WO2008109110A2 - System for regulating blood pressure - Google Patents

Abstract

A system is provided for evaluating a quantitative trait locus (QTL) or functional fragment derived thereof, where the QTL at least partially controls blood pressure and/or at least partially is indicative of a potential for developing a hypertensive disease.

Description

TITLE

SYSTEM FOR REGULATING BLOOD PRESSURE

Inventor: Bina Joe

CROSS-REFERENCE TO RELATED APPLICATIONS AND STATEMENT REGARDING SPONSORED RESEARCH

[0001] The present invention claims the benefit of the provisional patent application Ser. No. 60/905,186 filed March 6, 2007. This invention was made with government support under NIH Grant No. 5R01HL075414. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0002] Essential hypertension, which is the most common cardiovascular abnormality, is characterized by sustained blood pressure (BP) with no identifiable cause ^l. Genetic susceptibility factors account for approximately 27% of an individual's risk of developing essential hypertension ^~. Mapping experiments in humans is by far the only way by which one could attempt to identify the genetic role players in elevated BP. Such studies have successfully identified genetic determinants of monogenic forms of hypertension ^J~* , but few have emerged successful in defining the underlying susceptibility factors for essential hypertension, which represents 95% of all forms of hypertension. This void has a bearing on current clinical management practices for essential hypertension, which are limited to symptomatic treatments with select hypotensive agents.

[0003] Unlike mapping studies in humans, studies in experimental rat models that mimic essential hypertension are not limited to linkage analysis. Follow-up genetic dissections using recombinant congenic strains have allowed for rapid fine-mapping of several BP quantitative trait loci (QTLs) ^lj'13, one of which was previously identified in our laboratory using the Dahl salt-sensitive (S) rat ^16r17.

[0004] Thus, what are needed are improved methods to treat essential hypertension, to decrease fatality and to decrease the appearance and/or severity of the consequent debilitating symptoms associated with hypertension. What is also needed is a method to determine the susceptibility of individuals to hypertension, in efforts to prevent the appearance and/or severity of symptoms. What is also needed is a method to identify those individuals for whom hypertension appears to be genetic.

[0005] The present invention provides an improved system for defining underlying susceptibility factors for essential hypertension.

SUMMARY OF THE INVENTION

[0006] In one aspect, there is provided a method for selecting an animal model of disease for having desired genotypic properties. The method includes testing the animal for the presence of a parentally imprinted quantitative trait locus (QTL) that at least partially controls blood pressure and/or at least partially is indicative of a potential for developing a hypertensive disease. In certain embodiments, the method further includes testing a nucleic acid sample from the animal for the presence of QTL. The animal can be a rat and the QTL is located at chromosome 17. The QTL is mapping at around position IpI 1. In other embodiments, the animal is a mouse and the QTL is located at chromosome- 13.

[0007] The QTL is related to the potential to develop hypertension. In a particular embodiment, wherein the QTL comprises at least a part of Adamtslό gene. Also, in the QTL of the rat can comprise a marker marked with an asterisk (*), as identified in Table 1.

[0008] In another aspect, there is provided an isolated and/or recombinant nucleic acid sequence comprising a quantitative trait locus (QTL) or functional fragment derived thereof, where the QTL at least partially controls blood pressure and/or at least partially is indicative of a potential for developing a hypertensive disease.

[0009] In another aspect, there is provided an isolated and/or recombinant nucleic acid sequence comprising a synthetic quantitative trait locus (QTL) derived from at least one chromosome or functional fragment derived thereof where the QTL at least partially controls blood pressure and/or at least partially is indicative of a potential for developing a hypertensive disease. The isolated and/or recombinant nucleic acid sequence can be at least partly derived from a rat chromosome 1, a mouse chromosome, 13, or a human chromosome 5. In a particular embodiment, the QTL comprises at least a part of an Adamtslό gene.

[0010] In another aspect, there is provided a method for selecting an animal for having desired genotypic properties. The method includes: testing the animal for the presence of a quantitative trait locus (QTL) with an isolated and/or recombinant nucleic acid sequence comprising a QTL or functional fragment derived therefrom, where the QTL at least partially controls blood pressure and/or at least partially is indicative of a potential for developing a hypertensive disease. The method can include selecting a non-human animal having desired genotypic or potential phenotypic properties.

[0011] In another aspect, there is provided an animal selected for by the methods described herein. In a particular embodiment, the animal is homozygous for an allele at a QTL The QTL can be at least partially controls blood pressure and/or at least partially is indicative of a potential for developing a hypertensive disease, and/or wherein the QTL comprises at least a part of an Adamtslό allele.

[0012] In another aspect, there is provided an animal, which is transgenic, where the animal comprising the nucleic acid of claim 11. The animal can be a male or is a female. In another aspect, there is provided a sperm or an embryo from the animal.

[0013] In another aspect, there is provided an isolated and/or recombinant nucleic acid sequence, where the isolated and/or recombinant nucleic acid sequence is at least partly derived from a rat chromosome 1 from a region mapping at around position IpI 1.

[0014] In still another aspect, there is provided a method for determining the predisposition of an individual to hypertension. The method includes analyzing at least part of the DNA sequence of at least one allelic variant, fragment or derivative of the Adamtslό gene of the individual for the presence of at least one single nucleotide polymorphism (SNP) in the Adamtslό gene, wherein the SNP is selected from the group consisting of one or more of SNPs as described herein. In a particular embodiment, the predisposition is a predisposition to essential hypertension. Also, in a particular embodiment, the genomic sequence of the Adamtslό gene of the individual is analyzed. The genomic sequence of a part of the Adamtslό gene of the individual is analyzed. The determination of at least a part of the Adamtslό gene can be performed by hybridization of a nucleic acid to the Adamtsl 6 gene of the individual. Also, the hybridization can be performed with an allele-specifϊc oligonucleotide probe. The analysis can also be carried out by sequence analysis. The determination of the Adamtslό gene can be carried out by substitution mapping analysis.

[0015] In another aspect, there is provided a nucleic acid probe which specifically hybridizes to one or more SNPs in the Adamtslό gene wherein the SNP is selected from the group consisting of one or more of the SNPs described herein.

[0016] In another aspect, there is provided a method for determining whether an individual has, or is predisposed to developing, hypertension associated with a hypertensive haplotype. The method includes analyzing at least part of the DNA sequence of the Adamtslό gene of the individual for the presence of an allelic pattern, where each allele comprises at least one SNP selected from one or more of the SNPs described herein; and where the presence of the allelic pattern indicates that the individual is predisposed to the development of, or has, hypertension. In a particular embodiment, the predisposition is a predisposition to essential hypertension. Also, in certain embodiments, the genomic sequence of at least one allele of the Adamtslό gene of the individual is analyzed. The analysis can be performed by hybridization of at least one nucleic acid to the Adamtslό gene of the individual. Also, the hybridization can be performed with an allele-specific oligonucleotide probe. Further, the analysis can be carried out by sequence analysis.

[0017] In a particular embodiment, the determination of the Adamtslό gene is carried out by substitution mapping analysis. A part of the genomic sequence of at least one allele of the Adamtslό gene of the individual is analyzed. The analysis can be carried out by hybridization of a nucleic acid probe to at least one allele of the Adamtslό gene. Also, the analysis can be determined hybridization is with an allele-specific oligonucleotide probe, and analysis can be carried out by substitution mapping analysis.

[0018] In a particular embodiment, a part of the genomic sequence of the Adamtslό gene of the human is analyzed.

[0019] In another aspect, there is provided a method of determining the predisposition of an individual to hypertension which includes analyzing at least part of the DNA sequence of the Adamtslό gene of the individual for the presence of at least one haplotype for the Adamtslό gene. In a particular embodiment, the predisposition is a predisposition to essential hypertension.

[0020] Also, in a particular embodiment, the genomic sequence of at least one allele of the haplotypes for the Adamtslό gene of the individual is analyzed. A part of the genomic sequence at least two alleles of the haplotypes for the Adamtslό gene of the individual are analyzed. The analysis can be performed by hybridization of at least one nucleic acid to the Adamtslό gene of the individual. Also, hybridization can be performed with at least one allele-specific oligonucleotide probe. Further, the analysis can be carried out by sequence analysis or by substitution mapping analysis.

[0021] In a particular embodiment, a part of the genomic sequence of the haplotypes for the Adamtslό gene of the individual is analyzed. Also, the analysis can be carried out by hybridization of a nucleic acid probe to at least one allele of the haplotypes for the Adamtslό gene.

[0022] In another aspect there is provided a method of diagnosis of hypertension in a human. The method includes determining the genotype of at least one micro satellite region of the Adamtslό gene in the human, identifying whether the genotype is a risk genotype and, if a risk genotype is so identified, then diagnosing the human as hypertensive.

[0023] In another aspect, there is provided a method of identifying a human predisposed or susceptible to hypertension. The method includes determining the genotype of at least one microsatellite region of the Adamtslό gene in the human, identifying whether the genotype is a risk genotype and, if a risk genotype is so identified, then diagnosing the human as susceptible or predisposed to hypertension.

[0024] In a particular embodiment, the method can include one or more of: determining whether the human is homozygous or heterozygous for polymorphisms of the microsatellite region of the Adamtslό gene; and where the determination of the genotype of the region is accomplished by screening the region to identify a risk polymorphism in the region, the risk polymorphism being indicative of risk of hypertension or of predisposition or susceptibility to hypertension. The determining step can be accomplished by amplification of a nucleic acid sequence located within the microsatellite region; and the amplification can be accomplished by the polymerase chain reaction using one or more primers adapted to amplify a nucleic acid sequence located within the microsatellite.

[0025] In a particular embodiment, the primers have a nucleotide sequence selected from the group of nucleotide sequences described herein.

[0026] In another aspect, there is provided a method of diagnosis and treatment of a human susceptible or predisposed to hypertension. The method includes determining the genotype of one or more of the genomic regions of the Adamtslό gene that contain single nucleotide polymorphisms as genotypes and identifying whether the genotype is a risk genotype and, if a risk genotype is so identified, then diagnosing the human as susceptible or predisposed to hypertension, and administering treatment to reduce or delay or prevent hypertension. In a particular embodiment, the treatment is selected from the group consisting of administration of an effective amount of antihypertensive pharmaceutical, administration of an effective anti-hypertension therapy or administration of both an effective anti-hypertension therapy and an effective amount of antihypertensive pharmaceutical. The risk genotype can be one or more of the alleles described herein having a length 1 or >1 bp. [0027] In another aspect, there is provided a method of diagnosing hypertension or susceptibility to hypertension. The method includes determining the genotype of Adamtslό, identifying whether the genotype is a risk genotype and, if a risk genotype is so identified, then diagnosing the human as hypertensive or susceptible to hypertension, wherein an allele of 1 or >1 bp indicates a risk of hypertension or of susceptibility to hypertension and wherein an allele of 1 or >1 bp indicates the absence of risk of hypertension or of susceptibility to hypertension. In a particular embodiment, the risk genotype of the microsatellite region includes an allele of 1 or >1 bp.

[0028] Also, in a particular embodiment, the non-risk genotype of the D5S207microsatellite region includes an allele of 1 or >lbp.

