Human Chromosome 17 in Essential Hypertension

Transcription

1 Review Human Chromosome 17 in Essential Hypertension J. Knight, P. B. Munroe, J. C. Pembroke and M. J. Caulfield Clinical Pharmacology, The William Harvey Research Institute Bart s and The London Queen Mary, University of London Charterhouse Square, London, EC1M 6BQ, U.K. Summary Hypertension affects up to 30% of the adult population in Western societies and is a major risk factor for kidney disease, stroke and coronary heart disease. It is a complex trait thought to be influenced by a number of genes and environmental factors, although the precise aetiology remains unknown at this time. A number of methods have been successfully used to identify mutations that cause Mendelian traits and these are now being applied to the investigation of complex diseases. This review summarises the data gathered, using such approaches, that suggest there is a gene or genes on chromosome 17 causing human essential hypertension. Studies in rodent models are discussed first, followed by studies of human hypertension that include the investigation of pseudohypoaldosteronism type II, a monogenic trait that manifests with hypertension alongside other phenotypic variables. In addition, candidate gene studies, genome screens and linkage studies based on comparative mapping are outlined. To date no gene has been identified on human chromosome 17 that influences blood pressure and causes human essential hypertension. However, results of ongoing fine mapping and candidate gene studies in both rodents and man are eagerly awaited. Introduction Hypertension, defined as raised arterial pressure, is present in about 30% of the adult population in Western societies and is a major risk factor for kidney disease, stroke and coronary heart disease (Peterson et al. 2000). Blood pressure is a quantitative trait, with both systolic and diastolic measurements adopting a normal distribution. There is no natural demarcation between hypertensives and normotensives. The definition of an individual with high blood pressure is based upon the point at which intervention is beneficial to reduce risk of cardiac endpoints. In the United Kingdom the guidelines for therapy have been established by the British Hypertension Society; they recommend that hypertensive therapy is initiated in a person with a sustained systolic pressure equal to or greater than 160 mm Hg, or a sustained diastolic pressure equal to or Correspondence: J. Knight. Tel: J.Knight@iop.kcl.ac.uk greater than 100 mm Hg (Ramsay et al. 1999). These guidelines and thresholds for intervention are continually being refined, as new evidence suggests that intervention to treat lower levels of blood pressure significantly reduces the risk of cardiovascular disease in patients. From twin and adoption studies it has been demonstrated that hypertension has a hereditary component. Although the precise influence of genes on blood pressure remains uncertain, estimates from studies of familial aggregation imply that approximately 30% of blood pressure variance is due to inherited genes (Ward, 1995). The principal genetic strategies that can be employed to search for genes for hypertension in humans, or animal models, include genome-wide scans and candidate gene studies. Genome-wide scans are used to locate influential loci with no prior assumption about position or function, and offer the prospect of defining entirely novel mechanisms for disease. Possible candidate genes can be highlighted via a number of routes: they can C University College London 2003 Annals of Human Genetics (2003) 67,

2 J. Knight et al. be selected because of a suspected role of their protein products in a pathway affecting the trait under study, or due to information gleaned from intermediate phenotypes (i.e., biochemical traits or physiological parameters that tend to co-segregate with the trait in families). In addition, gene products that are targeted by drugs used to treat a disease are also thought to be good candidates, as are genes shown to influence complex traits in animal models. Furthermore, identification of mutations that cause rare Mendelian forms of hereditary hypertension can prove valuable in candidate gene selection for human studies of more complex phenotypes such as essential hypertension. There are several excellent hypertensive animal models, and comparative mapping has facilitated the application of knowledge gained from such models to human disease. This process involves identifying regions of synteny, i.e. areas where the same genes are present in the same order in the genomes of animal models and humans. Subsequently, studies can be designed to investigate the syntenic regions on the human chromosomes that have been shown to be of interest in animal models. This review describes the evidence that suggests there may be a gene or genes on human chromosome 17 involved in essential hypertension. Studies of this chromosome have been greatly facilitated by the fact that much of it is syntenic to rat chromosome 10. Indeed, the results from studies in hypertensive rats first indicated that genes on human chromosome 17 might be implicated in the aetiology of hypertension. These initial studies in rodent models triggered comparative linkage studies in human populations. Alongside these, genome-wide scans in family-based human hypertensive studies have been conducted, and investigations of specific candidate genes on human chromosome 17 have been performed. Interestingly, the Mendelian hypertensive trait pseudohypoaldosteronism type II (PHAII) also maps to chromosome 17. Last year an exciting study demonstrated that mutations in the serine threonine kinases PRKWNK1 (hwnk1) and PRKWNK4 (hwnk4), cause this trait (Wilson et al. 2001). The PRKWNK4 (hwnk4) gene, located on human chromosome 17, may affect ionic flux in gap junctions within the kidney, and represents an entirely novel mechanism for human hypertension. Experimental Models In the study of complex traits, inbred strains manifesting the disease of interest have proved a useful tool. The advantages of using these animals include their genetic homogeneity, i.e. the same experiment can be repeated on animals with the same genetic background many times. In addition, specific breeding programmes can be designed, and their environment can be controlled. Furthermore, it is anticipated that a reduced number of genes will play a role in complex traits in these strains, rendering their identification easier (Tanase, 1979). In the 1960s the first hypertensive inbred strain, the spontaneously hypertensive rat (SHR), was developed (Okamoto, 1969). Many other strains have now been established through selective breeding for high blood pressure and some of these manifest specific sub-phenotypes, for example the Dahl salt sensitive strain (S). Further information about these rat strains can be found at the following web-site: ( Initial Support for Hypertensive Genes on Rodent Chromosome 10 The primary indication that genes on human chromosome 17 are involved in hypertension came from two genome scans in hypertensive rats which provided evidence of a quantitative trait locus (QTL) on chromosome 10, the region known to be syntenic to human chromosome 17. The scans published by Hilbert et al. (1991) and Jacob et al. (1991) had both been carried out on the F2 generation of a cross between the stroke prone spontaneously hypertensive rat (SHRSP) and the normotensive Wistar-Kyoto rats (WKY). In both studies linkage of sodium loaded, diastolic and systolic blood pressure was established to markers in an interval including the angiotensin converting enzyme (Ace) gene. Indeed, in both studies the locus was responsible for more than 20% of the sodium loaded blood pressure variation. Basal diastolic blood pressure was also found to be significantly linked to this locus; however, it is of interest that basal systolic blood pressure was not. These studies were not independent of each other, as both groups were studying the same cross. However, 194 Annals of Human Genetics (2003) 67, C University College London 2003

3 Chromosome 17 and Hypertension different statistical methods were used; Jacob et al. (1991) used the MAPMAKER computer package and Hilbert et al. (1991) used analysis of variance. The identification of loci by both groups therefore provided corroborative evidence for a gene in this region. Investigation of Candidate Genes on Rat Chromosome 10 As a natural development of these initial observations by Hilbert et al. (1991) and Jacob et al. (1991), candidate genes within the linked region have been evaluated in rodent models. An important putative candidate is the Ace gene, which encodes a key enzyme in the renin angiotensin system. This cardiovascular cascade generates angiotensin II by Ace enzymatic cleavage of angiotensin I. As the effector peptide of the cascade angiotensin II causes vasoconstriction, leads to sodium retention in the distal nephron, augments sympathetic tone and may alter vascular remodelling. The involvement of Ace and other markers in the surrounding region has been extensively investigated using a variety of different strains (see Table 1). Nara et al. demonstrated cosegregation between Ace and hypertension in Like Hilbert et al. (1991) and Jacob et al. (1991) they studied the F2 generation of a SHRSP and WKY cross, but importantly they used an independent colony of these rats. Subsequent direct sequencing of the Ace gene failed to demonstrate any functionally active variant in SHRSP rats (Koike et al. 1994). Another important candidate located within the linked rat chromosome 10 region is phenylethanolamine n-methyltransferase (Pnmt). This enzyme catalyses biosynthesis of epinephrine (adrenaline) and norepinephrine (noradrenaline) within the sympathetic nervous system, which has a major influence upon vascular tone. The sequence of the Pnmt gene was compared between SHRSP and WKY rats but no causative variant was identified (Koike et al. 1995). Rodent Genome Screens and Congenic Studies Support Linkage to Chromosome 10 Additional support for a hypertension locus or loci on rat chromosome 10 emanates from genome-wide scans using a number of different inbred strains (summarised in Table 1). Table 1 Studies on rat chromsome 10 are tabulated below. (This includes the strain in which work was carried out and the genes, or markers, that demonstrated linkage with blood pressure. References for the work are also given.) Strains Genes/markers Reference Candidate gene/region studies S & MNS Ace & Npr1 Deng & Rapp, 1992 SHRSP & WKY Ace Nara et al GH& BN Ace Harris et al S & MNS 46cM of chr 10 Deng & Rapp, 1995 S & WKY Nos2, Not Ace SHR & WKY Ace Zhang et al SHR & BB/OK Ace-Shbg Kovacs et al Genome screens SHRSP & WKY Gh1 Jacob et al SHRSP & WKY Gh1, Ngfr & Ace Hilbert et al SHRSP & WKY RR1023 & Ace Nara et al S& Lewis D10Wox6 Garrett et al SHRSP & WKY RR1023 Nara et al S & WKY D10Mgh6 Kato et al S&BN Il4 S & MNS Vamp2 SHRSP & WKY RR1023 Mashimo et al Rat strain acronyms: S: Dahl salt sensitive strain, MNS: Milan Normotensive strain, SHRSP: Stroke prone spontaneously hypertensive, WKY: Wistar-Kyoto, GH: New Zealand Genetically Hypertensive, BN: Brown Norway, SHR: spontaneously hypertensive, BB/OK Spontaneously diabetic but normotensive. Abbreviations of markers at known genes. Ace: angiotensin converting enzyme; Npr1: natriuretic peptide receptor 1; Nos2: nitric oxide synthase 2; Shbg: sex hormone binding globulin; Gh1: growth hormone 1; Ngfr: nerve growth factor receptor; Il4: interleukin 4; Vamp2: vesicle-associated membrane protein 2 (synaptobrevin 2). Congenic strains have been used to refine the region on chromosome 10, in an attempt to identify the causative gene and evaluate potential interactions with other genes. A congenic strain is bred to differ from the original inbred strain by only the section of the genome which is suspected to harbour the QTL under study. The results from this work, shown in Table 2, are perhaps the most interesting studies published in hypertensive models to date. A substantial body of work on congenic rats for chromosome 10 has been performed by, or in collaboration with, the group led by Rapp, who have refined the position of two QTLs on chromosome 10 (Garrett et al. 2001). By using two existing congenic strains which already had regions of chromosome 10 from normotensive rats introgressed onto the genetic background of Dahl salt-sensitive rats, it was possible to systematically C University College London 2003 Annals of Human Genetics (2003) 67,

4 J. Knight et al. Strains Flanking markers Phenotype Reference SHRSP & WKY Asgr1-Aldoc High BP Kreutz et al S & MNS Proximal to distal Dukhanina et al S.M(10a) Vamp2- RR1023 Low BP S.M(10b) Aldoc- RR1023 Low BP S.M(10c) D10Mco10-Aldoc No QTL S.M(10d) Aldoc-Ngfr No QTL S& Lewis D10Mit10-D10Mco6 Low BP Garrett et al S & WKY (chr2) Rapp et al S & MNS (chr10) Aldoc-RR1023 Low BP Table 2 Information of congenic studies on rat chromosome 10 is tabulated alongside. (This includes the strain in which work was carried out, the genes or markers that flank the congenic region, and the phenotype influenced by the QTL. References for the work are also given.) Most definitions of gene symbols and acronyms of the rat strains are present in the legend of Table 1. Hence only additional abbreviations are defined here. Asgr1: asialoglycoprotein receptor 1; Aldoc: aldolase C. breed sub-strains with smaller segments of the congenic section. This allowed the areas harbouring QTL1+QTL2 to be narrowed to two regions of about 2.6 cm and 3.2 cm in size on rat chromosome 10. These regions are about 24 cm apart at cray positions and respectively. In earlier studies the same group used a double congenic animal with two separate regions from different chromosomes introgressed, to demonstrate that a QTL on chromosome 10 interacts epistatically with the QTL on rodent chromosome 2 (Rapp et al. 1998). This type of gene-gene interaction study considerably strengthens the justification for comparative mapping, by implying that the observations are indeed the result of genuine hypertensive loci in animal models. Murine Models Mouse models of hypertension are increasingly being employed to investigate genetic influences on the trait. In part, this is due to much improved methods of blood pressure measurement, which had previously proved to be technically difficult. One genome scan provided evidence for the presence of a QTL for salt induced high blood pressure on murine chromosome 11 (Paigen et al. 1999). This region is syntenic to human chromosome 17 and rat chromosome 10. Human Studies Recently, the extent of synteny between human chromosome 17, rat chromosome 10 and mouse chromosome 11 was further investigated by Bihoreau et al. (2001). This group produced several important comparative maps which are available from their website ( mapping resources). However, even before this excellent resource became available, the exciting results from studies of rat chromosome 10 led researchers to investigate whether these observations would translate across species. In 1997 Julier et al. published the first comparative linkage study indicating support for linkage to a region of human chromosome 17. Additional evidence came from genomewide scans, candidate gene investigations and studies of pseudohypoaldosteronism Type II (PHAII). The positions of putative loci on chromosome 17 vary between studies, although the interval between 60 and 67cM from the proximal telomere (17q21-22) has been most consistently highlighted by the published data. Human Linkage Studies Based on Comparative Mapping Comparative linkage studies using markers in the syntenic region have predominantly been carried out using affected sibling pairs. Table 3 gives some basic information from these studies. Three studies have been carried out in white Europeans, and two in people of African origin; the populations in these studies are of variable size and have different phenotypes, as detailed below. Julier et al. (1997) studied 357 white European families consisting largely of affected siblings pairs. The population was derived from four cohorts, two originally recruited on the basis of hypertension and two on the basis of diabetes. All affected subjects had hypertension in the top 5% of the distribution. After defining a 110 cm region of homology between the human genome and 196 Annals of Human Genetics (2003) 67, C University College London 2003

