Case Report: unmasking pseudo-homozygosity in CYP21A2: intergenerational gene conversion expansion and the necessity...

CYP21A2 gene mutations causing Congenital Adrenal Hyperplasia (CAH) exhibit high allelic complexity. Rare meiotic events, such as de novo expansion of deletion boundaries, can obscure true genotypes, leading to 'pseudo-homozygosity' and diagnostic pitfalls. This study characterizes a rare intergenerational expansion of a maternal CYP21A2 gene conversion and demonstrates the critical role of multimodal genetic testing in resolving complex inheritance paradoxes during prenatal counseling. A family with classical salt-wasting CAH, involving parents who are second cousins, was investigated. Initial screening suggested p.Ile173Asn homozygosity in their third child (designated as V-3 in the pedigree chart), who exhibited severe birth virilization and was initially assigned male but later reassigned female. In-house Multiplex Ligation-dependent Probe Amplification (MLPA) and Sanger sequencing using two independent unique primer sets were integrated to assess copy number variations and resolve parental discrepancies. Maternal probe analysis revealed a heterozygous Exon 1-3 gene conversion, leaving the p.Ile173Asn locus intact on her wild-type allele. However, this conversion meiotically expanded to Exons 1-4 in the affected child (V-3). This expansion physically eliminated the maternal wild-type primer template, causing PCR allele dropout and a false pseudo-homozygous Sanger pattern, which biologically corresponds to true functional hemizygosity in vivo. The father's suspected deletion was ruled out as probe-binding interference from his heterozygous p.Ile173Asn mutation. Guided by these resolved genotypes, early prenatal dexamethasone management and subsequent twelfth-week Chorionic Villus Sampling successfully confirmed a normal wild-type female fetus (46,XX), resulting in a healthy term delivery. In conclusion, the CYP21A2 locus is highly dynamic, and integrating dosage-sensitive assays with unique primers is mandatory for accurate genetic counseling and successful prenatal management in high-risk pregnancies.