[0029] In another aspect, there is provided a method for prevention or treatment of a hypertensive disease. The method includes administering to a mammalian subject in need of such treatment an effective amount of a compound in a pharmaceutically acceptable carrier enhancing or reducing biological activity of one or several polypeptides encoded by Adamtslό or a variant, fragment or derivative thereof; and/or enhancing or reducing activity of one or several biological networks and/or metabolic pathways related to the polypeptides.

[0030] The treatment can be gene therapy or gene transfer. Also, the treatment can includes treating regulator}' regions and/or gene containing region of one or more the Adamtslό gene or variants, fragments or derivatives thereof in somatic cells of the subject.

[0031] In a particular embodiment, the treatment includes treating regulatory regions and/or gene containing region of one or more the Adamtslό gene or variants, fragments or derivatives thereof in stem cells. The compound can be a recombinant polypeptide encoded by the Adamtslό gene or variant, fragment or derivative thereof. Also, the treatment can be based on siRNA hybridizing to mRNA and/or to hnRNA of the Adamtslό gene. In another embodiment, the method of treating is a dietary treatment or a vaccination.

[0032] In a particular aspect, the method includes a therapy restoring, at least partially, the observed alterations in biological activity of one or several polypeptides encoded by the Adamtslό gene in the subject, when compared with HT free healthy subjects.

[0033] In yet another aspect, there is provided a method of identifying subjects at risk of developing a hypertensive disease. The method includes: a) providing nucleic acid from a subject, wherein the nucleic acid comprises an Adamtslό gene; b) detecting the presence or absence of one or more variations in the Adamtslό gene, wherein the variation is selected using at least one marker from the group shown in Table 1 ; and, c) determining if the subject is at risk of developing a hypertensive disease based on the presence or absence of the one or more variations. In a particular embodiment, the detecting in step b) is accomplished by substitution mapping analysis or by hybridization analysis. Also, in a particular embodiment, the detecting in step b) comprises comparing the sequence of the nucleic acid to the sequence of a wild-type Adamtslό nucleic acid.

[0034] In another aspect, there is provided a method of identifying a variant, fragment or derivative of Adamtslό gene. The method includes: a) providing nucleic acid from a subject, wherein the nucleic acid comprises a Adamtslό gene; and b) detecting the presence or absence of one or more variations in the Adamtslό gene, wherein the variation is selected using at least one marker shown in Table 1. hi a particular embodiment, the method further includes: step c) determining if the subject is at risk of carrying a hypertensive disease allele based on the presence or absence of the one or more variations.

[0035] In another aspect, there is provided a screening method of preventive and therapeutic agents for diseases caused by the over- or under- expression of the Adamtslό gene or a variant, fragment or derivative thereof, where an animal, or tissues, organs or cells derived from the animal and a test substance are used.

[0036] The test substance can be administered to the animal, and the blood pressure level of the animal is measured and estimated. In a particular embodiment, the disease caused by the overexpression of Adamtslό is a dysfunction of the kidney. The disease can be caused by the overexpression of regucalcin is essential hypertension.

[0037] In another aspect, there is provided a preventive or therapeutic agent for diseases caused by the over- or under- expression of the Adamtslό gene or a variant, fragment or derivative thereof obtained by a screening method described herein.

[0038] In another aspect, there is provided a screening method of causative agents of diseases caused by the over- or under- expression of the Adamtslό gene or a variant, fragment or derivative thereof, where an animal or tissues, organs or cells derived from the animal and a test substance are used. The test substance can be administered to the animal, and the blood pressure level of the animal is measured and estimated, hi a particular embodiment, the disease is a hypertensive disease.

[0039] In yet another aspect, there is provided an animal model where the animal model is a non-human animal that over- or under- expresses the Adamtslό gene or a variant, fragment or derivative thereof, and shows a hypertensive pathology. The animal model can be selected by a morphological measurement estimation of blood pressure levels. In a particular embodiment, the animal has characteristics of hypertensive pathology that are stable through many generations. The non-human animal can be a female or a male. In a particular embodiment, the non-human animal is a rat.

[0040] In another aspect, there is provided a screening method of preventive and therapeutic agents for hypertensive diseases where a test substance is administered to an animal model, and a morphological measurement estimation of blood pressure and/or a biochemical measurement estimation of over- or under- expression of Adamtslό or a variant, fragment or derivative thereof are performed. In a particular embodiment, the ^' screening method of preventive and therapeutic agents is useful where the hypertensive disease is essential hypertension.

[0041] In still another aspect, there is provided a method for identification of an individual who has an altered risk of or susceptibility for developing a hypertensive disease. The method includes: providing a biological sample taken from the individual; collecting personal and clinical information of the individual; determining the nucleotides present in one or several of the polymorphic sites described herein in the individual's nucleic acid; and, combining the SNP marker data with personal and clinical information to assess the risk of an individual to develop the hypertensive disease. In a particular embodiment, the altered risk is an increased risk of the hypertensive disease. In another embodiment, the altered risk is a decreased risk of the hypertensive disease.

[0042] Also, in a particular aspect, the polymorphic sites are those present in the haplotypes described herein. The polymorphic sites are associated with the SNP markers set forth herein. The polymorphic sites are in complete linkage disequilibrium with the SNP markers set forth herein.

[0043] In another aspect, there is provided a method for identification of an individual who has an altered risk of or susceptibility for developing a hypertensive disease. The method includes: providing a biological sample taken from a subject; determining the nucleotides present in one or several of the polymorphic sites as set forth herein in the individual's nucleic acid: and, combining the SNP marker data to assess the risk of an individual to develop the hypertensive disease. In a particular embodiment, the altered risk is an increased risk of the hypertensive disease. In another embodiment, the altered risk is a decreased risk of the hypertensive disease. The polymorphic sites are those present in one or more of the haplotypes presented herein. The polymorphic sites are associated with one or more of the SNP markers set forth herein. In a particular embodiment, the polymorphic sites are in complete linkage disequilibrium with one or more of the SNP markers set forth herein.

[0044] In a particular aspect, one or several polymorphic sites reside within a hypertensive risk gene or genes as set forth herein. Also, one or several of the SNP markers are selected from the group shown in Table 2.

[0045] In another aspect, there is provided a method for assessing susceptibility or predisposition to a hypertensive disease in an individual. The method includes determining alteration of expression levels of one or several of the markers of Table 1 in the individual, wherein a difference in expression is indicative of susceptibility to a hypertensive disease. Alteration of expression levels can be determined by assessing transcription levels of one or several of the genes of Table 1 in the individual. Also, alteration of expression levels can be determined by assessing translation of mRNAs encoded by one or several of the genes of Table 1 in the individual.

[0046] In another aspect, there is provided a test kit based on a method described herein for assessment of an altered risk of or susceptibility for the hypertensive disease in a subject. The test kit can be useful for determining the nucleotides present in one or several of the SNP markers as set forth herein in the individual's nucleic acid for assessment of an altered risk of a hypertensive disease in a subject.

[0047] In another aspect, there is provided a test kit for determining the nucleotides present in one or several of the SNP markers as set forth herein tables in the individual's nucleic acid for assessment of an altered risk of a hypertensive disease in a subject, containing: a) reagents and materials for assessing nucleotides present in one or several SNP markers as set forth herein; and b) software to interpret the results of the determination. The test kit can further include a PCR primer set for amplifying nucleic acid fragments containing one or several SNP markers as set forth herein from the nucleic acids of the subject. Also, the test kit can include a capturing nucleic acid probe set specifically binding to one or several SNP markers present in hypertensive disease associated markers and haplotype regions as set forth herein. Further, the test kit can include a microarray or multiwell plate to assess the genotypes. Still further, the test kit can include a questionnaire for obtaining patient information concerning age. gender, height, weight, waist and hip circumference, skinfold and adipose tissue thicknesses, the proportion of adipose tissue in the body, the family history of diabetes and obesity, the medical history concerning hypertension. The test kit can also include a capturing nucleic acid probe set specifically binding to one or several SNP markers present in the Adamtslό gene. The test kit can include a microarray or multiwell plate to assess the genotypes.

[0048] In another aspect, there is provided an isolated nucleic acid comprising an Adamtsl6-like gene comprising a polymorphism at position 820 as defined by the position shown in Figs. 3 and 9A where the amino acid corresponding to position 274 is not a proline. In a particular embodiment, the nucleotide at position 820 is T. In another particular embodiment, the amino acid at position 274 is serine.

[0049] In still another particular aspect, there is provided an isolated nucleic acid molecule comprising a sequence complementary to the isolated nucleic acid molecule described herein.

[0050] In another aspect, there is provided an isolated nucleic acid comprising an Adamtsl6-like gene comprising a polymorphism at position 3508 as defined by the position shown in Figs. 3 and 9B, where the amino corresponding to position 1170 is not a threonine. In a particular embodiment, the nucleotide at position 3508 is T. In another particular embodiment, the amino acid at position 1170 is a serine.

[0051] In another aspect, there is provided an isolated nucleic acid molecule comprising a sequence complementary to the isolated nucleic acid molecules described herein.

[0052] In still another aspect, there is provided a method for detection of at least one single nucleotide polymorphism (SNP) in a human Adamtsl6-like gene. The method includes determining a nucleotide at position 820 in the human Adamtsl6-like gene, and detecting absence or presence of at least one SNP. In a particular embodiment, the amino acid encoded by the single polymorphism at position 820 is histidine.

[0053] In another aspect, there is provided a method for detection of at least one single nucleotide polymorphism (SNP) in a human Adamtsl6-like gene. The method includes determining a nucleotide at position 3508 in the human Adamtsl6-like gene as shown in Fig. 7, and thereby detecting absence or presence of at least one SNP. In a particular embodiment, the amino acid encoded by the single polymorphism at position 3508 is L.

[0054] In another aspect, there is provided an allele-specific nucleic acid primer that comprises between 100 -350 nucleotides which hybridizes to and detects an Adamtslό- like gene polymorphism at position 820 in the Adamtsl6-like gene as defined by the positions in Figs. 3 and 9A.

[0055] Also, in a particular embodiment, the T allele is indicative of increased levels of blood pressure in an animal.

[0056] Various objects and advantages of this invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1. Comprehensive representation of mapping BP QTL2. LOD plot for BP using the F2 (S x LEW) population⁴¹ is shown at the top followed by the congenic strains constructed to map the QTL to a 2.73Mb region shown in red. Linkage map of chromosome 1 is drawn to scale except for distances marked with an asterisk. The current location of this region on the physical map of the rat genome is between the markers shown on the cytogenetic map of rat chromosome 17. The inventor believes that it should lie within IpI 1 of chromosome 1, which is also shown in the diagram. All the iterations of substitution mapping¹⁸'⁴²'⁴³ are shown by congenic strains with BP lowering effect shown in green and the ones without the BP lowering effect shown in grey. White boxes around the congenic strain schematics represent regions of recombination. The region highlighted in purple denotes the current fine-mapped interval of 789kb. Predicted genes within the critical interval are shown by blue arrows.

