Characterization of a Novel Pathogenic Mutation Causing Mitochondrial Neurogastrointestinal Encephalopathy

Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a rare autosomal recessive disorder caused by mutations in TYMP gene, encoding nuclear thymidine phosphorylase (TP). MNGIE mainly presents with gastrointestinal symptoms and is mostly misdiagnosed in many patients as malabsorption syndrome, inflammatory bowel disease, anorexia nervosa, and intestinal pseudo-obstruction. Up to date, more than 80 pathogenic and likely pathogenic mutations associated with the disease have been reported in patients from a wide range of ethnicities. The objective of this study was to investigate the underlying genetic abnormalities in a 25-year-old woman affected with MNGIE. Multiple alignment species.

2 Abstract Background Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a rare autosomal recessive disorder caused by mutations in TYMP gene, encoding nuclear thymidine phosphorylase (TP). MNGIE mainly presents with gastrointestinal symptoms and is mostly misdiagnosed in many patients as malabsorption syndrome, inflammatory bowel disease, anorexia nervosa, and intestinal pseudo-obstruction. Up to date, more than 80 pathogenic and likely pathogenic mutations associated with the disease have been reported in patients from a wide range of ethnicities. The objective of this study was to investigate the underlying genetic abnormalities in a 25-year-old woman affected with MNGIE.

Case Presentation
The patient was a 25-year-old female referred to our center with the chief complaint of severe abdominal pain and diarrhea for two years that had worsened from two months prior to admission. The clinical and para-clinical findings were in favor of mitochondrial neurogastrointestinal encephalopathy syndrome. Subsequent genetic studies revealed a novel, private, homozygous nonsense mutation in TYMP gene (c. 1013 C>A, p.S338X).
Sanger sequencing confirmed the new mutation in the proband. Multiple sequence alignment showed high conservation of amino acids of this protein across different species.

Conclusion
The detected new nonsense mutation in the TYMP gene would be very important for genetic counseling and subsequent early diagnosis and initiation of proper therapy. This novel pathogenic variant would help us establish future genotype-phenotype correlations and identify different pathways related to this disorder. 3 Background Mitochondrial neurogastroinstestinal encephalopathy (MNGIE -OMIM# 603041) is a rare multisystem autosomal recessive disorder caused by homozygous or compound heterozygous mutations in the nuclear-encoded thymidine phosphorylase gene (TYMP; 131222) on chromosome 22q13, the first gene whose role was defined at molecular level in the defects of intergenomic communication [1]. Impairment in this enzyme with resultant decreased enzyme activity leads to accumulation of the enzyme's substrates, thymidine and deoxyuridine, which in turn leads to an imbalance in the intra-mitochondrial nucleotide pool and multiple deletions, point-specific mutations and depletions in mitochondrial DNA (mtDNA) [2,3]. Recent evidence has suggested that the accumulation of these nucleosides is the main culprit for the development of the molecular and phenotypic aberrations reported in this disorder [4].
First described in 1976, MNGIE usually presents in the first to fifth decades of life, with a progressive clinical course leading to death at the mean age of 37 [5][6][7]. The clinical manifestations of MNGIE are severe gastrointestinal dysmotility, cachexia, extraocular muscle weakness with resultant ptosis and ophthalmoplegia, sensorimotor neuropathy, and leukoencephalopathy [8]. More than 120 patients with diagnostic features consistent with MNGIE have so far been reported in the literature, with more than 80 pathogenic and likely pathogenic mutations (https://www.ensembl.org) associated with the disease identified in patients from a wide range of ethnicities [9, 10].
Herein, we report on a patient with MNGIE with a novel homozygous mutation in TYMP gene, along with the clinical, laboratory and imaging findings.

Case Presentation
Clinical Presentation 4 A 25-year-old female was referred to our center with the chief complaint of severe abdominal pain and diarrhea for two years that had worsened from two months prior to admission. She had significant weight loss during this period; weighing 36.5 kg with a height of 160 cm, her body mass index (BMI) was 14.3 kg/m 2 at the time of admission. Her past medical and surgical history was only significant for one undocumented episode of seizure at the age of three and appendectomy three years before. On interview, she denied fear of weight gain, laxative abuse, and self-induced vomiting. Her parents were Echocardiography was normal except for mild pericardial effusion.
The electrodiagnostic evaluation showed a neurogenic pattern on needle electromyography (EMG), conduction block in sensory nerves, and decreased compound muscle action potential (CMAP) in motor nerves with decreased conduction velocity and prolonged F-wave latency. Brain MRI with contrast showed leukoencephalopathy with diffusely increased T 2 signal intensity in both cerebral hemispheres white matter.
Hypersignal intensity in splenium of corpus callosum was also observed ( Figure 1).

