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A novel thymidine phosphorylase mutation in a Spanish MNGIE patient
Journal of the Neurological Sciences 228 (2005) 35 – 39 www.elsevier.com/locate/jns
A novel thymidine phosphorylase mutation in a Spanish MNGIE patient Josep Gameza, Maria Carmen Larab, Fermin Mearinc, Carlos Oliveras-Leyd, Nuria Raguere, Montse Olivef, Andres T. Leistg, Antonia Perelloc, Monica Peronac, Carlos Cerveraa, Antonio Luis Andreub, Ramon Martı´b, Michio Hiranoh,* a
Department of Neurology, Hospital Universitari Vall d’ Hebron, Barcelona, Spain b CIBBIM, Hospital Universitari Vall d’ Hebron, Barcelona, Spain c Department of Gastroenterology, Centro Medico Teknon, Barcelona, Spain d Department of Neurology, Centro Medico Teknon, Barcelona, Spain e Department of Neurophysiology, Hospital Universitari Vall d’ Hebron, Barcelona, Spain f Department of Neurophysiology, Centro Medico Teknon, Barcelona, Spain g Department of Radiology, Centro Medico Teknon, Barcelona, Spain h Department of Neurology, Columbia University College of Physicians and Surgeons, 630 West 168th St., P and S 4-443, New York, NY 10032, USA Received 4 June 2004; received in revised form 9 September 2004; accepted 10 September 2004 Available online 12 November 2004
Abstract A 29-year-old Spanish man presented with chronic intestinal pseudo-obstruction, progressive external ophthalmoplegia, peripheral neuropathy, and diffuse leukoencephalopathy. This combination of clinical features is characteristic of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Genetic analysis revealed a novel 18-base pair (bp) duplication (5044–5061dup) in exon 8 of the thymidine phosphorylase (TP) gene. The mutation is predicted to produce a 6 amino acid insertion in the alpha–beta-domain of the protein. This 18-bp insertion in the thymidine phosphorylase gene is the first duplication mutation identified in MNGIE. D 2004 Elsevier B.V. All rights reserved. Keywords: Mitochondrial encephalomyopathies; MNGIE; Thymidine phosphorylase; Mutation; Intergenomic communication defect; Duplication
1. Introduction Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive multisystem disorder, characterized by gastrointestinal, extraocular muscle, peripheral nerve, and cerebral white matter involvement [1–3]. Although the onset of symptoms is typically between the second and fifth decades of life, some individuals have presented in infancy with recurrent vomiting and difficulty gaining weight. Although the exact incidence and prevalence of the disease in the general population is unknown, disproportionately large numbers of
* Corresponding author. Tel.: +1 212 305 1048; fax: +1 212 305 3986. E-mail address: [email protected] (M. Hirano). 0022-510X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2004.09.034
patients have been identified in Mediterranean, Hispanic, Iranian–Jewish, and Ashkenazi–Jewish populations [3]. While neurological and gastrointestinal features are uniformly present in MNGIE patients, symptoms and signs of visceral neuropathy and myopathy are usually the most prominent and debilitating manifestations of the disease. Consequently, MNGIE patients complain of early satiety, borborygmi, vomiting, diarrhea, constipation, chronic abdominal pain, and episodic intestinal pseudo-obstruction. Gastrointestinal involvement leads to a severe weight loss in patients, who rapidly acquire a cachexic appearance, with signs of malnutrition including hypoproteinemia. The prognosis for MNGIE patients is related to the severity of gastrointestinal manifestations. The age at death has ranged between 18 and 58 years old, with a mean of 37 years old [3]. Mutations in the gene encoding thymidine phosphorylase (TP) have been identified as the cause of the disease [4]. TP
36
J. Gamez et al. / Journal of the Neurological Sciences 228 (2005) 35–39
catalyses the reversible phosphorolysis of thymidine (dThd) to thymine and is an essential enzyme in maintaining homeostasis of cellular nucleotide pools [5]. As a consequence of the absence of TP, high levels of dThd and deoxyuridine (dUrd) are present in the plasma of patients [6,7]. It has been hypothesized that excess dThd and dUrd alter mitochondrial nucleoside/nucleotide pools, which, in turn, impair mitochondrial DNA replication and/or repair [4,6,7]. MNGIE thus belongs to the group of mitochondrial encephalomyopathies caused by defects in intergenomic communication [2,8–10]. Although fewer than 75 MNGIE patients have been reported, the molecular genetic basis of the disease is heterogeneous [3,11,12]. Thirty different mutations in the TP gene have been identified and no predominant mutation has been reported [3,11,12]. Most of the mutations are missense mutations. Deletions, insertions, and frameshift and splice-site mutations have also been reported. To date, and to the best of our knowledge, no duplications have been described [2–4,11–13]. In this study, we report on the clinical, biochemical, and genetic characteristics of a family with MNGIE and a novel microduplication mutation in the TP gene.