[0058] Figure 2. Predicted Transcript analysis. The horizontal lines at the top are schematic representations of predicted transcripts (RGSC 3.4, Dec 2004) of LOC306664 at the NCBI and Ensembl databases. Primers were designed at the locations specified as A through D in the directions depicted by the arrows. Primers were used in multiple combinations to PCR amplify DNA from a mixed cDNA pool obtained from S or LEW rats. The combinations of primers are as follows: 1) B + E; expected size = 6,24 lbp; 2) B+D; expected size = 6,589bp; 3) B + C; expected size = 5,304 bp; 4) A + C: expected size= 2,700bp; 5) A + D: expected size = 4,053 bp 6) A + E; 3666bp. Results of the PCR reactions are shown by the ethidium bromide stained agarose gel picture. M = molecular weight marker; S = S rat mixed cDNA pool. L= LEW rat mixed cDNA pool. Numbers alongside the strain names depict the combination of primers used as detailed herein. [0059] Figure 3. Sequencing of LOC306664. Predicted candidate genes for the 2.73Mb QTL interval are shown at the top of the figure as blue colored boxes within the region flanked by the markers DlRat211 and DlRatl2, as shown in Fig. 1. This information was extracted from the NCBI database. Arrows above the boxes indicate the orientation of the gene sequence on the chromosome. The exons and introns of LOC306664, which codes for Adamtsl <5_predicted, are shown above the corresponding transcript sequence. Locations of transcript variants detected between S and LEW are numbered on the transcript sequence. These transcript variations in S versus LEW are as follows: (1) T/C⁸²⁰, (2) C/T¹⁴⁸², (3) C/T¹⁷⁸⁵, (4) A/G¹⁹²⁶, (5) T/C²²²⁶, (6) C/T²⁶⁴⁶, (7) T/ A³³⁰⁸. Resultant amino acid changes and their locations on the schematic diagram of the predicted polypeptide domain of Adamtsl 6 are shown at the bottom of the figure. Sequences disclosed as SEQ ID NOS 1-4, respectively, in order of appearance.

[0060] Figure 4. Tissue-specific^" expression of Adamtsl 6. The transcript of Adamtsl 6 was amplified from cDNA obtained from various tissues of the LEW rat. RT-PCR was performed as described under materials and methods. PCR products of Adamtsl 6 and Actin as resolved on a 1.5% agarose gel and stained with ethidium bromide are shown in the top and bottom panels respectively.

[0061] Figure 5. Fine-mapping the QTL. (a) Congenic substrains that retain smaller regions within the 2.73Mb BP QTL are schematically represented alongside the physical map of the rat in Mbs. The 2.73MB BP QTL is shown by the red line on the physical map. Congenic strains are depicted underneath the physical map. Green colored areas indicate the introgressed homozygous LEW alleles of the congenic substrain and the white boxes flanking the green-colored boxes are the regions of cross-over, (b) Histograms represent the difference in BP of the congenic substrains compared to the BP of S rats. These measurements were obtained in separate experiments by the tail-cuff method. 2% Salt diet = High salt diet containing 2% NaCl; 0.4% Salt diet = Low salt diet containing 0.4% NaCl. (c) BP of the congenic substrains (DlMco4xlx3Bxl) compared to S by the telemetry method as described under materials and methods. Age of rats at day 1 on the X-axis = 54-57 days. Arrow indicates the time point which is the same as when the tail- cuff measurements would have been obtained.

[0062] Figure 6. Mapping of the homologous mouse segment. An overview of Chromosome 13 for the mouse is shown, along with the DNA(contigs), markers, Ensemble Genes, ncRNA genes, and EST genes.

[0063] Figure 7. Mapping of the homologous human segment. An overview of Chromosome 5 for the human is shown, along with the DNA(contigs). markers. Ensemble Genes, ncRNA genes, and EST genes.

[0064] Figure 8. Comparison of Rat Adamtslβ gene to Human Adamtslβ gene. Shown is a comparison of rat Adamtslό polypeptide domain organization to human Adamtslό polypeptide domain organization. Shown are the exons, protein domains, the furin recognition site, TS thrombospondin site, and the protease and lacunin domain (PLAC). Indicated with arrows are: the rat nonsynonymous SNP / amino acid variant location, the corresponding location of rat nonsynonymous SNP / amino acid variant location, the rat synonymous SNP / amino acid location, corresponding location of Rat synonymous SNP / amino acid location, and human nonsynonymous SNP / amino acid variant location.

[0065]^" Figures 9A and 9B. Shown are amino acid sequences for human (SEQ ID NO: 5), splice (SEQ ID NO: 6), mouse (SEQ ID NO: 7), rat (S) (SEQ ID NO: 8), rat (LEW) (SEQ ID NO: 9) and cow (SEQ ID NO: 10). The positions 270 and 1170 are boxed in red.

[0066] Figure 10. Table 4 shows human ADAMST 16 variants with significant (p<0.05) associations to systolic, diastolic and mean arterial blood pressure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S) [0067] In one aspect, there is provided methods to determine the susceptibility of individuals to hypertension, and to identify those individuals for whom the disease appears to be genetic. It is, therefore, one further object of the present invention to provide improved methods to treat hypertension.

[0068] These objectives and others are met by the present invention, which in some embodiments provides a method of identifying subjects at risk of developing hypertension comprising: providing nucleic acid from a subject, where the nucleic acid comprises an Adamtslό gene; and detecting the presence or absence of one or more variations in the Adamtslβ gene. In other embodiments, the method further comprises determining if the subject is at risk of developing hypertension based on the presence or absence of the one or more variations.

[0069] In yet other embodiments, in the method of the present invention the variation is a single nucleotide polymorphism that causes a frameshift mutation in Adamtslό. or the variation causes a splice mutation in Adamtslό. or the variation causes a nonconservative amino acid substitution Adamtsl 6. In certain embodiments, the variation is selected from the group consisting of the mutations shown in Table 1. In some embodiments, the detecting is accomplished by hybridization analysis. In further embodiments, the detecting step comprises comparing the sequence of the nucleic acid to the sequence of a wild-type Adamtslό nucleic acid.

[0070] The present invention also provides a method of identifying subjects at risk of developing hypertension comprising: providing a blood or other biological sample from a subject, wherein the blood or other biological sample comprises an Adamtsl 6 protease; and detecting the presence or absence of one or more variants of the Adamtsl 6 protease. In some embodiments, the detecting step is accomplished by an antibody assay.

[0071] The invention also provides a method of identifying subjects at risk of carrying an allele for hypertension comprising: providing nucleic acid from a subject, wherein the nucleic acid comprises an Adamtsl 6 gene; and detecting the presence or absence of one or more variations in the Adamtslό gene. In other embodiments, the method of the present invention further comprises a step of determining if the subject is at risk of carrying a hypertensive disease based on the presence or absence of the one or more variations.

[0072] The present invention also provides an isolated nucleic acid comprising a sequence encoding a polypeptide of Adamtsl 6.

[0073] The present invention also provides a method of treating a patient with a hypertensive disease, comprising administering a therapeutically effective amount of an Adamtsl 6 protease such that the symptoms of the hypertensive disease are alleviated. The Adamtslό protease is selected from the group consisting: recombinant Adamtslό; synthetic Adamtslό; mutants, variants, fragments, and fusions of recombinant Adamtsl 6; and mutants, variants, fragments, and fusions of synthetic Adamtsl 6.

[0074] The present invention also provides a kit for determining if a subject is at risk of developing hypertension comprising a detection assay. The detection assay is capable of specifically detecting a variant Adamtslό allele. In some embodiments, the detection assay comprises a nucleic acid probe that hybridizes under stringent conditions to a nucleic acid sequence comprising at least one mutation selected from the group consisting of the mutations shown in Table 1.

[0075] The invention also provides an isolated nucleic acid comprising at least one marker sequence noted with an asterisk (*) as shown in Table 1. In some embodiments, the sequence is operably linked to a heterologous promoter. In further embodiments, the invention provides a vector comprising the isolated sequence. In yet further embodiments, the invention provides a host cell comprising the vector. In some embodiments, the host cell is selected from the group consisting of animal and plant cells: in other embodiments, the host cell is located in an organism.

[0076] The invention also provides an isolated nucleic acid sequence comprising at least one sequence noted with an asterisk^*) as shown in Table 1. In some embodiments, the invention provides a computer readable medium encoding a representation of the nucleic acid sequence. The invention also provides an isolated polypeptide comprising an amino acid sequence encoded by the sequence noted with an asterisk^*) as shown in Table 1.

[0077] The identification of the association between the Adamtslό gene and hypertension permits the screening of individuals to determine a predisposition to hypertension. Those individuals who are identified at risk for the development of the disease may benefit from dietary sodium restriction, can have their blood pressure more closely monitored and be treated at an earlier time in the course of the disease. Such blood pressure monitoring and treatment ma}' be performed using conventional techniques well known in the art.

[0078] The invention is based, in part, upon the finding that the activity the Adamtslό gene is central to the etiology of hypertension and hypertension-related disorders in mammals. Accordingly, the invention provides methods for treating hypertension using specific inhibitors of Adamtslό expression and/or activity, particularly the expression and/or activity of the Adamtslό gene.

[0079] Quantitative Trait Loci TOTLs) of Adamtslό

[0080] Metalloproteases are a class of enzymes that have been implicated in a large number of cellular processes. Despite the fact that these enzymes differ widely in their sequence and structure, they all rely on the action of a catalytically active metal atom, hi most instances this metal is a zinc atom, but examples are known which contain cobalt or nickel. Metalloproteases use the catalytic metal atom to catalyze hydrolysis of peptide bonds. Currently, metalloproteases have been divided into at least five distinct families based on structure and activity.

[0081] The members of one family of metalloproteases known as:" Adam " (A Disintegrin And Metalloprotease) display the common characteristic of being type I transmembrane glycoproteins that are important in diverse biologic processes. Structurally, the Adam metalloproteases consist of a prodomain that blocks protease activity; a zinc-binding metalloprotease domain: disintegrin and cysteine-rich domains with adhesion activity; an epidermal growth factor-like domain with cell fusion activity; a transmembrane domain; and a phosphorylated cytoplasmic regulatory domain.

[0082] A subfamily of the Adam metalloproteases can be distinguished from other the Adam metalloproteases family members based on the multiple copies of thrombospondin 1 -like repeats they carry.

[0083] In a particular aspect, there is described herein the fine-mapping of a rat BP QTL that is homologous to a BP QTL detected on human chromosome 5 ¹⁹. Also, positional cloning strategies were used to identify the gene underlying the rat BP QTL region that is associated with blood pressure. Quite distinct from other studies, wherein differential segments between two congenic strains with opposite effects is inferred as the QTL containing region^20"22 or multiple candidate genes with nonsynonyinous variants and/or differential expression patterns are within the QTL interval^23"23, the present invention described herein shows that the BP QTL2 region was isolated independently in a congenic strain spanning 789 kb and contained only two expressed transcripts. Of these, DNA sequencing coupled with transcript structure and expression analysis detected Adamtslό as the gene harboring nonsynonymous variants that potentially affect the structure of the protein product of Adamtslό, rather than quantitative differences in expression.

[0084] Adamts 16 is a member of a family of metalloproteinases, the function of which is unknown^{2 "} . Also, none of the Adamts proteins are previously implicated in blood pressure regulation.

[0085] There is provided here the culmination of sustained application of substitution mapping for over a decade and constitutes the first report of defining a novel BP locus entirely based on the substitution mapping approach, other than 11-β hydroxylase, which was also previously mapped as a BP QTL by our laboratory ' ' .

