1997 Oxford University Press

Human Molecular Genetics, 1997, Vol. 6, No. 12 20772085

Genetic susceptibility for human familial essential hypertension in a region of homology with blood pressure linkage on rat chromosome 10
Ccile Julier1,*, Marc Delpine1, Bernard Keavney1, Joseph Terwilliger1,2, S. Davis3, Daniel E. Weeks1,3, Thuan Bui1, Xavier Jeunematre4, Gilberto Velho5, Philippe Froguel6, Peter Ratcliffe1, Pierre Corvol4, Florent Soubrier5 and G. Mark Lathrop1
Wellcome Trust Centre for Human Genetics, University of Oxford, Windmill Road, Oxford OX3 7LD, UK, University Department of Psychiatry and Columbia Genome Centre, 722 West 168th Street, New York, NY 10032, USA, 3Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA 15261, USA, 4Laboratory of Experimental Medicine, INSERM U36, Collge de France, 3 rue dUlm, 75005 Paris, France, 5INSERM Unit 358, Hpital St Louis, 16 rue de la Grange aux Belles, 75010 Paris, France and 6CNRS EP 10, Institut Pasteur of Lille, 1 rue Calmette, 59019 Lille, France
2Columbia Received June 13, 1997; Revised and Accepted August 26, 1997 1The

Hypertension is a significant risk factor for heart attack and stroke and represents a major public health burden because of its high prevalence (e.g. 1520% of the European and American populations). Although blood pressure is known to have a strong genetic determination, the genes responsible for susceptibility to essential hypertension are mostly unknown. Loci involved in blood pressure regulation have been found by linkage in experimental hereditary hypertensive rat strains, but their relationship to human hypertension has not been extensively investigated. One of the principal blood pressure loci has been mapped to rat chromosome 10 and we have undertaken an investigation of the homologous region on human chromosome 17 in familial essential hypertension. Affected sib-pair analysis and parametric analysis with ascertainment correction gave significant evidence of linkage (P <0.0001 in some analyses) near two closely linked microsatellite markers, D17S183 and D17S934, that reside 18 cM proximal to the ACE locus in the homology region. Our results indicate that chromosome 17q could contain a susceptibility locus for human hypertension and show that comparative mapping may be a useful approach for identification of such loci in humans. INTRODUCTION The multifactorial basis of hypertension is well established and 3050% of the variation in blood pressure between individuals is

attributed to genetic factors (1). However, the genes responsible for susceptibility to hypertension and blood pressure variation are mostly unknown. Some progress towards their identification has been made, using two principal approaches: linkage analyses in families segregating for rare forms of familial hypertension and exploration of candidate genes. The first of these has led to the detection of linkage and the identification of genetic alterations at four genes: the 11-hydroxylase in glucocorticoid-suppressible hyperaldosteronism (2); the and subunits of the epithelial sodium channel in Liddles syndrome (pseudoaldosteronism) (3,4); the 11-hydroxysteroid dehydrogenase gene in the syndrome of apparent mineralocorticoid excess (5). However, these syndromes account for a small fraction of hypertension, with quasi-Mendelian inheritance and particular phenotypes, and it is therefore expected that other genes are also involved in the disorder. Using a candidate gene approach, the angiotensinogen gene (AGT) has been reported to contribute to susceptibility in some affected sib-pair studies (6,7) but not others (E.Brand, N.Chatelain, G.Bianchi, T.de Bruijn, M.Caufield, J.Connell, B.Keavney, S.Schmidt, H.Schunkert, H.Schuster, A.Sharma, X.Jeunemaitre and F.Soubrier, in preparation). It is unlikely that AGT or any other candidate genes that have been tested to date account for a majority of the genetic susceptibility to hypertension. Genetic studies of hereditary hypertension in the rat, and particularly genome-wide scans, have led to the detection of several loci involved in blood pressure regulation (812). Regions of homology in human represent candidate regions for containing susceptibility genes for hypertension, whose exploration is an alternative to investigation of rare syndromes or candidate genes. One of the principal loci identified in crosses involving spontaneously hypertensive stroke-prone (SHR/SP) and Dahl salt-sensitive hypertensive rats has been localised near

*To whom correspondence should be addressed. Tel: +44 1865 740 017; Fax: +44 1865 742 196; Email: cecile@well.ox.ac.uk +The SIBPAIR program is part of the ANALYZE programme package which is available by anonymous ftp from flemming.well.ox.ac.uk. MOD score analysis has been implemented as part of the VITESSE program package which is available from watson.hgen.pitt.edu.

