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

Information about the samples used in qPCR.

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

Information about the samples used in the miRNA profiling.

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

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

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

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

Tau immunohistochemistry from a <i>GRN</i>+/A152T+ patient.

<p>(A) Mild immunoreactivity in the neuronal cytoplasm and threads in the cerebral cortex, x200. (B) Isolated immunoreactivity in the neuronal cytoplasm and processes in the basal ganglia, x200. (C) Mild immunoreactivity in the cytoplasm and processes in the amygdala, x200. (D) Mild immunoreactivity in neuronal processes and lack of immunoreactivity in neuronal cytoplasm in the dentate gyrus of the hippocampus, x200.</p

Tau immunohistochemistry from a <i>GRN</i>+/A152T+ patient diagnosed with unclassifiable tauopathy.

<p>(A) Strong diffuse neuronal cytoplasm immunoreactivity and threads in the cerebral cortex, x200. (B) Mild immunoreactivity in the neuronal cytoplasm and processes in the basal ganglia, x200. (C) Moderate diffuse neuronal cytoplasm immunoreactivity, some neurofibrillary tangles and grains in the neuronal processes in the amygdala, x200. (D) Moderate diffuse neuronal cytoplasm immunoreactivity in the neuronal cytoplasm of the dentate gyrus and threads in the hippocampus, x200.</p

Comparison of demographic and clinical features between groups.

<p>Comparison of demographic and clinical features between groups.</p

Semi-quantitative analysis of tau pathology in individual autopsies.

<p>Semi-quantitative analysis of tau pathology in individual autopsies.</p