- Email: [email protected]
Genetics of hypertension
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Genetics of hypertension Patricia B Munroe* and Mark J Caulfield In the past year, substantial progress has been made in both mapping and fine mapping the genes involved in blood pressure regulation. Genome scans have been carried out in humans and mice and these reveal many new potential chromosomal locations for blood pressure susceptibility loci. The chromosomal regions containing blood pressure genes for many of the inbred hypertensive rat models have been refined using new congenic strains. Further genetic studies support a role for angiotensinogen, aldosterone synthase and a region close to the epithelial sodium channel in blood pressure regulation. Finally, comprehensive single-nucleotide polymorphism analysis of cardiovascular genes has been undertaken using chip technology. Addresses Department of Clinical Pharmacology, St. Bartholomew’s, and The Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK *e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:325–329 0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations ACE angiotensin-converting enzyme AGT angiotensinogen ENaC epithelial sodium channel GWS genome-wide scanning QTLs quantitative trait loci SNPs single-nucleotide polymorphisms
Introduction Human essential hypertension is a complex disorder which is thought to be determined by genetic and environmental factors (sodium salt, alcohol excess and obesity). The condition affects 10–20% of the populace in western nations and operates as an important risk factor for stroke and coronary artery disease mortality [1]. Previous workers have attempted to quantify the influence of genes upon blood pressure variation. At present, it is widely accepted that approximately 30–50% of this quantitative trait can arise from genetic susceptibility [2]. Thus, it is reasonable to propose that hypertension geneticists may be searching for either a small number of loci with substantial influences or multiple loci with very modest influence. These researchers can draw upon a burgeoning literature from experimental models of hypertension to guide their studies in humans. In the past year, important data from genomic screens in such models has been reported. In addition, we have seen the publication of the first whole-genome screens for human blood pressure genes. If observers of the past decade have been disappointed that we have not made more dramatic progress in identifying the genes for hypertension, we believe the past year has some encouraging new pointers. The scope of this review is to describe some of the most promising research on
the genetics of hypertension published during the past year. The scope of this review is to describe some of the most promising research on the genetics of hypertension published during the past year.
Candidate genes influencing sodium homeostasis Candidate gene analysis remains one of the main approaches to determining high blood pressure genes, and loci that control sodium handling are amongst the many candidates studied. During the past year, new functional data on angiotensinogen (AGT) variants have been reported, together with additional support for a role of aldosterone synthase and the epithelial sodium channel genes in blood pressure regulation. Despite AGT being one of the most studied candidate genes, the identity of AGT variant(s) predisposing to high blood pressure has not yet been defined. The gene has many polymorphisms, especially in the promoter region. These have been the focus of functional analyses to determine whether they influence protein activity and whether there is any correlation between genotype, AGT levels and blood pressure. This year, Zhao et al. [3•] carried out in vitro studies on the A-20C promoter polymorphism of the AGT gene. They found that when adenine (A) is at position –20 the sequence binds to the oestrogen receptor-α, whereas when cytosine (C) is present a viral transcription factor binds preferentially and there is increased basal transcriptional activity. In support of these molecular observations, an association between the A-20C polymorphism and plasma AGT levels has also been reported [4]. At present, the exact interaction between the A-20C polymorphism with the downstream A-6G promoter polymorphism, which has also been shown to affect basal transcription of AGT [5], remains to be established. Another promoter polymorphism (C-532T) and a rare mutation (Arg-30Pro) in the signal peptide of the AGT gene have also been found to be associated with plasma AGT levels. Paillard et al. [6] found a significant correlation between the C-532T polymorphism and increased plasma AGT levels, and Nakajimi et al. [7] found that the Arg-30Pro mutation in its heterozygous state results in mildly reduced plasma AGT levels. Their work lends further support for a relationship between AGT gene polymorphisms and plasma angiotensinogen levels; however, any direct link to hypertension still remains to be established. The dissection of variants affecting AGT function also provides investigators with the most important polymorphisms to assess in hypertensive subjects. In 1998, data were reported that support a minor role for the aldosterone synthase gene (CYP11B2) in essential hypertension [8]. The associated polymorphisms, C-344T
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Table 1 Mapped QTLs and potential candidate genes from genome-wide screens in man. Human populations Caucasian
Chromosome
Nearest marker
2
D2S1788
5
D5S1471
6
D6S1009
15
D15S652
3 11
D3S2387 D11S2019
15 16 17 15
D15S657 D16S3396 D17S1303 D15S203
Potential candidate genes
Reference
Calmodulin 2 Sodium—calcium exchanger 1 Alpha 1B adrenergic receptor Dopamine receptor type 1A Phospholamban Estrogen receptor Aminopeptidase N
[12••]
ATP-ase Ca2+-transporting plasma membrane Angiotensin receptor-like 1 Angiotensinase C Cholinergic receptor Thiazide-sensitive Na-Cl cotransporter
[14••]
Insulin-like growth factor 1
[15••]†
Chinese*
*In this genome scan markers on chromosomes 3, 11, 16 and 17 are linked with systolic blood pressure, chromosome 15 is linked with diastolic blood pressure. † Fine mapping linkage results of the diastolic blood pressure locus from Xu et al. [14••].
