This study aimed to resolve diagnostic discrepancies in which expanded carrier screening (ECS) in a healthy population suggested hemophilia A carrier status due to F8 intron 22 inversion (Inv22), while further analysis revealed a true distal Xq28 duplication. We evaluated the clinical utility of integrated genetic technologies in distinguishing between these two entities. A healthy pregnant woman underwent ECS, with long-distance polymerase chain reaction (LD-PCR) specifically employed for F8 Inv22 detection. Given the identified heterozygote of F8 inversion, prenatal diagnosis was conducted, integrating LD-PCR, single-nucleotide polymorphism array (SNP array), optical genome mapping (OGM), third-generation long-read sequencing, and X-chromosome inactivation (XCI) analysis to characterize the variant thoroughly. Initial LD-PCR suggested F8 Inv22 in both the fetus and the mother. SNP array revealed a 0.5-Mb Xq28 duplication spanning F8 exons 1-22. OGM and long-read sequencing confirmed the occurrence of duplication while excluding the F8 inversion, revealing int22h-1/int22h-2-mediated nonallelic homologous recombination as the mechanism. XCI analysis revealed skewed inactivation (18.1%) of the duplicated allele in the asymptomatic mother, whereas the fetus exhibited a random XCI pattern. This study highlights a critical diagnostic pitfall: even well-established techniques such as LD-PCR for F8 Inv22 may yield misleading results when applied to general population screening beyond their original diagnostic context. This underscores the importance of accurate interpretation of ECS results through appropriate analytic validation. We propose a tiered strategy: LD-PCR screening followed by OGM or long-read sequencing to differentiate F8 inversions from Xq28 duplications in the general population.
Yang et al. (Wed,) studied this question.