Lysosomal dysfunction and overload of nucleosides in thymidine phosphorylase deficiency of MNGIE.

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.

Mitochondrial Neurogastrointestinal Encephalomyopathy Syndrome Treated with Stem Cell Transplant: A Case Series and Literature Review.

Mitochondrial neurogastrointestinal encephalomyopathy syndrome is a rare autosomal recessive multisystem disorder caused by nuclear TYMP gene mutations, which leads to deficiency in thymidine phosphorylase enzyme. This deficiency then leads to mitochondrial dysfunction, which causes the features characteristic of this syndrome, including severe muscle wasting, gastrointestinal dysmotility, leukoencephalopathy, peripheral neuropathy, and ophthalmoplegia. Here, we present a case series of 3 patients with mitochondrial neurogastrointestinal encephalomyopathy from Saudi Arabia who underwent allogeneic stem cell transplant at King Faisal Specialist Hospital (Riyadh, Saudi Arabia). Two patients died within the first year of transplant, and the third is still alive but without improvement in clinical features. Allogeneic hematopoietic stem cell transplant-related mortality appears to be high; this may at least be partially related to established end-organ effects with decreased performance status. Although allogeneic hematopoietic stem cell transplant clearly affects correction of genetic and biochemical defects in mitochondrial neurogastrointestinal encephalomyopathy, its ability to reverse or improve established clinical manifestations has not been proven.

Transplantation of enteric nervous system stem cells rescues nitric oxide synthase deficient mouse colon.

Enteric nervous system neuropathy causes a wide range of severe gut motility disorders. Cell replacement of lost neurons using enteric neural stem cells (ENSC) is a possible therapy for these life-limiting disorders. Here we show rescue of gut motility after ENSC transplantation in a mouse model of human enteric neuropathy, the neuronal nitric oxide synthase (nNOS-/-) deficient mouse model, which displays slow transit in the colon. We further show that transplantation of ENSC into the colon rescues impaired colonic motility with formation of extensive networks of transplanted cells, including the development of nNOS+ neurons and subsequent restoration of nitrergic responses. Moreover, post-transplantation non-cell-autonomous mechanisms restore the numbers of interstitial cells of Cajal that are reduced in the nNOS-/- colon. These results provide the first direct evidence that ENSC transplantation can modulate the enteric neuromuscular syncytium to restore function, at the organ level, in a dysmotile gastrointestinal disease model.

Loss-of-Function Variants in MYLK Cause Recessive Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital disorder characterized by loss of smooth muscle contraction in the bladder and intestine. To date, three genes are known to be involved in MMIHS pathogenesis: ACTG2, MYH11, and LMOD1. However, for approximately 10% of affected individuals, the genetic cause of the disease is unknown, suggesting that other loci are most likely involved. Here, we report on three MMIHS-affected subjects from two consanguineous families with no variants in the known MMIHS-associated genes. By performing homozygosity mapping and whole-exome sequencing, we found homozygous variants in myosin light chain kinase (MYLK) in both families. We identified a 7 bp duplication (c.3838_3844dupGAAAGCG [p.Glu1282_Glyfs∗51]) in one family and a putative splice-site variant (c.3985+5C>A) in the other. Expression studies and splicing assays indicated that both variants affect normal MYLK expression. Because MYLK encodes an important kinase required for myosin activation and subsequent interaction with actin filaments, it is likely that in its absence, contraction of smooth muscle cells is impaired. The existence of a conditional-Mylk-knockout mouse model with severe gut dysmotility and abnormal function of the bladder supports the involvement of this gene in MMIHS pathogenesis. In aggregate, our findings implicate MYLK as a gene involved in the recessive form of MMIHS, confirming that this disease of the visceral organs is heterogeneous with a myopathic origin.

Mitochondrial Neurogastrointestinal Encephalopathy: Clinical, Biochemical and Molecular Study in Three Egyptian Patients.