5 Chromosome 17 and Hypertension Table 3 Linkage studies on human chromosome 17. (For each positive candidate region study the name of the markers to which linkage was demonstrated are presented. Negative studies are also cited and references as well as the number of families used and the population studied are given.) Population Families Linkage p-value Reference White Europeans 357 D17S Julier et al White Europeans 177 D17S Rutherford et al White Europeans 859 Negative - Knight et al White Americans 74 D17S Baima et al African American 45 Negative - African Caribbean 63 Negative - Knight et al the rat genome, based on comparative maps from rat chromosome 10, mouse 11 and human 17, Julier et al. (1997) studied markers spanning this region and found linkage in the region from 64 cm to 91 cm. The most significant support for linkage emerged with the microsatellite D17S934 at 63.7 cm, using two-point analysis. Baima et al. (1999) studied 74 white American families with hypertension, primarily consisting of affected sibling pairs, using only five markers to cover the region from 60 cm to 65 cm, and one additional marker at 91 cm. Linkage to blood pressure was established with two markers 0.7 cm apart, but not for a marker 1.2 cm proximal, or a marker 0.8 cm distally. The investigation of chromosome 17 by the Medical Research Council British Genetics of Hypertension Study (MRC BRIGHT Study) utilised 859 families (Knight et al. 2001) based upon affected sibling pairs, drawn from the upper 5% of the population blood pressure distribution. In this study simple sequence repeats spanning the 35 cm region which was linked to hypertension in the study by Julier et al. (1997) were genotyped, but no evidence of linkage was found. In 2001 Rutherford et al. studied 177 families and genotyped markers along chromosome 17. Although they found no evidence of linkage to markers between cm they did demonstrate weak linkage to a marker 95 cm from the distal telomere. In addition, there was some evidence of excess allele sharing at a marker 33 cm from the proximal telomere. The population studied had less severe hypertension than those studied by Julier et al. (1997) and Knight et al. (2001), but diabetics were excluded. A further study by Knight et al. (2000) investigated linkage of markers between 60 67cM in an African Caribbean population of 63 families. Baima et al. (1999) performed a similar analysis in an African American population of 45 families. The results of both of these investigations were negative. Due to the small size of these two studies they cannot be said to exclude a gene that plays an influential role in the aetiology of hypertension in this population. It is of note that populations of White European ancestry provide conflicting results. Two studies demonstrated significant linkage of markers in the interval cm from the proximal telomere (Julier et al. 1997; Baima et al. 1999). However, the study by Rutherford et al. (2001) only found linkage to a more distal region on chromosome 17. Moreover, the largest study performed to date provided no evidence of linkage of blood pressure to chromosome 17 (Knight et al. 2001). There are many factors that could lead to discrepancies in the results from comparative linkage studies, and these will be discussed in a latter section comparing and contrasting the results of the comparative studies with the results from the genome scans. Genome Wide Scans Results for Chromosome 17 Genome scans for QTLs involved in the aetiology of hypertension have been carried out in a number of populations. The results of these studies are summarised in Table 4. The three genome-wide scans that provided support for linkage to markers on chromosome 17 are discussed first. The scan published by Levy et al. (2000) demonstrated linkage to hypertension at two markers, one located at 60 cm and the other at 67 cm on chromosome 17. Microsatellite markers across the chromosome were typed as part of a 10 cm genome scan in 1702 subjects taken from 332 large two-generational pedigrees recruited for the Framingham study. The use of the Framingham study enabled blood pressure to be studied as a quantitative trait; this approach could highlight genes influencing all levels of blood pressure. Systolic C University College London 2003 Annals of Human Genetics (2003) 67,

6 J. Knight et al. Population Marker Reference American 69 discordant D2S Krushkal et al sib pairs D5S D6S1009 < D15S British 169 affected sib pairs D11S Sharma et al Chinese 564 sib pairs D3S Xu et al (207 discordant, D11S high concordant, D16S low concordant) D17S D15S Finnish 47 affected sib pairs AT1 a 3.38 (M.L.S) b Perola et al D22S (M.L.S) b DXS (M.L.S) b Canadian 335 unselected D2S Rice et al sib pairs D5S American 332 unselected D17S (M.L.S) b Levy et al extended families ATC6A06 c 3.1 (M.L.S) b a AT1 is on chromosome 3. b Perola et al. (2000) and Levy et al. (2000) published only the Maximum LOD scores (M.L.S). c ATC6A06 is on chromosome 17. Table 4 Genome scans for blood pressure QTLs humans. The population used for the screen, markers that show linkage, the p-value of the results and the reference are shown. pressure was calculated from all blood pressure recordings taken between the ages of 25 to 75 and for the diastolic pressure recordings taken between the ages of 25 and 54 were used. The blood pressures actually used were longitudinal measurements and these were only derived for subjects with at least three readings, with at least 10 years between the initial and final reading. The average of these measures for each subject was adjusted for age and body mass index (BMI) to yield a residual blood pressure; adjustments were performed independently for each sex to counter gender effects. Interestingly, the longitudinal blood pressure measurements were shown to have a greater level of heritability than single measurements. Furthermore, a non-parametric algorithm was used to estimate the blood pressure level as if the subject was not on anti-hypertensive therapy. There are risks to adjusting data for treatment effect; in any cohort of treated hypertensives each individual response to the same agent, at the same dose, may vary. Within this Framingham cohort there are likely to be complete responders, partial responders and even non-responders, rendering interpretation of such adjustments potentially hazardous. Recognising this could lead to a degree of misclassification the data were analysed without adjustment for