2. Patients and methods 2.1. Case report The patient, a 29-year-old man (case III:5 in Fig. 1), was referred in May 2003 for evaluation of chronic intestinal pseudo-obstruction, associated with severe weight loss and hypoalbuminemia for 12 months.
In retrospect, he had suffered from postprandial abdominal pain, early satiety, borborygmi, nausea, recurrent vomiting, and poor weight gain since infancy. Despite being 1.78 m tall, he had never weighed more than 50 kg. His parents were not consanguineous. In August 1999, he developed mild symmetric distal limb weakness. Three months later, he noted paresthesias in the hands and feet. Electrophysiological studies suggested a demyelinating polyneuropathy. CSF showed normal proteins and no cells. On admission in May 2003, he was markedly cachectic and weighed 44 kg. He had mild bilateral external ophthalmoparesis and enophthalmos, but no ptosis. Mild muscle weakness was present affecting the deltoids, biceps, and iliopsoas muscles (Medical Research Council scale 4+/ 5). Distal muscle strength was normal. Tendon reflexes were absent. A radionuclide gastric emptying study 3 h after ingestion revealed gastroparesis with remnant material. Upper gastrointestinal series showed distended jejuneum and ileum without obstruction. Esophagogastroduodenoscopy, ileoscopy, and colonoscopy were normal. Gastrointestinal manometry showed a neuropathic pattern affecting the distal half of the stomach and the duodenum. An increase in nonperistaltic duodenal motility was observed after the administration of octreotide. Abdominal CT showed no alterations. Rectal biopsy was normal. T2-weighted brain magnetic resonance (MRI) showed a diffuse leukoencephalopathy affecting both cerebral hemispheres, the brain stem and the cerebellum, but sparing the corpus callosum and basal ganglia. Electrophysiological studies revealed a severe sensory motor demyelinating neuropathy, and a mild myogenic pattern in the proximal muscles in all four limbs. Electrocardiogram showed a sinus
Fig. 1. Family Pedigree. The proband is indicated by an arrow. Genotypes are shown below the symbols. WT, wild-type. dup=nts. 5044–5061dup.
J. Gamez et al. / Journal of the Neurological Sciences 228 (2005) 35–39
37
Table 1 TP activities and nucleoside levels in the proband and family members
2.3. Molecular genetic studies
Case
Genotype
TP in buffy coat (nmol/h/mg protein)a
Plasma dThd (AM)a
Plasma dUrd (AM)a
III:5 (proband) III:4 II:3 II:4
dup/dup WT/dup WT/dup WT/dup
8 ND ND ND
3.2 b0.05 b0.05 b0.05
9.5 b0.05 b0.05 b0.05
Informed consent for blood samples was obtained from the members of the family studied, under an IRB-approved protocol. DNA was isolated from peripheral blood leukocytes using standard methods. The entire coding region of the TP gene plus intronic segments flanking the exons was PCR amplified as described [4]. The PCR products were purified and sequenced directly, using the ABI Prism BigDye Terminator Cycle Sequencing Kit and an ABI Prism 310 sequencer Genetic Analyser (Applied Biosystems, Foster City, CA).
