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(Hypertension. 1995;25:6-13.)
© 1995 American Heart Association, Inc.

Articles

Evaluation of the SA Locus in Human Hypertension

Toru Nabika; Alain Bonnardeaux; Michael James; Cecile Julier; Xavier Jeunemaitre; Pierre Corvol; Mark Lathrop; Florent Soubrier

From INSERM U358, Hôpital St Louis (T.N., M.J., C.J., M.L.), and INSERM U36, College de France (T.N., A.B., X.J., P.C., F.S.), Paris, France.

Correspondence to Florent Soubrier, MD, PhD, INSERM U36, College de France, 3 rue d'Ulm, 75005, Paris, France.

	Abstract

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Abstract The SA gene is expressed at 10-fold greater levels in the kidney of the spontaneously hypertensive rat compared with the normotensive Wistar-Kyoto rat. The gene is linked to blood pressure levels in a number of crosses involving the spontaneously hypertensive rat and other strains of genetically hypertensive rats. To assess its role in human hypertension, a human SA cDNA was cloned from a liver library. The cDNA was 1513 bp in length and exhibited a high identity with the published rat SA cDNA sequence in the coding region. A microsatellite marker was developed from a yeast artificial chromosome clone containing SA and mapped by linkage to human chromosome 16p13.11-12.3. Polymerase chain reaction amplification of human genomic DNA revealed two introns located in the SA gene, one of which contains a frequent polymorphism due to a single nucleotide substitution (cytosine to thymidine at residue 79 of the intron). Association and linkage studies in a large sample of hypertensive patients, normotensive control subjects, and multiplex sibships with these markers and other microsatellites in close proximity to SA revealed no evidence favoring involvement of the gene in the disease in humans. The methodology used in this study can be applied to the evaluation of other novel candidate genes obtained from investigations of experimental models of hereditary hypertension.

Key Words: hypertension, essential • genetics • sibling relations • rat, inbred SHR

	Introduction

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Essential hypertension is one of the most prevalent genetic disorders in developed countries. The causes of the disease are heterogeneous, and interactions between multiple genetic and environmental factors are thought to be involved in its pathogenesis.¹ Because of the complexity of the disease, rodent models of hereditary hypertension have been used in genetic linkage studies to determine which loci should be evaluated in humans. This strategy makes it possible to avoid heterogeneity in pathogenesis of the disease and to increase analytical power by making hundreds of F2 progeny.² Recent genetic studies have shown linkage between blood pressure and several candidate genes or genomic regions in the rat. These include the renin,³ the angiotensin-converting enzyme,^{4 5} the carboxypeptidase B,⁶ and the guanylyl cyclase A receptor loci.⁷ In some instances, these localizations have motivated the study of corresponding candidate loci in human hypertension.^{8 9}

Another approach to determine genetic loci that could be involved in hypertension consists of the isolation of new candidate genes that are differentially expressed in target tissues from hypertensive and normotensive rat strains. The example of the SA gene illustrates how the mapping of such candidate genes can be combined with genetic linkage studies of blood pressure to obtain new information on the loci potentially involved in the disease. SA is a gene of unknown function identified in screening for genes with increased expression in the kidney of the spontaneously hypertensive rat (SHR) compared with the kidney of the normotensive Wistar-Kyoto rat at the early hypertensive stage.¹⁰ The SA gene exhibits a 10-fold difference in expression in the target tissue from the two strains and is also expressed in the liver. The gene was mapped to rat chromosome 1,^{11 12} and SA or genetic markers of the same region were shown to be linked to blood pressure levels in a number of different crosses. These include crosses involving the SHR,^{13 14 15} the stroke-prone SHR,^{11 16} and the Dahl salt-sensitive hypertensive rat.¹⁷

These observations by independent groups suggest that SA is a likely candidate for a causative gene of hypertension in some models of hereditary hypertension in the rat. Since these hypertensive rat strains share many pathophysiological characteristics of the human disease,¹⁸ it is of interest to examine the possible relation of the SA gene to essential hypertension in humans. Alternatively, the SA gene in the rat may be linked to another locus involved in blood pressure regulation. Therefore, it is also of interest to test other markers that could be in the region of conserved synteny around the human SA gene.

