Mutations In TYMP Research Articles

Deoxynucleoside stress exacerbates the phenotype of a mouse model of mitochondrial neurogastrointestinal encephalopathy.

Balanced pools of deoxyribonucleoside triphosphate precursors are required for DNA replication, and alterations of this balance are relevant to human mitochondrial diseases including mitochondrial neurogastrointestinal encephalopathy. In this disease, autosomal recessive TYMP mutations cause severe reductions of thymidine phosphorylase activity; marked elevations of the pyrimidine nucleosides thymidine and deoxyuridine in plasma and tissues, and somatic multiple deletions, depletion and site-specific point mutations of mitochondrial DNA. Thymidine phosphorylase and uridine phosphorylase double knockout mice recapitulated several features of these patients including thymidine phosphorylase activity deficiency, elevated thymidine and deoxyuridine in tissues, mitochondrial DNA depletion, respiratory chain defects and white matter changes. However, in contrast to patients with this disease, mutant mice showed mitochondrial alterations only in the brain. To test the hypothesis that elevated levels of nucleotides cause unbalanced deoxyribonucleoside triphosphate pools and, in turn, pathogenic mitochondrial DNA instability, we have stressed double knockout mice with exogenous thymidine and deoxyuridine, and assessed clinical, neuroradiological, histological, molecular, and biochemical consequences. Mutant mice treated with exogenous thymidine and deoxyuridine showed reduced survival, body weight, and muscle strength, relative to untreated animals. Moreover, in treated mutants, leukoencephalopathy, a hallmark of the disease, was enhanced and the small intestine showed a reduction of smooth muscle cells and increased fibrosis. Levels of mitochondrial DNA were depleted not only in the brain but also in the small intestine, and deoxyribonucleoside triphosphate imbalance was observed in the brain. The relative proportion, rather than the absolute amount of deoxyribonucleoside triphosphate, was critical for mitochondrial DNA maintenance. Thus, our results demonstrate that stress of exogenous pyrimidine nucleosides enhances the mitochondrial phenotype of our knockout mice. Our mouse studies provide insights into the pathogenic role of thymidine and deoxyuridine imbalance in mitochondrial neurogastrointestinal encephalopathy and an excellent model to study new therapeutic approaches.

The Role of Brain MRI in Mitochondrial Neurogastrointestinal Encephalomyopathy

Leukoencephalopathy is a hallmark of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) a devastating disorder characterized by ptosis, ophthalmoparesis, gastrointestinal dysfunction and polyneuropathy. To characterize MNGIE-associated leukoencephalopathy and to correlate it with clinical, biochemical and molecular data, four MNGIE patients with heterogeneous clinical phenotypes (enteropathic arthritis, exercise intolerance, CIDP-like phenotype and typical presentation) were studied by magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). Diffusion weighted imaging (DWI) with apparent diffusion coefficient (ADC) maps were also obtained. In two patients we also investigated the role of brain MRI in monitoring the evolution of leukoencephalopathy by performing follow-up imaging studies at an interval of one and two years. The extension and distribution of leukoencephalopathy were not clearly linked with age, phenotype or disease severity, and did not seem to be related to TYMP mutations, enzyme activity or pyrimidine levels. In the studied patients MRS revealed reduced N-acetyl-aspartate and increased choline signals. Although DWI appeared normal in all patients but one, ADC maps always showed moderate increased diffusivity. Leukoencephalopathy worsened over a two-year period in two patients, regardless of the clinical course, indicating a lack of correlation between clinical phenotype, size and progression of white matter abnormalities during this period. Brain MRI should be considered a very useful tool to diagnose both classical and atypical MNGIE. Serial MRIs in untreated and treated MNGIE patients will help to establish whether the leukoencephalopathy is a reversible condition or not.

Possible toxicity of tuberculostatic agents in a patient with a novel TYMP mutation leading to mitochondrial neurogastrointestinal encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive multisystemic disorder caused by TYMP gene mutations. Here, we report on the first MNGIE patient diagnosed in Bulgaria who carries a novel homozygous TYMP mutation (p.Leu347Pro). The patient presented with gastrointestinal complaints, cachexia, hearing loss, ptosis, ophthalmoparesis, polyneuropathy, cognitive impairment and leukoencephalopathy on MRI examination of the brain. The patient´s motor capacity declined significantly leading to wheelchair dependence several months following the administration of tuberculostatic treatment suggesting mitochondrial toxicity of these agents.

Mitochondrial DNA depletion syndromes: review and updates of genetic basis, manifestations, and therapeutic options.

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 2years 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 20years 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.

