each underlying disease. Next, calculating odds ratios for ESRD patients by comparing them to individuals with just the primary disease but no kidney disease (ie HTN or NIDDM) can be useful in dissecting which alleles are necessary for progression to end-stage kidney failure. Using an allele-specific odds ratio of 1.5 or greater as a practical level of significance (see Austin H. et al., discussed above), the following observations, which are summarized in Table 5, can be made. For black women with hypertension, the odds ratio for the G allele was 2.5 &lsqb;(95% CI, 1.9-3.4), p<2.9E-9&rsqb;. The odds ratio for the homozygote (G/G) was less than 1.0, while the odds ratio for the heterozygote (G/A) was 3.6 (95% CI, 2.5-5.1). These data suggest that the G allele acts in a co-dominant manner in this patient population. These data further suggest that the ecNOS gene is significantly associated with hypertension alone in black women, i.e. abnormal activity of the ecNOS gene predisposes black women to hypertension. For black women with ESRD due to hypertension, the odds ratio for the G allele was 2.4 &lsqb;(95% CI, 1.8-3.3), p<7.3E-9&rsqb;, compared to black women with hypertension alone. The odds ratio for the homozygote (G/G) was 22.5 H &lsqb;the superscript “H” indicates the Haldane correction was employed&rsqb; (95% CI, 0.4-1325.4). The odds ratio for the heterozygote (G/A) was 7.2 (95% CI, 4.4-11.9). These data suggest that the G allele acts in a dominant manner in this patient population with a greater than additive effect of allele dosage &lsqb;22.5>13.4&equals;(7.2&plus;7.2-1.0)&rsqb; (Goldstein A M and Andrieu N, Monogr. Natl. Cancer Inst. 26: 49-54, 1999). These data further suggest that the ecNOS gene is significantly associated with ESRD due to hypertension in black women, i.e. abnormal activity of the ecNOS gene predisposes black women with hypertension to ESRD. For black men with ESRD due to hypertension, the odds ratio for the G allele was 2.6 &lsqb;(95% CI, 2-3.4), p<4.0E-14&rsqb;, compared to black men with hypertension alone. The odds ratio for the homozygote (G/G) was less than 1.0, while the odds ratio for the heterozygote (G/A) was 12.6 (95% CI, 7.8-20.5). These data suggest that the G allele acts in a co-dominant manner in this patient population. These data further suggest that the ecNOS gene is significantly associated with ESRD due to hypertension in black men, i.e. abnormal activity of the ecNOS gene predisposes black men with hypertension to ESRD. For white men with ESRD due to hypertension, the odds ratio for the G allele was 1.5 &lsqb;(95% CI, 1.1-2.0), p&equals;0.01&rsqb;, compared to white men with hypertension alone. The odds ratio for the homozygote (G/G) was less than 1.0, while the odds ratio for the heterozygote (G/A) was 2.7 (95% CI, 1.8-4.2). These data suggest that the G allele acts in a co-dominant manner in this patient population. These data further suggest that the ecNOS gene is significantly associated with ESRD due to hypertension in white men, i.e. abnormal activity of the ecNOS gene predisposes white men with hypertension to ESRD. For black men with NIDDM alone, the odds ratio for the G allele was 1.7 &lsqb;(95% CI, 1.3-2.1), p<0.00002&rsqb;. The odds ratio for the homozygote (G/G) was less than 1.0, while the odds ratio for the heterozygote (G/A) was 3.0 (95% CI, 2.2-4). These data suggest that the G allele acts in a co-dominant manner in this patient population. These data further suggest that the ecNOS gene is significantly associated with NIDDM in black men, i.e. abnormal activity of the ecNOS gene predisposes black men to NIDDM. For black men with ESRD due to NIDDM, the odds ratio for the A allele was 4.2 &lsqb;(95% CI, 3.0-6.0), p<7.9E-18&rsqb;, compared to black men with NIDDM alone. Data were not sufficient to generate genotypic odds ratios of 1.5 or greater. These data further suggest that the ecNOS gene is significantly associated with ESRD due to NIDDM in black men, i.e. abnormal activity of the ecNOS gene predisposes black men with NIDDM to ESRD. For black women with NIDDM, the odds ratio for the G allele was 4.4 &lsqb;(95% CI, 3.4-5.8), p<1.5E-26&rsqb;. The odds ratio for the homozygote (G/G) was less than 1.0, while the odds ratio for the heterozygote (G/A) was 10.1 (95% CI, 6.9-14.6). These data suggest that the G allele acts in a co-dominant manner in this patient population. These data further suggest that the ecNOS gene is significantly associated with NIDDM in black women, i.e. abnormal activity of the ecNOS gene predisposes black women to NIDDM. For black women with ESRD due to NIDDM, the odds ratio for the A allele was 3.1 &lsqb;(95% CI, 2.3-4.3), p<2.2E-12&rsqb;, compared to black women with NIDDM alone. Data were not sufficient to generate genotypic odds ratios of 1.5 or greater. These data further suggest that the ecNOS gene is significantly associated with ESRD due to NIDDM in black women, i.e. abnormal activity of the ecNOS gene predisposes black women with NIDDM to ESRD. For white men with ESRD due to NIDDM the odds ratio for the G allele was 1.7 &lsqb;(95% CI, 1.3-2.3), p<0.0002&rsqb;, compared to white men with NIDDM alone. The odds ratio for the homozygote (G/G) was 6.3 (95% CI, 2.6-15.2), while the odds ratio for the heterozygote (G/A) was 1.6 (95% CI, 1.1-2.4). These data suggest that the G allele acts in a dominant manner in this patient population, with a greater than multiplicative effect of allele dosage &lsqb;6.3>2.56&equals;(1.6) (1.6)&rsqb;. These data further suggest that the ecNOS gene is significantly associated with ESRD due to NIDDM in white men, i.e. abnormal activity of the ecNOS gene predisposes white men with NIDDM to ESRD. 7 TABLE 5 SUSCEPTIBILITY ALLELE CAUCASIAN AFRICAN-AMERICAN DISEASE Men Women Men Women HTN A A G G** ESRD/HTN G* G G** G** NIDDM G A G* G** ESRD/NIDDM G* A A** A** **&equals; p < 5E-8; *&equals; p < 0.05 According to commercially available software (GENOMATIX MatInspector Professional), the G2548→A SNP is predicted to have the following effects on transcription of the ecNOS gene. One predicted effect is disruption of an NF-1 (nuclear factor 1) site (5′-AGATG G CACAGAACTACA-3′; SEQ ID NO: 5) beginning at position &plus;2543 on the (&plus;) strand. This polymorphism would result in replacement of the indicated G by an A. NF-1 sites occur relatively frequently in the genome: 4.11 occasions per 1000 base pairs of random genomic sequence in vertebrates. Since NF-1 is a positive transcriptional regulator disruption of its binding site is expected to result in a decreased rate of transcription of the ecNOS gene. If