To assess these points, we first isolated and sequenced a human SA cDNA that was cloned from a liver cDNA library. This was used to screen for a human yeast artificial chromosome (YAC) clone containing the SA gene. A highly polymorphic microsatellite marker was isolated from the YAC and mapped to human chromosome 16 by linkage. This marker and other microsatellites from the same region of chromosome 16 were examined for linkage to hypertension in affected sib pairs. A frequent polymorphism was detected by single-strand conformation polymorphism (SSCP) analysis in one of the introns of the SA gene, and this was examined for association with the disease by comparison of genotype and allele frequencies in hypertensive patients and normotensive control subjects. This study illustrates the methodology that can be applied to evaluate novel candidate loci in human hypertension after identification from studies of rat models.

	Methods

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Subjects
This study was approved by an institutional review committee, and the subjects gave informed consent. The hypertensive patients, sibships, and normotensive control subjects studied here have been described elsewhere.¹⁹ Briefly, hypertensive probands were selected according to the following criteria: (1) age older than 20 years; (2) onset of hypertension at younger than 60 years; (3) established hypertension as defined either by long-term treatment or by a diastolic blood pressure (DBP) greater than 95 mm Hg on two consecutive visits for those untreated; (4) absence of secondary forms of hypertension through extensive workup when indicated; (5) family history of hypertension (occurring before age 60 years) with at least one parent and one sibling being affected; and (6) absence of excess alcohol intake (more than three drinks per day), oral contraceptive therapy, diabetes mellitus, or renal failure. Data on family members were collected for 173 hypertensive probands in Paris and 33 from Toulouse and Bordeaux. This led to ascertainment of 125 sibships in which two or more offspring were hypertensive. A total of 85 affected pairs, 33 trios, 5 quartets, and 2 quintets were thus evaluated, yielding 234 sib pairs. Two hundred ninety-two unrelated, normotensive control subjects were collected in preventive medicine centers in Paris and Nancy. Control subjects had systolic blood pressure (SBP) less than 140 mm Hg, DBP less than 90 mm Hg, and no history of antihypertensive treatment or of other chronic diseases such as diabetes mellitus and chronic renal failure. All individuals included in the study were whites of French origin. Their clinical characteristics are listed in Table 1.

View this table: [in this window] [in a new window]   Table 1. Clinical Parameters of the Populations Studied

Polymerase Chain Reaction Amplification
We performed polymerase chain reaction (PCR) amplifications using various primers under the following conditions: 2.5 ng/µL of genomic DNA, 0.5 nmol/L of each primer, 1.5 mmol/L MgCl₂, 0.02 U/mL of Taq polymerase (Boehringer-Mannheim), 1x Taq polymerase buffer provided by the supplier, and 63 nmol/L of dNTPs in 10 or 20 µL of reaction mixture. PCR was started with the initial denaturing step at 96°C for 4 minutes, followed by 35 cycles, each of which consisted of 1 minute at 94°C, 1 minute at annealing temperatures depending on primers, and 10 seconds at 72°C.

Cloning of the Human SA cDNA
A human liver cDNA library constructed in gt11 was screened with a fragment of rat SA cDNA as a probe. This was made by reversed transcription of total RNA extracted from the rat kidney. Primers (5'-ATGGCAATGTTACTTCGTG-3', 5'-AGGATGTCTTTCTGGGTCA-3') were designed based on the published sequence of rat SA cDNA to amplify a 460-bp DNA fragment positioned between nucleotide 289 and 749.¹⁰ Reverse transcription was done for 60 minutes at 37°C in a reaction mixture containing 3 µg of total RNA, 25 pmol of the designed primers, 25 nmol dNTPs, and 200 U Moloney-murine leukemia virus reverse transcriptase (Gibco BRL). PCR amplification was then performed on the synthesized cDNA with the same primers. The amplified fragment was purified on agarose gel and sequenced.

