Targeted long-read sequencing identifies missing pathogenic variant in unsolved 11β-hydroxylase deficiency

Purpose 11β-hydroxylase deficiency (11β-OHD), caused by homozygosity or compound heterozygosity CYP11B1 mutations, is the second most common cause of congenital adrenal hyperplasia (CAH). Due to the high degree of sequence identity between CYP11B1 and CYP11B2, chimeric genes, and complex structural variants (SVs), the conventional approach to gene testing for 11β-OHD is facing challenges. The study aimed to clarify the underlying genetic causes of two siblings of a Chinese family with 11β-OHD. Methods Peripheral blood samples and clinical information were collected from subjects and their family members. Sex steroid concentrations were measured using LC-MS/MS. Long-range PCR-based next-generation sequencing (NGS), PCR assay and target long-read sequencing were used to detect the pathogenic mutations. Results Early onset hypertension, increased serum levels of adrenocorticotropin (ACTH), progesterone, testosterone, and decreased cortisol and potassium were detected in both affected siblings. Long-range PCR-based NGS identified a heterozygous missense mutation (NM_000497.4:c.281C>T, p.Pro94Leu) in CYP11B1 gene in the two siblings. PCR detected no chimeric CYP11B2 / CYP11B1 gene. We finally identified a second pathogenic variant in CYP11B1 gene via target long-read sequencing (T-LRS). This novel variant was a deletion-insertion mutation and located chr8:143957269-143957579 (hg19) with the insertion of ‘ACAG’ (NM_000497.4:c.954+78_980delinsACAG), which was in trans with CYP11B1 : c.281C>T. Conclusion Our study suggests that the integrated long-range PCR-based NGS and T-LRS seem to be the most reliable and accurate method for 11β-OHD genetic diagnosis and carrier sequencing.