treatment, and reassuringly linkage of blood pressure to chromosome 17 was still evident. An additional advantage of the study by Levy et al. (2000) over other studies, specifically those looking at sibling pairs, is its ability to extract more complete genetic information. Because parental genotypes are available one is often able to determine whether siblings are sharing alleles from a common ancestor or not, thus providing the analysis with extra power. In another genome-wide screen Xu et al. (1999) studied Chinese sib pairs derived from population screening of 200,000 people in Anqing Province, which yielded a family resource of 207 discordant sib pairs, 258 sib pairs with high concordant blood pressures, and 99 with low concordant blood pressure. This genome scan did not find linkage to any chromosomal region that reached the genome-wide significance level recommended by Lander & Kruglyak (1995) (p-value <0.0001). However, some promising regions were found on chromosomes 3, 11, 16, and 17 that demonstrated suggestive linkage (unadjusted p-values of <0.001) to systolic blood pressure, and a region on chromosome 15 gave suggestive evidence of linkage to diastolic blood pressure (Xu et al. 1999). The marker with the most significant result on chromosome 17 was D17S1303 (117 cm) with an unadjusted p-value of resulting from the analysis of siblings who were highly concordant for systolic blood pressure. This marker is outside the regions exhibiting support for linkage that have already been 198 Annals of Human Genetics (2003) 67, C University College London 2003

7 Chromosome 17 and Hypertension described (Julier et al. 1997; Baima et al. 1999; Rutherford et al. 2001; Levy et al. 2000). The results of a genome scan on two groups of families from the Quebec Family study were published in 2000 (Rice et al. 2000). One cohort comprised 80 families of obese individuals (BMI >32), the other cohort was 121 random individuals (i.e. those not selected on the basis of weight or blood pressure). In this study multi-point variance-component linkage analysis of blood pressure was performed. Linkage is determined by comparisons of the file of different models models that include genetic data and models that do not. The genotypes were analysed from both groups independently, and then from the two groups combined. In analysis using the combined population the two loci that demonstrated most evidence of linkage to hypertension were D2S2972 and D5S1986. Analysis of the random population indicated that two regions on chromosome 17, one at the ACE locus (85 cm, p-value of <0.014) and the other at D17S1303 (117 cm, p-value of <0.02), although not satisfying the criteria for genome-wide significance may merit further study with additional markers. In contrast, three published genome-wide scans have not found evidence of linkage for hypertension to markers on chromosome 17 (Sharma et al. 2000; Perola et al. 2000; Krushkal et al. 1999). Two are based on affected sibling pair approaches (Sharma et al. 2000; Perola et al. 2000) and the other on a discordant sibling approach (Krushkal et al. 1999). All the studies found evidence of linkage at other loci, as shown in Table 4. Comparison of the Comparative Mapping Studies and the Genome Scans The comparative mapping studies and six genome screens outlined above provide conflicting evidence relating to the presence or absence of a blood pressure locus on human chromosome 17. Hence, it is important to consider what factors may have caused such discrepancies, and to consider the strengths and weaknesses, and thus the reliability, of the studies. There are clear distinctions in the phenotypes of the populations used in the comparative studies that demonstrated evidence of linkage between cm on human chromosome 17 (Julier et al. 1997; Baima et al. 1999) and those that did not (Rutherford et al. 2001; Knight et al. 2000). The studies with positive results had diabetic hypertensives included in the cohort, whereas the negative studies did not. The study by Julier et al. (1997) included four groups of patients, two of which were derived from diabetic cohorts; the study by Baima et al. (1999) also included diabetics (13% of the cohort); the studies by Rutherford et al. (2001) and Knight et al. (2000) excluded diabetics. The frequent concurrence of diabetes and hypertension is well documented, and may be the result of a different aetiology than essential hypertension alone (Beevers & MacGregor, 1999). Julier et al. (1997) addressed this issue in their discussion, by highlighting that each cohort independently gives suggestive evidence of linkage between hypertension and chromosome 17. Nevertheless, it is worth noting that the greatest statistical support for a locus on chromosome 17 in that study is produced by linkage analysis of the groups initially recruited on the basis of diabetes. The MRC BRIGHT study is the largest of the affected sibling pair studies and so it is particularly important to consider its reliability further. To ensure high accuracy, and to avoid the mis-specification of alleles that can occur in sibling pair studies due to the lack of parental genotype a number of techniques were employed. Positive controls were run on all gels and a quality control tool was designed in Microsoft Access to perform a sophisticated series of automated checks on the genotypes (Caulfield et al. 2000). All genotypes demonstrating inconsistencies in patterns of Mendelian inheritance were further investigated. In addition, the application of multiple statistical programmes should have ensured that if linkage was present it would have been detected. In the study by Baima et al. (1999) the loss of support for linkage over a small chromosomal area of less than 1 cm implies that their results should be treated with the utmost caution. This group suggested that linkage to a narrower region than that found by Julier et al. (1997) could be due to increased power gained by studying markers closer to the gene, and claimed that they had refined the candidate region. However, such rapid decay in linkage could be due to inaccurate marker placement and hence inter-marker distance. Another possible C University College London 2003 Annals of Human Genetics (2003) 67,

