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1.  Functional genomic analysis of human mitochondrial RNA processing 
Cell reports  2014;7(3):918-931.
Both strands of human mitochondrial DNA (mtDNA) are transcribed in continuous, multi-genic units that are cleaved into the mature rRNAs, tRNAs, and mRNAs required for respiratory chain biogenesis. We sought to systematically identify nuclear-encoded proteins that contribute to processing of mitochondrial RNAs (mt-RNAs) within the organelle. First, we devised and validated a multiplex “MitoString” assay that quantitates 27 mature and precursor mtDNA transcripts. Second, we applied MitoString profiling to evaluate the impact of silencing each of 107 mitochondrial-localized, predicted RNA-binding proteins. With the resulting dataset, we rediscover the roles of recently identified RNA processing enzymes, detect unanticipated roles of known disease genes in RNA processing, and identify new regulatory factors. We demonstrate that one such factor, FASTKD4, modulates half-lives of a subset of mt-mRNAs and associates with mitochondrial RNAs in vivo. MitoString profiling may be useful in diagnosing and deciphering the pathogenesis of mtDNA disorders.
PMCID: PMC4289146  PMID: 24746820
Cell  2013;155(1):21-26.
Technologies for genome-wide sequence interrogation have dramatically improved our ability to identify loci associated with complex human disease. However, a chasm remains between correlations and causality that stems, in part, from a limiting theoretical framework derived from Mendelian genetics, and an incomplete understanding of disease physiology. Here we propose a set of criteria, akin to Koch’s postulates for infectious disease, for assigning causality between genetic variants and human disease phenotypes.
PMCID: PMC4244836  PMID: 24074858
3.  Mutations in LYRM4, encoding iron–sulfur cluster biogenesis factor ISD11, cause deficiency of multiple respiratory chain complexes 
Human Molecular Genetics  2013;22(22):4460-4473.
Iron–sulfur clusters (ISCs) are important prosthetic groups that define the functions of many proteins. Proteins with ISCs (called iron–sulfur or Fe–S proteins) are present in mitochondria, the cytosol, the endoplasmic reticulum and the nucleus. They participate in various biological pathways including oxidative phosphorylation (OXPHOS), the citric acid cycle, iron homeostasis, heme biosynthesis and DNA repair. Here, we report a homozygous mutation in LYRM4 in two patients with combined OXPHOS deficiency. LYRM4 encodes the ISD11 protein, which forms a complex with, and stabilizes, the sulfur donor NFS1. The homozygous mutation (c.203G>T, p.R68L) was identified via massively parallel sequencing of >1000 mitochondrial genes (MitoExome sequencing) in a patient with deficiency of complexes I, II and III in muscle and liver. These three complexes contain ISCs. Sanger sequencing identified the same mutation in his similarly affected cousin, who had a more severe phenotype and died while a neonate. Complex IV was also deficient in her skeletal muscle. Several other Fe–S proteins were also affected in both patients, including the aconitases and ferrochelatase. Mutant ISD11 only partially complemented for an ISD11 deletion in yeast. Our in vitro studies showed that the l-cysteine desulfurase activity of NFS1 was barely present when co-expressed with mutant ISD11. Our findings are consistent with a defect in the early step of ISC assembly affecting a broad variety of Fe–S proteins. The differences in biochemical and clinical features between the two patients may relate to limited availability of cysteine in the newborn period and suggest a potential approach to therapy.
PMCID: PMC3888131  PMID: 23814038
4.  Large area micropatterning of cells on polydimethylsiloxane surfaces 
Precise spatial control and patterning of cells is an important area of research with numerous applications in tissue engineering, as well as advancing an understanding of fundamental cellular processes. Poly (dimethyl siloxane) (PDMS) has long been used as a flexible, biocompatible substrate for cell culture with tunable mechanical characteristics. However, fabrication of suitable physico-chemical barriers for cells on PDMS substrates over large areas is still a challenge.