Large heterozygous deletions on 22q13.33 in combination with TYMP or SCO2 point mutations in two patients with mitochondrial disorders

Large heterozygous deletions on 22q13.33 in combination with TYMP or SCO2 point mutations in two patients with mitochondrial disorders

099 Erythrocyte entrapped thymidine phosphorylase (EE-TP) therapy for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

MNGIE is a fatal, autosomal recessive disorder caused by mutations in TYMP, a nuclear gene encoding thymidine phosphorylase (TP), leading to accumulation of thymidine (dThd) and deoxyuridine (dUrd) in all...

Detection of uniparental isodisomy in autosomal recessive mitochondrial DNA depletion syndrome by high-density SNP array analysis

Mitochondrial DNA (mtDNA) depletion syndrome encompasses a heterogeneous group of disorders characterized by a reduction in the mtDNA copy number. We identified two patients with clinical presentations consistent with mtDNA depletion syndrome (MDS), who were subsequently found to have apparently homozygous point mutations in TYMP and DGUOK, two of the nine nuclear genes commonly associated with these disorders. Further sequence analyses of parents indicated that in each case only one parent; the mother of the first and the father of the second, was a heterozygous carrier of the mutation identified in the affected child. The presence of underlying deletions was ruled out by use of a custom target array comparative genomic hybridization (CGH) platform. A high-density single-nucleotide polymorphism (SNP) array analysis revealed that the first patient had a region of copy-neutral absence of heterozygosity (AOH) consistent with segmental isodisomy for an 11.3 Mb region at the long-arm terminus of chromosome 22 (including the TYMP gene), and the second patient had results consistent with complete isodisomy of chromosome 2 (where the DGUOK gene is located). The combined sequencing, array CGH and SNP array approaches have demonstrated the first cases of MDS due to uniparental isodisomy. This diagnostic scenario also demonstrates the necessity of comprehensive examination of the underlying molecular defects of an apparently homozygous mutation in order to provide patients and their families with the most accurate molecular diagnosis and genetic counseling.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)-like phenotype: an expanded clinical spectrum of POLG1 mutations

The aim of the study was to determine the prevalence of MNGIE-like phenotype in patients with recessive POLG1 mutations. Mutations in the POLG1 gene, which encodes for the catalytic subunit of the mitochondrial DNA polymerase gamma essential for mitochondrial DNA replication, cause a wide spectrum of mitochondrial disorders. Common phenotypes associated with POLG1 mutations include Alpers syndrome, ataxia-neuropathy syndrome, and progressive external ophthalmoplegia (PEO). Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by severe gastrointestinal dysmotility, cachexia, PEO and/or ptosis, peripheral neuropathy, and leukoencephalopathy. MNGIE is caused by TYMP mutations. Rare cases of MNGIE-like phenotype have been linked to RRM2B mutations. Recently, POLG1 mutations were identified in a family with clinical features of MNGIE but no leukoencephalopathy. The coding regions and exon-intron boundaries of POLG1 were sequence analyzed in patients suspected of POLG1 related disorders. Clinical features of 92 unrelated patients with two pathogenic POLG1 alleles were carefully reviewed. Three patients, accounting for 3.3% of all patients with two pathogenic POLG1 mutations, were found to have clinical features consistent with MNGIE but no leukoencephalopathy. Patient 1 carries p.W748S and p.R953C; patient 2 is homozygous for p.W748S, and patient 3 is homozygous for p.A467T. In addition, patient 2 has a similarly affected sibling with the same POLG1 genotype. POLG1 mutations may cause MNGIE-like syndrome, but the lack of leukoencephalopathy and the normal plasma thymidine favor POLG1 mutations as responsible molecular defect.

Clinical and genetic spectrum of mitochondrial neurogastrointestinal encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy is a rare multisystemic autosomic recessive disorder characterized by: onset typically before the age of 30 years; ptosis; progressive external ophthalmoplegia; gastrointestinal dysmotility; cachexia; peripheral neuropathy; and leucoencephalopathy. The disease is caused by mutations in the TYMP gene encoding thymidine phosphorylasethymine phosphorylase. Anecdotal reports suggest that allogeneic haematopoetic stem cell transplantation may be beneficial for mitochondrial neurogastrointestinal encephalomyopathy, but is associated with a high mortality. After selecting patients who fulfilled the clinical criteria for mitochondrial neurogastrointestinal encephalomyopathy and had severe thymidine phosphorylase deficiency in the buffy coat (<10% of normal activity), we reviewed their medical records and laboratory studies. We identified 102 patients (50 females) with mitochondrial neurogastrointestinal encephalomyopathy and an average age of 32.4 years (range 11-59 years). We found 20 novel TYMP mutations. The average age-at-onset was 17.9 years (range 5 months to 35 years); however, the majority of patients reported the first symptoms before the age of 12 years. The patient distribution suggests a relatively high prevalence in Europeans, while the mutation distribution suggests founder effects for a few mutations, such as c.866A>G in Europe and c.518T>G in the Dominican Republic, that could guide genetic screening in each location. Although the sequence of clinical manifestations in the disease varied, half of the patients initially had gastrointestinal symptoms. We confirmed anecdotal reports of intra- and inter-familial clinical variability and absence of genotype-phenotype correlation in the disease, suggesting genetic modifiers, environmental factors or both contribute to disease manifestations. Acute medical events such as infections often provoked worsening of symptoms, suggesting that careful monitoring and early treatment of intercurrent illnesses may be beneficial. We observed endocrine/exocrine pancreatic insufficiency, which had not previously been reported. Kaplan-Meier analysis revealed significant mortality between the ages of 20 and 40 years due to infectious or metabolic complications. Despite increasing awareness of this illness, a high proportion of patients had been misdiagnosed. Early and accurate diagnosis of mitochondrial neurogastrointestinal encephalomyopathy, together with timely treatment of acute intercurrent illnesses, may retard disease progression and increase the number of patients eligible for allogeneic haematopoetic stem cell transplantation.

