Linkage Analysis of Familial Hyperaldosteronism Type II--Absence of Linkage to the Gene Encoding the Angiotensin II Receptor Type 1

Recently, Davies et al. (1) reported that somatic mutations of the coding region of the angiotensin II type 1 receptor (AT1) gene were not found in 17 aldosterone producing adenomas (APA) studied by single strand conformation polymorphism (SSCP) and direct sequencing, although other genetic defects of the 5′ and 3′ regulatory regions of the gene were not excluded by this study. A previous study showed no abnormalities, on SSCP analysis of the coding region of the AT1 gene, in angiotensin II responsive and angiotensin II unresponsive APAs (2). Recently, we have performed linkage analysis with a polymorphism located within 15kb of the AT1 gene in a 29-member family that included 7 individuals affected with familial hyperaldosteronism type II (FH II). FH II is characterized by primary hyperaldosteronism, dominant transmission, nonsuppressibility of aldosterone by dexamethasone, and absence of the hybrid CYP11B1/CYP11B2 gene known to be the cause of glucocorticoid remediable hyperaldosteronism (or FH I) (3, 4). Angiotensin II stimulates aldosterone production through activation of the G-protein coupled 7 transmembrane domain receptor, AT1. Like Davies et al., we hypothesized that a germ line mutation causing constitutive activation of the AT1 receptor could be the cause of FH II. Genomic DNA from 29 family members, including all 7 affected individuals, was extracted from blood leukocytes, and PCR amplification of a marker containing a dinucleotide (CA) repeat polymorphism present in a 0.9 kb EcoRI fragment approximately 15 kb downstream from the 3′ end of the coding sequence of the AT1 gene was performed (5). Linkage analysis for this marker revealed an LOD score of −2.24 at a recombination fraction of 0.01 (Table 1), excluding this gene as a cause of FH II in this family. Figure 1demonstrates an informative recombination, where affected individuals 2 and 3 do not share an allele for this gene. Since a LOD score of less than −2 was achieved with this (Table 1) and other markers nearby (data not shown), mutations of AT1 do not appear to be the cause of familial hyperaldosteronism type II. A segment of the 29 member pedigree with familial hyperaldosteronism type II. Genomic DNA was extracted from peripheral blood leukocytes and PCR amplification of a CA repeat polymorphic marker close to the angiotensin II receptor type 1 was performed, one primer was radiolabelled (γ-32P) with T4 kinase. The PCR products were electrophoresed on a 6% polyacrylamide denaturing gel, the dried gel was exposed to X-ray film for 12–16 h. Individual 2 (alleles a/b) and individual 3 (alleles c/c) have the disease but do not share an allele for this marker for the AT1 gene. Individual 4 is of uncertain affectation status. The affected mother has the blc genotype. A segment of the 29 member pedigree with familial hyperaldosteronism type II. Genomic DNA was extracted from peripheral blood leukocytes and PCR amplification of a CA repeat polymorphic marker close to the angiotensin II receptor type 1 was performed, one primer was radiolabelled (γ-32P) with T4 kinase. The PCR products were electrophoresed on a 6% polyacrylamide denaturing gel, the dried gel was exposed to X-ray film for 12–16 h. Individual 2 (alleles a/b) and individual 3 (alleles c/c) have the disease but do not share an allele for this marker for the AT1 gene. Individual 4 is of uncertain affectation status. The affected mother has the blc genotype. LOD scores at different recombination fractions (θ) for a microsatellite marker for the angiotensin II receptor type 1 gene analyzed in a family with seven members affected with familial hyperaldosteronism type II LOD scores at different recombination fractions (θ) for a microsatellite marker for the angiotensin II receptor type 1 gene analyzed in a family with seven members affected with familial hyperaldosteronism type II