2078 Human Molecular Genetics, 1997, Vol. 6, No. 12 the angiotensin I converting enzyme gene (ACE) on rat chromosome 10 (8,9,13,14). This locus was estimated to account for >20% of the total variance of the systolic and diastolic blood pressures after NaCl loading in the F2(SHRSP WKY) cross (8). Based on comparative mapping data, the human homologues of the corresponding gene (or genes) should reside within a contiguous segment of human chromosome 17q, which contains the ACE gene. Although ACE has been examined genetically for linkage to human hypertension in one study with negative results (15), a systematic investigation of the complete homology region has not been made. Based on such an investigation in a large number of human hypertension families, we present evidence of linkage to a putative susceptibility locus for hypertension within the homology region on chromosome 17q but distinct from ACE. RESULTS Comparative mapping of rat chromosome 10 and human chromosome 17 The region of homology to the rat hypertension susceptibility loci BP-SP1 was defined based on comparative maps of rat chromosome 10, mouse chromosome 11 and human chromosome 17, in part from the compilations provided by the Mouse Genome Database that are available on the World Wide Web (16). As shown in Figure 1, the chromosomal region that spans this location is extensively conserved between these species, extending from MYHSE/Myhse/MYH3 on human 17p to the distal end of human 17q. Because of the wide confidence interval for the placement of the blood pressure locus in the rat, we decided to explore the whole homology region for a putative susceptibility locus for hypertension and selected 21 microsatellite markers to screen (Fig. 1). The markers cover 110 cM and extend over the region of homology between rat chromosome 10 and human chromosome 17p and 20 cM distally to GH on 17q. The mean heterozygosity of the markers is 0.75 (0.490.94) and the mean recombination distance between adjacent loci is 5.9% (0.115.0%). A higher density of markers was selected in the region proximal to or nearby GH/ACE, with a mean recombination frequency of 2.9% between the 13 markers in the D17S946D17S789 interval (30 cM). Family panels and linkage methods We studied a panel of 357 French and UK families containing 384 sibships with one or more hypertensive offspring and a total of 518 affected sib-pairs. Strict diagnostic criteria were applied so that the disease phenotype would correspond to the upper 15% of the blood pressure distribution (see Materials and Methods). A small number of families were ascertained through probands with non-insulin-dependent diabetes mellitus (29 families) or insulin-dependent diabetes mellitus (27 families); these families contained at least one hypertensive patient in addition to the diabetic proband. Further details on the families and clinical phenotypes are provided in Table 1. The relative power of standard non-parametric test statistics and their behaviour under the absence of linkage have been found to vary when compared in different data sets (S.Davis and D.E.Weeks, in preparation). Because of this, we decided to apply four statistical methods to the chromosome 17q data so that the results could be compared. These were: (i) affected sib-pair analysis with the SIBPAL program (17); (ii) affected sib-pair analysis with the SIBPAIR program (18,19); (iii) parametric

Figure 1. Comparative maps of rat chromosome 10, mouse chromosome 11 and human chromosome 17 define the target homology region. Comparative maps of the rat, mouse and human are from published sources with additional data from the Mouse Genome Database. The bars on the left represent the 100:1 odds support intervals for the placement of a blood pressure locus on rat chromosome 10 for the analysis of blood pressure under basal conditions (thick bar) or after NaCl loading (thin bar) (from ref. 8). The markers shown on the right were initially characterized in the HTE, HTN and HTD panels.

MOD score analysis in which maximum likelihood analysis is performed conditional on the phenotype status of family members as an ascertainment correction (20); (iv) affected sib-pair analysis with the GENEHUNTER program (21). Simulations were used to examine the behaviour of the four statistical methods in our family panel under various genetic models, with or without linkage to a marker locus. Figures 2 and 3 illustrate the results of these studies for a marker with allele frequencies identical to those estimated for D17S934 (one of the markers exhibiting the strongest evidence of linkage in the real data) and for a hypothetical, fully informative marker locus. The first issue to be examined was the adequacy of large sample or other theoretical approximations for the calculation of P values. In the absence of linkage to the marker, Figure 2 shows that the empirical distributions were either closely approximated by the theoretical distributions (SIBPAL and SIBPAIR) or that the latter gave conservative P values (parametric analysis and GENE-

