Patent Application: US-53819803-A

Abstract:
the invention provides a method and kit for detecting or diagnosing a risk of or predisposition to diabetes or a metabolic syndrome in a subject , the method comprising the steps of providing a biological sample of the subject to be tested and detecting the presence or absence of a variant genotype of the human α 2b . adrenoceptor in the biological sample . the presence of the variant genotype indicates an increased risk of diabetes or a metabolic syndrome in said subject . the invention also relates to a method for the treatment of type 2 diabetes .

Description:
the present invention relates to a dna molecule encoding a variant human α 2b - adrenoceptor , said variant α 2b - adrenoceptor protein and a method to assess the risk of individuals to develop hypertension in mammals as well as a method for the targeting treatment for hypertension . the concept “ a deletion of at least 1 glutamate from a glutamic acid repeat element of 12 glutamates ” refers to any deletion of 1 to 12 glutamates irrespective of the specific location in , or how many glutamates from said repeat element of 12 glutamates , amino acids 298 - 309 ( seq id no : 4 ), in an acidic stretch of 18 amino acids 294 - 311 located in the 3 rd intracellular loop of the receptor polypeptide are deleted . the concept “ deletion / deletion ( d / d ) genotype of the human α 2b - adrenoceptor ”, in short “ d / d genotype ”, refers to a genotype of an individual having both α 2b - adrenoceptor alleles code for a variant α 2b - adrenoceptor with a deletion of at least 1 glutamate from a glutamic acid repeat element of 12 glutamates , amino acids 298 - 309 , in an acidic stretch of 18 amino acids 294 - 311 ( seq id no : 4 ), located in the 3 rd intracellular loop of the receptor polypeptide . correspondingly “ deletion / insertion ( d / i ) genotype ” refers to a genotype having one of the gene alleles code for an α 2b - adrenoceptor with a said deletion and the other without a said deletion , i . e . with a respective insertion , and thus the “ insertion / insertion ( i / i ) genotype ” refers to a genotype having both alleles code for an α 2b - adrenoceptor without said deletion or deletions . a common variant form ( seq id no : 1 ) of the human α 2b - ar gene ( seq id no : 3 ) was recently identified ( heinonen et al . 1999 ). this variant gene encodes a receptor protein ( seq id no : 2 ) with a deletion of 3 glutamates , amino acids 307 - 309 , from a glutamic acid ( glu ) repeat element of 12 glutamates , amino acids 298 - 309 , in an acidic stretch of 18 amino acids 294 - 311 ( seq id no : 4 ), located in the 3 rd intracellular loop of the receptor polypeptide . this variant gene ( seq id no : 1 ) was associated with decreased basal metabolic rate ( bmr ) in a group of obese finnish subjects ( heinonen et al . 1999 ). of the 166 obese subjects , 47 ( 28 %) were homozygous for the long 12 glutamate repeat element ( glu 12 / glu 12 ), whereas 90 ( 54 %) were heterozygous ( glu 12 / glu 9 ) and 29 ( 17 %) were homozygous for the short form ( glu 9 / glu 9 ). the results to be presented below show that in a population sample of 912 finnish middle - aged men subjects homozygous for the short form ( glu 9 / glu 9 ) described above , thus representing a deletion / deletion ( d / d ) genotype of the α 2b - adrenoceptor , have a significantly elevated risk for hypertension . based on these results and previous publications referred to above it can be postulated that this dad genotype is related to an impaired capacity to downregulate α 2b - adrenoceptor function during sustained receptor activation . since altered α 2b - adrenoceptor function seems to be of relevance in the pathogenesis of hypertension , we believe it could also be of relevance in subjects with the insertion / deletion ( i / d ) ( heterozygous glu 12 / glu 9 ) and insertion / insertion ( i / i ) ( homozygous glu 12 / glu 12 ) genotypes when other risk factors for hypertension are present . further , since this specific deletion of 3 glutamates from said glutamic acid repeat element of 12 glutamates , amino acids 298 - 309 , in said acidic stretch of 18 amino acids 294 - 311 , located in the 3 rd intracellular loop of the receptor polypeptide seems to be of relevance in hypertension