Here, we present an improved technique which integrates photolithography and cell culture on PDMS substrates wherein the barriers to cell adhesion are formed using the photo-activated graft polymerization of polyethylene glycol diacrylate (PEG-DA). PDMS substrates with varying stiffness were prepared by varying the base to crosslinker ratio from 5:1 to 20:1. All substrates show controlled cell attachment confined to fibronectin coated PDMS microchannels with a resistance to non-specific adhesion provided by the covalently immobilized, hydrophilic PEG-DA.
Using photolithography, it is possible to form patterns of high resolution stable at 37°C over 2 weeks, and microstructural complexity over large areas of a few cm2. As a robust and scalable patterning method, this technique showing homogenous and stable cell adhesion and growth over macroscales can bring microfabrication a step closer to mass production for biomedical applications.
Electronic supplementary material
The online version of this article (doi:10.1186/1754-1611-8-24) contains supplementary material, which is available to authorized users.
PMCID: PMC4223844  PMID: 25383093
Cell micropatterning; Poly (dimethyl siloxane); Photolithography; Poly (ethylene glycol)
5.  Speeding up Permutation Testing in Neuroimaging 
Multiple hypothesis testing is a significant problem in nearly all neuroimaging studies. In order to correct for this phenomena, we require a reliable estimate of the Family-Wise Error Rate (FWER). The well known Bonferroni correction method, while simple to implement, is quite conservative, and can substantially under-power a study because it ignores dependencies between test statistics. Permutation testing, on the other hand, is an exact, non-parametric method of estimating the FWER for a given α-threshold, but for acceptably low thresholds the computational burden can be prohibitive. In this paper, we show that permutation testing in fact amounts to populating the columns of a very large matrix P. By analyzing the spectrum of this matrix, under certain conditions, we see that P has a low-rank plus a low-variance residual decomposition which makes it suitable for highly sub–sampled — on the order of 0.5% — matrix completion methods. Based on this observation, we propose a novel permutation testing methodology which offers a large speedup, without sacrificing the fidelity of the estimated FWER. Our evaluations on four different neuroimaging datasets show that a computational speedup factor of roughly 50× can be achieved while recovering the FWER distribution up to very high accuracy. Further, we show that the estimated α-threshold is also recovered faithfully, and is stable.
PMCID: PMC4193680  PMID: 25309108
6.  MicroRNAs: key regulators of stem cells 
The hallmark of a stem cell is its ability to self-renew and to produce numerous differentiated cells. This unique property is controlled by dynamic interplays between extrinsic signalling, epigenetic, transcriptional and post-transcriptional regulations. Recent research indicates that microRNAs (miRNAs) have an important role in regulating stem cell self-renewal and differentiation by repressing the translation of selected mRNAs in stem cells and differentiating daughter cells. Such a role has been shown in embryonic stem cells, germline stem cells and various somatic tissue stem cells. These findings reveal a new dimension of gene regulation in controlling stem cell fate and behaviour.
PMCID: PMC4118578  PMID: 19165214
7.  Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer 
Nature communications  2014;5:3128.
Metabolic remodeling is now widely regarded as a hallmark of cancer, but it is not clear whether individual metabolic strategies are frequently exploited by many tumours. Here we compare messenger RNA profiles of 1,454 metabolic enzymes across 1,981 tumours spanning 19 cancer types to identify enzymes that are consistently differentially expressed. Our meta-analysis recovers established targets of some of the most widely used chemotherapeutics, including dihydrofolate reductase, thymidylate synthase and ribonucleotide reductase, while also spotlighting new enzymes, such as the mitochondrial proline biosynthetic enzyme PYCR1. The highest scoring pathway is mitochondrial one-carbon metabolism and is centred on MTHFD2. MTHFD2 RNA and protein are markedly elevated in many cancers and correlated with poor survival in breast cancer. MTHFD2 is expressed in the developing embryo, but is absent in most healthy adult tissues, even those that are proliferating. Our study highlights the importance of mitochondrial compartmentalization of one-carbon metabolism in cancer and raises important therapeutic hypotheses.
PMCID: PMC4106362  PMID: 24451681
8.  EMRE is an essential component of the mitochondrial calcium uniporter complex 
Science (New York, N.Y.)  2013;342(6164):1379-1382.