[0086] Evidence indicating that a gene, Adamtslό, with nonsynonymous variants underlies the BP QTL2 on rat chromosome 1, is presented.

[0087] QTL2 is localized to a 2.73 Mb region, as determined on the basis of the differential segment between two congenic strains (see Fig. 1), one with the BP effect and the other without the BP effect ¹⁸.

[0088] There is now presented herein two congenic substrains where the QTL is captured in its entirety. This QTL exerts a phenotypic effect independently, i.e., without genetic interaction from introgressed LEW alleles of the previously reported strain with the BP effect, S.LEW(DlMco4xlx3A) ^{I 8}.

[0089] Fig. 1 provides a comparison between substitution mapping and the evidence obtained by genetic linkage analysis of the same region. The location of the QTL gene is far removed from the confidence intervals of LOD peaks with suggestive linkage (Fig 1). This comparison reiterates the difficulty of mapping QTL genes solely based on linkage analysis as is done in humans. This comparison also exemplifies the power of substitution mapping in animal models to resolve QTLs by isolating them as 'monogenic' QTL effectors. Since substitution mapping relies on location on the genome, any bias of previous knowledge of physiological function of a gene related to the phenotype of interest is removed, allowing for identification of novel QTL genes. One advantage of using this method is exemplified by the data presented herein because the fine-mapped BP QTL did not encompass any gene with previously known function related to BP.

[0090] Accuracy of mapping QTLs and detecting underlying genetic determinants relies heavily on the quality of assembly of genomes and gene predictions. Early impediments to the positional cloning described herein included the dislocation of the QTL region on rat chromosome 17 and multiple, discordant gene predictions of Adamtsl 6. The data presented in Fig. 1 and Table 1 allow for remedying the error in sequence assembly by the correct placement of the region spanned by markers DlRat211 and Dl Rat 12 on chromosome 1.

[0091] Table 1 shows the physical map locations of micro satellite markers that are linked to RNOl by genetic linkage analysis of F2(S x LEW).

Table 1

Microsatellite markers are those that are placed in order on the genetic linkage map of F2(S x LEW) (Physiol Genomics. 2001, 4:201-214). *Newly developed markers.

Physical map locations on chromosome 1 and 17 are obtained from the rat genome sequence assembly 3.4 at the Ensembl website. [0092] Similarly, the biological data allows for sorting the discrepancies among the available predictions for gene and transcript sequences of rat Adamtslό. The most recent version of the predicted Adamtslό gene at the NCBI rat genome resources website (Build 4.1) concurs with the evidence provided herein.

[0093] In addition to other tissues, Adamtslό was highly expressed in the kidney, which is a major organ involved in BP homeostasis. While not wishing to be held to theory, the inventor believes that, based on the roles of the Adamts family members and based on the presence of a metalloproteinase domain within Adamtslό, it is likely that Adamtslό functions as a metalloproteinase in vivo.

[0094] The single nucleotide polymorphisms described herein do not reside within the metalloproteinase domain of Adamtslό. They are located within a potential furin- cleavage site and a thrombospondin-motif, respectively, which may or may not influence the metalloproteinase activity of Adamtslό. Furin-cleavage is known to relieve enzyme latency of other members of the Adamts family of proteins ^31~36. Alternately, thrombospondin-motifs have protein-protein interactions with integrins, which in turn are implicated in vascular remodeling ³⁷. Identifying substrates and/or binding partners for Adamtslό is an important step in linking the physiological role of Adamtslό to blood pressure regulation.

[0095] In the genetic analysis of hypertension, salt- sensitivity is considered as an important environmental factor because it aggravates the elevation in BP. However, the S rat is documented to develop elevated blood pressure even when not fed a high salt containing diet ^⁸. In this context, the BP effects observed even under a low salt diet regimen is interesting and implies that Adamts 16 represents a susceptibility factor that contributes to the salt-insensitive development of hypertension, which may or may not have segregated during the original selection of S rats from the outbred Sprague-Dawley rats because the selection process involved a dietary salt challenge (8% NaCl). Two lines of evidence indicate that this is true - 1) There was no BP QTL on rat chromosome 1 that was detected in the genetic linkage analysis of S and R rats, which are the two contrasting lines with high and normal BP, that were derived from Sprague-Dawley rats; and 2) While screening for polymorphisms we detected that the nonsynonymous variants of the QTL gene, Adamtslό, are present in Sprague-Dawley rats. However they are not different between S and R.

[0096] In addition to the screening for polymorphisms of S and R, the data from other inbred strains indicated that there is a stronger association of genetically hypertensive rats with the S alleles of Adamtslό at positions 820 and 3508 (Table 2 and Fig. 3).

[0097] Table 2. Adamtsl 6 SNPs in outbred, inbred and wild rats

Table 2

MHS=Milan hypertensive strain, SHR=spontaneously hypertensive strain, LH-Lyon hypertensive strain, R=Dahl salt-resistant strain, WKY= Wistar Kyoto rat, LN=Lyon normotensive rat, LL=Lyon low-tensive or hypotensive rat, MNS=Milan normotensive strain, BN=Brown Norway rat, SBN=Sabra hypertension resistant, SBH=Sabra hypertension prone, MWF=Munich Wistar Fromter rat, SD = Sprague Dawley rat. Further details on the strains are available at the rat genome database. Wild rat samples were from Prof. Holmdahl's laboratory in Sweden. Corresponding amino acids predicted to be encoded based on the SNPs of Adamtsl 6 transcript are given in parenthesis next to the SNPs. [0098] In lieu of the difficulty to define what constitutes a good standard for identifying a gene underlying a QTL, The Complex Trait Consortium suggested that a candidate gene should meet more than one of eight criteria including analysis in transgenic and knock-in animals³⁹. These criteria are a 'necessary detour' applicable to situations wherein the QTL is mapped to a small region (usually less than a Mb) and the challenge is to identify the underlying genetic determinant for a QTL from among several closely linked candidate genes. In a recent review on the genetic dissection of hypertension¹³, Cowley indicates that the gold standard for validation of a potential candidate gene for hypertension is that the rescue of the phenotype must be done in the same species and with the same genetic background that was used to identify the QTL.

[0099] In one aspect, the present invention provides a 'gold standard' because (1) The QTL is fine-mapped to a small region solely based on natural recombination events, thus closely mimicking the performance of a natural allele in the rat as opposed to artificially genetically manipulated systems such as transgenics and knock-ins; and (2) it is now possible to map the QTL to a gene-sparse region as confirmed by comparative mapping in rats, mice and humans¹⁸, thus allowing for testing of the two expressed candidate transcripts, only one of which had coding sequence variants.

[00100] In addition to the variants within Adamtslό, there may be other variants within inter-genic or intra-genic regions of the critical QTL interval that contribute to the QTL effect. Like in any positional cloning study, regardless of any criteria, such possibilities cannot be ruled out. However, with the view of improving the clinical management strategies for high BP including the identification of novel and effective drug targets, the present inventive system shows that Adamtslό is a suitable candidate for use as the BP QTL on human chromosome 5. [00101 ] Materials and Methods [00102] Animals

[00103] All animal experiments were conducted as per pre-approved protocols by the Institutional animal care and use committee (IACUC) of the University of Toledo Health Science Campus (UTHSC). Dahl salt-sensitive (SS/Jr) inbred rats were from our colony maintained at UTHSC. LEW/NCrlBR (LEW rat) was originally obtained from the Charles River Laboratories (Wilmington, MA) and maintained in our colony. The congenic substrain S.LEW(D1MCO4X1X3B) was derived from S.LEW (D1MCO4X1) ¹⁸ as the parental strain contributing to the introgressed LEW alleles. Congenic substrains S.LEW(D1MCO4X1X3BX1) and S.LEW(D1MCO4X1X3BX2) were derived from S.LEW(D1MCO4X1X3B) as the progenitor with LEW alleles at the QTL region. The congenic substrains were constructed by crossing the progenitor congenic strain to the S rat to obtain a heterozygous Fl population. An Fl X Fl intercross then yielded an F2 population. The recombinant F2 animals were selected for varying sizes of the LEW RNOl introgressed region and backcrossed to the S rats. The resulting heterozygous progeny were genotyped to identify LEW alleles and then intercrossed to duplicate the recombinant chromosome. Finally the animals were selectively bred to generate a homozygous congenic rat strain containing the desired LEW chromosomal segment on the S genomic background.

[00104] Microsatellite Marker Development and tracking recombination events in congenic strains

[00105] Microsatellite repeats in the region of interest were identified on the rat genomic sequence (version 3.1) at the NCBI website. In order to amplify these microsatellite repeats, primers were designed using the PrimerS software available and synthesized by Integrated DNA technologies. DNA was isolated with the QIAamp Tissue Kit (Qiagen Inc.) from S, LEW and congenic substrains. PCR reactions were set up using the primers designed to amplify microsatellite repeats from S and LEW rats. Polymorphic repeats were used as markers to genotype DNA from tail biopsies of the relevant rat populations.

[00106] Predicted Genomic and Transcript analysis

[00107] Predicted gene sequences within the QTL region were downloaded from the rat genome sequence at the NCBI website. Primers to amplify exons were designed using the ExonPrimer software. Primers tagged with M- 13 sequences were synthesized by Integrated DNA technologies (IDT). All primer sequences mentioned in our work are available. Genomic DNA was amplified using these primers and sequenced by MWG Biotech Inc.

[00108] Predicted transcript sequences were extracted from the NCBI database and/or Ensembl. Primers were designed encompassing the start and stop codons of each predicted transcript using Primer3 or Oligonucleotide properties calculator. Messenger RNA from S and LEW kidney, liver, heart, lung, thyroid, pancreas, and spleen was extracted using TRIzol Reagent (Life Technologies). cDNA of the predicted transcripts within the QTL region was obtained by reverse transcription using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen). Standard polymerase chain reaction, using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and gene- specific primers, was used to amplify the cDNA. S and LEW gene amplicons were cloned into the Topo-TA-Cloning System (Invitrogen) as per the company recommended procedures. Plasmid DNA was purified using the QIAprep-miniprep (Qiagen). The full- length S and LEW alleles for each gene within the QTL region were sequenced by MWG (High Point. NC). using vector based and gene-specific primers. AU sequence data was analyzed using DNA Star (DNAStar Inc.) and/or Sequencher (Genecodes Corp.) [00109] Blood Pressure Measurements

[00110] Each set of congenic substrains (n=20 males) and control S rats (n=20 males) were bred, housed, and studied concomitantly to minimize environmental effects. Rats were weaned at 30 days of age and given a low salt diet (0.4% NaCl, Harlan Teklad diet TD7034). At 40-42 days of age rats on a high salt dietary study only were fed 2% NaCl diet (Harlan Teklad diet TD94217) for 24 days. Rats on a low salt dietary study were continued on the 0.4% NaCl in their diet. Systolic BP was measured using the tail-cuff microphonic method commencing on the 25^l day⁴ . Briefly, conscious restrained rats warmed to 28°C. The BP of each rat was measured for four consecutive days by two blinded operators. BP values for each day were the mean of three to four consistent readings. The final BP value used was the mean of the^" four daily BP values. The day after the last BP measurements, rats were euthanized and heart weights were recorded. Statistical Analysis was as previously reported ¹⁸ using SPSS software. [00111] BP was also collected using a telemetry system (Data Sciences International, St. Paul, MN) as explained in detail previously . Briefly, 6-week old rats S (n=6) and congenic rats (n=6) on a low salt diet were surgically implanted with a transmitter into the left flank and the probe was inserted through the femoral artery and advanced to the lower abdominal aorta. Rats were allowed to recover from surgery for a week before the transmitters were turned on and BP data collected for 24 hrs at a time over several weeks. [00112] Examples