The cDNA library was screened following a standard protocol.²⁰ Hybridized replica filters were washed in 1x SSC/0.1% sodium dodecyl sulfate (SDS) at 55°C. One positive clone was identified, which was subcloned into pBluescript (Stratagene) and sequenced.

Detection of SSCP
Two introns were identified in the SA gene by PCR amplification of human genomic DNA with primers obtained from the cDNA sequence (introns a and b in Fig 1). Primers for the SSCP study were designed based on sequences of the introns and of their flanking exons of the SA gene to obtain overlapping fragments (Table 2). PCR fragments were labeled either with ³²P–end-labeled primers or with [³²P]dCTP included in the reaction mixture. Fragments that were greater than 300 bp in length were digested with restriction enzymes listed in Table 2 to maximize the probability of detecting SSCP.^{21 22} Samples were analyzed on nondenaturing 6% polyacrylamide/methylene-bis-acrylamide (49:1) gel with or without 5% glycerol as described previously.²¹ Forty-seven unrelated individuals were screened to search for SSCP.

View larger version (61K): [in this window] [in a new window]   Figure 1. The sequence of human SA cDNA. Putative initiation (ATG) and termination (TGA) codons are shown in bold letters. Points a and b represent the insertion points of two introns that were identified through the study. Underlines with arrowheads indicate primers used in detection of the introns. The diverged sequence from the rat SA cDNA is indicated with a broken line. Sequences different from the other report28 are marked with underlines. The European Molecular Biology Laboratory Data Library accession numbers for human SA cDNA, introns a and b are X80062, X80063, and X80064, respectively.

View this table: [in this window] [in a new window]   Table 2. Primers Used in the Single-Strand Conformation Polymorphism Study

Isolation of a Microsatellite Marker at the SA Locus
The Centre d'Etude du Polymorphisme Humain (CEPH) (Paris, France) YAC library²³ (23 700 clones, 8 haploid genome equivalent) was screened with primers used in the SSCP study. One positive clone, 933F8 (insert size, 1.1 megabase [Mb]), was identified, and DNA was extracted in agarose plugs. The YAC was subcloned into cosmids by constructing a cosmid library in the triple helix vector (Stratagene) using Sau3AI partially digested clone DNA. The first screening of the library was done with ³²P-labeled human genomic DNA. Positive colonies were picked up and were secondarily screened with ³²P-end-labeled oligomers (CA)₁₅ and (GA)₁₅. Hybridization with the oligomers was performed in 6x SSC, 0.5% SDS, and 5x Denhardt's solution at 55°C, and filters were washed in 0.1x SSC/0.1% SDS at 55°C or 60°C. Nine positive clones were identified and were digested with a combination of Alu I, Hae III, and Rsa I for plasmid library construction. After screening of these libraries with ³²P-labeled (CA)₁₅ and (GA)₁₅, positive clones were picked up and sequenced.

Genotype Characterization
Primers and annealing temperatures for microsatellite markers are listed in Table 3. PCR reactions were made with ³²P-labeled primers, and the products were analyzed on standard sequencing gels. SSCP analysis (as described above) or allele-specific oligonucleotide hybridizations were performed for characterization of a single-nucleotide substitution in the SA gene (cytosine to thymidine substitution in intron b). Allele-specific oligonucleotide hybridizations were done as follows. Based on the sequence data of homozygotes of the two alleles, two oligomers, 5'-ATAATCATAAGGAGACTG-3' (reverse strand) and 5'-GTCTCCTCATGATTA-3', were designed for alleles 1 and 2 (see Fig 3), respectively. PCR products were denatured in 0.4N sodium hydroxide, and half of the volume was separately spotted on two membranes. Membranes were then neutralized in 2x SSC and cross-linked with UV light. Each membrane was hybridized in 7% polyethylene glycol/10% SDS at 42°C for 3 to 12 hours, with one of the two oligomers end labeled with [³²P]ATP. At the end of hybridization, membranes were rinsed in 1x SSC/0.1% SDS at room temperature and once for 10 minutes either at 50°C for allele-1 or at 45°C for allele-2 oligomer. After autoradiography, membranes were dehybridized in boiling water and rehybridized with the other oligomer to avoid misreading of genotypes. Genotypes obtained by SSCP and by allele-specific oligonucleotide hybridization were in complete accordance in 48 individuals screened by both methods.

