<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

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 point mutations identified at <i>DMD</i> exons.

<p>Asterisks indicate exons containing CpG codons.</p

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 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

NKCC1 and KCC2 Cotransporters in the CSF of Rett Syndrome (RTT) Patients and Controls.

<p>(A) Immunoblot results in RTT patients and controls. Numbers refer to the patient ID numbers of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068851#pone-0068851-t001" target="_blank">Table 1</a>. Comparison of patients and age matched controls. (B) Mean Optic Densities of NKCC1 and KCC2 Cotransporter Proteins for Rett Patients and Controls suggesting discrepant cotransporter levels between Patients and Controls supported by the respective MANCOVA (<i>F</i> (1, 73) = 6.99, <i>p</i><.01, <i>η_p²</i> = .087). Error bars represent 95% Confidence Interval. *<i>p</i><.01.</p

Clinical and laboratory features of Rett Syndrome patients included in the study.

<p>DEL: Deletion. LP: Lumbar Puncture. AED: Anti epileptic drugs. VPA: Valproic Acid. CBZ: Carbamazepine. LEV: Levetiracetam. TPM: Topiramate.</p