Untargeted metabolomics as an unbiased approach to the diagnosis of inborn errors of metabolism of the non-oxidative branch of the pentose phosphate pathway.

Inborn errors of metabolism (IEM) involving the non-oxidative pentose phosphate pathway (PPP) include the two relatively rare conditions, transketolase deficiency and transaldolase deficiency, both of which can be difficult to diagnosis given their non-specific clinical presentations. Current biochemical testing approaches require an index of suspicion to consider targeted urine polyol testing. To determine whether a broad-spectrum biochemical test could accurately identify a specific metabolic pattern defining IEMs of the non-oxidative PPP, we employed the use of clinical metabolomic profiling as an unbiased novel approach to diagnosis. Subjects with molecularly confirmed IEMs of the PPP were included in this study. Targeted quantitative analysis of polyols in urine and plasma samples was accomplished with chromatography and mass spectrometry. Semi-quantitative unbiased metabolomic analysis of urine and plasma samples was achieved by assessing small molecules via liquid chromatography and high-resolution mass spectrometry. Results from untargeted and targeted analyses were then compared and analyzed for diagnostic acuity. Two siblings with transketolase (TKT) deficiency and three unrelated individuals with transaldolase (TALDO) deficiency were identified for inclusion in the study. For both IEMs, targeted polyol testing and untargeted metabolomic testing on urine and/or plasma samples identified typical perturbations of the respective disorder. Additionally, untargeted metabolomic testing revealed elevations in other PPP metabolites not typically measured with targeted polyol testing, including ribonate, ribose, and erythronate for TKT deficiency and ribonate, erythronate, and sedoheptulose 7-phosphate in TALDO deficiency. Non-PPP alternations were also noted involving tryptophan, purine, and pyrimidine metabolism for both TKT and TALDO deficient patients. Targeted polyol testing and untargeted metabolomic testing methods were both able to identify specific biochemical patterns indicative of TKT and TALDO deficiency in both plasma and urine samples. In addition, untargeted metabolomics was able to identify novel biomarkers, thereby expanding the current knowledge of both conditions and providing further insight into potential underlying pathophysiological mechanisms. Furthermore, untargeted metabolomic testing offers the advantage of having a single effective biochemical screening test for identification of rare IEMs, like TKT and TALDO deficiencies, that may otherwise go undiagnosed due to their generally non-specific clinical presentations.

Opening a window on lysosomal acid lipase deficiency: Biochemical, molecular, and epidemiological insights.

Lysosomal acid lipase deficiency (LAL-D) is a multi-organ autosomal recessive disease caused by mutations in LIPA. We reviewed data from 681 samples (white blood cells [WBC] n = 625, fibroblasts = 30, liver = 4, amniocytes = 13, chorionic villus = 9) received for analysis of lysosomal acid lipase (LAL) activity over a 15-year period. LIPA sequencing was performed in 49 patients with reduced (n = 26) or deficient (n = 23) LAL activity. The Exome Aggregation Consortium and Genome Aggregation Database dataset were used for LAL-D prevalence calculations. LAL WBC activity was reduced in 67 patients (10.72%) and deficient in 37 (5.92%). The average of LAL activity ± margin of error (CI 95%) was 19.32 ± 0.86 pmol/min/mg for reduced activity patients and 5.90 ± 1.42 pmol/min/mg for deficient patients. The average age at diagnosis for LAL-D was 23.6 years with several patients older than age 30. The correlation between the age at diagnosis and LAL activity showed a significant moderate direct correlation (Pearson's r = 0.46, P < 0.005). Homozygous or compound heterozygous mutations were identified in 9 out of 23 patients with deficient results (detection rate 39.1%). The average LAL activity in molecularly confirmed patients was 4.02 ± 2.02 pmol/min/mg protein, while in molecularly negative patients was 13.886 ± 1.49 pmol/min/mg (P < 0.0001). Twenty-two different mutations were identified including two novel variants (c.309C>A and c.856G>C). A carrier frequency of approximately 1 in 350 was inferred. LAL activity in WBC is a validated tool for LAL-D diagnosis. Higher residual enzymatic activity might result in a milder phenotype leading to diagnosis delay. A cut-off below 12 pmol/min/mg protein might be useful to discriminate patients with LIPA mutations.