ND = not determined. WT = wild-type. dup = nts.5044–5061dup. a Control values: TP activity, mean = 607 nmol/h/mg protein, range = 544 – 692 ; plasma dThd and dUrd levels were undetectable (b0.05 AM) in controls.
tachycardia. Echocardiogram results revealed mitral valve prolapse. Neurological evaluation of his sister (III:4), including nerve conduction studies and electromyography, was normal. 2.2. Biochemical studies TP activity in buffy coat was assessed using the previously described method [6]. Plasma dThd and dUrd levels were determined using an HPLC method, as previously described [6,7].
3. Results TP activity in buffy coat was virtually abolished in the proband (8 nmol/h/mg protein; 1.3% of the average control value level; Table 1). Plasma dThd in the patient was 3.2 AM (normalb0.05 AM) and dUrd was 9.5 AM (normalb 0.05 AM; Table 1). Sequence analysis of the TP gene revealed the presence of a homozygous 18-base pair (bp) microduplication in exon 8 spanning nucleotides (nts) 5044–5061 (GCGGCGCTGGACGACGGC; GenBank accession # M58602). The same
Fig. 2. Electropherograms showing the homozygous 18-bp duplication (upper panel) and the wild-type sequences (lower panel).
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J. Gamez et al. / Journal of the Neurological Sciences 228 (2005) 35–39
mutation was found in a heterozygous state in the patient’s parents (individuals II:3 and II:4) and sister (individual III:4). The mutation was not detected in DNA from 100 healthy individuals (Fig. 2).
4. Discussion Previously, we have shown that MNGIE can be diagnosed definitively by biochemical analyses demonstrating severely reduced thymidine phosphorylase activity in buffy coat (V8% of control mean) or increased dThd (N3 Amol/L) and dUrd levels (N5 Amol/L) in blood [14] or by identification of thymidine phosphorylase gene mutation(s). We confirmed the diagnosis in our patient by detecting severely reduced thymidine phosphorylase activity in buffy coat, dramatically increased plasma thymidine and deoxyuridine, and a novel homozygous TP gene mutation in our patient. It is important for clinicians to recognize that the clinical diagnosis of MNGIE can be secured by blood tests and a muscle biopsy is not required. In fact, because skeletal muscle may be misleading because this tissue is usually mildly affected in this disease and typical markers of mitochondrial alterations such as ragged-red and cytochrome c oxidase (COX) deficient fibers, and multiple deletions of mtDNA may be absent [12,13]. The pathogenicity of the TP mutation (nts.5044– 5061dup) is based on the following criteria: (i)
The 18-bp DNA duplication is predicted to cause a duplication of six amino acids (ALA ALA LEU ASP ASP GLY) between alanine (ALA) 332 and serine (SER) 339. (ii) The six amino acid insertion is located in the helix #14 in the alpha-beta-domain of the protein. As consequence, the amino acids immediately following the duplication are displaced. The altered COOH terminus may disrupt the three-dimensional structure of the enzyme, reducing its stability and activity. (iii) The g.5044–5061dup mutation was not found in 60 other MNGIE patients, or 100 unrelated healthy individuals. (iv) TP activity is virtually absent in the patient’s buffy coat and plasma dThd and dUrd levels are markedly increased. Thirty different mutations have been identified in nine of the ten exons. Pathogenic mutations have never been identified in exon 1, which is a noncoding exon [3,11,12]. Eleven of the mutations described in the TP gene are missense. The other reported mutations are microdeletions, single nucleotide insertions, and frameshift and splice-site mutations. To date, no duplications have been described. This six amino acid insertion is therefore the largest TP gene mutation.
