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(Hypertension. 1999;34:1186.)
© 1999 American Heart Association, Inc.

Scientific Contributions

Association and Linkage Analyses of Glucocorticoid Receptor Gene Markers in Essential Hypertension

Ruby C. Y. Lin; William Y. S. Wang; Brian J. Morris

From the Hypertension Gene Laboratory, Department of Physiology and Institute for Biomedical Research, The University of Sydney, New South Wales, Australia.

Correspondence to Brian J. Morris, DSc, Hypertension Gene Laboratory, Department of Physiology and Institute for Biomedical Research, Building F13, The University of Sydney, NSW 2006, Australia. E-mail brianm{at}physiol.usyd.edu.au

	Abstract

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Abstract—Suggestive evidence has been obtained in a "4-corners" study for involvement of the glucocorticoid receptor gene (GRL) in genetic variation in blood pressure. Therefore, we tested markers at the GRL locus for association and linkage with essential hypertension (HT). For the association study, we used a well-characterized group of 129 white Australians of Anglo-Celtic extraction who had HT, a strong family history of HT (2 parents with the disease), and early-onset moderate-to-severe disease. Controls were 195 normotensive white subjects whose parents were normotensive past the age of 50 years. For the linkage study, we used 175 sibling pairs of similar ancestry. The case-control groups were genotyped for an Asn363Ser variant in exon 2, a G/T variant in intron 4, and a microsatellite marker (D5S207) tightly linked (<200 kb) to GRL. For the groups as a whole, no association or linkage was observed after analysis of data by a variety of statistical tests. Analysis of sibling-pair data gave an exclusion score of -3.8 for the logarithm of the odds for linkage, indicating significant nonlinkage. However, in females, weak association of the intron 4 polymorphism with HT (P=0.03), as well as with systolic and diastolic blood pressure in all subjects (P=0.04 and 0.03), was observed, and in the case of the D5S207 marker, association with HT was apparent in males (P=0.0001). Thus, although our results provide no overall support for GRL in HT etiology, apparent gender-specific associations could exist in this genomic region, possibly reflecting correlated occurrence with (an)other metabolic syndrome disorder(s).

Key Words: glucocorticoids • genes • cross-sectional studies • whites • hypertension, essential • microsatellites

	Introduction

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Glucocorticoids can affect blood pressure (BP) in humans, as demonstrated most strikingly in Cushing’s syndrome. In essential hypertension (HT), vasoconstrictor sensitivity to glucocorticoids is increased.¹ Glucocorticoids are able to modulate the pressor responsiveness of vascular tissue to angiotensin II² and upregulate vascular AT₁ receptors,³ although blockade of glucocorticoid receptors with RU486, a progesterone receptor antagonist, does not prevent adrenocorticotropic hormone–induced HT in rats.⁴ Such raised sensitivity could represent the primary stimulus leading to HT and has been invoked in a fetal programming process that could underlie raised BP later in life.⁵ On the other hand, patients with low renin HT have lower glucocorticoid sensitivity.⁶ A genetically mediated difference in glucocorticoid receptor (GR) function or expression could thus be a possible contributor to HT in at least some patients.

Mutations in the C-terminal hormone-binding domain of the glucocorticoid receptor gene (GRL,⁷ chromosome 5q31⁸ ) result in reduced ligand affinity,^{9 10} and a splice-site deletion reduces receptor number.¹¹ The result is at least partial glucocorticoid resistance at the level of the hypothalamus that then responds by increasing corticotropin, with an ensuing rise in cortisol and other steroids. The symptoms differ, however, from those seen in essential HT. Moreover, other, more common, polymorphisms do not explain glucocorticoid resistance, which is relatively rare.¹² GRL primary transcripts undergo differential splicing in all tissues to yield 2 receptor subforms that differ by the inclusion of amino acids encoded either by exon 9 (GR) or by exon 10 (GRß).⁷ GRß does not bind glucocorticoids and acts as a dominant negative inhibitor of the classic receptor (GR) by forming a heterodimer with it.¹³ However, there is no evidence of abnormalities in the relative expression of each subform.¹⁴

