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1.  Poor Outcome in a Mitochondrial Neurogastrointestinal Encephalomyopathy Patient with a Novel TYMP Mutation: The Need for Early Diagnosis 
Case Reports in Neurology  2012;4(3):248-253.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a devastating autosomal recessive disorder due to mutations in TYMP, which cause loss of function of thymidine phosphorylase (TP), nucleoside accumulation in plasma and tissues and mitochondrial dysfunction. The clinical picture includes progressive gastrointestinal dysmotility, cachexia, ptosis and ophthalmoparesis, peripheral neuropathy and diffuse leukoencephalopathy, which usually lead to death in early adulthood. Therapeutic options are currently available in clinical practice (allogeneic hematopoietic stem cell transplantation and carrier erythrocyte entrapped TP therapy) and newer, promising therapies are expected in the near future. However, successful treatment is strictly related to early diagnosis. We report on an incomplete MNGIE phenotype in a young man harboring the novel heterozygote c.199 C>T (Q67X) mutation in exon 2, and the previously reported c.866 A>C (E289A) mutation in exon 7 in TYMP. The correct diagnosis was achieved many years after the onset of symptoms and unfortunately, the patient died soon after diagnosis because of multiorgan failure due to severe malnutrition and cachexia before any therapeutic option could be tried. To date, early diagnosis is essential to ensure that patients have the opportunity to be treated. MNGIE should be suspected in all patients who present with both gastrointestinal and nervous system involvement, even if the classical complete phenotype is lacking.
PMCID: PMC3551392  PMID: 23341816
Mitochondrial neurogastrointestinal encephalomyopathy; Thymidine phosphorylase; TYMP
2.  Clinical and biochemical improvements in a patient with MNGIE following enzyme replacement 
Neurology  2013;81(14):1269-1271.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive metabolic disorder caused by a deficiency of thymidine phosphorylase (TP, EC2.4.2.4) due to mutations in the nuclear gene TYMP. TP deficiency leads to plasma and tissue accumulations of thymidine and deoxyuridine which generate imbalances within the mitochondrial nucleotide pools, ultimately leading to mitochondrial dysfunction.1 MNGIE is characterized clinically by leukoencephalopathy, external ophthalmoplegia, peripheral polyneuropathy, cachexia, and enteric neuromyopathy manifesting as gastrointestinal dysmotility. The condition is relentlessly progressive, with patients usually dying from a combination of nutritional and neuromuscular failure at an average age of 37 years.2 Allogeneic hematopoietic stem cell transplantation (AHSCT) offers a permanent cure. Clinical and biochemical improvements following AHSCT have been reported but it carries a high mortality risk and is limited by matched donor availability.3 A consensus proposal for standardizing AHSCT recommends treatment of patients without irreversible end-stage disease and with an optimally matched donor; a majority of patients are ineligible and thus there is a critical requirement for an alternative treatment.4
PMCID: PMC3795612  PMID: 23966250
3.  Liver as a Source for Thymidine Phosphorylase Replacement in Mitochondrial Neurogastrointestinal Encephalomyopathy 
PLoS ONE  2014;9(5):e96692.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive mitochondrial disease associated with mutations in the nuclear TYMP gene. As a result, the thymidine phosphorylase (TP) enzyme activity is markedly reduced leading to toxic accumulation of thymidine and therefore altered mitochondrial DNA. MNGIE is characterized by severe gastrointestinal dysmotility, neurological impairment, reduced life expectancy and poor quality of life. There are limited therapeutic options for MNGIE. In the attempt to restore TP activity, allogenic hematopoietic stem cell transplantation has been used as cellular source of TP. The results of this approach on ∼20 MNGIE patients showed gastrointestinal and neurological improvement, although the 5-year mortality rate is about 70%. In this study we tested whether the liver may serve as an alternative source of TP. We investigated 11 patients (7M; 35–55 years) who underwent hepatic resection for focal disorders. Margins of normal liver tissue were processed to identify, quantify and localize the TP protein by Western Blot, ELISA, and immunohistochemistry, and to evaluate TYMP mRNA expression by qPCR. Western Blot identified TP in liver with a TP/GAPDH ratio of 0.9±0.5. ELISA estimated TP content as 0.5±0.07 ng/μg of total protein. TP was identified in both nuclei and cytoplasm of hepatocytes and sinusoidal lining cells. Finally, TYMP mRNA was expressed in the liver. Overall, our study demonstrates that the liver is an important source of TP. Orthotopic liver transplantation may be considered as a therapeutic alternative for MNGIE patients.