No correlation has been found between specific mutations and the severity of clinical features [3]. As previously noted, skeletal muscle may show no morphological abnormalities of skeletal muscle [12,13]. Even more surprising than normal skeletal muscle is a lack of gastrointestinal symptoms in another MNGIE patient with TP-deficiency and TP gene mutations [15]. Intrafamilial phenotypic variability has also been described [13]. Genetic and clinical characterization of MNGIE families around the world is therefore necessary to further understand the genotypephenotype relationship of TP mutations and their relative frequency among different ethnic groups. An important clinical aspect of MNGIE is that patients’ survival and prognosis are generally related to the degree of gastrointestinal involvement, particularly weight loss. Patients often die as a result of cachexia, peritonitis, intestinal rupture, or esophageal bleeding related to cirrhosis, or aspiration pneumonia. Their mean age of death is 37 years old, and no effective therapy is available for this disorder. Nevertheless, parenteral nutrition can transiently ameliorate patients’ nutritional status [2]. Because excess dThd and dUrd levels are hypothesized to alter mitochondrial nucleoside and nucleotide pools leading to impaired mitochondrial DNA replication and/or repair, attempts to reduce dThd and dUrd levels may be a promising therapeutic strategy for MNGIE patients [6,7,16]. Alternatively, administration of exogenous TP enzyme could be a promising treatment.
Acknowledgements This study has been supported by a grant from the Spanish Fondo de Investigaciones Sanitarias (FIS 02/0648 and 03/0343), NIH grant P01NS1176, and a grant from the Muscular Dystrophy Association. The authors are indebted to the members of the family for their cooperation.
References [1] Hirano M, Silvestri G, Blake DM, Lombes A, Minetti C, Bonilla E, et al. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): clinical, biochemical, and genetic features of an autosomal recessive mitochondrial disorder. Neurology 1994;44:721 – 7. [2] Nishino I, Spinazzola A, Papadimitriou A, Hammans S, Steiner CD, Hahn CD, et al. MNGIE: an autosomal recessive disorder due to thymidine phosphorylase mutations. Ann Neurol 2000;47: 792 – 800. [3] Hirano M, Nishigaki Y, Martı´ R. MNGIE: a disease of two genomes. Neurologist 2004;10:8 – 17. [4] Nishino I, Spinazzola A, Hirano M. Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder. Science 1999; 283:689 – 92. [5] Brown NS, Bicknell R. Thymidine phosphorylase, 2-deoxy-d-ribose and angiogenesis. Biochem J 1998;334:1 – 8. [6] Spinazzola A, Marti R, Nishino I, Andreu A, Naini A, Tadesse S, et al. Altered thymidine metabolism due to defects of thymidine phosphorylase. J Biol Chem 2002;277:4128 – 33.
J. Gamez et al. / Journal of the Neurological Sciences 228 (2005) 35–39 [7] Martı´ R, Nishigaki Y, Hirano M. Elevated plasma deoxyuridine in patients with thymidine phosphorylase deficiency. Biochem Biophys Res Commun 2003;303:14 – 8. [8] Hirano M, Vu TH. Defects of intergenomic communication: where do we stand? Brain Pathol 2000;10:451 – 61. [9] Hirano M, Marti R, Ferreiro-Barros C, Vila` MR, Tadesse S, Nishigaki Y, et al. Defects of intergenomic communication: autosomal disorders that cause multiple deletions and depletion of mitochondrial DNA. Semin Cell Dev Biol 2001;12:417 – 27. [10] DiMauro S, Schon EA. Mitochondrial respiratory-chain diseases. N Engl J Med 2003;348:2656 – 68. [11] Kocaefe YC, Erdem S, Ozguc M, Tan E. Four novel thymidine phosphorylase gene mutations in mitochondrial neurogastrointestinal encephalomyopathy syndrome (MNGIE) patients. Eur J Hum Genet 2003;11:102 – 4.
