Long Read genomic and epigenomic profiling enhances timely comprehensive variant discovery in hypotonia and muscle weakness

Background Identifying the genetic basis of hypotonia and muscle weakness is critical for patient management and family counseling. However, diagnosis is often hindered by diverse genomic alterations, including repeat expansions, structural variants, and methylation defects across multiple loci. Standard-of-care testing, largely based on short-read sequencing, is limited in its ability to detect this heterogeneous genomic and epigenomic landscape, leaving many patients undiagnosed or requiring lengthy sequential testing. Methods We retrospectively analyzed 227 patients with hypotonia to assess diagnostic yield, time-to-diagnosis, and costs associated with standard-of-care testing. A long-read whole-genome sequencing (LR-WGS) workflow was developed to detect, filter, and prioritize pathogenic single-nucleotide variants, structural and copy number variants, repeat expansions, and targeted methylation changes at key disease loci. The workflow was validated in a reference-positive cohort with known diagnoses (n = 15) and applied to an unsolved cohort with unexplained hypotonia (n = 14). Variant interpretation followed ACMG guidelines and was confirmed with orthogonal methods. Results Standard-of-care testing achieved a diagnostic yield of 42% with an average time-to-diagnosis of 68.7 days, but 30% of diagnosed patients experienced significant delays (average 169 days) due to sequential testing across 19 technology combinations. LR-WGS (~ 30× coverage, N50 11.1 kb) identified all known pathogenic variants in the positive cohort, including SMN1 deletions, methylation defects at 15q11.2/Prader-Willi locus, FMR1 repeat expansions, and sequence and copy number variants in > 100 genes underlying myopathies and muscular dystrophies. The LR-WGS pipeline reduced genome-wide variant calls prioritized for interpretation by 97.9–99.9% and, in the unsolved cohort, yielded one definitive diagnosis ( de novo COL6A3 deletion) and one possible diagnosis (aberrant methylation and copy number at POMK ), for an additional 14% yield. Among patients diagnosed only after sequential testing (n = 29), LR-WGS is expected to reduce time-to-diagnosis by 85% and decrease cumulative diagnostic delays by 10.37–11.5 years, with projected healthcare cost savings of $396,000–439,000. Across the 227-patient cohort, LR-WGS is anticipated to reduce testing costs by 36% yielding an average savings of $611 per patient. Conclusions LR-WGS enables timely and comprehensive discovery of genomic and epigenomic variants in hypotonia and muscle weakness, improving diagnostic yield, shortening diagnostic timelines, and reducing costs compared with current standard-of-care testing.