antonella Spinazzola | Medical Research Council (original) (raw)

Papers by antonella Spinazzola

Research paper thumbnail of Amino Acid Starvation Has Opposite Effects on Mitochondrial and Cytosolic Protein Synthesis

PLoS ONE, 2014

Amino acids are essential for cell growth and proliferation for they can serve as precursors of p... more Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation.

Research paper thumbnail of Amino Acid Starvation Has Opposite Effects on Mitochondrial and Cytosolic Protein Synthesis

Research paper thumbnail of Oligomycin Induces a Decrease in the Cellular Content of a Pathogenic Mutation in the Human Mitochondrial ATPase 6 Gene

Journal of Biological Chemistry, 1999

A T 3 G mutation at position 8993 in human mitochondrial DNA is associated with the syndrome neur... more A T 3 G mutation at position 8993 in human mitochondrial DNA is associated with the syndrome neuropathy, ataxia, and retinitis pigmentosa and with a maternally inherited form of Leigh's syndrome. The mutation substitutes an arginine for a leucine at amino acid position 156 in ATPase 6, a component of the F 0 portion of the mitochondrial ATP synthase complex. Fibroblasts harboring high levels of the T8993G mutation have decreased ATP synthesis activity, but do not display any growth defect under standard culture conditions. Combining the notions that cells with respiratory chain defects grow poorly in medium containing galactose as the major carbon source, and that resistance to oligomycin, a mitochondrial inhibitor, is associated with mutations in the ATPase 6 gene in the same transmembrane domain where the T8993G amino acid substitution is located, we created selective culture conditions using galactose and oligomycin that elicited a pathological phenotype in T8993G cells and that allowed for the rapid selection of wild-type over T8993G mutant cells. We then generated cytoplasmic hybrid clones containing heteroplasmic levels of the T8993G mutation, and showed that selection in galactose-oligomycin caused a significant increase in the fraction of wild-type molecules (from 16 to 28%) in these cells.

Research paper thumbnail of Altered Thymidine Metabolism Due to Defects of Thymidine Phosphorylase

Journal of Biological Chemistry, 2002

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human dis... more Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human disease due to mutations in the thymidine phosphorylase (TP) gene. TP enzyme catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxy-D-ribose 1-phosphate. We present evidence that thymidine metabolism is altered in MNGIE. TP activities in buffy coats were reduced drastically in all 27 MNGIE patients compared with 19 controls. All MNGIE patients had much higher plasma levels of thymidine than normal individuals and asymptomatic TP mutation carriers. In two patients, the renal clearance of thymidine was ϳ20% that of creatinine, and because hemodialysis demonstrated that thymidine is ultrafiltratable, most of the filtered thymidine is likely to be reabsorbed by the kidney. In vitro, fibroblasts from controls catabolized thymidine in medium; by contrast, MNGIE fibroblasts released thymidine. In MNGIE, severe impairment of TP enzyme activity leads to increased plasma thymidine. In patients who are suspected of having MNGIE, determination of TP activity in buffy coats and thymidine levels in plasma are diagnostic. We hypothesize that excess thymidine alters mitochondrial nucleoside and nucleotide pools leading to impaired mitochondrial DNA replication, repair, or both. Therapies to reduce thymidine levels may be beneficial to MNGIE patients.

Research paper thumbnail of Thymidine Phosphorylase Deficiency Causes MNGIE: An Autosomal Recessive Mitochondrial Disorder

Nucleosides Nucleotides & Nucleic Acids, 2004

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder ... more Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the gene encoding thymidine phosphorylase (TP). The disease is characterized clinically by impaired eye movements, gastrointestinal dysmotility, cachexia, peripheral neuropathy, myopathy, and leukoencephalopathy. Molecular genetic studies of MNGIE patients' tissues have revealed multiple deletions, depletion, and site-specific point mutations of mitochondrial DNA. TP is a cytosolic enzyme required for nucleoside homeostasis. In MNGIE, TP activity is severely reduced and consequently levels of thymidine and deoxyuridine in plasma are dramatically elevated. We have hypothesized that the increased levels of intracellular thymidine and deoxyuridine cause imbalances of mitochondrial nucleotide pools that, in turn, lead to the mtDNA abnormalities. MNGIE was the first molecularly characterized genetic disorder caused by abnormal mitochondrial nucleoside/nucleotide metabolism. Future studies are likely to reveal further insight into this expanding group of diseases.