39
[12] Szigeti K, Wong LJ, Perng CL, Saifi GM, Eldin K, Adesina AM, et al. MNGIE with lack of skeletal muscle involvement and a novel TP splice site mutation. J Med Genet 2004;41:125 – 9. [13] Gamez J, Ferreiro C, Accarino ML, Guarner L, Tadesse S, Marti RA, et al. Phenotypic variability in a Spanish family with MNGIE. Neurology 2002;59:455 – 7. [14] Marti R, Spinazzola A, Tadesse S, Nishino I, Nishigaki Y, Hirano M. Definitive diagnosis of mitochondrial neurogastrointestinal encephalomyopathy by biochemical assays. Clin Chem 2004;50:120 – 4. [15] Martin MA, Bla´zquez S, Martı´ R, Bautista J, Lara MC, Cabello A, et al. Lack of gastrointestinal symptoms in a 60 year-old patient with MNGIE. Neurology 2004;63:1536 – 7. [16] Nishigaki Y, Marti R, Copeland WC, Hirano M. Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency. J Clin Invest 2003;111:1913 – 21.
A novel thymidine phosphorylase mutation in a Spanish MNGIE patient Josep Gameza, Maria Carmen Larab, Fermin Mearinc, Carlos Oliveras-Leyd, Nuria Raguere, Montse Olivef, Andres T. Leistg, Antonia Perelloc, Monica Peronac, Carlos Cerveraa, Antonio Luis Andreub, Ramon Martı´b, Michio Hiranoh,* a
Department of Neurology, Hospital Universitari Vall d’ Hebron, Barcelona, Spain b CIBBIM, Hospital Universitari Vall d’ Hebron, Barcelona, Spain c Department of Gastroenterology, Centro Medico Teknon, Barcelona, Spain d Department of Neurology, Centro Medico Teknon, Barcelona, Spain e Department of Neurophysiology, Hospital Universitari Vall d’ Hebron, Barcelona, Spain f Department of Neurophysiology, Centro Medico Teknon, Barcelona, Spain g Department of Radiology, Centro Medico Teknon, Barcelona, Spain h Department of Neurology, Columbia University College of Physicians and Surgeons, 630 West 168th St., P and S 4-443, New York, NY 10032, USA Received 4 June 2004; received in revised form 9 September 2004; accepted 10 September 2004 Available online 12 November 2004
Abstract A 29-year-old Spanish man presented with chronic intestinal pseudo-obstruction, progressive external ophthalmoplegia, peripheral neuropathy, and diffuse leukoencephalopathy. This combination of clinical features is characteristic of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Genetic analysis revealed a novel 18-base pair (bp) duplication (5044–5061dup) in exon 8 of the thymidine phosphorylase (TP) gene. The mutation is predicted to produce a 6 amino acid insertion in the alpha–beta-domain of the protein. This 18-bp insertion in the thymidine phosphorylase gene is the first duplication mutation identified in MNGIE. D 2004 Elsevier B.V. All rights reserved. Keywords: Mitochondrial encephalomyopathies; MNGIE; Thymidine phosphorylase; Mutation; Intergenomic communication defect; Duplication
1. Introduction Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive multisystem disorder, characterized by gastrointestinal, extraocular muscle, peripheral nerve, and cerebral white matter involvement [1–3]. Although the onset of symptoms is typically between the second and fifth decades of life, some individuals have presented in infancy with recurrent vomiting and difficulty gaining weight. Although the exact incidence and prevalence of the disease in the general population is unknown, disproportionately large numbers of
* Corresponding author. Tel.: +1 212 305 1048; fax: +1 212 305 3986. E-mail address: [email protected] (M. Hirano). 0022-510X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2004.09.034
patients have been identified in Mediterranean, Hispanic, Iranian–Jewish, and Ashkenazi–Jewish populations [3]. While neurological and gastrointestinal features are uniformly present in MNGIE patients, symptoms and signs of visceral neuropathy and myopathy are usually the most prominent and debilitating manifestations of the disease. Consequently, MNGIE patients complain of early satiety, borborygmi, vomiting, diarrhea, constipation, chronic abdominal pain, and episodic intestinal pseudo-obstruction. Gastrointestinal involvement leads to a severe weight loss in patients, who rapidly acquire a cachexic appearance, with signs of malnutrition including hypoproteinemia. The prognosis for MNGIE patients is related to the severity of gastrointestinal manifestations. The age at death has ranged between 18 and 58 years old, with a mean of 37 years old [3]. Mutations in the gene encoding thymidine phosphorylase (TP) have been identified as the cause of the disease [4]. TP
