Contribution of private mtDNA mutations to ID in ASD.

<p>Contribution of private mtDNA mutations to ID in ASD.</p

Characteristics of mtDNA variants.

<p><b>(A)</b> The nonsynonymous-synonymous rate ratio (hN/hS) for all possible nucleotide substitutions, observed heteroplasmies, and homoplasmies on mtDNA. hN is the number of nonsynonymous substitutions divided by the total number of possible nonsynonymous substitutions on mtDNA; similarly, hS is the number of synonymous substitutions divided by the total number of possible synonymous substitutions on mtDNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006391#pgen.1006391.ref034" target="_blank">34</a>]. The ratio of hN and hS is indicative of purifying selection if it is significantly less than one [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006391#pgen.1006391.ref034" target="_blank">34</a>]. <b>(B)</b> The box plot of CADD pathogenicity scores for all possible substitutions, observed heteroplasmies and homoplasmies at nonsynonymous sites on mtDNA. <b>(C, D)</b> hN/hS <b>(C)</b> and CADD pathogenicity scores of nonsynonymous substitutions <b>(D)</b> for heteroplasmies at non-polymorphic sites, heteroplasmies at polymorphic sites and homoplasmies at non-heteroplasmic sites. Each nucleotide substitution was only counted at most once. ***<i>P</i><2x10^-4; **<i>P</i><2x10^-3; <i>P</i>: p values for Chi-squared test <b>(A,C)</b> or Mann-Whitney test <b>(B,D)</b>.</p

Transmission pattern of pathogenic mtDNA mutations.

<p>The proportion of untransmitted (red), transmitted (purple) and <i>de novo</i> (blue) mutations that were nonsynonymous or predicted pathogenic was shown in (<b>A</b>) for mother-sibling pairs and in (<b>B)</b> for mother-proband pairs. Only mtDNA sites (n = 448) detected with high-confidence heteroplasmies (MAF ≥5%) or <i>de novo</i> homoplasmies were used for calculation. Untransmitted mutations were defined as mutations present in the mother, but were undetectable or had DAF <2% in the child. Likewise, <i>de novo</i> mutations were defined as those present in the child, but were undetectable or had DAF <2% in the mother. Transmitted mutations were mutations shared between the mother and the child with DAF ≥2%. The total number of untransmitted, transmitted and <i>de novo</i> mutations was indicated in parentheses for mother-sibling pairs and mother-proband pairs in the legend. Error bars represent the 95% confidence interval of the proportion estimated based on 10,000 bootstrap samples. CADD: mutations predicted pathogenic with CADD Phred score >15 or >20. Results in the main text were based on “CADD > 15”; <i>P</i>: p values for Fisher’s exact test.</p

3xFLAG-Morgue is expressed and functional in fly tissues.

