Expanding the genetic landscape of DSD: deep intronic <em>CYB5A</em> variant in 46,XY gonadal dysgenesis — ASN Events
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  3. Expanding the genetic landscape of DSD: deep intronic CYB5A variant in 46,XY gonadal dysgenesis

Expanding the genetic landscape of DSD: deep intronic CYB5A variant in 46,XY gonadal dysgenesis (#316)

Shirin Moradi Fard 1 2 , Thanh Nha Uyen Le 1 , Vu Chi Dung 3 , Vincent Harley 1
  1. Hudson Institute, Clayton, VICTORIA, Australia
  2. Department of Molecular and Translational Science, Monash University, Melbourne, Victoria, Australia
  3. National Children Hospital, Hanoi, Vietnam

Aims: Differences of sex development (DSD) affect around 1% of live births and arise from disruptions in chromosomal, gonadal, or genital development. Many individuals experience long and inconclusive diagnostic journeys, and the diagnostic yield for 46,XY DSD remains low at about 50%. Whole‑genome sequencing (WGS) can detect both coding and non‑coding variants, offering a more comprehensive approach and improving the likelihood of identifying previously missed pathogenic changes. 

Methods: We performed WGS on a family with two siblings presenting with 46,XY DSD, bilateral inguinal cryptorchidism, low testosterone and estradiol to identify potential variants. Patients were recruited from the National Children’s Hospital in Vietnam. DNA was extracted and sequencing data were processed using an in‑house bioinformatics pipeline. Variant calling, annotation, and prioritization were conducted to identify rare or novel variants potentially associated with DSD. To assess the functional impact of a deep intronic variant, we performed a minigene assay in HEK293T cells to evaluate its effect on splicing.

Results: WGS analysis identified compound‑heterozygous variants in the CYB5A gene: a paternally inherited missense variant (p.Val34Glu) and a maternally inherited deep intronic deletion (c.129+862_129+863del). Both siblings carried both variants. The missense variant lies within a highly conserved region and is predicted to be likely pathogenic by AlphaMissense. Minigene assays demonstrated that the intronic deletion creates cryptic splice sites, leading to inclusion of a pseudoexon and resulting in a premature stop codon.

Conclusion: CYB5A is an essential co‑factor for the steroidogenic enzyme CYP17A1 and is required for its 17,20‑lyase activity. Loss of this activity leads to reduced testosterone and estradiol, consistent with the hormone profile observed in our patients. Two novel CYB5A variants established the genetic diagnosis in both siblings, expanding the spectrum of pathogenic CYB5A‑related DSD.This work highlights the value of WGS and functional assays in identifying clinically relevant non‑coding variants in DSD.