8 J. Knight et al. explanation could be allele mis-specification leading to a false positive result, but this seems less likely as quality control procedures were in place to reduce the risk of genotype errors. The phenotypes of the populations used for the genome wide screens vary, but there is not a consistent difference between those in which evidence for linkage of hypertension to chromosome 17 was present and those in which it was not. Moreover, only one of the studies demonstrated evidence of linkage to markers between 60 and 67 cm (Levy et al. 2000). This may be due to additional power gained by the size of the study, the longitudinal blood pressure measurements and the high level of genetic information (all previously discussed). It is important to note that two of the genome screens that failed to find evidence of linkage in the cm interval were small affected sib-pair studies. Accordingly, it is possible that they did not have the power to identify the putative locus on chromosome 17 (Sharma et al. 2000; Perola et al. 2000). Other studies may also have had insufficient power to detect the putative locus on chromosome 17, for example, the study by Rice et al. (2000). Moreover, in this study many of the subjects are obese and hence the population could be enriched for a certain subset of hypertensives in which the aetiological variant on chromosome 17 is not as frequent. Although Krushkal et al. (1999) used the discordant sibling pair approach, which is thought to be more efficient at detecting QTLs (Risch & Zhang, 1995) only 69 sibpairs were analysed and again the study may have lacked power. Xu et al. (1999) applied the discordant sib-pair approach to a larger population but still did not find linkage. This could have occurred because a different ethnic population was studied, where the putative influential variant has either absent or only present at a low frequency, and therefore undetectable by linkage analysis. The conflicting evidence in relation to the presence of a blood pressure QTL on human chromosome 17, including studies offering strong evidence of such a locus (Levy et al. 2000), and equally convincing results excluding a major influential locus in the families from the MRC BRIGHT study (Knight et al. 2001), renders it difficult to determine whether such a locus exists. However, as outlined above there are major differences between the studies. Furthermore, there is a cluster of markers, at an interval 60 67cM from the proximal telomere on chromosome 17, that demonstrates significant evidence of linkage to hypertension in a number of the studies (Figure 1). It is feasible that there is a gene in this region that has a small effect and/or is only influential in a sub-set of the population such as diabetic hypertensives. Depending on the size and phenotype of a population under study, such a genetic effect may or may not be discernible. Until the genetic aetiology and architecture of hypertension is more fully understood it may be difficult to determine conclusively the presence or absence of QTLs. Human Candidate Gene Studies Most candidate gene studies for hypertension relate to ACE, although it is not within the syntenic chromosome 17 region that has been implicated in studies of essential hypertension. Other candidates include phenylethanolamine n-methyltransferase (PNMT) and solute carrier family 4 anion exchanger member 1 (SLC4A1). Both genes are biologically plausible candidates, as they code for proteins that act in pathways that are involved in the control of blood pressure. PNMT is involved in catecholamine biosynthesis, and SLC4A1 is a major glycoprotein of the erythrocyte membrane that is also present in the collecting ducts of the kidney and other tissues (Tanner, 1993). It is involved in chloride-bicarbonate exchange and the transport of glucose and water. However, though PNMT and SLC4A1 are often cited as candidate genes, to our knowledge there are no published studies that investigate their role in the aetiology of human essential hypertension. With further progress in physical mapping and gene identification it is probable that more candidate genes will become apparent in the region. A search of the NCBI database performed in early February 2002 detected about 120 known genes in the region 60 67cM from the proximal telomere on chromosome 17. Many are of unknown function, but others could be potential candidates, such as the corticotropin releasing hormone receptor 1 (CRHR1) gene. 200 Annals of Human Genetics (2003) 67, C University College London 2003