The mitochondrial uniporter is a highly selective calcium channel in the organelle’s inner membrane. Its molecular components include the EF-hand containing proteins mitochondrial calcium uptake 1 (MICU1) and MICU2 and the pore forming subunit mitochondrial calcium uniporter (MCU). We sought to achieve a full molecular characterization of the uniporter holocomplex (uniplex). Quantitative mass spectrometry of affinity-purified uniplex recovered MICU1 and MICU2, MCU and its paralog MCUb, and essential MCU regulator (EMRE), a previously uncharacterized protein. EMRE is a 10 kD, metazoan specific protein with a single transmembrane domain. In its absence, uniporter channel activity was lost despite intact MCU expression and oligomerization. EMRE was required for the interaction of MCU with MICU1 and MICU2. Hence, EMRE is essential for in vivo uniporter current and additionally bridges the calcium-sensing role of MICU1 and MICU2 with the calcium conducting role of MCU.
PMCID: PMC4091629  PMID: 24231807
9.  MICU1 controls both the threshold and cooperative activation of the mitochondrial Ca2+ uniporter 
Cell metabolism  2013;17(6):976-987.
Mitochondrial Ca2+ uptake via the uniporter is central to cell metabolism, signaling and survival. Recent studies identified MCU as the uniporter’s likely pore and MICU1, an EF-hand protein, as its critical regulator. How this complex decodes dynamic cytoplasmic [Ca2+] ([Ca2+]c) signals, to tune out small [Ca2+]c increases yet permit pulse transmission, remains unknown. We report that loss of MICU1 in mouse liver and cultured cells causes mitochondrial Ca2+ accumulation during small [Ca2+]c elevations, yet an attenuated response to agonist-induced [Ca2+]c pulses. The latter reflects loss of positive cooperativity, likely via the EF-hands. MICU1 faces the intermembrane space and responds to [Ca2+]c changes. Prolonged MICU1 loss leads to an adaptive increase in matrix Ca2+ binding, yet cells show impaired oxidative metabolism and sensitization to Ca2+ overload. Collectively, the data indicate that MICU1 senses the [Ca2+]c to establish the uniporter’s threshold and gain, thereby allowing mitochondria to properly decode different inputs.
PMCID: PMC3722067  PMID: 23747253
calcium signaling; mitochondria; Ca2+ uniporter; oxidative metabolism; cell death; MCU
10.  Targeted exome sequencing of suspected mitochondrial disorders 
Neurology  2013;80(19):1762-1770.
To evaluate the utility of targeted exome sequencing for the molecular diagnosis of mitochondrial disorders, which exhibit marked phenotypic and genetic heterogeneity.
We considered a diverse set of 102 patients with suspected mitochondrial disorders based on clinical, biochemical, and/or molecular findings, and whose disease ranged from mild to severe, with varying age at onset. We sequenced the mitochondrial genome (mtDNA) and the exons of 1,598 nuclear-encoded genes implicated in mitochondrial biology, mitochondrial disease, or monogenic disorders with phenotypic overlap. We prioritized variants likely to underlie disease and established molecular diagnoses in accordance with current clinical genetic guidelines.
Targeted exome sequencing yielded molecular diagnoses in established disease loci in 22% of cases, including 17 of 18 (94%) with prior molecular diagnoses and 5 of 84 (6%) without. The 5 new diagnoses implicated 2 genes associated with canonical mitochondrial disorders (NDUFV1, POLG2), and 3 genes known to underlie other neurologic disorders (DPYD, KARS, WFS1), underscoring the phenotypic and biochemical overlap with other inborn errors. We prioritized variants in an additional 26 patients, including recessive, X-linked, and mtDNA variants that were enriched 2-fold over background and await further support of pathogenicity. In one case, we modeled patient mutations in yeast to provide evidence that recessive mutations in ATP5A1 can underlie combined respiratory chain deficiency.
The results demonstrate that targeted exome sequencing is an effective alternative to the sequential testing of mtDNA and individual nuclear genes as part of the investigation of mitochondrial disease. Our study underscores the ongoing challenge of variant interpretation in the clinical setting.
PMCID: PMC3719425  PMID: 23596069
11.  Dissecting the pathways that destabilize mutant p53 
Cell Cycle  2013;12(7):1022-1029.
