Regular Article

CMO I Deficiency Caused by a Point Mutation in Exon 8 of the HumanCYP11B2Gene Encoding Steroid 18-Hydroxylase (P450_C18)

Humans have two isozymes with 11β-hydroxylase activity that are respectively required for cortisol and aldosterone synthesis. CYP11B1 (11β-hydroxylase) converts 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone, is expressed at high levels, and is regulated by ACTH. CYP11B2 (aldosterone synthase) is normally expressed at low levels and is regulated mainly by angiotensin II and potassium levels. The latter enzyme also has 18-hydroxylase and 18-oxidase activities and thus can synthesize aldosterone from deoxycorticosterone. Mutations in the CYP11B1 gene cause steroid 11β-hydroxylase deficiency, a form of congenital adrenal hyperplasia. Mutations in CYP11B2 result in aldosterone synthase deficiency, which can cause hyponatremia, hyperkalemia, and hypovolemia in infancy. These are both recessive disorders. Unequal crossing over between the CYP11B genes can generate a duplicated chimeric gene, causing glucocorticoid-suppressible hyperaldosteronism, an autosomal dominant form of hypertension. Frequent polymorphisms in these genes can affect aldosterone secretion and risk of hypertension.

Aldosterone synthase deficiency (ASD) is a rare autosomal recessive disorder characterized by severe hyperkalemia, salt loss, vomiting, severe dehydration and failure to thrive. ASD is a life-threatening electrolyte imbalance in infants resulting from mutations in CYP11B2. We described ASD in a Chinese male infant with vomiting, poor feeding and failure to thrive. He was mildly dehydrated, with a weight of 6 kg (−3.45 SDS) and length of 67 cm (−3.10 SDS). Laboratory tests showed hyponatremia (119 mmol/L), serum potassium 5.4 mmol/L, low plasma aldosterone and plasma renin activity (PRA) levels. Next-generation sequencing of his DNA revealed compound heterozygous mutations in CYP11B2, a known variant c.1391_1393delTGC (p.Leu464del, rs776404064) and a novel variant c.1294delA (p.Arg432Glyfs*37). The HEK-293T expression system was used to investigate the variants, demonstrating negligible aldosterone synthesis compared with WT CYP11B2. The patient started fludrocortisone and subsequently gained 3.2 kg of weight and normalized serum sodium (137 mmol/L). We further reviewed reported cases of ASD, summarizing clinical features and CYP11B2 mutations; missense and nonsense mutations are most frequent. Fludrocortisone treatment is essential for ASD, and the need for mineralocorticoid replacement wanes with age; eventually, therapy can be discontinued for many affected children. Our study broadens the ASD phenotypic spectrum and shows the efficiency of next-generation sequencing for patients with atypical clinical manifestations.

Humans have two isozymes with 11β-hydroxylase activity that are respectively required for cortisol and aldosterone synthesis. CYP11B1 (11β-hydroxylase) converts 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone, is expressed at high levels and is regulated by ACTH. CYP11B2 (aldosterone synthase) is normally expressed at low levels and is regulated mainly by angiotensin II and potassium levels. The latter enzyme also has 18-hydroxylase and 18-oxidase activities and thus can synthesize aldosterone from deoxycorticosterone. Mutations in the CYP11B1 gene cause steroid 11β-hydroxylase deficiency, a form of congenital adrenal hyperplasia. Mutations in CYP11B2 result in aldosterone synthase deficiency, which can cause hyponatremia, hyperkalemia and hypovolemia in infancy. These are both recessive disorders. Unequal crossing over between the CYP11B genes can generate a duplicated chimeric gene, causing glucocorticoid-suppressible hyperaldosteronism, an autosomal dominant form of hypertension. Frequent polymorphisms in these genes can affect aldosterone secretion and risk of hypertension.

The aldosterone synthase, CYP11B2, catalyses the conversion of 11-deoxycorticosterone to aldosterone, a process that requires three steps: a hydroxylation at position 11β to form corticosterone, another one at position 18 to produce 18-hydroxycorticosterone, and, finally, an oxidation at position 18 to form aldosterone. Aldosterone synthase deficiency usually finds its expression in infancy as a life-threatening electrolyte imbalance, caused by mutations in the CYP11B2 gene. Therefore, in depth studies of mutations and their enzymatic activities will provide information for the diagnosis and management of hypoaldosteronism caused by CYP11B2 deficiencies. Here, we report the development of a fast and cheap whole-cell technology for the enzymatic characterisation of CYP11B2 mutations. The principle of the new system is the heterologous expression of the mutants of CYP11B2 in fission yeast (Schizosaccharomyces pombe) followed by steroid bioconversion assays for the enzymatic characterisation of the investigated mutants.

The new system was validated and 10 known mutations of CYP11B2 have been investigated, two of them for the first time concerning their effect on the CYP11B2 three-step reaction. The results of the fission yeast system were in good agreement with the cell culture results presenting this new system as an alternative non radioactive method that can be applied for the enzymatic characterisation of CYP11B2 mutations.