we believe that also other deletions , i . e . deletions of at least 1 glutamate , from said glutamic acid repeat element of 12 glutamates , amino acids 298 - 309 , could be of relevance in the pathogenesis of hypertension , because the 3 rd intracellular loop of the receptor polypeptide it is located in seems to have an essential role in the down - regulation of the α 2b - adrenoceptor . thus persons with the did genotype have chronically up - regulated α 2b - adrenoceptors , leading to the elevation of systemic blood pressure . α 2b -- adrenoceptors mediate contraction of arteries , and genetic polymorphism present in the α 2b - adrenoceptor gene renders some subjects more susceptible to α 2b - adrenoceptor mediated vasoconstriction of the blood pressure regulating resistance arteries ( arteriolies ) and associated clinical disorders such as hypertension . these subjects will especially benefit from treatment with an α 2b - adrenoceptor antagonist , and will be at increased risk for adverse effects if subtype - nonselective α 2 - agonists are administered to them . therefore , a gene test recognizing subjects with a deletion variant of the α 2b - adrenoceptor gene will be useful in diagnostics and patient selection for specific therapeutic procedures and clinical drug testing trials . a gene test recognizing the d / d genotype of the α 2b - adrenoceptor is useful in assessing an individual &# 39 ; s risk to develop hypertension related to the d / d genotype . the test can be used to set a specific subdiagnosis of hypertension , based on its genetic etiology . furthermore , a gene test recognizing the d / d genotype of the α 2b - adrenoceptor is useful in selecting drug therapy for patients with hypertension . such drugs are e . g . a drug modulating , inhibiting or activating the vascular alpha - or beta - adrenargic receptors of the subjects either directly or through central nervous system effects , for example pindolol , propranolol , sotalol , timolol , acebutolol , atenol , betaxolol , bisoprol , esmolol , metoprolol , seliprol , carvedilol , labetalol , clonidine , moxonidine , prazosin , or indapamid , including α - adrenoceptor antagonists ( α 2b - selective or nonselective ). for instance , as angiotensin ii causes an increase of noradrenaline sensitivity , and this effect is at least in part mediated by α - adrenoceptors ( datte et al . 2000 ), the blood pressure lowering effect of drugs acting through angiotensin ii inhibition , such as the angiotensin ( at ) receptor blockers , is conceivably enhanced in persons with the d / d genotype of the α 2b - adrenoceptor . such drugs are for example captopril , cinapril , enalapril , imidapril , lisinopril , moexipril , perindopril , ramipril , trandolapril , candesartan , eprosartan , irbesartan , losartan , valsartan or telmisartan . a gene test recognizing the d / d genotype of the α 2b - adrenoceptor is useful in selecting drug therapy for patients who might be at increased risk for adverse effects of α 2 - adrenergic agonists ; either it will be possible to avoid the use of α 2 - agonists in such patients , or it will be possible to include a specific α 2b - antagonist in their therapeutic regimen . on the other hand , it is conceivable that the patients with other than the d / d genotype will benefit more from other antihypertensive drugs . the dna sequence can be used for screening a subject to determine if said subject is a carrier of a variant gene . the determination can be carried out either as a dna analysis according to well known methods , which include direct dna sequencing of the normal and variant gene , allele specific amplification using the polymerase chain reaction ( pcr ) enabling detection of either normal or variant sequence , or by indirect detection of the normal or variant gene by various molecular biology methods including e . g . pcr - single stranded conformation polymorphism ( sscp ) method or denaturing gradient gel electrophoresis ( dgge ). determination of the normal or variant gene can also be done by using a restriction fragment length polymorphism ( rflp ) method , which is particularly suitable for genotyping large numbers of samples . similarly , a test based on gene chip or array technology can be easily developed in analogy with many currently existing