One fundamental feature of mutant forms of p53 consists in their accumulation at high levels in tumors. At least in the case of neomorphic p53 mutations, which acquire oncogenic activity, stabilization is a driving force for tumor progression. It is well documented that p53 mutants are resistant to proteasome-dependent degradation compared with wild-type p53, but the exact identity of the pathways that affect mutant p53 stability is still debated. We have recently shown that macroautophagy (autophagy) provides a route for p53 mutant degradation during restriction of glucose. Here we further show that in basal conditions of growth, inhibition of autophagy with chemical inhibitors or by downregulation of the essential autophagic genes ATG1/Ulk1, Beclin-1 or ATG5, results in p53 mutant stabilization. Conversely, overexpression of Beclin-1 or ATG1/Ulk1 leads to p53 mutant depletion. Furthermore, we found that in many cell lines, prolonged inhibition of the proteasome does not stabilize mutant p53 but leads to its autophagic-mediated degradation. Therefore, we conclude that autophagy is a key mechanism for regulating the stability of several p53 mutants. We discuss plausible mechanisms involved in this newly identified degradation pathway as well as the possible role played by autophagy during tumor evolution driven by mutant p53.
PMCID: PMC3646859  PMID: 23466706
p53; mutant; mutations; autophagy; proteasome; ubiquitin tumor; cancer
12.  Management of Infantile Hemangiomas: Current Trends 
Infantile hemangiomas (IH) are common vascular tumours. IH have a characteristic natural course. They proliferate rapidly during the early infantile period followed by a period of gradual regression over several years. Most of the uncomplicated IH undergo spontaneous involution, with a small proportion of cases requiring intervention. These are children with IH in life-threatening locations, local complications like haemorrhage, ulceration and necrosis and functional or cosmetic disfigurements. Systemic corticosteroids have been the first line of treatment for many years. Recently, non-selective beta-blockers, such as oral propranalol and topical timolol, have emerged as promising and safer therapies. Other treatment options include interferon α and vincristine which are reserved for life-threatening haemangiomas that are unresponsive to conventional therapy. This review mainly focuses on the current trends and evidence-based approach in the management of IH.
PMCID: PMC4134656  PMID: 25136206
Infantile hemangioma; steroids; propranolol; timolol; pulsed dye laser
13.  Next generation sequencing with copy number variant detection expands the phenotypic spectrum of HSD17B4-deficiency 
BMC Medical Genetics  2014;15:30.
D-bifunctional protein deficiency, caused by recessive mutations in HSD17B4, is a severe, infantile-onset disorder of peroxisomal fatty acid oxidation. Few affected patients survive past two years of age. Compound heterozygous mutations in HSD17B4 have also been reported in two sisters diagnosed with Perrault syndrome (MIM # 233400), who presented in adolescence with ovarian dysgenesis, hearing loss, and ataxia.
Case presentation
An adult male presented with cerebellar ataxia, peripheral neuropathy, hearing loss, and azoospermia. The clinical presentation, in combination with biochemical findings in serum, urine, and muscle biopsy, suggested a mitochondrial disorder. Commercial genetic testing of 18 ataxia and mitochondrial disease genes was negative. Targeted exome sequencing followed by analysis of single nucleotide variants and small insertions/deletions failed to reveal a genetic basis of disease. Application of a computational algorithm to infer copy number variants (CNVs) from exome data revealed a heterozygous 12 kb deletion of exons 10–13 of HSD17B4 that was compounded with a rare missense variant (p.A196V) at a highly conserved residue. Retrospective review of patient records revealed mildly elevated ratios of pristanic:phytanic acid and arachidonic:docosahexaenoic acid, consistent with dysfunctional peroxisomal fatty acid oxidation.
Our case expands the phenotypic spectrum of HSD17B4-deficiency, representing the first male case reported with infertility. Furthermore, it points to crosstalk between mitochondria and peroxisomes in HSD17B4-deficiency and Perrault syndrome.
