Genomic organization of the human MBNL1 gene.

<p>(A) Scale representation of 198 kb of the human MBNL1 locus. Green boxes correspond to exons and black lines to introns. Tested promoter regions are indicated as P1 and P2; a yellow circle denotes a predicted CpG island. (B) Schematic representation of H3K27Ac marks, typical of promoter regions, on seven human cell lines. (C) 5′-Ends of ESTs mapping to the MBNL1 locus support two potential transcription start sites for the gene. Data according to the UCSC Genome Browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093125#pone.0093125-Meyer1" target="_blank">[58]</a>. (E,F,H,I) Direct visualization of the eGFP reporter under the control of the ME enhancer. (D–F) Enhancer-less Hsa-P1 construct is a negative control. (G–I) Flies carrying the ME enhancer upstream of the human MBNL1 promoter (Hsa-P1) reproduce Muscleblind expression in the somatic musculature. (E,H) Lateral and (F,I) ventral views of late embryos. All micrographs were taken at 200× magnification. Anterior is to the left and dorsal up, unless otherwise stated.</p

ME reproduces <i>muscleblind</i> expression in the embryonic somatic musculature.

<p>(A) Localization of the putative <i>cis</i>-regulatory modules M1, M2, ML, H and M3, indicated as orange boxes, in the context of the <i>muscleblind</i> genomic locus. Fluorescence confocal images of lateral (B,E,H,) and ventral (C,F,I,) views of late <i>Drosophila</i> embryos. (D,G) Schematic representation of the reporter constructs used to transform the germline of <i>Drosophila</i>. In control <i>yw</i> flies (B,C) an anti-Mbl antibody detects robust expression in the somatic musculature and in the CNS (green). Direct visualization of the GFP reporter under the control of the ME enhancer in the pH-Stinger vector (E,F,H,I). Promoter-less ME constructs (D–F) do not activate GFP expression and serve as negative controls. Flies carrying the ME enhancer upstream of <i>Hsp70</i> (G–I) reproduce Muscleblind expression in the somatic musculature but not in the CNS. All micrographs were taken at 200× magnification. Anterior is to the left and dorsal up unless otherwise stated.</p

NE reproduces <i>muscleblind</i> expression in the central nervous system.

<p>(A) Schematic representation of the reporter construct used to transform the <i>Drosophila</i> germline. Fluorescence confocal images of lateral (B–D) and ventral (E–J) views of late <i>Drosophila</i> embryos expressing construct (A) co-stained with anti-GFP (green) and anti-Mbl antibodies (red). NE drives expression in the CNS (arrows; C,I) overlapping Muscleblind expression (B,E,H; D,G,J shows the merge in yellow). No signal of the reporter was observed in tissues other than CNS. Endogenous Muscleblind expression in the muscles is in focus in (E,G,H,J). Micrographs were taken at 200× (B–G) and 400× magnification (H–J). Anterior is to the left and dorsal up unless otherwise stated.</p

Sequence and melting temperature (Tm) of PCR primers used for cloning genomic DNA.

<p>Restriction sites are highlighted in bold.</p

Six Serum miRNAs Fail to Validate as Myotonic Dystrophy Type 1 Biomarkers

<div><p>Myotonic dystrophy type 1 (DM1) is an autosomal dominant genetic disease caused by expansion of a CTG microsatellite in the 3’ untranslated region of the <i>DMPK</i> gene. Despite characteristic muscular, cardiac, and neuropsychological symptoms, CTG trinucleotide repeats are unstable both in the somatic and germinal lines, making the age of onset, clinical presentation, and disease severity very variable. A molecular biomarker to stratify patients and to follow disease progression is, thus, an unmet medical need. Looking for a novel biomarker, and given that specific miRNAs have been found to be misregulated in DM1 heart and muscle tissues, we profiled the expression of 175 known serum miRNAs in DM1 samples. The differences detected between patients and controls were less than 2.6 fold for all of them and a selection of six candidate miRNAs, <i>miR-103</i>, <i>miR-107</i>, <i>miR-21</i>, <i>miR-29a</i>, <i>miR-30c</i>, and <i>miR-652</i> all failed to show consistent differences in serum expression in subsequent validation experiments.</p></div

Validation by q-PCR did not reveal differences in miRNA expression levels between controls and myotonic dystrophy type 1 patients.

<p>(A, B) Graphical representation of the results generated by two algorithms, geNorm and NormFinder, to identify the optimal normalisation miRNA from among all of the candidates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150501#pone.0150501.s005" target="_blank">S4 Table</a>). (C) Analysis of the relative expression levels of <i>miR-103</i>, <i>miR-107</i>, <i>miR-21</i>, <i>miR-29a</i>, <i>miR-30c</i>, and <i>miR-652</i> by quantitative PCR on the serum samples of nine DM1 patients and nine healthy individuals. All data were normalised to <i>miR-15</i> expression levels but no significant differences were observed between either group. Graph bars represent average fold-changes of miRNA expression on a logarithmic scale, calculated using the 2^-∆∆Ct method, as well as their confidence intervals. Graph bars represent average fold changes of miRNA expression, calculated using the 2^-∆∆Ct method, along with their standard error.</p

Information about the samples used in qPCR.

<p>Information about the samples used in qPCR.</p

The ratio of <i>miR-130a</i> and <i>miR-21</i> failed as a myotonic dystrophy type 1 biomarker.

<p>(A) The ratio of <i>miR-130a</i> and <i>miR-21</i> according to expression levels obtained from the profiling performed with serum samples from nine DM1 patients and nine healthy controls. (B) The same ratio was calculated after measuring <i>miR-130a</i> and <i>miR-21</i> expression levels by quantitative PCR on serum samples from 21 DM1 and 17 control individuals. No statistically-significant differences were observed. Graph bars represent the average ∆Cts (<i>miR-130a</i>--<i>miR-2</i>1) and their standard errors.</p

Information about the samples used in the miRNA profiling.

<p>Information about the samples used in the miRNA profiling.</p

Profiling of miRNA expression levels in myotonic dystrophy type 1 patients and controls.

<p>(A) Heat map graphical representation and clustering analysis of miRNA expression from 9 DM1 patients (P01-P10, excluding P03) and 9 healthy controls (C11-C20 excluding C18). Blue and yellow indicate statistically significant down- and upregulated miRNAs compared to controls, respectively (t-test α  =  0.05). Data is presented as a dendrogram, with the closest branches of the tree showing samples with less dissimilar expression patterns. (B) Statistical analysis of the miRNA profiling carried out with the G*Power tool. These miRNAs have the highest fold-change and Power∼1 statistics in the sample pool. (C) Graphical representation of the expression levels of the miRNAs selected via G*Power analysis. Only <i>miR-21</i> showed a statistically-significant difference when Bonferroni correction was applied. Graph bars represent average fold changes and their standard errors. <i>P</i> > 0.05.</p