View this table: [in this window] [in a new window]   Table 3. Markers Used in Genotyping of Hypertensive Sibships

View larger version (34K): [in this window] [in a new window]   Figure 3. The single-strand conformational polymorphism (SSCP) in intron b. A, An SSCP found in the fragment P29/30 of intron b (see Table 2). The polymorphism was analyzed on nondenaturing acrylamide gel with 5% glycerol run at room temperature. B, The single-nucleotide substitution at nucleotide 79 of intron b. PCR-amplified fragments of two homozygotes were directly sequenced. Cytosine in the genotype 22 was replaced with thymidine in the genotype 11.

Statistical Analyses
We performed two-point and multipoint linkage analyses with LINKAGE²⁴ using the CEPH database version 6. Linkages between hypertensive state and selected markers in sibships were analyzed with the affected sib pair method described by Lange using Hodge's weighting for different sibship sizes^{25 26} and by a method developed by C. Amos as implemented in the program SIBPAL.²⁷ The SIBPAL program provides estimates of the identity-by-descent (IBD) probabilities for affected sib pairs (ie, the probability that affected siblings receive the same allele from either parent), taking into account all information on the markers in each family, including information on genotypes of parents and unaffected siblings when these are available. In the presence of linkage, we expect that the IBD probability will be greater than 0.5; otherwise it should be equal to 0.5. Estimates and SEMs of the IBD probabilities were obtained with the SIBPAL program. For a biallelic marker, comparisons of genotype distributions and of allelic frequencies were assessed by the ² test with 2 and 1 df, respectively. Deviation from Hardy-Weinberg equilibrium was tested by the ² test with 1 df.

	Results

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Complementary DNA of the Human SA
Fig 1 shows the sequence of a cDNA cloned from a human liver cDNA library. The isolated human cDNA is 1513 bp long and has an open reading frame of 1290 bp. The sequence of the human cDNA exhibited a high similarity (85%) with the rat kidney SA cDNA sequence over 1200 bp of the open reading frame. After nucleotide position 1201 of the human cDNA, however, the sequence similarity abruptly disappears, although the open reading frame continues over 90 nucleotides and finishes with a stop codon (TGA) at position 1290. The consensus sequence for exon-intron junction is found at the disappearance of the homology, suggesting that this represents a differently spliced form of the SA gene in the human liver. Eighty-three percent homology with the rat SA was observed in the first 400 amino acids deduced from the nucleotide sequence.

Localization of the SA Gene and Identification of Flanking Microsatellite Markers
Microsatellite markers were obtained from YAC clone 933F8 containing the SA gene as described above. Four distinct dinucleotide repeats [3 (CA)_n and 1 (GA)_n] were identified, and one of these (p4546) was further characterized. Primers for genotyping p4546 were designed to amplify a 108-bp fragment constituting the repeat (Table 3). Members of 12 families from the CEPH panel were characterized for this marker, and lod scores were calculated for markers in the CEPH database. Several markers in the region of chromosome 16p13.11-12.3 showed highly significant evidence of linkage with p4546. The localization of the 933F8 YAC clone to chromosome 16p was confirmed by fluorescent in situ hybridization (data not shown). The placement of p4546 was estimated with respect to chromosome 16 markers in the recent Généthon map by multilocus linkage analysis.²⁹ These results showed that the SA gene lies in the interval spanned by markers AFM165yb6 (D16S410) and AFM191wb10 (D16S412) with odds greater than 1000:1 (Fig 2).

