Biallelic CYB5A disruptions in 46, XY DSD: Novel missense and deep intronic variants

Cytochrome b5, encoded by CYB5A, is an essential co-factor for CYP17A1-mediated 17,20-lyase activity. CYB5A deficiency is a rare cause of 46,XY DSD. We report two siblings with 46,XY DSD from a Vietnamese family, presenting with phenotypic female.

Clinical evaluation and hormone profiling were conducted. Whole genome sequencing (WGS) was performed on extracted DNA from both affected siblings and identified variants were classified according to the ACMG/AMP 2015 guidelines. A minigene RNA splicing assay in HEK293T cells was used to evaluate the functional impact of the CYB5A intronic variant.

The two patients' hormone profile showed low testosterone and estradiol. WGS identified novel compound heterozygous CYB5A variants: a paternally inherited missense variant (p.Val34Glu) and a maternally inherited deep intronic deletion (c.129 + 862_129 + 863del) which predicted to create the cryptic splice sites by SpliceAI. Minigene assays confirmed the activation of the cryptic splice sites, resulting in an inclusion of a pseudo-exon between exon 1 and 2. This aberrant insertion introduced a premature stop codon, leading to predicted nonsense-mediated decay of the transcript. Both variants were classified as likely pathogenic according to the ACMG/AMP guideline.

Two novel CYB5A variants established the genetic diagnosis in both siblings, expanding the spectrum of pathogenic CYB5A-related DSD. Our findings show that a deep intronic variant can lead to null protein due to disrupted splicing. These results highlight the value of WGS and functional assays for detecting and validating clinically relevant non-coding variants in DSD.