and an intronic conversion in intron 2, have subsequently been studied in a Scottish case-control population [9]. Although the numbers were small in both groups within the Scottish study (~130), significant association was observed between both polymorphisms and hypertension. These results differ from the study by Brand et al. [8], in which only the polymorphism C-344T was found to be associated with the disease. These researchers also reported an association between the -344T allele and a higher excretion rate of the metabolite tetrahydroaldosterone in this study [8]. A relationship between the C-344T polymorphism with both urinary and plasma aldosterone levels has now been found in a number of studies [6,8,9]; therefore, raised aldosterone levels might be a good intermediate phenotype to aid selection of patients with alterations in CYP11B2. An alternative to carrying out candidate gene analysis in pre-selected hypertensive groups is to study blood pressure as a quantitative trait in families or a randomly selected population. Recently, microsatellite markers flanking the epithelial sodium channel gene (ENaC) have been studied in 286 families comprising at least both parents and two children [10]. Results from this study indicate linkage between three of the markers studied (D16S412, D16S403 and D16S420) and systolic blood pressure. Some support for these findings comes from a recent study involving 66 pairs of dizygotic twins, in which linkage between markers mapping close to ENaC and systolic blood pressure was also reported [11]. These studies do not prove that ENaC subunits account for the linkage observed, as it is feasible that linkage is between another locus in this region and the disorder. In light of these new observations, further studies assessing the chromosomal region containing ENaC in different populations are warranted, possibly in individuals with blood pressure in response to salt loading.
The first human genome screens In the past year, two groups have performed genome-wide scanning (GWS) to determine the location of blood pressure loci in humans. Kruskal et al. [12••] used a highly discordant sib-pair design, an approach that offers greater statistical power than conventional concordant sib-pair analysis, to determine genes influencing interindividual variation of systolic blood pressure. They studied 69 discordant sib-pairs and found 4 regions with significant linkage (P < 0.01) located on chromosomes 2p, 5q, 6q and 15q (see Table 1). A key statistical issue within this study is the selected threshold for acceptance of linkage. Kruskal et al. [12••] indicate that their statistically accepted threshold does not coincide with that proposed by Lander and Kruglyak [13], implying that these observations must be treated cautiously. Xu et al. [14••] have carried out GWS in discordant, highly concordant and low concordant Chinese sib-pairs for systolic and diastolic blood pressure. Their genome scan did not reveal any chromosomal regions that reached a 5% genomewide significance level; however, there were some promising regions with suggestive linkage to systolic blood pressure (closest markers being D11S2019, D3S2387, D16S3396 and D17S1303) and one region (closest marker D15S657) with suggestive linkage to diastolic blood pressure (Table 1). There does not appear to be an overlap of regions containing blood pressure quantitative trait loci (QTLs) between the two GWS studies, although marker D15S652 from the scan by Krushkal et al. [12••] has not been confidently mapped. The lack of concordance between the mapping results could be due to differences in the ethnicity of the populations studied or in the selection criteria used to define the study populations. Krushkal et al. [12••] studied discordant siblings from the upper and lower 20% of the systolic blood pressure distribution that were adjusted for age
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and sex. The discordant group studied by Xu et al. [14••] were drawn from the upper and lower 10% of systolic blood pressure distribution and were adjusted only for age. Of great interest however, is a follow-up study of the putative diastolic blood pressure QTL on chromosome 15 [15••]. Xu et al. [15••] have been able to confirm linkage between low diastolic blood pressure and a region on the long arm of chromosome 15, near marker D15S203, in a re-defined concordant hypotensive sib-pair resource and a new collection of sib-pairs from the same population. There are six possible candidate genes in this region, including the insulin-like growth factor 1 receptor (Table 1). This approach of studying hypotensive siblings appears to have yielded a new blood pressure locus and provides support for a combination of approaches to dissect genes influencing blood pressure.
Mouse genome screening Mouse models of hypertension also exist, although until recently very few genetic studies had been carried out owing to the difficulties in assessing phenotypes. A GWS for QTLs of systolic blood pressure has recently been carried out using the inbred hypertensive mouse [16••]. Significant linkage with systolic blood pressure was found with a region on chromosome 10 in the F2 inter-cross and with a region on chromosome 13 in the back-cross. Suggestive evidence for linkage was observed on chromosomes 2, 6, 8 and 18. None of the mouse loci appear to map to human or rat syntenic regions, indicating that analysis in the mouse might detect new genes regulating blood pressure.
