Benchmarking long-read sequencing approaches to resolve facioscapulohumeral dystrophy locus complexity.

Area of Science:

• Genetics
• Molecular Biology
• Epigenetics

Background:

• Facioscapulohumeral dystrophy (FSHD) is linked to D4Z4 macrosatellite array contractions at 4q35.
• FSHD1 involves D4Z4 contraction (1-10 units) and reduced DNA methylation.
• FSHD2 results from SMCHD1 variants causing epigenetic deregulation of 4q35.

Purpose of the Study:

• To analyze complex structural variants at 4q35/10q26 in FSHD patients lacking defined genetic causes.
• To resolve the architecture and methylation patterns of these variants using long-read sequencing.
• To evaluate the diagnostic relevance of these structural variants for atypical FSHD cases.

Main Methods:

• High-resolution long-read sequencing (Oxford Nanopore, PacBio) on 7 representative FSHD cases.
• Detailed structural and epigenetic analyses of 4q35 and 10q26 loci.
• Comparison of long-read sequencing strategies for resolving complex rearrangements.

Main Results:

• Identified intrachromosomal recombination between D4Z4 and subtelomeric elements causing variable deletions.
• Resolved nucleotide-level architecture and methylation patterns of complex structural variants.
• Demonstrated that these variants are undetectable by standard methods like Bionano Optical Genome Mapping.

Conclusions:

• Complex structural variants at 4q35/10q26 are a significant cause of FSHD in patients with atypical genetic profiles.
• In-depth molecular characterization is essential for diagnosing FSHD patients negative for FSHD1/FSHD2.
• Structural variants may be pathogenic in the absence of SMCHD1 variants, aiding diagnosis and genetic counseling.