36
J. Gamez et al. / Journal of the Neurological Sciences 228 (2005) 35–39
catalyses the reversible phosphorolysis of thymidine (dThd) to thymine and is an essential enzyme in maintaining homeostasis of cellular nucleotide pools [5]. As a consequence of the absence of TP, high levels of dThd and deoxyuridine (dUrd) are present in the plasma of patients [6,7]. It has been hypothesized that excess dThd and dUrd alter mitochondrial nucleoside/nucleotide pools, which, in turn, impair mitochondrial DNA replication and/or repair [4,6,7]. MNGIE thus belongs to the group of mitochondrial encephalomyopathies caused by defects in intergenomic communication [2,8–10]. Although fewer than 75 MNGIE patients have been reported, the molecular genetic basis of the disease is heterogeneous [3,11,12]. Thirty different mutations in the TP gene have been identified and no predominant mutation has been reported [3,11,12]. Most of the mutations are missense mutations. Deletions, insertions, and frameshift and splice-site mutations have also been reported. To date, and to the best of our knowledge, no duplications have been described [2–4,11–13]. In this study, we report on the clinical, biochemical, and genetic characteristics of a family with MNGIE and a novel microduplication mutation in the TP gene.
2. Patients and methods 2.1. Case report The patient, a 29-year-old man (case III:5 in Fig. 1), was referred in May 2003 for evaluation of chronic intestinal pseudo-obstruction, associated with severe weight loss and hypoalbuminemia for 12 months.
In retrospect, he had suffered from postprandial abdominal pain, early satiety, borborygmi, nausea, recurrent vomiting, and poor weight gain since infancy. Despite being 1.78 m tall, he had never weighed more than 50 kg. His parents were not consanguineous. In August 1999, he developed mild symmetric distal limb weakness. Three months later, he noted paresthesias in the hands and feet. Electrophysiological studies suggested a demyelinating polyneuropathy. CSF showed normal proteins and no cells. On admission in May 2003, he was markedly cachectic and weighed 44 kg. He had mild bilateral external ophthalmoparesis and enophthalmos, but no ptosis. Mild muscle weakness was present affecting the deltoids, biceps, and iliopsoas muscles (Medical Research Council scale 4+/ 5). Distal muscle strength was normal. Tendon reflexes were absent. A radionuclide gastric emptying study 3 h after ingestion revealed gastroparesis with remnant material. Upper gastrointestinal series showed distended jejuneum and ileum without obstruction. Esophagogastroduodenoscopy, ileoscopy, and colonoscopy were normal. Gastrointestinal manometry showed a neuropathic pattern affecting the distal half of the stomach and the duodenum. An increase in nonperistaltic duodenal motility was observed after the administration of octreotide. Abdominal CT showed no alterations. Rectal biopsy was normal. T2-weighted brain magnetic resonance (MRI) showed a diffuse leukoencephalopathy affecting both cerebral hemispheres, the brain stem and the cerebellum, but sparing the corpus callosum and basal ganglia. Electrophysiological studies revealed a severe sensory motor demyelinating neuropathy, and a mild myogenic pattern in the proximal muscles in all four limbs. Electrocardiogram showed a sinus
Fig. 1. Family Pedigree. The proband is indicated by an arrow. Genotypes are shown below the symbols. WT, wild-type. dup=nts. 5044–5061dup.
J. Gamez et al. / Journal of the Neurological Sciences 228 (2005) 35–39
37
Table 1 TP activities and nucleoside levels in the proband and family members
2.3. Molecular genetic studies
Case
Genotype
TP in buffy coat (nmol/h/mg protein)a
Plasma dThd (AM)a
Plasma dUrd (AM)a
III:5 (proband) III:4 II:3 II:4
dup/dup WT/dup WT/dup WT/dup
8 ND ND ND
3.2 b0.05 b0.05 b0.05
9.5 b0.05 b0.05 b0.05
Informed consent for blood samples was obtained from the members of the family studied, under an IRB-approved protocol. DNA was isolated from peripheral blood leukocytes using standard methods. The entire coding region of the TP gene plus intronic segments flanking the exons was PCR amplified as described [4]. The PCR products were purified and sequenced directly, using the ABI Prism BigDye Terminator Cycle Sequencing Kit and an ABI Prism 310 sequencer Genetic Analyser (Applied Biosystems, Foster City, CA).