The possibility that glucocorticoid effects might explain in part the genetics of raised BP was highlighted in a "4-corners" study, involving healthy young adults (mean age 21 years) of both genders grouped according to family history.¹⁵ High-BP offspring of high-BP parents (high/high) had 26% higher plasma cortisol (P=0.02) than did low-BP offspring of low-BP parents (low/low).¹⁵ In this regard, high urinary cortisol has also been related to salt-resistant HT.¹⁶ In the 4-corners study, the frequency of the minor (4.5-kb) allele of a biallelic (4.5- and 2.3-kb alleles) BclI restriction fragment length polymorphism (RFLP) of GRL was 36% in low/low compared with 50% in high/high subjects, although this difference did not quite reach statistical significance (P=0.055).¹⁵ The minor allele appeared, moreover, to track with elevation in personal BP.¹⁷ Furthermore, vascular tissue of subjects homozygous for the minor allele displayed enhanced in vivo responsiveness to glucocorticoids.¹⁸ The location of the BclI polymorphism is not known, although could be in an unsequenced region of the first or second intron or upstream DNA.^{19 20} Moreover, it has been suggested that the BclI marker is more likely to be in linkage disequilibrium with a variant that affects expression rather than one that produces an alteration in affinity.¹⁸ However, it has also been found that high/high subjects have increased sensitivity to and ligand-binding affinity for glucocorticoids, suggesting there could also be an abnormality in the GR.²¹ This "defect" was then suggested to contribute to insulin resistance in HT. In this regard, variation in glucocorticoid sensitivity has been associated with a polymorphism in exon 2 (transactivation domain) that causes an Asn363Ser alteration in GRL.^{22 23} The minor allele of the BclI RFLP has also shown an association with severe hyperinsulinemic obesity²⁴ as well as with abdominal visceral fat in the leaner subjects from a general population.²⁰

To more fully address the question of whether variation at the GRL locus is involved in HT, we carried out both association and linkage studies using GRL polymorphisms. Our case-control subjects were the hypertensive (HT) offspring of 2 HT parents and the normotensive (NT) offspring of NT parents; ie, they were the equivalent of the high/high and low/low groups of the 4-corners approach referred to above, but in affected adults. Rather than the BclI RFLP, whose detection requires laborious Southern blotting because of lack of sequence information to develop a polymerase chain reaction (PCR)-RFLP method, we chose markers that were more amenable to testing, namely, the Asn363Ser variant referred to above, an intron 4 polymorphism,¹² and a microsatellite marker, D5S207, located within 200 kb telomeric of GRL.²⁵ Moreover, the case-control groups^{26 27 28} and the cohort of affected sibships²⁹ had the capacity for demonstrating association and linkage with HT. The present study represents the first to test GRL in HT etiology.

	Methods

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Subjects
All subjects were of Anglo-Celtic descent and were obtained by community advertising in and near Sydney. HT was defined as a systolic/diastolic BP of >140/90 mm Hg on 3 separate occasions before the initiation of treatment and lack of diabetes, renal disease, or secondary causes of HT. NT subjects were required to have BP <140/90 mm Hg and parents who were NT past the age of 50 years. The study had ethical approval, and all subjects gave informed consent. Each subject provided a 50 mL blood sample, which was collected while the patient was in the sitting position.

Two groups, with contrasting phenotypes, were used for a case-control study. These consisted of 129 HTs who had 2 HT parents and 195 NTs. The number of subjects we used should have provided sufficient power, as judged from previous estimates³⁰ and from our positive results for variants of other genes in the same subjects, whereas our use of only HTs with 2 HT parents may have increased the likelihood of revealing an association.^{26 27 28} Characteristics of the subjects are shown in Table 1. Plasma parameters (mean±SE), determined by methods detailed previously,³¹ were as follows (for the HT versus NT groups, respectively): total cholesterol (mmol/L), 5.8±0.1 versus 5.2±0.1 (P=0.0003); triglycerides (mmol/L), 2.5±0.1 versus 1.5±0.1 (P=0.0001); HDL cholesterol (mmol/L), 1.1±0.05 versus 1.4±0.04 (P=0.0001); LDL cholesterol (mmol/L), 3.6±0.1 versus 3.2±0.1 (P=0.006); angiotensinogen (pmol/mL), 1402±40 versus 1172±182 (P=0.0001); renin (pmol angiotensin I · mL^-1 · h^-1), 10.3±1.4 versus 8.3±0.6; and angiotensin-converting enzyme (nmol Gly-Gly · min^-1 · mL^-1), 82±4 versus 85±3.

View this table: [in this window] [in a new window]   Table 1. Characteristics of HT and NT Groups

A second group of HTs involved a cohort of 239 affected individuals from 104 sibships (2 affected sibs) that included 16 trios, 6 quartets, and 1 quintet, giving an effective sibling-pair number of 175 after weighting.³² Their characteristics are also shown in Table 1.