2079 Human Genetics, 1997, 6, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 12 2079 HUNTER). These results justify the use of the theoretical approximations as a conservative approach for assigning significance to linkage data from these families. Figure 3 illustrates the power to detect linkage with P 0.001 or P 0.0001 as a function of the affected sib-pair identity-by-descent (IBD) probabilities under various genetic models. As explained in Materials and Methods, the simulations assumed a disease prevalence of 5% and a dominant susceptibility locus with two alleles, one of which conferred increased risk for hypertension. The penetrance associated with the susceptibility genotypes was varied to obtain IBD probabilities in the interval 5159% (compared with 50% in the absence of linkage). With any of the first three statistical methods, the power to detect a susceptibility locus tightly linked to D17S934 at a significance level of P 0.001 was 70% when the average affected sib-pair IBD in the sample was 5758%. For a fully informative marker, greater power was found even with the more stringent condition of P 0.0001 for similar IBD. Since the simulations assumed no recombination between the marker and disease loci, these are estimates of the maximum power that could be achieved under these genetic models with a dense map covering the homology region.

Table 1(a). Number of families and affected sib-pairs divided into subsets according to ascertainment criteria Family panel HTE HTO HTD HTN Totals No. with affected parent(s) Number of affected individuals in sibship 1 2 3 4 5 23 89 26 5 143 37 101 56 9 12 178 30 39 15 0 6 60 12 7 4 0 4 15 2 3 1 0 0 4 0 Number of nuclear families 174 166 36 27 402 81 Number of pedigreesa 154 147 29 27 357 NA Number of affected sib-pairs 305 135 24 57 518 78

6 1 0 1 0 2 0

HTE, panel of essential hypertension from France; HTO, panel of essential hypertension from Oxford; HTD, panel with hypertension and insulin-dependent diabetes; HTN, hypertension and non-insulin-dependent diabetes. Further details on the different family panels is provided in Materials and Methods. aSome multi-generational pedigrees contain two or more nuclear families Table 1(b). Phenotypes in the four groups of families as a function of hypertension status for individuals with a positive or negative diagnosis of hypertension
Disease status HTE (French) HT+ (n = 567) HT (n = 141) HTO (Oxford) HT+ (n = 207) HT (n = 256) HTD (French) HT+ (n = 57) HT (n = 112) HTN (French) HT+ (n = 67) HT (n = 21) SBP untreated (mmHg) 161.1 21.3a (n = 111) 125.4 10.2 162.8 15.8a (n = 95) 122.6 10.1 188.3 19.9a (n = 45) 126.2 9.9 186.9 17.2a (n = 59) 163.8 25.6 (n = 8) 161.8 18.5 (n = 12) 153.8 24.0 (n = 112) SBP treated (mmHg) 154.9 22.3 (n = 456) DBP untreated (mmHg) 103.2 11.0a (n = 111) 80.0 6.8 102.0 7.7a (n = 95) 75.2 8.1 100.2 13.5a (n = 43) 73.6 7.5 102.5 10.8a (n = 59) 92.5 13.9 (n = 8) 90.0 10.4 (n = 12) 89.7 13.0 (n = 111) DBP treated (mmHg) 94.7 12.5 (n = 456) Age at diagnosis 40.2 10.8 NA 44.4 9.9 NA 44.3 10.3 NA 50.6 .7.2 NA Age at time of study 53.0 11.8a 44.8 12.0 52.0 10.4a 43.8 10.7 53.6 11.6c 47.4 12.3 60.9 8.7 57.1 12.8 BMI (kg/m2) 25.7 3.9a 23.5 3.5 26.9 3.9a 25.3 3.8 25.4 4.3c 23 73.2 29.3 4.7c 26.2 2.9

131.9 17.2

75.0 7.6

Significance of the difference between HT+ and HT groups: aP < 0.0001; bP < 0.001; cP < 0.01. n, number of individuals. HT+, hypertensives; HT, normotensives; SBP: systolic blood pressure; DBP: diastolic blood pressure. Table 1(c). Phenotypes in HTD and HTN families as a function of diabetes and hypertension status for individuals with a positive or negative diagnosis of hypertension
Disease status IDDM patients HT+ (n = 20) HT (n = 30) NIDDM patients HT+ (n = 71) HT (n = 27) Non-diabetics HT+ (n = 33) HT (n = 76) SBP untreated (mmHg) 191.2 16.9a (n = 17) 127.0 10.6 188.1 19.4a (n = 60) 131.5 15.6 183.9 18.1a (n = 27) 125.5 9.7 178.3 23.2 (n = 6) 157.3 17.9 (n = 11) SBP treated (mmHg) 150.7 11.0 (n = 3) DBP untreated (mmHg) 100.6 11.4a (n = 17) 74.1 7.9 101.8 11.2a (n = 59) 75.2 7.1 101.8 14.3a (n = 26) 73.7 8.0 96.7 8.2 (n = 6) 89.1 13.8 (n = 11) DBP treated (mmHg) 86.7 5.8 (n = 3) Age at onset (years) 38.8 9.2 NA 50.7 6.9 NA 46.6 10.2 NA Age (years) 47.7 9.3 45.1 9.1 60.5 8.4 55.7 13.1 57.2 12.4b 47.9 13.3 BMI (kg/m2) 23.9 3.3 23.7 3.6 29.4 4.8c 26.0 3.5 25.6 4.2c 23.6 2.9

