Targeted gene panel sequencing for molecular diagnosis of congenital adrenal hyperplasia

Abstract

CONTEXT\nCongenital adrenal hyperplasia (CAH) is a group of autosomal recessive genetic diseases caused by genetic deficiency in nine genes encoding steroidogenesis enzymes and cofactors.\n\n\nOBJECTIVE\nTo establish a targeted next-generation sequencing (NGS) assay for all nine CAH candidate genes.\n\n\nMETHODS\nWe developed a customized targeted NGS assay of CAH candidate genes (CYP21A2, CYP17A1, CYP11B1, StAR, CYP11A1, POR, HSD3B2, H6PD, CYP11B2) and apply this assay plus MLPA of CYP21A2 in a total of 469 patients with CAH like signs and symptoms.\n\n\nRESULTS\nWe totally identified 125 variants with seven variant types in eight genes. Variant types included missense variant (46.8%), splicing variant (21.5%), small indel (12.5%), large structure variation (11.8%), nonsense variant (4.1%), UTR variant (2.9%), synonymous variant (0.3%). Successful genotyping, defined as biallelic pathogenic or likely pathogenic variants, was achieved in 98.5% (336/341) of cases, including biallelic variants in CYP21A2 (n\u2009=\u2009254), CYP17A1 (n\u2009=\u200945), CYP11B1 (n\u2009=\u200923), StAR (n\u2009=\u20097), HSD3B2 (n\u2009=\u20094), POR (n\u2009=\u20091), CYP11A1 (n\u2009=\u20091) and CYP11B2 (n\u2009=\u20091) gene. Importantly, the assay found one patient with CYP11B1 deficiency, one patient with non-classic POR deficiency and two patients with non-classic CYP17A1 deficiency while clinically diagnosed differently.\n\n\nCONCLUSIONS\nOur NGS-based assay plus MLPA of CYP21A2 is a useful tool to genotype all subtypes of CAH. The test successfully achieved genotype in 98.5% of patients with clinically determined CAH. It also efficiently facilitated the diagnosis of CAH in patients with rare subtypes as well as non-classic phenotypes.