[00113] Location of the QTL on the rat genome

[00114] BP QTL2 was fine-mapped to a 2.73Mb region flanked by the microsatellite markers DlRat211 and Dl Rat 12. Table 1 lists the microsatellite markers in and around this 2.73Mb region that are linked to the map of chromosome 1. Fig. 1 is a comprehensive representation of markers genotyped for linkage analysis and for the construction and genetic analysis of congenic strains, which lead to the fine-mapping of BP QTL2 to 2.73Mb. In Figure 1, the 2.73Mb BP QTL2 region is represented as a red highlighted band on rat chromosome 17 instead of on rat chromosome 1. This is because there is a discrepancy between the mapping of the genomic fragment encompassing DlRat211 and DlRatl2. As can be seen in Table 1, the physical map locations of the relevant markers in and around the 2.73Mb BP QTL2 region are distributed between rat chromosomes 1 and 17. Genotyping data shows that this fragment is incorrectly placed on the physical map of the rat genome. It is currently placed towards the p-terminus of the physical map of rat

11 chromosome 17, whereas, the correct location of the critical 2.73Mb BP QTL2 flanked by DlRat211 and DlRatl2 should be between the markers DlMco4 and DlMco8 on the physical map of rat chromosome 1 (Fig. 1 and Table 1). [00115] Predicted gene sequence analysis

[00116] The 2.73 Mb QTL region had 19 annotated genes¹⁸. Because homologous regions of this QTL in mice and humans encompass BP QTLs¹⁹'²⁹, we chose to focus on sequencing Rat genes that have orthologues in both mouse and human. Two out of the four orthologous gene sequences annotated on the rat genome database at NCBI (LOC306664 and LOC290935) were predicted as associated with Adamts 16, an orphan disintegrin-like metalloproteinase with thrombospondin motif 16¹⁸. We sequenced all the exons predicted by the NCBI database. Seven nucleotide substitutions within predicted exon sequences of Adamts 16 were detected. Since the exon predictions of Adamts 16 were different between the NCBI and Ensembl databases, the issue of whether these polymorphic variants resulted in amino acid substitutions was not immediately discernable.

[00117] Predicted transcript sequence and expression analysis of Adamts 16 [00118] Due of the differences in coding sequence predictions of Adamts 16 by NCBI and Ensembl, there were also discrepancies between the transcript sequence predictions at the two websites. To test the validity of the gene and transcript sequence prediction of Adamts 16, we sought to amplify the expressed rat transcript of Adamts 16 using several sets of random primers encompassing each of the transcript sequence predictions (Fig..2) and used these to amplify transcripts from mixed pools of reverse-transcribed RNA from S and LEW rats. Only one out of the six primer combinations tested resulted in a transcribed product of size between 3000 and 4000bp (Fig. 2). The PCR amplified samples from both S and LEW rats were cloned and sequence confirmed as the transcript of Adamtsl, which confirmed the NCBI predicted transcript. Only two out of the seven polymorphisms detected by genomic DNA sequence analysis of LOC306664 were nonsynonymous variants that resulted in amino acid substitutions of the predicted mature polypeptide chain of Adamts 16 from Pro²⁷⁴ and Thr¹¹⁷⁰ in LEW rats to Ser²⁷⁴ and Ser¹¹⁷⁰ in S rats (Fig. 3). The variant at position 274 maps to no particular domain of Adamtslό, but lies in between the propeptide and metalloproteinase domains (Fig. 3). The variant at 1 170 maps to the C-terminal thrombospondin domain of Adamts 16 (Fig. 3). [00119] A gene Underlying a QTL may either have coding-sequence differences that change the function of the protein it encodes or may have differences in regulatory- sequences that cause a difference in gene expression between the parental strains or both. To test the latter two possibilities, we had to locate the tissues where Adamtslό is expressed. Reverse-transcription PCR of cDNA samples from various tissues of the LEW rat were probed with primers designed to amplify the full length Adamtsl 6 transcript. (Fig. 4). Expression of Adamtsl 6 was prominent in the rat kidney, but detected in other tissues including the heart, pituitary, brain and lung (Fig. 4). Real-time PCR of kidney and heart samples revealed that Adαmtslό is not differentially expressed in these organs (data not shown).

[00120] Recombinant congenic substrains for high resolution mapping of the QTL "[00121] To test the effect of Adamts 16 as a candidate gene for the QTL, further substitution mapping was conducted. F2 populations were derived from congenic strains containing LEW alleles from the QTL region. These rats (n> 1000) were screened for recombinations within the 2.73 Mb region. Three recombinant animals obtained were fixed homozygous for LEW alleles and are depicted as S.LEW (DlMco4xlx3B), S.LEW (DlMco4xlx3Bxl) and S.LEW (DlMco4xlx3Bx2) in Figure 5a. The two congenic substrains, S.LEW(DlMco4xlx3Bxl) and S.LEW(DlMco4xlx3Bx2) are not distinguishable from each other based on our current characterization of these strains. This is because the regions of cross-over between the introgressed LEW alleles and the background S genome occurred between the markers DlMco86 and DlMco83 on one end and between DlMco84 and DlMco85 on the other end of both of these congenic strains (Fig. 5a). Nevertheless, both of these strains span 789kb and contains only two genes, one of which is the full-length coding sequence of Adαmtslό (Fig. 5a). [00122] Other gene sequences and expression analysis

[00123] Apart from the complete coding sequence of Adαmtslό, the introgressed region of the minimal congenic substrains S.LEW (DlMco4xlx3Bxl) and S.LEW (DlMco4xlx3Bx2) contained only one other predicted gene, LOC306665. According to the previous version of the rat genome annotation at the NCBI website (version 3.1) the first 4 exons of LOC502107 were also within the congenic interval of the two substrains. Transcript analysis indicated that the predicted gene LOC306665 encoded a transcript, but LOC502107 did not (data not shown). Subsequent version of the rat genome annotation (version 4.1) has also discontinued the record for LOC502107. Nevertheless, sequence comparisons between S and LEW of all the predicted exons within the 789kb QTL2 region revealed no polymorphic variants within either LOC306665 or the former

LOC502107.

[00124] In vivo evidence that Adamts 16 underlies BP OTL2

[00125] Blood pressure measurements of each of the congenic substrains containing the

LEW variants ofAdamtslό weτe tested after animals were administered a high salt (2%

NaCl) containing diet for four weeks. Data obtained indicates that the BP of the congenic substrains S.LEW (DlMco4xlx3B), S.LEW (DlMco4xlx3Bxl) and S.LEW

(DlMco4xlx3Bx2) were significantly lower by 19, 24 and 18 mm of Hg, respectively, than that of the hypertensive S rat (Figs. 5 a,b; Table 3).

[00126] ^*The values were the averages of two separate batches of experiments done on different days. Values in brackets are SEM. **Effect = Congenic value - S value.

Negative values indicate a decrease in the congenic effect compared to the S rat. Number of rats in each group ranged from 20 to 30.

[00127] Therefore, the smallest congenic interval shared by these strains, i.e., 789kb flanked by DlMco86 and DlMco85, was inferred as adequate to account for the effect of

BP QTL2 (Fig. 5a and Fig. 1).

[00128] Dietary salt as an environmental factor of the QTL effect

[00129] To test whether the BP effect elicited by the alleles underlying this QTL are dependent or independent of the dietary high salt challenge, we compared the BP of the two congenic substrains S.LEW (DlMco4xlx3Bxl) and S.LEW (DlMco4xlx3Bx2) to that of the S rat in the absence of a high salt diet. As can be seen from Fig. 5b and Table 3, both of the congenic substrains fed with low salt (0.4% NaCl) had significantly lower BP by 18 and 14 mm of Hg, respectively, compared to the low salt fed S rat. Heart weights of all the congenic strains were also significantly lower than that of the S rat (Table 3). Blood pressure differences on the low salt diet were also observed in a separate group of S and S.LEW(DlMco4xlx3Bxl) rats monitored by telemetry. Similar differences in BP of the S versus the congenic strain were observed (Fig. 5c) at the time point of the telemetry measurement (day 17) that was comparable to the time point of the tail-cuff measurements.

[00130] Assessment of Adamts 16 polymorphisms in outbred. inbred and wild rats [00131] The two nonsynonymous polymorphisms in Adamts 16 were sequenced in outbred Sprague-Dawley rats, other inbred rat strains and in wild rats (Table 2). Results obtained indicate that the polymorphisms exist as natural variants in all the three groups and are not mutations that occurred only in hypertensive S rats. Interestingly however, the same two polymorphisms as in S and LEW rats were observed between the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) (highlighted in Table 2), which may explain the co-localization of our QTL with a BP QTL that was identified using these two strains³⁰. All hypertensive strains tested except SBH had both of the nonsynonymous polymorphisms like in the S rat (Table 2). Among the normotensive strains, all but three of them (R, WKY and LN), had at least one SNP like the normotensive LEW (Table 2). [00132] Applications

[00133] Commercial embodiments of the present invention include, for example, the areas of diagnostics, drug discovery and therapy. Accordingly, a first aspect of the present invention is directed to methods for identifying individuals predisposed to essential hypertension. The methods can be conducted using a sample of cells that express the Adamts 16 gene or a promoter thereof, isolated from the individual. The cells are assayed to determine the extent of post-translational modification, where a change relative to cells isolated from a normotensive individual is indicative of predisposition to essential hypertension. Alternatively, a nucleic acid sample can be isolated from the individual in order to analyze the Adamts 16 gene or fragment thereof to detect Adamts 16 associated with essential hypertension. [00134] Also provided is a method for identifying a human subject as having an increased risk of developing essential hypertension, comprising correlating the presence of a nucleic acid sequence at nucleotides -820 through -3508 in a human Adamts 16 gene with an increased risk of developing essential hypertension; and determining the nucleic acid sequence of nucleotides -820 through -3508 of the subject's Adamtsl όgene, whereby a subject having a nucleic acid sequence at nucleotides -820 through -3508 in the Adamtslόgene correlated with an increased risk of developing essential hypertension is identified as having an increased risk of developing essential hypertension. [00135] Furthermore, a method of identifying an allele in the promoter region of the human Adamtslό gene correlated with an increased risk of developing essential hypertension is also provided, comprising determining the nucleic acid sequence of the nucleotides at positions -820 through -3508 in the human Adamtslό gene from a subject; and correlating the presence of the nucleic acid sequence of the subject with the diagnosis of essential hypertension in the subject, whereby the nucleic acid sequence of the nucleotides at positions -820 through -3508 in the promoter region of the human Adamtslό gene identifies an allele correlated with an increased risk of developing essential hypertension.