View larger version (17K): [in this window] [in a new window]   Figure 2. Line graph shows location (Loc) score for the placement of the SA and the p4546 loci with respect to selected chromosome 16 markers characterized in the Centre d'Etude du Polymorphisme Humain (Paris, France) reference families. The maximum likelihood placement (maximum of the location score curve) for SA is indicated by an arrow. Odds against alternative orders are indicated in the figure.

SSCP Polymorphism of the SA Gene
Two introns were identified by PCR amplification of human genomic DNA with primers selected from the sequence of the human SA cDNA (see Fig 1). The introns were located at residues 406 and 1119 of the cDNA sequence (at points a and b in Fig 1) and were 1323 bp and 969 bp in length, respectively. Primers for the SSCP study were designed based on sequences of the introns and the flanking exons (Table 2). Among 11 fragments studied, one from intron b exhibited a frequent polymorphism (Fig 3A). Direct sequencing of homozygotes for the two alleles indicated that residue 79 of intron b was thymidine in allele 1 (the upper band on SSCP gel) instead of cytosine in allele 2 (the lower on SSCP gel) (Fig 3). The allele frequencies of this polymorphism (designated CT79) in 292 normotensive whites of French origin were 0.45 and 0.55 for alleles 1 and 2, respectively. Although we found four other variants in the two introns, these were not analyzed further because in each case the frequency of one of the two alleles was less than 5%.

Eighty parents of CEPH reference families were genotyped for the CT79 polymorphism using SSCP gels. Members of 17 informative families were characterized for CT79, and no recombination events were observed with p4546, which confirms that the two markers detect the same locus.

Linkage and Association Studies
Genotypes in the hypertensive sibships were characterized for the SA microsatellite p4546 and for three other Généthon microsatellite markers (AFM113xa9, AFM165yb6, and AFM191wb10) from the region that spans the segment of chromosome 16 containing the gene (Fig 2).²⁹ As shown in Table 4, no significant excess of the identity-by-state statistics were observed for the four microsatellites. An association study was also performed by comparing genotype and allele frequencies for polymorphism CT79 in hypertensive patients and normotensive control subjects; no significant differences were observed between the two populations (Table 5). The results were not changed when comparisons were made between control subjects and subgroups of hypertensive subjects stratified by one of the following criteria: SBP greater than 170 mm Hg and DBP greater than 100 mm Hg, age of onset of hypertension younger than 35 years, or body mass index less than 25.0 kg/m² (results not shown).

View this table: [in this window] [in a new window]   Table 4. Identity-by-State Tests for Deviations From Expectations for the Absence of Linkage and Estimates of Identity-by-Descent Probabilities and Their SEMs for Microsatellite Markers in the Regions of the SA Gene

View this table: [in this window] [in a new window]   Table 5. Genotype and Allele Frequencies of CT79 Polymorphism in Hypertensive and Normotensive Populations

The genotype frequencies in the normotensive control population were found to deviate significantly from Hardy-Weinberg expectations (²=9.77, P<.002). Since the normotensive control subjects were selected from two different regions of France, we further subdivided this population according to the city of origin (Table 5). The genotype frequencies were similar in the two groups, and both exhibited a deficit in heterozygotes compared with that expected under Hardy-Weinberg equilibrium (Paris: ²=5.61 [P<.02]; Nancy: ²=4.72 [P<.05]). Although the reasons for this deviation remain unclear, genotype and allelic frequencies were not significantly different from the hypertensive sample for either control group. The hypertensive population showed no deviation from Hardy-Weinberg expectations.

