Characterization of novel CAPN3 isoforms in white blood cells: an alternative approach for limb-girdle muscular dystrophy 2A diagnosis

Blázquez, Lorea; Azpitarte, Margarita; Sáenz, Amets; Goicoechea, Maria; Otaegui, David; Ferrer, Xavier; Illa, Isabel; Gutiérrez-Rivas, Eduardo; Vilchez, Juan J.; Munáin, Adolfo López de

doi:10.1007/s10048-008-0129-1

Cited by 34 publications

(35 citation statements)

References 29 publications

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“…16 Our study supported the alternative approach of performing the molecular diagnosis by using mRNA from peripheral blood as p94-calpain isoform was found to be expressed in human circulating peripheral blood mononuclear cells. 37 Unexpectedly, we also observed the existence of an alternatively spliced form of CAPN3 mRNA with a missing region spanning from exon 1 to exon 7.…”

Section: Intronic Alterations Affect Splicing Of Capn3 Genesupporting

confidence: 73%

“…15 Many CAPN3 intronic variants have been identified during diagnostic screening: they account for about 15% of all variants listed on the Leiden Database, and for about 25% of the mutations reported in other studies. [15][16][17] For the majority of intronic variants the consequences on mRNA splicing have been only inferred by in-silico analysis, whereas experimental demonstration of their pathogenicity has been obtained by mRNA studies for only 1% of them (Leiden Database). [15][16][17][18] Although deep intronic sequences were originally believed to be non-functional, because they do not code for proteins, it has been suggested that some of these sequences do indeed have relevance.…”

Section: Introductionmentioning

confidence: 99%

“…[15][16][17] For the majority of intronic variants the consequences on mRNA splicing have been only inferred by in-silico analysis, whereas experimental demonstration of their pathogenicity has been obtained by mRNA studies for only 1% of them (Leiden Database). [15][16][17][18] Although deep intronic sequences were originally believed to be non-functional, because they do not code for proteins, it has been suggested that some of these sequences do indeed have relevance. 19 In most cases, deep intronic disease-causing variations or rearrangements could affect gene splicing directly by disruption of pre-existing intronic splicing-regulatory elements.…”

Section: Introductionmentioning

confidence: 99%

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CAPN3 mRNA processing alteration caused by splicing mutation associated with novel genomic rearrangement of Alu elements

et al. 2011

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Recessive mutations of CAPN3 gene are reported to be responsible for limb girdle muscular dystrophy type 2A (LGMD2A). In all, 15-25% of intronic nucleotide changes identified in this gene were investigated by in silico analysis, but occasionally supported by experimental data or reported in some cases as a polymorphism. We report here genetic and transcriptional analyses in three Tunisian patients belonging to the same consanguineous family sharing the same mutation c.1194-9 A4G and Alu repeats insertion in intron 7 of CAPN3 gene. Reverse transcriptase-PCR experiments performed on total RNA from the patient's muscle biopsy showed retention of the eight last nucleotides of intron 9 in the CAPN3 transcript lacking the first seven exons. Our results provide evidence regarding the potential involvement of Alu elements in aberrant processing of pre-mRNA owing to the disruption of pre-existing intronic splicing regulatory elements. We also demonstrated variable mRNA alternative splicing among tissues and between LGMD2A patients. A deep intronic variation and rearrangement have been reported in the literature as causing genetic diseases in humans. However, this is the first report on a potential pathogenic CAPN3 gene mutation resulting from an Alu insertion.

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Section: Intronic Alterations Affect Splicing Of Capn3 Genesupporting

confidence: 73%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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CAPN3 mRNA processing alteration caused by splicing mutation associated with novel genomic rearrangement of Alu elements

et al. 2011

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“…Mutations that involve the expression of pseudoexons have been identified in other genes associated with muscular dystrophy including dystrophin35–37 and calpain 3 38,39. However, in contrast with the present DYSF mutation, these generally lead to frameshifts and premature stop codons that prevent protein expression.…”

Section: Discussionmentioning

confidence: 84%

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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“…All carried pathogenic mutations in CAPN3 (table 3). 28 29 33 In these four patients, western blots for calpain 3 protein in muscle were normal, and muscle biopsies were characterised by mild dystrophic features.…”

Section: Resultsmentioning

confidence: 89%

Patients with a phenotype consistent with facioscapulohumeral muscular dystrophy display genetic and epigenetic heterogeneity

et al. 2011

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Objective To identify the genetic and epigenetic defects in patients presenting with a facioscapulohumeral (FSHD) clinical phenotype without D4Z4 contractions on chromosome 4q35 tested by linear gel electrophoresis (LGE) and Southern blot analysis. Design and patients We studied 16 patients displaying an FSHD-like phenotype, with normal cardiovascular and respiratory function, a myopathic pattern on EMG, and a muscle biopsy being normal or displaying only mild and a specific dystrophic changes. We sequenced the genes calpain 3 (CAPN3), valosin containing protein (VCP) and four and a half LIM domains protein 1 (FHL1) and we analyzed the D4Z4 repeat arrays by extensive genotyping and DNA methylation analysis. Results We identified one patient carrying a complex rearrangement in the FSHD locus that masked the D4Z4 contraction associated with FSHD1 in standard genetic testing, one patient with somatic mosaicism for the D4Z4 4q35 contraction, six patients that were diagnosed with FSHD2, four patients with CAPN3 mutations, two patients with a VCP mutation, No mutations were detected in FHL1, and in two patients we could not identify the genetic defect. Conclusions In patients presenting an FSHD-like clinical phenotype with a negative molecular testing for FSHD consider: 1) detailed genetic testing including D4Z4 contraction of permissive hybrid D4Z4 repeat arrays, p13E-11 probe deletions, D4Z4 hypomethylation in absence of repeat contraction as observed in FSHD2, 2) mutations in CAPN3 even in the absence of protein deficiency on western blot analysis 3) VCP mutations even in the absence cognitive impairment, Paget disease and typical inclusion in muscle biopsy.

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Characterization of novel CAPN3 isoforms in white blood cells: an alternative approach for limb-girdle muscular dystrophy 2A diagnosis

Cited by 34 publications

References 29 publications

CAPN3 mRNA processing alteration caused by splicing mutation associated with novel genomic rearrangement of Alu elements

CAPN3 mRNA processing alteration caused by splicing mutation associated with novel genomic rearrangement of Alu elements

A novel dysferlin mutant pseudoexon bypassed with antisense oligonucleotides

Patients with a phenotype consistent with facioscapulohumeral muscular dystrophy display genetic and epigenetic heterogeneity

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