Genotyping
DNA was isolated from whole-blood DNA by use of a kit (Qiagen). Genotypes for polymorphisms in or near GRL were determined by PCR. A microsatellite marker, D5S207, located 0 to 10 cM³³ (within 200 kb²⁵ ) of GRL, was tested by using primers as follows: forward, 5'-TTG GAA GCC TTA GGA AGT GC-3'; reverse, 5'-AAG AAT TC TAG TTT CAA TAC CG-3'. The forward primer was fluorescently labeled at the 5' end with the tetrachlorinated analogue of 6-carboxyfluorescein during synthesis by Bresagen. Other polymorphisms tested were within GRL and were biallelic. One was an exon 2 variant (A1218G) that altered the codon (Asn363Ser). This variant resides outside the ligand binding domain and does not impair GR function.²² It was detected with primers described previously¹² : forward, 5'-AGT ACC TCT GGA GGA CAG AT-3'; reverse, 5'-GTC CAT TCT TAA GAA ACA GG-3'. However, restriction digestion (see below) was used instead of single-strand conformation polymorphism analysis to detect the variant. The other polymorphism was in intron 4 and involved a G to T substitution 16 nucleotides upstream from exon 5. This was detected by using the following primers: forward, 5'-GAA TAA ACT GTG TAG CGC AG-3'; reverse, 5'-TAG TCC CCA GAA CTA AGA GA-3'.¹² Amplification was carried out on a PTC-200 Programmable Thermal Controller (MJ Research). The PCR protocol for each marker involved 10 cycles of 1 minute each at 94°C, 65°C, and 72°C, followed by 15 cycles for 1 minute each at 94°C, 60°C, and 72°C, and then 20 cycles for 1 minute each at 94°C, 58°C, and 72°C, finishing with a step at 72°C for 30 minutes. The reaction mixture consisted of 20 µL of 0.3 pmol of each primer, 0.2 mmol/L of each dNTP, 0.1 U AmpliTaq DNA polymerase (Perkin-Elmer), 56 mmol/L KCl, 11 mmol/L Tris-HCl (pH 8.3), and 2 mmol/L MgCl₂. To determine genotypes of the Asn363Ser polymorphism, we digested relevant PCR products at 65°C for 15 hours with 1 U Tsp509I and NEbuffer 1 supplied by the manufacturer of the enzyme (New England BioLabs). This digestion gave fragments of 19 bp and 134 bp (Asn363 variant) or did not cut the PCR product (153-bp band), in the case of the Ser363 variant. An invariant band of 95 bp was seen for all samples. For intron 4 genotype detection, we incubated the PCR products from this region at 37°C for 15 hours with 1 U HinfI and NEbuffer 2 (New England BioLabs). When G was at the polymorphic site, the size of the uncut PCR product was 259 bp, and for T at this position, the PCR product was cut to give fragments of 116 and 143 bp; an invariant band of 211 bp was also seen for all samples. Bands were visualized by ethidium bromide staining after electrophoresis on a 3% high-resolution agarose gel. For D5S207 genotyping, PCR products were electrophoresed on an ABI 377 automated sequencer, and genotypes were assigned by use of ABI Genotyper software. To confirm the accuracy of genotype assignment, genotyping was performed at least twice for all markers.

Statistical Analysis
In the case-control study, StatView (Abacus Concepts) was used to test for differences by ² analysis with 1 df for allele data or 2 df for genotype data in the case of the biallelic markers and 4 df for the microsatellite data after merging values for the rare (131-bp) allele (n=0 to 2) with those for the adjacent allele. Because significance values can be inaccurate for sparse contingency tables containing rare alleles, we also performed CLUMP analysis.³⁴ Determination of linkage disequilibrium between the polymorphisms involved analysis of haplotype frequencies in the largest group.³⁵ Comparison of various parameters across genotypes involved 1-way ANOVA using StatView.

For the linkage data, allele sharing by descent (identity by descent) or by state (identity by state) was determined by using linkage programs suitable for complex traits in order to test whether there was concordant sibling-pair sharing of alleles more often than expected under random mendelian segregation. Because the various programs available have relative advantages and disadvantages, the use of several has been advocated.³⁶ We used SPLINK,³⁷ which generates allele-shared identity-by-descent estimates for all possible pairs in a sibship and computes probabilities for each marker genotype when parents are not available, and MAPMAKER/SIBS.³⁸ The significance thresholds we set for acceptance or rejection of linkage were as recommended.³⁹

	Results

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Association Study
Hardy-Weinberg equilibrium was observed for genotype data for each polymorphism. Allele frequencies were similar to what others have described previously for white populations: 6% for the Asn363Ser variant,²³ 31% for the T allele of the intron 4 polymorphism,¹² and 10%, 15%, 36%, 37%, and 1% for alleles of the D5S207 marker,⁴⁰ where we found a rare extra allele 2 bp smaller than the smallest reported previously (Table 2). Linkage disequilibrium between alleles of these 3 polymorphisms was not observed.