Significance of the difference between HT+ and HT groups: aP <0.0001; bP <0.001; cP <0.01. n, number of individuals. HT+, hypertensives; HT, normotensives; SBP: systolic blood pressure; DBP: diastolic blood pressure.

2080 Human Molecular Genetics, 1997, Vol. 6, No. 12 a a

Figure 2. Empirical (observed) P values for critical values corresponding to the theoretical large sample P values for four test statistics in 1500 replicates of the family panel simulated under the null hypothesis of no linkage for: (a) D17S934; (b) a fully informative marker locus. THEOR=EMPIR defines a diagonal line at which the empirical P value is exactly equal to the P value calculated from the theoretical distribution. The test is judged to be conservative at a particular point if the empirical P value is smaller than the theoretical P value, i.e. for values to the right and below the diagonal line.

It was striking that the GENEHUNTER statistic had considerably less power than the other three methods in these simulations, particularly when the marker was less than fully informative. The conservative behaviour of the GENEHUNTER test statistic has been noted previously (21; S.Davis and D.E.Weeks, in preparation) and similar power comparisons have been found in other settings (S.Davis and D.E.Weeks, in preparation). Despite lower power, possibly due to its conservative features, the GENEHUNTER approach is of interest here because it (and the parametric method) can be applied to multilocus analysis. Chromosome 17 linkage The 24 microsatellite markers shown in Figure 1 were initially characterised in French families from the HTE, HTN and HTD panels. Several markers between D17S946 and D17S789 provided suggestive evidence of linkage (P <0.01) and the subset of markers included in this interval were characterised in the total family panel (Table 2a). The strongest support for linkage with the SIBPAL and SIBPAIR affected sib-pair methods was found at D17S183 (P = 0.0001 with the SIBPAIR program). Although D17S183 has only 47% heterozygosity, it is closely linked to D17S934 (no obligate recombinants in the family panel), a marker with 83% heterozygosity. D17S934 produced similar, although slightly less significant results, with the affected sib-pair methods. The parametric analyses provided broadly similar evidence for linkage,

Figure 3. Power to detect linkage as a function of the average affected sib-pair IBD with four test statistics in 500 simulation replicates under different genetic models for: (a) D17S934 at P <0.001; (b) D17S934 at P <0.0001; (c) a fully informative marker locus at P <0.001; (d) a fully informative marker locus at P <0.0001. The power is shown as a function of the IBD probabilities averaged over all affected sib-pairs in the 500 simulation replicates of each model tested.

2081 Human Genetics, 1997, 6, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 12 2081 but here the most significant result was at D17S934: P = 0.0002 (or P = 0.00004) assuming a 5% (or 1%) disease prevalence. As observed in the simulations, the GENEHUNTER NPL statistic was often much less significant than those obtained with the other methods, possibly because of its conservative behaviour. However, the strongest evidence of linkage from GENEHUNTER was also obtained at D17S934 (P = 0.008). The simulation results in Figure 3 suggest that it might be possible to obtain additional support for linkage by combining D17S934 or D17S183 with other markers to increase the information content. To examine this possibility, we undertook further linkage tests with pairs of markers using the parametric and GENEHUNTER approaches for multilocus analysis. Table 2b shows the results of multilocus analyses with all marker pairs that included D17S934. The strongest evidence for linkage was obtained in parametric analysis when D17S934 was combined with GH, the marker with the highest heterozygosity. These tests achieved a significance of P = 0.00002 at 5% prevalence and P = 0.000004 at 1% prevalence. The maximum likelihood placement of the susceptibility locus was in the interval between D17S934 and GH at 13 cM from the latter (Fig. 4). Assuming 5% prevalence, the parameter estimates at this location were 0.02 for the frequency of the susceptibility allele and 0.21 for the penetrance of the susceptibility genotypes. All four family panels contributed to the support for linkage within the interval D17S934GH (P = 0.05, HTE; P = 0.04, HTO; P = 0.01, HTN; P = 0.02, HTD, at 5% prevalence). Thus, there appears to be no evidence of heterogeneity between families from France and Oxford or between families with and without diabetic probands. Other analysis methods also led to the same conclusion.