Hypertensive rodent model QTLs Genome-wide scanning has been widely used to map blood pressure QTLs in inbred hypertensive rat strains and more recently to determine modifier loci [17,18•]. Indeed, last year a new blood pressure QTL was mapped to rat chromosome 12 in the Dahl salt-sensitive strain [19]. After mapping a QTL, congenic strains are developed to confirm its presence, and congenic substitution mapping is then used to reduce the chromosomal region in which the QTL resides. In the past year, the QTLs on rat chromosomes 1 and 19 have been confirmed and the critical regions refined [20,21]. A congenic strain of the spontaneously hypertensive rat containing part of chromosome 1q from the Brown Norway control strain has been developed as a model for hypertension-induced renal injury [22]. With the development of improved rat maps [23•], the time taken to fine map QTLs should be reduced when combined with expression profiling of candidates in the critical region.
Comparative mapping — experimental models to man Reproducible rat and mouse QTLs present good candidates for comparative mapping in human hypertension. Rat chromosome 10 contains two blood pressure QTLs [24,25] and is syntenic to human chromosome 17. Julier et al. [26] have found evidence for linkage of markers on human chromosome 17 with hypertension. Baima et al. [27•] recently published data supporting these observations in a smaller
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study group and claim to have refined the critical region of linkage; however, their study had fewer sib-pairs than that of Julier et al. [26] and this may have prevented adequate statistical power to establish linkage at other markers. Both studies provide some support for comparative mapping as a method of supplying candidate regions for the human disorder; however, until the causative variants have been determined in both rats and humans we will not know for certain the validity of this approach.
Single-nucleotide polymorphisms and blood pressure homeostasis In May 1999, an innovative academic/industry consortium was established, comprising 10 major pharmaceutical companies and the Wellcome Trust (UK), to identify and map 300,000 of the most abundant human polymorphisms, on the basis of single-nucleotide changes, for subsequent deposit into public domain databases [28]. With dense maps of these single-nucleotide polymorphisms (SNPs) distributed amongst our estimated 100,000 genes, we may be able to assemble informative haplotypes on genes of interest and rapidly home in on causative individual variants or haplotypes. Several publications report new SNPs within candidate genes that might be important in blood pressure regulation [29•,30•,31••]. The most interesting publications have illustrated the value of SNP detection when combined with chip-based technology [30•,31••]. One of these reports focused upon SNP detection in coding regions of candidates for cardiovascular, neuropyschiatric, endocrine diseases and cancer [30•]. The most interesting and relevant paper, by Haluska et al., [31••] reported a systematic survey of coding and non-coding regions of 75 candidate genes for blood pressure homeostasis. These candidates were drawn from the known physiology or pharmacology of blood pressure regulation, and the SNPs were identified with variant detection arrays using chip technology. A surprising 54% of coding, or exonic SNPs, predicted amino-acid changes, which implies considerably greater diversity in peptide sequence than has been previously thought. However, it is noteworthy that many of the coding region SNPs had a low frequency of the minor allele (less than 10%). A key observation reported by Haluska and colleagues [31••] is that individuals of African ancestry possess an average of two extra SNPs per gene. At face value this appears to be intuitive given the anthropological divergence of different ancestral ethnic groups. This finding is particularly germane to the search for hypertension genes because people of west African origin tend to have higher prevalence of hypertension and concomitant target organ damage. Certain studies imply physiological differences that could stem from different susceptibility genes or haplotypes. Both of these papers give encouraging insight into the uses of novel, high-throughput technology for SNP detection.
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The capability to generate large numbers of haplotypes will be critical in understanding the susceptibility genes for all common disorders.
Angiotensin-converting enzyme gene haplotypes The angiotensin-converting enzyme (ACE) converts angiotensin I to the effector peptide angiotensin II; this mediates vasoconstriction, alters sodium homeostasis and influences vascular remodelling within the renin angiotensin system [32]. Several reports have associated the ACE gene with coronary artery disease, blood pressure and cardiovascular structure, but other studies have been unable to replicate these findings [33–35]. One consistent observation is an important genetic influence on the level of ACE enzyme activity that is associated with an intronic insertion/deletion polymorphism within the ACE gene. Although no functional influence of the ACE I/D polymorphism has been demonstrated, it is assumed that there is an as yet unidentified functional variant. There are two publications on the ACE gene that underscore the value of comprehensive SNP detection in candidates of interest. The first by Rieder et al. [36•] evaluated sequence variation within the regulatory regions, exons and introns of the ACE gene in small numbers of African Americans and white Europeans. The authors reported 13 distinct haplotypes and concluded that, by using the ACE I/D polymorphism, haplotypes could be sorted into 2 distinct groups or clades. The second report by Farrall et al. [37•] draws upon previous work on the ACE gene QTL [38] in larger numbers of African Caribbeans and white Europeans. They had previously suggested that there are three distinct haplotype groups or clades (clades 1, 2 and 3) within the ACE gene. By using information on additional SNPs detected by Rieder et al. [36•], they were able to confirm an ancestral intragenic recombination event between clades 2 and 3. This made it possible to determine a variant that increases levels of ACE activity to be within exons 1–5 and introns 1–4 (and part of 5). Furthermore, the tight linkage disequilibrium within the three clades means that selection of a single SNP to represent each clade may allow significant economy in the numbers of genotypes required for analysis of the ACE gene. This could be a very valuable finding for large-scale genotyping projects of other genes and supports comprehensive SNP detection throughout coding and non-coding regions to unambiguously assign haplotypes.