A metabolomic map of Zellweger spectrum disorders reveals novel disease biomarkers.

PURPOSE: Peroxisome biogenesis disorders-Zellweger spectrum disorders (PBD-ZSD) are metabolic diseases with multisystem manifestations. Individuals with PBD-ZSD exhibit impaired peroxisomal biochemical functions and have abnormal levels of peroxisomal metabolites, but the broader metabolic impact of peroxisomal dysfunction and the utility of metabolomic methods is unknown. METHODS: We studied 19 individuals with clinically and molecularly characterized PBD-ZSD. We performed both quantitative peroxisomal biochemical diagnostic studies in parallel with untargeted small molecule metabolomic profiling in plasma samples with detection of >650 named compounds. RESULTS: The cohort represented intermediate to mild PBD-ZSD subjects with peroxisomal biochemical alterations on targeted analysis. Untargeted metabolomic profiling of these samples revealed elevations in pipecolic acid and long-chain lysophosphatidylcholines, as well as an unanticipated reduction in multiple sphingomyelin species. These sphingomyelin reductions observed were consistent across the PBD-ZSD samples and were rare in a population of >1,000 clinical samples. Interestingly, the pattern or "PBD-ZSD metabolome" was more pronounced in younger subjects suggesting studies earlier in life reveal larger biochemical changes. CONCLUSION: Untargeted metabolomics is effective in detecting mild to intermediate cases of PBD-ZSD. Surprisingly, dramatic reductions in plasma sphingomyelin are a consistent feature of the PBD-ZSD metabolome. The use of metabolomics in PBD-ZSD can provide insight into novel biomarkers of disease.

Elucidation of the complex metabolic profile of cerebrospinal fluid using an untargeted biochemical profiling assay.

We sought to determine the molecular composition of human cerebrospinal fluid (CSF) and identify the biochemical pathways represented in CSF to understand the potential for untargeted screening of inborn errors of metabolism (IEMs). Biochemical profiles for each sample were obtained using an integrated metabolomics workflow comprised of four chromatographic techniques followed by mass spectrometry. Secondarily, we wanted to compare the biochemical profile of CSF with those of plasma and urine within the integrated mass spectrometric-based metabolomic workflow. Three sample types, CSF (N=30), urine (N=40) and EDTA plasma (N=31), were analyzed from retrospectively collected pediatric cohorts of equivalent age and gender characteristics. We identified 435 biochemicals in CSF representing numerous biological and chemical/structural families. Sixty-three percent (273 of 435) of the biochemicals detected in CSF also were detected in urine and plasma, another 32% (140 of 435) were detected in either plasma or urine, and 5% (22 of 435) were detected only in CSF. Analyses of several metabolites showed agreement between clinically useful assays and the metabolomics approach. An additional set of CSF and plasma samples collected from the same patient revealed correlation between several biochemicals detected in paired samples. Finally, analysis of CSF from a pediatric case with dihydropteridine reductase (DHPR) deficiency demonstrated the utility of untargeted global metabolic phenotyping as a broad assessment to screen samples from patients with undifferentiated phenotypes. The results indicate a single CSF sample processed with an integrated metabolomics workflow can be used to identify a large breadth of biochemicals that could be useful for identifying disrupted metabolic patterns associated with IEMs.

Expanding the phenotypic spectrum of Succinyl-CoA ligase deficiency through functional validation of a new SUCLG1 variant.

