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1.
J Transl Med ; 22(1): 449, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38741129

RESUMO

Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.


Assuntos
DNA Mitocondrial , Fibroblastos , Lisossomos , Mitocôndrias , Encefalomiopatias Mitocondriais , Nucleosídeos , Timidina Fosforilase , Humanos , Lisossomos/metabolismo , Timidina Fosforilase/metabolismo , Timidina Fosforilase/deficiência , Timidina Fosforilase/genética , Encefalomiopatias Mitocondriais/metabolismo , Encefalomiopatias Mitocondriais/patologia , Encefalomiopatias Mitocondriais/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Nucleosídeos/metabolismo , Pseudo-Obstrução Intestinal/metabolismo , Pseudo-Obstrução Intestinal/patologia , Pseudo-Obstrução Intestinal/enzimologia , Pseudo-Obstrução Intestinal/genética , Oftalmoplegia/metabolismo , Oftalmoplegia/patologia , Oftalmoplegia/congênito , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patologia , Masculino , Feminino , Pele/patologia , Pele/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo
4.
J Mol Med (Berl) ; 101(10): 1237-1253, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37603049

RESUMO

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in the TYMP gene, which encodes thymidine phosphorylase (TP). As a cytosolic metabolic enzyme, TP defects affect biological processes that are thought to not be limited to the abnormal replication of mitochondrial DNA. This study aimed to elucidate the characteristic metabolic alterations and associated homeostatic regulation caused by TYMP deficiency. The pathogenicity of novel TYMP variants was evaluated in terms of clinical features, genetic analysis, and structural instability. We analyzed plasma samples from three patients with MNGIE; three patients with m.3243A > G mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS); and four healthy controls (HC) using both targeted and untargeted metabolomics techniques. Transcriptomics analysis and bioenergetic studies were performed on skin fibroblasts from participants in these three groups. A TYMP overexpression experiment was conducted to rescue the observed changes. Compared with controls, specific alterations in nucleosides, bile acids, and steroid metabolites were identified in the plasma of MNGIE patients. Comparable mitochondrial dysfunction was present in fibroblasts from patients with TYMP deficiency and in those from patients with the m.3243A > G mutation. Distinctively decreased sterol regulatory element binding protein (SREBP) regulated cholesterol metabolism and fatty acid (FA) biosynthesis as well as reduced FA degradation were revealed in fibroblasts with TYMP deficiency. The restoration of thymidine phosphorylase activity rescued the observed changes in MNGIE fibroblasts. Our findings indicated that more widespread metabolic disturbance may be caused by TYMP deficiency in addition to mitochondrial dysfunction, which expands our knowledge of the biochemical outcome of TYMP deficiency. KEY MESSAGES: Distinct metabolic profiles in patients with TYMP deficiency compared to those with m.3243A > G mutation. TYMP deficiency leads to a global disruption of nucleoside metabolism. Cholesterol and fatty acid metabolism are inhibited in individuals with MNGIE. TYMP is functionally related to SREBP-regulated pathways. Potential metabolite biomarkers that could be valuable clinical tools to improve the diagnosis of MNGIE.


Assuntos
DNA Mitocondrial , Timidina Fosforilase , Humanos , Timidina Fosforilase/genética , Timidina Fosforilase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Mutação , DNA Mitocondrial/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Colesterol , Ácidos Graxos
5.
Neurogastroenterol Motil ; 35(10): e14643, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37448106

RESUMO

BACKGROUND: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare mitochondrial disease caused by mutations in TYMP, encoding thymidine phosphorylase. Clinically it is characterized by severe gastrointestinal dysmotility associated with cachexia and a demyelinating sensorimotor polyneuropathy. Even though digestive manifestations are progressive and invariably lead to death, the features of gastrointestinal motor dysfunction have not been systematically evaluated. The objective of this study was to describe gastrointestinal motor dysfunction in MNGIE using state-of-the art techniques and to evaluate the relationship between motor abnormalities and symptoms. METHODS: Prospective study evaluating gastrointestinal motor function and digestive symptoms in all patients with MNGIE attended at a national referral center in Spain between January 2018 and July 2022. KEY RESULTS: In this period, five patients diagnosed of MNGIE (age range 16-46 years, four men) were evaluated. Esophageal motility by high-resolution manometry was abnormal in four patients (two hypoperistalsis, two aperistalsis). Gastric emptying by scintigraphy was mildly delayed in four and indicative of gastroparesis in one. In all patients, small bowel high-resolution manometry exhibited a common, distinctive dysmotility pattern, characterized by repetitive bursts of spasmodic contractions, without traces of normal fasting and postprandial motility patterns. Interestingly, objective motor dysfunctions were detected in the absence of severe digestive symptoms. CONCLUSIONS AND INFERENCES: MNGIE patients exhibit a characteristic motor dysfunction, particularly of the small bowel, even in patients with mild digestive symptoms and in the absence of morphological signs of intestinal failure. Since symptoms are not predictive of objective findings, early investigation is indicated.