ND = not determined. WT = wild-type. dup = nts.5044–5061dup. a Control values: TP activity, mean = 607 nmol/h/mg protein, range = 544 – 692 ; plasma dThd and dUrd levels were undetectable (b0.05 AM) in controls.
tachycardia. Echocardiogram results revealed mitral valve prolapse. Neurological evaluation of his sister (III:4), including nerve conduction studies and electromyography, was normal. 2.2. Biochemical studies TP activity in buffy coat was assessed using the previously described method [6]. Plasma dThd and dUrd levels were determined using an HPLC method, as previously described [6,7].
3. Results TP activity in buffy coat was virtually abolished in the proband (8 nmol/h/mg protein; 1.3% of the average control value level; Table 1). Plasma dThd in the patient was 3.2 AM (normalb0.05 AM) and dUrd was 9.5 AM (normalb 0.05 AM; Table 1). Sequence analysis of the TP gene revealed the presence of a homozygous 18-base pair (bp) microduplication in exon 8 spanning nucleotides (nts) 5044–5061 (GCGGCGCTGGACGACGGC; GenBank accession # M58602). The same
Fig. 2. Electropherograms showing the homozygous 18-bp duplication (upper panel) and the wild-type sequences (lower panel).
38
J. Gamez et al. / Journal of the Neurological Sciences 228 (2005) 35–39
mutation was found in a heterozygous state in the patient’s parents (individuals II:3 and II:4) and sister (individual III:4). The mutation was not detected in DNA from 100 healthy individuals (Fig. 2).
4. Discussion Previously, we have shown that MNGIE can be diagnosed definitively by biochemical analyses demonstrating severely reduced thymidine phosphorylase activity in buffy coat (V8% of control mean) or increased dThd (N3 Amol/L) and dUrd levels (N5 Amol/L) in blood [14] or by identification of thymidine phosphorylase gene mutation(s). We confirmed the diagnosis in our patient by detecting severely reduced thymidine phosphorylase activity in buffy coat, dramatically increased plasma thymidine and deoxyuridine, and a novel homozygous TP gene mutation in our patient. It is important for clinicians to recognize that the clinical diagnosis of MNGIE can be secured by blood tests and a muscle biopsy is not required. In fact, because skeletal muscle may be misleading because this tissue is usually mildly affected in this disease and typical markers of mitochondrial alterations such as ragged-red and cytochrome c oxidase (COX) deficient fibers, and multiple deletions of mtDNA may be absent [12,13]. The pathogenicity of the TP mutation (nts.5044– 5061dup) is based on the following criteria: (i)
The 18-bp DNA duplication is predicted to cause a duplication of six amino acids (ALA ALA LEU ASP ASP GLY) between alanine (ALA) 332 and serine (SER) 339. (ii) The six amino acid insertion is located in the helix #14 in the alpha-beta-domain of the protein. As consequence, the amino acids immediately following the duplication are displaced. The altered COOH terminus may disrupt the three-dimensional structure of the enzyme, reducing its stability and activity. (iii) The g.5044–5061dup mutation was not found in 60 other MNGIE patients, or 100 unrelated healthy individuals. (iv) TP activity is virtually absent in the patient’s buffy coat and plasma dThd and dUrd levels are markedly increased. Thirty different mutations have been identified in nine of the ten exons. Pathogenic mutations have never been identified in exon 1, which is a noncoding exon [3,11,12]. Eleven of the mutations described in the TP gene are missense. The other reported mutations are microdeletions, single nucleotide insertions, and frameshift and splice-site mutations. To date, no duplications have been described. This six amino acid insertion is therefore the largest TP gene mutation.