Research paper thumbnail of Thymidine Phosphorylase Gene Mutations in MNGIE, a Human Mitochondrial Disorder

Science, 1999

9. Phylum-level signatures occurred at the following positions (Escherichia coli numbering): 358 ... more 9. Phylum-level signatures occurred at the following positions (Escherichia coli numbering): 358 (C), 359 (U), 361 (A), 396 (G), and 555 (U). Treponema genuslevel signatures occurred at 108 (G) and 111 (U) [B. J. Paster et al., J. Bacteriol. 173, 6101 (1991)]. 10. E. Stackebrandt and B. M. Goebel, Int. J. Syst. Bacteriol. 44, 846 (1994). 11. RFS clones [from Reticulitermes flavipes (Kollar) (Rhinotermitidae)] and clone ZAS89 were from T. G. Lilburn, T. M. Schmidt, and J. A. Breznak (in preparation); sp40 clones were from Mastotermes darwiniensis Froggatt (Mastotermitidae) (5); and clone NL1 was from Nasutitermes lujae (Wasmann) (Nasutitermitinae) (2). 11. YA was prepared by allowing 56 g of dry baker's yeast (Red Star Yeast and Products, Milwaukee, WI) to autolyze in 200 ml of distilled water for 24 hours at 56°C. Cell debris was removed by centrifugation, and the supernatant was neutralized with 5N NaOH and filter sterilized. Cofactor solution contained pyridoxal HCl and pyridoxal phosphate (250 g/ml each); calcium folinic acid, ␤-NAD, coenzyme A, and FAD (50 g/ml each); nicotinamide (25 g/ml); folic acid (2.5 g/ml); riboflavin (0.5 g/ml); hemin (in 10 mM NaOH) (65 g/ml); and thiamine pyrophosphate (2500 g/ml); and was filter sterilized. Replacement of rumen fluid and nutrient broth in modified JM-4 medium (6) with 2% (by volume) each of YA and cofactor solution yielded 2YACo medium; increasing YA to 4% yielded 4YACo medium. 13. Commercial preparations tested included yeast extract (Difco Laboratories,

Research paper thumbnail of EFNS guidelines on the molecular diagnosis of mitochondrial disorders

European Journal of Neurology, 2009

Objectives: These European Federation of Neurological Sciences (EFNS) guidelines are designed to... more Objectives: These European Federation of Neurological Sciences (EFNS) guidelines are designed to provide practical help for the general neurologist to make appropriate use of molecular genetics for diagnosing mitochondrial disorders (MIDs), which gain increasing attention and are more frequently diagnosed due to improved diagnostic tools.Background: Since the publication of the first EFNS guidelines on the molecular diagnosis of inherited neurological diseases in 2001, rapid progress has been made in this field, necessitating the creation of an updated version.Search strategy: To collect data about the molecular diagnosis of MIDs search for literature in various electronic databases, such as Cochrane library, MEDLINE, OMIM, GENETEST or Embase, were carried out and original papers, meta-analyses, review papers, and guideline recommendations were reviewed.Results: The guidelines summarise the possibilities and limitations of molecular genetic diagnosis of MIDs and provide practical recommendations and diagnostic criteria in accordance with the EFNS Scientific Committee to guide the molecular diagnostic work-up of MIDs.Recommendations: The proposed guidelines suggest an approach to the molecular diagnosis of MIDs in a manner accessible to general neurologists.

Research paper thumbnail of Mitochondrial myopathy and ophthalmoplegia in a sporadic patient with the 5698G→A mitochondrial DNA mutation

Neuromuscular Disorders, 2004

A 21-year-old woman described proximal muscle weakness since early childhood. At age 16, she deve... more A 21-year-old woman described proximal muscle weakness since early childhood. At age 16, she developed bilateral ptosis, progressive external ophthalmoplegia, and exercise intolerance. She harbored a heteroplasmic G12315A mutation in the mitochondrial DNA tRNA Leu(CUN) gene, which disrupts a highly conserved G-C base pair in the TCC stem of the molecule. Mutant mitochondrial DNA was 62% of total in muscle and 17% in blood. The mutation was undetectable in blood, urinary sediment, and hair follicles from the patient's mother. This second patient with G12315A and progressive external ophthalmoplegia confirms the pathogenicity of the mutation and helps to define the correlation between genotype and phenotype. q

Research paper thumbnail of Lack of founder effect for an identical mtDNA depletion syndrome (MDS)-associated MPV17 mutation shared by Navajos and Italians

Neuromuscular Disorders, 2008

Navajo neurohepatopathy is a hepato-cerebral variant of mitochondrial DNA depletion syndrome due ... more Navajo neurohepatopathy is a hepato-cerebral variant of mitochondrial DNA depletion syndrome due to a specific mutation in MPV17, a gene located on human chromosome 2p. The same mutation was reported in an Italian family. To understand whether the MPV17 mutation was transmitted by descent from a common ancestor to Navajos and Italians we constructed a dense haplotype of the MPV17 locus using suitable single nucleotide polymorphisms. Complete discordance between Italian and Navajo haplotypes rules out the former hypothesis, suggesting that the mutation occurred independently in the two populations.

