Additional file 6 of Controlling bias and inflation in epigenome- and transcriptome-wide association studies using the empirical null distribution

Additional Excel Table S3c. Additional Excel Table S3c with output of significant results of the TWAS meta-analyses of age: effect sizes, standard error, P values, test statistics of all cohorts, and the meta-analysis results. (121 KB XLSX

Muscle fiber size, degree of central nucleation and percentage of fibrotic/necrotic tissue.

<p>A. Regenerating and hypertrophic fibers were mainly observed in the <i>mdx</i> mice. A dystrophin level depend trend towards wild type distribution was observed for <i>mdx-Xist</i>^Δhs mice where <15% dystrophin already resulted in improvement. B. Dystrophin levels between 15–30% and >30% resulted in a reduction of the percentage of centralized nuclei of 40% and 60% respectively. C. The quadriceps of all mice was significantly more severely affected compared to <i>Xist</i>^Δhs mice. D. The diaphragm was the most severely affected muscle with on average 20% fibrotic/necrotic tissue in <i>mdx</i> mice. All mice were significantly more severely affected compared to <i>Xist</i>^Δhs mice. <i>Mdx-Xist</i>^Δhs mice with >30% dystrophin had less fibrotic/necrotic tissue than <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice with <15% dystrophin, but this difference was only significant between both <i>mdx-Xist</i>^Δhs groups. # indicates a significant difference of that bar with all the other groups. Single asterisks indicate a <i>P</i><0.05 and double asterisks indicate a <i>P</i><0.01.</p

Expression of several genes involved in disease pathology.

<p>A and B. For most biomarkers a clear dystrophin level dependent restoration of gene expression levels was observed in the <i>mdx-Xist</i>^Δhs mice, where intermediate dystrophin levels resulted in low expression of genes involved in disease pathology. For some genes, dystrophin levels <15% were enough to reduce gene expression while for other genes >30% was necessary. C. In the heart, even dystrophin levels of <15% decreased expression of fibrotic biomarkers like <i>Timp-1</i>. Since the mice were young, no difference in expression levels in heart function was observed, except for <i>Nppa</i>. Double asterisks indicate a <i>P</i><0.01.</p

<i>Mdx-Xist</i>^Δhs mice.

<p>A. To breed mice with low dystrophin levels, female <i>Xist</i>^Δhs mice, carrying a mutation in the <i>Xist</i> promoter which coordinates X-inactivation, were crossed with dystrophin negative <i>mdx</i> males. During embryogenesis, the maternal X-chromosome encoding a functional dystrophin gene will be preferentially (60–90%) inactivated as a result of the mutated <i>Xist</i> promoter. The <i>Xist</i>^Δhs mice were a kind gift from N. Brockdorff (MRC Clinical Sciences Center, London, UK, current affiliation Department of Biochemistry, University of Oxford, UK). B. Picture of a representative Western blot. The percentage of dystrophin was determined for the quadriceps of all <i>mdx-Xist</i>^Δhs mice by Western blot (2–9 technical replicates per mouse). The percentage of individual mice was determined using a concentration curve made from wild type samples. Myosin was used as a loading control. C. Skewed X-inactivation resulted in dystrophin levels of 3–47% (mean 22.7, stdev 12.1, <i>n</i> = 24) (as determined by Western blot) in the female <i>mdx-Xist</i>^Δhs offspring. Each bar represents the dystrophin level of an individual mouse. The dystrophin levels of the individual mice belonging to the three dystrophin groups can be appreciated from this graph. D. Dystrophin levels determined by Western blot and manual counting of dystrophin positive fibers demonstrate a strong correlation (R = 0.97). E. Longitudinal sections of a quadriceps stained with dystrophin (green) and spectrin (red). From the pictures it can be appreciated that dystrophin expression is not homogeneously expressed across the fiber but rather confined to certain nuclear domains.</p

Exercise induced histopathology and biomarker gene expression.

<p>A. Wild type mice were less severely affected than <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice. Due to high variation between individual <i>mdx</i> mice, the difference between <i>mdx</i> and wild type mice was not significant. <i>Mdx-Xist</i>^Δhs mice had slightly less fibrotic tissue than <i>mdx</i> mice. B. No difference in expression of genes involved in disease pathology was found between <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice. Both mouse strains did differ significantly from wild type mice, of which the expression levels were low. Single and double asterisks indicate a <i>P</i><0.05 and <i>P</i><0.01, respectively. # Indicates a significant difference from all other groups, average dystrophin levels of <i>mdx-Xist</i>^Δhs mice was 21% (2%–45% median 25.8).</p

Functional performance measured directly after treadmill exercise.

<p>A. Longest hanging time with the two limb hanging test was achieved by wild type and <i>mdx-Xist</i>^Δhs mice which both performed significantly (<i>P</i><0.001) better than <i>mdx</i> mice. B. Fore limb grip strength of <i>mdx-Xist</i>^Δhs mice was significantly (<i>P</i><0.001) better than that of <i>mdx</i> mice. C. Wild type mice outperformed <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice on the rotarod, while no significant difference was observed between <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice. Average dystrophin level of <i>mdx-Xist</i>^Δhs mice was 21% (2%–45% median 25.8).</p

Serum and plasma biomarkers assessed before and directly after treadmill running.

<p>A. CK levels assessed before and directly after treadmill running. CK levels collected before exercise were significantly (<i>P</i><0.01) lower than those collected directly after exercise in <i>mdx-Xist</i>^Δhs and <i>mdx</i> mice. This exercise induced increase was absent in wild type mice. B. CK levels determined before exercise were significantly (<i>P</i><0.01) elevated in <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice compared to wild type, but this was less pronounced for the <i>mdx-Xist</i>^Δhs mice as <i>mdx</i> mice had significantly (<i>P</i><0.05) higher CK levels. C. Plasma collected directly after exercise contained extremely high CK levels, in both <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice but not in wild type mice. D. Serum levels of MMP-9 were elevated in <i>mdx</i> mice compared to <i>Xist</i>^Δhs mice at both 8 and 14 weeks of age, while levels were normalized in <i>mdx-Xist</i>^Δhs mice. E. TIMP-1 levels were elevated in serum of both <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice. In wild type mice we found an age related increase of the serum TIMP-1 level. Interestingly, at the age of 8 weeks, levels of <i>mdx-Xist</i>^Δhs mice were significantly lower than those of <i>mdx</i> mice. F. <i>Mdx</i> mice had significantly elevated OPN levels compared to both wild type and <i>mdx-Xist</i>^Δhs at 8 weeks of age. Single asterisks indicate a <i>P</i><0.05, average dystrophin level of <i>mdx-Xist</i>^Δhs mice was 21% (2%–45% median 25.8).</p

Additional file 2: Table S1. of Molecular dissection of germline chromothripsis in a developmental context using patient-derived iPS cells

Primer sequences used for each viewpoint in the 4C-seq experiments. Primer sequences are shown without the sequences of the sequencing primer and the P5 and P7 Illumina adaptors. (XLSX 10 kb

Additional file 5: Figure S3. of A SNP panel for identification of DNA and RNA specimens

Individual heterozygosity across loci for each sample determined by GenAlEx software. (DOC 46 kb

Additional file 4: Figure S1. of A SNP panel for identification of DNA and RNA specimens

Plot of the expected heterozygosity (x-axis) and observed heterozygosity (y-axis) for the 50 SNPs in the panel. Pearson correlation is 0.98. (DOC 54 kb