Conclusions The past year has seen encouraging progress with early reports of human genome screens and more sophisticated approaches to cardiovascular SNP detection. These studies lay important foundations for large-scale human hypertension research. Within the next few years, we anticipate case-control analyses shifting from reports on single gene variants to description of haplotypes with multiple SNPs. Indeed, it may be possible with genotyping technologies such as mass spectrometry, microarrays or chips to present comprehensive SNP
screens of cardiovascular candidates. The current and new genomic screening programmes may describe novel chromosomal regions for detailed mapping. We predict that new regions will be further characterised with the identification of specific disease-causing haplotypes, allowing functional genomics and proteomics to succeed.
Acknowledgements We are grateful to Joanne Knight and Hannah Mitchison for critically reading the manuscript. We would also like to thank The Special Trustees of St. Bartholomew’s Hospital, The Medical Research Council and The British Heart Foundation for financial support.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ••of outstanding interest 1.
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12. Krushkal J, Ferrell R, Mockrin S, Turner S, Sing CF, Boerwinkle E: •• Genome-wide linkage analysis of systolic blood pressure using highly discordant siblings. Circulation 1999, 99:1407-1410. This rapid communication reports data on the first published genome screen for blood pressure genes in humans. A discordant sib-pair design is used, and four chromosomal regions are found to have significant statistical linkage to systolic blood pressure. 13. Lander E, Kruglyak L: Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 1995, 11:241-247. 14. Xu X, Rogus JJ, Terwedow HA, Yang J, Wang Z, Chen C, Niu T, •• Wang B, Xu H, Weiss S et al.: An extreme-sib-pair genome scan for genes regulating blood pressure. Am J Hum Genet 1999, 64:1694-1701. This paper describes a systematic search for genes controlling systolic and diastolic blood pressure in high concordant, low concordant and discordant sib-pairs selected from a screen of more than 200 000 adults from Anqing in China. No chromosomal regions reach statistical significance, but five regions give suggestive linkage to blood pressure and eight regions are regarded as promising. 15. Xu X, Yang J, Rogus J, Chen C, Schork N, Xu X. Mapping of a blood •• pressure quantitative locus to chromosome 15q in a Chinese population. Hum Mol Genet 1999, 8:2551-2555. This paper describes confirmation of a diastolic blood pressure QTL mapping to the long arm of chromosome 15 in Chinese concordant hypotensive sib-pairs. 16. Wright FA, O’Connor DT, Roberts E, Kutey G, Berry CC, Yoneda LU, •• Timberlake D, Schlager S: Genome scan for blood pressure loci in mice. Hypertension 1999, 34:625-630. This paper describes the results from the first genome screen for genes involved in regulating blood pressure in the mouse. Significant linkage is found on two chromosomal regions; four regions have suggestive evidence of linkage to blood pressure. There are other regions with suggestive evidence of linkage to pulse rate and haemocrit levels. 17.
Dominiczak AF, Clark JS, Jeffs B, Anderson NH, Negrin CD, Lee WK, Brosnan MJ: Genetics of experimental hypertension. J Hypertension 1998, 16:1859-1869.
18. Kantachuvesiri S, Haley CS, Fleming S, Kurian K, Whitworth CE, • Wenham P, Kotelevtsev Y, Mullins JJ: Genetic mapping of modifier loci affecting malignant hypertension in TGRmRen2 rats. Kidney Int 1999, 56:414-420. This paper describes the application of a transgene being used as a main gene to determine modifier loci. The transgenic rat (TGRmRen2) develops malignant hypertension; however, the severity varies according to the control rat with which it is bred. Genome-wide screening and QTL analysis reveal the location of potential modifier genes in the Fischer and Lewis control rats. 19. Kato N, Hyne G, Bihoreau MT, Gauguier D, Lathrop M, Rapp JP: Complete genome searches for quantitative trait loci controlling blood pressure and related traits in four segregating populations derived from Dahl sensitive hypertensive rats. Mammalian Genome 1999, 10:259-265.