Background: Mitochondrial Neurogastrointestinal Encephalopathy syndrome is a rare autosomal recessive disorder. The disease is caused by mutations in the thymidine phosphorylase gene. This article reports the clinical, biochemical and molecular findings in three Egyptian patients with Mitochondrial Neurogastrointestinal Encephalopathy sundrome from two different pedigrees. Subjects and Methods: The three patients were subjected to thorough neurologic examination. Brain Magtnetic Resonance Imaging. Histochemical and biochemical assay of the mitochondrial respiratory chain complexes in muscle homogenate was performed (1/3). Thymidine Phosphorylase enzyme activity was performed in 2/3 patients and Thymidine Phosphorylase gene sequencing was done (2/3) to confirm the diagnosis. Results: All patients presented with symptoms of severe gastrointestinal dysmotility with progressive cachexia, neuropathy, sensory neural hearing loss, asymptomatic leukoencephalopathy. Histochemical analysis of themuscle biopsy revealed deficient cytochrome C oxidase and mitochrondrial respiratory chain enzyme assay revealed isolated complex 1 deficiency (1/3). Thymidine Phosphorylase enzyme activity revealed complete absence of enzyme activity in 2/3 patients. Direct sequencing of Thymidine Phosphorylase gene revealed c.3371 A>C homozygous mutation. Molecular screening of both families revealed heterozygous mutation in both parents and 4 siblings. Conclusions: Mitochondrial Neurogastrointestinal Encephalopathy syndrome is a rare mitochondrial disorder with an important diagnostic delay. In case of pathogenic mutations in Thymidine Phosphorylase gene in the family, carrier testing and prenatal diagmosis of at risk members is recommended for early detection. The possibility of new therapeutic options makes it necessary to diagnose the disease in an early state.

[Mitochondrial neurogastrointestinal encephalopathy syndrome].

The article presents a case report of clinical observations of syndrome mitochondrial neurogastroenterology encephalomyopathy (MNGIE syndrome).

Preclinical toxicity evaluation of erythrocyte-encapsulated thymidine phosphorylase in BALB/c mice and beagle dogs: an enzyme-replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy.

Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is currently under development as an enzyme replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disorder caused by a deficiency of thymidine phosphorylase. The rationale for the development of EE-TP is based on the pathologically elevated metabolites (thymidine and deoxyuridine) being able to freely diffuse across the erythrocyte membrane where the encapsulated enzyme catalyses their metabolism to the normal products. The systemic toxic potential of EE-TP was assessed when administered intermittently by iv bolus injection to BALB/c mice and Beagle dogs for 4 weeks. The studies consisted of one control group receiving sham-loaded erythrocytes twice weekly and two treated groups, one dosed once every 2 weeks and the other dosed twice per week. The administration of EE-TP to BALB/c mice resulted in thrombi/emboli in the lungs and spleen enlargement. These findings were also seen in the control group, and there was no relationship to the number of doses administered. In the dog, transient clinical signs were associated with EE-TP administration, suggestive of an immune-based reaction. Specific antithymidine phosphorylase antibodies were detected in two dogs and in a greater proportion of mice treated once every 2 weeks. Nonspecific antibodies were detected in all EE-TP-treated animals. In conclusion, these studies do not reveal serious toxicities that would preclude a clinical trial of EE-TP in patients with MNGIE, but caution should be taken for infusion-related reactions that may be related to the production of nonspecific antibodies or a cell-based immune response.

Assessment of thymidine phosphorylase function: measurement of plasma thymidine (and deoxyuridine) and thymidine phosphorylase activity.

We describe detailed methods to measure thymidine (dThd) and deoxyuridine (dUrd) concentrations and thymidine phosphorylase (TP) activity in biological samples. These protocols allow the detection of TP dysfunction in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Since the identification of mutations in TYMP, the gene encoding TP, as the cause of MNGIE (Nishino et al. Science 283:689-692, 1999), the assessment of TP dysfunction has become the best screening method to rule out or confirm MNGIE in patients. TYMP sequencing, to find the causative mutations, is only needed when TP dysfunction is detected. dThd and dUrd are measured by resolving these compounds with high-performance liquid chromatography (HPLC) followed by the spectrophotometric monitoring of the eluate absorbance at 267 nm (HPLC-UV). TP activity can be measured by an endpoint determination of the thymine formed after 1 h incubation of the buffy coat homogenate in the presence of a large excess of its substrate dThd, either spectrophotometrically or by HPLC-UV.

Chronic intestinal pseudo-obstruction and neurological manifestations in early adulthood: considering MNGIE syndrome in differential diagnosis.

The mitochondrial neurogastrointestinal encephalomyopathy syndrome (MNGIE) is a rare and life-threatening, autosomal recessive, multisystem disorder, caused by the mutations in the thymidine phosphorylase gene. Herein, we report a case of a 21 year-old male with a long history of intestinal pseudo-obstruction who was diagnosed with MNGIE syndrome after an extensive examination. In this case, our objective was to bring the gastroenterologist's attention to this difficult to diagnose syndrome in the coexistence of intestinal pseudo-obstruction and neurologic manifestations. The patient was a member of a consanguineous family of six children, in whom two sisters had died due to this disorder and one sister was affected and is still alive. The patient presented with cachexia, abdominal pain, diarrhea and muscle weakness, and was previously considered to have gluten sensitive enteropathy and treated with dietary solutions.

Biochemical abnormalities in a patient with thymidine phosphorylase deficiency with fatal outcome.