the rate of translation is tied to the level of messenger RNA, as is the case for most proteins, then less gene product (ecNOS enzyme) will be the result, ultimately leading to less nitric oxide (NO) produced in tissues such as endothelial cells in patients with the A allele. The polymorphism also can cause disruption of an MYOD (myoblast determining factor) binding site, which consists of 5′-G C CATCTGAG-3′ (SEQ ID NO: 6), ending at position &plus;2540 on the (−) strand. Thus, this polymorphism results in replacement of the indicated C by a T on the (−) strand, since T is complementary to the polymorphic base, A, at this position on the (&plus;) strand. MYOD binding sites are less frequent than NF1 sites, occurring 0.96 times per 1000 base pairs of random genomic sequence. MYOD is increasingly recognized as a potent transcriptional activator of more tissues than merely those destined to become skeletal muscle, in which context it was originally discovered. This association suggests an unexpected biochemical mechanism for diabetic or hypertensive renal failure, e.g. in black women, who express a higher frequency of the A allele. MYOD may operate in endothelial cells. It is possible that ecNOS production by smooth muscle cells, which are known to express MYOD, is important in regulation of renal blood flow and apoptosis of down-stream cellular elements. Another predicted effect is disruption of an LMO2COM (complex of Lmo2 bound to Tal-1, E2A protein) binding site, which consists of the sequence 5′-CCTCAGATG G CA-3′ (SEQ ID NO: 7), beginning at position &plus;2539 on the (&plus;) strand. This polymorphism results in the replacement of the indicated G with an A. LMO2COM binding sites occur with a frequency of 1.11 times per 1000 base pairs of random genomic sequence, which is relatively frequent. The E2A protein is an adenoviral “early” protein, for which no cellular homolog is yet known. Also predicted is the disruption of a TAL1ALPHAE47 (Tal-1alpha/E47 heterodimer) binding site, which consists of the sequence 5′-CCCCTCAGATG G CACA-3′ (SEQ ID NO: 8), beginning at position&plus;2537 on the (&plus;) strand. This polymorphism results in the replacement of the indicated G with an A. TAL1ALPHAE47 binding sites occur quite infrequently, at the rate of 0.14 times per 1000 base pairs of random genomic sequence in vertebrates. The less frequently that the binding site occurs in random genomic DNA, the more likely that the binding site is specifically involved in transcription of this gene. Association of disease with this site thus suggests a novel mechanism for ecNOS regulation in cells whose identity is not yet known, but which could include endothelial, smooth muscle, mesangial, or tubular epithelial cells, for example. The Tal-1beta (or alpha)/E47 heterodimer can behave as a transcriptional activator, so replacement of the indicated G with an A is predicted to result in a lower rate of transcription of the ecNOS gene and thus a lower level of nitric oxide production in tissues. Another predicted effect is the disruption of a TAL1BETAE47 (Tal-1beta/E47 heterodimer) binding site, which consists of the sequence 5′-CCCCTCAGATG G CACA-3′ (SEQ ID NO: 8), beginning at position &plus;2537 on the (&plus;) strand. This polymorphism results in the replacement of the indicated G with an A. TAL1BETAE47 binding sites also occur quite rarely, at the rate of 0.11 times per 1000 base pairs of random genomic sequence. Association of disease with this site thus suggests a novel mechanism for ecNOS regulation in cells whose identity is not yet known, but which could include, for example, endothelial, smooth muscle, mesangial, or tubular epithelial cells. If Tal-1beta (or alpha)/E47 heterodimer behaves as a transcriptional activator, then replacement of the indicated G with an A is predicted to result in a lower rate of transcription of the ecNOS gene and thus a lower level of nitric oxide production in tissues. 
 Example 2 
 C to T Transition at Position 2684 Methods of DNA amplification, sequencing and data analysis were essentially as described in Example 1. A substitution mutation (transition) was found in which the C found at position 2684 in the reference sequence (SEQ ID NO: 1) was replaced with a T. Data analysis produced the following results. 8 TABLE 6 ALLELE FREQUENCIES C T CONTROL Black men (n &equals; 84 chromosomes) 10 (12%) 74 (88%) Black women (n &equals; 74 chromosomes) 18 (24%) 56 (76%) White men (n &equals; 76 chromosomes) 29 (38%) 47 (62%) White women (n &equals; 94 chromosomes) 29 (31%) 65 (69%) DISEASE BREAST CANCER Black women (n &equals; 40 chromosomes) 7 (18%) 33 (82%) White women (n &equals; 38 chromosomes) 12 (32%) 26 (68%) LUNG CANCER Black men (n &equals; 40 chromosomes) 21 (53%) 19 (48%) Black women (n &equals; 32 chromosomes) 6 (19%) 26 (81%) White men (n &equals; 40 chromosomes) 17 (43%) 23 (58%) White women (n &equals; 22 chromosomes) 8 (36%) 14 (64%) PROSTATE CANCER Black men (n &equals; 40 chromosomes) 9 (23%) 31 (77%) White men (n &equals; 38 chromosomes) 17 (45%) 21 (55%) NIDDM Black men (n &equals; 4 chromosomes) 1 (25%) 3 (75%) Black women (n &equals; 6 chromosomes) 3 (50%) 3 (50%) White men (n &equals; 8 chromosomes) 0 (0%) 8 (100%) White women (n &equals; 18 chromosomes) 14 (78%) 4 (22%) ESRD due to NIDDM Black men (n &equals; 12 chromosomes) 1 (8%) 11 (92%) Black women (n &equals; 16 chromosomes) 2 (13%) 14 (88%) White men (n &equals; 10 chromosomes) 2 (20%) 8 (80%) White women (n &equals; 8 chromosomes) 2 (25%) 6 (75%) HYPERTENSION (HTN) Black men (n &equals; 24 chromosomes) 3 (13%) 21 (88%) Black women (n &equals; 24 chromosomes) 2 (8%) 22 (92%) White men (n &equals; 22 chromosomes) 7 (32%) 15 (68%) White women (n &equals; 20 chromosomes) 8 (40%) 12 (60%) ESRD due to HTN Black men (n &equals; 20 chromosomes) 4 (20%) 16 (80%) Black women (n &equals; 18 chromosomes) 0 (0%) 18 (100%) White men (n &equals; 18 chromosomes) 5 (28%) 13 (72%) White women (n &equals; 18 chromosomes) 3 (17%) 15 (83%) MYOCARDIAL INFARCTION White women (n &equals; 14 chromosomes) 5 (36%) 9 (64%) 9 TABLE 7 GENOTYPE FREQUENCIES C/C C/T T/T CONTROLS Black men (n &equals; 42) 0 (0%) 10 (24%) 32 (76%) Black women (n &equals; 37) 2 (5%) 14 (38%) 21 (57%) White men (n &equals; 38) 5 (13%) 19 (50%) 14 (37%) White women (n &equals; 47) 2 (4%) 25 (53%) 20 (43%) DISEASE BREAST CANCER Black women (n &equals; 20) 0 (0%) 7 (35%) 13 (65%) White women (n &equals; 19) 1 (5%) 10 (53%) 8 (42%) LUNG CANCER Black men (n &equals; 20) 8 (40%) 5 (25%) 7 (35%) Black women (n &equals; 16) 0 (0%) 6 (38%) 10 (63%) White men (n &equals; 20) 2 (10%) 13 (65%) 5 (25%) White women (n &equals; 11) 2 (18%) 4 (36%) 5 (45%) PROSTATE CANCER Black men (n &equals; 20) 0 (0%) 9 (45%) 11 (55%) White men (n &equals; 19) 2 (11%) 13 (68%) 4 (21%) NIDDM Black men (n &equals; 2) 0 (0%) 1 (50%) 1 (50%) Black women (n &equals; 3) 1 (33%) 1 (33%) 1 (33%) White men (n &equals; 4) 0 (0%) 0 (0%) 4 (100%) White women (n &equals; 9) 6 (67%) 2 (22%) 1 (11%) ESRD due to NIDDM Black men (n &equals; 6) 0 (0%) 1 (17%) 5 (83%) Black women (n &equals; 8) 0 (0%) 2 (25%) 6 (75%) White men (n &equals; 5) 0 (0%) 2 (40%) 3 (60%) White women (n &equals; 4) 0 (0%) 2 (50%) 2 (50%) HYPERTENSION (HTN) Black men (n &equals; 12) 0 (0%) 3 (25%) 9 (75%) Black women (n &equals; 14) 0 (0%) 2 (17%) 12 (83%) White men (n &equals; 11) 1 (9%) 5 (45%) 5 (45%) White women (n &equals; 10) 1 (10%) 6 (60%) 3 (30%) ESRD due to HTN Black men (n &equals; 10) 1 (10%) 2 (20%) 7 (70%) Black women (n &equals; 9) 0 (0%) 0 (0%) 9 (100%) White men (n &equals; 9) 0 (0%) 5 (56%) 4 (44%) White women (n &equals; 9) 0 (0%) 3 (33%) 6 (67%) MYOCARDIAL INFARCTION White women (n &equals; 7) 0 (0%) 5 (71%) 2 (29%) In Table 8, the susceptibility allele is indicated, as well as the odds ratio (OR). Haldane's correction was used if the denominator was zero. If the odds ratio (OR) was &gE;1.5, the 95% confidence interval (C.I.) is also given. An odds ratio of 1.5 was chosen as the threshold of significant based on the recommendation of Austin et al. in Epideminol. Rev., 16:65-76, (1994). Odds ratio of 1.5 or high-lighted below. 10 TABLE 8 ALLELE-SPECIFIC ODDS RATIOS SUSCEPTIBILITY DISEASE ALLELE OR 95% C.I. Breast Cancer Black women T 1.5 0.6-4.0 White women C 1.0 Lung Cancer Black men C 8.2 3.3-20 Black women T 1.4 White men T 0.8 White women C 1.3 Prostate Cancer Black men C 2.1 0.8-5.8 White men C 0.8 NIDDM Black men C 2.5 0.2-26 Black women C 3.1 0.6-17 White men T 10.6 1.4-81 White women C 7.8 2.4-26 ESRD due to NIDDM* Black men T 3.7 0.2-78 Black women T 7.0 0.8-62 White men C 5.0 0.5-47 White women T 10.5 1.5-74 Hypertension (HTN) Black men C 1.1 Black women T 3.5 0.8-17 White men T 1.3 White women C 1.5 0.6-40 ESRD due to HTN* 1 Black men C 1.8 0.3-9.0 Black women T 4.1 0.5-37 White men T 1.2 White women T 2.3 0.5-11 Myocardial Infarction White women C 1.2 *Compared to group with NIDDM alone. * 1 Compared to group with HTN alone. 
 Genotype-Specific Odds Ratios In Table 9, the susceptibility allele (S) is indicated, and the alternative allele at this locus is defined as the protective allele (P). Also presented is the odds ratio (OR) for the SS and SP genotypes. The odds ratio for the PP genotype is 1 by definition, since it is the reference group, and is not presented in the table below. For odds ratios &gE;1.5, the asymptotic 95% confidence interval (C.I.) is also given, in parentheses. An odds ratio of 1.5 was chosen as the threshold of significance based on the recommendation of Austin et al., in Epidemiol. Rev., 16:65-76 (1994). Odds ratios of 1.5 or higher are high-lighted below. Haldane's correction was used when the denominator was zero. To minimize confusion, genotype-specific odds ratios are presented only for diseases in which the allele-specific odds ratio was at least 1.5. 11 TABLE 9 GENOTYPE-SPECIFIC ODDS RATIOS SUSCEPTIBILITY DISEASE ALLELE OR(SS) OR(SP) Breast Cancer Black women T 3.1 (0.3-28) 2.6 (0.3-24) Lung Cancer Black men C 74 (9.1-598) 2.3 (0.9-5.7) Prostate Cancer Black men C 2.8 (0.2-47) 2.6 (1.2-5.6) NIDDM Black men C 22 (1.1-437) 3.1 (0.6-17) Black women C 11 (0.5-240) 1.5 (0.1-26) White men T 3.4 (0.4-30) 0.3 White women C 60 (4.6-782) 1.6 (0.1-19) ESRD due to NIDDM* Black men T 3.7 (0.2-78) 1.0 Black women T 13 (1.0-173) 5.0 (0.3-73) White men C 1.3 6.4 (0.6-68) White women T 22 (1.8-261) 13 (1.2-141) Hypertension (HTN) Black women T 2.9 (0.3-26) 0.9 White women C 3.3 (0.2-49) 1.6 (0.4-7.2) ESRD due to HTN* 1 Black men C 3.8 (0.4-40) 0.9 Black women T 0.8 0.2 White women T 5.6 (0.5-64) 1.6 (0.1-19) *Compared to group with NIDDM alone. * 1 Compared to group with HTN alone. The control samples agree with Hardy-Weinberg equilibrium, as follows: A frequency of 0.12 for the C allele (“p”) and 0.88 for the T allele (“q”) among black male control individuals predicts genotype frequencies of 1% C/C, 22% C/T, and 77% T/T at Hardy-Weinberg equilibrium (p 2 &plus;2pq&plus;q 2 &equals;1). The observed genotype frequencies were 0% C/C, 24% C/T, and 76% T/T, in excellent agreement with those predicted for Hardy-Weinberg equilibrium. A frequency of 0.24 for the C allele (“p”) and 0.76 for the T allele (“q”) among black female control individuals predicts genotype frequencies of 6% C/C, 36% C/T, and 58% T/T at Hardy-Weinberg equilibrium (p 2 &plus;2pq&plus;q 2 &equals;1). The observed genotype frequencies were 5% C/C, 38% C/T, and 57% T/T, in excellent agreement with those predicted for Hardy-Weinberg equilibrium. A frequency of 0.38 for the C allele (“p”) and 0.62 for the T allele (“q”) among white male control individuals predicts genotype frequencies of 14% C/C, 48% C/T, and 38% T/T at Hardy-Weinberg equilibrium (p 2 &plus;2pq&plus;q 2 &equals;1). The observed genotype frequencies were 13% C/C, 50% C/T, and 37% T/T, in excellent agreement with those predicted for Hardy-Weinberg equilibrium. A frequency of 0.31 for the C allele (“p”) and 0.69 for the T allele (“q”) among white female control individuals predicts genotype frequencies of 10% C/C, 42% C/T, and 48% T/T at Hardy-Weinberg equilibrium (p 2 &plus;2pq&plus;q 2 &equals;1). The observed genotype frequencies were 4% C/C, 53% C/T, and 43% T/T, in fair agreement with those predicted for Hardy-Weinberg equilibrium. Using an allele-specific odds ratio of 1.5 or greater as a practical level of significance, the following observations can be made. Among black women with breast cancer, the odds ratio for the T allele at this locus was 1.5 (95% CI, 0.6-4.0). The odds ratio for the TC heterozygote was 2.6 (95% CI, 0.3-24), and 3.1 (95% CI, 0.3-28) for the TT homozygote. The genotype-specific odds ratios suggest that the T allele behaves as a dominant susceptibility allele. For black men with lung cancer, the odds ratio for the C allele at this locus was 8.2 (95% CI, 3.3-20). The odds ratio for the CT heterozygote was 2.3 (95% CI, 0.9-5.7), and 74 (95% CI, 9.1-598) for the CC homozygote. The genotype-specific odds ratios suggest that the T allele behaves as a dominant susceptibility allele, since the heterozygote (with one allele copy) has an odds ratio of 2.3. However, there is a pronounced (more than multiplicative) effect of gene dosage, since the homozygote with two