Table 2(a). Results of single marker linkage analyses on the whole family panel for markers from a selected portion of the homology region Locus symbol D17S946 D17S932 D17S951 D17S183 D17S934 D17S806 D17S788 D17S787 D17S808 D17S948 GH D17S807 D17S789 Locus name 283zb9 248yg9 298wg5 SCG43 256vb9 234td2 095zd11 095tc5 238yf8 291vb9 GH 234xc9 107yb8 Locus heterozygosity 0.76 0.80 0.76 0.47 0.83 0.87 0.63 0.77 0.66 0.76 0.94 0.84 0.79 Distance () 0.04 <0.01 <0.01 <0.01 0.02 0.05 0.02 0.10 0.02 0.01 0.05 0.04 SIBPAIR IBD estimate 0.54 0.55 0.56 0.61 0.56 0.55 0.55 0.56 0.55 0.56 0.55 0.56 0.56 SIBPAIR P value 0.05 0.01 0.005 0.0001 0.0004 0.01 0.03 0.007 0.01 0.009 0.003 0.01 0.009 SIBPAL P value 0.06 0.02 0.006 0.0003 0.0007 0.01 0.03 0.01 0.01 0.009 0.003 0.02 0.01 GENE HUNTER P value n.s. 0.06 0.05 0.02 0.008 0.03 n.s. 0.03 0.08 n.s. 0.04 0.03 0.05 Parametric P values with prevalence 5% 1% 0.07 0.04 0.02 0.005 0.0002 0.03 0.06 0.04 0.06 0.02 0.002 0.03 0.02 0.06 0.02 0.006 0.003 0.00004 0.02 0.05 0.10 0.07 0.01 0.003 0.02 0.01

Locus heterozygosity has been computed from the estimated allele frequencies. n.s., nonsignificant. Table 2(b). Results from linkage tests that include D17S934 and a second marker locus Second marker Locus symbol D17S946 D17S932 D17S951 D17S183 D17S934 D17S806 D17S788 D17S787 D17S808 D17S948 GH D17S807 D17S789 Locus name 283zb9 248yg9 298wg5 SCG43 256vb9 234td2 095zd11 095tc5 238yf8 291vb9 GH 234xc9 107yb8 0.76 0.80 0.76 0.47 0.83 0.87 0.63 0.77 0.66 0.76 0.94 0.84 0.79 0.04 <0.01 <0.01 <0.01 0.02 0.05 0.02 0.10 0.02 0.01 0.05 0.04 0.003 0.004 0.005 0.004 0.006 0.003 0.005 0.006 0.002 0.0002 0.002 0.00007 0.00008 0.00002 0.00007 0.00007 0.001 0.00005 0.0009 0.00002 0.00001 0.000004 0.00001 0.000007 0.004 0.003 0.011 0.007 Locus heterozygosity Distance () GENE HUNTER P value Parametric P values with prevalence 5% 1% 0.0004 0.001 0.004 0.0009 0.0001 0.0003 0.002 0.0003

2082 Human Molecular Genetics, 1997, Vol. 6, No. 12

Figure 4. Test statistics from parametric linkage analysis shown as a function of the position of the putative trait locus for D17S934 paired with six other markers having heterozygosity >75%: (a) assuming 1% disease prevalence; (b) assuming 5% disease prevalence.

Figure 4 shows the multilocus test statistics as a function of the position of the putative susceptibility loci for D17S934 paired with other marker loci having heterozygosity >75%. The significance of a test statistic at a given position can be calculated by comparison with a 2 distribution with 2 degrees of freedom. Similar results were obtained with combinations involving D17S183. For example, the combination D17S183+GH gave a maximum test statistic with a significance of P = 0.00002 at both

1% and 5% prevalence. The two-locus GENEHUNTER results were less significant than those obtained from the parametric analysis (Table 2b), as previously seen with single marker loci. Even when all the markers were included in the multilocus analysis, the GENEHUNTER program gave a maximum test statistic for linkage with a significance of only P = 0.004. Computational limitations did not permit us to undertake a comparable multilocus parametric analysis.

