CYP21A2 , the causative gene for 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH), is molecularly challenging to analyse due to high sequence homology with its pseudogene. PathWest’s current testing strategy is to perform multiplex ligation-dependent probe amplification (MLPA; MRC Holland P050-D1) for deletions, duplications and gene conversion, and Sanger sequencing of the functional gene. Sequencing of the promoter region is performed when there is a reduction of the MLPA 307nt probe (-113 wild-type SNP). A local case worked up for non-classic CAH (biochemical carrier) was referred for molecular testing of CYP21A2. No parental samples were available. Discrepant results were heterozygous variants on MLPA wild-type probes for exon 3 I2G but homozygous wild-type sequence variants from genomic sequence data. Promoter region sequencing showed the following pathogenic promoter haplotype variants: c.-210T>C, c.-199C>T, c.-190dup, c.-126C>T, c.-113G>A, c.-110T>C, c.-103A>G. Interrogation of the long-range PCR primers for the functional gene showed the c.-199C>T variant is at the last base which the forward primer (5′-CCTTGCTTCTTGATGGGT*G*A*T*C-3′) anneals, likely leading to allele dropout (* = modified thiophosphate). These findings suggest the gene conversion event of the promoter region between the functional and pseudogene and the formation of a gene hybrid not amplified on sequencing explaining the apparent wild-type homozygosity on long-range PCR.
Estrella et al. (Sun,) studied this question.