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

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Abstract

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder caused by mutations in the CAPN3 gene. Its definitive diagnosis is laborious, since the clinical phenotype is often similar to other types of muscular dystrophy and since the CAPN3 gene encompasses a large genomic region with more than 300 pathogenic mutations described to date. In fact, it is estimated that nearly 25% of the cases with a phenotype suggestive of LGMD2A do not have mutations in the CAPN3 gene and that, in up to 22% of the cases, only one mutation is identified. In the present work, we have characterised CAPN3 messenger RNA (mRNA) expression in peripheral blood, and we have performed a retrospective diagnostic study with 26 LGMD2A patients, sequencing a transcript of CAPN3 present in white blood cells (WBCs). The 25% of the mutations presented in this paper (7/28) act modifying pre-mRNA splicing of the CAPN3 transcript, including the first deep-intronic mutation described to date in the CAPN3 gene. Our results determine that the sequencing of CAPN3 transcripts present in WBCs could be applied as a new approach for LGMD2A diagnosis. This method improves and simplifies diagnosis, since it combines the advantages of mRNA analysis in a more accessible and rapidly regenerated tissue. However, the lack of exon 15 in the CAPN3 isoforms present in blood, and the presence of mRNA degradation make it necessary to combine mRNA and DNA analyses in some specific cases.

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Acknowledgements

We thank all patients and family members who participated in this study. We are very grateful to Dr. Valcárcel and Dr. Martins de Araujo for their helpful advice and to A. Iribarren, O. Zuriarrain, M. Martínez-Isasi and N. Coll for their technical assistance. We also wish to thank C. Elsden for the editing of the manuscript. This work was supported by the Association Française contre les Myopathies (Ref. 12642), by the Spanish Ministry of Health (FIS PI06/1018) and by the Ilundain Foundation. L. Blázquez is supported by the Department of Education, University and Research of the Basque Government. A. Sáenz and M. Goicoechea are researchers from the Spanish Ministry of Health (FIS) and Ilundain Foundation respectively. D. Otaegui has a contract as a technician from the Spanish Ministry of Health (FIS). All the experiments performed in this work comply with the current Spanish laws.

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Authors and Affiliations

  1. Experimental Unit, Hospital Donostia, San Sebastián, Spain

    L. Blázquez, M. Azpitarte, A. Sáenz, M. Goicoechea & D. Otaegui

  2. Department of Neurology, Hospital Donostia, Dr. Begiristain s/n, San Sebastián, 20014, Spain

    A. López de Munain

  3. Ilundain Foundation, San Sebastián, Spain

    M. Goicoechea & A. López de Munain

  4. Department of Neurology, CHU de Bordeaux, Bordeaux, France

    X. Ferrer

  5. Department of Neurology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

    I. Illa

  6. Department of Neurology, Hospital Universitario 12 de Octubre, Madrid, Spain

    E. Gutierrez-Rivas

  7. Department of Neurology, Hospital La Fe, Valencia, Spain

    J. J. Vilchez

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  1. L. Blázquez
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  2. M. Azpitarte
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  10. A. López de Munain
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Correspondence to L. Blázquez.

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Fig. 1

Pathological effect of splice-site mutations described at cDNA level. A c.632 + 4A>G mutation causes exon 4 skipping at mRNA level. B c.802–9G>A mutation causes the insertion of the last seven nucleotides of intron 5 in mature mRNA. C In patient 14, CAPN3 mRNA allele, which carries c.1524 + 1G>C mutation, is degraded and therefore p.Arg490Gln mutation, in heterozygosis in DNA, is seen as pseudo-homozygous in cDNA. D c.2185–12_2194del mutation causes the retention of intron 20 at mRNA level. E c.1992 + 1G>T mutation causes the retention of the first 31 nucleotides of intron 17. F In patient 26, the CAPN3 mRNA allele, which carries c.309G>A mutation, is degraded and therefore p.Thr139Ile mutation in exon 3 is seen as pseudo-homozygous. (JPG 308 kb)

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Blázquez, L., Azpitarte, M., Sáenz, A. et al. Characterization of novel CAPN3 isoforms in white blood cells: an alternative approach for limb-girdle muscular dystrophy 2A diagnosis. Neurogenetics 9, 173–182 (2008). https://doi.org/10.1007/s10048-008-0129-1

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