Thymidine Phosphorylase Gene Mutations in MNGIE, a Human Mitochondrial Disorder

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Science  29 Jan 1999:
Vol. 283, Issue 5402, pp. 689-692
DOI: 10.1126/science.283.5402.689


Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human disease associated with multiple deletions of skeletal muscle mitochondrial DNA (mtDNA), which have been ascribed to a defect in communication between the nuclear and mitochondrial genomes. Examination of 12 MNGIE probands revealed homozygous or compound-heterozygous mutations in the gene specifying thymidine phosphorylase (TP), located on chromosome 22q13.32-qter. TP activity in leukocytes from MNGIE patients was less than 5 percent of controls, indicating that loss-of-function mutations in TP cause the disease. The pathogenic mechanism may be related to aberrant thymidine metabolism, leading to impaired replication or maintenance of mtDNA, or both.

Mutations in mtDNA have been associated with a wide spectrum of mitochondrial diseases (1), and more than 50 pathogenic mtDNA point mutations have been identified as causes of maternally inherited mitochondrial encephalomyopathies. Another group of diseases are typically associated with multiple deletions of mtDNA, but show autosomal transmission and thus have been attributed to defective communication between the nuclear and mitochondrial genomes. Among these diseases are autosomal dominant progressive external ophthalmoplegia, which has been linked to two chromosomal loci, 10q23.3-q24.3 and 3p14.1-p21.2 (2), and an autosomal recessive disease associated with multiple deletions, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE; Mendelian Inheritance in Man number 550900, Genome Database accession number 9835128), linked to chromosome 22q13.32-qter (3).

MNGIE is characterized clinically by onset between the second to fifth decades, ptosis, progressive external ophthalmoplegia, gastrointestinal dysmotility, thin body habitus, peripheral neuropathy, myopathy, leukoencephalopathy, and lactic acidosis (4). Laboratory studies show various mitochondrial abnormalities in skeletal muscle, including ragged-red fibers with ultrastructurally abnormal mitochondria, decreased activities of respiratory chain enzymes, and multiple mtDNA deletions or mtDNA depletion, or both (4, 5).

Linkage to chromosome 22q13.32-qter was confirmed in a total of seven families with MNGIE (3, 6). In this region (7), the gene encoding thymidine phosphorylase (TP; E.C. appeared particularly interesting. TP catalyzes the reversible phosphorolysis of thymidine (thymidine + phosphate ↔ thymine + 2-deoxy d-ribose 1-phosphate) and is likely to have an important role in nucleoside metabolism by regulating the availability of thymidine for DNA synthesis (8).

To screen for TP mutations, we designed primers to amplify and sequence all the TP coding exons and the flanking regions (9). We sequenced DNA from 12 unrelated MNGIE probands and one control, and compared the data to a reference sequence (10). All the probands had homozygous or compound-heterozygous mutations (Table 1). Southern blot analysis of skeletal muscle DNA revealed that seven of nine patients had multiple mtDNA deletions. We were unable to study skeletal muscle DNA in three probands.

Table 1

TP mutations with MNGIE. Exons 2 through 10 from 12 probands were sequenced. Patients 1 to 4 are the original families used for linkage analysis and correspond to individuals IV-3, II-1, V-3, and II-1, respectively in (3). Southern blot analyses of skeletal muscle were performed with Bam HI and Pvu II (24). Multiple deletions of mtDNA were not found in patients 8 and 9; this might be due to partial mtDNA depletion (6). 4-bp del, deletion of nts 3527–3530; 6-bp del, deletion of nts 3895–3900; ins4196C, insertion of C at nt 4196; Pi, phosphate; aa, amino acid; ND, not determined.

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We identified 10 different mutations: four missense, three splice-site, two deletions, and a single-nucleotide insertion (Table 1 and Fig. 1). We confirmed segregation of these point mutations with the disease in families 1 to 7. Three of the missense mutations, G1419A, G1443A, and A3371C, were found in multiple families.

Figure 1

Representative electropherograms showing TP mutations in patients with MNGIE. (A) Homozygous G1419A in patient 6. (B) Homozygous G1443A in patient 12. (C) Homozygous A3371C in patient 1. (D) Heterozygous T1504C in patient 2 (top), which is designated “N” by Sequence Analysis version 3.0 software (Perkin-Elmer). The mutated splice-donor site (GN) is underlined. RT-PCR produced two bands, which were excised separately from the gel. Direct sequencing of the shorter fragment revealed exon 4 skipping (bottom).

