Special Issue “Genetic Advances in Neuromuscular Disorders: From Gene Identification to Gene Therapy”

Since the gene responsible for Duchenne muscular dystrophy was first described in 1987 [...

Transcription-terminating mutation in telethonin causing autosomal recessive muscular dystrophy type 2G in a European patient

A 27-year-old woman of Moldavian origin presented at the age of 15 with progressive proximal limb weakness and painful cramps in her calf muscles. Clinical examination revealed prominent muscle weakness in proximal muscles of the lower extremities and distal anterior compartment of legs, and mild weakness in shoulder girdle muscles. In addition, she had marked calf hypertrophy, muscle atrophy involving the anterior and posterior compartments of the thighs, and the distal anterior compartment of legs, as well as mild scapular winging and hyperlordosis. A muscle biopsy taken from the biceps brachii showed mild dystrophic changes, absent vacuoles, and abundant lobulated fibers. Immunofluorescence and Western blot assays demonstrated complete telethonin deficiency. Molecular analysis revealed a homozygous Trp25X mutation in the telethonin (TCAP) gene resulting in termination of transcription at an early point. Four families from Brazil with telethonin deficiency have previously been reported and classified as LGMD2G, but the actual frequency of this disease is unknown. With this current identification of a case outside the Brazilian population, telethonin mutation-associated LGMD should be considered worldwide

Genetic diagnosis of Duchenne and Becker muscular dystrophy through mRNA analysis : New splicing events

Background Up to 7% of patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) remain genetically undiagnosed after routine genetic testing. These patients are thought to carry deep intronic variants, structural variants or splicing alterations not detected through multiplex ligation-dependent probe amplification or exome sequencing. Methods RNA was extracted from seven muscle biopsy samples of patients with genetically undiagnosed DMD/BMD after routine genetic diagnosis. RT-PCR of the DMD gene was performed to detect the presence of alternative transcripts. Droplet digital PCR and whole-genome sequencing were also performed in some patients. Results We identified an alteration in the mRNA level in all the patients. We detected three pseudoexons in DMD caused by deep intronic variants, two of them not previously reported. We also identified a chromosomal rearrangement between Xp21.2 and 8p22. Furthermore, we detected three exon skipping events with unclear pathogenicity. Conclusion These findings indicate that mRNA analysis of the DMD gene is a valuable tool to reach a precise genetic diagnosis in patients with a clinical and anatomopathological suspicion of dystrophinopathy that remain genetically undiagnosed after routine genetic testing

<i>DMD</i> Mutations in 576 Dystrophinopathy Families: A Step Forward in Genotype-Phenotype Correlations

<div><p>Recent advances in molecular therapies for Duchenne muscular dystrophy (DMD) require precise genetic diagnosis because most therapeutic strategies are mutation-specific. To understand more about the genotype-phenotype correlations of the <i>DMD</i> gene we performed a comprehensive analysis of the <i>DMD</i> mutational spectrum in a large series of families. Here we provide the clinical, pathological and genetic features of 576 dystrophinopathy patients. <i>DMD</i> gene analysis was performed using the MLPA technique and whole gene sequencing in blood DNA and muscle cDNA. The impact of the DNA variants on mRNA splicing and protein functionality was evaluated by <i>in silico</i> analysis using computational algorithms. DMD mutations were detected in 576 unrelated dystrophinopathy families by combining the analysis of exonic copies and the analysis of small mutations. We found that 471 of these mutations were large intragenic rearrangements. Of these, 406 (70.5%) were exonic deletions, 64 (11.1%) were exonic duplications, and one was a deletion/duplication complex rearrangement (0.2%). Small mutations were identified in 105 cases (18.2%), most being nonsense/frameshift types (75.2%). Mutations in splice sites, however, were relatively frequent (20%). In total, 276 mutations were identified, 85 of which have not been previously described. The diagnostic algorithm used proved to be accurate for the molecular diagnosis of dystrophinopathies. The reading frame rule was fulfilled in 90.4% of DMD patients and in 82.4% of Becker muscular dystrophy patients (BMD), with significant differences between the mutation types. We found that 58% of DMD patients would be included in single exon-exon skipping trials, 63% from strategies directed against multiexon-skipping exons 45 to 55, and 14% from PTC therapy. A detailed analysis of missense mutations provided valuable information about their impact on the protein structure.</p></div

Mutation rate per nuclotide and type of mutation.

<p>Mutational type target was calculated in nucleotides according to muscular isoform Dp427m (NM_004006). μ_x is the mutational rate per nucleotide and per generation for each mutation type.</p

Distribution of point mutations distribution along the dystrophin domains.

<p>Mutations in DMD in red, in IMD in green, and in BMD in blue. Mutations detected in female isolated carriers in black. CH1-2: <i>calponin homology</i> domains binding actine ABD1; H1–H4: <i>hinge regions</i>; R1-24: spectrin-like repeats; WW: domain containing two tryptophans; EF-1-2: putative calcium binding sites; ZZ: <i>zinc-finger</i> domain.</p

Distribution of intronic breakpoints in large intragenic rearrangements in the <i>DMD</i> gene.

<p>(A) Breakpoints in deletions. (B) Breakpoints in duplications. 5’ breakpoints in grey and 3’ breakpoints in white.</p

Exceptions to the reading frame rule in large intragenic rearrangements.

<p>(A) In-frame mutations in DMD patients. (B) Frameshift mutations in BMD patients. Asterisk indicates mutations identified in both phenotypes.</p

Identified mutations and phenotypic groups.

<p>Identified mutations and phenotypic groups.</p

Distribution of identified point mutations and phenotypic groups.

<p>Distribution of identified point mutations and phenotypic groups.</p