Additional file 1: of Whole-exome sequencing identifies novel pathogenic mutations and putative phenotype-influencing variants in Polish limb-girdle muscular dystrophy patients

Clinical characteristics and WES results (including evidence of pathogenicity according to ACMG criteria) of 85 patients studied. (XLSX 24 kb

Additional file 2: of Whole-exome sequencing identifies novel pathogenic mutations and putative phenotype-influencing variants in Polish limb-girdle muscular dystrophy patients

Lists of rare (< 1%) variants in genes related with “muscle phenotype” for individual patients. (XLSX 3003 kb

Additional file 4: of Whole-exome sequencing identifies novel pathogenic mutations and putative phenotype-influencing variants in Polish limb-girdle muscular dystrophy patients

List of copy number variants (CNVs) identified in the vicinity of LGMD-related genes. (XLS 2184 kb

Additional file 5: of Whole-exome sequencing identifies novel pathogenic mutations and putative phenotype-influencing variants in Polish limb-girdle muscular dystrophy patients

Selected genes with reported skeletal muscle expression which could contribute to LGMD. (DOCX 16 kb

Additional file 3: of Whole-exome sequencing identifies novel pathogenic mutations and putative phenotype-influencing variants in Polish limb-girdle muscular dystrophy patients

List of variants in genes expressed in the human muscle and in genes whose mouse homologs are expressed in muscle, which are in the interactome of known LGMD- and MFM-associated genes. (XLSX 1451 kb

Two Desmin Gene Mutations Associated with Myofibrillar Myopathies in Polish Families

<div><p>Desmin is a muscle-specific intermediate filament protein which forms a network connecting the sarcomere, T tubules, sarcolemma, nuclear membrane, mitochondria and other organelles. Mutations in the gene coding for desmin (<i>DES</i>) cause skeletal myopathies often combined with cardiomyopathy, or isolated cardiomyopathies. The molecular pathomechanisms of the disease remain ambiguous. Here, we describe and comprehensively characterize two <i>DES</i> mutations found in Polish patients with a clinical diagnosis of desminopathy. The study group comprised 16 individuals representing three families. Two mutations were identified: a novel missense mutation (Q348P) and a small deletion of nine nucleotides (A357_E359del), previously described by us in the Polish population. A common ancestry of all the families bearing the A357_E359del mutation was confirmed. Both mutations were predicted to be pathogenic using a bioinformatics approach, including molecular dynamics simulations which helped to rationalize abnormal behavior at molecular level. To test the impact of the mutations on <i>DES</i> expression and the intracellular distribution of desmin muscle biopsies were investigated. Elevated desmin levels as well as its atypical localization in muscle fibers were observed. Additional staining for M-cadherin, α-actinin, and myosin heavy chains confirmed severe disruption of myofibrill organization. The abnormalities were more prominent in the Q348P muscle, where both small atrophic fibers as well large fibers with centrally localized nuclei were observed. We propose that the mutations affect desmin structure and cause its aberrant folding and subsequent aggregation, triggering disruption of myofibrils organization.</p></div

Quantification of desmin content in muscle by immunoblotting.

<p>Equal volumes of homogenates (A) and supernatants (B) from control (C1-C4), patient IV:2, ZP family (P1, A357_E359del mutation), and patient III:3, DP family (P2, Q348P mutation) muscle were subjected to SDS polyacrylamide gel electrophoresis, blotted on nitrocellulose membrane and probed with anti-desmin and anti-GAPDH antibodies, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115470#s3" target="_blank">Materials and Methods</a>. Lower panels in A and B, densitometric analyses of desmin content in the examined muscles. For controls, the data are presented as mean ± SEM for n = 4.</p

Histogram of helix bending angle at residue 348.

<p>WT helix is naturally bent, about 175°, in a dimer coiled-coil conformation. For the Q348P mutation the maximum of the helix bending angle is nearly the same, about 172°, but a long tail of this plot with a local maximum at 158° indicates a high flexibility of helix at this residue in the mutant structure. The structure of A357-E359del also demonstrates a higher flexibility than WT.</p

Electron microscopy analysis of the longitudinal sections of probands's muscle biopsies.

<p>Bars, 1 µm.</p

Pedigree diagram of family ZP.

<p>Open diamond (IV:17). indicates sex unknown. Open triangle (V:4) indicates miscarriage. The other symbols indications as above. Family ZP consisted of 52 members, five of which participated in the study. Genetic analyses were performed for individuals III:7, IV:2, IV:4, IV:5, and IV:14.</p