PMCID: PMC4015298  PMID: 24602372
HSD17B4; DBP; D-bifunctional protein deficiency; Perrault syndrome; Next-generation sequencing; Exome sequencing; Copy number variants; CNV; Mitochondria; Mitochondrial disorders; Mitochondrial disease; Mendelian disorders; Human genetics; Ataxia; Multi-system disorders; Peroxisomal defects
14.  Glycoprotein folding and quality-control mechanisms in protein-folding diseases 
Disease Models & Mechanisms  2014;7(3):331-341.
Biosynthesis of proteins – from translation to folding to export – encompasses a complex set of events that are exquisitely regulated and scrutinized to ensure the functional quality of the end products. Cells have evolved to capitalize on multiple post-translational modifications in addition to primary structure to indicate the folding status of nascent polypeptides to the chaperones and other proteins that assist in their folding and export. These modifications can also, in the case of irreversibly misfolded candidates, signal the need for dislocation and degradation. The current Review focuses on the glycoprotein quality-control (GQC) system that utilizes protein N-glycosylation and N-glycan trimming to direct nascent glycopolypeptides through the folding, export and dislocation pathways in the endoplasmic reticulum (ER). A diverse set of pathological conditions rooted in defective as well as over-vigilant ER quality-control systems have been identified, underlining its importance in human health and disease. We describe the GQC pathways and highlight disease and animal models that have been instrumental in clarifying our current understanding of these processes.
PMCID: PMC3944493  PMID: 24609034
N-glycosylation; Glycoprotein folding; ER quality control; ER-associated degradation; ER export
15.  SLC25A1, or CIC, is a novel transcriptional target of mutant p53 and a negative tumor prognostic marker 
Oncotarget  2014;5(5):1212-1225.
Mutations of the p53 gene hallmark many human cancers. Several p53 mutant proteins acquire the capability to promote cancer progression and metastasis, a phenomenon defined as Gain of Oncogenic Function (GOF). The downstream targets by which GOF p53 mutants perturb cellular programs relevant to oncogenesis are only partially known. We have previously demonstrated that SLC25A1 (CIC) promotes tumorigenesis, while its inhibition blunts tumor growth. We now report that CIC is a direct transcriptional target of several p53 mutants. We identify a novel interaction between mutant p53 (mutp53) and the transcription factor FOXO-1 which is responsible for regulation of CIC expression levels. Tumor cells harboring mutp53 display higher CIC levels relative to p53 null or wild-type tumors, and inhibition of CIC activity blunts mutp53-driven tumor growth, partially overcoming GOF activity. CIC inhibition also enhances the chemotherapeutic potential of platinum-based agents. Finally, we found that elevated CIC levels predict poor survival outcome in tumors hallmarked by high frequency of p53 mutations. Our results identify CIC as a novel target of mutp53 and imply that the employment of CIC inhibitors may improve survival rates and reduce chemo-resistance in tumors harboring these types of mutations, which are among the most intractable forms of cancers.
PMCID: PMC4012738  PMID: 24681808
SLC25A1; CIC; citrate; cancer; p53 mutations; mutant; FOXO-1; survival; prognostic; prognosis; marker
16.  Proteomic Mapping of Mitochondria in Living Cells via Spatially-Restricted Enzymatic Tagging 
Science (New York, N.Y.)  2013;339(6125):1328-1331.
Microscopy and mass spectrometry (MS) are complementary techniques: the former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins, while the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here we introduce technology that combines these strengths by offering spatially- and temporally-resolved proteomic maps of endogenous proteins within living cells. The method relies on a genetically-targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix. The specificity of live-cell peroxidase-mediated proteomic mapping combined with its ease of use offers biologists a powerful tool for understanding the molecular composition of living cells.
PMCID: PMC3916822  PMID: 23371551
17.  Circulating Branched-chain Amino Acid Concentrations Are Associated with Obesity and Future Insulin Resistance in Children and Adolescents 
Pediatric obesity  2012;8(1):52-61.
Branched-chain amino acid (BCAA) concentrations are elevated in response to overnutrition, and can affect both insulin sensitivity and secretion. Alterations in their metabolism may therefore play a role in the early pathogenesis of type 2 diabetes in overweight children.
To determine whether pediatric obesity is associated with elevations in fasting circulating concentrations of branched-chain amino acids (isoleucine, leucine, and valine), and whether these elevations predict future insulin resistance.