PMCID: PMC4011889  PMID: 24802030
4.  Limited dCTP Availability Accounts for Mitochondrial DNA Depletion in Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE) 
PLoS Genetics  2011;7(3):e1002035.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a severe human disease caused by mutations in TYMP, the gene encoding thymidine phosphorylase (TP). It belongs to a broader group of disorders characterized by a pronounced reduction in mitochondrial DNA (mtDNA) copy number in one or more tissues. In most cases, these disorders are caused by mutations in genes involved in deoxyribonucleoside triphosphate (dNTP) metabolism. It is generally accepted that imbalances in mitochondrial dNTP pools resulting from these mutations interfere with mtDNA replication. Nonetheless, the precise mechanistic details of this effect, in particular, how an excess of a given dNTP (e.g., imbalanced dTTP excess observed in TP deficiency) might lead to mtDNA depletion, remain largely unclear. Using an in organello replication experimental model with isolated murine liver mitochondria, we observed that overloads of dATP, dGTP, or dCTP did not reduce the mtDNA replication rate. In contrast, an excess of dTTP decreased mtDNA synthesis, but this effect was due to secondary dCTP depletion rather than to the dTTP excess in itself. This was confirmed in human cultured cells, demonstrating that our conclusions do not depend on the experimental model. Our results demonstrate that the mtDNA replication rate is unaffected by an excess of any of the 4 separate dNTPs and is limited by the availability of the dNTP present at the lowest concentration. Therefore, the availability of dNTP is the key factor that leads to mtDNA depletion rather than dNTP imbalances. These results provide the first test of the mechanism that accounts for mtDNA depletion in MNGIE and provide evidence that limited dNTP availability is the common cause of mtDNA depletion due to impaired anabolic or catabolic dNTP pathways. Thus, therapy approaches focusing on restoring the deficient substrates should be explored.
Author Summary
Mitochondria are subcellular organelles that constitute the main energy supply within the cell. They contain their own DNA, which should be continuously replicated to ensure the correct mitochondrial function. Several mitochondrial diseases are caused by genetic defects that compromise this replication and result in mitochondrial DNA depletion. In most cases, these genetic defects block the synthesis of dATP, dGTP, dCTP, and dTTP, the 4 nucleotides needed for mitochondrial DNA replication. However, for one of these disorders (mitochondrial neurogastrointestinal encephalomyopathy, MNGIE), the biochemical pathways needed to synthesize them are intact, but degradation of dTTP is genetically blocked, leading to dTTP accumulation. We investigated the biochemical mechanisms through which the dTTP excess leads to mitochondrial DNA depletion in MNGIE, and we found that the delay of mitochondrial DNA replication rate observed when dTTP is in excess is not caused by this excess in itself. Instead, the dTTP overload produces a secondary dCTP depletion that actually delays mitochondrial DNA replication. Therefore, the common factor accounting for mitochondrial DNA depletion in these disorders is the limited availability of one or more nucleotides. This indicates that strategies to provide nucleotides to patients' mitochondria should be explored as a possible treatment for these fatal disorders.
PMCID: PMC3069123  PMID: 21483760
5.  CoQ10 deficiencies and MNGIE: Two Treatable Mitochondrial Disorders 
Biochimica et Biophysica Acta  2012;1820(5):625-631.
Although causative mutations have been identified for numerous mitochondrial disorders, few disease-modifying treatments are available. Two examples of treatable mitochondrial disorders are coenzyme Q10 (CoQ10 or ubiquinone) deficiency and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).
Scope of Review
Here, we describe clinical and molecular features of CoQ10 deficiencies and MNGIE and explain how understanding their pathomechanisms have led to rationale therapies. Primary CoQ10 deficiencies, due to mutations in genes required for ubiquinone biosynthesis, and secondary deficiencies, caused by genetic defects not directly related to CoQ10 biosynthesis, often improve with CoQ10 supplementation. In vitro and in vivo studies of CoQ10 deficiencies have revealed biochemical alterations that may account for phenotypic differences among patients and variable responses to therapy. In contrast to the heterogeneous CoQ10 deficiencies, MNGIE is a single autosomal recessive disease due to mutations in the TYMP gene encoding thymidine phosphorylase (TP). In MNGIE, loss of TP activity causes toxic accumulations of the nucleosides thymidine and deoxyuridine that are incorporated by the mitochondrial pyrimidine salvage pathway and cause deoxynucleoside triphosphate pool imbalances, which, in turn cause mtDNA instability. Allogeneic hematopoetic stem cell transplantation to restore TP activity and eliminate toxic metabolites is a promising therapy for MNGIE.
CoQ10 deficiencies and MNGIE demonstrate the feasibility of treating specific mitochondrial disorders through replacement of deficient metabolites or via elimination of excessive toxic molecules.