[00136] Additionally provided is a method of identifying an allele in the promoter region of a human Adamtslό gene correlated with an increased risk of developing essential hypertension, comprising determining the nucleic acid sequence of the nucleotides at positions -820 through -3508 in the promoter region of the human Adamtslό gene; cloning the nucleic acid sequence encoding the promoter region of the human Adamtslό gene into an expression vector having a reporter gene; expressing the reporter gene to produce a gene product under the direction of the promoter region of the human Adamtslό gene; and determining the amount of reporter gene product produced, an amount of reporter gene product less than the amount of reporter gene product produced under the direction of a Adamtslό gene promoter containing an allele of a nucleic acid sequence of the nucleotides at position -820 through -3508 of the human Adamtslό gene where an allele correlated with an increased risk of developing essential hypertension.

[00137] Further provided is a method for identifying a human subject as having an increased risk of developing essential hypertension, comprising cloning the nucleic acid sequence encoding the promoter region of the human Adamtslό gene of the subject into an expression vector having a reporter gene; expressing the reporter gene to produce a gene product under the direction of the promoter region of the human Adamts 16 gene; and determining the amount of reporter gene product produced, an amount of reporter gene product less than the amount of reporter gene product produced under the direction of a Adamts 16 gene promoter containing the nucleotide sequence of one or more alleles that identify a subject as having an increased risk of developing essential hypertension. [00138] In addition, a method for identifying an allele in the promoter region of the human Adamts 16 gene is provided, comprising determining the nucleic acid sequence of the nucleotides at positions -820 through -3508.

[00139] The present invention further provides an isolated nucleic acid consisting of the promoter region of the human Adamts 16 gene, the complementary sequence of the isolated nucleic acid, vectors containing the nucleic acid and cells expressing the vector. [00140] The present invention also provides a method of identifying a subj ect at reduced risk of having essential hypertension, comprising determining the presence in the subject of an allele in the promoter region of the subject's Adamtsl6 gene correlated with a reduced risk of developing essential hypertension, the presence of the allele identifying the subject as having a reduced risk of developing essential hypertension. [00141] Additionally provided in the present invention is a method for identifying a human subject as having a reduced risk of developing essential hypertension, comprising correlating the presence of a nucleic acid sequence at nucleotides -820 through -3508 in a human Adamts 16 gene with a reduced risk of developing essential hypertension; and determining the nucleic acid sequence of nucleotides -820 through -3508 of the subject's Adamts 16 gene, whereby a subject having a nucleic acid sequence at nucleotides -820 through -3508 in the Adamtsl6 gene correlated with a reduced risk of developing essential hypertension is identified as having a reduced risk of developing essential hypertension. [00142] A method is also provided for identifying an allele in the promoter region of the human Adamts 16 gene correlated with a reduced risk of developing essential hypertension comprising determining the nucleic acid sequence of the nucleotides at positions -820 through -3508 in the human Adamtsl6 gene from a subject; and correlating the presence of the nucleic acid sequence of the subject with the absence of essential hypertension in the subject, whereby the nucleic acid sequence of the nucleotides at positions -820 through -3508 in the promoter region of the human Adamts 16 gene identifies an allele correlated with a reduced risk of developing essential hypertension. [00143] The present invention further provides a method of identifying an allele in the promoter region of a human Adamts 16 gene correlated with a reduced risk of developing essential hypertension, comprising: determining the nucleic acid sequence of the nucleotides at positions -820 through -3508 in the promoter region of the human Adamts 16 gene; cloning the nucleic acid sequence encoding the promoter region of the human Adamts 16 gene into an expression vector having a reporter gene; expressing the reporter gene to produce a gene product under the direction of the promoter region of the human Adamts 16 gene; determining i) the amount of reporter gene product produced, ii) an amount of reporter gene product greater than the amount of reporter gene product produced under the direction^' of a Adamts 16 gene promoter containing the nucleotide sequence of one or more alleles thereof; and identifying a nucleic acid sequence of the nucleotides at position -820 through - 3508 of the human Adamts 16 gene as an allele correlated with a reduced risk of developing essential hypertension.

[00144] Also provided is a method for identifying a human subject as having a reduced risk of developing essential hypertension, comprising cloning the nucleic acid sequence encoding the promoter region of the human Adamts 16 gene of the subject into an expression vector having a reporter gene; expressing the reporter gene to produce a gene product under the direction of the promoter region of the human Adamts 16 gene; and determining the amount of reporter gene product produced, an amount of reporter gene product greater than the amount of reporter gene product produced under the direction of a Adamts 16 gene promoter, and identifying a subject as having a reduced risk of developing essential hypertension.

[00145] In addition to the embodiments described above, the present invention provides a method of identifying a human subject as having an increased likelihood of regulating blood pressure with dietary sodium intake, comprising determining the presence in the subject of a Adamts 16 promoter allele genotype correlated with an increased likelihood of regulating blood pressure with dietary sodium intake, whereby the presence of the genotype identifies the subject as having an increased likelihood of regulating blood pressure by dietary sodium intake.

[00146] Additionally provided in this invention is a method of identifying a human subject as having an increased likelihood of regulating blood pressure with dietary sodium intake, comprising a) correlating the presence of a Adamts 16 promoter allele genotype with an increased likelihood of regulating blood pressure with dietary sodium intake; and b) determining the Adamts 16 promoter allele genotype of the subject, whereby a subject having a Adamts 16 promoter allele genotype correlated with an increased likelihood of regulating blood pressure with dietary sodium intake is identified as having an increased likelihood of regulating blood pressure with dietary sodium intake. [00147] Furthermore, a method is provided for identifying a Adamts 16 promoter allele genotype correlated with an increased likelihood of regulating blood pressure with dietary sodium intake, comprising a) determining the Adamts 16 promoter allele genotype of a subject; and b) correlating the presence of the genotype of step (a) with the ability of the subject to regulate blood pressure by dietary sodium intake, thereby identifying a genotype correlated with an increased likelihood of regulating blood pressure by dietary sodium intake.

[00148] Additionally, the present invention provides a method for identifying a human subject as having a reduced likelihood of regulating blood pressure with dietary sodium intake, comprising determining the presence in the subject of a Adamts 16 promoter allele genotype correlated with a reduced likelihood of regulating blood pressure with dietary sodium intake, whereby the presence of the genotype identifies the subject as having a reduced likelihood of regulating blood pressure with dietary sodium intake. [00149] Additionally provided in this invention is a method of identifying a human subject as having a reduced likelihood of regulating blood pressure with dietary sodium intake, comprising a) correlating a Adamts 16 promoter allele genotype with a reduced likelihood of regulating blood pressure with dietary sodium intake; and b) determining the Adamts 16 promoter allele genotype of the subject, whereby a subject having a Adamts 16 promoter allele genotype correlated with a reduced likelihood of regulating blood pressure with dietary sodium intake is identified as having a reduced likelihood of regulating blood pressure with dietary sodium intake.

[00150] Also, a method is provided of identifying a Adamts 16 promoter allele genotype correlated with a reduced likelihood of regulating blood pressure with dietary sodium intake, comprising a) determining the Adamts 16 promoter allele genotype of a subject: and b) correlating the presence of the genotype of step (a) with the inability of the subject to regulate blood pressure by dietary sodium intake, thereby identifying the genotype correlated with a reduced likelihood of regulating blood pressure by dietary sodium intake. [00151] In a further embodiment, the present invention provides a method of identifying a human subject as having an increased likelihood of regulating blood pressure with a diuretic, comprising determining the presence in the subject of a. Adamtsl 6 promoter allele genotype correlated with an increased likelihood of regulating blood pressure with a diuretic, whereby the presence of the genotype identifies the subject as having an increased likelihood of regulating blood pressure with a diuretic. [00152] Additionally provided is a method of identifying a human subject as having an increased likelihood of regulating blood pressure with a diuretic, comprising a) correlating the presence of &Adamtsl6 promoter allele genotype with an increased likelihood of regulating blood pressure with a diuretic; and b) determining the Adamtsl 6 promoter allele genotype of the subject, whereby a subject having a Adamtsl 6 promoter allele genotype correlated with an increased likelihood of regulating blood pressure with a diuretic is identified as having an increased likelihood of regulating blood pressure with a diuretic. [00153] Also provided herein is a method of identifying a Adamtsl 6 promoter allele genotype correlated with an increased likelihood of regulating blood pressure with a diuretic, comprising a) determining the Adamtsl 6 promoter allele genotype of a subject; and b) correlating the presence of the genotype of step (a) with the ability of the subject to regulate blood pressure with a diuretic, thereby identifying a genotype correlated with an increased likelihood of regulating blood pressure with a diuretic. [00154] A method of identifying a human subject as having a reduced likelihood of regulating blood pressure with a diuretic is also provided, comprising determining the presence in the subject of a Adamtsl 6 promoter allele genotype correlated with a reduced likelihood of regulating blood pressure with a diuretic, whereby the presence of the genotype identifies the subject as having a reduced likelihood of regulating blood pressure with a diuretic.

[00155] Furthermore, the present invention provides a method of identifying a human subject as having a reduced likelihood of regulating blood pressure with a diuretic, comprising a) correlating a. Adamtsl 6 promoter allele genotype with a reduced likelihood of regulating blood pressure with a diuretic; and b) determining the Adamtsl 6 promoter allele genotype of the subject, whereby a subject having a Adamtsl 6 promoter allele genotype correlated with a reduced likelihood of regulating blood pressure with a diuretic is identified as having a reduced likelihood of regulating blood pressure with a diuretic. [00156] Also provided is a method of identifying a Adamtsl 6 promoter allele genotype correlated with a reduced likelihood of regulating blood pressure with a diuretic, comprising a) determining the Adamtsl 6 promoter allele genotype of a subject; and b) correlating the presence of the genotype of step (a) with the inability of the subject to regulate blood pressure with a diuretic, thereby identifying the genotype as correlated with a reduced likelihood of regulating blood pressure with a diuretic. [00157] In one specific embodiment, the present invention relates to identification of specific polymorphisms of the Adamtsl 6 gene associated with human hypertension. The analysis of the Adamtsl 6 gene for these polymorphisms will identify subjects with a genetic predisposition to develop essential hypertension or pregnancy-induced hypertension. The management of hypertension in these subjects could then be more specifically managed, e.g., by dietary sodium restriction, by carefully monitoring blood pressure and treating with conventional drugs, by the administration of certain inhibitors or by the administration of drugs to inhibit the synthesis of Adamtsl 6. The analysis of the Adamtsl 6 gene is performed by comparing the DNA sequence of an individual's Adamtsl 6 gene with the DNA sequence of the native, non- variant Adamtsl 6 gene. [00158] In one embodiment, the invention provides several new polymorphisms as described herein that can be can be used to determine the predisposition to hypertension. It has further been found that some of these polymorphisms occur in linkage disequilibrium with the variants T/C(-820), T/A(-35086), and other molecular variants, as described in further detail herein. Accordingly, in another embodiment the invention provides a method of that which can be used in place of, or in addition to, an analysis based upon the previously known molecular variants.