copies of the C allele displayed a more than 30-fold larger odds ratio. For black men with prostate cancer, the odds ratio for the C allele at this locus was 2.1 (95% CI, 0.8-5.8). The odds ratio for the heterozygote (2.6, 95% CI, 1.2-5.6) was essentially the same as for the CC homozygote (2.8, 95% CI, 0.2-47), suggesting that the C allele behaves in a dominant fashion. For black men with NIDDM, the odds ratio for the C allele at this locus was 2.5 (95% CI, 0.2-26). The odds ratio for the heterozygote was 3.1 (95% CI, 0.6-17), and for the CC homozygote was 22 (95% CI, 1.1-437). The genotype-specific odds ratios suggest that the C allele behaves as a dominant susceptibility allele, since the heterozygote (with one allele copy) had an odds ratio of 3.1. However, there was a pronounced effect of gene dosage, since the homozygote with two copies of the C allele displayed a more than 7-fold larger odds ratio than the heterozygote. For black women with NIDDM, the odds ratio for the C allele at this locus was 3.1 (95% CI, 0.6-17). The odds ratio for the heterozygote was 1.5 (95% CI, 0.1-26), and for the CC homozygote was 11 (95% CI, 0.5-240). The genotype-specific odds ratios suggest that the C allele behaves as a dominant susceptibility allele, since the heterozygote (with one allele copy) had an odds ratio of 1.5. However, there is a pronounced (more than multiplicative) effect of gene dosage, since the homozygote with two copies of the C allele displayed a more than 7-fold larger odds ratio than the heterozygote. For white men with NIDDM, the odds ratio for the T allele at this locus was 10.6 (95% CI, 1.4-81). The odds ratio for the heterozygote was actually less than one (0.3), but for the TT homozygote was 3.4 (95% CI, 0.4-30). The genotype-specific odds ratios suggest that the T allele behaves as a recessive susceptibility allele. For white women with NIDDM, the odds ratio for the C allele at this locus was 7.8 (95% CI, 2.4-26). The odds ratio for the heterozygote was 1.6 (95% CI, 0.1-19), and for the CC homozygote was 60 (95% CI, 4.6-782). The genotype-specific odds ratios suggest that the C allele behaves as a dominant susceptibility allele, since the heterozygote (with one allele copy) had an odds ratio of 1.6. However, there is a pronounced (more than multiplicative) effect of gene dosage, since the homozygote with two copies of the C allele displayed a more than 37-fold larger odds ratio than the heterozygote. For black men with ESRD due to NIDDM, the odds ratio for the T allele at this locus was 3.7 (95% CI, 0.2-78), compared with black men with NIDDM but no renal disease. The odds ratio for the heterozygote was 1.0, but for the TT homozygote was 3.7 (95% CI, 0.2-78). The genotype-specific odds ratios suggest that the T allele behaves as a recessive susceptibility allele. For black women with ESRD due to NIDDM, the odds ratio for the T allele at this locus was 7.0 (95% CI, 0.8-62), compared with black women with NIDDM but no renal disease. The odds ratio for the heterozygote was 5.0 (95% CI, 0.3-73), and for the TT homozygote was 13 (95% CI, 1.0-173). The genotype-specific odds ratios suggest that the T allele behaves as a dominant susceptibility allele. However, there is a pronounced (more than additive) effect of gene dosage, since the homozygote with two copies of the C allele displayed a more than two-fold larger odds ratio than the heterozygote. For white men with ESRD due to NIDDM, the odds ratio for the C allele at this locus was 5.0 (95% CI, 0.5-47) vs. white men with NIDDM but no renal disease. Inspection of the genotype-specific odds ratios suggests that the C allele is codominant, since the heterozygote had a much higher odds ratio (6.4, 95% CI 0.6-68) than the CC homozygote (1.3) or the reference TT genotype (odds ratio 1, by definition). For white women with ESRD due to NIDDM, the odds ratio for the T allele at this locus was 10.5 (95% CI, 1.5-74) vs. white women with NIDDM but no renal disease. The odds ratio for the heterozygote was 13 (95% CI, 1.2-141), and the TT homozygote was 22 (95% CI, 1.8-261). The genotype-specific odds ratios suggest that the T allele behaves as a dominant susceptibility allele. However, there is a pronounced (approximately additive) effect of gene dosage, since the homozygote with two copies of the T allele displayed a roughly two-fold larger odds ratio than the heterozygote. For black women with hypertension, the odds ratio for the T allele at this locus was 3.5 (95% CI, 0.8-17). The odds ratio for the heterozygote was 0.9, but for the TT homozygote was 2.9 (95% CI, 0.3-26). The genotype-specific odds ratios suggest that the T allele behaves as a recessive susceptibility allele. For white women with hypertension, the odds ratio for the C allele at this locus was 1.5 (95% CI, 0.6-40). The odds ratio for the heterozygote was 1.6 (95% CI, 0.4-7.2), and for the CC homozygote was 3.3 (95% CI, 0.2-49). The genotype-specific odds ratios suggest that the C allele behaves in a dominant fashion, with a strictly additive effect of allele dosage, since 1.6&plus;1.6˜3.3. For black men with ESRD due to hypertension (HTN), the odds ratio for the C allele at this locus was 1.8 (95% CI, 0.3-9.0) relative to black men with HTN but no renal failure. The odds ratio for the heterozygote was 0.9, but for the CC homozygote was 3.8 (95% CI, 0.4-40). The genotype-specific odds ratios suggest that the C allele behaves in a recessive fashion. For black women with ESRD due to HTN, the odds ratio for the T allele was 4.1 (95% CI, 0.5-37) relative to black women with HTN alone. The genotype-specific odds ratios were found to be unhelpful, so no inference can be drawn about whether the T allele behaves in a dominant, recessive, or codominant fashion. For white women with ESRD due to HTN, the odds ratio for the T allele was 2.3 (95% CI, 0.5-11) relative to white women with HTN alone. The odds ratio for the heterozygote was 1.6 (95% CI, 0.1-19), and for the TT homozygote was 5.6 (95% CI, 0.5-64). The genotype-specific odds ratios suggest that the C allele behaves in a dominant fashion, with a more than multiplicative effect of allele dosage, since 5.6/(1.6) 2 &equals;5.6/3.56&equals;1.6>1. According to commercially available software &lsqb;GENOMATIX MatInspector Professional; http://genomatix.qsf.de/cqi-bin/matinspector/matinspector.pl; Quandt et al., Nucleic Acids Res. 23: 4878-4884 (1995)&rsqb;, the C2684→T SNP is predicted to have the following potential effects on transcription of the ecNOS gene: a. Disruption of an NF1 (nuclear factor 1) binding site, which consists of the sequence 5′-CCCTGGC C