<p><b>A.</b> The P[Elav-Gal4] or P[<i>da</i>-Gal4] lines were used to express Morgue-3xFLAG (P[UAS-Morgue:3xFlag]) or 3xFLAG-Morgue (P[UAS-3xFlag:Morgue]) in different independent insertion lines. Cell free lysate were analyzed by SDS/PAGE and anti-FLAG Western blots. No anti-FLAG immunoreactive band was observed in a negative control using P[<i>elav</i>-Gal4] and P[UAS-<i>morgue</i>]. <b>B.</b> Anti-Flag immunostaining of stage 16 embryos carrying a copy of P[<i>elav</i>-Gal4] and P[UAS-3XFlag:Morgue]. 3XFLAG-Morgue protein is highly expressed in the embryonic central (arrow) and peripheral (arrowhead) nervous system. <b>C.</b> Viability of flies expressing 3xFLAG-Morgue via P[<i>da</i>-Gal4]. Progeny from crosses between flies carrying different combinations of P[<i>da</i>-Gal4] and P[UAS-3xFlag:Morgue] or P[UAS-Morgue:3xFlag] were analyzed. Flies homozygous for P[<i>da</i>-Gal4] and P[UAS-Morgue:3xFlag] are completely lethal. Flies with reduced copies of P[<i>da</i>-Gal4] or P[UAS-Morgue:3xFlag] exhibit variable effects ranging from nearly full lethality to nearly full viability. <b>D.</b> Expression of Morgue-3xFLAG and 3xFLAG-Morgue proteins induce a similar cell death enhancement phenotype as that observed for wild-type Morgue when co-expressed with R/Grim. LacZ-P[GMR-Gal4],P[UAS-R/Grim] and P[UAS-LacZ]: Morgue-P[GMR-Gal4],P[UAS-R/Grim] and P[UAS-Morgue]: 3xFLAG-Morgue-P[GMR-Gal4],P[UAS-R/Grim] and P[UAS-3xFlag:Morgue]: and Morgue3xFLAG-P[GMR-Gal4],P[UAS-R/Grim] and P[UAS-Morgue:3xFlag]. Both Morgue-3x-FLAG and 3x-FLAG-Morgue proteins exhibit a similar ability to enhance R/Grim induced cell death in the eye as wild-type Morgue. All photos were taken of 1–3 day old flies.</p

Both the NH₂ and COOH regions of Morgue protein exhibit association with polyubiquitin.

<p><b>A.</b> Schematic representation of MBP (Maltose binding protein) and 6xHis-tagged full length Morgue (MBP:Morgue:6xHis) as well as either the Morgue NH2 (MBP-MorgueN:6xHis) or COOH (MBP:MorgueC:6xHis) region. Note that MorgueN contains the zinc finger and F box while MorgueC contains the UEV domain. <b>B.</b> Western blot analysis of <i>in vitro</i> immunoprecipitation assays using ubiquitin (anti-Ub) or MBP (anti-MBP) antisera and native or tagged Morgue proteins. Note presence of ubiquitin immunoreactive bands at approximate sizes of ubiquitin dimers and tetramers that associate with tagged Morgues but not native Morgue protein. Also, MBP immunoreactive bands are observed for each tagged Morgue but not native Morgue. The distinct sizes of these bands correspond to distinct sizes of tagged Morgue proteins. <b>C.</b> Semi-quantitative analysis of Morgue association with ubiquitin. The tagged MorgueN protein exhibits slightly stronger association to polyubiquitin than the tagged MorgueC protein. Neither exhibits the full level of association seen for full length Morgue. Minimal association is observed between MBP and polyubiqutin.</p

Overexpresion of Morgue deletion (MorgueΔ) mutants exhibit variable viability.

<p>Effects of widespread expression of Morgue deletion mutants on fly viability. Counts of homozygous non-balancer (+) and heterozygote balancer progeny derived from genetic crosses where P[<i>da</i>-Gal4] was used to drive expression of P[UAS-GFP] or various P[UAS-MorgueΔ] strains. Homozygote (non-balancer) and heterozygote (with either a TM3 or both a CyO and a TM3 balancer) progeny derived from: P[<i>da</i>-Gal4], P[UAS-MorgueΔ]/TM3, P[<i>da</i>-Gal4], P[UAS-GFP]/TM3, or P[UAS-MorgueΔ]/CyO, P[<i>da</i>-Gal4]/TM3 parent flies were counted. Expression of MorgueΔZF, MorgueΔFB, MorgueΔUEV, and MorgueΔZF-FB resulted in reduced Morgue-induced lethality. In contrast, removal of the F box alone retained near complete Morgue-induced lethality. Note that if the homozygotes are fully viable, the expected percentage of viable flies is either 50% or 25% depending on whether one or two balancer chromosomes are present.</p

Substitutions of the Morgue Gly421 residue do not affect Morgue-induced lethality or cell death.