General Significance
Studies of CoQ10 deficiencies and MNGIE illustrate how understanding the pathogenic mechanisms of mitochondrial diseases can lead to meaningful therapies.
PMCID: PMC3319470  PMID: 22274133
6.  Biochemical abnormalities in a patient with thymidine phosphorylase deficiency with fatal outcome 
Deficiency of the cytosolic enzyme thymidine phosphorylase (TP) causes a multisystem disorder called mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome. Clinical symptoms are gastrointestinal dysfunction, muscle involvement and neurological deterioration. TP deficiency is biochemically characterised by accumulation of thymidine and deoxyuridine in body fluids and compromised mitochondrial deoxyribose nucleic acid (mtDNA) integrity (depletion and multiple deletions). In this report we describe a patient with the clinical and biochemical features related to the end stage of the disease. Home parenteral nutrition had started to improve the clinical condition and preparations were initiated for stem cell transplantation (SCT) as a last resort treatment. Unfortunately, the patient died during the induction phase of SCT. This report shows that TP deficiency is a severe clinical condition with a broad spectrum of affected tissues. TP deficiency can be easily determined by the measurement of pyrimidine metabolites in body fluids and TP activity in peripheral blood leucocytes. Early detection and treatment may prevent the progress of the clinical symptoms and, therefore, should be considered for inclusion in newborn screening programmes.
PMCID: PMC3757267  PMID: 20151198
7.  Mitochondrial DNA Depletion Syndromes: Review and Updates of Genetic Basis, Manifestations, and Therapeutic Options 
Neurotherapeutics  2013;10(2):186-198.
Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a genetically and clinically heterogeneous group of autosomal recessive disorders that are characterized by a severe reduction in mtDNA content leading to impaired energy production in affected tissues and organs. MDS are due to defects in mtDNA maintenance caused by mutations in nuclear genes that function in either mitochondrial nucleotide synthesis (TK2, SUCLA2, SUCLG1, RRM2B, DGUOK, and TYMP) or mtDNA replication (POLG and C10orf2). MDS are phenotypically heterogeneous and usually classified as myopathic, encephalomyopathic, hepatocerebral or neurogastrointestinal. Myopathic MDS, caused by mutations in TK2, usually present before the age of 2 years with hypotonia and muscle weakness. Encephalomyopathic MDS, caused by mutations in SUCLA2, SUCLG1, or RRM2B, typically present during infancy with hypotonia and pronounced neurological features. Hepatocerebral MDS, caused by mutations in DGUOK, MPV17, POLG, or C10orf2, commonly have an early-onset liver dysfunction and neurological involvement. Finally, TYMP mutations have been associated with mitochondrial neurogastrointestinal encephalopathy (MNGIE) disease that typically presents before the age of 20 years with progressive gastrointestinal dysmotility and peripheral neuropathy. Overall, MDS are severe disorders with poor prognosis in the majority of affected individuals. No efficacious therapy is available for any of these disorders. Affected individuals should have a comprehensive evaluation to assess the degree of involvement of different systems. Treatment is directed mainly toward providing symptomatic management. Nutritional modulation and cofactor supplementation may be beneficial. Liver transplantation remains controversial. Finally, stem cell transplantation in MNGIE disease shows promising results.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-013-0177-6) contains supplementary material, which is available to authorized users.
PMCID: PMC3625391  PMID: 23385875
Mitochondrial myopathy; Mitochondrial encephalomyopathy; Hepatocerebral syndrome; Mitochondrial neurogastrointestinal (MNGIE) disease; Alpers-Huttenlocher syndrome
8.  Emergency surgery in chronic intestinal pseudo-obstruction due to mitochondrial neurogastrointestinal encephalomyopathy: case reports 
Chronic intestinal pseudo-obstruction (CIPO) is a syndrome characterized by recurrent clinical episodes of intestinal obstruction in the absence of any mechanical cause occluding the gut. There are multiple causes related to this rare syndrome. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is one of the causes related to primary CIPO. MNGIE is caused by mutations in the gene encoding thymidine phosphorylase. These mutations lead to an accumulation of thymidine and deoxyuridine in blood and tissues of these patients. Toxic levels of these nucleosides induce mitochondrial DNA abnormalities leading to an abnormal intestinal motility.
Herein, we described two rare cases of MNGIE syndrome associated with CIPO, which needed surgical treatment for gastrointestinal complications. In one patient, intra-abdominal hypertension and compartment syndrome generated as a result of the colonic distension forced to perform emergency surgery. In the other patient, a perforated duodenal diverticulum was the cause that forced to perform surgery. There is not a definitive treatment for MNGIE syndrome and survival does not exceed 40 years of age. Surgery only should be considered in some selected patients.
