Introduction: UDP N‐acetylglucosamine2‐epimerase/N‐acetylmannosamine‐kinase (GNE) gene mutations can cause mostly autosomal‐recessive myopathy with juvenile‐onset known as hereditary inclusion‐body myopathy (HIBM). Methods: We describe a family of a patient showing an unusual HIBM with both vacuolar myopathy and myositis without quadriceps‐sparing, hindering diagnosis. We show how genetic testing with functional assays, clinical transcriptome sequencing (RNA‐seq) in particular, helped facilitate both the diagnosis and a better understanding of the genotype‐phenotype relationship. Results: We identified a novel 7.08 kb pathogenic deletion upstream of GNE using array comparative genomic hybridization (aCGH) and a common Val727Met variant. Using RNA‐seq, we found only monoallelic (Val727Met‐allele) expression, leading to ~50% GNE reduction in muscle. Importantly, α‐dystroglycan is hypoglycosylated in the patient muscle, suggesting HIBM could be a “dystroglycanopathy.” Conclusions: Our study shows the importance of considering aCGH for GNE‐myopathies, and the potential of RNA‐seq for faster, definitive molecular diagnosis of unusual myopathies. Muscle Nerve, 2019

Background Aberrant splicing of individual genes is a well-known mechanism promoting pathology for a wide range of conditions, but disease is less commonly attributed to global disruption of exon usage. To explore the possible association of aberrant splicing with inflammatory bowel disease, we developed a pipeline for quantifying transcript abundance and exon inclusion transcriptome-wide and applied it to a dataset of ileal and rectal biopsies, both obtained in duplicate from 34 pediatric or young adult cases of ulcerative colitis and Crohn’s disease. Results Expression and splicing covary to some extent, and eight individuals exhibited aberrant profiles that can be explained by altered ratios of epithelial to stromal and immune cells. Ancestry-related biases in alternative splicing accounting for 5% of the variance were also observed, in part also related to cell-type proportions. In addition, two individuals were identified who had 284 exons with significantly divergent percent spliced in exons, including in the established IBD risk gene CEACAM1, which caused their ileal samples to resemble the rectum. Conclusions These results imply that quantitative differences in splice usage contribute to the pathology of inflammatory bowel disease in a previously unrecognized manner.

Despite increased use of whole exome sequencing (WES) for the clinical analysis of rare disease, overall diagnostic yield for most disorders hovers around 30%. Previous studies of mRNA have succeeded in increasing diagnoses for clearly defined disorders of monogenic inheritance. We asked if targeted RNA sequencing could provide similar benefits for primary immunodeficiencies (PIDs) and very early-onset inflammatory bowel disease (VEOIBD), both of which are difficult to diagnose due to high heterogeneity and variable severity. We performed targeted RNA sequencing of a panel of 260 immune-related genes for a cohort of 13 patients (seven suspected PID cases and six VEOIBD) and analyzed variants, splicing, and exon usage. Exonic variants were identified in seven cases, some of which had been previously prioritized by exome sequencing. For four cases, allele specific expression or lack thereof provided additional insights into possible disease mechanisms. In addition, we identified five instances of aberrant splicing associated with four variants. Three of these variants had been previously classified as benign in ClinVar based on population frequency. Digenic or oligogenic inheritance is suggested for at least two patients. In addition to validating the use of targeted RNA sequencing, our results show that rare disease research will benefit from incorporating contributing genetic factors into the diagnostic approach.

No abstract

Purpose50-60% of neuromuscular-disease patients remain undiagnosed even after extensive genetic testing that hinders precision-medicine/clinical-trial-enrollment. Importantly, those with DNA-based molecular diagnosis often remain without known molecular mechanism driving different degrees of disease severity that hinders patient stratification and trial-readiness. These are due to: a) clinical-genetic-heterogeneity (eg: limb-girdle-muscular-dystrophies(LGMDs)>30-subtypes); b) high-prevalence of variants-of-uncertain-significance (VUSs); (c) unresolved genotype-phenotype-correlations for patient stratification, and (d) lack of minimally-invasive biomarker-driven-assays. We therefore implemented a combinatorial phenotype-driven blood-biomarker functional-genomics approach to enhance diagnostics and trial-readiness by elucidating disease mechanisms of a neuromuscular-disease patient-cohort clinically-suspected of Dysferlinopathy/related-LGMD, the second-most-prevalent LGMD in the US.MethodsWe used CD14+monocyte protein-expression-assay on 364 Dysferlinopathy/related-LGMD-suspected patient-cohort without complete molecular-diagnosis or genotype-phenotype correlation; and then combined with blood-based targeted-transcriptome-sequencing (RNA-Seq) with tiered-analytical-algorithm correlating with clinical-measurements for a subset of patients.ResultsOur combinatorial-approach significantly increased the diagnostic-yield from 25% (N=326; 18%-27%; 95%CI) to 82% (N=38; 69.08% to 84.92%; 95% CI) by combining monocyte-assay with enhanced-RNA-Seq-analysis and clinical-correlation, following ACMG-AMP-guidelines. The tiered-analytical-approach detected aberrant-splicing, allele-expression-imbalance, nonsense-mediated-decay, and compound-heterozygosity without parental/offspring-DNA-testing, leading to VUS-reclassifications, identification of variant-pathomechanisms, and enhanced genotype-phenotype resolution including those with carrier-range Dysferlin-protein-expression and milder-symptoms, allowing patient-stratification for better trial-readiness. We identified uniform-distribution of pathogenic-variants across DYSF-gene-domains without any hotspot suggesting the relevance of upcoming gene-(full-DYSF-cDNA)-therapy trials.ConclusionOur results show the relevance of using a clinically-driven multi-tiered-approach utilizing a minimally-invasive biomarker-functional-genomic platform for precision-medicine-diagnostics, trial-recruitment/monitoring, elucidating pathogenic-mechanisms for patient stratification to enhance better trial outcomes, which in turn, will guide more rational use of current-therapeutics and development of novel-interventions for neuromuscular-disorders, and applicable to other genetic-disorders.

No abstract

For inherited diseases, obtaining a definitive diagnosis is critical for proper disease management, family planning, and participation in clinical trials. This can be challenging for dysferlinopathy due to the significant clinical overlap between the 30+ subtypes of limb–girdle muscular dystrophy (LGMD) and the large number of variants of unknown significance (VUSs) that are identified in the dysferlin gene, DYSF. We performed targeted RNA-Seq using a custom gene-panel in 77 individuals with a clinical/genetic suspicion of dysferlinopathy and evaluated all 111 identified DYSF variants according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. This evaluation identified 11 novel DYSF variants and allowed for the classification of 87 DYSF variants as pathogenic/likely pathogenic, 8 likely benign, while 16 variants remained VUSs. By the end of the study, 60 of the 77 cases had a definitive diagnosis of dysferlinopathy, which was a 47% increase in diagnostic yield over the rate at study onset. This data shows the ability of RNA-Seq to assist in variant pathogenicity classification and diagnosis of dysferlinopathy and is, therefore, a type of analysis that should be considered when DNA-based genetic analysis is not sufficient to provide a definitive diagnosis.