Deficiency of the TCA cycle enzyme Succinyl-CoA Synthetase/Ligase (SCS), due to pathogenic variants in subunits encoded by SUCLG1 and SUCLA2, causes mitochondrial encephalomyopathy, methylmalonic acidemia, and mitochondrial DNA (mtDNA) depletion. In this study, we report an 11year old patient who presented with truncal ataxia, chorea, hypotonia, bilateral sensorineural hearing loss and preserved cognition. Whole exome sequencing identified a heterozygous known pathogenic variant and a heterozygous novel missense variant of uncertain clinical significance (VUS) in SUCLG1. To validate the suspected pathogenicity of the novel VUS, molecular and biochemical analyses were performed using primary skin fibroblasts from the patient. The patient's cells lack the SUCLG1 protein, with significantly reduced levels of SUCLA2 and SUCLG2 protein. This leads to essentially undetectable SCS enzyme activity, mtDNA depletion, and cellular respiration defects. These abnormal phenotypes are rescued upon ectopic expression of wild-type SUCLG1 in the patient's fibroblasts, thus functionally confirming the pathogenic nature of the SUCLG1 VUS identified in this patient and expanding the phenotypic spectrum for SUCLG1 deficiency.

Functional cellular analyses reveal energy metabolism defect and mitochondrial DNA depletion in a case of mitochondrial aconitase deficiency.

Defects in the tricarboxylic acid cycle (TCA) are associated with a spectrum of neurological phenotypes that are often difficult to diagnose and manage. Whole-exome sequencing (WES) led to a rapid expansion of diagnostic capabilities in such disorders and facilitated a better understanding of disease pathogenesis, although functional characterization remains a bottleneck to the interpretation of potential pathological variants. We report a 2-year-old boy of Afro-Caribbean ancestry, who presented with neuromuscular symptoms without significant abnormalities on routine diagnostic evaluation. WES revealed compound heterozygous missense variants of uncertain significance in mitochondrial aconitase (ACO2), which encodes the TCA enzyme ACO2. Pathogenic variants in ACO2 have been described in a handful of families as the cause of infantile cerebellar-retinal degeneration syndrome. Using biochemical and cellular assays in patient fibroblasts, we found that ACO2 expression was quantitatively normal, but ACO2 enzyme activity was <20% of that observed in control cells. We also observed a deficiency in cellular respiration and, for the first time, demonstrate evidence of mitochondrial DNA depletion and altered expression of some TCA components and electron transport chain subunits. The observed cellular defects were completely restored with ACO2 gene rescue. Our findings demonstrate the pathogenicity of two VUS in ACO2, provide novel mechanistic insights to TCA disturbances in ACO2 deficiency, and implicate mitochondrial DNA depletion in the pathogenesis of this recently described disorder.

Aromatic L-amino acid decarboxylase deficiency diagnosed by clinical metabolomic profiling of plasma.

Aromatic L-amino acid decarboxylase (AADC) deficiency is an inborn error of metabolism affecting the biosynthesis of serotonin, dopamine, and catecholamines. We report a case of AADC deficiency that was detected using the Global MAPS platform. This is a novel platform that allows for parallel clinical testing of hundreds of metabolites in a single plasma specimen. It uses a state-of-the-art mass spectrometry platform, and the resulting spectra are compared against a library of ~2500 metabolites. Our patient is now a 4 year old boy initially seen at 11 months of age for developmental delay and hypotonia. Multiple tests had not yielded a diagnosis until exome sequencing revealed compound heterozygous variants of uncertain significance (VUS), c.286G>A (p.G96R) and c.260C>T (p.P87L) in the DDC gene, causal for AADC deficiency. CSF neurotransmitter analysis confirmed the diagnosis with elevated 3-methoxytyrosine (3-O-methyldopa). Metabolomic profiling was performed on plasma and revealed marked elevation in 3-methoxytyrosine (Z-score +6.1) consistent with the diagnosis of AADC deficiency. These results demonstrate that the Global MAPS platform is able to diagnose AADC deficiency from plasma. In summary, we report a novel and less invasive approach to diagnose AADC deficiency using plasma metabolomic profiling.