Deficiency of the cytosolic enzyme thymidine phosphorylase (TP) causes a multisystem disorder called mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome. Clinical symptoms are gastrointestinal dysfunction, muscle involvement and neurological deterioration. TP deficiency is biochemically characterised by accumulation of thymidine and deoxyuridine in body fluids and compromised mitochondrial deoxyribose nucleic acid (mtDNA) integrity (depletion and multiple deletions). In this report we describe a patient with the clinical and biochemical features related to the end stage of the disease. Home parenteral nutrition had started to improve the clinical condition and preparations were initiated for stem cell transplantation (SCT) as a last resort treatment. Unfortunately, the patient died during the induction phase of SCT. This report shows that TP deficiency is a severe clinical condition with a broad spectrum of affected tissues. TP deficiency can be easily determined by the measurement of pyrimidine metabolites in body fluids and TP activity in peripheral blood leucocytes. Early detection and treatment may prevent the progress of the clinical symptoms and, therefore, should be considered for inclusion in newborn screening programmes.

Inhibition of protein kinase A in murine enteric neurons causes lethal intestinal pseudo-obstruction.

A number of in vitro studies suggest that many important developmental and functional events in the enteric nervous system are regulated by the intracellular signaling enzyme cAMP protein kinase A (PKA). To evaluate the in vivo significance of these observations, a Cre-inducible, dominant-negative, mutant regulatory subunit (RIalphaB) of PKA was activated in enteric neurons by either a Proteolipid protein-Cre transgene or a Hox11L1-Cre "knock-in" allele. In both models, RIalphaB activation resulted consistently in profound distension of the proximal small intestine within 2 weeks after birth. Intestinal transit of radio-opaque tracers was severely retarded in the double-transgenic animals, which died shortly after weaning. In the enteric nervous system, recombination was restricted to neurons as demonstrated by histochemical analysis and confocal microscopic colocalization of a Cre recombinase-dependent reporter gene with the neuronal marker Hu(C/D), in contrast with the glial marker S100. Histochemical analysis of beta-galactosidase expression and acetylcholinesterase activity, as well as neuronal counts, demonstrated that intestinal dysmotility was not associated with obvious malformation of the myenteric plexus. However, inhibition of PKA activity in enteric neurons disrupted the major motor complexes of isolated intestinal segments in vitro. These results provide strong evidence that PKA activity plays a critical role in enteric neurotransmission in vivo, and highlight neuronal PKA or related signaling molecules as potential therapeutic targets in gastrointestinal motility disorders.

Study of the pathogenesis of paralytic ileus in animal models of experimentally induced postoperative and septic ileus.

Paralytic ileus is defined as an inhibition of propulsive intestinal motility. Postoperative ileus is the most common type, however, also during sepsis and critical illness paralytic ileus is a common finding. The pathogenesis of paralytic ileus is still debated. It is believed to result from the activation of inhibitory neural reflex pathways and activation of inflammatory processes. It is generally accepted that postoperative ileus results from the activation of an inhibitory neural reflex pathway. In our rat model we showed that different degrees of nociceptive stimulation activate different reflex pathways: laparatomy activates an adrenergic inhibitory reflex pathway, whereas manipulation results in additional activation of inhibitory NANC neurons releasing NO and VIP as neurotransmitters. We also demonstrated that blockade of the afferent limb of the reflex pathway by peripheral kappa-opioid agonists or by non-steroidal anti-inflammatory drugs ameliorated postoperative ileus. However, the use of prokinetics lead to disappointing results. In the murine septic model we demonstrated an important role for activation of inducible NO synthase in the endotoxin-induced delay in gastric emptying and small intestinal transit. We hypothesise that activation of the residential macrophages in the gut wall leads to the production of iNOS and other inflammatory mediators. These mediators will attract more inflammatory cells and influence smooth muscle contractility. Next, we provide evidence that production of iNOS results in the activation of guanylyl cyclase leading to the production of cGMP and smooth muscle relaxation. However, a parallel mechanism of action for NO via oxidative stress needs further investigation.

COX-2 dependent inflammation increases spinal Fos expression during rodent postoperative ileus.