	Discussion

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The SA gene was initially identified because of its differential expression in kidneys from genetically hypertensive and normotensive rats. Its physiological role in blood pressure regulation, however, remains unclear at present. To characterize this gene in humans and to perform genetic studies of this locus in essential hypertension, we first isolated a human cDNA for the SA gene. We observed 85% sequence similarity between the rat and human genes on a portion of 1200 bp of the coding region, confirming that it is conserved in both species. However, the structure of the cDNA cloned from a human liver cDNA library completely diverged after nucleotide position 1201 (human numbering). Interestingly, this divergence occurs at a consensus sequence for exon-intron junction. Recently, Iwai et al²⁸ reported the sequence of another SA cDNA clone from human liver similar to the rat kidney cDNA that also diverges from our sequence at nucleotide 1201. These results suggest the occurrence of a differential splicing of this gene, with at least two possible mRNAs, in the liver. The high degree of conservation of the gene sequence between rats and humans also suggests that this gene is functional and under high selection pressure.

To investigate the involvement of the gene in human hypertension, we looked for polymorphic nucleotide sequences by the SSCP method in a group of selected hypertensive subjects. Although we did not identify all of the introns in the gene, we found several polymorphisms in the two introns that we studied, and the most informative was used for a cross-sectional study based on a large series of hypertensive patients and normotensive control subjects. The most striking difference between the hypertensive and normotensive populations was the finding of significant deviations from Hardy-Weinberg equilibrium at the CT79 polymorphism, located in intron b, in the normotensive group. As shown in Table 5, this is due to a deficiency of heterozygotes in normotensive subjects compared with the number expected under Hardy-Weinberg equilibrium. Interestingly, this was observed in normotensive populations from both Paris and Nancy. Because a deficiency of heterozygotes is a known consequence of population admixture, our observation could be due to a previously undetected source of heterogeneity in these samples. However, this has not been observed with many markers previously characterized in the same control samples,^{19 30 31} and we cannot exclude the possibility that the SA locus has an effect on blood pressure that leads to a decrease in heterozygote frequency in the normotensive samples. If this effect exists, it is not strong enough to produce a significant difference in genotype distributions observed in the hypertensive and normotensive samples that we have studied.

To perform a linkage study with hypertensive families, we isolated a YAC clone containing the SA gene and subcloned it into cosmids to identify highly informative multiallelic markers. One CA repeat was further characterized and was found to be highly informative (83% heterozygosity). In the CEPH families, no recombination was found between the CT79 polymorphism and this microsatellite, confirming the localization of the CA repeat in immediate proximity to the SA coding region. The absence of chimerism in this YAC clone was verified by fluorescent in situ hybridization (results not shown). The search for linkage in the hypertensive families was performed by means of two different affected sib pair methods, and no significant evidence of linkage was found. Since the 95% confidence interval gave an upper boundary of 0.54 on IBD probability at the SA microsatellite, the results suggest that this locus is not a major determinant of increased familial risk of hypertension.

There are two possible explanations for the differences between the observed genetic effects of the SA locus in rat and human hypertension. The first is that genetic variation at the SA locus plays a role in blood pressure regulation in the rat but not in human hypertension. This hypothesis implies that, in rats, (1) the gene is important for blood pressure regulation by a mechanism that is as yet unknown and (2) the polymorphism of the gene existed in outbred rat populations from which hypertensive and control strains were constructed. One of these two conditions may not be fulfilled in humans. The gene might not be important for human blood pressure control in contrast to the rat, but this discrepancy is quite unlikely. Another possibility is that the SA gene is not involved in blood pressure variance because there is no functional polymorphism altering the regulatory or the coding part of the gene in humans. In contrast to this hypothesis, a recent case-control study performed in Japan provided evidence for an association between a restriction fragment length polymorphism (RFLP) of the human SA gene and hypertension.²⁸ Our preliminary results indicate that this RFLP is also observed in whites, while allele frequencies are not significantly different between hypertensive and normotensive subjects (the frequencies of minor allele were 0.15 and 0.14 for 13 hypertensive and 18 normotensive subjects, respectively). If these results can be confirmed, they would suggest that the association of this RFLP with hypertension is only observed in some ethnic groups or might be also observed in some particular subgroups of hypertensive subjects.