such tests for single - nucleotide polymorphisms . the determination can also be carried out at the level of rna by analyzing rna expressed at tissue level using various methods . allele specific probes can be designed for hybridization . hybridization can be done e . g . using northern blot , rnase protection assay or in situ hybridization methods . rna derived from the normal or variant gene can also be analyzed by converting tissue rna first to cdna and thereafter amplifying cdna by an allele specific pcr method . the kit for use in the method according to the invention preferably contains the various components needed for carrying out the method packaged in separate containers and / or vials and including instructions for carrying out the method . thus , for example , some or all of the various reagents and other ingredients needed for carrying out the determination , such as buffers , primers , enzymes , control samples or standards etc can be packaged separately but provided for use in the same box . instructions for carrying out the method can be included inside the box , as a separate insert , or as a label on the box and / or on the separate vials . the kit may also contain the necessary software needed to interpret the results obtained with the kit , or for utilizing the results from a gene chip used in the method . the invention will be described in more detail in the experimental section . the polymerase chain reaction - single stranded conformational analysis ( pcr - ssca ) used to identify the genomic alleles encoding the α 2b -- adrenoceptor was carried out as follows : the genomic dna encoding the α 2b - adrenergic receptor was amplified in two parts specific for the intronless α 2b - adrenoceptor gene sequence ( lomasney et al . 1990 ). the pcr primer pairs for pcr amplification were as follows : pair 1 : 5 ′- ggggcgacgctcttgtcta - 3 ′ ( seq id no : 5 ) and 5 ′- ggtctccccctcctccttc - 3 ′ ( seq id no : 6 ) ( product size 878 bp ), pair 2 : 5 ′- gcagcaaccgcagaggtc - 3 ′ ( seq id no : 7 ) and 5 ′- gggcaagaagcagggtgac - 3 ′ ( seq id no : 8 ) ( product size 814 bp ). the primers were delivered by kebolab ( helsinki , finland ). pcr amplification was conducted in a 5 μl volume containing 100 ng genomic dna ( isolated from whole blood ), 2 . 5 mmol / l of each primer , 1 . 0 mmol / l deoxy - ntps , 30 nmol / l 33 p - dctp and 0 . 25 u amplitaq dna polymerase ( perkin elmer cetus , norwalk , conn .). pcr conditions were optimized using the pcr optimizer ™ kit ( invitrogen , san diego , calif .). samples were amplified with a geneamp pcr system 9600 ( perkin elmer cetus ). pcr products were digested with restriction enzymes for ssca analysis . the product of primer pair 1 was digested with dde i and dra iii ( promega corp ., madison , wis .). the product of primer pair 2 was digested with alu i and hinc ii ( promega corp .). the digested samples were mixed with ssca buffer containing 95 % formamide , 10 mmol / l naoh , 0 . 05 % xylene cyanol and 0 . 05 % bromophenol blue ( total volume 25 μl ). before loading , the samples were denatured for 5 min at 95 ° c . and kept 5 min on ice . three microliters of each sample were loaded on mde ™ high - resolution gel ( fmc , bioproducts , rockland , mass .). the gel electrophoresis was performed twice , at two different running conditions : 6 % mde gel at + 4 ° c . and 3 % mde gel at room temperature , both at 4 w constant power for 16 h . the gels were dried and autoradiography was performed by apposing to kodak biomax mr film for 24 h at room temperature . dna samples migrating at different rates in ssca were sequenced with the thermo sequenase ™ cycle sequencing kit ( amersham life science , cleveland , ohio ). for genotyping the identified 3 - glutamic acid deletion , dna was extracted from peripheral blood using standard methods . the α 2b - ar i / d genotype was determined by separating pcr - amplified dna fragments with . electrophoresis . based on the nature of the i / d variant , identification of the long and short alleles was achieved by their different electrophoretic migration rates due to their 9 bp size difference . the region of interest was amplified using a sense primer 5 ′- agg - gtg - ttt - gtg - ggg - cat - ct - 3 ′ ( seq id no : 9 ) and an anti - sense primer 5 ′- caa - gct - gag - gcc - gga - gac - act - 3 ′ ( seq id no : 10 )( oligold , eurogentec , belgium ), yielding a product size of 112 bp for the long allele ( i ) and 103 bp for the short allele ( d ). pcr amplification was conducted in a 10 μl volume containing ˜ 100 ng genomic dna , 1 × buffer g ( invitrogen , san diego , calif ., usa ), 0 . 