Diagnostic overview of blood‐based dysferlin protein assay for dysferlinopathies

Ankala, Arunkanth; Nallamilli, Babi Ramesh Reddy; Rufibach, Laura; Hwang, Esther; Hegde, Madhuri

doi:10.1002/mus.24195

Cited by 17 publications

(22 citation statements)

References 30 publications

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“…One other sibling is a nonsymptomatic carrier of this mutation. DYSF protein expression in blood monocytes is absent or negligible in Patients 1 and 2 as expected from previous analyses of dysferlinopathy patients 25. The segregation of all DYSF intron 44i allelic variants in the immediate family members is shown in Table 2.…”

Section: Resultssupporting

confidence: 75%

“…Included in this set was the index family, patients P1 and P2. In a second phase of the study, we were provided DNA samples from eight unrelated MM individuals who were DYSF negative in monocyte assays and for whom at least one of the pathogenic DYSF mutations had not been identified by exon sequencing (some of which previously described25). DNAs from an additional 724 individuals without muscle disease (either clinically normal or with unrelated conditions) were used in these studies.…”

Section: Methodsmentioning

confidence: 99%

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A novel dysferlin mutant pseudoexon bypassed with antisense oligonucleotides

Dominov

Uyan

Sapp

et al. 2014

Ann Clin Transl Neurol

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ObjectiveMutations in dysferlin (DYSF), a Ca2+-sensitive ferlin family protein important for membrane repair, vesicle trafficking, and T-tubule function, cause Miyoshi myopathy, limb-girdle muscular dystrophy type 2B, and distal myopathy. More than 330 pathogenic DYSF mutations have been identified within exons or near exon–intron junctions. In ~17% of patients who lack normal DYSF, only a single disease-causing mutation has been identified. We studied one family with one known mutant allele to identify both the second underlying genetic defect and potential therapeutic approaches.MethodsWe sequenced the full DYSF cDNA and investigated antisense oligonucleotides (AONs) as a tool to modify splicing of the mRNA transcripts in order to process out mutant sequences.ResultsWe identified a novel pseudoexon between exons 44 and 45, (pseudoexon 44.1, PE44.1), which inserts an additional 177 nucleotides into the mRNA and 59 amino acids within the conserved C2F domain of the DYSF protein. Two unrelated dysferlinopathy patients were also found to carry this mutation. Using AONs targeting PE44.1, we blocked the abnormal splicing event, yielding normal, full-length DYSF mRNA, and increased DYSF protein expression.InterpretationThis is the first report of a deep intronic mutation in DYSF that alters mRNA splicing to include a mutant peptide fragment within a key DYSF domain. We report that AON-mediated exon-skipping restores production of normal, full-length DYSF in patients’ cells in vitro, offering hope that this approach will be therapeutic in this genetic context, and providing a foundation for AON therapeutics targeting other pathogenic DYSF alleles.

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Section: Resultssupporting

confidence: 75%

Section: Methodsmentioning

confidence: 99%

A novel dysferlin mutant pseudoexon bypassed with antisense oligonucleotides

Dominov

Uyan

Sapp

et al. 2014

Ann Clin Transl Neurol

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“…Cases suspected of dysferlinopathy, as in LGMD2B or Miyoshi myopathy with DYSF (OMIM# 603009) variant(s), were analyzed for DYSF protein expression using our established blood PBMC assay 25, 29, 30…”

Section: Methodsmentioning

confidence: 99%

Genetic landscape and novel disease mechanisms from a large LGMD cohort of 4656 patients

Nallamilli

Chakravorty

Kesari

et al. 2018

Ann Clin Transl Neurol

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165

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ObjectiveLimb‐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.MethodsA 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.ResultsMolecular 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.InterpretationOverall, 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.

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“…The same investigators also evaluated dysferlin stability in peripheral blood stored at 4°C and determined that dysferlin in whole blood remains stable until about 48 h after blood collection without a 200‐kDa non‐specific protein. Furthermore, it is detectable even at day 6 post‐collection . We did not find the 200‐kDa protein by WB (data not shown); this allowed us to obtain reliable quantitative data of dysferlin expression in healthy subjects.…”

Section: Discussionmentioning

confidence: 80%

“…The majority of patients we described lived locally and, for out‐of‐town patients, samples were processed at their home location when possible. Other samples were transported as whole blood on ice, taking into account the 48‐h stability of dysferlin after blood collection . Our positive controls were patients with previously identified DYSF mutations who agreed to participate in the study.…”

Section: Discussionmentioning

confidence: 99%