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Functional Analysis of Two Recurrent Amino Acid Substitutions in the CYP21 Gene from Italian Patients with Congenital Adrenal Hyperplasia

Department of Pediatrics, University of Bologna and S. Orsola-Malpighi Hospital (M.B., L.B., A.B., P.P., A.C., E.C.), 40139 Bologna, Italy; and Department of Molecular Medicine (S.L., A.W.), Center for Molecular Medicine, Karolinska Hospital, 17176 Stockholm, Sweden

Address all correspondence and requests for reprints to: Michela Barbaro, Department of Molecular Medicine, Center for Molecular Medicine, Karolinska Hospital, 171 76 Stockholm, Sweden. E-mail: michela.barbaro{at}cmm.ki.se; or Prof. Alessandro Cicognani, Department of Pediatrics, Via Massarenti 11, 40138 Bologna, Italy. E-mail: cicogna{at}alma.unibo.it.

Congenital adrenal hyperplasia (CAH) is most commonly due to 21-hydroxylase deficiency and presents a wide spectrum of clinical manifestations from a severe classical form to a milder late-onset form with a variable severity of hyperandrogenic symptoms. A limited number of mutations account for the majority of the mutated alleles, but additional rare mutations are responsible for the symptoms in some patients. By CYP21 gene analysis, we identified a chimeric CYP21P/CYP21 gene with the fusion breakpoint downstream of the common P30L mutation as well as a GCC to ACC change at codon 15 (A15T) in two subjects with classical CAH and a CCC to TCC change at codon 482 (P482S) in seven subjects referred for nonclassical CAH, precocious pubarche, menstrual irregularities, or hypertrichosis.

The two amino acid substitutions were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells, and enzyme activity toward the two natural substrates (17-hydroxyprogesterone and progesterone) was determined. The A15T mutant exhibited no significant difference in activity compared with the wild-type protein, whereas the P482S mutation reduced enzyme activity to 70% of normal. This impairment of activity was confirmed in vivo by detection of heterozygote carriers by the ACTH test.

This work was supported in part by the Alma Mater Studiorum-University of Bologna (Progetto Marco Polo) and Pharmacia-Italy (to M.B.); the Society for Child Care, the Frimurare Barnhuset, and the Ronald McDonald Child Fund (to S.L.); the Swedish Research Council (Grant 12198); and the Novo Nordisk Foundation (to A.W.).

Abbreviations: 4-A, 4-Androstenedione; BA, bone age; BL, birth length; BW, birth weight; CAH, congenital adrenal hyperplasia; HCASD, Height for Chronological Age Standard Deviation Score; NC-CAH, nonclassical CAH; 17OHP, 17-hydroxyprogesterone; P450c21, 21-hydroxylase; SV-CAH, simple-virilizing CAH; V_max, maximum velocity.

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