8 mm dnts , 0 . 3 μm of each primer and 0 . 25 units of amplitaq dna polymerase ( perkin elmer cetus , norwalk , conn ., usa ). samples were amplified with a geneamp pcr system 9600 ( perkin elmer cetus ). after initial denaturation at 94 ° c . for 2 minutes , the samples were amplified over 35 cycles . pcr amplification conditions were 96 ° c . ( 40 s ), 69 ° c . ( 30 s ) and 72 ° c . ( 30 s ) followed by final extension at 72 ° c . for 6 minutes . the pcr products representing the long and short alleles were identified by two alternative methods . 1 ) the amplified samples were mixed with 4 μl of stop solution ( thermo sequenase ™ cycle sequencing kit ), heated to 95 ° c . for 2 min , and loaded hot onto sequencing gels ( long ranger ™, fmc ). the gels were dried and autoradiography was performed as previously described . 2 ) separation of the amplified pcr products was performed with electrophoresis on a high - resolution 4 % metaphor agarose gel ( fmc bioproducts , rockland , me .) and the bands were visualized by ethidium bromide staining . in both methods , the long ( glu 12 ) and short ( glu 9 ) alleles were identified based on their different electrophoretic migration rates . the above referred population study of 912 finnish middle - aged men subjects including 192 subjects with a specific deletion / deletion ( d )/ d ) genotype of the α 2b - adrenoceptor is described in more detail in the following : knowing the vasoconstrictive property of α 2b - ar in mice and the possible involvement of the investigated acidic region in the desensitization mechanism of the receptor we hypothesized that the observed insertion / deletion allelic variation could be associated with hypertension . to test this hypothesis , we carried out a population study in 912 middle - aged finnish men with no prior history of coronary heart disease . the study was carried out as part of the kuopio ischemic heart disease risk factor study ( kmid ), which is an ongoing population - based study designed to investigate risk factors for cardiovascular diseases and related outcomes in men from eastern finland ( salonen 1988 ). this area is known for its homogenous population ( sajantila et al . 1996 ) and high coronary morbidity and mortality rates ( keys 1980 ). of the 912 subjects , 192 ( 21 %) had the d / d genotype , 256 ( 28 %) had the i / i genotype and 464 ( 51 %) were heterozygous i . e . i / d . this genotype distribution is in hardy - weinberg equilibrium ( p = 0 . 46 ). four hundred and seventeen men had no family history of hypertension , and 495 had hypertension in the family ( either parents or siblings or both ). it was assumed that genetic traits would have a stronger association with hypertension in the subjects who had a history of hypertension in the family , and thus the association between the α 2b - adrenoceptor genotype and hypertension was analyzed separately in men with and without family history ( tables 1 and 2 ). in a multivariate linear regression model , men with the dd genotype had on the average a higher mean systolic blood pressure ( bp ) as compared with the other genotypes ( p = 0 . 021 ) among men with a family history ( table 1 ). among those with a family history of hypertension , dd homozygous men had , in a multivariate logistic model , a 2 . 04 - fold ( 95 % confidence interval 1 . 06 to 3 . 93 , p = 0 . 