<p><b>A.</b> Schematic representation of Morgue point mutant proteins with Alanine, Cysteine, or Serine substitutions of the active site Glycine (residue 421). <b>B.</b> The progeny derived from P[<i>da</i>-Gal4], P[UAS-MorgueG421X]/TM3 parent flies were examined and the percentage of homozygotes/heterozygotes was determined. As for native Morgue, expression of each Morgue point mutant essentially resulted in completely lethality. Note that the expected percentage of homozygotes/heterozygotes is 50% if the homozygotes are fully viable. <b>C.</b> Enhancement of eye cell death in P[GMR-Gal4], P[UAS-R/Grim] flies by Morgue point mutants. Compared to GFP co-expression of native Morgue with R/Grim results in enhanced levels of eye cell death (evidenced by additional loss of pigment cells). Similar enhancement is observed for co-expression of the MorgueG421A, MorgueG421C, and MorgueG421S point mutants. <b>D.</b> Enhancement of Reaper-induced CNS midline cell death by Morgue Gly421 mutant proteins. The P[52a-Gal4] line was used to drive expression of P[UAS-LacZ] and P[UAS-Reaper] in embryonic CNS midline cells. Expression of Reaper alone does not induce significant amounts of cell death. Co-expression of native Morgue induces increased levels of CNS midline cell death. This enhanced death is somewhat enhanced by Morgue421A but is not significantly altered by either the MorgueG421C or MorgueG421S point mutant proteins. All views are sagittal with anterior to left. Stage 12 (P[UAS-Reaper] and PUAS-Morgue421A]), stage 16 (P[UAS-Morgue and P[UAS-MorgueG421C]), and stage15 (P[UAS-Reaper] and P[UAS-MorgueG421S]) embryos are shown.</p

Purification of three Morgue-associated proteins.

<p><b>A.</b> Silver staining of an analytical SDS polyacrylamide gel separating proteins from whole fly extracts that associate with Morgue. Protein bands corresponding to Morgue-3xFLAG (∼60 kD) (black arrowhead) as well as the anti-FLAG Ig heavy chain (∼50 kD) are indicated (gray arrowhead). Lanes 1–4 correspond to material purified via the anti-FLAG resin from the following flies: Lane 1: P[<i>da</i>-Gal4]; Lane 2: P[UAS-Morgue3xFlag]; Lane 3: P[<i>da</i>-Gal4], P[UAS-3xFlag:Morgue]; Lane 4: P[<i>da</i>-Gal4], P[UAS-Morgue3xFlag]; Lane 5: Protein molecular weight markers. The three major bands corresponding to Morgue-associated proteins purified are indicated (arrows) and correspond to polypeptides migrating between 28 kD and 20 kD. <b>B.</b> Coomassie Brilliant Blue staining of a preparatory SDS polyacrylamide gel separating Morgue-associated proteins from extracts of P[<i>da</i>-Gal4], P[UAS-Morgue:3xFlag] adult flies purified via anti-FLAG resin. Three major bands (arrows) were excised from the gel and the corresponding polypeptides analyzed via mass spectrometry. Lane on right corresponds to protein molecular weight markers.</p

Overexpression of Morgue results in lethality.

<p>Effects of widespread Morgue expression on fly viability. Counts of heterozygote (balancer) and homozygote non-balancer (+) progeny derived from genetic crosses where P[<i>da</i>-Gal4] was used to drive expression of P[UAS-GFP] or P[UAS-Morgue]. Homozygous P[<i>da</i>-Gal4] or P[<i>da</i>-Gal4],P[UAS-GFP] flies are viable while P[<i>da</i>-Gal4],P[UAS-Morgue] homozygotes are completely lethal. Flies containing two copies of P[<i>da</i>-Gal4] and one copy of P[UAS-Morgue] or vice versa exhibit either complete or significant lethality. UAS-morgue1 and UAS-morgue2 represent independent insertions of P[UAS-Morgue].</p

Additional file 3: Table S5. of Independent impacts of aging on mitochondrial DNA quantity and quality in humans

Associations between mtDNA copy number and 32 phenotypic traits. (XLSX 11 kb