Assuntos
Gastroenteropatias , Pseudo-Obstrução Intestinal , Encefalomiopatias Mitocondriais , Masculino , Humanos , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Estudos Prospectivos , Pseudo-Obstrução Intestinal/genética , Encefalomiopatias Mitocondriais/diagnóstico , Encefalomiopatias Mitocondriais/genética , Mutação , Gastroenteropatias/genética
6.
Neurol India ; 71(2): 323-325, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37148061

RESUMO

Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is an autosomal recessive disease associated with the mutation of the TYMP gene. MNGIE causes gastrointestinal and neurological symptoms, and the gastrointestinal symptoms are usually notable, which may lead to misdiagnosis. However, we herein report a 29-year-old female who presented with prominent neurological symptoms, while her gastrointestinal symptoms were mild. Brain MRI revealed prominent diffused leukoencephalopathy and peripheral neuropathy was confirmed by the nerve conduction velocity test. Biochemical tests showed elevated plasma thymidine, deoxyuridine, and lactate levels. Molecular genetic testing demonstrated a novel homozygous TYMP c. 447 dupG mutation and the patient's mother was heterozygous for the mutation but had no clinical features. MNGIE was diagnosed based on the results. Unlike other patients who had notable gastrointestinal symptoms, this patient presented with more prominent neurological symptoms than gastrointestinal symptoms, which might have been caused by the novel mutation in the TYMP gene.


Assuntos
Encefalomiopatias Mitocondriais , Humanos , Feminino , Adulto , Encefalomiopatias Mitocondriais/complicações , Encefalomiopatias Mitocondriais/diagnóstico , Encefalomiopatias Mitocondriais/genética , Timidina Fosforilase/genética , Mutação/genética , Testes Genéticos
7.
Clin Neurol Neurosurg ; 229: 107712, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37084649

RESUMO

Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a well-known mitochondrial depletion syndrome. Since Van Goethem et al. described MNGIE syndrome with pathogenic POLG1 mutations in 2003, POLG1 gene became a target for MNGIE patients. Cases with POLG1 mutations strikingly differ from classic MNGIE patients due to a lack of leukoencephalopathy. Here we present a female patient with very early onset disease and leukoencephalopathy compatible with classic MNGIE disease who turned out to have homozygous POLG1 mutation compatible with MNGIE-like syndrome, mitochondrial depletion syndrome type 4b.


Assuntos
Leucoencefalopatias , Encefalomiopatias Mitocondriais , Humanos , Feminino , Encefalomiopatias Mitocondriais/complicações , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/patologia , Timidina Fosforilase/genética , Mutação/genética , Leucoencefalopatias/genética , Leucoencefalopatias/complicações , Síndrome
8.
Pediatr Blood Cancer ; 70(7): e30334, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37046411

RESUMO

BACKGROUND: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disorder due to mutations in the TYMP gene. Clinical findings are characterized by neurologic manifestations and severe gastrointestinal dysfunction. The syndrome is usually fatal, the most effective treatment appears to be hematopoietic stem cell transplantation (HSCT). PROCEDURE: In this retrospective study, we evaluated HSCT that was performed using a reduced toxicity myeloablative conditioning regimen in patients with MNGIE at our center. RESULTS: A total of six allogeneic transplant procedures were performed in four patients. Three patients had fully matched donors, and one patient had a haploidentical donor. Treosulfan-based myeloablative conditioning regimen was applied in five of six transplants. Bone marrow was used as a stem cell source. One patient is being followed up in the 4th year of posttransplant with full chimeric and without graft versus host disease (GVHD). One patient died of acute stage IV gastrointestinal system GVHD. Two patients underwent second transplantation due to engraftment failure, one of which was the patient who had a haploidentical transplant. CONCLUSIONS: Treosulfan-based regimen is well tolerated, although engraftment failure with this conditioning regimen can be a significant problem. We share our haploidentical transplant experience, which will be the first reported case in the literature.


Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Estudos Retrospectivos , Transplante Homólogo/métodos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Doença Enxerto-Hospedeiro/etiologia , Condicionamento Pré-Transplante/métodos
10.
J Biol Chem ; 299(3): 103002, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36773803

RESUMO

Plasma thymidine levels in rodents are higher than in other mammals including humans, possibly due to a different pattern and lower level of thymidine phosphorylase expression. Here, we generated a novel knock-in (KI) mouse line with high systemic expression of human thymidine phosphorylase to investigate this difference in nucleotide metabolism in rodents. The KI mice showed growth retardation around weaning and died by 4 weeks of age with a decrease in plasma thymidine level compared with the litter-control WT mice. These phenotypes were completely or partially rescued by administration of the thymidine phosphorylase inhibitor 5-chloro-6-(2-iminopyrrolidin-1-yl) methyl-2,4(1H,3H)-pyrimidinedione hydrochloride or thymidine, respectively. Interestingly, when thymidine phosphorylase inhibitor administration was discontinued in adult animals, KI mice showed deteriorated grip strength and locomotor activity, decreased bodyweight, and subsequent hind-limb paralysis. Upon histological analyses, we observed axonal degeneration in the spinal cord, muscular atrophy with morphologically abnormal mitochondria in quadriceps, retinal degeneration, and abnormality in the exocrine pancreas. Moreover, we detected mitochondrial DNA depletion in multiple tissues of KI mice. These results indicate that the KI mouse represents a new animal model for mitochondrial diseases and should be applicable for the study of differences in nucleotide metabolism between humans and mice.


Assuntos
Encefalomiopatias Mitocondriais , Miopatias Mitocondriais , Animais , Humanos , Camundongos , DNA Mitocondrial/metabolismo , Transtornos do Crescimento/genética , Mamíferos/metabolismo , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/patologia , Nucleotídeos , Timidina , Timidina Fosforilase/genética , Timidina Fosforilase/metabolismo
11.
Handb Clin Neurol ; 194: 9-21, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36813323

RESUMO

Progressive external ophthalmoplegia (PEO), characterized by ptosis and impaired eye movements, is a clinical syndrome with an expanding number of etiologically distinct subtypes. Advances in molecular genetics have revealed numerous pathogenic causes of PEO, originally heralded in 1988 by the detection of single large-scale deletions of mitochondrial DNA (mtDNA) in skeletal muscle of people with PEO and Kearns-Sayre syndrome. Since then, multiple point variants of mtDNA and nuclear genes have been identified to cause mitochondrial PEO and PEO-plus syndromes, including mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and sensory ataxic neuropathy dysarthria ophthalmoplegia (SANDO). Intriguingly, many of those nuclear DNA pathogenic variants impair maintenance of the mitochondrial genome causing downstream mtDNA multiple deletions and depletion. In addition, numerous genetic causes of nonmitochondrial PEO have been identified.


Assuntos
Oftalmoplegia Externa Progressiva Crônica , Oftalmoplegia , Humanos , Oftalmoplegia Externa Progressiva Crônica/complicações , Oftalmoplegia Externa Progressiva Crônica/genética , Oftalmoplegia Externa Progressiva Crônica/patologia , DNA Mitocondrial/genética , Oftalmoplegia/genética , Oftalmoplegia/patologia , Músculo Esquelético/patologia , Síndrome
12.
J Med Case Rep ; 16(1): 363, 2022 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-36192783

RESUMO

BACKGROUND: Mitochondrial neurogastrointestinal encephalopathy is a rare multisystem autosomal recessive disease caused by mutations in the TYMP gene, that encodes for thymidine phosphorylase. Mitochondrial neurogastrointestinal encephalopathy is a progressive degenerative disease characterized by a distinctive tetrad of gastrointestinal dysmotility, peripheral neuropathy, ophthalmoplegia with ptosis, and asymptomatic leukoencephalopathy. It provides a diagnostic dilemma to physicians in regions like Pakistan because of a lack of genetic study availability and associated financial constraints of the population. However, with careful examination and a few basic investigations, mitochondrial neurogastrointestinal encephalopathy can be diagnosed by ruling out most of the close differentials. CASE PRESENTATION: We report the case of a 23-year-old Asian female whose chief complaints were epigastric pain, bilious emesis, weight loss for 3 months, and bilateral lower limb weakness for 20 days. All clinical signs and symptoms along with relevant investigations including nerve conduction studies, electromyography, and magnetic resonance imaging of the brain were highly suggestive of mitochondrial neurogastrointestinal encephalopathy syndrome. Because of financial constraints, genetic studies could not be performed. The patient was managed with a multidisciplinary approach involving gastroenterology, physiotherapy, and nutrition departments. Currently, therapeutic options for the disease include allogeneic hematopoietic stem cell transplant and carrier erythrocyte entrapped thymidine phosphorylase; however, these could not be provided to the patient owing to certain limitations. CONCLUSIONS: As misdiagnosis and delayed diagnosis are quite common in this disease, the prime objective of this case report is to increase the basic understanding of this disease, especially its signs and symptoms, and address the limitations regarding the diagnostic investigations and management of patients with mitochondrial neurogastrointestinal encephalopathy.


