Western blot analysis and immunohistochemistry are important tools in the initial diagnosis of primary dysferlinopathy due to their low cost and convenience in clinical practice.[sup] However, severe reduction of DYSF can also be observed in other skeletal muscle diseases, such as calpainopathy, caveolinopathy, and anoctaminopathy, which are classified as secondary dysferlinopathies.[sup] In addition, false-negative results can occur in western blot analysis when DYSF has accumulated in the cytoplasm.[sup] Therefore, gene analysis is necessary and still remains the “gold standard” for diagnosis.[sup]
The DYSF geneis located on chromosome 2p13, which spans a genomic region of more than 230 kbp and comprises 55 exons.[sup], It encodes a transmembrane protein DYSF which has been linked to membrane repair,[sup] Ca [sup]2+ signaling,[sup] cell adhesion,[sup] and angiogenesis.[sup] To date, 510 different mutations in this gene have been reported in the Leiden muscular dystrophy database worldwide (Leiden Muscular Dystrophy pages [c] www.dmd.nl).
In addition, we performed MLPA assay of DYSF gene in patients with only one pathogenic mutation to confirm the existence of exonic rearrangements in Chinese patients.
Immunohistochemical analyses were performed using primary antibodies for DYSF, sarcoglycans, and dystrophin (all from Novocastra Laboratories, Newcastle, UK).
In 41 patients, all 55 exons and the intron/exon boundary of the DYSF gene were amplified by PCR as previously described.[sup] The PCR products were directly sequenced using an ABI 3730XL automatic sequencing machine (Applied Biosystems, Life Technologies, Carlsbad, CA, USA).
These mutations span the whole length of the DYSF gene.
As previously reported, the mutations found in this study span the whole length of the DYSF gene, and no mutational hot spots were identified.
Interestingly, the missense mutations identified in this study were located mainly in the C2B domain and inner DysF domain.
At present, there is no information available on the percentage of defects in the DYSF gene caused by deletions/duplications of complete exons.
In conclusion, DYSF mutations in Chinese patients clustered in the N-terminal region of the DYSF gene.
Gln832X and c.663 1G>C) account for about a third of the DYSF mutations in Korean patients with dysferlinopathy.
Analysis of the DYSF mutational spectrum in a large cohort of patients.