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Objective: Limb‐girdle muscular dystrophies (LGMDs), one of the most heterogeneous neuromuscular disorders (NMDs), involves predominantly proximal‐muscle weakness with >30 genes associated with different subtypes. The clinical‐genetic overlap among subtypes and with other NMDs complicate disease‐subtype identification lengthening diagnostic process, increases overall costs hindering treatment/clinical‐trial recruitment. Currently seven LGMD clinical trials are active but still no gene‐therapy‐related treatment is available. Till‐date no nation‐wide large‐scale LGMD sequencing program was performed. Our objectives were to understand LGMD genetic basis, different subtypes' relative prevalence across US and investigate underlying disease mechanisms. Methods: A total of 4656 patients with clinically suspected‐LGMD across US were recruited to conduct next‐generation sequencing (NGS)‐based gene‐panel testing during June‐2015 to June‐2017 in CLIA‐CAP‐certified Emory‐Genetics‐Laboratory. Thirty‐five LGMD‐subtypes‐associated or LGMD‐like other NMD‐associated genes were investigated. Main outcomes were diagnostic yield, gene‐variant spectrum, and LGMD subtypes' prevalence in a large US LGMD‐suspected population. Results: Molecular diagnosis was established in 27% (1259 cases; 95% CI, 26–29%) of the patients with major contributing genes to LGMD phenotypes being: CAPN3(17%), DYSF(16%), FKRP(9%) and ANO5(7%). We observed an increased prevalence of genetically confirmed late‐onset Pompe disease, DNAJB6‐associated LGMD subtype1E and CAPN3‐associated autosomal‐dominant LGMDs. Interestingly, we identified a high prevalence of patients with pathogenic variants in more than one LGMD gene suggesting possible synergistic heterozygosity/digenic/multigenic contribution to disease presentation/progression that needs consideration as a part of diagnostic modality. Interpretation: Overall, this study has improved our understanding of the relative prevalence of different LGMD subtypes, their respective genetic etiology, and the changing paradigm of their inheritance modes and novel mechanisms that will allow for improved timely treatment, management, and enrolment of molecularly diagnosed individuals in clinical trials. [ABSTRACT FROM AUTHOR]

Wagner syndrome and erosive vitreoretinopathy together constitute the phenotypic continuum of an autosomal dominant vitreoretinopathy, with clinical findings typically isolated to the eye. The disease is caused by pathogenic variants in the VCAN gene and all such variants reported to date are those that plausibly result in haploinsufficiency of exon 8 containing vcan transcripts. Here, we report the molecular findings and long‐term follow‐up of a 16‐year‐old female with a history of retinal detachments and pigmentary retinal changes. Next‐generation sequencing and microarray analysis of 141 genes established a diagnosis of Wagner syndrome in this individual, by detection of an 11.7 kilobase (kb) deletion encompassing exon 8 of VCAN. In light of the emerging functions and roles of versican protein in human disease, we discuss how variants within exon 8 of the VCAN gene can be compared to those in exon 2 of the COL2A1 gene that cause atypical Stickler syndrome and propose that variants in other regions of the gene can be expected to present with a more systemic disease. The distinctive facial features and atypical gastrointestinal symptoms observed in this long‐term follow‐up study support the possibility that individuals with VCAN‐related vitreoretinopathy may have extra‐ocular clinical features. [ABSTRACT FROM AUTHOR]

Introduction: Nemaline myopathy (NM) is a congenital neuromuscular disorder often characterized by hypotonia, facial weakness, skeletal muscle weakness, and the presence of rods on muscle biopsy. A rare form of nemaline myopathy known as Amish Nemaline Myopathy has only been seen in a genetically isolated cohort of Old Order Amish patients who may additionally present with tremors in the first 2-3 months of life.Methods: We describe an Hispanic male diagnosed with nemaline myopathy histopathologically and subsequently confirmed by next generation gene sequencing.Results: Direct sequencing revealed that he is homozygous for a pathogenic nonsense variant c.323C>G (p.S108X) in exon 9 of the TNNT1 gene.Conclusions: This report describes a novel pathogenic variant in the TNNT1 gene and represents a nemaline myopathy-causing variant in the TNNT1 gene outside of the Old Order Amish and Dutch ancestry. [ABSTRACT FROM AUTHOR]

