Noninvasive detection of F8 int22h-related inversions and sequence variants in maternal plasma of hemophilia carriers

Abstract

Direct detection of <i>F8</i> and <i>F9</i> sequence variants in maternal plasma of hemophilia carriers has been demonstrated by microfluidics digital PCR. Noninvasive prenatal assessment of the most clinically relevant group of sequence variants among patients with hemophilia, namely, those involving <i>int22h</i>-related inversions disrupting the <i>F8</i> gene, poses additional challenges because of its molecular complexity. We investigated the use of droplet digital PCR (ddPCR) and targeted massively parallel sequencing (MPS) for maternal plasma DNA analysis to noninvasively determine fetal mutational status in pregnancies at risk for hemophilia. We designed family-specific ddPCR assays to detect causative sequence variants scattered across the <i>F8</i> and <i>F9</i> genes. A haplotype-based approach coupled with targeted MPS was applied to deduce fetal genotype by capturing a 7.6-Mb region spanning the <i>F8</i> gene in carriers with <i>int22h</i>-related inversions. The ddPCR analysis correctly determined fetal hemophilia status in 15 at-risk pregnancies in samples obtained from 8 to 42 weeks of gestation. There were 3 unclassified samples, but no misclassification. Detailed fetal haplotype maps of the <i>F8</i> gene region involving <i>int22h</i>-related inversions obtained through targeted MPS enabled correct diagnoses of fetal mutational status in 3 hemophilia families. Our data suggest it is feasible to apply targeted MPS to interrogate maternally inherited <i>F8 int22h</i>-related inversions, whereas ddPCR represents an affordable approach for the identification of <i>F8</i> and <i>F9</i> sequence variants in maternal plasma. These advancements may bring benefits for the pregnancy management for carriers of hemophilia sequence variants; in particular, the common <i>F8 int22h</i>-related inversions, associated with the most severe clinical phenotype.