GGCTGACCCT-3′ (SEQ ID NO: 9), beginning at position&plus;2677 on the (&plus;) strand. This polymorphism replaces the indicated C with a T, which should result in a weaker binding site for NF1, a transcriptional activator of ecNOS. NF1 binding sites occur rather frequently, 4.11 times per 1000 base pairs of random genomic sequence. Since NF-1 is a positive transcriptional regulator, disruption of its binding site is expected to result in a decreased rate of transcription of the ecNOS gene. If the rate of translation is tied to the level of messenger RNA, as is the case for most proteins, then less gene product (ecNOS enzyme) will be the result, ultimately leading to less nitric oxide (NO) produced in tissues such as endothelial cells. b. Disruption of an ER (estrogen receptor) binding site, which consists of the sequence 5′-CCCTGGC C GGCTGACCCT-3′ (SEQ ID NO: 9), beginning at position &plus;2677 on the (&plus;) strand. This polymorphism replaces the indicated C with a T, which should result in a weaker binding site for the estrogen receptor, a transcriptional activator of ecNOS. ER binding sites occur moderately frequently, at the rate of 1.73 sites per 1000 base pairs of random genomic sequence. Since the estrogen receptor is a transcriptional activator, disruption of its binding site is expected to result in a decreased rate of transcription of the ecNOS gene. If the rate of translation is tied to the level of messenger RNA, as is the case for most proteins, then less gene product (ecNOS enzyme) will be the result, ultimately leading to less nitric oxide (NO) produced in tissues such as endothelial cells. In rodents, androgens have been shown to accelerate renal failure. Thus, it is intriguing that this polymorphism might interfere with the effect of estrogen, essentially tilting the balance towards androgens. c. Disruption of a TCF11 (TCF11/KCR-F1/Nrf1 homodimer) binding site, which consists of the sequence 5′-GTCAGCC G GCCAG-3′ (SEQ ID NO: 10), which ends at position &plus;2679 on the (−) strand. This polymorphism replaces the C on the (&plus;) strand by a T on the (&plus;) strand. The complementary base on the (−) strand is thus changed from the reference sequence G , indicated in TCF11's binding site, above, to an A, complementary to the T of the polymorphism. The TCF11 binding site occurs rather frequently, at the rate of 4.63 times per 1000 base pairs of random genomic sequence. Involvement of the TCF11 homodimer in regulation of ecNOS has not previously been demonstrated. d. Disruption of an AP4 (activator protein 4) binding site, which consists of the sequence 5′-GTCAGCCG G C-3′ (SEQ ID NO: 11), which ends at position &plus;2682 on the (-) strand. The C2684→T polymorphism replaces the C on the (&plus;) strand by a T on the (&plus;) strand. The complementary base on the (−) strand thus becomes A, rather than the reference sequence G , as indicated immediately above. AP4 is a potent transcriptional activator. Its sites occur with only moderate frequency in genomic DNA: 0.96 times per 1000 base pairs in a random genomic sequence of vertebrates. Disruption of an AP4 site is predicted to lead to a decrease in transcription of the ecNOS gene, with a resultant decrease in tissue nitric oxide production. e. Disruption of a VMAF (v-Maf) binding site, which consists of the sequence 5′-GC C GGCTGACCCTGCCTCA-3′ (SEQ ID NO: 12), beginning at position&plus;2682 on the (&plus;) strand. Thus, the C2684→T polymorphism replaces the indicated C by a T. VMAF sites occur moderately frequently, i.e., 0.99 times per 1000 base pairs of random genomic sequence in vertebrates. At the moment, very little is known about the regulation of ecNOS by the cellular homolog of v-Maf. Sim et al., Mol. Genet. Metab., 65: 562 (1998), reported a disruption of a MspI restriction site in the ecNOS gene. However, the specific MspI site reported in Sim et al., was not further identified by sequencing, and there are 11 MspI restriction sites predicted in the sequence we have examined (GenBank Accession Number AF032908). 
 Example 3 
 C to T Transition at Position 2575 Methods of DNA amplification, sequencing and data analysis were essentially as described in Example 1 except that the forward primer was 5′ gagtctggccaacacaaatcc 3′ (SEQ ID NO: 13) and the reverse primer was 5′ ctctagggtcatgcaggttctc 3′ (SEQ ID NO: 14). A substitution mutation (transition) was found in which the C found in the reference sequence (SEQ ID NO: 1) was replaced with a T. Data analysis produced the following results. 12 TABLE 9 ALLELE FREQUENCIES C T CONTROL Black men (n &equals; 64 chromosomes) 61 (95%) 3 (5%) Black women (n &equals; 70 chromosomes) 70 (100%) 0 (0%) White men (n &equals; 84 chromosomes) 84 (100%) 0 (0%) White women (n &equals; 102 102 (100%) 0 (0%) chromosomes) DISEASE BREAST CANCER Black women (n &equals; 40 chromosomes) 38 (95%) 2 (5%) White women (n &equals; 38 chromosomes) 38 (100%) 0 (0%) LUNG CANCER Black men (n &equals; 38 chromosomes) 38 (100%) 0 (0%) Black women (n &equals; 32 chromosomes) 30 (94%) 2 (6%) White men (n &equals; 40 chromosomes) 40 (100%) 0 (0%) White women (n &equals; 22 chromosomes) 22 (100%) 0 (0%) PROSTATE CANCER Black men (n &equals; 40 chromosomes) 39 (98%) 1 (3%) White men (n &equals; 40 chromosomes) 40 (100%) 0 (0%) NIDDM Black men (n &equals; 4 chromosomes) 4 (100%) 0 (0%) Black women (n &equals; 8 chromosomes) 8 (100%) 0 (0%) White men (n &equals; 8 chromosomes) 8 (100%) 0 (0%) White women (n &equals; 6 chromosomes) 6 (100%) 0 (0%) ESRD DUE TO NIDDM Black men (n &equals; 12 chromosomes) 12 (100%) 0 (0%) Black women (n &equals; 16 chromosomes) 16 (100%) 0 (0%) White men (n &equals; 10 chromosomes) 10 (100%) 0 (0%) White women (n &equals; 8 chromosomes) 8 (100%) 0 (0%) HYPERTENSION (HTN) Black men (n &equals; 22 chromosomes) 21 (95%) 1 (5%) Black women (n &equals; 16 chromosomes) 12 (75%) 4 (25%) White men (n &equals; 20 chromosomes) 20 (100%) 0 (0%) White women (n &equals; 18 chromosomes) 18 (100%) 0 (0%) ESRD DUE TO HTN Black men (n &equals; 14 chromosomes) 14 (100%) 0 (0%) Black women (n &equals; 12 chromosomes) 12 (100%) 0 (0%) White men (n &equals; 14 chromosomes) 14 (100%) 0 (0%) White women (n &equals; 8 chromosomes) 8 (100%) 0 (0%) MYOCARDIAL INFARCTION White women (n &equals; 14 chromosomes) 14 (100%) 0 (0%) 13 TABLE 10 GENOTYPE FREQUENCIES C/C C/T T/T CONTROLS Black men (n &equals; 32) 29 (91%) 3 (9%) 0 (0%) Black women (n &equals; 35) 35 (100%) 0 (0%) 0 (0%) White men (n &equals; 42) 42 (100%) 0 (0%) 0 (0%) White women (n &equals; 51) 51 (100%) 0 (0%) 0 (0%) DISEASE BREAST CANCER Black women (n &equals; 20) 18 (90%) 