2083 Human Genetics, 1997, 6, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 12 2083 DISCUSSION Our linkage results support the hypothesis of a susceptibility locus for human hypertension on chromosome 17q. We selected chromosome 17q for study because of its homology to a region of rat chromosome 10 that contains one of the principal loci implicated in the regulation of blood pressure in experimental models of hereditary hypertension. The blood pressure locus on rat chromosome 10 was first detected in an F2 cohort derived from a SHRSP WKY cross (8,9), where it was estimated to contribute >20% of the total variance of the systolic and diastolic blood pressures after NaCl loading (8). Subsequently, the same region has been found to be linked to blood pressure variation in several other crosses involving SHR/SP (13) or Dahl hypertensive rats (14). Rather than test a single marker or candidate gene as in past studies of the ACE region in human hypertension, e.g. Jeunemaitre et al. (15), we took account of the imprecise localisation of the blood pressure locus and examined markers throughout the region of homology of rat chromosome 10 and human chromosome 17. Families containing a total of 518 affected sib-pairs were ascertained for the study and strict diagnostic criteria were applied to obtain a phenotype that corresponded to 15% population prevalence of the disease. By increasing the stringency of the phenotype definition, we increased the power to detect linkage. Moreover, a substantial number of families contained three or more affected sib-pairs or one or more affected parent. In simulations, this ascertainment led to a greater average affected sib-pair IBD probability in the family panel compared with the expectation in randomly selected affected sib-pairs, also increasing the power of the study (results not shown). For a dominant susceptibility allele with 0.05 frequency and a highly informative marker tightly linked to the susceptibility locus, simulations suggested that it would be possible to detect linkage with at least 70% power when the expected affected sib-pair IBD probability in the sample was in the range 0.570.58 or greater. Nominal evidence of linkage (P <0.01) to hypertension was found at several markers spanning the region of homology. The strongest evidence came from two closely linked markers, D17S183 and D17S934, that map 18 cM proximal to the ACE locus, where we obtained P 0.0001 in some affected sib-pair and parametric analyses. The combination of D17S183 or D17S934 with GH, the most informative of the markers studied, gave test statistics with a significance of P <0.00001 in multilocus parametric linkage analysis. Many other pairwise marker analyses were also significant with P <0.0001, some of which are shown in Table 2b. On the other hand, non-parametric linkage analysis with the GENEHUNTER method did not reach the same level of significance. Variation in the performance of different statistical methods in the presence of linkage has been described previously (S.Davis and D.E.Weeks, in preparation). In particular, the GENEHUNTER statistic has been found to be less powerful, often giving non-significant test statistics when other methods are significant, similar to the pattern observed here in simulation studies. However, because of our observation that the GENEHUNTER statistic appears to be conservative based on these simulation studies, it might be possible to modify the method in order to obtain empirical P values that may be similar to those obtained in the other methods. It should also be noted that the pairwise marker data analysed with the GENEHUNTER and the parametric methods were not identical, as it was necessary to reduce the size of some pedigrees in the GENEHUNTER calculations, as discussed in Material and Methods. When parametric analysis was performed on the reduced data for a subset of loci pairs, the tests were found to be less significant, although still more so than GENEHUNTER (results not shown). These comparisons point to the need for careful evaluation of statistical methods as part of linkage studies of multifactorial diseases. ACE maps to the region of blood pressure linkage on rat chromosome 10 and it is also in the homology region that we have targeted in human hypertension. Because of the important role of ACE in the reninangiotensin system, it has been studied previously as a candidate gene in hypertension, but the results have been largely negative (22). In particular, Jeunemaitre et al. (15) found no evidence for linkage to hypertension with the GH microsatellite, near to the ACE locus, in an independent panel of families. In our family panel, which contained a larger number of sib-pairs and which was selected under a different protocol, we obtained P = 0.003 by affected sib-pair analysis with GH. However, the strongest evidence of linkage was found with D17S183 and D17S934, which are 18 cM proximal to ACE. In the parametric analyses for the marker pairs D17S183GH and D17S934GH the maximum likelihood placement of the putative susceptibility locus was 13 cM from GH/ACE. The latter were outside the 95% support interval, suggesting that another gene (or genes) from the homology region could be implicated in hypertension. Congenic strains bred from Dahl salt-sensitive hypertensive rats gave evidence for a QTL with a major effect on blood pressure localised within a 31 cM segment that includes ACE (23). The congenic segment maps to the homology region in which we have found linkage in human hypertension families and lends support to the hypothesis that the same genes could be implicated in hypertension in humans. Recent data from crosses involving inbred hypertensive strains of rat suggest that a blood pressure locus on rat chromosome 10 may reside at a distance from the ACE gene or that two linked loci may be involved (24,25). Other candidate genes, e.g. phenylethanolamine N-methyltransferase (26), and transcripts have been mapped to the interval, but at present no evidence is available to implicate any of these in hypertension in either the rat or human. The identification of the gene or genes responsible for hypertension susceptibility will most likely require a systematic investigation of the region in both species. If homologous genes are involved, a powerful approach will be to combine fine mapping of congenic lines in the rat and human families. Traditional approaches to gene identification have been successful for rare Mendelian syndromes associated with severe hypertension (27), but to date these genes have not been implicated in essential hypertension. Previous genetic investigations of essential hypertension have mostly focused on key candidate genes from the reninangiotensin system (28) and significant evidence of linkage in more than one family panel has been reported for angiotensinogen (6,7). Our study illustrates an alternative approach which, as a first stage, targets homology regions based on blood pressure linkage in the rat and does not rely on a priori testing of candidate genes. The next step will be to seek confirmation of these linkage results in other large panels of families containing multiple cases of hypertension with a similar disease definition to those used here.