Molecular Analysis
After obtaining informed consent, a blood sample was obtained from the patient. The whole blood sample was prepared in EDTA tube. Genomic DNA was then extracted from peripheral leukocytes using the QIAamp DNA Blood Mini Kit (Qiagen, Germany).
To reveal the conservation of amino acid sequence of TYMP protein across various species, multiple sequence alignment analysis by BLAST available on ExPASy  Progressive external ophthalmoplegia (PEO) was the first disease identified in this group caused by defects in the intergenomic communication [14]. MNGIE, a rare progressive multisystem autosomal recessive disorder caused by a mutation in TYMP gene is also a member of this group of disorders.
TYMP gene which encodes the cytosolic enzyme named thymidine phosphorylase, TP, is located at chromosome 22q13.33. This protein catalyzes phosphorylation of mitochondrial dThd and dUrd to thymine and uridine, respectively (Figure 4). TP plays an important role in the metabolic pathways of various cells including those in the brain, muscle, RBCs, WBCs, and bone marrow, relying on the salvage pathway for recovering nucleosides [15,16]. MNGIE usually presents with symptoms of gastrointestinal dysfunction, such as gastrointestinal motility disorders, gastro-esophageal reflux, dysphagia, abdominal pain and distention, and diarrhea leading to severe weight loss and cachexia [17]. At this stage of the disease, most of the patients are misdiagnosed as malabsorption syndrome, inflammatory bowel disease (IBD), anorexia nervosa, or intestinal pseudo-obstruction, often leading to unnecessary medical interventions and delay in diagnosis of up to 10 years [6,[18][19][20]. Ptosis, ophthalmoparesis, hearing loss, and sensory-motor neuropathy constitute the most common neurologic features of patients with MNGIE [8]. Due to the high metabolic activity of extraocular muscles, deterioration in their function resulting in ophthalmoplegia or ophthalmoparesis occurs early in the course of the disease that parallels the disease progression [21]. Neuroimaging studies such as brain MRI and magnetic resonance spectroscopy (MRS) might yield a clue about the diagnosis of MNGIE, with the absence of leukoencephalopathy ruling out MNGIE in most cases. Unlike the patient reported here, who had involvement of splenium of the corpus callosum, it is relatively spared in most individuals [8,10,22]. Our patient was found to have leukoencephalopathy, with diffuse T 2 hyperintensity in both cerebral hemispheres white 8 matter on brain MRI, though, she had normal cognitive function. This, in turn, is likely to be due to the impaired blood-brain barrier function in these patients leading to edema in lieu of demyelination [23].
Other disorders with phenotypes similar to MNGIE, caused by mutations in RRM2B and POLG genes have been reported [24,25]. Therefore, it is prudent to test the individuals suspected of having MNGIE for these genes as well. In our patient whole exome sequencing was done and no mutations in these genes were detected.
To the best of our knowledge, around 80 pathogenic mutations in the TYMP gene have so far been reported. Attempts to draw a genotype-phenotype correlation in this disorder have mostly been discouraging, except for c. 622G>A variant (p.Val208Met), producing less severe TP dysfunction, leading to a late-onset disease [26][27][28].
Current treatment modalities for MNGIE mainly focus on restoration of the activity of TP and lowering the circulatory levels of the nucleoside substrates. Hematopoietic stem cell transplantation (HSCT) has so far been used to restore TP enzyme activity in patients with MNGIE. A retrospective analysis of 24 patients who underwent HSCT for the treatment of MNGIE revealed a survival rate of 37.5% after a median follow-up of almost four years.
Most of the deaths were attributed to the transplantation with MNGIE complications leading to death in a quarter of the patients [29]. It was found that younger patients without gastrointestinal dysmotility and liver disease receiving HSCT from an HLAmatched donor would benefit mostly from this type of treatment, highlighting the importance of diagnosis in the momentous days early in the course of the illness, when HSCT would change the course of the disease [29]. Hemodialysis and peritoneal dialysis have also been proposed as treatment modalities in these patients intending to remove the nucleosides from the peripheral circulation. A prospective study evaluating a 29-yearold patient with MNGIE who underwent extensive hemodialysis for one year also revealed that it has only a transient effect on the serum and urine levels of nucleosides with no long-term effects; there were no changes in the level of the toxic metabolites in the CSF in both short-term (within 24 hours) and long-term (at months 6 and 12) [30]. Our patient in this report underwent hemodialysis with only mild improvements in the gastrointestinal symptoms. These findings cast doubt on the efficacy of dialysis in the treatment of MNGIE.
Other therapeutic modalities including platelet infusion, which was also performed in our patient, and orthotopic liver transplantation have also been reported for the treatment of this disease in the literature [4,31].

Consent for publication
The patient consented to the publication of the case and accompanying clinical and genetics data. Written informed consent was obtained from the patient.

Availability of data and materials
All data are available from the corresponding author on request.  Figure 1 Brain MRI revealing diffuse white matter signal changes.

Figure 2
Sanger sequencing chromatogram of the novel identified variant in this study.
The arrow shows the position of homozygous mutation in TYMP (c. 1013 C>A, p.S338X).
18 Figure 3 Multiple sequence alignment of the whole amino acid sequence of TYMP with other TYMP orthologs across different species.
19 Figure 4 The main roles of TYMP in pyrimidine metabolism extracted from the KEGG database (ko00240). The red numbers (2.4.2.4) shows the positions of the enzyme functions in pyrimidine metabolism. As depicted, this enzyme with its phosphorolysis activity on thymidine (TdR) is able to catalyze TdR to thymine and 2-deoxy-D-ribose 1-phosphate (dR-1-P). TYMP also catalyze the thymidine formation from thymine and dR-1-P. TP also in another catalectic activity can phosphorolyze the deoxyuridine to uracil and dR-1-P, indicating its vital roles in biological metabolism.

Supplementary Files
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