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Baima J, Nicolaou M, Schwartz F, DeStefano AL, Manolis A, Gavras I, Laffer C, Elijovich F, Farrer L, Baldwin C, Gavras H: Evidence for linkage between essential hypertension and a putative locus on human chromosome 17. Hypertension 1999, 34:4-7. This paper confirms linkage between a small region on human chromosome 17q and blood pressure in Caucasian hypertensive sib-pairs. No support for linkage of this region is found in black hypertensive sib-pairs. 28. Marshall E: Drug firms to create public database of genetic mutations. Science 1999, 284:406-407. 29. Cambien F, Poirier O, Nicaud V, Herrmann S-M, Mallet C, Ricard S, • Behague I, Hallet V, Blanc H, Loukaci V et al.: Sequence diversity in 36 candidate genes for cardiovascular disorders. Am J Hum Genet 1999, 65:183-191. This paper describes the molecular screening of 36 candidate genes for cardiovascular diseases. DNA variations are discovered using single-stranded conformation polymorphism analysis and allele frequencies are determined in a cohort of 750 individuals. 30. Cargill M, Altshuler D, Ireland J, Sklar P, Ardlie K, Patil N, Lane CR, • Lim EP, Kalyanaraman N, Nemesh J et al.: Characterisation of singlenucleotide polymorphisms in coding regions of human genes. Nat Genet 1999, 22:231-238. This paper describes the molecular variation in the coding region of 106 genes that are relevant to cardiovascular disease, neuropyschiatry and endocrine disorders. Single-nucleotide polymorphism detection is carried out by using chip technology and denaturing high performance liquid chromotography. 31. Halushka MK, Fan J-B, Bentley K, Hsie L, Shen N, Weder A, •• Cooper R, Lipshutz R, Chakravarti A: Patterns of single-nucleotide polymorphisms in candidate genes for blood pressure homeostasis. Nat Genet 1999, 22:239-247. This paper describes the molecular variation in 75 candidate genes for blood pressure homeostasis and hypertension in individuals of northern European and African descent. Single-nucleotide polymorphism detection is performed using chip technology. 32. MacGregor GA, Markandu ND, Roulston JE, Jones JC, Morton JJ: Maintenance of blood pressure by the renin-angiotensin system in normal man. Nature 1981, 291:329-331. 33. Cambien F, Poirer O, Lecerl L, Evans A, Cambous J-P, Arvelier D, Luc G, Bard J-M, Bara L, Ricard S et al.: Deletion polymorphism in the gene for the angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature 1992, 359:641-644.
20. Hubner N, Lee AE, Lindpaintner K, Ganten D, Kreutz R: Congenic substitution mapping excludes Sa as a candidate gene locus for a blood pressure quantitative trait locus on rat chromosome 1. Hypertension 1999, 34:643-648.
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36. Rieder MJ, Taylor SL, Clark AG, Nickerson DA: Sequence variation in • the human angiotensin converting enzyme. Nat Genet 1999, 22:59-62. This paper describes the complete genomic sequence and polymorphisms of the angiotensin-converting enzyme. Seventy-eight variants are found and these are used to determine the haplotypes associated with an insertion/deletion polymorphism in intron 16 and circulating concentrations of the enzyme.
23. Watanabe TK, Bihoreau MT, McCarthy LC, Kiguwa SL, Hishigaki H, • Tsuji A, Browne J, Yamasaki Y, Mizoguchi-Miyakita A, Oga K et al.: A radiation hybrid map of the rat genome containing 5,255 markers. Nat Genet 1999, 22:27-36. This paper describes new resources for genomic studies in the rat. It includes a whole-genome radiation hybrid map, a high-resolution map containing over 3 000 new microsatellites and a comparative map with over 500 genes localised to the radiation hybrid map from mapping information available from the mouse and human maps.
Farrall M, Keavney B, McKenzie C, Delepine M, Matsuda F, Lathrop GM: Fine-mapping of an ancestral recombination breakpoint in DCP1. Nat Genet 1999, 23:270-271. The authors describe the refinement of the location of an ancestral recombination breakpoint in the angiotensin-converting enzyme that allows the region of the gene harbouring the quantitative trait variant to be localised. The polymorphisms described by Rieder et al. [36•] are used.
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Genetics of hypertension Patricia B Munroe* and Mark J Caulfield In the past year, substantial progress has been made in both mapping and fine mapping the genes involved in blood pressure regulation. Genome scans have been carried out in humans and mice and these reveal many new potential chromosomal locations for blood pressure susceptibility loci. The chromosomal regions containing blood pressure genes for many of the inbred hypertensive rat models have been refined using new congenic strains. Further genetic studies support a role for angiotensinogen, aldosterone synthase and a region close to the epithelial sodium channel in blood pressure regulation. Finally, comprehensive single-nucleotide polymorphism analysis of cardiovascular genes has been undertaken using chip technology. Addresses Department of Clinical Pharmacology, St. Bartholomew’s, and The Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK *e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:325–329 0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations ACE angiotensin-converting enzyme AGT angiotensinogen ENaC epithelial sodium channel GWS genome-wide scanning QTLs quantitative trait loci SNPs single-nucleotide polymorphisms
Introduction Human essential hypertension is a complex disorder which is thought to be determined by genetic and environmental factors (sodium salt, alcohol excess and obesity). The condition affects 10–20% of the populace in western nations and operates as an important risk factor for stroke and coronary artery disease mortality [1]. Previous workers have attempted to quantify the influence of genes upon blood pressure variation. At present, it is widely accepted that approximately 30–50% of this quantitative trait can arise from genetic susceptibility [2]. Thus, it is reasonable to propose that hypertension geneticists may be searching for either a small number of loci with substantial influences or multiple loci with very modest influence. These researchers can draw upon a burgeoning literature from experimental models of hypertension to guide their studies in humans. In the past year, important data from genomic screens in such models has been reported. In addition, we have seen the publication of the first whole-genome screens for human blood pressure genes. If observers of the past decade have been disappointed that we have not made more dramatic progress in identifying the genes for hypertension, we believe the past year has some encouraging new pointers. The scope of this review is to describe some of the most promising research on
the genetics of hypertension published during the past year. The scope of this review is to describe some of the most promising research on the genetics of hypertension published during the past year.