Assuntos
Pseudo-Obstrução Intestinal , Encefalomiopatias Mitocondriais , Distrofia Muscular Oculofaríngea , Adulto , Feminino , Humanos , Pseudo-Obstrução Intestinal/complicações , Pseudo-Obstrução Intestinal/diagnóstico , Pseudo-Obstrução Intestinal/terapia , Encefalomiopatias Mitocondriais/diagnóstico , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/terapia , Distrofia Muscular Oculofaríngea/complicações , Distrofia Muscular Oculofaríngea/diagnóstico , Oftalmoplegia/congênito , Paquistão , Timidina Fosforilase/genética , Adulto Jovem
13.
Methods Mol Biol ; 2546: 431-437, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36127610

RESUMO

Inborn errors of pyrimidine metabolism result from deficiencies in pyrimidine de novo synthesis, degradation, and salvage pathways. Enzymatic deficiencies in pyrimidine catabolism lead to mitochondrial neurogastrointestinal encephalopathy (MNGIE), pyrimidinuria, dihydropyrimidinuria, ureidopropionic aciduria, and other disorders. While MNGIE presents with gastrointestinal dysmotility, cachexia and leukoencephalopathy, pyrimidinuria, and dihydropyrimidinuria may show symptoms of epilepsy, autism, mental retardation, and dysmorphic features. The application of HPLC-MS/MS facilitates rapid screening of pyrimidine metabolites. Here we describe a sensitive and reliable LC-MS/MS method for quantitative determination of uracil, thymine, thymidine, dihydrouracil, and dihydrothymine in urine that are diagnostic biomarkers of MNGIE, pyrimidinuria, and dihydropyrimidinuria.


Assuntos
Deficiência da Di-Hidropirimidina Desidrogenase , Espectrometria de Massas em Tandem , Biomarcadores , Cromatografia Líquida , Humanos , Pirimidinas , Espectrometria de Massas em Tandem/métodos , Timidina , Timina , Uracila
14.
Int J Mol Sci ; 23(15)2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35955927

RESUMO

mitochondrial neuro-gastrointestinal encephalomyopathy (MNGIE) is a rare genetic disorder characterized by thymidine phosphorylase (TP) enzyme defect. The absence of TP activity induces the imbalance of mitochondrial nucleotide pool, leading to impaired mitochondrial DNA (mtDNA) replication and depletion. Since mtDNA is required to ensure oxidative phosphorylation, metabolically active tissues may not achieve sufficient energy production. The only effective life-saving approach in MNGIE has been the permanent replacement of TP via allogeneic hematopoietic stem cell or liver transplantation. However, the follow-up of transplanted patients showed that gut tissue changes do not revert and fatal complications, such as massive gastrointestinal bleeding, can occur. The purpose of this study was to clarify whether the reintroduction of TP after transplant can recover mtDNA copy number in a normal range. Using laser capture microdissection and droplet-digital-PCR, we assessed the mtDNA copy number in each layer of full-thickness ileal samples of a naive MNGIE cohort vs. controls and in a patient pre- and post-TP replacement. The treatment led to a significant recovery of gut tissue mtDNA amount, thus showing its efficacy. Our results indicate that a timely TP replacement is needed to maximize therapeutic success before irreversible degenerative tissue changes occur in MNGIE.