2 (10%) 0 (0%) White women (n &equals; 19) 19 (100%) 0 (0%) 0 (0%) LUNG CANCER Black men (n &equals; 19) 19 (100%) 0 (0%) 0 (0%) Black women (n &equals; 16) 14 (88%) 2 (13%) 0 (0%) White men (n &equals; 20) 20 (100%) 0 (0%) 0 (0%) White women (n &equals; 11) 11 (100%) 0 (0%) 0 (0%) PROSTATE CANCER Black men (n &equals; 20) 19 (95%) 1 (5%) 0 (0%) White men (n &equals; 20) 20 (100%) 0 (0%) 0 (0%) NIDDM Black men (n &equals; 2) 2 (100%) 0 (0%) 0 (0%) Black women (n &equals; 4) 4 (100%) 0 (0%) 0 (0%) White men (n &equals; 4) 4 (100%) 0 (0%) 0 (0%) White women (n &equals; 3) 3 (100%) 0 (0%) 0 (0%) ESRD due to NIDDM Black men (n &equals; 6) 6 (100%) 0 (0%) 0 (0%) Black women (n &equals; 8) 8 (100%) 0 (0%) 0 (0%) White men (n &equals; 5) 5 (100%) 0 (0%) 0 (0%) White women (n &equals; 4) 4 (100%) 0 (0%) 0 (0%) HYPERTENSION (HTN) Black men (n &equals; 11) 10 (91%) 1 (9%) 0 (0%) Black women (n &equals; 8) 4 (50%) 4 (50%) 0 (0%) White men (n &equals; 10) 10 (100%) 0 (0%) 0 (0%) White women (n &equals; 9) 9 (100%) 0 (0%) 0 (0%) ESRD due to HTN Black men (n &equals; 7) 7 (100%) 0 (0%) 0 (0%) Black women (n &equals; 6) 6 (100%) 0 (0%) 0 (0%) White men (n &equals; 7) 7 (100%) 0 (0%) 0 (0%) White women (n &equals; 4) 4 (100%) 0 (0%) 0 (0%) MYOCARDIAL INFARCTION White women (n &equals; 7) 7 (100%) 0 (0%) 0 (0%) 
 Allele-Specific Odds Ratios The susceptibility allele is indicated, as well as the odds ratio (OR). Haldane's correction was used if the denominator was zero. If the odds ratio (OR) is &gE;1.5, the 95% confidence interval (C.I.) is also given. An odds ratio of 1.5 was chosen as the threshold of significance based on the recommendation of Austin et al., in Epidemiol. Rev., 16:65-76, (1994). “&lsqb;E&rsqb;pidemiology in general and case-control studies in particular are not well suited for detecting weak associations (odds ratios <1.5).” Id. at 66. Odds ratios of 1.5 or higher are high-lighted below. 14 TABLE 11 ALLELE-SPECIFIC ODDS RATIOS SUSCEPTIBILITY DISEASE ALLELE OR 95% C.I. Breast Cancer Black women T 9.2 1.1-80 White women C 1.0 Lung Cancer Black men C 4.4 0.5-36 Black women T 11.6 1.3-101 White men C 1.0 White women C 1.0 Prostate Cancer Black men C 1.9 0.2-19 White men C 1.0 NIDDM Black men C 2.0 0.2-18 Black women C 1.0 White men C 1.0 White women C 1.0 ESRD due to NIDDM* Black men C 1.0 Black women C 1.0 White men C 1.0 White women C 1.0 Hypertension (HTN) Black men C 0.8 Black women T 50.8 6.2-418 White men C 1.0 White women C 1.0 ESRD due to HTN* 1 Black men C 2.0 0.2-20 Black women C 9.0 1.1-76 White men C 1.0 White women C 1.0 Myocardial Infarction White women C 1.0 *Compared to group with NIDDM alone. * 1 Compared to group with HTN alone. 
 Genotype-Specific Odds Ratios In Table 12, the susceptibility allele (S) is indicated; the alternative allele at this locus is defined as the protective allele (P). Also presented is the odds ratio (OR) for the SS and SP genotypes. The odds ratio for the PP genotype is 1 by definition, since it is the reference group, and is not presented in the table below. For odds ratios &gE;1.5, the asymptotic 95% confidence interval (C.I.) is also given, in parentheses. Odds ratios of 1.5 or higher are high-lighted below. Haldane's correction was used when the denominator was zero. To minimize confusion, genotype-specific odds ratios are presented only for diseases in which the allele-specific odds ratio was at least 1.5. 15 TABLE 12 GENOTYPE-SPECIFIC ODDS RATIOS SUSCEPT- IBILITY DISEASE ALLELE OR(SS) OR(SP) Breast Cancer Black women T 1.9 (0.1-32) 9.6 (1.1-85) Lung Cancer Black men T* 1.5 (0.1-25) 0.2 (0-1.8) Black women T 2.4 (0.1-41) 12.2 (1.4-109) Prostate Cancer Black men T* 1.5 (0.1-25) 0.6 (0.2-2.7) NIDDM Black men T 11.8 (0.6-218) 1.7 (0.2-17) Hypertension (HTN) Black women T 7.9 (0.5-137) 71 (8.0-628) ESRD due to HTN Black men *1 T* 3.9 (0.2-67) 0.6 (0.1-4.9) Black women *1 T* 5.5 (0.3-93) 5.5 (0.3-93) *C, not T, is the susceptibility allele according to the allele-specific odds ratio (see table above). *1 Compared to group with HTN alone. The control samples agree with Hardy-Weinberg equilibrium, as follows: A frequency of 0.95 for the C allele (“p”) and 0.05 for the T allele (“q”) among black male control individuals predicts genotype frequencies of 90% C/C, 10% C/T, and 0% T/T at Hardy-Weinberg equilibrium (p 2 &plus;2pq&plus;q 2 &equals;1). The observed genotype frequencies were 91% C/C, 9% C/T, and 0% T/T, in excellent agreement with those predicted for Hardy-Weinberg equilibrium. A frequency of 1.0 for the C allele (“p”) and 0 for the T allele (“q”) among black female control individuals predicts genotype frequencies of 100% C/C, 0% C/T, and 0% T/T at Hardy-Weinberg equilibrium (p 2 &plus;2pq&plus;q 2 &equals;1). The observed genotype frequencies were 100% C/C, 0% C/T, and 0% T/T, in perfect agreement with those predicted for Hardy-Weinberg equilibrium. A frequency of 1.0 for the C allele (“p”) and 0 for the T allele (“q”) among white male control individuals predicts genotype frequencies of 100% C/C, 0% C/T, and 0% T/T at Hardy-Weinberg equilibrium (p 2 &plus;2pq&plus;q 2 &equals;1). The observed genotype frequencies were 100% C/C, 0% C/T, and 0% T/T, in perfect agreement with those predicted for Hardy-Weinberg equilibrium. A frequency of 1.0 for the C allele (“p”) and 0 for the T allele (“q”) among white female control individuals predicts genotype frequencies of 100% C/C, 0% C/T, and 0% T/T at Hardy-Weinberg equilibrium (p 2 &plus;2pq&plus;q 2 &equals;1). The observed genotype frequencies were 100% C/C, 0% C/T, and 0% T/T, in perfect agreement with those predicted for Hardy-Weinberg equilibrium. Using an allele-specific odds ratio of 1.5 or greater as a practical level of significance, the following observations can be made. Among black women with breast cancer, the odds ratio for the T allele at this locus was 9.2 (95% CI, 1.1-80). The odds ratio for the TC heterozygote was 9.6 (95% CI, 1.1-85), considerably higher than for the TT homozygote, which was 1.9 (95% CI, 0.1-32). When the heterozygote has a different odds ratio than either homozygote, the alleles are said to be codominant (Khoury et al., Fundamentals of Genetic Epidemiology , Oxford University Press: 33 (1993)). For black men with lung cancer, the odds ratio for the C allele at this locus was 4.4 (95% CI, 0.5-36). However, in this case the genotype-specific odds ratios were unhelpful in suggesting whether the C allele functions as a recessive, dominant, or codominant allele because the C allele no longer appears as the susceptibility allele. For black women with lung cancer, the odds ratio for the T