Reclassification of Duplications as Benign: Recommendations for Cautious Interpretation of Variants Identified in Prenatal Screening.

Duplications are the main type of dystrophin gene () variants, which typically cause dystrophinopathies such as Duchenne muscular dystrophy and Becker muscular dystrophy. Maternally inherited exon duplication in in fetuses is a relatively common finding of genetic screening in clinical practice. However, there is no standard strategy for interpretation of the pathogenicity of duplications during prenatal screening, especially for male fetuses, in which maternally inherited pathogenic variants more frequently cause dystrophinopathies. Here, we report three non-contiguous duplications identified in a woman and her male fetus during prenatal screening. Multiplex ligation probe amplification and long-read sequencing were performed on the woman and her family members to verify the presence of duplications. Structural rearrangements in the gene were mapped by long-read sequencing, and the breakpoint junction sequences were validated using Sanger sequencing. The woman and her father carried three non-contiguous duplications. Long-read and Sanger sequencing revealed that the woman's father carried an intact copy and a complex structural rearrangement of the gene. Therefore, we reclassified these three non-contiguous duplications, one of which is listed as pathogenic, as benign. We postulate that breakpoint analysis should be performed on identified duplication variants, and the pathogenicity of the duplications found during prenatal screening should be interpreted cautiously for clinical prediction and genetic/reproductive counseling.