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A Novel Semiquantitative Polymerase Chain Reaction/Enzyme Digestion-Based Method for Detection of Large Scale Deletions/Conversions of the CYP21 Gene and Mutation Screening in Turkish Families with 21-Hydroxylase Deficiency

Department of Pediatrics, Divisions of Medical Genetics (T.T.) and Pediatric Endocrinology (F.B., H.G.), Istanbul Medical Faculty, and Divisions of Medical Genetics (O.U., M.Y.-A., H.K., B.W.) and Auxology (N.S.), Child Health Institute, Istanbul University, 34390 Istanbul, Turkey; and Department of Pediatrics, Weill Medical College, Cornell University (J.Q.W., D.X.S., R.C.W., M.I.N.), New York, New York 10029

Address all correspondence and requests for reprints to: Dr. Turgut Tukel, Department of Human Genetics, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1498, Room 14-52, New York, New York 10029. E-mail: turgut.tukel{at}mssm.edu. Or to: Dr. Bernd Wollnik, Institute of Child Health, Division of Medical Genetics, Istanbul University, Millet Cad., Capa, 34390 Istanbul, Turkey. E-mail: .

21-Hydroxylase deficiency is a recessively inherited disorder resulting from mutations in the CYP21 gene. The CYP21 gene is located along with the CYP21P pseudogene in the human leukocyte antigen major histocompatibility complex region on chromosome 6. Molecular diagnosis is difficult due to the 98% similarity of CYP21 and CYP21P genes and the fact that almost all frequently reported mutations reside on the pseudogene. Allele-specific PCR for the 8 most frequently reported point mutations was performed in 31 Turkish families with at least a single 21-hydroxylase-deficient individual. The allele frequencies of the point mutations were as follows: P30L, 0%; IVS2 (AS,A/C-G,-13), 22.5%; G1108nt, 3.2%; I172N, 11.4%; exon 6 cluster (I236N, V237E, M239K), 3.2%; V281L, 0%; Q318X, 8%; and R356W, 9.6%. Large deletions and gene conversions were detected by Southern blot analysis, and the allele frequencies were 9.6% and 22.5%, respectively. Sequence analysis of the gene, performed on patients with only 1 mutated allele, revealed 2 missense mutations (R339H and P435S). A novel semiquantitative PCR/enzyme digestion-based method for the detection of large scale deletions/conversions of the gene was developed for routine diagnostic purposes, and its accuracy was shown by comparison with the results of Southern blot analysis.

This work was supported by the Research Fund of Istanbul University (Project 1246/181298).

Abbreviations: CAH, Congenital adrenal hyperplasia; d, deoxy; NC, nonclassical; SV, simple virilizing; SW, salt-wasting.

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