View this table: [in this window] [in a new window]   Table 2. Association Analyses of GRL Variants in HT

No significant association with HT was seen for any of the markers for the group as a whole (Table 2). Using a relative risk of 1.6, the present study had 86.7% power to detect significant association of the Asn363Ser marker with HT, given the negative significance we obtained, and for the intron 4 marker, this power was 99.7%.⁴¹

For the DS5207 data, CLUMP analysis followed by Monte-Carlo simulations (T1) gave ² 9.3 (reached 87 times in 1000 simulations). Analysis after collapsing the columns with small expected values together (T2) gave ² 9.2 (47 times in 1000). Comparing each column against the rest (T3) gave ² 5.1 (104 times in 1000). A 2x2 contingency table clumped to produce maximum (T4) gave ² 6.5 (107 times in 1000).

The possible association of the Asn363Ser variant with elevation in body mass index (BMI) seen previously by others²³ was also evident from our data (not shown). However, we could see no association with BP. The lack of association with HT was based on ² analysis, which approximates the Fisher exact test for a 2x2 contingency table when expected values exceed 5, as applies in Table 2. In the case of the intron 4 polymorphism, female patients exhibited a weak (P<0.05) association of the G allele with HT (Table 2) and elevation in BP, but only after combining data for the HT and NT groups (Table 3). For D5S207, we saw an association with HT in males (Table 2), with the common 137-bp allele of this microsatellite marker being in excess. HT carriers of the 137-bp allele (n=63) had BP that was slightly, but not significantly, higher (174±25/112±20 [mean±SD] mm Hg) than the 49 lacking the 137-bp allele (172±24/108±13 [mean±SD] mm Hg). In HTs with the 137-bp allele, plasma cholesterol (6.0±0.15 versus 5.6±0.16 [mean±SE] mmol/L) was elevated (P=0.015), as was LDL cholesterol (3.7±0.17 versus 3.3±0.18, P=0.049).

View this table: [in this window] [in a new window]   Table 3. Tracking of BP With Genotype of Intron 4 G/T Polymorphism in Females

Sibling-Pair Linkage Study
The marker we used was selected for proximity to GRL and was informative with an average heterozygosity of 0.68. Allele frequencies in the HT siblings were 0.01, 0.06, 0.07, 0.44, 0.36, and 0.06 for the respective alleles. Two-point SPLINK (weighted and unweighted) gave P=0.58 and an associated logarithm of the odds (LOD) score of 0.00. By use of MAPMAKER/SIBS, a maximum LOD score of 0.00 was obtained. SPLINK results for male-male pairs (n=19) were P=0.57. In addition, we calculated exclusion values using MAPMAKER/SIBS under the assumption of no dominance variance for a hypothetical locus with relative risk to a sibling (_s) of 1.6 and using a conventional exclusion threshold of LOD -2. The exclusion LOD score we obtained (-3.8) was indicative of an absence of linkage of D5S207 with HT.

	Discussion

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The present study found no association or linkage of markers in and near GRL with HT in the respective study groups of HTs as a whole. However, evidence for possible gender-specific associations was obtained in the case-control HTs.

In considering the findings, it should be noted that linkage and association approaches test different things and that each has different strengths and weaknesses. Linkage studies prefer multiallelic markers to better track inheritance, whereas in association studies, biallelic markers are preferred. Linkage studies test for correlated transmission (concordant inheritance) of a disease and an allele within a pedigree, whereas association studies test for correlated occurrence in a population.⁴² Linkage can be seen without association, as can happen when there are many independent trait-causing loci in a population, so that association with any particular allele is weak. Association can be detected without linkage, such as when an allele explains only a minor proportion of the variance for a trait, meaning that even though the allele is more frequent in affected individuals, it does not predict disease status within a pedigree very well.⁴² This situation could apply to HT, considering its relatively small relative risk value and complexity and the expectation that many HT susceptibility loci may each contribute to a relatively minor proportion of the variance. Our negative result by both association and linkage analysis strengthens the argument for lack of a role for GRL in HT etiology. However, the differences stated above could be part of the explanation for our negative linkage in the face of positive association findings, albeit confined to gender-specific subgroups.

