Preclinical diagnosis and identification of the chimeric CYP11B1/CYP11B2 gene in two pediatric cases of a Japanese family with glucocorticoid-remediable aldosteronism

Abstract

Glucocorticoid-remediable aldosteronism (GRA), also known as familial aldosteronism type 1, is a form of aldosteronism caused by unequal crossing over between the CYP11B1 gene, which encodes 11β-hydroxylase, and the CYP11B2 gene, which encodes aldosterone synthase (AS). Although GRA accounts for ~0.7% of primary aldosteronism cases worldwide [1], it is extremely rare in Japan, with only five cases in two pedigrees reported to date [2, 3]. GRA is inherited in an autosomal-dominant fashion and causes hypertension at an early age, with a high incidence of cerebrovascular complications at a young age [4]. We previously reported three cases of GRA in a Japanese family [2]. Nearly 20 years later, the proband has two sons. Here, the genetic diagnosis and identification of crossover sites of the chimeric gene in the descendants are described. When the proband was 21 years old, she, along with her sister and mother, was incidentally found to have familial hypertension [2]. Examination of the proband and her sister revealed hypokalemia and a high plasma aldosterone concentration (PAC) with suppressed plasma renin activity (PRA) in addition to adrenocorticotropin (ACTH)dependent changes in PAC and suppression of PAC by dexamethasone. Genetic testing by long-range PCR revealed the CYP11B1/CYP11B2 chimeric gene in these three individuals, leading to the diagnosis of GRA. The proband was initially treated with dexamethasone and subsequently with a calcium-channel blocker. Over 20 years, she successfully delivered two boys while using hydralazine during pregnancy. A few years later, she requested genetic testing for GRA for her sons and visited our department. Both children, a 5-year-old and a 3-yearold, had a normal perinatal and developmental status and normal to slightly elevated blood pressure with normokalemia (4.0, 4.6 mEq/L) in addition to high PAC (348, 367 pg/mL) and reduced PRA (0.1, 0.3 ng/mL/h). Genetic testing for GRA was performed with permission after genetic counseling. To detect the chimeric gene, genomic DNA was extracted from the peripheral blood leukocytes of the proband, her two sons, and a normal control subject and subjected to long-range PCR. In the first reaction, a sense primer for intron 1 (primer A) and an antisense primer for intron 6 (primer B) were used to detect CYP11B2. In the second reaction, a sense primer for intron 1 of CYP11B1 (primer C) and primer B was used to detect the chimeric gene. The protocol was as follows. Touchdown PCR with ten cycles of 98 °C for 10 s, 71–66 °C (lowered by 0.5 °C each cycle) for 30 s and 72 °C for 150 s for the CYP11B2 gene, followed by 25 cycles of 98 °C for 10 s, 66 °C for 30 s and 72 °C for 150 s for the CYP11B1/CYP11B2 chimeric gene and a two-step PCR with 35 cycles of 98 °C for 10 s and 72 °C for 150 s with a high-fidelity DNA polymerase (NEB Japan) were performed. The chimeric gene was detected only in the proband and her sons (Fig. 1); CYP11B2 was detected in the proband, her sons, and the control subject, indicating the two boys to be carriers of the GRA gene. * Fumio Otsuka [email protected]