Transglutaminase 2 reprogramming of glucose metabolism in mammary epithelial cells via activation of inflammatory signaling pathways.

Aberrant glucose metabolism characterized by high levels of glycolysis, even in the presence of oxygen, is an important hallmark of cancer. This metabolic reprogramming referred to as the Warburg effect is essential to the survival of tumor cells and provides them with substrates required for biomass generation. Molecular mechanisms responsible for this shift in glucose metabolism remain elusive. As described herein, we found that aberrant expression of the proinflammatory protein transglutaminase 2 (TG2) is an important regulator of the Warburg effect in mammary epithelial cells. Mechanistically, TG2 regulated metabolic reprogramming by constitutively activating nuclear factor (NF)-κB, which binds to the hypoxia-inducible factor (HIF)-1α promoter and induces its expression even under normoxic conditions. TG2/NF-κB-induced increase in HIF-1α expression was associated with increased glucose uptake, increased lactate production and decreased oxygen consumption by mitochondria. Experimental suppression of TG2 attenuated HIF-1α expression and reversed downstream events in mammary epithelial cells. Moreover, downregulation of p65/RelA or HIF-1α expression in these cells restored normal glucose uptake, lactate production, mitochondrial respiration and glycolytic protein expression. Our results suggest that aberrant expression of TG2 is a master regulator of metabolic reprogramming and facilitates metabolic alterations in epithelial cells even under normoxic conditions. A TG2-induced shift in glucose metabolism helps breast cancer cells to survive under stressful conditions and promotes their metastatic competence.

Mitochondrial myopathy, lactic acidosis, and sideroblastic anemia (MLASA) plus associated with a novel de novo mutation (m.8969G>A) in the mitochondrial encoded ATP6 gene.

Mitochondrial myopathy, lactic acidosis and sideroblastic anemia (MLASA) is a rare mitochondrial disorder that has previously been associated with mutations in PUS1 and YARS2. In the present report, we describe a 6-year old male with an MLASA plus phenotype. This patient had features of MLASA in the setting of developmental delay, sensorineural hearing loss, epilepsy, agenesis of the corpus callosum, failure to thrive, and stroke-like episodes. Sequencing of the mitochondrial genome identified a novel de novo, heteroplasmic mutation in the mitochondrial DNA (mtDNA) encoded ATP6 gene (m.8969G>A, p.S148N). Whole exome sequencing did not identify mutations or variants in PUS1 or YARS2 or any known nuclear genes that could affect mitochondrial function and explain this phenotype. Studies of fibroblasts derived from the patient revealed a decrease in oligomycin-sensitive respiration, a finding which is consistent with a complex V defect. Thus, this mutation in MT-ATP6 may represent the first mtDNA point mutation associated with the MLASA phenotype.

Differential targeting of Tetrahymena ORC to ribosomal DNA and non-rDNA replication origins.

The Tetrahymena thermophila origin recognition complex (ORC) contains an integral RNA subunit, 26T RNA, which confers specificity to the amplified ribosomal DNA (rDNA) origin by base pairing with an essential cis-acting replication determinant--the type I element. Using a plasmid maintenance assay, we identified a 6.7 kb non-rDNA fragment containing two closely associated replicators, ARS1-A (0.8 kb) and ARS1-B (1.2 kb). Both replicators lack type I elements and hence complementarity to 26T RNA, suggesting that ORC is recruited to these sites by an RNA-independent mechanism. Consistent with this prediction, although ORC associated exclusively with origin sequences in the 21 kb rDNA minichromosome, the interaction between ORC and the non-rDNA ARS1 chromosome changed across the cell cycle. In G(2) phase, ORC bound to all tested sequences in a 60 kb interval spanning ARS1-A/B. Remarkably, ORC and Mcm6 associated with just the ARS1-A replicator in G(1) phase when pre-replicative complexes assemble. We propose that ORC is stochastically deposited onto newly replicated non-rDNA chromosomes and subsequently targeted to preferred initiation sites prior to the next S phase.