Candidate genes influencing sodium homeostasis Candidate gene analysis remains one of the main approaches to determining high blood pressure genes, and loci that control sodium handling are amongst the many candidates studied. During the past year, new functional data on angiotensinogen (AGT) variants have been reported, together with additional support for a role of aldosterone synthase and the epithelial sodium channel genes in blood pressure regulation. Despite AGT being one of the most studied candidate genes, the identity of AGT variant(s) predisposing to high blood pressure has not yet been defined. The gene has many polymorphisms, especially in the promoter region. These have been the focus of functional analyses to determine whether they influence protein activity and whether there is any correlation between genotype, AGT levels and blood pressure. This year, Zhao et al. [3•] carried out in vitro studies on the A-20C promoter polymorphism of the AGT gene. They found that when adenine (A) is at position –20 the sequence binds to the oestrogen receptor-α, whereas when cytosine (C) is present a viral transcription factor binds preferentially and there is increased basal transcriptional activity. In support of these molecular observations, an association between the A-20C polymorphism and plasma AGT levels has also been reported [4]. At present, the exact interaction between the A-20C polymorphism with the downstream A-6G promoter polymorphism, which has also been shown to affect basal transcription of AGT [5], remains to be established. Another promoter polymorphism (C-532T) and a rare mutation (Arg-30Pro) in the signal peptide of the AGT gene have also been found to be associated with plasma AGT levels. Paillard et al. [6] found a significant correlation between the C-532T polymorphism and increased plasma AGT levels, and Nakajimi et al. [7] found that the Arg-30Pro mutation in its heterozygous state results in mildly reduced plasma AGT levels. Their work lends further support for a relationship between AGT gene polymorphisms and plasma angiotensinogen levels; however, any direct link to hypertension still remains to be established. The dissection of variants affecting AGT function also provides investigators with the most important polymorphisms to assess in hypertensive subjects. In 1998, data were reported that support a minor role for the aldosterone synthase gene (CYP11B2) in essential hypertension [8]. The associated polymorphisms, C-344T
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Table 1 Mapped QTLs and potential candidate genes from genome-wide screens in man. Human populations Caucasian
Chromosome
Nearest marker
2
D2S1788
5
D5S1471
6
D6S1009
15
D15S652
3 11
D3S2387 D11S2019
15 16 17 15
D15S657 D16S3396 D17S1303 D15S203
Potential candidate genes
Reference
Calmodulin 2 Sodium—calcium exchanger 1 Alpha 1B adrenergic receptor Dopamine receptor type 1A Phospholamban Estrogen receptor Aminopeptidase N
[12••]
ATP-ase Ca2+-transporting plasma membrane Angiotensin receptor-like 1 Angiotensinase C Cholinergic receptor Thiazide-sensitive Na-Cl cotransporter
[14••]
Insulin-like growth factor 1
[15••]†
Chinese*
*In this genome scan markers on chromosomes 3, 11, 16 and 17 are linked with systolic blood pressure, chromosome 15 is linked with diastolic blood pressure. † Fine mapping linkage results of the diastolic blood pressure locus from Xu et al. [14••].
and an intronic conversion in intron 2, have subsequently been studied in a Scottish case-control population [9]. Although the numbers were small in both groups within the Scottish study (~130), significant association was observed between both polymorphisms and hypertension. These results differ from the study by Brand et al. [8], in which only the polymorphism C-344T was found to be associated with the disease. These researchers also reported an association between the -344T allele and a higher excretion rate of the metabolite tetrahydroaldosterone in this study [8]. A relationship between the C-344T polymorphism with both urinary and plasma aldosterone levels has now been found in a number of studies [6,8,9]; therefore, raised aldosterone levels might be a good intermediate phenotype to aid selection of patients with alterations in CYP11B2. An alternative to carrying out candidate gene analysis in pre-selected hypertensive groups is to study blood pressure as a quantitative trait in families or a randomly selected population. Recently, microsatellite markers flanking the epithelial sodium channel gene (ENaC) have been studied in 286 families comprising at least both parents and two children [10]. Results from this study indicate linkage between three of the markers studied (D16S412, D16S403 and D16S420) and systolic blood pressure. Some support for these findings comes from a recent study involving 66 pairs of dizygotic twins, in which linkage between markers mapping close to ENaC and systolic blood pressure was also reported [11]. These studies do not prove that ENaC subunits account for the linkage observed, as it is feasible that linkage is between another locus in this region and the disorder. In light of these new observations, further studies assessing the chromosomal region containing ENaC in different populations are warranted, possibly in individuals with blood pressure in response to salt loading.