Assuntos
Transplante de Fígado , Erros Inatos do Metabolismo , Encefalomiopatias Mitocondriais , DNA Mitocondrial/genética , Humanos , Íleo , Microdissecção e Captura a Laser , Lasers , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/terapia
15.
Int J Dev Neurosci ; 82(7): 626-638, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35841120

RESUMO

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE; OMIM 603041) is a rare inherited metabolic disorder mostly caused by mutations in TYMP gene encoding thymidine phosphorylase (TP) protein that affects the mitochondrial nucleotide metabolism. TP, functionally active as a homodimer, is involved in the salvage pathway of pyrimidine nucleosides. MNGIE-like syndrome having an overlapping phenotype of MNGIE was also described and has been associated with mutations in POLG and RRM2B genes. In the present study, we report the molecular investigation of a consanguineous family including two patients with clinical features suggestive of MNGIE syndrome. Bioinformatics analyses were carried out in addition to mtDNA deletion screening and copy number quantification in the blood of the two patients. Whole exome sequencing and Sanger sequencing analyses revealed the segregation in the affected family a novel mutation c.1205T>A (p.L402Q) within the exon 9 of the TYMP gene. In addition, mtDNA analysis revealed the absence of mtDNA deletions and a decrease of the copy number in the blood of the two patients of the studied family. The p.Leu402Gln mutation was located in a conserved amino acid within the α/ß domain of the TP protein and several software supported its pathogenicity. In addition, and based on docking and molecular dynamic simulation analyses, results revealed that L402Q caused a conformational change in TP mutated structure and could therefore alter its flexibility and stability. These changes prevent also the formation of stable homodimer leading to non-functional protein with partial or complete loss of its catalytic activity.


Assuntos
Encefalomiopatias Mitocondriais , Timidina Fosforilase , Humanos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Encefalomiopatias Mitocondriais/genética , Simulação de Acoplamento Molecular , Mutação , Timidina/metabolismo , Timidina Fosforilase/genética , Timidina Fosforilase/metabolismo , Linhagem , Masculino , Feminino
16.
Mol Genet Metab Rep ; 30: 100829, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34926160

RESUMO

We describe the case of a Greek female patient with the Classic form of the ultra- rare and fatal autosomal recessive disorder Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and the impact of allogeneic hematopoietic stem cell transplantation on the biochemical and clinical aspects of the disease. The patient presented at the age of 15 years with severe gastrointestinal symptoms, cachexia, peripheral neuropathy and diffuse leukoencephalopathy. The diagnosis of MNGIE disease was established by the increased levels of thymidine and deoxyuridine in plasma and the complete deficiency of thymidine phosphorylase activity. The novel c.[978dup] (p.Ala327Argfs*?) variant and the previously described variant c.[417 + 1G > A] were identified in TYMP. The donor for the allogeneic hematopoietic stem cell transplantation was her fully compatible sister, a carrier of the disease. The patient had a completely uneventful post- transplant period and satisfactory PB chimerism levels. A marked and rapid decrease in thymidine and deoxyuridine plasma levels and an increase of the thymidine phosphorylase activity to the levels measured in her donor sister was observed and is still present sixteen months post-transplant. Disease symptoms stabilized and some improvement was also observed both in her neurological and gastrointestinal symptoms. Follow up studies will be essential for determining the long term impact of allogeneic hematopoietic stem cell transplantation in our patient.

17.
J Clin Med ; 10(22)2021 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-34830516

RESUMO

Mitochondrial disorders are a remarkably complex group of diseases caused by impairment of the mitochondrial respiratory chain (or electron transport chain) [...].

18.
Hum Gene Ther ; 32(19-20): 1210-1223, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34498979

RESUMO

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare disease caused by recessive mutations in the TYMP gene, which encodes the enzyme thymidine phosphorylase (TP). In this study, the efficient integration of a TYMP transgene into introns of the Tymp and Alb loci of hepatocytes in a murine model of MNGIE was achieved by the coordinated delivery and activity of CRISPR/Cas9 and a TYMP cDNA. CRISPR/Cas9 was delivered either as mRNA using lipid nanoparticle (LNP) or polymeric nanoparticle, respectively, or in an AAV2/8 viral vector; the latter was also used to package the TYMP cDNA. Insertion of the cDNA template downstream of the Tymp and Alb promoters ensured transgene expression. The best in vivo results were obtained using LNP carrying the CRISPR/Cas9 mRNAs. Treated mice showed a consistent long-term (1 year) reduction in plasma nucleoside (thymidine and deoxyuridine) levels that correlated with the presence of TYMP mRNA and functional enzyme in liver cells. In mice with an edited Alb locus, the transgene produced a hybrid Alb-hTP protein that was secreted, with supraphysiological levels of TP activity detected in the plasma. Equivalent results were obtained in mice edited at the Tymp locus. Finally, some degree of gene editing was found in animals treated only with AAV vectors containing the DNA templates, in the absence of nucleases, although there was no impact on plasma nucleoside levels. Overall, these results demonstrate the feasibility of liver-directed genome editing in the long-term correction of MNGIE, with several advantages over other methods.