9 Chromosome 17 and Hypertension 55 D17S793 D17S798 D17S1293 Julier Levy Baima D17S946 D17S1814 D17S250, D17S1299 and D17S 800 D17S932 D17S934 D17S806 and D17S D17S941 D17S788 D17S D17S808, D17S948 and GH D17S807 The markers studied by Julier, Levy, and Baima, are shown. The solid circles, hollow squares, and crosses, indicate significant, suggestive, and no support for linkage, respectively. ACE at 85 cm and other markers that showed suggestive linkage in the studies are distal to the region in the figure. Figure 1 Position of markers linked to hypertension. Angiotensin Converting Enzyme (ACE) and Human Hypertension The ACE gene is a good candidate physiologically, and based on the rodent comparative mapping studies discussed above. Both linkage and association studies have been used in an attempt to establish if polymorphisms in the ACE gene have any influence on hypertension and other cardiovascular related traits. An insertion/deletion polymorphism in this gene has been used to investigate its relationship with blood pressure in a variety of populations, such as those studied by Barley et al. (1996) and Borecki et al. (1997). Results have been disparate and some may have arisen due to incorrect typing of the marker caused by preferential amplification of one of the alleles (Shanmugam et al. 1993). Two large studies have been published that support a role for ACE in hypertension (O Donnell et al. 1998; Fornage et al. 1998). O Donnell et al. (1998) found evidence of linkage and association with hypertension and diastolic blood pressure in men but not in women; they performed their analysis on 3095 participants from the Framingham study. Fornage et al. (1998) investigated the influence of the ACE gene on blood pressure levels in 1488 siblings. Sex-specific analysis also demonstrated that variations in the ACE gene had a significant effect on systolic, diastolic and mean arterial blood pressure in males but not in females. These results suggest a relationship between this gene and hypertension, but they are not conclusive. Zhu et al. (2001) performed association and linkage analysis in a collection of 1343 Nigerians from 322 families, typing 13 intragenic markers in the ACE gene. These included one dinucleotide repeat (in the 5 untranslated region), the intronic insertion/deletion polymorphism, and 11 SNPs, five of which are in the 3 untranslated region, three are intronic, and three lie within exons. Both linkage and association was present between the majority of these markers and the concentration of circulating ACE. However, there was no evidence of linkage to blood pressure between A-262T C University College London 2003 Annals of Human Genetics (2003) 67,

10 J. Knight et al. and A11860G even though they were most significantly linked to ACE plasma levels. It should be noted that these SNPS did demonstrate association with blood pressure when analysis was performed under the assumption that another gene (as well as ACE) was involved in blood pressure regulation. The evidence for both linkage and association of the ACE gene to hypertension in a number of different populations implies that it may play some role in the trait. However, as the results from such studies are not consistently positive, and none of the variants have been proved to have functional significance, it remains unclear whether this gene influences hypertension or not. Monogenic Traits Monogenic traits can provide information about possible pathological pathways causing common diseases. In addition, it is thought that subtle variations in the genes responsible for these disorders could be involved in the aetiology of complex traits that share some aspects of the phenotype. Thus, genes that harbour causative mutations for these disorders are frequently studied as candidate genes. The genes for a number of monogenic disorders with phenotypes that include hypertension have been identified (Lifton et al. 2001). However, none of these genes have been shown to cause human essential hypertension. Interest in chromosome 17 has been promoted by results from studies of pseudohypoaldosteronism type II (PHAII), also known as Gordon s syndrome and familial hyperkalaemic hypertension. Despite an autosomal dominant mode of inheritance this disease exhibits genetic heterogeneity, with linkage in different families to human chromosomes 1q31-42, 12p13.3 and 17p11- q21 (Mansfield et al. 1997; O Shaughnessy et al. 1998; Disse-Nicodeme et al. 2000). Patients present with hyperkalaemia, normal renal glomerular filtration rate, hypertension and mild metabolic acidosis. In August 2001, Wilson et al. described mutations in two genes (PRKWNK1 and PRKWNK4) that cause PHAII in some families. The group, led by Lifton, studied a new PHAII family in which they found a deletion close to the telomere of the short arm of chromosome 12, which lay entirely within the large first intron of the human ortholog of rat Wnk1. Another family, in whom PHAII had previously been shown to be linked to chromosome 12, also had a deletion in the same intron of human PRKWNK1. Interestingly, real time polymerase chain reaction demonstrated that patients with intron 1 deletions had a five-fold increase in the expression level of the transcript compared to unaffected individuals. This suggested a functional effect of the causative mutation. Paralogs of PRKWNK1 were found on human chromosomes 9, X and 17. The paralog on 17 (PRKWNK4) is between 61 and 63 cm from the top of the chromosome, within the region to which the trait had been previously mapped. Four different mutations were found in this gene in four pedigrees that show evidence of linkage between chromosome 17 and PHAII. The PRKWNK1 and PRKWNK4 proteins are both expressed in the kidney, in the distal nephron (the distal convoluted tubule and the corticol collecting duct respectively). Both show distinct localisation patterns; PRKWNK4 co-localises with a known tight junction protein, and mutations in components of the tight junction have previously been shown to alter specific paracellular ionic fluxes. It is proposed that the mutations in PRKWNK4 may lead to increased salt reabsorption and intravascular volume, thereby increasing blood pressure, but further functional studies of this gene are required. The study by Wilson et al. (2001) has led to PRKWNK4 being considered as a promising candidate gene for essential hypertension, and it is therefore certain to come under