PMCID: PMC3018369  PMID: 21143863
9.  MNGIE Syndrome: Liver Cirrhosis Should Be Ruled Out Prior to Bone Marrow Transplantation 
JIMD Reports  2012;10:41-44.
Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is an autosomal recessive mitochondriopathy caused by loss-of-function mutations in the thymidine phosphorylase gene. The disease leads to premature death and is characterized by gastrointestinal dysmotility and cachexia, external ophthalmoplegia, a sensorimotor neuropathy, and leukoencephalopathy. Bone marrow transplantation (BMT) is the only potentially curative treatment that can achieve a sustained biochemical correction of the metabolic imbalances.
We report a 23-year-old male homozygous for the c.866A > C, p.Glu289Ala mutation of the TYMP gene, who presented with fatty liver and cachexia. Laboratory examinations were unremarkable except for increased transaminase activities. Grade II fibrosis and steatosis was found in an initial and a follow-up liver biopsy 4 years later. Myeloablative conditioning and BMT was performed 10 years after initial presentation due to the progressive weight loss and polyneuropathy. Pre-transplant liver staging was normal except for an elevated transient elastography of 31.6 kPa. Severe ascites developed after transplantation and liver function deteriorated progressively to liver failure. Despite engraftment on day +15, the patient died on day +18 from liver failure. Autopsy revealed micronodular liver cirrhosis, and postmortem diagnosis of acute-on-chronic liver failure was done.
This case illustrates the difficulties and importance of diagnosing liver cirrhosis in MNGIE. Before BMT, patients must be carefully evaluated by transient elastography, liver biopsy, or assessment of hepatic venous pressure gradient. In patients with liver cirrhosis, further studies should evaluate if liver transplantation may be an alternative to BMT. Considerable amounts of thymidine phosphorylase are expressed in liver tissue which may prevent accumulation of toxic metabolites.
PMCID: PMC3755566  PMID: 23430799
10.  Mitochondrial Neurogastrointestinal Encephalopathy Due to Mutations in RRM2B 
Archives of neurology  2009;66(8):1028-1032.
Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a progressive neurodegenerative disorder associated with thymidine phosphorylase deficiency resulting in high levels of plasma thymidine and a characteristic clinical phenotype.
To investigate the molecular basis of MNGIE in a patient with a normal plasma thymidine level.
Clinical, neurophysiological, and histopathological examinations as well as molecular and genetic analyses.
Nerve and muscle center and genetic clinic.
A 42-year-old woman with clinical findings strongly suggestive for MNGIE.
Main Outcome Measures
Clinical description of the disease and its novel genetic cause.
Identification of mitochondrial DNA depletion in muscle samples (approximately 12% of the control mean content) prompted us to look for other causes of our patient’s condition. Sequencing of genes associated with mitochondrial DNA depletion—POLG, PEO1, ANT1, SUCLG1, and SUCLA2—did not reveal deleterious mutations. Results of sequencing and array comparative genomic hybridization of the mitochondrial DNA for point mutations and deletions in blood and muscle were negative. Sequencing of RRM2B, a gene encoding cytosolic p53-inducible ribonucleoside reductase small subunit (RIR2B), revealed 2 pathogenic mutations, c.329G>A (p.R110H) and c.362G>A (p.R121H). These mutations are predicted to affect the docking interface of the RIR2B homodimer and likely result in impaired enzyme activity.
This study expands the clinical spectrum of impaired RIR2B function, challenges the notion of locus homogeneity of MNGIE, and sheds light on the pathogenesis of conditions involved in the homeostasis of the mitochondrial nucleotide pool. Our findings suggest that patients with MNGIE who have normal thymidine levels should be tested for RRM2B mutations.
PMCID: PMC2747647  PMID: 19667227
11.  Thymidine and deoxyuridine accumulate in tissues of patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) 
FEBS letters  2007;581(18):3410-3414.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease due to ECGF1 gene mutations causing thymidine phosphorylase (TP) deficiency. Analysis of post-mortem samples of five MNGIE patients and two controls, revealed TP activity in all control tissues, but not in MNGIE samples. Converse to TP activity, thymidine and deoxyuridine were absent in control samples, but present in all tissues of MNGIE patients. Concentrations of both nucleosides in the tissues were generally higher than those observed in plasma of MNGIE patients. Our observations indicate that in the absence of TP activity, tissues accumulate nucleosides, which are excreted into plasma.