Research paper thumbnail of Mitochondrial disorders

Current Neurology and Neuroscience Reports, 2003

We present here a discussion on the most relevant recent publications on mitochondrial disease. I... more We present here a discussion on the most relevant recent publications on mitochondrial disease. In addition to many papers concerning the description of the genotype-to-phenotype correlations in mitochondrial DNA-related disorders, this very broad area of neurogenetics includes a number of novel observations on the basic aspects of mitochondrial biogenesis that can be relevant in explaining the molecular mechanisms of mitochondrial abnormalities. The completion of the human genome project and the wealth of knowledge gained on the genetics of oxidative phosphorylation in yeast have promoted a substantial acceleration in the discovery of a remarkable number of nuclear genes associated with specific mitochondrial disorders. A further development of these contributions has been the generation of several cellular and animal models of disease that can now be exploited for testing both pathogenetic hypotheses and therapeutic strategies. Most of the latter are based on the use of chemical compounds aimed at reducing the negative impact of mitochondrial defects on both energy production and generation of reactive oxygen species. The first successful attempts for gene therapy of some mitochondrial diseases have recently been achieved and will hopefully increase in the near future.

Research paper thumbnail of Clinical and molecular features of mitochondrial DNA depletion syndromes

Journal of Inherited Metabolic Disease, 2009

Mitochondrial DNA depletion syndromes (MDSs) form a group of autosomal recessive disorders charac... more Mitochondrial DNA depletion syndromes (MDSs) form a group of autosomal recessive disorders characterized by profoundly decreased mitochondrial DNA copy numbers in affected tissues. Three main clinical presentations are known: myopathic, encephalomyopathic and hepatocerebral. The first is associated with mutations in thymidine kinase 2 (TK2) and p53-induced ribonucleotide reductase B subunit (RRM2B); the second with mutations in succinate synthase A (SUCLA2) and B (SUCLG1); the third with mutations in Twinkle (PEO1), pol-γA (POLG1), deoxyguanosine kinase (DGUOK) and MPV17 (MPV17). In this work, we review the MDS-associated phenotypes and present our own experience of 32 MDS patients, with the aim of defining the mutation frequency of the known genes, the clinical spectrum of the diseases, and the genotype–phenotype correlations. Five of our patients carried previously unreported mutations in one of the eight MDS genes.

Research paper thumbnail of Glucose metabolism and diet-based prevention of liver dysfunction in MPV17 mutant patients

Journal of Hepatology, 2009

Research paper thumbnail of Definitive Diagnosis of Mitochondrial Neurogastrointestinal Encephalomyopathy by Biochemical Assays

Background: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations ... more Background: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in the gene encoding thymidine phosphorylase (TP). The clinical manifestations of MNGIE are recognizable and homogeneous, but in the early stages, the disease is often misdiagnosed. This study assesses the reliability of biochemical assays to diagnose MNGIE. Methods: We studied 180 patients with clinical features suggestive of MNGIE, 14 asymptomatic TP mutation carriers, and 20 controls. TP enzyme activity in the buffy coat was determined by a fixed-time method, and the plasma nucleosides thymidine (dThd) and deoxyuridine (dUrd) were assessed by a gradient-elution reversed phase HPLC method. TP was sequenced through standard procedures in patients who met the clinical criteria for MNGIE. Results: Twenty-five of the 180 patients fulfilled the clinical criteria for MNGIE and had homozygous or compound heterozygous TP mutations. All had drastically decreased TP activity [mean (SD), 10 (15) nmol thymine formed ⅐ h ؊1 ⅐ (mg protein) ؊1 vs 634 (217) nmol thymine formed ⅐ h ؊1 ⅐ (mg protein) ؊1 for the controls]. Relative to the control mean, TP activities were reduced to 35% in mutation carriers and 65% in MNGIE-like patients. All 25 MNGIE patients had detectable plasma dThd [8.6 (3.4) mol/L] and dUrd [14.2 (4.4) mol/L].

Research paper thumbnail of EFNS guidelines on the molecular diagnosis of neurogenetic disorders: general issues, Huntington’s disease, Parkinson’s disease and dystonias

European Journal of Neurology, 2009

Background and purpose: These EFNS guidelines on the molecular diagnosis of neurogenetic disorde... more Background and purpose: These EFNS guidelines on the molecular diagnosis of neurogenetic disorders are designed to provide practical help for the general neurologist to make appropriate use of molecular genetics in diagnosing neurogenetic disorders. Since the publication of the first two EFNS-guideline papers on the molecular diagnosis of neurological diseases in 2001, rapid progress has been made in this field, necessitating an updated series of guidelines.Methods: Literature searches were performed before expert members of the task force wrote proposals, which were discussed in detail until final consensus had been reached among all task force members.Results and conclusion: This paper provides updated guidelines for molecular diagnosis of Huntington’s disease, Parkinson’s disease and dystonias as well as a general introduction to the topic. Possibilities and limitations of molecular genetic diagnosis of these disorders are evaluated and recommendations are provided.

