P804: Verification of a software and amplification-based nanopore sequencing solution to characterize complex variants in 11 challenging, high-frequency carrier genes*

treatment of CAH to mitigate virilization in affected females; however, the safety of prenatal DEX administration remains controversial.This study aims to develop the prenatal cell-free DNA screening for CAH at early gestational weeks, enabling selective prenatal DEX administration to maximize the benefits for high-risk CAH pregnancies.Methods: This study prospectively recruited twenty high-risk core families for CAH, in which both parents were confirmed heterozygotes of CYP21A2 gene mutations.CAH-specific targeted hybrid capture probes were designed for this study.Genomic DNA (gDNA) from family members and cell-free fetal DNA (cffDNA) from maternal plasma were extracted.The hybrid capture probes were used to selectively enrich high-frequency single nucleotide variant (SNV) loci within 1 Mb upstream and downstream of the CYP21A2 gene, followed by high-throughput sequencing.Haplotype phasing of affected individuals was achieved through family-based linkage analysis.Fetal haplotype inheritance was inferred using relative haplotype dosage (RHDO) combined with the Bayes factor (BF) model, which accurately determined the fetal genotype.The accuracy of prenatal cfDNA screening results was validated through invasive prenatal diagnosis.For families where prenatal cfDNA screening identified the fetus as a female CAH patient, prenatal DEX treatment was recommended as an option, which was applied until delivery.Postnatal follow-up was conducted to observe the degree of virilization of the external genitalia.Results: This study included twenty high-risk pregnant persons with a median gestational age of 8 +1 (range: 7 +4 -12 +3 ).The initial pass rate of prenatal cfDNA screening was 90% (18/20), the reporting rate was 95% (19/20), and the accuracy was 100% (19/19).In family F9, the first test yielded a "nocall" result for maternal haplotype inheritance due to a low fetal fraction.Resampling of blood was performed one week later, providing a definitive diagnosis.For family F11, a limited number of informative SNVs resulted in the "nocall" for maternal prenatal cfDNA screening result, and chorionic villus sampling conducted two weeks later diagnosed the fetus as affected.Ultimately, prenatal cfDNA screening successfully identified one female-affected fetus, four male-affected fetuses, nine heterozygotes, and five normal fetuses.The fetus in F5 was diagnosed as a female CAH patient at a gestational age of 8 +3 weeks.DEX treatment was initiated upon reporting and continued until delivery.Postnatal follow-up revealed no significant virilization of the external genitalia in the newborn.Therefore, the precise intrauterine treatment avoided 88.9% (8/9) of overtreatment of female fetuses.Conclusion: Prenatal cfDNA screening is an early, safe, and accurate method that not only reduces the risks of invasive sampling but also enables the prenatal diagnosis of CAH as early as 7 weeks of gestation.This approach facilitates the identification of female CAH-affected fetuses, allowing for precise intrauterine treatment, thereby minimizing the risks of widespread DEX use and providing valuable guidance for clinical management.