Comprehensive genetic analysis of 182 unrelated families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Abstract

Background: Genetic analysis is commonly performed in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency.

Study objective: The objective of the study was to describe comprehensive CYP21A2 mutation analysis in a large cohort of CAH patients.

Methods: Targeted CYP21A2 mutation analysis was performed in 213 patients and 232 parents from 182 unrelated families. Complete exons of CYP21A2 were sequenced in patients in whom positive mutations were not identified by targeted mutation analysis. Copy number variation and deletions were determined using Southern blot analysis and PCR methods. Genotype was correlated with phenotype.

Results: In our heterogeneous U.S. cohort, targeted CYP21A2 mutation analysis did not identify mutations on one allele in 19 probands (10.4%). Sequencing identified six novel mutations (p.Gln262fs, IVS8+1G>A, IVS9-1G>A, p.R408H, p.Gly424fs, p.R426P) and nine previously reported rare mutations. The majority of patients (79%) were compound heterozygotes and 69% of nonclassic (NC) patients were compound heterozygous for a classic and a NC mutation. Duplicated CYP21A2 haplotypes, de novo mutations and uniparental disomy were present in 2.7% of probands and 1.9 and 0.9% of patients from informative families, respectively. Genotype accurately predicted phenotype in 90.5, 85.1, and 97.8% of patients with salt-wasting, simple virilizing, and NC mutations, respectively.

Conclusions: Extensive genetic analysis beyond targeted CYP21A2 mutational detection is often required to accurately determine genotype in patients with CAH due to the high frequency of complex genetic variation.

Figure 1

Schematic diagram of RCCX modules and representative Southern blots and PCR results for detecting CYP21A2 duplications and unusual haplotypes. Active genes are shown in gray. Sizes and locations of the restriction fragments are annotated with arrows. C4 genes can be long (C4L) or short (C4S). The schematic is shown for C4L only. The size of sequence in the dashed frame is 30 kb. The schematic diagram shows a common bimodular RCCX (A); an uncommon bimodular RCCX with an additional copy of CYP21A2 gene linked to the pseudogene XA, and PCR amplification using primer pairs P3S/2R and P3S/3R are positive for duplicated CYP21A2 allele but negative for common RCCX modules (7) (B); a monomodular RCCX with 21A2/21A1P fusion gene resulting from a 30-kb deletion, in which junction sites can vary, and an 8515-bp PCR product amplified by primers CYP779f/Tena32F was digested by TaqI to detect fusion genes (20) (C); RCCX haplotype was demonstrated by TaqI restriction fragment length polymorphism (RFLP; upper panel) and confirmed by PshAI RFLP for the RP gene (lower panel). For the C4 gene, the TaqI digest for C4L form results in fragment sizes of 7.0 and 6.0 kb, and C4S results in fragments of 6.4 and 5.4 kb. Focusing on the CYP21 genes, patient 1 demonstrates equal signal densities for CYP21A2 and CYP21A1P. Patient 2 carries a CYP21A2/CYP21A1P fusion gene (30 kb deletion), in which the CYP21A2 to CYP21A1P ratio is reduced. Patient 3 has three copies of CYP21A2, shown by an increased CYP21A2 to CYP21A1P ratio. The CYP21A2 duplication allele was inherited from patient 4 (the father) (D). E, Two pairs of primers specifically amplify the CYP21A2 duplication allele. Patients 1 and 2 are negative for the duplication, whereas patient 3 and subject 4 (the father) are positive. F, TaqI digestion patterns of 8515-bp PCR products for detecting large gene deletion that can result in TNXB/XA or CYP21A2/CYP21A1P fusion genes. Subject 0 is a patient who was previously reported to carry a gene deletion involving both CYP21A2 and TNXB genes that was demonstrated by four bands (54). Subjects 1–3 are all carriers of a gene deletion involving only CYP21A2 that was shown by two bands for CYP21 and a single band for TNXB. Subject 4 is negative for gene deletion. 21A2, Active gene CYP21A2; 21A1P, pseudogene CYP21A1P; RP1, serine/threonine nuclear kinase gene; RP2, a gene fragment of RP1; TNXB, extracellular matrix protein tenascin-X gene; TNXA (or XA), truncated tenascin pseudogene.