Clinical utility of RNA sequencing to resolve unusual GNE myopathy with a novel promoter deletion

Chakravorty, Samya; Berger, Kiera; Arafat, Dalia; Nallamilli, Babi Ramesh Reddy; Subramanian, Hari Prasanna; Joseph, Soumya; Anderson, Mary E.; Campbell, Kevin P.; Glass, Jonathan D.; Gibson, Greg; Hegde, Madhuri

doi:10.1002/mus.26486

Cited by 14 publications

(15 citation statements)

References 28 publications

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“…To date, >250 genes are associated with various inherited neuromuscular disorders (NMDs) ( 1 – 4 ) comprising a broad heterogeneous group of genetic myopathies. However, significant numbers of affected patients remain without a definitive molecular diagnosis due to novel gene or multiple gene associations with disease ( 5 , 6 ).…”

Section: Introductionmentioning

confidence: 99%

Clinical and Genomic Evaluation of 207 Genetic Myopathies in the Indian Subcontinent

et al. 2020

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Objective: Inherited myopathies comprise more than 200 different individually rare disease-subtypes, but when combined together they have a high prevalence of 1 in 6,000 individuals across the world. Our goal was to determine for the first time the clinical-and gene-variant spectrum of genetic myopathies in a substantial cohort study of the Indian subcontinent. Methods: In this cohort study, we performed the first large clinical exome sequencing (ES) study with phenotype correlation on 207 clinically well-characterized inherited myopathy-suspected patients from the Indian subcontinent with diverse ethnicities. Results: Clinical-correlation driven definitive molecular diagnosis was established in 49% (101 cases; 95% CI, 42-56%) of patients with the major contributing pathogenicity in either of three genes, GNE (28%; GNE-myopathy), DYSF (25%; Dysferlinopathy), and CAPN3 (19%; Calpainopathy). We identified 65 variant alleles comprising 37 unique variants in these three major genes. Seventy-eight percent of the DYSF patients were homozygous for the detected pathogenic variant, suggesting the need for carrier-testing for autosomal-recessive disorders like Dysferlinopathy that are common in India. We describe the observed clinical spectrum of myopathies including uncommon and rare subtypes in India:

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Section: Introductionmentioning

confidence: 99%

Clinical and Genomic Evaluation of 207 Genetic Myopathies in the Indian Subcontinent

et al. 2020

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“…The current diagnostic pathway for Dysferlinopathy typically only assesses Dysferlin protein expression and DYSF DNA-sequencing [35], but here we show that by using a tiered analytical approach we can tease apart all possible aberrations at RNA-transcript structural-, exonic-, allelic-, gene-, and protein-expression levels that will lead to not only a higher diagnostic yield but also important help enable patient stratification by connecting molecular underpinnings to clinical severities. One limitation of this study is that the prior DNA-testing were done non-uniformly (Exome/Gene-Panel/Sanger) based on respective physician discretion, outcome measurements, as well as novel therapy discoveries [10,24].…”

Section: Discussionmentioning

confidence: 99%

“…We reasoned that whole-blood targeted-RNA-Seq of 274 NMD-associated genes expressed in skeletal muscle (Table S1) [24] with high read-depth in a tiered-analysis (Tiers1/2/3; DYSF isoform-expression differences between blood and muscle are unlikely to hinder analysis since alternate exons are in-frame and well-expressed in blood except for exon 17 (Table 3 of [25]). 51 Dysferlinopathy-suspected patients who provided consents underwent RNA-Seq, among whom 38 patients' %DYSF and/or DNA-sequencing were unable to provide a complete molecular diagnosis and in 13 patients the pathomechanism of the identified DYSF-variants were unclear.…”

Section: Combinatorial Biomarker Approach Enhances Genotype-phenotypementioning

confidence: 99%

Combinatorial clinically driven blood biomarker functional genomics significantly enhances genotype-phenotype resolution and diagnostics in neuromuscular disease

Chakravorty

Berger

Rufibach

et al. 2021

Preprint

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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.

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“…Recently, Chakravorty et al reported that a patient with a deletion in the promoter of one allele GNE and having a single mutation in the other allele showed GNE myopathy phenotype 6 . This shows that heterozygous knockout or non-functional one allele may lead to GNE Myopathy.…”

Section: Characterization Of Gne Exon-3 Knockout Cell Line (Skm-gnehz)mentioning

confidence: 99%

“…Both homozygous and compound heterozygous mutations have been reported in patients with predominantly M743T in Persian Jews, A207V in Japanese, I618T in Roma Gypsies and V727M in Indian subcontinent [1][2][3][4][5] . A patient with deletion in promoter region of one allele and single mutation in other allele has also been reported with GNE Myopathy 6 . Though it is a slowly progressive disease, progressivity also depends on the nature of biallelic mutation 7 .…”

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Generation and Characterization of Skeletal Muscle Cell based Model for GNE Myopathy: Implications in Actin Dynamics

Devi¹,

Yadav²,

Mashangva³

et al. 2020

Preprint

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UDP-N-acetyl glucosamine-2 epimerase/ N-acetyl mannosamine kinase (GNE) catalyzes key enzymatic reactions in the biosynthesis of sialic acid. Mutation in GNE gene causes GNE-Myopathy characterized by adult onset muscle weakness and degeneration. GNE is involved in different cellular processes like adhesion, apoptosis, ER stress and autophagy. Lack of appropriate model system limits drug and treatment options for GNE Myopathy as GNE knock out was found to be embryonically lethal. In the present study, we have generated L6 rat skeletal muscle cell-based model system for GNE Myopathy where GNE gene is knocked out at exon 3 using AAV mediated SEPT homology recombination. The cell line was heterozygous for GNE gene with one wild type and one truncated allele as confirmed by sequencing. The phenotype showed reduced GNE epimerase activity with little reduction in sialic acid content. In addition, the GNE knock out cell line revealed altered cytoskeletal organization with disrupted actin filament. The signaling cascade regulating actin dynamics such as Rho A, Cofilin, FAK and Src were altered leading to reduced cell migration in GNE heterozygous cells. Our study indicates possible role of GNE in regulating actin dynamics and cell migration of skeletal muscle cell. The skeletal muscle cell based system offers great potential in understanding pathomechanism and target identification for GNE Myopathy.

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Clinical utility of RNA sequencing to resolve unusual GNE myopathy with a novel promoter deletion

Cited by 14 publications

References 28 publications

Clinical and Genomic Evaluation of 207 Genetic Myopathies in the Indian Subcontinent

Clinical and Genomic Evaluation of 207 Genetic Myopathies in the Indian Subcontinent

Combinatorial clinically driven blood biomarker functional genomics significantly enhances genotype-phenotype resolution and diagnostics in neuromuscular disease

Generation and Characterization of Skeletal Muscle Cell based Model for GNE Myopathy: Implications in Actin Dynamics

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