Validation of miRNA/mRNA interactions in DM2 patients.

<p>Validation by qPCR of the expression of randomly selected mRNAs either up-(<b>A</b>) and down-(<b>B</b>) regulated in DM2 patients vs CTR. Expression level compared to CTR of the targeting miRNA is indicated for each mRNA (DM2 = 12, CTR = 10;*p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001).</p

Correlation of atrophy and hypertrophy with DM2 miRNAs score.

<p>A) Representative stainings of transverse sections of frozen DM2 muscle biopsies. Top panel shows a hematoxylin and eosin staining; bottom panel shows an immunostaining for fast myosin heavy chain. Atrophic (<i>blue arrows</i>) and hypertrophic (<i>asterisks</i>) myofibers, as well as nuclear clumps (<i>red arrows</i>) and internal nuclei (<i>black arrows</i>) are highlighted. B) Spearman’s correlation between DM2 miRNAs score and Atrophy (AR) or Hypertrophy (HR) indexes in fast and slow fibers (DM2 n = 13; CTR n = 9).</p

Validation of miRNA modulations in DM1 and DM2 patients.

<p>In the heat map on the left, the mean values of miRNA expression in both DM1 and DM2 compared to controls are shown in a log₂ scale (-ΔΔCt), where red and green indicate positive and negative modulation respectively. On the right, the table shows the same values in a linear scale; statistically significant differences are highlighted in bold (DM2 n = 13, DM1 = 16, CTR n = 13; *p≤0.05 **p≤0.01 ***p≤0.001).</p

Experimental Plan.

<p>miRNA (blue) and mRNA (red) expression patterns were determined in DM2 and CTR, and significantly modulated miRNAs and mRNAs were identified. Validation was performed by qPCR for both miRNAs and mRNAs in 13 DM2 patients and 13 CTR. miRNA targets among the mRNAs identified by Class Comparison analysis were predicted by dedicated softwares. Only miRNA/mRNA couples displaying inverse significant correlation were selected and then analyzed by Ingenuity Pathway Analysis software that allowed to identify enriched molecular pathways and functions.</p

Profiling of miRNAs in DM2 patients and CTR.

<p>In the heat map on the left, mean miRNAs expression values are shown in a log₂ scale (-ΔΔCt), where red and green indicate positive and negative modulation respectively. The table on the right shows the same values in a linear scale (DM2 = 13, CTR  = 13; *p≤0.05 **p≤0.01).</p

Pathways and Functions Analysis by IPA.

<p> <b>Table legend:</b></p><p><b>ratio</b> = number of modulated genes/total number of genes present in the relevant IPA category.</p><p><b>enrichment index</b> =  ratio between observed genes and number of genes expected by chance in each category.</p

Characteristics of Profiling Cohorts (Mean±Se).

<p><b>Abbreviations: MRC scale</b> = Medical Research Council scale (0–5 grade); <b>CPK</b> = Creatine PhosphoKinase; <b>FT3</b> = free-triiodothyronine; <b>FT4</b> = free-thyroxine; <b>TSH</b> = thyroid stimulating hormone; <b>ECG</b> =  electrocardiogram; <b>QRS duration</b> = QRS complex of ECG corresponds to the depolarization of the right and left ventricles of the human heart; <b>EF</b> = ejection fraction; <b>ICM</b> = ischemic cardiomyopathy.</p

Data_Sheet_1_Case report: Dihydropyridine receptor (CACNA1S) congenital myopathy, a novel phenotype with early onset periodic paralysis.docx

IntroductionCACNA1S related congenital myopathy is an emerging recently described entity. In this report we describe 2 sisters with mutations in the CACNA1S gene and the novel phenotype of congenital myopathy and infantile onset episodic weakness.Clinical descriptionBoth sisters had neonatal onset hypotonia, muscle weakness, and delayed walking. Episodic weakness started in infancy and continued thereafter, provoked mostly by cold exposure. Muscle imaging revealed fat replacement of gluteus maximus muscles. Next generation sequencing found the missense p.Cys944Tyr variant and the novel splicing variant c.3526-2A>G in CACNA1S. Minigene assay revealed the splicing variant caused skipping of exon 28 from the transcript, potentially affecting protein folding and/or voltage dependent activation.ConclusionThis novel phenotype supports the notion that there are age related differences in the clinical expression of CACNA1S gene mutations. This expands our understanding of mutations located in regions of the CACNA1S outside the highly conserved S4 segment, where most mutations thus far have been identified.</p

Receptor and post-receptor abnormalities contribute to insulin resistance in myotonic dystrophy type 1 and type 2 skeletal muscle

<div><p>Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant multisystemic disorders caused by expansion of microsatellite repeats. In both forms, the mutant transcripts accumulate in nuclear foci altering the function of alternative splicing regulators which are necessary for the physiological mRNA processing. Missplicing of insulin receptor (IR) gene (<i>INSR</i>) has been associated with insulin resistance, however, it cannot be excluded that post-receptor signalling abnormalities could also contribute to this feature in DM. We have analysed the insulin pathway in skeletal muscle biopsies and in myotube cultures from DM patients to assess whether downstream metabolism might be dysregulated and to better characterize the mechanism inducing insulin resistance. DM skeletal muscle exhibits alterations of basal phosphorylation levels of Akt/PKB, p70S6K, GSK3β and ERK1/2, suggesting that these changes might be accompanied by a lack of further insulin stimulation. Alterations of insulin pathway have been confirmed on control and DM myotubes expressing fetal <i>INSR</i> isoform (<i>INSR-A</i>). The results indicate that insulin action appears to be lower in DM than in control myotubes in terms of protein activation and glucose uptake. Our data indicate that post-receptor signalling abnormalities might contribute to DM insulin resistance regardless the alteration of <i>INSR</i> splicing.</p></div

Clinical data on DM patients used in this study.

a<p>Medical Research Council, scale for muscle strength; scale (0–5 grade) on 15 muscles at both sides in the upper and lower limbs for a total of 150 maximum score.</p>b<p>Electrocardiogram, included first-degree atrio-ventricular block, incomplete or complete bundle-branch block.</p>c<p>Muscle Impairment Rating Scale, stage of the disease for DM1 patients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083777#pone.0083777-Mathieu1" target="_blank">[73]</a>.</p