No correlation has been found between specific mutations and the severity of clinical features [3]. As previously noted, skeletal muscle may show no morphological abnormalities of skeletal muscle [12,13]. Even more surprising than normal skeletal muscle is a lack of gastrointestinal symptoms in another MNGIE patient with TP-deficiency and TP gene mutations [15]. Intrafamilial phenotypic variability has also been described [13]. Genetic and clinical characterization of MNGIE families around the world is therefore necessary to further understand the genotypephenotype relationship of TP mutations and their relative frequency among different ethnic groups. An important clinical aspect of MNGIE is that patients’ survival and prognosis are generally related to the degree of gastrointestinal involvement, particularly weight loss. Patients often die as a result of cachexia, peritonitis, intestinal rupture, or esophageal bleeding related to cirrhosis, or aspiration pneumonia. Their mean age of death is 37 years old, and no effective therapy is available for this disorder. Nevertheless, parenteral nutrition can transiently ameliorate patients’ nutritional status [2]. Because excess dThd and dUrd levels are hypothesized to alter mitochondrial nucleoside and nucleotide pools leading to impaired mitochondrial DNA replication and/or repair, attempts to reduce dThd and dUrd levels may be a promising therapeutic strategy for MNGIE patients [6,7,16]. Alternatively, administration of exogenous TP enzyme could be a promising treatment.
Acknowledgements This study has been supported by a grant from the Spanish Fondo de Investigaciones Sanitarias (FIS 02/0648 and 03/0343), NIH grant P01NS1176, and a grant from the Muscular Dystrophy Association. The authors are indebted to the members of the family for their cooperation.
References [1] Hirano M, Silvestri G, Blake DM, Lombes A, Minetti C, Bonilla E, et al. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): clinical, biochemical, and genetic features of an autosomal recessive mitochondrial disorder. Neurology 1994;44:721 – 7. [2] Nishino I, Spinazzola A, Papadimitriou A, Hammans S, Steiner CD, Hahn CD, et al. MNGIE: an autosomal recessive disorder due to thymidine phosphorylase mutations. Ann Neurol 2000;47: 792 – 800. [3] Hirano M, Nishigaki Y, Martı´ R. MNGIE: a disease of two genomes. Neurologist 2004;10:8 – 17. [4] Nishino I, Spinazzola A, Hirano M. Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder. Science 1999; 283:689 – 92. [5] Brown NS, Bicknell R. Thymidine phosphorylase, 2-deoxy-d-ribose and angiogenesis. Biochem J 1998;334:1 – 8. [6] Spinazzola A, Marti R, Nishino I, Andreu A, Naini A, Tadesse S, et al. Altered thymidine metabolism due to defects of thymidine phosphorylase. J Biol Chem 2002;277:4128 – 33.
J. Gamez et al. / Journal of the Neurological Sciences 228 (2005) 35–39 [7] Martı´ R, Nishigaki Y, Hirano M. Elevated plasma deoxyuridine in patients with thymidine phosphorylase deficiency. Biochem Biophys Res Commun 2003;303:14 – 8. [8] Hirano M, Vu TH. Defects of intergenomic communication: where do we stand? Brain Pathol 2000;10:451 – 61. [9] Hirano M, Marti R, Ferreiro-Barros C, Vila` MR, Tadesse S, Nishigaki Y, et al. Defects of intergenomic communication: autosomal disorders that cause multiple deletions and depletion of mitochondrial DNA. Semin Cell Dev Biol 2001;12:417 – 27. [10] DiMauro S, Schon EA. Mitochondrial respiratory-chain diseases. N Engl J Med 2003;348:2656 – 68. [11] Kocaefe YC, Erdem S, Ozguc M, Tan E. Four novel thymidine phosphorylase gene mutations in mitochondrial neurogastrointestinal encephalomyopathy syndrome (MNGIE) patients. Eur J Hum Genet 2003;11:102 – 4.
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