Assuntos
Edição de Genes , Encefalomiopatias Mitocondriais , Animais , Modelos Animais de Doenças , Lipossomos , Camundongos , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/terapia , Nanopartículas , Timidina Fosforilase
19.
Brain ; 144(5): 1451-1466, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-33855352

RESUMO

Abnormal gut motility is a feature of several mitochondrial encephalomyopathies, and mutations in genes such as TYMP and POLG, have been linked to these rare diseases. The human genome encodes three DNA ligases, of which only one, ligase III (LIG3), has a mitochondrial splice variant and is crucial for mitochondrial health. We investigated the effect of reduced LIG3 activity and resulting mitochondrial dysfunction in seven patients from three independent families, who showed the common occurrence of gut dysmotility and neurological manifestations reminiscent of mitochondrial neurogastrointestinal encephalomyopathy. DNA from these patients was subjected to whole exome sequencing. In all patients, compound heterozygous variants in a new disease gene, LIG3, were identified. All variants were predicted to have a damaging effect on the protein. The LIG3 gene encodes the only mitochondrial DNA (mtDNA) ligase and therefore plays a pivotal role in mtDNA repair and replication. In vitro assays in patient-derived cells showed a decrease in LIG3 protein levels and ligase activity. We demonstrated that the LIG3 gene defects affect mtDNA maintenance, leading to mtDNA depletion without the accumulation of multiple deletions as observed in other mitochondrial disorders. This mitochondrial dysfunction is likely to cause the phenotypes observed in these patients. The most prominent and consistent clinical signs were severe gut dysmotility and neurological abnormalities, including leukoencephalopathy, epilepsy, migraine, stroke-like episodes, and neurogenic bladder. A decrease in the number of myenteric neurons, and increased fibrosis and elastin levels were the most prominent changes in the gut. Cytochrome c oxidase (COX) deficient fibres in skeletal muscle were also observed. Disruption of lig3 in zebrafish reproduced the brain alterations and impaired gut transit in vivo. In conclusion, we identified variants in the LIG3 gene that result in a mitochondrial disease characterized by predominant gut dysmotility, encephalopathy, and neuromuscular abnormalities.


Assuntos
DNA Ligase Dependente de ATP/genética , Gastroenteropatias/genética , Motilidade Gastrointestinal/genética , Encefalomiopatias Mitocondriais/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Animais , Feminino , Gastroenteropatias/patologia , Humanos , Masculino , Encefalomiopatias Mitocondriais/patologia , Mutação , Linhagem , Peixe-Zebra
20.
J Mol Neurosci ; 71(12): 2526-2533, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33825174

RESUMO

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a multi-system disorder caused by several homozygous or compound heterozygous mutations, mostly in the nuclear gene of TYMP. Our current knowledge on the underlying pathology of the disease is derived through the study of about 200 cases of different ethnicities. Clinical presentations include severe cachexia, weakness, ptosis, diplopia, abdominal cramps or digestive tract disorders, hearing impairment, and paresthesia.Herein, we aim to present five novel mutations of the nuclear gene of TYMP in six Iranian patients diagnosed with MNGIE. In our population, age at the time of diagnosis was 18 to 49 years, while the onset of the symptoms varied from 13 to 20 years. We detected two pathogenic non-frameshift nonsense premature stop codon mutations (c.1013C > A, and c.130C > T), one variant of uncertain significance (VUS) non-frameshift missense mutation (c.345G > T), one likely pathogenic frameshift insertion (c.801_802insCGCG), and one likely benign homozygous non-frameshift deletion (c.1176_1187del) from two siblings. Our findings also confirm the autosomal recessive inheritance pattern of MNGIE in the Iranian population. The lack of knowledge in the area of nuclear gene-modifier genes shadows the genotype-phenotype relationships of MNGIE.


Assuntos
Pseudo-Obstrução Intestinal/genética , Distrofia Muscular Oculofaríngea/genética , Oftalmoplegia/congênito , Fenótipo , Timidina Fosforilase/genética , Adolescente , Códon sem Sentido , Feminino , Genes Recessivos , Humanos , Pseudo-Obstrução Intestinal/patologia , Irã (Geográfico) , Masculino , Distrofia Muscular Oculofaríngea/patologia , Oftalmoplegia/genética , Oftalmoplegia/patologia , Timidina Fosforilase/metabolismo , Adulto Jovem
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