The present study demonstrates that the marked reduction of Dysferlin observed in LGMD/MM patients is not genetically heterogeneous. Given the location of Dysferlin, close to the muscle membrane, its interactions with other proteins, and cumulative data about the existence of secondary dysferlinopathies, this is surprising, because in other membrane complexes, such as sarcoglycans, a marked secondary reduction of each component is common. A Dysferlin reduction has been observed in primary calpain-3 deficiency (LGMD2A9
) or Caveolin-3 deficiency (LGMD1C10
). An LGMD/MM phenotype has also been observed in patients carrying mutations in the Anoctamin-5 gene (LGMD2L11
Our results derive from an exhaustive analysis of RNA and DNA from 65 patients having a severe Dysferlin reduction. In theory, it has been claimed that a DNA analysis is necessary to diagnose a primary dysferlinopathy.39
In practice, however, it is not easy. The dysferlin gene is huge and composed of 55 exons. It spans 233
bp of genomic DNA and generates a 6.9-kb-wide transcript. In our mutation-screening flowchart (), the DNA analysis was first carried out by a DHPLC of all exons and flanking introns. DHPLC is cheaper, but it can produce false-negative/positive results and therefore it can only have a screening value.40
A second problem with DHPLC is with regard to the huge number of polymorphisms and variants that are present in this gene (Supplementary Table S4). These are located in all exons and confound an interpretation of the results with many heteroduplex shifts per patient. We therefore used a second technique based on the sequence analysis of the dysferlin cDNA. When a muscle biopsy was not available, we were able to analyse the patients' mRNA from blood, as Dysferlin is highly expressed in the monocytes. This method28, 35
is less invasive and can provide an adequate amount of mRNA: the analysis helped us to understand the pathogenic role of the two intronic variants identified by DNA analysis, both leading to an alteration of the splicing mechanism ().
Despite the larger number of cases identified by mRNA analysis, this method alone can be faulty when the mutated allele is not expressed. We showed that the mechanism of nonsense-mediated mRNA decay
(NMD) also occurred in dysferlinopathy.16
In six out of 65 patients () we identified a homozygous mutation in cDNA, a mutation that was heterozygous in gDNA. By direct sequencing of gDNA, we identified in 3/6 patients (X584, X674, X676) an additional frameshift mutation that was missed by DHPLC. For the three other patients (X267, X268, X675), we failed to identify the primary cause of the missing mRNA expression of the second allele; however, the NMD confirmed the primary involvement of the dysferlin gene.
This confirms that mRNA analysis alone can be faulty, as true homozygote patients cannot be distinguished from compound heterozygote patients with important consequences in respect of genetic counselling.
Third, we resequenced all the relevant genomic regions, and three additional mutations were found. Furthermore, we used three additional methods: long PCR, real-time PCR and array CGH.
Particularly noteworthy was the first evidence of a non
mutation as a new pathological mechanism involved in the dysferlinopathies. Patient X295 carries a homozygous 8-bp (g.6233_6240del, p. P2078fsNON STOP) deletion that was identified in exon 55 of the dysferlin gene. The deletion led to a frameshift in the reading frame with the loss of the stop codon (). We supposed that the new reading frame could give rise to the translation of 97 additional amino acids through the 3′end of the mRNA. The patient showed a residual expression of <10% of larger-sized Dysferlin in the skeletal muscle (). We hypothesize that the mutation could cause a mechanism of non
-stop mRNA decay
. Indeed, it was demonstrated that in the eukaryotes there is a mechanism of degradation of mRNA lacking the stop codon.41, 42, 43, 44 Non-stop
mutation has previously been identified in the ACTA1 gene.45
Figure 4 Non-stop mutation. (a and b) Analysis of DNA sequence obtained from a control (a) and a patient (b). The mutated base is highlighted in blue. The sequence of exon 55 shows a homozygous deletion of 8bp (g. 6233_6240del; p. Pro2078LeufsNON STOP). (more ...)
Many groups have questioned the value of protein analysis in carrying out a correct diagnosis. Fanin et al26
observed that the levels of Dysferlin were reduced to 50% of those of the controls in the carriers of LGMD2B. They showed that a reduction of 50% indicated both familial and isolated LGMD2B heterozygotes, and suggested the use of Dysferlin protein testing to select muscle biopsies from suspected carriers for a subsequent mutation analysis.26
Our data support the dysferlin gene as the unique cause of Dysferlin deficiency between 0 and 20% by WB analysis. Although for three patients (3484, 4132 and X147) the second allele was not identified, this was only because of incomplete testing for insufficient DNA. However, this does not affect the main conclusions of the study, because these patients show sure causative alleles (frameshift/duplication) that cannot be coincidental.
This marked reduction is necessary to affect muscle membrane repair. We cannot exclude the presence of other functional mutations, but a direct proof of pathogenicity is always required, as the dysferlin gene shows a large number of variants and polymorphisms that can be misleading. In these cases, the possible lack of mutations in the dysferlin gene may be because of incomplete genetic testing.
The results obtained in this present paper have an immediate diagnostic application: a Dysferlin reduction to 20% (which can also be measured from blood monocytes28, 35
) can be used to identify LGMD2B with 100% accuracy. In the case of LGMD2B this observation is noteworthy. When a rapid Dysferlin blood testing will be available, important decisions will derive, such as to avoid steroids (that are ineffective/deleterious in LGMD2B in contrast with other forms of muscular dystrophy) and any distressing sport in children.7, 46
Although it is generally agreed that an extensive molecular analysis has a high cost, a precise determination of the dysferlin gene is, however, particularly important from a diagnostic/counselling perspective and in view of the development of a future therapeutic strategy. A successful recognition of all the mutations demonstrates the power of a combined diagnostic strategy. More importantly, a complete genetic testing should be applied to all other LGMD cases.47