considerable scrutiny over the months and possibly years ahead. Conclusions and Future Work The studies outlined in this review provide considerable, although not conclusive, evidence of the presence of a gene on human chromosome 17 that is involved in the aetiology of essential hypertension. As outlined above, the results from some linkage studies in humans indicate the presence of an influential gene between 60 67cM from the proximal telomere of the chromosome. A considerable number of candidates lie within this region including PRKWNK4, the gene recently demonstrated to play a causative role in PHAII (Wilson et al. 2001). Furthermore, a QTL of 3.2 cm defined by Garrett et al. (2001) in rodent models of hypertension appears to be in a syntenic region. However, there have been a number 202 Annals of Human Genetics (2003) 67, C University College London 2003

11 Chromosome 17 and Hypertension of studies that have failed to find evidence of a QTL in this region (e.g. Knight et al. 2001), and until a causative gene is identified or the region is conclusively excluded from harbouring influential genetic variation, the cause of the disparities in the results will probably remain obscure. The uncertainty of the presence of a QTL in the region is by no means unusual. No gene has yet been proved to be involved in the aetiology of essential hypertension. It is possible that the methods outlined in this review will identify genes given time. Indeed, it is also conceivable that the methods will need further refinement in order to locate the genes involved in the aetiology of complex traits. A recent paper by Corvol et al. (1999) discusses the merits and pitfalls of the candidate gene approach. Several problems including limited power, inconsistent phenotypes between studies, and inadequate consideration of other variables (age, sex etc), are proposed as explanations for disparities between studies. Corvol et al. (1999) also suggest that more work needs to be performed in order to assess the contribution of inbred strains to the understanding of the aetiology of essential hypertension. The relevance of studies of monogenic traits is also not yet completely clear. Despite the fact that genes coding for different sub-units of the epithelial sodium channel (EnaC) have been conclusively linked to monogenic traits such as Liddle s syndrome, there is no conclusive evidence of the involvement of these genes (SCNN1B, SCNN1G) in human essential hypertension (Lee et al. 2000). Since the role of the PRKWNK genes in PHAII was only discovered in the last year it is yet to be established whether they play a role in essential hypertension. Another suggested refinement of existing techniques is the simultaneous analysis of a number of genes involved in the same pathway. This may allow the identification of variants that combine to affect blood pressure that may not have been identified by individual studies (Bowden, 1999; Luft, 2000). New technology and advances in knowledge will not only improve the success of current methods but will also enable development of further new technologies. For example, the increasing availability of SNP s, and automated methods of typing them, makes genome wide association analysis a viable option. An approach that may be particularly useful for further investigation of the role of the putative locus on chromosome 17 is the investigation of sub-sets of people with essential hypertension who also manifest other associated phenotypes. For example, those with high potassium levels, as observed in PHAII. It is thought that such populations will be enriched for people carrying particular mutations, thereby increasing the power to detect their effect (Lander & Schork, 1994). In summary, the evidence for a gene on chromosome 17 involved in the aetiology of hypertension is mounting. However, even in large studies it has been difficult to replicate positive results. We expect the gene to be influential in only a subset of hypertensives and it is possible that the different populations studied represent different sub-sets, and this is what has led to the inconsistent results. Overall, these studies demonstrate that there are reasons to be optimistic about the possibility of identifying the genes involved in the aetiology of complex traits. Reference Baima, J., Nicolaou, M., Schwartz, F., DeStefano, A. L., Manolis, Gavras, I., Laffer, C., Elijovich, F., Farrer, L., Baldwin, C. T. & Gavras, H. (1999) Evidence for linkage between Essential Hypertension and a putative locus on human chromosome 17. Hypertension 34, 4 7. Barley, J., Blackwood, A., Miller, M., Markandu, N. D., Carter, N. D., Jeffery, S., Cappuccio, F. P., MacGregor, G. A. & Sagnella, G. A. (1996) Angiotensin converting enzyme gene I/D polymorphism, blood pressure and the renin-angiotensin system in Caucasian and Afro-Caribbean peoples. J Hum Hypertens 10, Beevers D, G. & MacGregor, G A. (1999) Hypertension in Practice 3. Bihoreau, M. T., Sebag-Montefiore, L., Godfrey, R. F., Wallis, R. H., Brown, J. H., Danoy, P. A., Collins, S. C., Rouard, M., Kaisaki, P. J., Lathrop, M. & Gauguier, D. (2001) A high-resolution consensus linkage map of the rat, integrating radiation hybrid and genetic maps. Genomics 75, Borecki, I. B., Province, M. A., Ludwig, E. H., Ellison, R. C., Folsom, A. R., Heiss, G., Lalouel, J. M., Higgins, M. & Rao, D. C. (1997) Associations of candidate loci angiotensinogen and angiotensin-converting enzyme with severe hypertension: The NHLBI Family Heart Study. Ann Epidemiol 7, Bowden, A. C. (1999) Metabolic control analysis in biotechnology and medicine. Nat Biotechnol 17, C University College London 2003 Annals of Human Genetics (2003) 67,

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