The Journal of clinical endocrinology and metabolism · 2026
Biallelic CYB5A disruptions in 46, XY DSD: Novel missense and deep intronic variants.
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Moradifard Shirin, Le Thanh Nha Uyen, Ha Nguyen Thu, Dung Vu Chi, Thao Bui Phuong, Harley Vincent
Аннотация
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.