Exclusion of Mutations in the Dysferlin Alternative Exons 1 of DYSF-v1,5a,and 40a in a Cohort of 26 Patients

Abstract

Mutations in the gene encoding dysferlin (DYSF; MIM# 603009, 2p13, GenBank NM_003494.2) cause primary dysferlinopathies, which are autosomal recessive muscular dystrophies. DYSF has a large mutational spectrum, and genetic diagnosis is complicated by incomplete mutation detection rates. Recently, novel dysferlin transcripts were characterized by identifying alternative exons 1 of DYSF-v1 (GenBank DQ267935), exon 5a (GenBank DQ976379), and exon 40a (GenBank EF015906). To evaluate the frequency of possible mutations in the newly identified DYSF alternative exons, we screened the corresponding genomic regions for mutations in a cohort of 26 patients, carrying only one mutation undoubtedly considered as disease causing in the 55 canonical DYSF exons. No disease-causing mutation was identified in alternative exons 1 of DYSF-v1, exon 5a, and exon 40a, demonstrating a low frequency of disease-causing mutations in these exons.

Introduction

Mutations in the large gene encoding dysferlin (DYSF; MIM# 603009, 2p13, GenBank NM_003494.2) cause primary dysferlinopathies, which are autosomal recessive diseases including a wide spectrum of phenotypes, mainly limb girdle muscular dystrophy type 2B (MIM# 253601) (Bashir et al., 1998) and Miyoshi myopathy (MIM# 254130) (Liu et al., 1998), but ranging from isolated hyperCPKemia to severe disability (Urtizberea et al., 2008). DYSF has a large mutational spectrum with more than 350 different sequence variants reported to date, spread along the entire coding sequence (Leiden Muscular Dystrophy pages^©, www.dmd.nl); the gene is divided into 55 canonical exons. One major problem in genetic diagnosis of dysferlinopathies is the incomplete mutation detection rate. In particular, in approximately 10% to 20% of patients, only one disease-causing mutation is identified.

In two recent publications, Pramono and colleagues reported the characterization of novel dysferlin (DYSF; MIM# 603009, 2p13, GenBank NM_003494.2) transcripts correlated to the identification of alternative exons within the DYSF genomic locus: exon 1 of DYSF-v1 (GenBank DQ267935) (Pramono et al., 2006), exon 5a (GenBank DQ976379), and exon 40a (GenBank EF015906) (Pramono et al., 2009). Importantly, as stated also by Pramono and colleagues, the identification of DYSF alternative exons 1 of DYSF-v1, 5a, and 40a raised the question of possible disease-causing mutations existing in these exons.

We recently analyzed the mutational data from a large cohort of 113 index patients, with diagnostic suspicion of primary dysferlinopathy (Krahn et al., 2009). In 26 index patients from this cohort, only 1 mutation undoubtedly considered as disease-causing was identified in the 55 canonical DYSF exons. To evaluate the frequency of possible mutations in DYSF alternative exons 1 of DYSF-v1, 5a, and 40a, we screened the corresponding genomic regions for mutations in these 26 patients.

Patients, Materials, and Methods

We included 26 index patients with a clinical phenotype suggestive of primary dysferlinopathy, together with a marked decrease or absence of dysferlin expression indicated by Western blotting and/or immunohistochemistry on muscle biopsy samples. In all patients, DYSF genomic denaturing high pressure liquid chromatography (DHPLC) mutational screening, performed as described and after informed consent (Krahn et al., 2009), previously showed only one mutation clearly interpreted as disease causing. Mutational analysis was based on polymerase chain reaction amplification of exons 1 of DYSF-v1, 5a, and 40a, and corresponding flanking intronic regions using specifically designed primer pairs (for exon 1 of DYSF-v1: forward primer 5′-CAGGCATTGGTCACTTCTCC-3′ and reverse primer 5′-GGTAGCACCCGAGCATGG-3′; for exon 5a: forward primer 5′-AGGGTTGGGGATGGAGGT-3′ and reverse primer 5′-AGGAGTCCTCTGCCACCTG-3′; for exon 40a: forward primer 5′-GGTGCCTGGTCAGAGAGC-3′ and reverse primer 5′-ACTCTCCCCTCCCATTTCC-3′), and subsequent direct sequencing on an ABI 3130xl Genetic Analyzer^® (Applied Biosystems, Carlsbad, CA). This study complies with the ethical rules of the institutions involved.

Results and Discussion

No disease-causing mutation of DYSF alternative exons 1 of DYSF-v1, 5a, and 40a was identified in the 26 included samples. The intronic single-nucleotide polymorphisms rs6752632 and rs61333801 (dbSNP build 130) were found at a homozygous state in one patient, and at a heterozygous state in five patients.

The cumulative DYSF coding sequence of NM_003494.2 (6243 bp), exons 1 of DYSF-v1 (232 bp), 5a (205 bp), and 40a (63 bp) corresponds to 6743 bp. Thus, the expected frequency of coding sequence variants in DYSF alternative exons 1 of DYSF-v1, 5a, and 40a was expected to be of 7.42%, corresponding to 1.92 expected coding sequence variants among the 26 included samples. As no mutation in DYSF alternative exons 1 of DYSF-v1, 5a, and 40a was found in these 26 patients, the extrapolated frequency of possible coding sequence variants in these exons is lower than 3.85%. As discussed by Pramono and colleagues (2009), transcripts carrying exons 5a and 40a are very infrequent in skeletal muscle, and therefore mutations in these exons are less likely to cause muscle disease in primary dysferlinopathy. Our results further sustain the discussion of Pramono and colleagues. In conclusion, if disease-causing mutations in DYSF alternative exons 1 of DYSF-v1, 5a, and 40a may exist in patients affected with primary dysferlinopathy, their frequency should be low. Genomic DYSF mutational screening should include exons 1 of DYSF-v1, 5a, and 40a, but without priority in the routine molecular diagnosis strategy, as these exons evidently are not mutational hotspots.

Footnotes

Acknowledgments

We sincerely thank the patients and clinicians for their invaluable cooperation. We thank the Jain Foundation, the Assistance Publique des Hôpitaux de Marseille, the French Direction de l'Hospitalisation et de l'Organisation des Soins, and the Association Française contre les Myopathies for supporting this work.

Disclosure Statement

The authors declare that no competing financial interests exist.

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