Research Design and Methods
Sixty-nine healthy subjects, ages 8 to18 years, were enrolled as a cross-sectional cohort. A subset who were pre- or early-pubertal, ages 8 to 13 years, were enrolled in a prospective longitudinal cohort for 18 months (n=17 with complete data).
Elevations in the concentrations of BCAA’s were significantly associated with BMI Z-score (Spearman’s Rho 0.27, p=0.03) in the cross-sectional cohort. In the subset of subjects followed longitudinally, baseline BCAA concentrations were positively associated with HOMA-IR measured 18 months later after controlling for baseline clinical factors including BMI Z-score, sex, and pubertal stage (p=0.046).
Elevations in the concentrations of circulating branched-chain amino acids are significantly associated with obesity in children and adolescents, and may independently predict future insulin resistance.
PMCID: PMC3519972  PMID: 22961720
branched-chain amino acids; insulin resistance; pediatric obesity; metabolomics; type 2 diabetes
18.  Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage–Induced Mutagenesis in Saccharomyces cerevisiae 
PLoS Biology  2014;12(1):e1001771.
After DNA damage, Def1 triggers degradation of the catalytic subunit of the replicative DNA polymerase at stalled replication forks, allowing special polymerases to take over DNA synthesis.
DNA damages hinder the advance of replication forks because of the inability of the replicative polymerases to synthesize across most DNA lesions. Because stalled replication forks are prone to undergo DNA breakage and recombination that can lead to chromosomal rearrangements and cell death, cells possess different mechanisms to ensure the continuity of replication on damaged templates. Specialized, translesion synthesis (TLS) polymerases can take over synthesis at DNA damage sites. TLS polymerases synthesize DNA with a high error rate and are responsible for damage-induced mutagenesis, so their activity must be strictly regulated. However, the mechanism that allows their replacement of the replicative polymerase is unknown. Here, using protein complex purification and yeast genetic tools, we identify Def1 as a key factor for damage-induced mutagenesis in yeast. In in vivo experiments we demonstrate that upon DNA damage, Def1 promotes the ubiquitylation and subsequent proteasomal degradation of Pol3, the catalytic subunit of the replicative polymerase δ, whereas Pol31 and Pol32, the other two subunits of polymerase δ, are not affected. We also show that purified Pol31 and Pol32 can form a complex with the TLS polymerase Rev1. Our results imply that TLS polymerases carry out DNA lesion bypass only after the Def1-assisted removal of Pol3 from the stalled replication fork.
Author Summary
DNA damages can lead to the stalling of the cellular replication machinery if not repaired on time, inducing DNA strand breaks, recombination that can result in gross chromosomal rearrangements, even cell death. In order to guard against this outcome, cells have evolved several precautionary mechanisms. One of these involves the activity of special DNA polymerases—known as translesion synthesis (TLS) polymerases. In contrast to the replicative polymerases responsible for faithfully duplicating the genome, these can carry out DNA synthesis even on a damaged template. For that to occur, they have to take over synthesis from the replicative polymerase that is stalled at a DNA lesion. Although this mechanism allows DNA synthesis to proceed, TLS polymerases work with a high error rate even on undamaged DNA, leading to alterations of the original sequence that can result in cancer. Consequently, the exchange between replicative and special polymerases has to be highly regulated, and the details of this are largely unknown. Here we identified Def1—a protein involved in the degradation of RNA polymerase II—as a prerequisite for error-prone DNA synthesis in yeast. We showed that after treating the cells with a DNA damaging agent, Def1 promoted the degradation of the catalytic subunit of the replicative DNA polymerase δ, without affecting the other two subunits of the polymerase. Our data suggest that the special polymerases can take over synthesis only after the catalytic subunit of the replicative polymerase is removed from the stalled fork in a regulated manner. We predict that the other two subunits remain at the fork and participate in TLS together with the special polymerases.
PMCID: PMC3897375  PMID: 24465179
19.  MPV17 Mutations Causing Adult-Onset Multisystemic Disorder With Multiple Mitochondrial DNA Deletions 
Archives of neurology  2012;69(12):1648-1651.
To identify the cause of an adult-onset multisystemic disease with multiple deletions of mitochondrial DNA (mtDNA).
