Long-read sequencing: An effective method for genetic analysis of CYP21A2 variation in congenital adrenal hyperplasia

The sequence similarity between CYP21A2 gene and its inactive pseudogene CYP21A1P, and copy number variation (CNV) caused by unequal crossover, make it challenging to characterize the CYP21A2 gene through traditional methods. This study aimed to evaluate the clinical utility of the long-read sequencing (LRS) method in carrier screening and genetic diagnosis of congenital adrenal hyperplasia (CAH) by comparing the efficiency of the LRS method with the conventional multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing approaches in CYP21A2 analysis. In a retrospective study, full sequence analysis of the CYP21A2 and CYP21A1P was performed for three pedigrees through long-range locus-specific PCR followed by LRS based on the Pacific Biosciences (PacBio, California, USA) single-molecule real-time (SMRT) platform, and the results were compared with those obtained from next-generation sequencing (NGS)-based whole exome sequencing (WES) and the traditional methods of MLPA plus Sanger sequencing. The LRS method successfully identified seven CYP21A2 variants, including three single nucleotide variants (NM_000500.9:c.1451G > C p.(Arg484Pro), c.293-13A/C > G (IVS2-13A/C > G), c.518 T > A p.(Ile173Asn)), one 111-bp polynucleotide insertion, one set of 3′URT variants (NM_000500.9:c.*368 T > C, c.*390A > G, c.*440C > T, c.*443 T > C) and two types of chimeric genes and straightforwardly depicted the inheritance patterns of these variants within families. Moreover, the LRS method enabled us to determine the cis–trans configuration of multiple variants in one assay, without the need to analyze additional family samples. Compared with traditional methods, this LRS method can achieve a precise, comprehensive and intuitive result in the genetic diagnosis of 21-hydroxylase deficiency (21-OHD). The LRS method is comprehensive in CYP21A2 analysis and intuitive in result presentation, which holds substantial promise in clinical application as a crucial tool for carrier screening and genetic diagnosis of CAH.