Comprehensive mutation analysis of the CYP21A2 gene: an efficient multistep approach to the molecular diagnosis of congenital adrenal hyperplasia

Abstract

Congenital adrenal hyperplasia, due to 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder of adrenal steroidogenesis caused by mutations in the CYP21A2 gene. Direct comparison of established and novel methodologies of CYP21A2 genetic analysis in a large cohort representing a wide range of genotypes has not been previously reported. We genotyped a cohort of 129 unrelated patients with 21-OHD, along with 145 available parents, using Southern blot (SB) analysis, multiplex ligation-dependent probe amplification (MLPA), PCR-based restriction fragment length polymorphism (RFLP) analysis, multiplex minisequencing and conversion-specific PCR, duplication-specific amplification, and DNA sequencing. CYP21A2 genotyping identified four duplicated CYP21A2 genes (1.53%) and 79 chimeric CYP21A1P/CYP21A2 genes (30.15%). Parental SB data were essential for determining the CYP21 haplotype in three cases, whereas PCR-based RFLP analysis was necessary for MLPA results to be accurately interpreted in the majority of cases. The comparison of different methods in detecting deletion and duplication showed that MLPA with PCR-based RFLP was comparable with SB analysis, with parental data of 100% sensitivity and specificity. DNA sequencing was required for the identification of 16 (6.1%) rare point mutations and determination of clinically significant chimera junction sites. MLPA with PCR-based RFLP analysis is an excellent substitute for SB analysis in detecting CYP21A2 deletion and duplication and a combination of MLPA, PCR-based RFLP, duplication-specific amplification, and DNA sequencing is a convenient and comprehensive strategy for mutation analysis of the CYP21A2 gene in patients with 21-OHD.

Figure 1

Southern blot analysis of RCCX module in two representative patients and their respective parents. A: Schematic of a typical RCCX bimodule allele. Dashed boxes denote pseudogenes; short vertical lines, TaqI restriction sites; numbers, fragment sizes (in kb). B and C: TaqI and PshAI Southern blots. Enzyme digested fragments with size (in kb) correspond to target gene(s). D: Densitometric ratios were measured in Southern blots. The ratios indicate gene dosages, which can be used to infer gene arrangement. Columns 1 and 1F represent patient 1 and the father, respectively; columns 2, 2M, and 2F represent patient 2, the mother, and the father, respectively.

Figure 2

MLPA, PCR-based RFLP, and PCR-based duplication analysis of two representative patients and their respective parents. A: Scheme shows MLPA probes and PCR primers. Dashed boxes indicate pseudogenes; white arrows, primers used to amplify duplicated CYP21A2 gene; black arrows, PCR-based RFLP primers; short vertical lines, Taq^αI restriction site of CYP779f/Tena32F amplified products, with numbers showing the fragment size (in kb); vertical arrows, hybridization sites of the MLPA probes. ex, exon; int, intron; UTR, untranslated region. B and C: Normalized MLPA data of families of patient 1 and patient 2 with probes on x axis. Columns correspond to normalized electropherogram peak areas calculated using Coffalyser software. Diamonds denote results of point mutations; circles denote results of chimeric genes. Ratio values (y axis) between 0.7 and 1.3 (dashed lines) indicate two copies, bottom dashed lines (one copy) and top dashed lines (two copies). 1 and 1F represent patient 1 and the father, respectively. 2, 2M, and 2F represent patient 2, the mother, and the father, respectively. D: Electropherogram of Taq^αI digested CYP779f/Tena32F amplified products. Target fragments are labeled with gene names and size. −, CYP21A2 deletion on one allele; +, no deletion; M, 1 kb DNA ladder; C1–C3, controls with two, one, and no CYP21A2 deletions, respectively. The 3.2 kb band for the CYP21A1P gene was not detected in samples 1F, 2, 2M, and 2F, which indicated no CYP21A2 deletion; sample 1 shows one 3.2 kb band for CYP21A1P gene, confirming CYP21A2 deletion. E: Electropherogram of products of duplication-specific amplification using primer 21BF1 and XA; no amplification is observed without CYP21A2 duplication. A positive 5.6 kb band for samples 1 and 1F indicates CYP21A2 duplication. The faint bands shown in samples 2, 2M, 2F, and C3 result from unspecific amplification. F: Genotyping results of patient 1 and 2. The following 21A2, 21A1P, XA, and XB stand for CYP21A2, CYP21A1P, TNXA, and TNXB, respectively. CYP21A2 genes identified with point mutations are set bold and underlined.

Figure 3

Molecular analysis strategy of the CYP21A2 gene in CAH patients. The target of this strategy is to identify mutations in deleted, duplicated, and normal CYP21A2 genes. Final genotyping result of each patient is given in the bottom row, including chimera type of CYP21A1P/CYP21A2 genes and point mutations in normal and duplicated CYP21A2 genes. Rectangles denote the methods; ovals, results from the upstream methods; diamonds, results of point mutations; circles, results of chimeric genes. The junction site of each chimeric gene was identified based on data from MLPA (dashed lines) and sequencing CYP779f/TenaF amplicon.