PMC:3873243 / 6703-11785 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3873243","sourcedb":"PMC","sourceid":"3873243","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3873243","text":"MitoExome sequencing identified a likely deleterious homozygous mutation in UQCC2\nWe studied a consanguineous Lebanese patient presenting with severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. Spectrophotometric enzyme assays revealed a severe complex III deficiency with residual activity of only 9% in skeletal muscle and 5% in skin fibroblasts when normalized to citrate synthase activity (Figure 1A). Complex I activity was reduced to 29% and 37% and complex IV activity was reduced to 51% and 53% in muscle and fibroblasts respectively, whereas complex II activity was normal. Secondary deficiency in complex I and complex IV have been described in patients with primary complex III deficiency previously [16]–[19]. In our own experience, skeletal muscle from 4 previous patients with pathogenic mutations in genes encoding complex III subunits or assembly factors (CYC1, UQCRC2, and two BCS1L) had residual activities for complexes I, II, III and IV of 54±17, 117±17, 16±6 and 55±13 (expressed as % of control mean relative to citrate synthase; mean ± S.E.M.). Given the severity of the complex III defect in regard to the activities of complexes I and IV, we thus regarded the patient as having a primary complex III defect. In order to uncover the molecular basis for the complex III defect, we performed “MitoExome” sequencing, involving the targeted capture and massively parallel sequencing (MPS) of the mtDNA and ∼1000 nuclear genes predicted to encode the entire mitochondrial proteome [20]. This approach identified 829 single nucleotide variants or small insertion/deletions in the patient, which were analyzed to select the disease gene (Figure 1B). Three genes, TYMP, MTCH1 and UQCC2, harbored rare (allele frequency \u003c0.005 in dbSNP version 132 [21] and the 1000 genomes project release 20100804 [22]) homozygous or compound heterozygous variants that were predicted to potentially impact protein function. Although TYMP is a known OXPHOS “disease gene”, the clinical and biochemical presentation of the patient, plus the consanguinity, suggested that the rare compound heterozygous TYMP mutations (c.242G\u003eA, c.148A\u003eC) were not causal. Patients with TYMP mutations typically present with combined OXPHOS deficiency associated with mtDNA deletions/depletion rather than a primary complex III deficiency, and have mitochondrial neurogastrointestinal encephalomyopathy (MNGIE, MIM 603041) as opposed to the primary lactic acidosis and renal tubulopathy observed in our patient [23]. Although the homozygous c.170C\u003eT mutation in the pro-apoptotic MTCH1 was not reported in dbSNP version 132 [21] or the 1000 genomes project release 20100804 [22], it was detected with a minor allele frequency of 0.005526 in the Exome Variant Server (NHLBI GO Exome Sequencing Project, http://evs.gs.washington.edu/EVS/ March 2013), suggesting it is likely to be too common to cause a rare mitochondrial disorder. Computational analyses suggested that the third candidate, UQCC2 (previously called MNF1, M19 or C6orf125) was likely causal based on 1) its orthology to the S. cerevisiae complex III assembly factor Cbp6p, and 2) its co-expression with complex III subunits (see below). Further analyses indicated a wider conservation of complex III assembly factors, as the interaction partner of S. cerevisiae Cbp6p, Cbp3p, also had a mammalian homolog, UQCC1 (previously called UQCC), which was co-expressed with UQCC2 and similarly co-expressed with complex III subunit genes. The homozygous c.214-3C\u003eG UQCC2 (NM_032340) mutation fell within a 30.7 Mb long contiguous stretch of homozygosity (LCSH) (Figure S1), consistent with both alleles being inherited from a common ancestor. The mutation was verified via Sanger sequencing (Figure 1C) and was not reported in dbSNP version 132 [21], the 1000 genomes project release 20100804 [22] or the Exome Variant Server. To investigate whether the c.214-3C\u003eG mutation might be a common variant found within the Lebanese population, a Sequenom assay was developed to genotype 86 Lebanese controls. The c.214-3C\u003eG variant was not detected, suggesting it is rare in the ethnically-matched population. To see if this gene might be the cause of complex III deficiency in other patients, we sequenced the coding regions of UQCC2 in 11 patients with confirmed complex III deficiency who lacked a molecular diagnosis. No potentially pathogenic changes were identified.\n10.1371/journal.pgen.1004034.g001 Figure 1 MitoExome sequencing identifies a homozygous mutation in UQCC2 in a patient with complex III deficiency.\n(A) The activity of complexes I–IV (CI-IV) as measured by spectrophotometric analysis and normalized to the activity of citrate synthase (CS), expressed as a percentage of control. Values are the average of duplicate assays. (B) Prioritization of single nucleotide variants (SNVs) and small insertion/deletions (indels) identified by MitoExome MPS. (C) Sequence chromatograms of UQCC2 in control and patient gDNA validating the c.214-3C\u003eG mutation detected by MitoExome sequencing.\n\nT","divisions":[{"label":"Title","span":{"begin":0,"end":81}},{"label":"Figure 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