PMCID: PMC1986782  PMID: 17612528
Mitochondria; MNGIE; thymidine phosphorylase; thymidine; deoxyuridine
12.  Defects in Mitochondrial DNA Replication and Human Disease 
Mitochondrial DNA (mtDNA) is replicated by the DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single stranded DNA binding protein, topoisomerase, and initiating factors. Nucleotide precursors for mtDNA replication arise from the mitochondrial salvage pathway originating from transport of nucleosides, or alternatively from cytoplasmic reduction of ribonucleotides. Defects in mtDNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mtDNA deletions, point mutations, or depletion which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mtDNA depletion syndromes (MDS) such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders, such as progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). This review focuses on our current knowledge of genetic defects of mtDNA replication (POLG, POLG2, C10orf2) and nucleotide metabolism (TYMP, TK2, DGOUK, and RRM2B) that cause instability of mtDNA and mitochondrial disease.
PMCID: PMC3244805  PMID: 22176657
DNA polymerase γ; mitochondrial DNA replication; nucleotide pools; mitochondrial DNA depletion syndrome; progressive external ophthalmoplegia; ataxia-neuropathy
13.  Sensory ataxic neuropathy with dysarthria/dysphagia and ophthalmoplegia (SANDO). Two case reports 
Acta Myologica  2011;30(3):188-190.
Case histories of two unrelated patients suffering from sensory ataxic neuropathy, dysarthria/ dysphagia and external ophthalmoplegia (SANDO) are reported. Both patients showed compound heterozygosity for POLG1 gene mutations, and presented with symptom of the clinical characteristics of SANDO. A patient with a p.A467T and p.W748S, well-known mutations showed a progressive course with early onset and multisystem involvement, including symptoms characteristics for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). The second patient showed a less well-known p.T251I and p.G848S mutations with late onset and dysphagia/dysarthria dominated, moderate symptoms. This later is the second published case history, when these POLG1 gene mutations are the possible background of late onset SANDO, dominantly presenting with bulbar symptoms.
PMCID: PMC3298093  PMID: 22616202
SANDO; heterozygote POLG1 mutations
14.  Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency 
Journal of Clinical Investigation  2003;111(12):1913-1921.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5′-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations.
PMCID: PMC161426  PMID: 12813027
15.  Pathogenesis and treatment of mitochondrial myopathies: recent advances  
Acta Myologica  2010;29(2):333-338.
In this brief review, I have highlighted recent advances in several areas of mitochondrial medicine, including mtDNA-related diseases, mendelian mitochondrial encephalomyopathies, and therapy. The pathogenic mechanisms of mtDNA mutations, especially those affecting mitochondrial protein synthesis, are still largely unknown. The pathogenicity of homoplasmic mtDNA mutations has become evident but has also called attention to modifying nuclear genes, yet another example of impaired intergenomic signaling. The functional significance of the homoplasmic changes associated with mitochondrial haplogroups has been confirmed. Among the mendelian disorders, a new form of “indirect hit” has been described, in which the ultimate pathogenesis is toxic damage to the respiratory chain. Three therapeutic strategies look promising: (i) allogeneic hematopoietic stem cell transplantation in MNGIE (mitochondrial neurogastrointestinal encephalomyopathy); (ii) bezafibrate, an activator of PGC-1α, has proven effective in animal models of mitochondrial myopathy; and (iii) pronucleus transfer into a normal oocyte is effective in eliminating maternal transmission of mtDNA, thus preventing the appearance of mtDNA-related disorders.
PMCID: PMC3040593  PMID: 21314015
mtDNA-related disorders; mendelian mitochondrial disorders; homoplasmy; pathogenesis; therapy
16.  Survey of human mitochondrial diseases using new genomic/proteomic tools 
Genome Biology  2001;2(6):research0021.1-research0021.16.
We have constructed Bayesian prior-based, amino-acid sequence profiles for the complete yeast mitochondrial proteome and used them to develop methods for identifying and characterizing the context of protein mutations that give rise to human mitochondrial diseases. (Bayesian priors are conditional probabilities that allow the estimation of the likelihood of an event - such as an amino-acid substitution - on the basis of prior occurrences of similar events.) Because these profiles can assemble sets of taxonomically very diverse homologs, they enable identification of the structurally and/or functionally most critical sites in the proteins on the basis of the degree of sequence conservation. These profiles can also find distant homologs with determined three-dimensional structures that aid in the interpretation of effects of missense mutations.
This survey reports such an analysis for 15 missense mutations, one insertion and three deletions involved in Leber's hereditary optic neuropathy, Leigh syndrome, mitochondrial neurogastrointestinal encephalomyopathy, Mohr-Tranebjaerg syndrome, iron-storage disorders related to Friedreich's ataxia, and hereditary spastic paraplegia. We present structural correlations for seven of the mutations.