[00159] DNA sequencing of the entire Adamtsl 6gene in a series of study subjects can lead to the identification of a number single nucleotide polymorphisms (SNPs) in the Adamtsl 6 gene. Typing of a number of these SNPs in larger series of subjects can provide the definition of the haplotype structure of the Adamtsl όgene, that is, the observed distribution of these genetic variants on human chromosomes. These data can provide the most common haplotypes that are sufficient to describe the majority of the variation observed in the Adamtsl 6 gene in either population. Thus, in another embodiment the invention provides a reduced set of SNPs that can be used to characterize such haplotypes by conventional DNA typing methods. Further evaluation of this variation aids in assessing predisposition for hypertension. The analysis of the Adamtsl 6 gene for molecular variants will identify subjects with a genetic predisposition to develop essential hypertension or pregnancy-induced hypertension.

[00160] The present invention also relates to the identification of haplotypes of the Adamtslό gene which can also be used to determine predisposition to hypertension. In accordance with this aspect of the present invention, the haplotype of an individual is analyzed for the alleles described herein and the presence of a particular haplotype is then associated with a predisposition to hypertension. [00161] Human SNP of ADAMSl 6

[00162] There is only one relevant study in humans wherein a BP QTL is reported on human chromosome 5 within the region homologous to the QTL that we have fine- mapped in the rat¹⁹. The inventor herein assessed the status of the human SNPs of ADAMTS 16 in this cohort. The data obtained is presented in the Table below, which indicates that two of the SNPs within ADAMTSl 6 are associated with human essential hypertension. This evidence forms the basis to further explore the structure-function relationship between ADAMTSl 6 and BP in S versus S.LEW congenic strains. [00163] In accordance with the provisions of the patent statutes, the principle and mode of operation of this invention have been explained and illustrated in its preferred embodiment. However, it must be understood that this invention may be practiced otherwise than as specifically explained and illustrated without departing from its spirit or scope.

[00164] References

[00165] The publication and other material used herein to illuminate the invention or provide additional details respecting the practice of the invention, are incorporated be reference herein, and for convenience are provided in the following bibliography.

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Claims

CLAIMS What is claimed is:

1. A method for selecting an animal model of disease for having desired genotypic properties, the method comprising: testing the animal for the presence of a parentally imprinted quantitative trait locus (QTL) that at least partially controls blood pressure and/or at least partially is indicative of a potential for developing a hypertensive disease.

2. The method according to claim 1, further comprising: testing a nucleic acid sample from the animal for the presence of QTL.

3. The method according to claim 1, wherein the animal is a rat and the QTL is located at chromosome 17.

4. The method according to claim 3, wherein the QTL is mapping at around position IpI 1.

5. The method according to claim 1, wherein the animal is a mouse and the QTL is located at chromosome 13

6. The method according to claim 1 , wherein the QTL is related to the potential to develop hypertension.

7. The method according to claim 1, wherein the QTL comprises at least a part of Adamts 16 gene.

8. The method according to claim 3, wherein in the QTL of the rat comprises a marker marked with an asterisk (*), as identified in Table 1.

9. An isolated and/or recombinant nucleic acid sequence comprising a quantitative trait locus (QTL) or functional fragment derived thereof, where the QTL at least partially controls blood pressure and/or at least partially is indicative of a potential for developing a hypertensive disease.

10. An isolated and/or recombinant nucleic acid sequence comprising a synthetic quantitative trait locus (QTL) derived from at least one chromosome or functional fragment derived thereof where the QTL at least partially controls blood pressure and/or at least partially is indicative of a potential for developing a hypertensive disease.

11. The isolated and/or recombinant nucleic acid sequence of claim 11 , at least partly derived from a rat chromosome I₅ a mouse chromosome, 13, or a human chromosome 5.

12. The isolated and/or recombinant nucleic acid sequence of claim 11 , wherein the QTL comprises at least a part of an Adamtslό gene.

13. A method for selecting an animal for having desired genotypic properties, the method comprising: testing the animal for the presence of a quantitative trait locus (QTL) with an isolated and/or recombinant nucleic acid sequence comprising a QTL or functional fragment derived therefrom, where the QTL at least partially controls blood pressure and/or at least partially is indicative of a potential for developing a hypertensive disease..

14. The method according to claim 18, to select a non-human animal having desired genotypic or potential phenotypic properties.

15. An animal selected for by the method according to claim 18.

16. The animal of claim 19, wherein the animal is homozygous for an allele at a QTL

17. The animal of claim 19. wherein the QTL at least partially controls blood pressure and/or at least partially is indicative of a potential for developing a hypertensive disease, and/or wherein the QTL comprises at least a part of an Adamts 16 allele.

18. An animal, which is transgenic, the animal comprising the nucleic acid of claim 11.

19. The animal of claim 22, which is a male.

20. The animal of claim 22, which is a female.

21. A sperm or an embryo from the animal of claim 22.

22. The isolated and/or recombinant nucleic acid sequence of claim 11 , wherein the isolated and/or recombinant nucleic acid sequence is at least partly derived from a rat chromosome 1 from a region mapping at around position IpI 1.

23. A method for determining the predisposition of an individual to hypertension which comprises analyzing at least part of the DNA sequence of at least one allelic variant, fragment or derivative of the Adamts 16 gene of the individual for the presence of at least one single nucleotide polymorphism (SNP) in the Adamts 16 gene, wherein the SNP is selected from the group consisting of one or more of SNPs as described herein.

24. The method of claim 23, wherein the predisposition is a predisposition to essential hypertension.

25. The method of claim 23, wherein the genomic sequence of the Adamts 16 gene of the individual is analyzed.

26. The method of claim 23 , wherein the genomic sequence of a part of the Adamts 16 gene of the individual is analyzed.

27. The method of claim 23. wherein the determination of at least a part of the Adamts 16 gene is performed by hybridization of a nucleic acid to the Adamts 16 gene of the individual.

28. The method of claim 27, wherein the hybridization is performed with an allele-specific oligonucleotide probe.

29. The method of claim 23, wherein the analysis is carried out by sequence analysis.

30. The method of claim 23, wherein the determination of the Adamtslό gene is carried out by substitution mapping analysis.

31. A nucleic acid probe which specifically hybridizes to one or more SNPs in the Adamtslό gene wherein the SNP is selected from the group consisting of one or more of the SNPs described herein.

32. A method for determining whether an individual has, or is predisposed to developing, hypertension associated with a hypertensive haplotype, the method comprising analyzing at least part of the DNA sequence of the Adamtslό gene of the individual for the presence of an allelic pattern, wherein each allele comprises at least one SNP selected from one or more of the SNPs described herein; and wherein the presence of the allelic pattern indicates that the individual is predisposed to the development of, or has, hypertension.

33. The method of claim 32, wherein the predisposition is a predisposition to essential hypertension.

34. The method of claim 32, wherein the genomic sequence of at least one allele of the Adamtslό gene of the individual is analyzed.

35. The method of claim 32, wherein the analysis is performed by hybridization of at least one nucleic acid to the Adamtslό gene of the individual.

36. The method of claim 35, wherein the hybridization is performed with an allele-specific oligonucleotide probe.

37. The method of claim 32, wherein the analysis is carried out by sequence analysis.

38. The method of claim 32, wherein the determination of the Adamtslό gene is carried out by substitution mapping analysis.

39. The method of claim 32, wherein a part of the genomic sequence of at least one allele of the Adamtslό gene of the individual is analyzed.

40. The method of claim 39, wherein the analysis is carried out by hybridization of a nucleic acid probe to at least one allele of the Adamtslό gene.

41. The method of claim 39, wherein the analysis is determined hybridization is with an allele-specific oligonucleotide probe.

42. The method of claim 39, wherein the analysis is carried out by substitution mapping analysis.

43. The method of claim 39, wherein a part of the genomic sequence of the Adamtslό gene of the human is analyzed.

44. A method of determining the predisposition of an individual to hypertension which comprises analyzing at least part of the DNA sequence of the Adamtslό gene of the individual for the presence of at least one haplotype for the Adamtslό gene.

45. The method of claim 44, wherein the predisposition is a predisposition to essential hypertension.

46. The method of claim 44, wherein the genomic sequence of at least one allele of the haplotypes for the Adamtslό gene of the individual is analyzed.

47. The method of claim 44, wherein a part of the genomic sequence at least two alleles of the haplotypes for the Adamtslό gene of the individual are analyzed.

48. The method of claim 44, wherein the analysis is performed by hybridization of at least one nucleic acid to the Adamtslό gene of the individual.

49. The method of claim 44, wherein the hybridization is performed with at least one allele-specific oligonucleotide probe.

50. The method of claim 44, wherein the analysis is carried out by sequence analysis.

51. The method of claim 44, wherein the analysis is carried out by substitution mapping analysis.

52. The method of claim 44, wherein a part of the genomic sequence of the haplotypes for the Adamtslό gene of the individual is analyzed.

53. The method of claim 44, wherein the analysis is carried out by hybridization of a nucleic acid probe to at least one allele of the haplotypes for the Adamtslό gene.

54. A method of diagnosis of hypertension in a human, the method comprising determining the genotype of at least one microsatellite region of the Adamtslό gene in the human, identifying whether the genotype is a risk genotype and, if a risk genotype is so identified, then diagnosing the human as hypertensive.

55. A method of identifying a human predisposed or susceptible to hypertension, the method comprising determining the genotype of at least one microsatellite region of the Adamtslό gene in the human, identifying whether the genotype is a risk genotype and, if a risk genotype is so identified, then diagnosing the human as susceptible or predisposed to hypertension.

56. The method of claim 54 or 55, comprising determining whether the human is homozygous or heterozygous for polymorphisms of the microsatellite region of the Adamts 16 gene.

57. The method of claim 54 or 55, wherein the determination of the genotype of the region is accomplished by screening the region to identify a risk polymorphism in the region, the risk polymorphism being indicative of risk of hypertension or of predisposition or susceptibility to hypertension.

58. The method of claim 55, wherein the determining is accomplished by amplification of a nucleic acid sequence located within the microsatellite region.

59. The method of claim 58, wherein the amplification is accomplished by the polymerase chain reaction using one or more primers adapted to amplify a nucleic acid sequence located within the microsatellite.

60. The method of claim 59, wherein the primers have a nucleotide sequence selected from the group of nucleotide sequences described herein.

61. A method of diagnosis and treatment of a human susceptible or predisposed to hypertension, comprising determining the genotype of one or more of the genomic regions of the Adamts 16 gene that contain single nucleotide polymorphisms as genotypes and identifying whether the genotype is a risk genotype and, if a risk genotype is so identified, then diagnosing the human as susceptible or predisposed to hypertension, and administering treatment to reduce or delay or prevent hypertension.

62. The method of claim 61, wherein the treatment is selected from the group consisting of administration of an effective amount of antihypertensive pharmaceutical, administration of an effective anti-hypertension therapy or administration of both an effective anti-hypertension therapy and an effective amount of antihypertensive pharmaceutical.

63. The method of claim 62, wherein the risk genotype is one or more of the alleles described herein having a length 1 or >1 bp.

64. A method of diagnosing hypertension or susceptibility to hypertension, the method comprising determining the genotype of Adamtslό, identifying whether the genotype is a risk genotype and, if a risk genotype is so identified, then diagnosing the human as hypertensive or susceptible to hypertension, wherein an allele of 1 or >1 bp indicates a risk of hypertension or of susceptibility to hypertension and wherein an allele of 1 or >1 bp indicates the absence of risk of hypertension or of susceptibility to hypertension.

