High-confidence assessment of functional impact of human mitochondrial non-synonymous genome variations by APOGEE

<div><p>24,189 are all the possible non-synonymous amino acid changes potentially affecting the human mitochondrial DNA. Only a tiny subset was functionally evaluated with certainty so far, while the pathogenicity of the vast majority was only assessed <i>in-silico</i> by software predictors. Since these tools proved to be rather incongruent, we have designed and implemented APOGEE, a machine-learning algorithm that outperforms all existing prediction methods in estimating the harmfulness of mitochondrial non-synonymous genome variations. We provide a detailed description of the underlying algorithm, of the selected and manually curated training and test sets of variants, as well as of its classification ability.</p></div

Performance evaluation calculated on 153 known and unbiased mitochondrial non-synonymous variants.

<p>Performance evaluation calculated on 153 known and unbiased mitochondrial non-synonymous variants.</p

Performance evaluation calculated on 864 known mitochondrial non-synonymous variants.

<p>Number of available predictions in last column.</p

List of assembled predictors and annotations in MitImpact.

<p>List of assembled predictors and annotations in MitImpact.</p

Known variants grouped by mitochondrial gene symbol and OXPHOS complex.

<p>Known variants grouped by mitochondrial gene symbol and OXPHOS complex.</p

Meta-predictors performance comparisons by receiver operating characteristic curves.

<p>Meta-predictors performance comparisons by receiver operating characteristic curves.</p

Immunoblot detection of circadian proteins in Huh-7 cells expressing the HCV core protein genotype 1b or 3a and GFP-expressing control cells.

<p>(<b>A</b>) 48 hours after transfection cells were lysed and equal amounts of proteins were loaded on a 10% polyacrylamide gel, separated by electrophoresis and immunoblotted with specific Rev-Erbα, Rorα, CLOCK, ARNTL, ARNTL2, PER1, PER2, CRY1 and CRY2 primary antibodies. β-actin expression served as loading control. (<b>B</b>) Densitometric quantification of CRY2, PER2 and CLOCK proteins normalized to β-actin expression of three different experiments.</p

qRT-PCR analysis of clock gene mRNA expression in OR6 control cells (cured, not expressing the HCV 1b full replicon) and in HCV replicating OR6 cells (HCV).

<p>Control (cured) OR6 cells and OR6 cells replicating HCV genotype 1b were serum shocked and RNA was extracted every 1 h, 4 h, 10 h, 16 h, 22 h and 28 h after serum shock. mRNA levels of Rev-Erbα, Rorα, ARNTL, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1 and CRY2 genes were assessed by qRT-PCR (<b>A</b> and <b>B</b>). Values were normalized against TBP as housekeeping control gene. Results are expressed as means ± SE of three independent experiments. * = p<0.05 in HCV replicating cells versus control cured OR6 cells.</p

qRT-PCR analysis of clock gene mRNAs expression levels in Huh-7 cells overexpressing the HCV core proteins genotype 1b and 3a.

<p>Huh-7 cells were transiently transfected with HCV core proteins 1b and 3a as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060527#pone.0060527-Abid1" target="_blank">[17]</a>, or with GFP. 48 hours after transfection mRNA levels of Rev-Erbα, Rorα, ARNTL, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1 and CRY2 genes were assessed by qRT-PCR. Values were normalized against TBP as housekeeping control gene. Light gray: GFP transfected cells; dark gray: HCV core protein genotype 3a transfected cells; black: HCV core protein genotype 1b transfected cells. Results are expressed as means ± SE of three independent experiments. * = p<0.05 in HCV core proteins transfected versus GFP transfected control cells.</p

Mutual antagonism between HCV genotype 1b replication and PER2 expression in OR6 cells.

<p>(<b>A</b>) Representative immunoblots of PER2 and CRY2 protein expression in HCV replicating OR6 cells versus control (cured) cells. 1 out of 3 blots is showed. β-actin was used as a loading control. (<b>B</b>) PER2 overexpression in HCV replicating OR6 cells using a Flag-tagged construct. HCV replicating OR6 cells or control OR6 cells were electroporated with Flag-PER2, 24 hours after electroporation protein lysates were processed for immunoblotting and Flag levels were detected with a specific antibody. β-actin was used as a loading control. (<b>C</b>) HCV replication was detected by a luciferase reporter assay in HCV replicating OR6 cells 24 hours post electroporation with a control (GFP) or Flag-PER2 construct. (<b>D</b>) qRT-PCR to detect HCV RNA levels upon control (GFP) or Flag-PER2 plasmid electroporation in HCV replicating OR6 cells HCV RNA values were normalized against TBP as housekeeping control gene (<b>C</b> and <b>D</b>). Results are expressed as means ± SE of three independent experiments. * = p<0.05, ** = p<0.01 in HCV replicating OR6 cells overexpressing Flag-PER2 versus HCV replicating OR6 cells overexpressing GFP.</p