Compound mutations of PEO1 and TYMP in a progressive external ophthalmoplegia patient with incomplete mitochondrial neurogastrointestinal encephalomyopathy phenotype

The Mendelian inherited progressive external ophthalmoplegia (PEO) and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) are genetically heterogeneous mitochondrial diseases caused by nuclear-mitochondrial intergenomic defects. The PEO1 and TYMP nuclear genes are closely related in the machinery of the mitochondrial DNA (mtDNA) replication. Mutations in PEO1 and TYMP genes usually cause autosomal dominant PEO, and autosomal recessive MNGIE. We identified a PEO family of Korean origin with additional phenotype of incomplete MNGIE symptom (Family ID: MT16). The entire mitochondrial genome and all coding exons of PEO1, TYMP, ANT1, POLG1, POLG2, DGUOK, and TK2 nuclear genes were sequenced. Clinical information was obtained through history taking, physical examinations, clinical observations, and electrophysiological investigations. Muscle biopsy of left biceps brachii and shoulder magnetic resonance imaging (MRI) were undertaken. We found two heterozygous mutations, Arg374Gln in PEO1 and Glu106Gln in TYMP from the proband who showed complex phenotypes of a typical PEO and late-onset incomplete MNGIE. The PEO1 Arg374Gln has been reported in several PEO patients, but TYMP Glu106Gln has not been reported. Neither large deletion nor causative point mutations were observed in the mtDNA. We suggest that the heterozygous TYMP mutation might affect complex phenotypes as a secondary genetic cause in the co-presence of PEO1 mutation.

Mitochondrial neurogastrointestinal encephalopathy in an Indian family with possible manifesting carriers of heterozygous TYMP mutation

Background Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a distinctive autosomal recessive disorder with mitochondrial alterations due to mutations TYMP gene encoding thymidine phosphorylase. Materials and methods Study of clinical and biochemical characteristics of a family with MNGIE. Results Index case was a 32 year old man presenting with recurrent vomiting, early satiety and progressive weight loss. He had ptosis, restricted eye movements, generalized muscle wasting, and absent tendon reflexes. Lactate levels were elevated in venous blood and CSF lactate. MRI brain showed diffuse leucoencephalopathy. Barium swallow showed near total obstruction at mid portion of vertical limb of duodenum with ileus. Esophageal manometry suggested myopathy. Muscle biopsy revealed moderate numbers of ragged blue and ragged red fibers as well as cytochrome c oxidase deficient fibers. An elder brother had similar symptoms and expired after a surgical procedure and a 28 year old brother has similar illness. The father had asymptomatic bilateral ptosis with mild ophthalmoparesis. The paternal grandfather and paternal aunt also had bilateral ptosis. Clinical diagnosis of MNGIE was confirmed in the two living brothers by demonstrating severe defects of thymidine phosphorylase activity in buffy coat, elevated thymidine and deoxyuridine in plasma, and a homozygous TYMP c.893 G > A mutation. Conclusions This family with biochemically and genetically confirmed mitochondrial neurogastrointestinal encephalopathy syndrome uncharacteristically included heterozygous TYMP mutation carriers manifesting extra-ocular weakness. It is important to identify MNGIE patients early because therapeutic options are emerging.

Limited dCTP Availability Accounts for Mitochondrial DNA Depletion in Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)

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.