Research paper thumbnail of MNGIE: from nuclear DNA to mitochondrial DNA

Neuromuscular Disorders, 2001

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a unique autosomal recessive dis... more Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a unique autosomal recessive disorder with mitochondrial DNA alterations. The disease is characterized clinically by ptosis, progressive external ophthalmoparesis, gastrointestinal dysmotility, cachexia, peripheral neuropathy, and leukoencephalopathy. Muscle biopsies typically reveal mitochondrial abnormalities including ragged-red ®bers and focal cytochrome c oxidase de®ciency. Analysis of mitochondrial DNA in skeletal muscle shows partial depletion, multiple deletions, or both. To identify the cause of MNGIE, we mapped the disease locus to chromosome 22q13.32±qter. Within this region, we identi®ed the gene encoding thymidine phosphorylase as the MNGIE gene. We have identi®ed homozygous or compound-heterozygous thymidine phosphorylase gene mutations in 35 MNGIE patients (21 families) from diverse ethnic groups, including: Ashkenazi Jewish, Western European, Jamaican, Hispanic, and Japanese. We con®rmed pathogenicity of the mutations by a spectrophotometric assay of thymidine phosphorylase activity with peripheral leukocytes of 15 MNGIE patients. Thymidine phosphorylase enzymatic activity was severely reduced, thus enabling us to conclude that the loss-of-function mutations in thymidine phosphorylase gene cause MNGIE. Thymidine phosphorylase catabolizes thymidine to thymine. In agreement with this notion, we noted that plasma thymidine level is increased more than 20-fold in MNGIE patients compared to controls. Therefore, we have hypothesized that increased thymidine causes mitochondrial nucleotide pool imbalance which, in turn, leads to motochondrial DNA alterations, via a mitochondria-speci®c thymidine salvage pathway. The identi®cation of the MNGIE gene has allowed us to classify MNGIE as a disease of nucleoside dysmetabolism. We may be entering a new era of research on mitochondrial nucleoside metabolism. q

Research paper thumbnail of Mitochondrial neurogastrointestinal encephalomyopathy and thymidine metabolism: results and hypotheses

Mitochondrion, 2002

impairment is limited. 4 While larger studies are required to give evidence-based recommendations... more impairment is limited. 4 While larger studies are required to give evidence-based recommendations, perhaps physical aspects cannot fully explain the risk of falls in older adults.

Research paper thumbnail of 64 Systematic identification of human mitochondrial disease genes through integrative genomics

Research paper thumbnail of Disorders of Nuclear-Mitochondrial Intergenomic Communication

Bioscience Reports, 2007

In the course of evolution, mitochondria lost their independence, and mtDNA became “slave” of nDN... more In the course of evolution, mitochondria lost their independence, and mtDNA became “slave” of nDNA, depending on numerous nucleus-encoded factors for its integrity, replication and expression. Mutations in any of these factors may alter the cross-talk between the two genomes and cause diseases that affect mtDNA integrity or expression, being inherited as mendelian traits.

Research paper thumbnail of Assay of mitochondrial ATP synthesis in animal cells

Methods in Cell Biology, 2001

Research paper thumbnail of Early-onset liver mtDNA depletion and late-onset proteinuric nephropathy in Mpv17 knockout mice

Human Molecular Genetics, 2008

In humans, MPV17 mutations are responsible for severe mitochondrial depletion syndrome, mainly af... more In humans, MPV17 mutations are responsible for severe mitochondrial depletion syndrome, mainly affecting the liver and the nervous system. To gain insight into physiopathology of MPV17-related disease, we investigated an available Mpv17 knockout animal model. We found severe mtDNA depletion in liver and, albeit to a lesser extent, in skeletal muscle, whereas hardly any depletion was detected in brain and kidney, up to 1 year after birth. Mouse embryonic fibroblasts did show mtDNA depletion, but only after several culturing passages, or in a serumless culturing medium. In spite of severe mtDNA depletion, only moderate decrease in respiratory chain enzymatic activities, and mild cytoarchitectural alterations, were observed in the Mpv17 2/2 livers, but neither cirrhosis nor failure ever occurred in this organ at any age. The mtDNA transcription rate was markedly increased in liver, which could contribute to compensate the severe mtDNA depletion. This phenomenon was associated with specific downregulation of Mterf1, a negative modulator of mtDNA transcription. The most relevant clinical features involved skin, inner ear and kidney. The coat of the Mpv17 2/2 mice turned gray early in adulthood, and 18-month or older mice developed focal segmental glomerulosclerosis (FSGS) with massive proteinuria. Concomitant degeneration of cochlear sensory epithelia was reported as well. These symptoms were associated with significantly shorter lifespan. Coincidental with the onset of FSGS, there was hardly any mtDNA left in the glomerular tufts. These results demonstrate that Mpv17 controls mtDNA copy number by a highly tissue-and possibly cytotype-specific mechanism.