Of the 19 mutations analyzed, 14 involved changes in very highly conserved parts of the affected proteins. Five out of seven structural correlations provided reasonable explanations for the malfunctions. As additional genetic and structural data become available, this methodology can be extended. It has the potential for assisting in identifying new disease-related genes. Furthermore, profiles with structural homologs can generate mechanistic hypotheses concerning the underlying biochemical processes - and why they break down as a result of the mutations.
PMCID: PMC33397  PMID: 11423010
17.  Nuclear factors involved in mitochondrial translation cause a subgroup of combined respiratory chain deficiency 
Brain  2010;134(1):183-195.
Mutations in several mitochondrial DNA and nuclear genes involved in mitochondrial protein synthesis have recently been reported in combined respiratory chain deficiency, indicating a generalized defect in mitochondrial translation. However, the number of patients with pathogenic mutations is small, implying that nuclear defects of mitochondrial translation are either underdiagnosed or intrauterine lethal. No comprehensive studies have been reported on large cohorts of patients with combined respiratory chain deficiency addressing the role of nuclear genes affecting mitochondrial protein synthesis to date. We investigated a cohort of 52 patients with combined respiratory chain deficiency without causative mitochondrial DNA mutations, rearrangements or depletion, to determine whether a defect in mitochondrial translation defines the pathomechanism of their clinical disease. We followed a combined approach of sequencing known nuclear genes involved in mitochondrial protein synthesis (EFG1, EFTu, EFTs, MRPS16, TRMU), as well as performing in vitro functional studies in 22 patient cell lines. The majority of our patients were children (<15 years), with an early onset of symptoms <1 year of age (65%). The most frequent clinical presentation was mitochondrial encephalomyopathy (63%); however, a number of patients showed cardiomyopathy (33%), isolated myopathy (15%) or hepatopathy (13%). Genomic sequencing revealed compound heterozygous mutations in the mitochondrial transfer ribonucleic acid modifying factor (TRMU) in a single patient only, presenting with early onset, reversible liver disease. No pathogenic mutation was detected in any of the remaining 51 patients in the other genes analysed. In vivo labelling of mitochondrial polypeptides in 22 patient cell lines showed overall (three patients) or selective (four patients) defects of mitochondrial translation. Immunoblotting for mitochondrial proteins revealed decreased steady state levels of proteins in some patients, but normal or increased levels in others, indicating a possible compensatory mechanism. In summary, candidate gene sequencing in this group of patients has a very low detection rate (1/52), although in vivo labelling of mitochondrial translation in 22 patient cell lines indicate that a nuclear defect affecting mitochondrial protein synthesis is responsible for about one-third of combined respiratory chain deficiencies (7/22). In the remaining patients, the impaired respiratory chain activity is most likely the consequence of several different events downstream of mitochondrial translation. Clinical classification of patients with biochemical analysis, genetic testing and, more importantly, in vivo labelling and immunoblotting of mitochondrial proteins show incoherent results, but a systematic review of these data in more patients may reveal underlying mechanisms, and facilitate the identification of novel factors involved in combined respiratory chain deficiency.
PMCID: PMC3707321  PMID: 21169334
mitochondrial translation; combined respiratory chain deficiency; early-onset encephalomyopathy
18.  Targeted Deletion of Both Thymidine Phosphorylase and Uridine Phosphorylase and Consequent Disorders in Mice 
Molecular and Cellular Biology  2002;22(14):5212-5221.
Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP−/− UP−/−) mice. TP activities were inhibited in TP−/− UP−/− mice, and the level of thymidine in the plasma of TP−/− UP−/− mice was higher than for TP−/− mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP−/− UP−/− mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T2 maps in the brain and axonal edema by electron microscopic study of the brain in TP−/− UP−/− mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.
PMCID: PMC139774  PMID: 12077348
19.  Mitochondrial enteropathy: the primary pathology may not be within the gastrointestinal tract 
Gut  2001;48(1):121-124.
BACKGROUND—Mitochondrial DNA (mtDNA) defects are an important cause of disease. Although gastrointestinal symptoms are common in these patients, their pathogenesis remains uncertain.
AIM—To investigate the role of the mtDNA defect in the production of gastrointestinal dysfunction.
PATIENT—A 20 year old woman who presented at 15 years of age with recurrent vomiting and pseudo-obstruction, who did not respond to conservative management and ultimately had subtotal gastrectomy and Roux-en-y reconstruction. She subsequently presented with status epilepticus and was found to have a mitochondrial respiratory chain disorder due to a pathogenic mtDNA point mutation (A3243G).