032 ) probability ( prevalence ) of hypertension ( either systolic bp at least 165 mmhg or diastolic bp at least 95 mmhg or antihypertensive medication , table 2 ). the association of the use of α - adrenoceptor antagonists such as prazosin with hypertension was analyzed among the dd homozygous men and other men , separately . the antihypertensive effect was estimated as the blood pressure difference between the specific drug type vs other drugs . in men with the dd genotype but not among the other men , the use of α - adrenoceptor antagonists was associated with a lowering of both systolic and diastolic blood pressure as well as decreased occurrence of a number of self - reported adverse effects . among 440 men who were hypertensive at the 11 - year follow - up ( systolic bp ≧ 165 mmhg or diastolic bp ≧ 95 mmhg or antihypertensive treatment ), among men with the d / d α 2b - adrenoceptor genotype , the means systolic bp was 111 mmhg in those treated with alpha - blocker and 137 mmhg in those treated with other drugs , whereas these means were 150 mmhg and 138 mmhg in men with other genotypes . there was a similar trend for beta - adrenoceptor antagonists ( beta - blockers ) such as atenolol , metoprolol and pindolol , as well as for angiotensin converting enzyme ( ace ) inhibitors such as captopril , enalapril and lisinopril . for example , among 344 men , who were hypertensive in the kihd baseline examination the mean systolic blood pressure was 11 years later among subjects with dad genotype 134 mmhg in those treated with beta - blocker and 141 mmhg among those treated with other drugs , whereas for men with other genotypes these means were 137 and 138 mmhg . among the 440 men who were hypertensive at the 11 - year follow - up , in those with the d / d genotype , the mean systolic bp was 133 in beta - blocker treated and 139 in others , whereas in men with other genotypes these means were 139 and 138 mmhg . among men who were treated with β - blockers , the mean systolic blood pressure was 128 . 8 ( sd 16 . 2 ) in those with the d / d genotype and 135 . 5 mmhg ( sd 19 . 3 ) in those with other genotypes ( p = 0 . 04 for difference ). in a linear covariance model adjusting for age and body - mass index ( kg / m2 ), the genotype - β - blocker interaction was statistically significant ( 1 - sided p = 0 . 04 ). the antihypertensive effect of antihypertensive drug types acting through other mechanisms than adrenoceptor or noradrenaline sensitivity modulation and was greater in men with other than the did genotype . for example , the blood pressure lowering effect of diuretics and calcium channel blockers was larger in α 2b - ar genotypes other than d / d . men with the dd genotype had an increased prevalence of adverse effects and a smaller antihypertensive response during α 2 - adrenoceptor agonist therapy such as clonidin . taken together , the known biological properties of the α 2b - ar , the homogeneity of the finnish population , the study design , the relatively large representative study population and the association of hypertension with one trait suggest that the d / d receptor allele is a causal genetic risk factor for hypertension . it will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments , only a few of which are disclosed herein . it will be apparent for the specialist in the field that other embodiments exist and do not depart from the spirit of the invention . thus , the described embodiments are illustrative and should not be construed as restrictive . datte j - y , gohlke p , pees c , ziegler a . short treatments of normotensive and hypertensive rats by angiotensin ii and nitric oxide inhibitor induce and increase of noradrenaline sensitivity in isolated vena portae preparations . pharmacol res 2000 ; 41 : 641 - 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805 . lomasney j w , lorenz w , allen l f , king k , regan j w , yang - feng t l , caron m c , lefkowitz r j : expansion of the alpha - 2 adrenergic receptor family : cloning and characterization of a human alpha - 2 adrenergic receptor subtype , the gene for which is located on chromosome 2 . proc natl acad sci usa . 1990 ; 87 : 5094 - 5098 . macdonald e , kobilka b k , scheinin m : gene targeting — homing in on α 2 - adrenoceptor subtype function . trends pharmacol sci 1997 ; 18 : 211 - 219 macmillan l b , hein l , smith m s , piascik m t , limbird l e : central hypotensive effects of the alpha2a - adrenergic receptor subtype . science 1996 ; 273 : 801 - 803 sajantila a , salem a h , savolainen p , bauer k , gierig c , paabo s : paternal and maternal dna lineages reveal a bottleneck in the founding of the finnish population . proc . natl . acad . sci . u . s . a . 1996 ; 93 : 12035 - 12039 salonen j t : is there a continuing need for longitudinal epidemiologic research ? the kuopio ischaemic heart disease risk factor study . ann . clin res 1988 ; 20 : 46 - 50