Research paper thumbnail of Amino Acid Starvation Has Opposite Effects on Mitochondrial and Cytosolic Protein Synthesis

PLoS ONE, 2014

Amino acids are essential for cell growth and proliferation for they can serve as precursors of p... more Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation.

Research paper thumbnail of Amino Acid Starvation Has Opposite Effects on Mitochondrial and Cytosolic Protein Synthesis

Research paper thumbnail of Oligomycin Induces a Decrease in the Cellular Content of a Pathogenic Mutation in the Human Mitochondrial ATPase 6 Gene

Journal of Biological Chemistry, 1999

A T 3 G mutation at position 8993 in human mitochondrial DNA is associated with the syndrome neur... more A T 3 G mutation at position 8993 in human mitochondrial DNA is associated with the syndrome neuropathy, ataxia, and retinitis pigmentosa and with a maternally inherited form of Leigh's syndrome. The mutation substitutes an arginine for a leucine at amino acid position 156 in ATPase 6, a component of the F 0 portion of the mitochondrial ATP synthase complex. Fibroblasts harboring high levels of the T8993G mutation have decreased ATP synthesis activity, but do not display any growth defect under standard culture conditions. Combining the notions that cells with respiratory chain defects grow poorly in medium containing galactose as the major carbon source, and that resistance to oligomycin, a mitochondrial inhibitor, is associated with mutations in the ATPase 6 gene in the same transmembrane domain where the T8993G amino acid substitution is located, we created selective culture conditions using galactose and oligomycin that elicited a pathological phenotype in T8993G cells and that allowed for the rapid selection of wild-type over T8993G mutant cells. We then generated cytoplasmic hybrid clones containing heteroplasmic levels of the T8993G mutation, and showed that selection in galactose-oligomycin caused a significant increase in the fraction of wild-type molecules (from 16 to 28%) in these cells.

Research paper thumbnail of Altered Thymidine Metabolism Due to Defects of Thymidine Phosphorylase

Journal of Biological Chemistry, 2002

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human dis... more Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human disease due to mutations in the thymidine phosphorylase (TP) gene. TP enzyme catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxy-D-ribose 1-phosphate. We present evidence that thymidine metabolism is altered in MNGIE. TP activities in buffy coats were reduced drastically in all 27 MNGIE patients compared with 19 controls. All MNGIE patients had much higher plasma levels of thymidine than normal individuals and asymptomatic TP mutation carriers. In two patients, the renal clearance of thymidine was ϳ20% that of creatinine, and because hemodialysis demonstrated that thymidine is ultrafiltratable, most of the filtered thymidine is likely to be reabsorbed by the kidney. In vitro, fibroblasts from controls catabolized thymidine in medium; by contrast, MNGIE fibroblasts released thymidine. In MNGIE, severe impairment of TP enzyme activity leads to increased plasma thymidine. In patients who are suspected of having MNGIE, determination of TP activity in buffy coats and thymidine levels in plasma are diagnostic. We hypothesize that excess thymidine alters mitochondrial nucleoside and nucleotide pools leading to impaired mitochondrial DNA replication, repair, or both. Therapies to reduce thymidine levels may be beneficial to MNGIE patients.

Research paper thumbnail of Thymidine Phosphorylase Deficiency Causes MNGIE: An Autosomal Recessive Mitochondrial Disorder

Nucleosides Nucleotides & Nucleic Acids, 2004

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder ... more Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the gene encoding thymidine phosphorylase (TP). The disease is characterized clinically by impaired eye movements, gastrointestinal dysmotility, cachexia, peripheral neuropathy, myopathy, and leukoencephalopathy. Molecular genetic studies of MNGIE patients' tissues have revealed multiple deletions, depletion, and site-specific point mutations of mitochondrial DNA. TP is a cytosolic enzyme required for nucleoside homeostasis. In MNGIE, TP activity is severely reduced and consequently levels of thymidine and deoxyuridine in plasma are dramatically elevated. We have hypothesized that the increased levels of intracellular thymidine and deoxyuridine cause imbalances of mitochondrial nucleotide pools that, in turn, lead to the mtDNA abnormalities. MNGIE was the first molecularly characterized genetic disorder caused by abnormal mitochondrial nucleoside/nucleotide metabolism. Future studies are likely to reveal further insight into this expanding group of diseases.