A negative sibling-pair linkage in a complex polygenic trait may indicate either that the locus tested is not linked to the condition or that the size of the study group was insufficient to provide sufficient power to reveal a small genetic contribution of any gene in this region to the trait. It has been estimated that in HT with strong family history and disease onset before 55 years of age, relative risk to a sibling (_s) may be 3.8.⁴³ Analyses by others have suggested that for a complex disease 100 sibling pairs would be sufficient to provide 90% power to show significant linkage at the LOD 3 (P=0.0001³⁹ ) level if its _s exceeds 3.5.⁴⁴ For a disease like HT with multiple weak contributing loci (eg, _s values of 1.6), the 175 siblings we tested should have 90% power to provide evidence for suggestive linkage, ie, LOD 2 (P=0.001).⁴⁴ That our siblings do indeed have sufficient power to reveal a locus for HT is evident from recent findings, in the same cohort, suggestive of significant linkage (by different tests) of 2 other chromosomal loci to HT.^{29 45} Moreover, our sibling number was comparable to that used in a study that showed linkage of the angiotensinogen gene to HT.⁴⁶ For our siblings, age of onset of HT was 43±12 [mean±SD] years, and when we confined our analyses to those with onset before 45 years of age, we still did not find significance. In fact, our data for the D5S207 marker (exclusion LOD score of -3.8) suggests that any gene, such as GRL, linked to D5S207 is not involved in the etiology of HT.

The Asn363Ser variant was relatively rare (6%), meaning that its power to detect association was less than the intron 4 variant (minor allele 47%). The suggestive association with HT seen for the intron 4 and D5S207 markers is somewhat unusual because it applied to different genders. We could find no evidence for these markers being in linkage disequilibrium. Thus, any effect of a causative variant in GRL that may be coinherited with alleles of D5S207 would be independent of effects of alleles of the intron 4 marker. Because D5S207 could be up to 200 kb from GRL, the linkage disequilibrium could involve another gene. It is also possible that at least one of the associations observed could be spurious, arising from subgroup analysis and multiple comparisons. That the apparent association in females might have been restricted to premenopausal or postmenopausal state was discounted because we found no difference after stratification of data by mean age of 52 years. Interestingly, sexual dimorphism in the phenotype of glucocorticoid resistance has been described.⁴⁷ Phenotypic differences, possibly extending to BP, can be ascribed to an imbalance of steroid hormones leading to inappropriate activation of transcription factors, whose interactions are complex.⁴⁸ It is also of possible relevance that urinary free cortisol has been found to be higher in male than in female HTs, particularly when sodium intake was controlled for.¹⁶ Higher ligand concentration confined to one gender in the face of, for example, genetically determined higher GR responsiveness, could synergize to produce HT. This would then offer a possible explanation for the association with HT seen in males in the case of the D5S207 data. The absence of any relation of urinary cortisol to BMI¹⁶ is consistent with an absence of gender difference in adiposity being a contributory factor in this argument.

A locus for interindividual variation in systolic BP of young white subjects has been described recently for chromosome 5.⁴⁹ However, this was remote from the marker we tested, being in a region that contained the ß₁-adrenoceptor gene (ADRB1), located 10 to 25 cM telomeric from D5S207. The ß₂-adrenoceptor gene (ADRB2) on the other hand maps 2 cM (3 Mb) telomeric from GRL,²⁵ which is sufficiently close for our findings to also exclude ADRB2 in HT.

In conclusion, we could find little evidence for the involvement of GRL, or any locus linked to it, in the etiology of essential HT. There is, moreover, nothing in our data to indicate an abnormality in glucocorticoid function. Our findings of mild gender-specific associations with HT could reflect the effect(s) of an(other) condition(s) prevalent in the patients studied. These findings could also reflect the involvement of different gene(s) in this region for each respective gender and require further validation in other larger groups of HTs, as well as in conditions such as obesity and diabetes.

Note Added in Proof
Since submission of our work, studies of a general white population in Melbourne, Australia, have confirmed an absence of linkage of the GRL D5S207 polymorphism with variation in blood pressure.⁵⁰ Moreover, D5S207 allele frequencies in that study (0.01, 0.06, 0.10, 0.49, 0.34, 0.004, respectively) were similar to what we found (S. Takami and S.B. Harrap, personal communication, 1999).

View this table: [in this window] [in a new window]   Table 2A. Continued

	Acknowledgments

 
This study was supported by a grant from the National Health and Medical Research Council of Australia.

Received June 11, 1999; first decision July 6, 1999; accepted August 9, 1999.

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