METHODS—Resected bowel was studied using light and electron microscopy and mtDNA analysed from both mucosal and muscular layers using polymerase chain reaction generated RFLP analysis. 
RESULTS— Histological and electron microscopic studies revealed no morphological abnormalities in the resected stomach, and molecular genetic analysis failed to identify the genetic defect in either the mucosal or muscle layers.
CONCLUSION—This study suggests that in some individuals with gastrointestinal symptoms associated with established mitochondrial DNA disease, the primary pathology of the mitochondrial enteropathy lies outside the gastrointestinal tract.

Keywords: mitochondrial encephalomyopathy; cyclical vomiting; pseudo-obstruction
PMCID: PMC1728165  PMID: 11115833
20.  Analysis of Mitochondrial DNA Sequences in Childhood Encephalomyopathies Reveals New Disease-Associated Variants 
PLoS ONE  2007;2(9):e942.
Mitochondrial encephalomyopathies are a heterogeneous group of clinical disorders generally caused due to mutations in either mitochondrial DNA (mtDNA) or nuclear genes encoding oxidative phosphorylation (OXPHOS). We analyzed the mtDNA sequences from a group of 23 pediatric patients with clinical and morphological features of mitochondrial encephalopathies and tried to establish a relationship of identified variants with the disease.
Methodology/Principle Findings
Complete mitochondrial genomes were amplified by PCR and sequenced by automated DNA sequencing. Sequencing data was analyzed by SeqScape software and also confirmed by BLASTn program. Nucleotide sequences were compared with the revised Cambridge reference sequence (CRS) and sequences present in mitochondrial databases. The data obtained shows that a number of known and novel mtDNA variants were associated with the disease. Most of the non-synonymous variants were heteroplasmic (A4136G, A9194G and T11916A) suggesting their possibility of being pathogenic in nature. Some of the missense variants although homoplasmic were showing changes in highly conserved amino acids (T3394C, T3866C, and G9804A) and were previously identified with diseased conditions. Similarly, two other variants found in tRNA genes (G5783A and C8309T) could alter the secondary structure of Cys-tRNA and Lys-tRNA. Most of the variants occurred in single cases; however, a few occurred in more than one case (e.g. G5783A and A10149T).
Conclusions and Significance
The mtDNA variants identified in this study could be the possible cause of mitochondrial encephalomyopathies with childhood onset in the patient group. Our study further strengthens the pathogenic score of known variants previously reported as provisionally pathogenic in mitochondrial diseases. The novel variants found in the present study can be potential candidates for further investigations to establish the relationship between their incidence and role in expressing the disease phenotype. This study will be useful in genetic diagnosis and counseling of mitochondrial diseases in India as well as worldwide.
PMCID: PMC1976591  PMID: 17895983
21.  Inherited Mitochondrial Diseases of DNA Replication 
Annual review of medicine  2008;59:131-146.
Mitochondrial genetic diseases can result from defects in mitochondrial DNA (mtDNA) in the form of deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These mutations may be spontaneous, maternally inherited, or a result of inherited nuclear defects in genes that maintain mtDNA. This review focuses on our current understanding of nuclear gene mutations that produce mtDNA alterations and cause mitochondrial depletion syndrome (MDS), progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). To date, all of these etiologic nuclear genes fall into one of two categories: genes whose products function directly at the mtDNA replication fork, such as POLG, POLG2, and TWINKLE, or genes whose products supply the mitochondria with deoxynucleotide triphosphate pools needed for DNA replication, such as TK2, DGUOK, TP, SUCLA2, ANT1, and possibly the newly identified MPV17.
PMCID: PMC2271032  PMID: 17892433
DNA polymerase γ; nucleotide pools; mitochondrial DNA depletion syndrome; progressive external ophthalmoplegia; ataxia-neuropathy
22.  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
23.  Natural history of MELAS associated with mitochondrial DNA m.3243A>G genotype 
Neurology  2011;77(22):1965-1971.
To describe the natural history of clinical and laboratory features associated with the m.3243A>G mitochondrial DNA point mutation. Natural history data are needed to obtain prognostic information and for clinical trial planning.
We included 85 matrilineal relatives from 35 families with at least 2 visits in this prospective cohort study. Thirty-one were fully symptomatic with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), and 54 were carrier relatives. Evaluations included standardized questionnaires (medical history and daily living functioning), physical examination, neuropsychological testing, and a battery of imaging and laboratory tests. We evaluated changes in clinical and laboratory features over time and survival. Outcomes are reported over a follow-up period of up to 10.6 years (mean 3.8 ± 2.2 years for patients and 5.5 ± 3.0 for carrier relatives).