Tetrahymena ORC contains a ribosomal RNA fragment that participates in rDNA origin recognition.

The Tetrahymena thermophila ribosomal DNA (rDNA) replicon contains dispersed cis-acting replication determinants, including reiterated type I elements that associate with sequence-specific, single-stranded binding factors, TIF1 through TIF4. Here, we show that TIF4, previously implicated in cell cycle-controlled DNA replication and rDNA gene amplification, is the T. thermophila origin recognition complex (TtORC). We further demonstrate that TtORC contains an integral RNA subunit that participates in rDNA origin recognition. Remarkably, this RNA, designated 26T, spans the terminal 282 nts of 26S ribosomal RNA. 26T RNA exhibits extensive complementarity to the type I element T-rich strand and binds the rDNA origin in vivo. Mutations that disrupt predicted interactions between 26T RNA and its complementary rDNA target change the in vitro binding specificity of ORC and diminish in vivo rDNA origin utilization. These findings reveal a role for ribosomal RNA in chromosome biology and define a new mechanism for targeting ORC to replication initiation sites.

Impaired mitochondrial complex I function as a candidate driver in the biological stress response and a concomitant stress-induced brain metabolic reprogramming in male mice.

Mitochondria play a critical role in bioenergetics, enabling stress adaptation, and therefore, are central in biological stress responses and stress-related complex psychopathologies. To investigate the effect of mitochondrial dysfunction on the stress response and the impact on various biological domains linked to the pathobiology of depression, a novel mouse model was created. These mice harbor a gene trap in the first intron of the Ndufs4 gene (Ndufs4GT/GT mice), encoding the NDUFS4 protein, a structural component of complex I (CI), the first enzyme of the mitochondrial electron transport chain. We performed a comprehensive behavioral screening with a broad range of behavioral, physiological, and endocrine markers, high-resolution ex vivo brain imaging, brain immunohistochemistry, and multi-platform targeted mass spectrometry-based metabolomics. Ndufs4GT/GT mice presented with a 25% reduction of CI activity in the hippocampus, resulting in a relatively mild phenotype of reduced body weight, increased physical activity, decreased neurogenesis and neuroinflammation compared to WT littermates. Brain metabolite profiling revealed characteristic biosignatures discriminating Ndufs4GT/GT from WT mice. Specifically, we observed a reversed TCA cycle flux and rewiring of amino acid metabolism in the prefrontal cortex. Next, exposing mice to chronic variable stress (a model for depression-like behavior), we found that Ndufs4GT/GT mice showed altered stress response and coping strategies with a robust stress-associated reprogramming of amino acid metabolism. Our data suggest that impaired mitochondrial CI function is a candidate driver for altered stress reactivity and stress-induced brain metabolic reprogramming. These changes result in unique phenomic and metabolomic signatures distinguishing groups based on their mitochondrial genotype.

Expansion of the Phenotypic Spectrum of Propionic Acidemia with Isolated Elevated Propionylcarnitine.

We report three patients with elevations of propionylcarnitine (C3), one without elevations of 2-methylcitrate and 3-hydroxypropionate in urine organic acid analysis, and the other two showing only mild elevations, all of whom were subsequently confirmed to have propionic acidemia by molecular analysis of PCCA and PCCB genes. To date, they have had a mild clinical course. These cases illustrate the importance of considering high C3 as the only biochemical abnormality in a diagnosis of propionic acidemia. Since mild C3 elevations may be overlooked and considered non-diagnostic in isolation, we advise considering a diagnosis of propionic acidemia even in the absence of significant elevations 2-methylcitrate or 3-hydroxypropionate in urine organic acid analysis.

Holocarboxylase synthetase deficiency pre and post newborn screening.

