Steven Laurie

Background Neuromuscular disorders (NMDs) affect approximately 1 in 1,000 individuals and are clinically and genetically heterogeneous. Despite advances in genomic diagnostics, many cases remain unsolved after initial sequencing. Bioinformatic reanalysis approaches provide opportunities to identify missed variants and establish novel disease genes. Methods Preexisting next-generation sequencing datasets from 101 undiagnosed NMD families were reanalyzed using the RD-Connect Genome-Phenome Analysis Platform. The data comprised clinical exome sequencing (45 families), whole exome sequencing (31 families), and whole genome sequencing (25 families). Variant prioritization incorporated population frequency, Human Phenotype Ontology terms, in silico predictions, and genotype–phenotype correlation. Results Reanalysis identified causative variants in 17 out of 101 previously unsolved families (16.83% diagnostic yield). Eight cases harbored coding variants in known NMD genes (RYR1, AGRN, SCN4A, TTN, MYH2, GOLGA2) consistent with the observed phenotype. In five cases, intronic variants in known NMD genes (COL6A3, SGCA, DOK7, DYSF, CHRND) were considered causative following in silico predictions and careful correlation with the phenotype. One case had an extended phenotype (PTPN11), and one case had a dual diagnosis (MYH2, KIF21A). A novel ATP2A2 missense variant was identified in two unrelated families, establishing ATP2A2 as a new NMD gene. Conclusion Research-based reanalysis of preexisting NGS data improved diagnostic yield of previously unsolved cases consistent with previous literature, reinforcing the utility of in-depth, phenotype-driven reanalysis with expert review. Our study provided 17 families with unsolved NMDs with a diagnosis after having waited for a decade. This supports the routine reanalysis of unsolved NGS data, highlighting its potential to reclassify variants of unknown significance and reveal novel genes and pathomechanisms in NMDs.

Genetic variants in RNU4-2 , which is transcribed into the U4 small nuclear RNA component of the major spliceosome, were recently shown to cause ReNU syndrome, a prevalent dominant neurodevelopmental disorder (NDD). These variants almost exclusively arise de novo and cluster within 18 nucleotides of RNU4-2 . Here we describe a new recessive NDD associated with homozygous and compound heterozygous variants in RNU4-2 . We identify 38 individuals with biallelic variants outside the 18-nucleotide ReNU syndrome region that cluster within other functionally important elements of U4: Stem II, the k-turn and the Sm protein binding site. We characterize the clinical phenotype in 31 individuals, demonstrating that the recessive disorder is clinically distinct from ReNU syndrome and is associated with distinctive white matter abnormalities, including enlarged perivascular spaces. Finally, we find reduced RNU4-2 transcript levels in individuals with the recessive disorder, suggesting a loss-of-function disease mechanism that is distinct from the mechanism underlying ReNU syndrome. Together, these findings expand the genotypic and phenotypic spectrum of RNU4-2 -associated NDDs.

PURPOSE: Hereditary cancers represent 5%-10% of all cancers, typically characterized by familial aggregation, early onset, and/or multiple primary tumors. Isolated cases with extreme early-onset or multiple unrelated cancers are rare and frequently underdiagnosed. This study aimed to improve genetic diagnostic yield in unresolved patients with strong clinical suspicion of hereditary cancer. Inclusion criteria were (1) ≥4 primary tumors in different organs (or ≥3 if two are rare), (2) adult-type cancers at ≤25 years, or (3) profuse gastrointestinal adenomatous polyposis before age 50 years or profuse polyposis of unknown type before age 30 years. METHODS: Germline DNA from 98 patients selected through ERN-GENTURIS underwent short-read whole-genome sequencing (WGS). Ninety had received negative results from standard genetic testing, whereas eight had not been tested. Variant analysis was performed using the RD-Connect GPAP and Solve-RD frameworks. RESULTS: Pathogenic or likely pathogenic variants in phenotype-related high-penetrance genes were found in 6% of patients, including APC and TP53 mosaicisms and germline variants in TP53 , BAP1 , BARD1 , and MBD4 (homozygous). Three patients carried pathogenic variants in hereditary cancer genes unrelated to their phenotype, and a heterozygous ERCC3 pathogenic variant was detected in a young patient with breast cancer. Suggestive variants of uncertain significance were identified, including a chromosome 3 inversion affecting VHL and MLH1 in a patient with renal and gastric cancers. Potentially regulatory noncoding variants in phenotype-associated genes were observed in 15% of patients (22 variants). CONCLUSION: Comprehensive resequencing of patients suspected of hereditary cancer increased the diagnostic yield by 6%. Detected pathogenic variants involved phenotype-related genes not previously analyzed or missed because of mosaicism. WGS further enables identification of novel gene associations and structural, deep intronic, or regulatory variants.

We report a family affected with childhood onset distal muscle weakness with a heterozygous chromosome 9q34 deletion encompassing the SPTAN1 gene. The deletion was detected through exome-sequencing based copy number variant (CNV) detection, segregates in four patients and is non-penetrant in two other relatives. Electromyography, muscle MRI and muscle biopsy revealed a myopathic disease phenotype. Cellular consequences of the deletion were investigated using qPCR and western blotting on patient-derived fibroblasts, which revealed a reduction of RNA but not protein levels. Immunocytochemistry was performed on muscle tissue which did not reveal reduction of α-II-spectrin. SPTAN1 loss-of-function variants have previously been reported to cause distal hereditary motor neuropathy and recently distal myopathy. Here, we confirm the role of SPTAN1 haploinsufficiency as a cause of distal myopathy. We propose an age-dependent lack of α-II-spectrin and suggest CNV detection in repurposed exome sequencing as an important diagnostic tool.

Despite advances in genomic diagnostics, the majority of individuals with rare diseases remain without a confirmed genetic diagnosis. The rapid emergence of advanced omics technologies, such as long-read genome sequencing, optical genome mapping and multiomic profiling, has improved diagnostic yield but also substantially increased analytical and interpretational complexity. Addressing this complexity requires systematic multidisciplinary collaboration, as recently demonstrated by targeted diagnostic workshops. Here, we highlight the experience of the Solve-RD consortium, a pan-European initiative, in implementing four structured workshops, termed 'Solvathons', as a regular and effective component of its operational workflow. We provide actionable insights, best practices and lessons learned for successful data integration, expert training and scalable collaborative diagnostics within large research consortia.

The major spliceosome contains five small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) essential for splicing. Variants in RNU4-2, encoding U4, cause a neurodevelopmental disorder called ReNU syndrome. We investigated de novo variants in 50 snRNA-encoding genes in a French cohort of 23,649 individuals with rare disorders and gathered additional cases through international collaborations. Altogether, we identified 145 previously unreported probands with (likely) pathogenic variants in RNU4-2 and 21 individuals with de novo and/or recurrent variants in RNU5B-1 and RNU5A-1, encoding U5. Pathogenic variants typically arose de novo on the maternal allele and cluster in regions critical for splicing. RNU4-2 variants mainly localize to two structures, the stem III and T-loop/quasi-pseudoknot, which position the U6 ACAGAGA box for 5′ splice site recognition and associate with different phenotypic severity. RNU4-2 variants result in specific defects in alternative 5′ splice site usage and methylation patterns (episignatures) that correlate with variant location and clinical severity. This study establishes RNU5B-1 as a neurodevelopmental disorder gene, suggests RNU5A-1 as a strong candidate and highlights the role of de novo variants in snRNAs.