Case report.
University hospitals.
A 65-year-old man with axonal sensorimotor peripheral neuropathy, ptosis, ophthalmoparesis, diabetes mellitus, exercise intolerance, steatohepatopathy, depression, parkinsonism, and gastrointestinal dysmotility.
Skeletal muscle biopsy revealed ragged-red and cytochrome-c oxidase–deficient fibers, and Southern blot analysis showed multiple mtDNA deletions. No deletions were detected in fibroblasts, and the results of quantitative polymerase chain reaction showed that the amount of mtDNA was normal in both muscle and fibroblasts. Exome sequencing using a mitochondrial library revealed compound heterozygous MPV17 mutations (p.LysMet88-89MetLeu and p.Leu143*), a novel cause of mtDNA multiple deletions.
In addition to causing juvenile-onset disorders with mtDNA depletion, MPV17 mutations can cause adult-onset multisystemic disease with multiple mtDNA deletions.
PMCID: PMC3894685  PMID: 22964873
20.  Mitochondrial Encephalomyopathy Due to a Novel Mutation in ACAD9 
JAMA neurology  2013;70(9):1177-1179.
Mendelian forms of complex I deficiency are usually associated with fatal infantile encephalomyopathy. Application of “MitoExome” sequencing (deep sequencing of the entire mitochondrial genome and the coding exons of >1000 nuclear genes encoding the mitochondrial proteome) allowed us to reveal an unusual clinical variant of complex I deficiency due to a novel homozygous mutation in ACAD9. The patient had an infantile-onset but slowly progressive encephalomyopathy and responded favorably to riboflavin therapy.
A 13-year-old boy had exercise intolerance, weakness, and mild psychomotor delay. Muscle histochemistry showed mitochondrial proliferation, and biochemical analysis revealed severe complex I deficiency (15% of normal). The level of complex I holoprotein was reduced as determined by use of Western blot both in muscle (54%) and in fibroblasts (57%).
The clinical presentation of complex I deficiency due ACAD9 mutations spans from fatal infantile encephalocardiomyopathy to mild encephalomyopathy. Our data support the notion that ACAD9 functions as a complex I assembly protein. ACAD9 is a flavin adenine dinucleotide–containing flavoprotein, and treatment with riboflavin is advisable.
PMCID: PMC3891824  PMID: 23836383
21.  Acetabular Distraction: An Alternative for Severe Defects with Chronic Pelvic Discontinuity? 
Stabilization of a pelvic discontinuity with a posterior column plate with or without an associated acetabular cage sometimes results in persistent micromotion across the discontinuity with late fatigue failure and component loosening. Acetabular distraction offers an alternative technique for reconstruction in cases of severe bone loss with an associated pelvic discontinuity.
We describe the acetabular distraction technique with porous tantalum components and evaluate its survival, function, and complication rate in patients undergoing revision for chronic pelvic discontinuity.
Between 2002 and 2006, we treated 28 patients with a chronic pelvic discontinuity with acetabular reconstruction using acetabular distraction. A porous tantalum elliptical acetabular component was used alone or with an associated modular porous tantalum augment in all patients. Three patients died and five were lost to followup before 2 years. The remaining 20 patients were followed semiannually for a minimum of 2 years (average, 4.5 years; range, 2–7 years) with clinical (Merle d’Aubigné-Postel score) and radiographic (loosening, migration, failure) evaluation.
One of the 20 patients required rerevision for aseptic loosening. Fifteen patients remained radiographically stable at last followup. Four patients had early migration of their acetabular component but thereafter remained clinically asymptomatic and radiographically stable. At latest followup, the average improvement in the patients not requiring rerevision using the modified Merle d’Aubigné-Postel score was 6.6 (range, 3.3–9.6). There were no postoperative dislocations; however, one patient had an infection, one a vascular injury, and one a bowel injury.
Acetabular distraction with porous tantalum components provides predictable pain relief and durability at 2- to 7-year followup when reconstructing severe acetabular defects with an associated pelvic discontinuity.
Level of Evidence
Level IV, therapeutic study. See Instructions for Authors for a complete description of levels of evidence.