Research paper thumbnail of Thymidine Phosphorylase Gene Mutations in MNGIE, a Human Mitochondrial Disorder

Science, 1999

9. Phylum-level signatures occurred at the following positions (Escherichia coli numbering): 358 ... more 9. Phylum-level signatures occurred at the following positions (Escherichia coli numbering): 358 (C), 359 (U), 361 (A), 396 (G), and 555 (U). Treponema genuslevel signatures occurred at 108 (G) and 111 (U) [B. J. Paster et al., J. Bacteriol. 173, 6101 (1991)]. 10. E. Stackebrandt and B. M. Goebel, Int. J. Syst. Bacteriol. 44, 846 (1994). 11. RFS clones [from Reticulitermes flavipes (Kollar) (Rhinotermitidae)] and clone ZAS89 were from T. G. Lilburn, T. M. Schmidt, and J. A. Breznak (in preparation); sp40 clones were from Mastotermes darwiniensis Froggatt (Mastotermitidae) (5); and clone NL1 was from Nasutitermes lujae (Wasmann) (Nasutitermitinae) (2). 11. YA was prepared by allowing 56 g of dry baker's yeast (Red Star Yeast and Products, Milwaukee, WI) to autolyze in 200 ml of distilled water for 24 hours at 56°C. Cell debris was removed by centrifugation, and the supernatant was neutralized with 5N NaOH and filter sterilized. Cofactor solution contained pyridoxal HCl and pyridoxal phosphate (250 g/ml each); calcium folinic acid, ␤-NAD, coenzyme A, and FAD (50 g/ml each); nicotinamide (25 g/ml); folic acid (2.5 g/ml); riboflavin (0.5 g/ml); hemin (in 10 mM NaOH) (65 g/ml); and thiamine pyrophosphate (2500 g/ml); and was filter sterilized. Replacement of rumen fluid and nutrient broth in modified JM-4 medium (6) with 2% (by volume) each of YA and cofactor solution yielded 2YACo medium; increasing YA to 4% yielded 4YACo medium. 13. Commercial preparations tested included yeast extract (Difco Laboratories,

Research paper thumbnail of EFNS guidelines on the molecular diagnosis of mitochondrial disorders

European Journal of Neurology, 2009

Objectives: These European Federation of Neurological Sciences (EFNS) guidelines are designed to... more Objectives: These European Federation of Neurological Sciences (EFNS) guidelines are designed to provide practical help for the general neurologist to make appropriate use of molecular genetics for diagnosing mitochondrial disorders (MIDs), which gain increasing attention and are more frequently diagnosed due to improved diagnostic tools.Background: Since the publication of the first EFNS guidelines on the molecular diagnosis of inherited neurological diseases in 2001, rapid progress has been made in this field, necessitating the creation of an updated version.Search strategy: To collect data about the molecular diagnosis of MIDs search for literature in various electronic databases, such as Cochrane library, MEDLINE, OMIM, GENETEST or Embase, were carried out and original papers, meta-analyses, review papers, and guideline recommendations were reviewed.Results: The guidelines summarise the possibilities and limitations of molecular genetic diagnosis of MIDs and provide practical recommendations and diagnostic criteria in accordance with the EFNS Scientific Committee to guide the molecular diagnostic work-up of MIDs.Recommendations: The proposed guidelines suggest an approach to the molecular diagnosis of MIDs in a manner accessible to general neurologists.

Research paper thumbnail of Mitochondrial myopathy and ophthalmoplegia in a sporadic patient with the 5698G→A mitochondrial DNA mutation

Neuromuscular Disorders, 2004

A 21-year-old woman described proximal muscle weakness since early childhood. At age 16, she deve... more A 21-year-old woman described proximal muscle weakness since early childhood. At age 16, she developed bilateral ptosis, progressive external ophthalmoplegia, and exercise intolerance. She harbored a heteroplasmic G12315A mutation in the mitochondrial DNA tRNA Leu(CUN) gene, which disrupts a highly conserved G-C base pair in the TCC stem of the molecule. Mutant mitochondrial DNA was 62% of total in muscle and 17% in blood. The mutation was undetectable in blood, urinary sediment, and hair follicles from the patient's mother. This second patient with G12315A and progressive external ophthalmoplegia confirms the pathogenicity of the mutation and helps to define the correlation between genotype and phenotype. q

Research paper thumbnail of Lack of founder effect for an identical mtDNA depletion syndrome (MDS)-associated MPV17 mutation shared by Navajos and Italians

Neuromuscular Disorders, 2008

Navajo neurohepatopathy is a hepato-cerebral variant of mitochondrial DNA depletion syndrome due ... more Navajo neurohepatopathy is a hepato-cerebral variant of mitochondrial DNA depletion syndrome due to a specific mutation in MPV17, a gene located on human chromosome 2p. The same mutation was reported in an Italian family. To understand whether the MPV17 mutation was transmitted by descent from a common ancestor to Navajos and Italians we constructed a dense haplotype of the MPV17 locus using suitable single nucleotide polymorphisms. Complete discordance between Italian and Navajo haplotypes rules out the former hypothesis, suggesting that the mutation occurred independently in the two populations.