Another possible explanation is that the SA gene is not itself implicated but is linked to another gene at which variation affects blood pressure regulation in the rat. Indeed, the differential screening strategy leads to the identification of genes for which a polymorphism of expression exists between the two strains of rats. The SA gene expression polymorphism might be therefore only a neutral marker, without any relation to blood pressure physiology. With regard to the low number of genes analyzed in the initial study (a total of three),¹⁰ the chances of finding a gene cosegregating with blood pressure is probably low. However, the possibility of identifying cosegregation at these loci still exists for two reasons. The first is that the number of loci that have a linkage with blood pressure could be higher than initially expected. More than eight blood pressure loci have been already identified to date, and the list keeps elongating rapidly.^{32 33 34} Second, the confidence interval for the localization of any quantitative trait locus identified through linkage in experimental crosses is extremely wide and will contain multiple candidate genes.

In the latter case, a homologous gene responsible for high blood pressure could exist in humans, but at a distance from the SA locus. For this reason we have also performed linkage studies with microsatellite markers flanking the SA gene. Using two markers spanning an interval of approximately 5 cM on each side of the SA gene, we have not been able to detect any linkage with hypertension. We may thus be able to eliminate linkage within a 10-cM interval around the SA gene. However, it is likely that this interval includes genes that reside near the SA gene in rats but are on chromosome 11p in humans.³⁵ Thus, a homologous locus in the human could be localized to regions other than chromosome 16p. Further studies will be needed to determine with precision the limits of the conserved synteny groups in humans that correspond to this region of rat chromosome 1 and to assess their relations to human hypertension.

As for every negative study, it is important to discuss the limitations of our study. We used several highly informative markers and large populations to obtain maximal power with the current approaches. However, we still could miss the effects of the SA locus on blood pressure as a quantitative trait because we studied blood pressure as a dichotomous trait (ie, hypertensive versus normotensive subjects). In addition, our study does not include any intermediate phenotypes related to the SA gene product, as it is not yet characterized. Using intermediate phenotypes may reinforce or add new information to such genetic studies, as was the case for angiotensinogen and angiotensin-converting enzyme genes.^{30 36} However, using the same study design, we successfully detected statistically significant linkage and/or association with hypertension on the angiotensinogen and angiotensin II receptor type I loci.^{19 30} Hence, the SA locus has only a minor effect, if it has any, on hypertension in the studied population.

In conclusion, our study of the SA gene illustrates a general approach that can be applied to evaluate candidate genes in humans. This requires cloning of the human equivalent of the gene, identification of YAC or other clones corresponding to the gene, development and mapping of microsatellite markers, and detection of polymorphisms within the gene by SSCP or by other techniques. In our case, the results coming from this strategy do not plead for the hypothesis that the SA gene itself plays a detectable role in hypertension among whites. More genetic studies in the rat will be required to further define the origin of the linkage of the SA locus, ie, the SA gene or a gene located at proximity of the SA gene, with blood pressure and to identify the physiological mechanism involved. The results of these investigations could lead to complementary investigations in humans.

Note added in proof. The insulin growth facter 2 (IGF₂) locus was also tested since this gene is close to the SA gene in the rat genome, but located in 11p15.5 in humans. Using a tetranucleotide microsatellite marker located in the first intron of the tyrosine hydroxylase gene, close to the IGF₂ gene, no linkage was observed in hypertensive families.

	Acknowledgments

 
This work was supported by INSERM, CNRS, and by a grant from the Groupement de Recherche et d'Etude sur les Génomes (GREG). Dr Nabika was supported by Foundation IPSEN and by the exchange program of scientists between INSERM, France and Japan Society for the Promotion of Science, Japan. We thank the CEPH YAC group for their provision of YAC screening materials, Dora Cherif (CEPH) for in situ hybridization of the YAC, and Marie-Therese Bihoreau for sequencing the microsatellites. We also thank Isabelle Fery for her skillful assistance.

Received May 9, 1994; first decision June 7, 1994; accepted October 3, 1994.

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