Neurologic examination, neuropsychological testing, and daily living scores significantly declined in all patients with MELAS, whereas no significant deterioration occurred in carrier relatives. Cerebral MRI scores declined significantly in patients with MELAS. Magnetic resonance spectroscopy estimates of lactate in the lateral ventricles increased over time, and high lactate was associated with increased mortality. Symptom onset in childhood often was associated with worse outcome. Patients with MELAS had a greater death rate than carrier relatives.
Patients with MELAS carrying the m.3243A>G mutation show a measurable decline in clinical and imaging outcomes. It is hoped that these data will be helpful in anticipating the disease course and in planning clinical trials for MELAS.
PMCID: PMC3235358  PMID: 22094475
24.  Recurrent ischemic strokes in a young celiac woman with MTHFR gene mutation 
Celiac disease (CD) is frequently associated with neurological disorders, but very few reports concern the association with ischemic stroke. A 26-year-old woman affected by CD with secondary amenorrhea, carrier of a homozygous 5,10-methylenetetrahydrofolate reductase mutation with hyperhomocysteinemia, was affected by two occipital ischemic strokes within a period of 5 mo. At the time of the second stroke, while she was being treated with folic acid, acetylsalicylic acid and a gluten-free diet, she had left hemianopsia, left hemiparesthesias, and gait imbalance. Brain magnetic resonance imaging showed a subacute right occipital ischemic lesion, which was extended to the dorsal region of the right thalamus and the ipsilateral thalamo-capsular junction. Antitransglutaminase and deamidated gliadin peptide antibodies were no longer present, while antinuclear antibodies, antineuronal antibodies and immune circulating complexes were only slightly elevated. Since the patient was taking folic acid, her homocysteine ​​levels were almost normal and apparently not sufficient alone to explain the clinical event. A conventional cerebral angiography showed no signs of vasculitis. Finally, rare causes of occipital stroke in young patients, such as Fabry’s disease and mitochondrial myopathy, encephalomyopathy, lactic acidosis and stroke-like symptoms, were also excluded by appropriate tests. Thus, the most probable cause for the recurrent strokes in this young woman remained CD, although the mechanisms involved are still unknown. The two main hypotheses concern malabsorption (with consequent deficiency of vitamins known to exert neurotrophic and neuroprotective effects) and immune-mediated mechanisms. CD should be kept in mind in the differential diagnosis of ischemic stroke in young patients.
PMCID: PMC3396202  PMID: 22807619
Celiac disease; Female; Methylenetetrahydrofolate reductase; Stroke; Vasculitis; Young
25.  Encephalomyopathies caused by abnormal nuclear-mitochondrial intergenomic cross-talk 
Acta Myologica  2009;28(1):2-11.
Autosomal dominant Progressive External Ophthalmoplegias are Mendelian disorders characterized by the accumulation of multiple deletions of mitochondrial DNA in critical tissues. Most of the Autosomal dominant Progressive External Ophthalmoplegias families carry heterozygous mutations in one of three genes: ANT1, encoding the muscle-heart specific mitochondrial adenine nucleotide translocator, Twinkle, encoding the mitochondrial DNA helicase, and POLG1, encoding the catalytic subunit of the mitochondrial DNA-specific polymerase. Mutations in both POLG1 alleles are also found in autosomal recessive Progressive External Ophthalmoplegias sibships with multiple affected members and in apparently sporadic cases. In addition, recessive POLG1 mutations are responsible for three additional diseases: Alpers-Huttenlocher hepatopathic poliodystrophy, Sensory-Ataxic Neuropathy Dysarthria and Ophthalmoplegia and juvenile SpinoCerebellar Ataxia-Epilepsy syndrome. Mitochondrial neuro-gastro-intestinal encephalomyopathy is an autosomal recessive disorder of juvenile onset, caused by mutations in the gene encoding Thymidine Phosphorylase. Thymidine Phosphorylase is involved in the control and maintenance of the pyrimidine nucleoside pool of the cell. Finally, mitochondrial DNA depletion syndrome is a heterogeneous group of disorders characterized by a reduction in mitochondrial DNA copy number. Clinically, they include a myopathic form, a more generalized encephalomyopathic form and a fatal infantile hepato-cerebral syndrome leading to rapidly progressive liver and brain failure. To date, eight genes have been associated with mitochondrial DNA depletion syndrome. Novel disease genes have recently been added to this list, including OPA1 and GFER, and new clinical variants add further complexity to this expanding area of mitochondrial medicine.
PMCID: PMC2859628  PMID: 19772189
Mitochondrial DNA; oxidative phosphorylation; mitochondrial disorders; MtDNA multiple deletions; MtDNA depletion

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