2084 Human Molecular Genetics, 1997, Vol. 6, No. 12 MATERIALS AND METHODS Families Patients were diagnosed with hypertension if: (i) multiple measurements of diastolic blood pressure exceeded 95 mm Hg at the time of examination; (ii) the patient had a clinical record with multiple diastolic blood pressure readings over 95 mm Hg prior to treatment; (iii) the patient was on long-term anti-hypertensive treatment with one or more drugs. All patients were 60 or younger at time of onset of hypertension. Subjects with diastolic blood pressure <90 and systolic blood pressure <140 at >30 years were considered unaffected; otherwise the phenotype was assumed unknown. Comparison with unpublished survey data from the UK suggests that the disease prevalence under these ascertainment criteria will be between 1 and 5% after adjustment for age and BMI. The families were divided into four panels based on additional ascertainment criteria: (i) HTE (essential hypertension from France), families ascertained through patients from the Hypertension Clinic at Broussais Hospital and from other centres in France (6); (ii) HTO (essential hypertension in families collected in the region of Oxford, UK), families ascertained from the hypertension clinic at the John Radcliffe Hospital, Oxford, or through general practices in the Oxford area and containing at least one individual with a diagnosis of hypertension; (iii) HTD (hypertension and insulin dependent diabetes), French families initially collected for studies of insulin-dependent diabetes mellitus (IDDM) with at least one offspring having been diagnosed with IDDM and at least one offspring (not necessarily diabetic) diagnosed with hypertension; (iv) HTN (hypertension and non-insulin-dependent diabetes mellitus), French families initially collected for studies of non-insulin-dependent diabetes mellitus (NIDDM) with at least two offspring having been diagnosed with NIDDM and at least one offspring (not necessarily diabetic) diagnosed with hypertension. In the HTE panel, families with a parental history of hypertension were preferentially included. In the HTO families, the third diagnostic criterion was replaced by the requirement of treatment with two drugs. In the HTD and HTN panels, criteria (i) or (ii) were required for a positive diagnosis of hypertension. Additional selection criteria applied to some probands were BMI <27 kg/m2 (HTE) or BMI <30 kg/m2 (HTO) and reported alcohol consumption 14 U/week for females or 21 U/week for males (HTE, HTO). Use of oral contraception or secondary causes of hypertension were exclusion criteria for the diagnosis of hypertension. Genotyping Genotype characterisations at microsatellites were performed as described (29) with some modifications. Linkage Reported variability in linkage results obtained with different non-parametric statistical methods and computer implementations (S.Davis and D.E.Weeks, in preparation) led us to apply four different methods of analysis to the data for the purpose of comparison: SIBPAL. The SIBPAL program (17) is one of the most commonly applied of the non-parametric methods. SIBPAL provides affected sib-pair and IBD regression statistics. We used only the former here. Information on half-sibs is incorporated into the affected sib-pair statistic, but the results cannot be reliably interpreted if the number of half-sibs is small, as it is in our sample. To remove the effect of half-sibs, we divided extended pedigrees into nuclear families prior to applying SIBPAL. The program estimates IBD sharing at a single marker locus from parental and offspring data for each affected sib-pair and treats pairs from within the same family as independent. SIBPAIR. The SIBPAIR program implements the algorithm described in Satsangi et al. (20) and Kuokkanen et al. (19). It is essentially equivalent to the calculation of lod scores under the assumption of a simple recessive disease model which takes into account non-independence of affected sib-pairs from the same family and missing data on parents. This method has been shown to conform closely to its asymptotic null distribution in small samples and was amongst the most powerful of the non-parametric test statistics examined by Davis and Weeks (in preparation). Parametric analysis. Parametric linkage analysis was undertaken with a modified version of the VITESSE (30) program. Parameter estimates and test statistics were calculated conditional on the trait phenotype status of all the family members (mod scores) as a correction for ascertainment (20). We assumed a two-allele, dominant model for the trait locus which allowed for incomplete, non-zero penetrance of the three genotypes while constraining the overall disease prevalence to a predetermined value (here either 5 or 1%, representing the extremes of the estimated prevalence for hypertension defined with our diagnosis criteria). For a single marker locus, this is equivalent to maximising the lod score over all genetic models satisfying these constraints. In the tests of linkage with a single marker locus, we imposed the additional constraint of no recombination between the putative susceptibility locus and the marker locus, as in Davies et al. (31); this constraint is used since the recombination rate may be confounded with other parameters. Under the hypothesis of dominance, we assumed that the susceptibility allele d had frequency p with penetrance fd for the two genotypes Dd and dd. The parameters p and fd were estimated and the penetrance fDD associated with the DD genotype was calculated as a function of these estimates and the fixed disease prevalence (r) as fDD = {r fd[1 (1 p)2]}/(1 p)2. A likelihood ratio test statistic was calculated against the null hypothesis in which the susceptibility allele frequency was 0 or, equivalently, q = f = r. In the parametric multilocus analysis the test statistic was calculated at different placement of the trait locus with respect to the markers. The likelihood ratio is calculated against the null hypothesis in which the susceptibility allele frequency is 0. The test statistic has been compared with a 2 distribution with 2 degrees of freedom to obtain significance levels. Simulations, some of which are described below, showed this to be conservative. GENEHUNTER. The NPL score statistic was calculated with the GENEHUNTER program (21) with the all pairs option. Because of the large size of some of the pedigrees in our study, the statistical calculations could not be made with the complete data. We divided the pedigrees into nuclear families and removed a number of individuals from the larger sibships to overcome this. Significance levels are evaluated with the perfect data approximation as explained in Kruglyak et al. (21). The parametric and GENEHUNTER programs were applied to analyse multilocus marker data. The SIBPAL and SIBPAIR programs are restricted in application to a single marker locus.