PMCID: PMC3462839  PMID: 23001499
22.  Next-generation sequencing reveals DGUOK mutations in adult patients with mitochondrial DNA multiple deletions 
Brain  2012;135(11):3404-3415.
The molecular diagnosis of mitochondrial disorders still remains elusive in a large proportion of patients, but advances in next generation sequencing are significantly improving our chances to detect mutations even in sporadic patients. Syndromes associated with mitochondrial DNA multiple deletions are caused by different molecular defects resulting in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia to multi-systemic disorders of variable severity. The mutations underlying these conditions remain undisclosed in half of the affected subjects. We applied next-generation sequencing of known mitochondrial targets (MitoExome) to probands presenting with adult-onset mitochondrial myopathy and harbouring mitochondrial DNA multiple deletions in skeletal muscle. We identified autosomal recessive mutations in the DGUOK gene (encoding mitochondrial deoxyguanosine kinase), which has previously been associated with an infantile hepatocerebral form of mitochondrial DNA depletion. Mutations in DGUOK occurred in five independent subjects, representing 5.6% of our cohort of patients with mitochondrial DNA multiple deletions, and impaired both muscle DGUOK activity and protein stability. Clinical presentations were variable, including mitochondrial myopathy with or without progressive external ophthalmoplegia, recurrent rhabdomyolysis in a young female who had received a liver transplant at 9 months of age and adult-onset lower motor neuron syndrome with mild cognitive impairment. These findings reinforce the concept that mutations in genes involved in deoxyribonucleotide metabolism can cause diverse clinical phenotypes and suggest that DGUOK should be screened in patients harbouring mitochondrial DNA deletions in skeletal muscle.
PMCID: PMC3501975  PMID: 23043144
DGUOK; mitochondrial DNA instability; autosomal recessive progressive external ophthalmoplegia; multiple mitochondrial DNA deletions
23.  Complementary RNA and Protein Profiling Identifies Iron as a Key Regulator of Mitochondrial Biogenesis 
Cell reports  2013;3(1):10.1016/j.celrep.2012.11.029.
Mitochondria are centers of metabolism and signaling whose content and function must adapt to changing cellular environments. The biological signals that initiate mitochondrial restructuring and the cellular processes that drive this adaptive response are largely obscure. To better define these systems, we performed matched quantitative genomic and proteomic analyses of mouse muscle cells as they performed mitochondrial biogenesis. We find that proteins involved in cellular iron homeostasis are highly coordinated with this process and that depletion of cellular iron results in a rapid, dose-dependent decrease of select mitochondrial protein levels and oxidative capacity. We further show that this process is universal across a broad range of cell types and fully reversed when iron is reintroduced. Collectively, our work reveals that cellular iron is a key regulator of mitochondrial biogenesis, and provides quantitative data sets that can be leveraged to explore posttranscriptional and posttranslational processes that are essential for mitochondrial adaptation.
PMCID: PMC3812070  PMID: 23318259
24.  Correction: Independent Component Analysis for Brain fMRI Does Indeed Select for Maximal Independence 
PLoS ONE  2013;8(10):10.1371/annotation/52c7b854-2d52-4b49-9f9f-6560830f9428.
PMCID: PMC3812296
25.  Meclizine Inhibits Mitochondrial Respiration through Direct Targeting of Cytosolic Phosphoethanolamine Metabolism* 
The Journal of Biological Chemistry  2013;288(49):35387-35395.
Background: Previous studies have shown that meclizine inhibits respiration in intact cells, but not in isolated mitochondria, via an unknown mechanism.
Results: Meclizine directly inhibits PCYT2 (CTP:phosphoethanolamine cytidylyltransferase).
Conclusion: Meclizine attenuates mitochondrial respiration by directly inhibiting the Kennedy pathway of phosphatidylethanolamine biosynthesis.
Significance: We identified a novel molecular target of meclizine, an over-the-counter antinausea drug, raising possibilities for new clinical applications.
We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.
PMCID: PMC3853286  PMID: 24142790
Energy Metabolism; Metabolomics; Mitochondria; Phosphatidylethanolamine; Respiration; Meclizine; Phosphoethanolamine

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