Research paper thumbnail of Mitochondrial disorders

Current Neurology and Neuroscience Reports, 2003

We present here a discussion on the most relevant recent publications on mitochondrial disease. I... more We present here a discussion on the most relevant recent publications on mitochondrial disease. In addition to many papers concerning the description of the genotype-to-phenotype correlations in mitochondrial DNA-related disorders, this very broad area of neurogenetics includes a number of novel observations on the basic aspects of mitochondrial biogenesis that can be relevant in explaining the molecular mechanisms of mitochondrial abnormalities. The completion of the human genome project and the wealth of knowledge gained on the genetics of oxidative phosphorylation in yeast have promoted a substantial acceleration in the discovery of a remarkable number of nuclear genes associated with specific mitochondrial disorders. A further development of these contributions has been the generation of several cellular and animal models of disease that can now be exploited for testing both pathogenetic hypotheses and therapeutic strategies. Most of the latter are based on the use of chemical compounds aimed at reducing the negative impact of mitochondrial defects on both energy production and generation of reactive oxygen species. The first successful attempts for gene therapy of some mitochondrial diseases have recently been achieved and will hopefully increase in the near future.

Research paper thumbnail of Clinical and molecular features of mitochondrial DNA depletion syndromes

Journal of Inherited Metabolic Disease, 2009

Mitochondrial DNA depletion syndromes (MDSs) form a group of autosomal recessive disorders charac... more Mitochondrial DNA depletion syndromes (MDSs) form a group of autosomal recessive disorders characterized by profoundly decreased mitochondrial DNA copy numbers in affected tissues. Three main clinical presentations are known: myopathic, encephalomyopathic and hepatocerebral. The first is associated with mutations in thymidine kinase 2 (TK2) and p53-induced ribonucleotide reductase B subunit (RRM2B); the second with mutations in succinate synthase A (SUCLA2) and B (SUCLG1); the third with mutations in Twinkle (PEO1), pol-γA (POLG1), deoxyguanosine kinase (DGUOK) and MPV17 (MPV17). In this work, we review the MDS-associated phenotypes and present our own experience of 32 MDS patients, with the aim of defining the mutation frequency of the known genes, the clinical spectrum of the diseases, and the genotype–phenotype correlations. Five of our patients carried previously unreported mutations in one of the eight MDS genes.

Research paper thumbnail of Glucose metabolism and diet-based prevention of liver dysfunction in MPV17 mutant patients

Journal of Hepatology, 2009

Research paper thumbnail of Definitive Diagnosis of Mitochondrial Neurogastrointestinal Encephalomyopathy by Biochemical Assays

Background: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations ... more Background: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in the gene encoding thymidine phosphorylase (TP). The clinical manifestations of MNGIE are recognizable and homogeneous, but in the early stages, the disease is often misdiagnosed. This study assesses the reliability of biochemical assays to diagnose MNGIE. Methods: We studied 180 patients with clinical features suggestive of MNGIE, 14 asymptomatic TP mutation carriers, and 20 controls. TP enzyme activity in the buffy coat was determined by a fixed-time method, and the plasma nucleosides thymidine (dThd) and deoxyuridine (dUrd) were assessed by a gradient-elution reversed phase HPLC method. TP was sequenced through standard procedures in patients who met the clinical criteria for MNGIE. Results: Twenty-five of the 180 patients fulfilled the clinical criteria for MNGIE and had homozygous or compound heterozygous TP mutations. All had drastically decreased TP activity [mean (SD), 10 (15) nmol thymine formed ⅐ h ؊1 ⅐ (mg protein) ؊1 vs 634 (217) nmol thymine formed ⅐ h ؊1 ⅐ (mg protein) ؊1 for the controls]. Relative to the control mean, TP activities were reduced to 35% in mutation carriers and 65% in MNGIE-like patients. All 25 MNGIE patients had detectable plasma dThd [8.6 (3.4) mol/L] and dUrd [14.2 (4.4) mol/L].

Research paper thumbnail of EFNS guidelines on the molecular diagnosis of neurogenetic disorders: general issues, Huntington’s disease, Parkinson’s disease and dystonias

European Journal of Neurology, 2009

Background and purpose: These EFNS guidelines on the molecular diagnosis of neurogenetic disorde... more Background and purpose: These EFNS guidelines on the molecular diagnosis of neurogenetic disorders are designed to provide practical help for the general neurologist to make appropriate use of molecular genetics in diagnosing neurogenetic disorders. Since the publication of the first two EFNS-guideline papers on the molecular diagnosis of neurological diseases in 2001, rapid progress has been made in this field, necessitating an updated series of guidelines.Methods: Literature searches were performed before expert members of the task force wrote proposals, which were discussed in detail until final consensus had been reached among all task force members.Results and conclusion: This paper provides updated guidelines for molecular diagnosis of Huntington’s disease, Parkinson’s disease and dystonias as well as a general introduction to the topic. Possibilities and limitations of molecular genetic diagnosis of these disorders are evaluated and recommendations are provided.