Normal oxidative phosphorylation in intestinal smooth muscle of childhood chronic intestinal pseudo-obstruction

Chronic intestinal pseudo-obstruction (CIPO) is a severe disease of the digestive tract motility. In pediatric population, CIPO remains of unknown origin for most patients. Chronic intestinal pseudo-obstruction is also a common feature in the course of mitochondrial oxidative phosphorylation disorders related for some patients to mutations in TYMP, POLG1, mtDNA tRNA(leu(UUR)) or tRNA(lys) genes. We hypothesized that CIPOs could be the presenting symptom of respiratory chain enzyme deficiency and thus we investigated oxidative phosphorylation in small bowel and/or colon smooth muscle of primary CIPO children. We studied eight children with CIPO and 12 pediatric controls. We collected clinical, radiological and pathological data and measured respiratory chain enzymatic activity in isolated smooth muscle of the small bowel and/or the colon. We also sequenced TYMP, POLG, mtDNA tRNA(leu(UUR)) and tRNA(lys) genes. Neither pathological nor radiological data were in favor of a mitochondrial dysfunction. No respiratory chain enzyme deficiency was detected in CIPO children. In myogenic CIPO, respiratory enzymes and citrate synthase activities were increased in small bowel and/or colon whereas no abnormality was noted in neurogenic and unclassified CIPO. Levels of enzyme activities were higher in control small bowel than in control colon muscle. Sequencing of TYMP, POLG, mtDNA tRNA(leu(UUR)) and tRNA(lys) genes and POLG gene did not reveal mutation for any of the patients. The normal enzymatic activities as the lack of radiological and genetic abnormalities indicate that, at variance with adult patients, oxidative phosphorylation deficiency is not a common cause of childhood CIPO.

A novel TYMP mutation in a French Canadian patient with mitochondrial neurogastrointestinal encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disorder characterized by gastrointestinal, extraocular muscle, peripheral nerve, and cerebral white matter involvement. Mutations in the nuclear gene TYMP encoding for thymidine phosphorylase (TP) cause loss of TP activity, systemic accumulation of its substrates in plasma and tissues, as well as alterations in mitochondrial DNA including deletions, depletion, and somatic point mutations. To date, more than 30 mutations have been reported in diverse ethnic populations. We present herein the clinical, neuroimaging, neuromuscular, and molecular findings of the first French Canadian patient with MNGIE caused by a novel homozygous invariant splicing site (IVS5 +1 G>A) mutation of the TYMP gene.

In vitro supplementation with dAMP/dGMP leads to partial restoration of mtDNA levels in mitochondrial depletion syndromes

Mitochondrial DNA depletion syndrome, a frequent cause of childhood (hepato)encephalomyopathies, is defined as a reduction of mitochondrial DNA copy number related to nuclear DNA. It was previously shown that mtDNA depletion can be prevented by dAMP/dGMP supplementation in deoxyguanosine kinase-deficient fibroblasts. We investigated myotubes of patients diagnosed with mtDNA depletion carrying pathogenic mutations in DGUOK, POLG1 (Alpers syndrome) and TYMP. Differentiating myotubes of all patients and controls were supplemented with different doses of dAMP/dGMP or dAMP/dGMP/dCMP in TYMP deficiency, and analysed for mtDNA/nDNA ratio and for cytochrome c oxidase (COX) activity. Serum deprivation and myotube formation triggered a decrease in mtDNA copy number in DGUOK or POLG1 deficient myotubes, but not in TYMP deficiency and healthy controls. Supplementation with dAMP/dGMP leads to a significant and reproducible rescue of mtDNA depletion in DGUOK deficiency. POLG1 deficient myotubes also showed a mild, not significant increase in mtDNA copy number. MtDNA depletion did not result in deficient COX staining in DGUOK and POLG1-deficient myotubes. Treatment with ethidium bromide resulted in very severe depletion and absence of COX staining in all cell types, and no recovery was observed after supplementation with dAMP/dGMP. We show that supplementation with dAMP/dGMP increases mtDNA copy number significantly in DGUOK deficient myotubes and, leads to a mild, non-significant improvement of mtDNA depletion in POLG1 deficiency. No adverse effect on mtDNA copy number was observed on high-dose supplementation in vitro. Further studies are needed to determine possible therapeutic implications of dAMP/dGMP supplementation for DGUOK deficiency in vivo.

Characterization of a novel TYMP splice site mutation associated with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

Mitochondrial neurogastrointestinal encephalomyopathy is an autosomal recessive disorder caused by loss-of-function mutations in the thymidine phosphorylase gene ( TYMP). We report here a patient compound heterozygous for two TYMP mutations: a novel g.4009G>A transition affecting the consensus splice donor site of intron 9, and a previously reported g.675G>C splice site mutation. The novel mutation causes exon 9 skipping but leaves the reading frame intact; however, TYMP protein was not detected by immunoblot analysis, suggesting that neither mutant allele is expressed as protein. The patient’s fibroblasts showed gradual loss of the mitochondrial DNA-encoded subunit I of cytochrome- c oxidase, suggesting a progressive mitochondrial DNA defect in culture.