BACKGROUND AND AIMS: Cyclooxygenase 2 (COX-2) and prostaglandins (PGs) participate in the pathogenesis of inflammatory postoperative ileus. We sought to determine whether the emerging neuronal modulator COX-2 plays a significant role in primary afferent activation during postoperative ileus using spinal Fos expression as a marker. METHODS: Rats, and COX-2(+/+) and COX-2(-/-) mice underwent simple intestinal manipulation. The effect of intestinal manipulation on Fos immunoreactivity (IR) in the L(5)-S(1) spinal cord, in situ circumference, and postoperative leucocytic infiltrate of the intestinal muscularis was measured. Postoperative PGE(2) production was measured in peritoneal lavage fluid. The dependence of these parameters on COX-2 was studied in pharmacological (DFU, Merck- Frosst, selective COX-2 inhibitor) and genetic (COX-2(-/-) mice) models. RESULTS: Postoperative Fos IR increased 3.7-fold in rats and 2.2-fold in mice. Both muscularis leucocytic infiltrate and the circumference of the muscularis increased significantly in rats and COX-2(+/+) mice postoperatively, indicating dilating ileus. Surgical manipulation markedly increased PGE(2) levels in the peritoneal cavity. DFU pretreatment and the genetic absence of COX-2(-/-) prevented dilating ileus, and leucocytic infiltrate was diminished by 40% with DFU and by 54% in COX-2(-/-) mice. DFU reversed postsurgical intra- abdominal PGE(2) levels to normal. Fos IR after intestinal manipulation was attenuated by approximately 50% in DFU treated rats and in COX-2(-/-) mice. CONCLUSIONS: Postoperatively, small bowel manipulation causes a significant and prolonged increase in spinal Fos expression, suggesting prolonged primary afferent activation. COX-2 plays a key role in this response. This activation of primary afferents may subsequently initiate inhibitory motor reflexes to the gut, contributing to postoperative ileus.

Familial mitochondrial intestinal pseudo-obstruction and neurogenic bladder.

Intestinal dysmotility and neurogenic bladder have been described as part of two autosomal-recessive mitochondrial disorders assumed to be due to a defect in communication between the nuclear and mitochondrial genomes: myoneurogastrointestinal encephalopathy (MNGIE) and diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (Wolfram syndrome). Partial cytochrome c oxidase deficiency has been described in both. We describe three Ashkenazi Jewish siblings with progressive intestinal dysmotility, neurogenic bladder, and autonomic manifestations but no central nervous system involvement. Cytochrome c oxidase deficiency was demonstrated in peripheral and multiple intestinal muscle biopsies. Mitochondrial DNA analysis of an intestinal biopsy of patient 1 showed heteroplasmy consisting of a normal 16.5-kb band and an approximately 28-kb band, suggestive of a duplication. Mitochondrial DNA analysis of a muscle biopsy of patient 2 showed multiple deletions, mainly 10- and 11-kb bands. We suggest that this unique combination of intestinal pseudo-obstruction and neurogenic bladder could comprise a new autosomal-recessive mitochondrial disorder.

Involvement of endogenous nitric oxide and c-kit-expressing cells in chronic intestinal pseudo-obstruction.

BACKGROUND/PURPOSE: Chronic intestinal pseudo-obstruction (CIP) in infants and children is a motility disorder without apparent mechanical cause. Nitric oxide (NO), an inhibitory neurotransmitter and c-kit cells, essential for the intestinal pacemaker activity, both play a key role in the intestinal motility function. In the current study, the authors investigated the distributive change in the intestinal nitric oxide synthase (NOS) and c-kit cells of patients with CIP. METHODS: Tissues were obtained from 4 patients undergoing bowel resection or biopsy for CIP at laparotomy. For controls, the intestinal specimens were obtained from 4 age-matched cases of intestinal stricture, intussusception, and autopsy with no evidence of gastrointestinal disease. Immunohistochemical studies were performed on paraffin-embedded tissue cross sections with neuronal NOS and inducible NOS monoclonal antibody as well as a rabbit polyclonal antibody against the human c-kit receptor. RESULTS: Under immunohistochemical staining, a greatly increased density of neuronal NOS immunoreactivity and an evidently increased number of intense NOS immunoreactive nerve fibers were observed in the myenteric plexus and circular muscle layers compared with the control sections. In the submucosal plexus and longitudinal muscle layer, there was no change in NOS immunoreactivity. Inducible NOS immunoreactivity was not detected in the control cases. However, in tissues of CIP, almost all the epithelial cells were positively and strongly labeled for inducible NOS immunoreactivity. For c-kit cells staining, the number of c-kit-positive cells in the myenteric plexus and circular muscle layers were greatly less than that in the controls, especially in the myenteric plexus region. CONCLUSION: These findings suggest that sustained production of NO by an increased NOS activity and a deficiency of c-kit cells in the intestine may be related to the pathogenesis of CIP.

Chronic intestinal pseudoobstruction with myopathy and ophthalmoplegia. A muscular biochemical study of a mitochondrial disorder.

The association of chronic intestinal pseudoobstruction with ophthalmoplegia has been reported previously in visceral myopathies. We report a case of this association in which muscle mitochondria had a crystalline appearance, a dense core, and decreased cytochrome c oxidase and succinate cytochrome c reductase activities. The absence of evident mitochondrial DNA deletion in the skeletal muscle of this patient does not exclude the possibility of localized deletion or mutation of mitochondrial DNA in digestive muscle.