Research paper thumbnail of MNGIE: from nuclear DNA to mitochondrial DNA

Neuromuscular Disorders, 2001

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a unique autosomal recessive dis... more Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a unique autosomal recessive disorder with mitochondrial DNA alterations. The disease is characterized clinically by ptosis, progressive external ophthalmoparesis, gastrointestinal dysmotility, cachexia, peripheral neuropathy, and leukoencephalopathy. Muscle biopsies typically reveal mitochondrial abnormalities including ragged-red ®bers and focal cytochrome c oxidase de®ciency. Analysis of mitochondrial DNA in skeletal muscle shows partial depletion, multiple deletions, or both. To identify the cause of MNGIE, we mapped the disease locus to chromosome 22q13.32±qter. Within this region, we identi®ed the gene encoding thymidine phosphorylase as the MNGIE gene. We have identi®ed homozygous or compound-heterozygous thymidine phosphorylase gene mutations in 35 MNGIE patients (21 families) from diverse ethnic groups, including: Ashkenazi Jewish, Western European, Jamaican, Hispanic, and Japanese. We con®rmed pathogenicity of the mutations by a spectrophotometric assay of thymidine phosphorylase activity with peripheral leukocytes of 15 MNGIE patients. Thymidine phosphorylase enzymatic activity was severely reduced, thus enabling us to conclude that the loss-of-function mutations in thymidine phosphorylase gene cause MNGIE. Thymidine phosphorylase catabolizes thymidine to thymine. In agreement with this notion, we noted that plasma thymidine level is increased more than 20-fold in MNGIE patients compared to controls. Therefore, we have hypothesized that increased thymidine causes mitochondrial nucleotide pool imbalance which, in turn, leads to motochondrial DNA alterations, via a mitochondria-speci®c thymidine salvage pathway. The identi®cation of the MNGIE gene has allowed us to classify MNGIE as a disease of nucleoside dysmetabolism. We may be entering a new era of research on mitochondrial nucleoside metabolism. q

Research paper thumbnail of Mitochondrial neurogastrointestinal encephalomyopathy and thymidine metabolism: results and hypotheses

Mitochondrion, 2002

impairment is limited. 4 While larger studies are required to give evidence-based recommendations... more impairment is limited. 4 While larger studies are required to give evidence-based recommendations, perhaps physical aspects cannot fully explain the risk of falls in older adults.

Research paper thumbnail of 64 Systematic identification of human mitochondrial disease genes through integrative genomics

Research paper thumbnail of Disorders of Nuclear-Mitochondrial Intergenomic Communication

Bioscience Reports, 2007

In the course of evolution, mitochondria lost their independence, and mtDNA became “slave” of nDN... more In the course of evolution, mitochondria lost their independence, and mtDNA became “slave” of nDNA, depending on numerous nucleus-encoded factors for its integrity, replication and expression. Mutations in any of these factors may alter the cross-talk between the two genomes and cause diseases that affect mtDNA integrity or expression, being inherited as mendelian traits.

Research paper thumbnail of Assay of mitochondrial ATP synthesis in animal cells

Methods in Cell Biology, 2001

Research paper thumbnail of Early-onset liver mtDNA depletion and late-onset proteinuric nephropathy in Mpv17 knockout mice

Human Molecular Genetics, 2008

In humans, MPV17 mutations are responsible for severe mitochondrial depletion syndrome, mainly af... more In humans, MPV17 mutations are responsible for severe mitochondrial depletion syndrome, mainly affecting the liver and the nervous system. To gain insight into physiopathology of MPV17-related disease, we investigated an available Mpv17 knockout animal model. We found severe mtDNA depletion in liver and, albeit to a lesser extent, in skeletal muscle, whereas hardly any depletion was detected in brain and kidney, up to 1 year after birth. Mouse embryonic fibroblasts did show mtDNA depletion, but only after several culturing passages, or in a serumless culturing medium. In spite of severe mtDNA depletion, only moderate decrease in respiratory chain enzymatic activities, and mild cytoarchitectural alterations, were observed in the Mpv17 2/2 livers, but neither cirrhosis nor failure ever occurred in this organ at any age. The mtDNA transcription rate was markedly increased in liver, which could contribute to compensate the severe mtDNA depletion. This phenomenon was associated with specific downregulation of Mterf1, a negative modulator of mtDNA transcription. The most relevant clinical features involved skin, inner ear and kidney. The coat of the Mpv17 2/2 mice turned gray early in adulthood, and 18-month or older mice developed focal segmental glomerulosclerosis (FSGS) with massive proteinuria. Concomitant degeneration of cochlear sensory epithelia was reported as well. These symptoms were associated with significantly shorter lifespan. Coincidental with the onset of FSGS, there was hardly any mtDNA left in the glomerular tufts. These results demonstrate that Mpv17 controls mtDNA copy number by a highly tissue-and possibly cytotype-specific mechanism.