65. The method of claim 57, wherein the risk genotype of the microsatellite region includes an allele of 1 or >1 bp.

66. The method of claim 57, wherein the non-risk genotype of the D5S207 microsatellite region includes an allele of 1 or >lbp

67. A method for prevention or treatment of a hypertensive disease comprising administering to a mammalian subject in need of such treatment an effective amount of a compound in a pharmaceutically acceptable carrier enhancing or reducing biological activity of one or several polypeptides encoded by Adamtslό or a variant, fragment or derivative thereof; and/or enhancing or reducing activity of one or several biological networks and/or metabolic pathways related to the polypeptides.

68. The method according to claim 67, wherein the treatment is gene therapy or gene transfer.

69. The method according to claim 67, wherein the treatment comprises treating regulatory regions and/or gene containing region of one or more the Adamtslό gene or variants, fragments or derivatives thereof in somatic cells of the subject.

70. The method according to claim 67, wherein the treatment comprises treating regulatory regions and/or gene containing region of one or more the Adamtslό gene or variants, fragments or derivatives thereof in stem cells.

71. The method according to claim 67, wherein the compound is a recombinant polypeptide encoded by the Adamtslό gene or variant, fragment or derivative thereof.

72. The method according to claim 67, wherein the treatment is based on siRNA hybridising to mRNA and/or to hnRNA of the Adamtslό gene.

73. The method according to claim 67, wherein the method of treating is a dietary treatment or a vaccination.

74. The method according to claim 67, comprising a therapy restoring, at least partially, the observed alterations in biological activity of one or several polypeptides encoded by the Adamtslό gene in the subject, when compared with HT free healthy subjects.

75. A method of identifying subjects at risk of developing a hypertensive disease comprising: a) providing nucleic acid from a subject, wherein the nucleic acid comprises an Adamtslό gene; b) detecting the presence or absence of one or more variations in the Adamtslό gene, wherein the variation is selected using at least one marker from the group shown in Table 1 ; and c) determining if the subject is at risk of developing a hypertensive disease based on the presence or absence of the one or more variations.

76. The method of claim 75, wherein the detecting in step b) is accomplished by substitution mapping analysis or by hybridization analysis.

77. The method of claim 75, wherein the detecting in step b) comprises comparing the sequence of the nucleic acid to the sequence of a wild-type Adamtslό nucleic acid.

78. A method of identifying a variant, fragment or derivative of Adamts 16 gene, comprising: a) providing nucleic acid from a subject, wherein the nucleic acid comprises a. Adamts 16 gene; and b) detecting the presence or absence of one or more variations in the Adamts 16 gene, wherein the variation is selected using at least one marker shown in Table 1.

79. The method of claim 78, further comprising step c) determining if the subject is at risk of carrying a hypertensive disease allele based on the presence or absence of the one or more variations.

80. A screening method of preventive and therapeutic agents for diseases caused by the over- or under- expression of the Adamts 16 gene or a variant, fragment or derivative thereof, wherein an animal, or tissues, organs or cells derived from the animal and a test substance are used.

81. The screening method according to claim 80, wherein the test substance is administered to the animal, and the blood pressure level of the animal is measured and estimated.

82. The screening method according to claim 80, wherein the disease caused by the overexpression of Adamts 16 is a dysfunction of the kidney.

83. The screening method according to claim 80, wherein the disease caused by the overexpression of regucalcin is essential hypertension.

84. A preventive or therapeutic agent for diseases caused by the over- or under- expression of the Adamts 16 gene or a variant, fragment or derivative thereof obtained by the screening method according to claim 80.

85. A screening method of causative agents of diseases caused by the over- or under- expression of the Adamts 16 gene or a variant, fragment or derivative thereof, wherein an animal or tissues, organs or cells derived from the animal and a test substance are used.

86. The screening method according to claim 85, wherein the test substance is administered to the animal, and the blood pressure level of the animal is measured and estimated.

87. The screening method according to claim 85, wherein the disease is a hypertensive disease.

88. An animal model wherein the animal model is a non-human animal that over- or under- expresses the Adamtslό gene or a variant, fragment or derivative thereof, and shows a hypertensive pathology.

89. The animal model according to claim 88, wherein the animal is selected by a morphological measurement estimation of blood pressure levels.

90. The animal model according to claim 88, wherein the characteristic of hypertensive pathology is stable through many generations.

91. The animal model of claim 88, wherein the non-human animal is a female non-animal.

92. The animal model of claim 88, wherein the non-human animal is a male non-animal.

93. The animal of claim 88, wherein the non-human animal is a rat.

94. A screening method of preventive and therapeutic agents for hypertensive diseases wherein a test substance is administered to a animal model according to claim 88, and a morphological measurement estimation of blood pressure and/or a biochemical measurement estimation of over- or under- expression of Adamtslό or a variant, fragment or derivative thereof are performed.

95. The screening method of claim 94, wherein the hypertensive disease is essential hypertension.

96. A preventive or therapeutic agent for a hypertensive disease obtained by the screening method according to any of claims 88 to 95.

97. A method for identification of an individual who has an altered risk of or susceptibility for developing a hypertensive disease, the method comprising: a) providing a biological sample taken from the individual; b) collecting personal and clinical information of the individual; c) determining the nucleotides present in one or several of the polymorphic sites described herein in the individual's nucleic acid; and d) combining the SNP marker data with personal and clinical information to assess the risk of an individual to develop the hypertensive disease.

98. The method according to claim 97, wherein the altered risk is an increased risk of the hypertensive disease.

99. The method according to claim 97, wherein the altered risk is a decreased risk of the hypertensive disease.

100. The method according to claim 97, wherein the polymorphic sites are those present in the haplotypes described herein.

101. The method according to claim 97, wherein the polymorphic sites are associated with the SNP markers set forth herein.

102. The method according to claim 101, wherein the polymorphic sites are in complete linkage disequilibrium with the SNP markers set forth herein.

103. A method for identification of an individual who has an altered risk of or susceptibility for developing a hypertensive disease, the method comprising: a) providing a biological sample taken from a subject; b) determining the nucleotides present in one or several of the polymorphic sites as set forth herein in the individual's nucleic acid; and c) combining the SNP marker data to assess the risk of an individual to develop the hypertensive disease.

104. The method according to claim 103, wherein the altered risk is an increased risk of the hypertensive disease.

105. The method according to claim 103, wherein the altered risk is a decreased risk of the hypertensive disease.

106. The method according to claim 103, wherein the polymorphic sites are those present in one or more of the haplotypes presented herein.

107. The method according to claim 103, wherein the polymorphic sites are associated with one or more of the SNP markers set forth herein.

108. The method according to claim 107, wherein the polymorphic sites are in complete linkage disequilibrium with one or more of the SNP markers set forth herein.

109. The method according to claim 97, wherein the one or several polymorphic sites reside within a hypertensive risk gene or genes as set forth herein.

110. The method according to claim 97, wherein one or several of the SNP markers are selected from the group shown in Table 2.

111. A method for assessing susceptibility or predisposition to a hypertensive disease in an individual, the method comprising determining alteration of expression levels of one or several of the markers of Table 1 in the individual, wherein a difference in expression is indicative of susceptibility to a hypertensive disease.

112. The method according to claim 11 1, wherein alteration of expression levels is determined by assessing transcription levels of one or several of the genes of Table 1 in the individual.

113. The method according to claim 111, wherein alteration of expression levels is determined by assessing translation of mRNAs encoded by one or several of the genes of Table 1 in the individual.

114. A test kit based on a method according to claim 1 for assessment of an altered risk of or susceptibility for the hypertensive disease in a subject.

115. A test kit for determining the nucleotides present in one or several of the SNP markers as set forth herein in the individual's nucleic acid for assessment of an altered risk of a hypertensive disease in a subject.

116. A test kit for determining the nucleotides present in one or several of the SNP markers as set forth herein tables in the individual's nucleic acid for assessment of an altered risk of a hypertensive disease in a subject, containing: a) reagents and materials for assessing nucleotides present in one or several SNP markers as set forth herein; and b) software to interpret the results of the determination.

117. A test kit according to any of the claims herein, further including a PCR primer set for amplifying nucleic acid fragments containing one or several SNP markers as set forth herein from the nucleic acids of the subject.

118. The test kit according to any of the preceding claims, including a capturing nucleic acid probe set specifically binding to one or several SNP markers present in hypertensive disease associated markers and haplotype regions as set forth herein.

119. The test kit according to any of the preceding claims, including a microarray or multiwell plate to assess the genotypes.

120. The test kit according to any of the preceding claims, including a questionnaire for obtaining patient information concerning age. gender, height, weight, waist and hip circumference, skinfold and adipose tissue thicknesses, the proportion of adipose tissue in the body, the family history of diabetes and obesity, the medical history concerning hypertension.

121. The test kit of any of the preceding claims, including a capturing nucleic acid probe set specifically binding to one or several SNP markers present in the Adamtslό gene.

122. The test kit of any of the preceding claims, including a microarray or multiwell plate to assess the genotypes.

123. An isolated nucleic acid comprising an Adamtslό-like gene comprising a polymorphism at position 820 as defined by the position shown in Figs. 3 and 9A, as wherein the amino acid corresponding to position 274 is not a proline.

124. The isolated nucleic acid molecule of claim 123, wherein the nucleotide at position 820 is T.

125. The isolated nucleic acid molecule of claim 123, wherein the amino acid at position 274 is serine.

126. An isolated nucleic acid molecule comprising a sequence complementary to the isolated nucleic acid molecule of claim 123.

127. An isolated nucleic acid comprising an Adamtsl6-like gene comprising a polymorphism at position 3508 as defined by the position shown in Figs. 3 and 9B, wherein the amino corresponding to position 1 170 is not a threonine.

128. The isolated nucleic acid molecule of claim 123, wherein the nucleotide at position 3508 is T.

129. The isolated nucleic acid molecule of claim 126, wherein the amino acid at position 1170 is a serine.

130. An isolated nucleic acid molecule comprising a sequence complementary to the isolated nucleic acid molecule of claim 127.

131. A method for detection of at least one single nucleotide polymorphism (SNP) in a human AdamtsJ6-\ike gene, which method comprises determining a nucleotide at position 820 in the human Adamtsl6-\ik& gene as shown in Fig. 7, and thereby detecting absence or presence of at least one SNP.

132. A method according to claim 131 , in which the amino acid encoded by the single polymorphism at position 820 is histidine.

133. A method for detection of at least one single nucleotide polymorphism (SNP) in a human Adamtsl6-\ike gene, which method comprises determining a nucleotide at position 3508 in the human AdamtslόΛike gene as shown in Fig. 7, and thereby detecting absence or presence of at least one SNP.

134. A method according to claim 131 , in which the amino acid encoded by the single polymorphism at position 3508 is L.

135. An allele-specific nucleic acid primer, comprising between 100 - 350 nucleotides which hybridizes to and

gene polymorphism at position 820 in the Adamtslόλike gene as defined by the positions in Figs. 3 and 9A.

136. The nucleic acid sequence of claim 123, wherein the T allele is indicative of increased levels of blood pressure in an animal.