The first human genome screens In the past year, two groups have performed genome-wide scanning (GWS) to determine the location of blood pressure loci in humans. Kruskal et al. [12••] used a highly discordant sib-pair design, an approach that offers greater statistical power than conventional concordant sib-pair analysis, to determine genes influencing interindividual variation of systolic blood pressure. They studied 69 discordant sib-pairs and found 4 regions with significant linkage (P < 0.01) located on chromosomes 2p, 5q, 6q and 15q (see Table 1). A key statistical issue within this study is the selected threshold for acceptance of linkage. Kruskal et al. [12••] indicate that their statistically accepted threshold does not coincide with that proposed by Lander and Kruglyak [13], implying that these observations must be treated cautiously. Xu et al. [14••] have carried out GWS in discordant, highly concordant and low concordant Chinese sib-pairs for systolic and diastolic blood pressure. Their genome scan did not reveal any chromosomal regions that reached a 5% genomewide significance level; however, there were some promising regions with suggestive linkage to systolic blood pressure (closest markers being D11S2019, D3S2387, D16S3396 and D17S1303) and one region (closest marker D15S657) with suggestive linkage to diastolic blood pressure (Table 1). There does not appear to be an overlap of regions containing blood pressure quantitative trait loci (QTLs) between the two GWS studies, although marker D15S652 from the scan by Krushkal et al. [12••] has not been confidently mapped. The lack of concordance between the mapping results could be due to differences in the ethnicity of the populations studied or in the selection criteria used to define the study populations. Krushkal et al. [12••] studied discordant siblings from the upper and lower 20% of the systolic blood pressure distribution that were adjusted for age
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and sex. The discordant group studied by Xu et al. [14••] were drawn from the upper and lower 10% of systolic blood pressure distribution and were adjusted only for age. Of great interest however, is a follow-up study of the putative diastolic blood pressure QTL on chromosome 15 [15••]. Xu et al. [15••] have been able to confirm linkage between low diastolic blood pressure and a region on the long arm of chromosome 15, near marker D15S203, in a re-defined concordant hypotensive sib-pair resource and a new collection of sib-pairs from the same population. There are six possible candidate genes in this region, including the insulin-like growth factor 1 receptor (Table 1). This approach of studying hypotensive siblings appears to have yielded a new blood pressure locus and provides support for a combination of approaches to dissect genes influencing blood pressure.
Mouse genome screening Mouse models of hypertension also exist, although until recently very few genetic studies had been carried out owing to the difficulties in assessing phenotypes. A GWS for QTLs of systolic blood pressure has recently been carried out using the inbred hypertensive mouse [16••]. Significant linkage with systolic blood pressure was found with a region on chromosome 10 in the F2 inter-cross and with a region on chromosome 13 in the back-cross. Suggestive evidence for linkage was observed on chromosomes 2, 6, 8 and 18. None of the mouse loci appear to map to human or rat syntenic regions, indicating that analysis in the mouse might detect new genes regulating blood pressure.
Hypertensive rodent model QTLs Genome-wide scanning has been widely used to map blood pressure QTLs in inbred hypertensive rat strains and more recently to determine modifier loci [17,18•]. Indeed, last year a new blood pressure QTL was mapped to rat chromosome 12 in the Dahl salt-sensitive strain [19]. After mapping a QTL, congenic strains are developed to confirm its presence, and congenic substitution mapping is then used to reduce the chromosomal region in which the QTL resides. In the past year, the QTLs on rat chromosomes 1 and 19 have been confirmed and the critical regions refined [20,21]. A congenic strain of the spontaneously hypertensive rat containing part of chromosome 1q from the Brown Norway control strain has been developed as a model for hypertension-induced renal injury [22]. With the development of improved rat maps [23•], the time taken to fine map QTLs should be reduced when combined with expression profiling of candidates in the critical region.
Comparative mapping — experimental models to man Reproducible rat and mouse QTLs present good candidates for comparative mapping in human hypertension. Rat chromosome 10 contains two blood pressure QTLs [24,25] and is syntenic to human chromosome 17. Julier et al. [26] have found evidence for linkage of markers on human chromosome 17 with hypertension. Baima et al. [27•] recently published data supporting these observations in a smaller
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study group and claim to have refined the critical region of linkage; however, their study had fewer sib-pairs than that of Julier et al. [26] and this may have prevented adequate statistical power to establish linkage at other markers. Both studies provide some support for comparative mapping as a method of supplying candidate regions for the human disorder; however, until the causative variants have been determined in both rats and humans we will not know for certain the validity of this approach.