allele at this locus was 11.6 (1.3-101). Inspection of the genotype-specific odds ratios suggests that the T allele is codominant, since the heterozygote has a much higher odds ratio (12.2, 95% CI 1.4-109) than the TT homozygote (2.4, 95% CI 0.1-41) or the reference CC genotype (odds ratio 1, by definition). For black men with prostate cancer, the odds ratio for the C allele at this locus was 1.9 (95% CI, 0.2-19). However, in this case the genotype-specific odds ratios are unhelpful in suggesting whether the C allele functions as a recessive, dominant, or codominant allele because the C allele no longer appears as the susceptibility allele. For black men with NIDDM, the odds ratio for the C allele at this locus was 2.0 (95% CI, 0.2-18). However, in this case the genotype-specific odds ratios are again unhelpful in suggesting whether the C allele functions as a recessive, dominant, or codominant allele because the C allele no longer appears as the susceptibility allele. For black women with hypertension (HTN), the odds ratio for the T allele at this locus was 50.8 (95% CI, 6.2-418). Inspection of the genotype-specific odds ratios suggests that the T allele is codominant, since the heterozygote had a much higher odds ratio (71, 95% CI 8.0-628) than the TT homozygote (7.9, 95% CI, 0.5-137) or the reference CC genotype (odds ratio 1, by definition). For black men with ESRD due to hypertension (HTN), the odds ratio for the C allele at this locus was 2.0 (95% CI, 0.2-20) when compared with black men with HTN. However, in this case the genotype-specific odds ratios were unhelpful in suggesting whether the C allele functions as a recessive, dominant, or codominant allele because the C allele no longer appears as the susceptibility allele. For black women with ESRD due to hypertension (HTN), the odds ratio for the C allele at this locus was 9.0 (95% CI, 1.1-76) when compared with black women with HTN. However, in this case the genotype-specific odds ratios were unhelpful in suggesting whether the C allele functions as a recessive, dominant, or codominant allele because the C allele no longer appears as the susceptibility allele. According to commercially available software &lsqb;GENOMATIX MatInspector Professional; http://genomatix.qsf.de/cqi-bin/matinspector/matinspector.pl; Quandt et al., Nucleic Acids Res. 23: 4878-4884 (1995)&rsqb;, the G2458→A SNP is predicted to have the following potential effects on transcription of the ecNOS gene: a. Disruption of a STAF — 01 (Se-Cys tRNA gene transcription activating factor 1) site (5′-AAACCCCAGCATGCA C TCTGGC-3′ (SEQ ID NO: 15) beginning at position 2560 on the (&plus;) strand. This polymorphism results in replacement of the indicated C by a T. STAF — 01 sites occur extremely rarely in the genome: 0.02 occasions per 1000 base pairs of random genomic sequence in vertebrates. STAF is a transcriptional activator possessing seven zinc finger domains. It belongs to a family of similar transcription factors (Myslinski et al., J. Biol. Chem., 273(34):21998-22006, 1998). Although originally described as an activator of transcription by RNA polymerase III from the selenocysteine tRNA gene in Xenopus and the mouse, and by RNA polymerase II from small nuclear RNA-type genes such as U6 snRNA in humans, STAF can also activate transcription of other genes by RNA polymerase II (Schuster et al., Mol. Cell Biol., 18(5):2650-2658, 1998). Since STAF is a positive transcriptional regulator, disruption of its binding site is expected to result in a decreased rate of transcription of the ecNOS gene. If the rate of translation is tied to the level of messenger RNA, as is the case for many proteins, then the T allele is expected to result in less gene product (ecNOS enzyme), ultimately leading to less nitric oxide (NO) produced in tissues such as endothelial cells. b. Disruption of a TH1E47 — 01 (Thing1/E47 heterodimer) site. Thing1 is also called Hxt, eHAND, or Hand1 (Scott et al., Mol. Cell. Biol., 20(2):530-541, 2000). The putative binding site for the heterodimer (5′-CATGCA C TCTGGCCTG-3′ (SEQ ID NO: 16) begins at position &plus;2569 on the (&plus;) strand. This polymorphism results in replacement of the indicated C by a T. TH1E47 — 01 sites occur relatively often in the genome: 2.04 occasions per 1000 base pairs of random genomic sequence in vertebrates. E47 usually functions as a transcriptional activator. Binding of E47 by Thing1/Hxt/eHAND/Hand1, which itself can be a transcriptional activator for trophoblast during development (Scott et al., op. cit.), may actually result in repression of E47's activity. As a further complication to predicting the nature of TH1E47's effect on the ecNOS gene, whether positive or negative, activity of the E47 homodimer is repressed by phosphorylation (Neufeld B et al., J. Biol. Chem., 275(27): 20239-42, 2000). Phosphorylation has not yet been reported to affect the activity of the Hand1/E47 heterodimer. c. Disruption of an NF1_Q6 (nuclear factor 1) site (5′- C TCTGGCCTGAAGTGCCT-3′ (SEQ ID NO: 17) beginning at position &plus;2575 on the (&plus;) strand. This polymorphism results in replacement of the indicated C by a T. NF1_Q6 sites occur relatively frequently in the genome: 4.11 sites per 1000 base pairs of random genomic sequence in vertebrates. NF1, usually a transcriptional activator, has not yet been shown to affect expression of the ecNOS gene. 
 Example 4 
 Deletion at Position 1272 Sample collection and DNA isolation were as described in Example 1. 
 DNA Amplification DNA encoding the eNOS promoter region was amplified by polymerase chain reaction (PCR). One hundred nanograms of purified genomic DNA was used in each PCR reaction. The forward primer was 5′ agcagtgcaccaaggaaaatgagg 3′ (SEQ ID NO: 18) and the reverse primer was 5′ agtgcagtggtgtgatcttggttc 3′ (SEQ ID NO: 19). The reaction mix consisted of 100 ng leukocyte genomic DNA, 10 pmol of each primer, 200 nM dNTPs, 1 U Taq DNA polymerase (Perkin-Elmer), 1X PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2 , and 0.01% &lsqb;w/v&rsqb; gelatin) and 3% (v/v) DMSO. The total reaction volume was 25 &mgr;l. The PCR protocol used consisted of 4 minutes at 95° C. followed by 29 cycles of a 40 second denaturation step at 95° C., a 20 second annealing step at 59° C. and a 1 minute extension step at 73° C. After the completion of the 29 cycles a final extension reaction was conducted at 73° C. for 4 minutes. The PCR product obtained was then purified using QIAquick 96 PCR purification kit (Qiagen, Inc. Valencia, Calif.) following the manufacturer's protocol. Purified PCR product was then used for sequencing. 
