Genetics

Head‑to‑head long‑ vs short‑read genome sequencing in pediatric neurogenetics

In 100 children and adolescents with neurologic phenotypes, singleton short‑ and long‑read genome sequencing produced identical overall diagnostic yield of 29%. However, long‑read sequencing provided additional clinically relevant information in 13 cases, clarifying 17 variants through phasing, STR sizing, and structural resolution. Long‑read data enabled phasing of SMN1 and autosomal recessive alleles, precise characterization of structural variants, and accurate short tandem repeat determination. Methylation calling from long‑read data detected pathogenic aberrant methylation in the Prader–Willi region and across an FMR1 expansion. The authors propose long‑read genome sequencing as a streamlined first‑line test that reduces follow‑up assays and refines variant interpretation in pediatric neurological disorders. 1

Integrated full‑genome analysis rescues diagnoses after negative standard testing

This study applied an in‑house Full‑Genome Analysis pipeline to 29 patients with unresolved suspected genetic disorders after conventional testing. The integrated approach combined short‑read whole‑genome sequencing, linked‑read sequencing, and optical genome mapping to jointly detect SNVs, CNVs, and complex structural variants. Molecular diagnoses were achieved in 12 of 29 patients (41.4%), including nine pathogenic SNVs, three CNVs, and two complex SVs. Two CNVs were missed by chromosomal microarray, and both complex SVs were undetectable by standard short‑read exome or genome sequencing. The data support comprehensive small‑ and large‑variant detection within a single workflow for difficult‑to‑diagnose rare disease cases. 2

Enhanced RNA‑seq of stimulated blood lymphocytes doubles yield in unsolved rare disease families

This study implemented an optimized RNA‑seq workflow using phytohemagglutinin‑activated peripheral blood cells from 55 families with negative WES/WGS. The pipeline integrated transcript isoform distribution analysis, MANE‑based realignment, and pharmacologic induction to detect cryptic splicing defects. RNA quality was higher and less variable than fresh PBMCs, and the approach improved diagnostic yield 2.2‑fold, from 9% to 20%. Positive rates were highest in families with prior VUS (71%) and suspected‑gene cases (40%), but low in unsolved families (5.2%). Among 11 positive families, 10 achieved definitive diagnoses that directly informed diverse reproductive decisions in preconception counseling. 3

CRISPR excision of NOTCH2NLC GGC expansions reverses NIID phenotypes in models

Investigators developed a CRISPR/spCas9 strategy to precisely excise expanded GGC repeats in the NOTCH2NLC 5′ UTR, the cause of NIID. Guide design and validation avoided detectable off‑target editing in closely related NOTCH2 and NOTCH2NL paralogs, despite high sequence similarity. In human cell lines, NIID patient iPSCs, and a transgenic NIID mouse model, repeat excision ameliorated neuropathology, molecular abnormalities, and behavioral deficits. These results provide preclinical proof‑of‑concept for repeat‑targeted genome editing as a therapeutic approach in NIID and related expansion disorders. 4