Direct detection of F8 and F9 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 hemophilia patients, namely those involving int22h-related inversions disrupting the F8 gene, poses additional challenges due to 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 F8 and F9 genes. A haplotype-based approach coupled with targeted MPS was applied to deduce fetal genotype by capturing a 7.6-Mb region spanning the F8 gene in carriers with int22h-related inversions. The ddPCR analysis correctly determined fetal hemophilia status in fifteen at-risk pregnancies in samples obtained from 8 to 42 weeks of gestation. There were three unclassified samples but no misclassification. Detailed fetal haplotype maps of the F8 gene region involving int22h-related inversions obtained through targeted MPS enabled correct diagnoses of fetal mutational status in three hemophilia families. Our data suggest that it is feasible to apply targeted MPS to interrogate maternally inherited F8 int22h-related inversions, while ddPCR represents an affordable approach for identification of F8 and F9 sequence variants in maternal plasma. These advancements may bring benefits for the pregnancy management for carriers of hemophilia sequence variants, in particular the common F8 int22h-related inversions, associated with the most severe clinical phenotype.
Irena Hudecova, Peiyong Jiang, Joanna Davies, Y. M. Dennis Lo, Rezan A. Kadir and Rossa W.K. Chiu
Blood 2017 :blood-2016-12-755017; doi: https://doi.org/10.1182/blood-2016-12-755017