Proband Nanopore Long-Read Genome Sequencing Facilitates Preimplantation Genetic Testing for Facioscapulohumeral...

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array at 4q35. Genetic diagnosis is particularly difficult in somatic mosaic cases, where conventional short-read sequencing cannot directly resolve pathogenic haplotypes. We aimed to establish a strategy using Nanopore ultra-long-read sequencing for accurate haplotype resolution and to demonstrate its clinical application in preimplantation genetic testing for monogenic disease (PGT-M).

A male patient with somatic mosaic FSHD1 underwent Nanopore ultra-long-read sequencing to directly span the D4Z4 repeat array, identify contraction events, and distinguish normal and pathogenic haplotypes. Embryos derived from the patient were subsequently screened using this approach combined with short-read sequencing and Sanger sequencing to determine carrier status. Finally, Bionano optical genome mapping (OGM) was performed for prenatal validation.

Nanopore sequencing revealed 3 haplotypes with 4, 16, and 33 D4Z4 repeat units on 4qA, consistent with somatic mosaicism. Ultra-long reads enabled direct identification of a pathogenic 4-unit haplotype and precise haplotype phasing despite limited downstream SNPs. Methylation profiling confirmed hypomethylation of the pathogenic allele. Of 8 biopsied embryos, 5 were definitively classified as unaffected, 2 showed upstream recombination within a 224-kb region, and 1 carried the pathogenic haplotype. Integration of Nanopore, short-read sequencing, and Sanger sequencing resolved uncertain cases and reduced misclassification risk. An unaffected embryo was transferred, leading to the birth of a healthy infant. Prenatal OGM confirmed 17 and 33 D4Z4 repeat units without contraction, concordant with preimplantation results.

Nanopore ultra-long-read sequencing provides accurate detection of D4Z4 repeat contractions and haplotype resolution in somatic mosaic FSHD1, overcoming the limitations of conventional short-read sequencing. Applied to PGT-M, this approach ensured reliable embryo diagnosis and successful prevention of disease transmission. These findings support the broader potential of ultra-long-read sequencing for genetic testing and diagnosis of neurologic disorders involving complex repeat-mediated loci.