 DNA Sequencing Purified PCR product was sequenced by cycle sequencing using a Perkin-Elmer dye terminator kit according to the manufacturer's protocol Briefly, 8 &mgr;l of terminator ready reaction mix (PE Applied Biosystems, Foster City, Calif.) was combined with 5 ng of PCR product obtained by the method of Example 1 which served as the template. To this was added 3.2 pmol of primers and deionized water to 10 &mgr;l. Primers used were the same as those used in the original PCR amplification. The cycling protocol consisted of 25 cycles of a 10 second denaturation step at 96° C., a 5 second annealing step at 50° C. and a 4 minute extension step at 60° C. After the last cycle, the reaction mixture was cooled to 4° C. until purification. Unincorporated dye was removed from the sequencing products by ethanol precipitation and loaded onto sequencing cells on either Applied Biosystems (ABI 377) or Licor automatic gel sequencers. Two &mgr;l samples in sample buffer (5:1 100% formamide:blue dextran dye) were loaded onto sequencing gels and run at 2.4 kV for 6 hours in 1X TBE running buffer. Laser scans of the gel were at a rate of 1200 per hour. Peaks generated were analyzed by eye for heterozygosity. On sample was run per lane of the gel. 
 Results A deletion polymorphism was found at position 1272 of SEQ ID NO: 1 in which the reference sequence C at position 1272 is deleted. This mutation was found in 27% of patients with ESRD due to NIDDM and 20% of patients with ESRD due to HTN, but not in the reference sequence. This deletion causes disruption of a potential NF-1 (nuclear factor 1) site (CTTTGGCACTAC C CAAAA) (SEQ ID NO: 20) beginning at position 1259 on the (−) strand. NF-1 sites occur relatively frequently with 4.11 sites per 1000 base pairs of random genomic DNA in vertebrates. Since NF-1 is a transcriptional activator, disruption of its binding site is expected to result in a decreased rate of transcription of the ecNOS gene. If the rate of translation is tied to the level of messenger RNA, as is the case for most proteins, then less gene product (ecNOS enzyme) will be the result, ultimately leading to less nitric oxide (NO) produced in tissues such as endothelial cells. This deletion also causes disruption of a potential BARBIE (barbiturate-inducible element) site (TGC C AAAGCGTAAGG) (SEQ ID NO: 21) beginning at position 1269 on the (&plus;) strand. BARBIE is a transcriptional regulator not yet linked with regulation of the ecNOS gene. BARBIE sites occur with considerably less frequency than NF-1 sites at a rate of 0.56 times per 1000 base pairs of random genomic sequence in vertebrates. 
 Example 5 
 T to A Substitution at Position 2841 DNA isolation, purification, amplification and sequencing were as described in Example 4 except the forward primer was 5′ gagtctggccaacacaaatcc 3′ (SEQ ID NO: 3) and the reverse primer was 5′ ctctagggtcatgcaggttctc 3′ (SEQ ID NO: 22). A substitution polymorphism (transversion) was found in which the reference sequence T at position 2841 of SEQ ID NO: 1 is replaced with an A. This polymorphism was found in 29% of patients with ESRD due to NIDDM, but not in the reference sequence or patients with ESRD due to HTN. This polymorphism disrupts the predicted binding site of NFY (nuclear factor Y), with sequence GCCCCA A TTTC, (SEQ ID NO: 23) ending at position 2837 on the (−) strand. The T2837→A polymorphism replaces the nucleotide T on the (&plus;) strand with an A. The corresponding reference sequence nucleotide on the (−) strand is therefore changed from the A , indicated in the NFY binding site sequence immediately above, to a T. Disruption of the NFY binding site is expected to result in reduced transcription of the ecNOS gene, since NFY is a potent transcriptional activator. NFY binding sites occur with extreme rarity, <0.01 sites per 1000 base pairs of random genomic sequence in vertebrates. Thus, finding a SNP at this site is strongly suggestive that it is a causal SNP in end-stage renal disease due to NIDDM. 
 Example 6 
 G to T Substitution at Position 2843 DNA isolation, purification, amplification and sequencing were as described in Example 5. A substitution polymorphism (transversion) was found in which the reference sequence G at position 2843 of SEQ ID NO: 1 is replaced with a T. This polymorphism was found in 29% of patients with ESRD due to NIDDM and 14% of patients with ESRD due to HTN, but not in the reference sequence. This polymorphism disrupts the predicted binding site of NFY (nuclear factor Y), GCCC C AATTTC, (SEQ ID NO: 23) ending at position 2837 of SEQ ID NO: 1 on the (-) strand. The G-630→T polymorphism replaces the reference sequence nucleotide G on the (&plus;) strand with a T. The corresponding nucleotide on the (−) strand is therefore changed from the C , indicated in the NFY binding site sequence immediately above, to an A. Disruption of the NFY binding site in this core region is expected to result in reduced transcription of the ecNOS gene, since NFY is a potent transcriptional activator. NFY binding sites occur with extreme rarity, <0.01 sites per 1000 base pairs of random genomic sequence in vertebrates. Thus, finding a SNP at this site is strongly suggestive that it is a causal SNP in end-stage renal disease due to NIDDM, and, to a lesser extent, hypertension. 
 Example 7 
 G to T Substitution at Position 3556 DNA isolation, purification, amplification and sequencing were as described in Example 4 except that the forward primer was 5′ atccttgctgggcctctat 3′ (SEQ ID NO: 24) and the reverse primer was 5′ tgcttgccgcacagcccaa3′ (SEQ ID NO: 25). A substitution polymorphism (transversion) was found in which the G at position 3556 of SEQ ID NO: 1 is replaced with a T. This polymorphism was found in 50% of patients with ESRD due to HTN, but not in the reference sequence or patients with ESRD due to NIDDM. This polymorphism produces a missense mutation of Glycine in exon 1 (encoded by G GG, codon 18) to Tryptophan (encoded by T GG). This G18W amino acid mutation replaces a small amino acid with a bulky hydrophobic one, which may interfere with protein conformation and ultimately enzymatic activity. Reduced enzymatic activity would result in decreased nitric oxide production in tissues, consistent with the results predicted for all of the above SNPs. 
 Conclusion In light of the detailed description of the invention and the examples presented above, it can be appreciated that the several aspects of the invention are achieved. It is to be understood that the present invention has been described in detail by way of illustration and example in order to acquaint others skilled in the art with the invention, its principles, and its practical application. Particular formulations and processes of the present invention are not limited to the descriptions of the specific embodiments presented, but rather the descriptions and examples should be viewed in terms of the claims that follow and their equivalents. While some of the examples and descriptions above include some conclusions about the way the invention may function, the inventor does not intend to be bound by those conclusions and functions, but puts them forth only as possible explanations. It is to be further understood that the specific embodiments of the present invention as set forth are not intended as being exhaustive or limiting of the invention, and that many alternatives, modifications, and variations will be apparent to those of ordinary skill in the art in light of the foregoing examples and detailed description. Accordingly, this invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and scope of the following claims.