Objective Neuromuscular diseases (NMDs) are a group of >200 highly genetically as well as clinically heterogeneous inherited genetic disorders that affect the peripheral nervous and muscular systems, resulting in gross motor disability. The clinical and genetic heterogeneities of NMDs make disease diagnosis complicated and expensive, often involving multiple tests. Methods To expedite the molecular diagnosis of NMDs, we designed and validated several next generation sequencing (NGS)-based comprehensive gene panel tests that include complementary deletion and duplication testing through comparative genomic hybridization arrays. Our validation established the targeted gene panel test to have 100% sensitivity and specificity for single nucleotide variant detection. To compare the clinical diagnostic yields of single gene (NMD-associated) tests with the various NMD NGS panel tests, we analyzed data from all clinical tests performed at the Emory Genetics Laboratory from October 2009 through May 2014. We further compared the clinical utility of the targeted NGS panel test with that of exome sequencing (ES). Results We found that NMD comprehensive panel testing has a 3-fold greater diagnostic yield (46%) than single gene testing (15-19%). Sanger fill-in of low-coverage exons, copy number variation analysis, and thorough in-house validation of the assay all complement panel testing and allow the detection of all types of causative pathogenic variants, some of which (about 18%) may be missed by ES. Interpretation Our results strongly indicate that for molecular diagnosis of heterogeneous disorders such as NMDs, targeted panel testing has the highest clinical yield and should therefore be the preferred first-tier approach. Ann Neurol 2015;77:206-214 [ABSTRACT FROM AUTHOR]

Introduction: Dysferlin deficiency causes dysferlinopathies. Among peripheral blood mononuclear cells (PBMCs), the dysferlin protein is expressed specifically in CD14+ monocytes. Methods: We quantified dysferlin protein levels in PBMC lysates of 77 individuals suspected clinically of having a dysferlinopathy to screen for true positives. Subsequent molecular confirmation was done by Sanger sequencing and comparative genomic hybridization arrays to establish diagnosis. Results: Of the 44 individuals who had significantly reduced dysferlin levels (⩽10%), 41 underwent molecular testing. We identified at least 1 mutation in 85% (35 of 41), and 2 mutations, establishing a dysferlinopathy diagnosis, in 61% (25 of 41) of these individuals. Among those with dysferlin protein levels of >10% (33 of 77), only 1 individual (of 14 who underwent molecular testing) had a detectable mutation. Conclusions: Our results suggest that dysferlin protein levels of ⩽10% in PBMCs, are highly indicative of primary dysferlinopathies. However, this assay may not distinguish carriers from those with secondary dysferlin reduction. [ABSTRACT FROM AUTHOR]

ABSTRACT Introduction Clinical heterogeneity of limb-girdle muscular dystrophies ( LGMDs, 24 known subtypes), which includes overlapping phenotypes and varying ages of onset and morbidity, adds complexity to clinical and molecular diagnoses. Methods To diagnose LGMD subtype, protein analysis, using immunohistochemistry ( IHC) and immunoblotting, was followed by gene sequencing through a panel approach (simultaneous sequencing of known LGMD genes) in 9 patients from unrelated families of the Indian Agarwal community. Haplotype studies were performed by targeted SNP genotyping to establish mutation segregation. Results We identified 2 founder mutations in CAPN3, a missense (c.2338G>C; p. D780H) and a splice-site (c.2099-1G>T) mutation, on 2 different haplotype backgrounds. The patients were either heterozygous for both or homozygous for either of these mutations. Conclusions Founder mutations have immediate clinical application, at least in selected population groups. Clinically available gene panels may provide a definitive molecular diagnosis for heterogeneous disorders such as LGMD. Muscle Nerve 47: 931-937, 2013 [ABSTRACT FROM AUTHOR]