Holocarboxylase synthetase deficiency is an autosomal recessive disorder of biotin metabolism resulting in multiple carboxylase deficiency. The typical presentation described in the medical literature is of neonatal onset within hours to weeks of birth with emesis, hypotonia, lethargy, seizures, metabolic ketolactic acidosis, hyperammonemia, developmental delay, skin rash and alopecia. The condition is screened for by newborn screening (NBS) tandem mass spectroscopy by elevated hydroxypentanoylcarnitine on dried blood spots. Urine organic acid profile may demonstrate elevated lactic, 3-OH isovaleric, 3-OH propionic, 3-MCC, methylcitric acids, and tiglylglycine consistent with loss of function of the above carboxylases. Here we describe a cohort of patients, 2 diagnosed pre-NBS and 3 post-NBS with broad differences in initial presentation and phenotype. In addition, prior to the advent of NBS, there are isolated reports of late-onset holocarboxylase synthetase deficiency in the medical literature, which describe patients diagnosed between 1 and 8 years of life, however to our knowledge there are no reports of late-onset HCLS being missed by NBS. Also we report two cases, each with novel pathogenic variants HCLS, diagnosed at age 3 years and 21 months respectively. The first patient had a normal newborn screen whilst the second had an abnormal newborn screen but was misdiagnosed as 3-methylcrotonylcarboxylase (3-MCC) deficiency and subsequently lost to follow-up until they presented again with severe metabolic acidosis.

Chronic Oral L-Carnitine Supplementation Drives Marked Plasma TMAO Elevations in Patients with Organic Acidemias Despite Dietary Meat Restrictions.

Recent studies have implicated trimethylamine N-oxide (TMAO) in atherosclerosis, raising concern about L-carnitine, a common supplement for patients with inborn errors of metabolism (IEMs) and a TMAO precursor metabolized, in part, by intestinal microbes. Dietary meat restriction attenuates carnitine-to-TMAO conversion, suggesting that TMAO production may not occur in meat-restricted individuals taking supplemental L-carnitine, but this has not been tested. Here, we mine a metabolomic dataset to assess TMAO levels in patients with diverse IEMs, including organic acidemias. These data were correlated with clinical information and confirmed using a quantitative TMAO assay. Marked plasma TMAO elevations were detected in patients treated with supplemental L-carnitine, including those on a meat-free diet. On average, patients with an organic acidemia had ~45-fold elevated [TMAO], as compared to the reference population. This effect was mitigated by metronidazole therapy lasting 7 days each month. Collectively, our data show that TMAO production occurs at high levels in patients with IEMs receiving oral L-carnitine. Further studies are needed to determine the long-term safety and efficacy of chronic oral L-carnitine supplementation and whether suppression or circumvention of intestinal bacteria may improve L-carnitine therapy.

Diagnosis of adenylosuccinate lyase deficiency by metabolomic profiling in plasma reveals a phenotypic spectrum.

Adenylosuccinate lyase (ADSL) deficiency is a rare autosomal recessive neurometabolic disorder that presents with a broad-spectrum of neurological and physiological symptoms. The ADSL gene produces an enzyme with binary molecular roles in de novo purine synthesis and purine nucleotide recycling. The biochemical phenotype of ADSL deficiency, accumulation of SAICAr and succinyladenosine (S-Ado) in biofluids of affected individuals, serves as the traditional target for diagnosis with targeted quantitative urine purine analysis employed as the predominate method of detection. In this study, we report the diagnosis of ADSL deficiency using an alternative method, untargeted metabolomic profiling, an analytical scheme capable of generating semi-quantitative z-score values for over 1000 unique compounds in a single analysis of a specimen. Using this method to analyze plasma, we diagnosed ADSL deficiency in four patients and confirmed these findings with targeted quantitative biochemical analysis and molecular genetic testing. ADSL deficiency is part of a large a group of neurometabolic disorders, with a wide range of severity and sharing a broad differential diagnosis. This phenotypic similarity among these many inborn errors of metabolism (IEMs) has classically stood as a hurdle in their initial diagnosis and subsequent treatment. The findings presented here demonstrate the clinical utility of metabolomic profiling in the diagnosis of ADSL deficiency and highlights the potential of this technology in the diagnostic evaluation of individuals with neurologic phenotypes.