2085 Human Genetics, 1997, 6, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 12 2085 Allele frequency estimates were obtained prior to linkage analysis based on the allele counts using one randomly selected individual for which genotypes were available from each pedigree. The analyses were performed in the combined panel of families, as well as in the four sub-panels. The linkage results on the sub-panels reported in the text were calculated with the allele frequency estimates from the combined family panel. Recalculation with estimates from the sub-panels did not modify these significantly, as the allele frequencies did not differ significantly between sub-panels of families. Simulations The performance of the test statistic was evaluated by simulations with SLINK (32) under a dominant single locus model with two alleles, d and D, having allele frequencies of 0.05 and 0.95 respectively. The simulations were made conditional on the observed phenotype status in the family panel. For the power calculations, we simulated 500 replicates under models in which the penetrance associated with the dd and dD genotypes was 0.10, 0.13, 0.15, 0.17 or 0.20. The penetrance of the DD genotype was adjusted so as to obtain an overall prevalence of 5% for the disease in the general population as discussed above. Simulations were made under the null hypothesis with the penetrance for all three genotypes equal to the population prevalence; 1500 replicates were made to examine the null distributions of the test statistics. In the power calculations shown here, the marker and trait loci are assumed to be completely linked. We considered two situations: one in which the marker was completely informative and the other in which the marker allele frequencies were equal to those at D17S934. The test statistics were calculated with the allele frequency estimates fixed at the values used in the simulations. In the results presented in Figures 2 and 3, the parametric analyses were undertaken assuming 5% prevalence; however, neither the power nor the performance of the statistic under the null hypothesis were substantially different when the simulated data were analysed with 1% prevalence. ACKNOWLEDGEMENTS We thank M.Marmot and H.Hemingway for providing the blood pressure survey data from the Whitehall II study. Financial support has been provided by the Wellcome Trust, INSERM, the French Ministry of Research and the EC Concerted Action Molecular Genetics of Hypertension. B.K. is supported by a MRC Training Fellowship. G.M.L. holds a Wellcome Trust Principal Fellowship. S.D. and D.E.W. were supported in part by NIH grant HG00719. Portions of the results of this paper were obtained using the program package SAGE.
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