Single-nucleotide polymorphisms and blood pressure homeostasis In May 1999, an innovative academic/industry consortium was established, comprising 10 major pharmaceutical companies and the Wellcome Trust (UK), to identify and map 300,000 of the most abundant human polymorphisms, on the basis of single-nucleotide changes, for subsequent deposit into public domain databases [28]. With dense maps of these single-nucleotide polymorphisms (SNPs) distributed amongst our estimated 100,000 genes, we may be able to assemble informative haplotypes on genes of interest and rapidly home in on causative individual variants or haplotypes. Several publications report new SNPs within candidate genes that might be important in blood pressure regulation [29•,30•,31••]. The most interesting publications have illustrated the value of SNP detection when combined with chip-based technology [30•,31••]. One of these reports focused upon SNP detection in coding regions of candidates for cardiovascular, neuropyschiatric, endocrine diseases and cancer [30•]. The most interesting and relevant paper, by Haluska et al., [31••] reported a systematic survey of coding and non-coding regions of 75 candidate genes for blood pressure homeostasis. These candidates were drawn from the known physiology or pharmacology of blood pressure regulation, and the SNPs were identified with variant detection arrays using chip technology. A surprising 54% of coding, or exonic SNPs, predicted amino-acid changes, which implies considerably greater diversity in peptide sequence than has been previously thought. However, it is noteworthy that many of the coding region SNPs had a low frequency of the minor allele (less than 10%). A key observation reported by Haluska and colleagues [31••] is that individuals of African ancestry possess an average of two extra SNPs per gene. At face value this appears to be intuitive given the anthropological divergence of different ancestral ethnic groups. This finding is particularly germane to the search for hypertension genes because people of west African origin tend to have higher prevalence of hypertension and concomitant target organ damage. Certain studies imply physiological differences that could stem from different susceptibility genes or haplotypes. Both of these papers give encouraging insight into the uses of novel, high-throughput technology for SNP detection.
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The capability to generate large numbers of haplotypes will be critical in understanding the susceptibility genes for all common disorders.
Angiotensin-converting enzyme gene haplotypes The angiotensin-converting enzyme (ACE) converts angiotensin I to the effector peptide angiotensin II; this mediates vasoconstriction, alters sodium homeostasis and influences vascular remodelling within the renin angiotensin system [32]. Several reports have associated the ACE gene with coronary artery disease, blood pressure and cardiovascular structure, but other studies have been unable to replicate these findings [33–35]. One consistent observation is an important genetic influence on the level of ACE enzyme activity that is associated with an intronic insertion/deletion polymorphism within the ACE gene. Although no functional influence of the ACE I/D polymorphism has been demonstrated, it is assumed that there is an as yet unidentified functional variant. There are two publications on the ACE gene that underscore the value of comprehensive SNP detection in candidates of interest. The first by Rieder et al. [36•] evaluated sequence variation within the regulatory regions, exons and introns of the ACE gene in small numbers of African Americans and white Europeans. The authors reported 13 distinct haplotypes and concluded that, by using the ACE I/D polymorphism, haplotypes could be sorted into 2 distinct groups or clades. The second report by Farrall et al. [37•] draws upon previous work on the ACE gene QTL [38] in larger numbers of African Caribbeans and white Europeans. They had previously suggested that there are three distinct haplotype groups or clades (clades 1, 2 and 3) within the ACE gene. By using information on additional SNPs detected by Rieder et al. [36•], they were able to confirm an ancestral intragenic recombination event between clades 2 and 3. This made it possible to determine a variant that increases levels of ACE activity to be within exons 1–5 and introns 1–4 (and part of 5). Furthermore, the tight linkage disequilibrium within the three clades means that selection of a single SNP to represent each clade may allow significant economy in the numbers of genotypes required for analysis of the ACE gene. This could be a very valuable finding for large-scale genotyping projects of other genes and supports comprehensive SNP detection throughout coding and non-coding regions to unambiguously assign haplotypes.
Conclusions The past year has seen encouraging progress with early reports of human genome screens and more sophisticated approaches to cardiovascular SNP detection. These studies lay important foundations for large-scale human hypertension research. Within the next few years, we anticipate case-control analyses shifting from reports on single gene variants to description of haplotypes with multiple SNPs. Indeed, it may be possible with genotyping technologies such as mass spectrometry, microarrays or chips to present comprehensive SNP
screens of cardiovascular candidates. The current and new genomic screening programmes may describe novel chromosomal regions for detailed mapping. We predict that new regions will be further characterised with the identification of specific disease-causing haplotypes, allowing functional genomics and proteomics to succeed.
Acknowledgements We are grateful to Joanne Knight and Hannah Mitchison for critically reading the manuscript. We would also like to thank The Special Trustees of St. Bartholomew’s Hospital, The Medical Research Council and The British Heart Foundation for financial support.
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