Fatty Acid Oxidation-Driven Src Links Mitochondrial Energy Reprogramming and Oncogenic Properties in Triple-Negative Breast Cancer.

Transmitochondrial cybrids and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in triple-negative breast cancer (TNBC). Analysis of cybrids and established breast cancer (BC) cell lines showed that metastatic TNBC maintains high levels of ATP through fatty acid ß oxidation (FAO) and activates Src oncoprotein through autophosphorylation at Y419. Manipulation of FAO including the knocking down of carnitine palmitoyltransferase-1A (CPT1) and 2 (CPT2), the rate-limiting proteins of FAO, and analysis of patient-derived xenograft models confirmed the role of mitochondrial FAO in Src activation and metastasis. Analysis of TCGA and other independent BC clinical data further reaffirmed the role of mitochondrial FAO and CPT genes in Src regulation and their significance in BC metastasis.

Increased COUP-TFII expression in adult hearts induces mitochondrial dysfunction resulting in heart failure.

Mitochondrial dysfunction and metabolic remodelling are pivotal in the development of cardiomyopathy. Here, we show that myocardial COUP-TFII overexpression causes heart failure in mice, suggesting a causal effect of elevated COUP-TFII levels on development of dilated cardiomyopathy. COUP-TFII represses genes critical for mitochondrial electron transport chain enzyme activity, oxidative stress detoxification and mitochondrial dynamics, resulting in increased levels of reactive oxygen species and lower rates of oxygen consumption in mitochondria. COUP-TFII also suppresses the metabolic regulator PGC-1 network and decreases the expression of key glucose and lipid utilization genes, leading to a reduction in both glucose and oleate oxidation in the hearts. These data suggest that COUP-TFII affects mitochondrial function, impairs metabolic remodelling and has a key role in dilated cardiomyopathy. Last, COUP-TFII haploinsufficiency attenuates the progression of cardiac dilation and improves survival in a calcineurin transgenic mouse model, indicating that COUP-TFII may serve as a therapeutic target for the treatment of dilated cardiomyopathy.

Screen for abnormal mitochondrial phenotypes in mouse embryonic stem cells identifies a model for succinyl-CoA ligase deficiency and mtDNA depletion.

Mutations in subunits of succinyl-CoA synthetase/ligase (SCS), a component of the citric acid cycle, are associated with mitochondrial encephalomyopathy, elevation of methylmalonic acid (MMA), and mitochondrial DNA (mtDNA) depletion. A FACS-based retroviral-mediated gene trap mutagenesis screen in mouse embryonic stem (ES) cells for abnormal mitochondrial phenotypes identified a gene trap allele of Sucla2 (Sucla2(SAßgeo)), which was used to generate transgenic mice. Sucla2 encodes the ADP-specific ß-subunit isoform of SCS. Sucla2(SAßgeo) homozygotes exhibited recessive lethality, with most mutants dying late in gestation (e18.5). Mutant placenta and embryonic (e17.5) brain, heart and muscle showed varying degrees of mtDNA depletion (20-60%). However, there was no mtDNA depletion in mutant liver, where the gene is not normally expressed. Elevated levels of MMA were observed in embryonic brain. SCS-deficient mouse embryonic fibroblasts (MEFs) demonstrated a 50% reduction in mtDNA content compared with wild-type MEFs. The mtDNA depletion resulted in reduced steady state levels of mtDNA encoded proteins and multiple respiratory chain deficiencies. mtDNA content could be restored by reintroduction of Sucla2. This mouse model of SCS deficiency and mtDNA depletion promises to provide insights into the pathogenesis of mitochondrial diseases with mtDNA depletion and into the biology of mtDNA maintenance. In addition, this report demonstrates the power of a genetic screen that combines gene trap mutagenesis and FACS analysis in mouse ES cells to identify mitochondrial phenotypes and to develop animal models of mitochondrial dysfunction.