Two missense mutations, G1419A and G1443A, change the second and tenth encoded amino acids within the thymidine/pyrimidine-nucleoside phosphorylase consensus sequence [Gly145 → Arg145 (G145R) and G153S (11), respectively] (12). The other two missense mutations, A2744G and A3371C, change amino acids K222S and E289A, respectively (Table 1 and Fig. 2). Lysine-222 in human TP corresponds to K191 in the Escherichia coli protein, which forms a hydrogen bond with a phosphate (13). Because serine has an uncharged alkyl group, the K222S mutant presumably cannot create a hydrogen bond with a phosphate. Although the functional importance of E289 is unknown, this residue is well conserved (Fig. 2). The four TP missense mutations were not present in DNA from 63 control individuals (14).

Figure 2

Amino acid conservation through evolution (11). All of the identified missense mutations replace the well-conserved amino acids (shaded residues). The thymidine/pyrimidine-nucleoside consensus signature (12) is underlined. Sequences of Bacillus stearothermophilus andB. subtilis encode portions of pyrimidine-nucleoside phosphorylase, which also has uridine phosphorylase activity in addition to thymidine phosphorylase activity, while other sequences are for thymidine phosphorylase. Multiple alignments and consensus sequences were obtained from the ProDom database (25). We also added the amino acid sequence ofMycobacterium tuberculosis TP predicted from the DNA sequence of M. tuberculosis H37Rv complete genome, segment 143/162 (European Molecular Biology Laboratory accessionAL021841).

Splice-site mutations in TP caused aberrant mRNA splicing. By reverse transcription–polymerase chain reaction (RT-PCR) analysis, we confirmed exon skipping in blood samples from two patients (15). The T1504C mutation disrupted the splice donor site sequence (GT) in intron 4, resulting in skipping of exon 4 (Fig. 1). The G3867C mutation disrupted the splice acceptor site sequence (AG) in intron 8, resulting in exon 9 skipping. Because a “leucine zipper pattern” sequence (12) (amino acids 417 to 438) spans the junction between exons 9 and 10 (Fig. 2), this mutation is predicted to delete an important segment of the protein.

The 6–base pair (bp) deletion in exon 9 would result in the deletion of L397 and A398. Although these amino acids are not strictly conserved, their loss could alter the structure and enzymatic activity of the protein. Insertion of a C at nucleotide (nt) 4196 shifts the reading frame from amino acid 471, which would remove an in-frame stop codon in the cDNA sequence.

We found a heterozygous 4-bp deletion in intron 7 of patient 8, who had the G1419A transition in the other allele. This microdeletion may be a neutral polymorphism or it may affect splicing. Alternatively, the second pathogenic mutation in patient 8 may be present in exon 1; or in another intron, resulting in alteration of the mRNA; or a regulatory sequence.

All of the patients harbored a homozygous A3673G polymorphism relative to the reference sequence. We also identified a T3576C polymorphism, which was homozygous in patients 3 to 6 and 11 and 12, and heterozygous in patients 8 and 10. Both polymorphisms are unlikely to be pathogenic, because a healthy control was also homozygous for the A3673G transition; the T3576C polymorphism is silent.

To investigate the functional effects of the TP mutations, we assayed TP activity in peripheral leukocytes from six probands and 19 normal controls. Patients 1, 2, and 4 had no detectable TP activity, and all others had activity that was <5% of that in controls (Fig. 3). This finding, and the fact that TP mutations segregated with MNGIE, lead us to conclude that the disease is caused by loss-of-function mutations in TP.

Figure 3

Assay of TP activity in leukocytes from patients with MNGIE. Patients 1, 2, 3, 4, 6, and 7 and 19 controls were studied. The rate of conversion of thymidine to thymine was measured spectrophotometrically as in (26). Peripheral leukocytes were homogenized in lysis buffer [50 mM tris-HCl (pH 7.2) containing 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, and 0.02% 2-mercaptoethanol] and subjected to brief sonication. Samples were centrifuged at 20,000g for 30 min at 4°C, and the supernatants were used for the enzyme assay. From each supernatant, 100 to 250 μg of protein was incubated with 0.1 M tris-arsenate buffer (pH 6.5) and 10 mM thymidine in a total volume of 0.1 ml. After 1 hour of incubation at 37°C, the reaction was stopped by adding 1 ml of 0.3 M NaOH. The amount of thymine formed was measured at 300 nm wavelength, based on a 3.4 × 103 difference in the molar extinction coefficient between thymidine and thymine at alkaline pH. Enzyme activity was expressed as micromoles of thymine generated per hour per milligram of protein. The protein content was determined as in (27). Results were obtained from two independent experiments performed in duplicate. Patients 1, 2, and 4 showed no detectable activity. The difference between patients and controls were statistically significant, as assessed by the two-tailed Student'st-test (P < 10–10). Bars indicate the mean value (M) and standard deviation (SD).

Human TP has been studied extensively and shown to possess at least three functions: catalysis (as thymidine phosphorylase), angiogenesis, and cell trophism (8). TP is also called platelet-derived endothelial cell growth factor (PD-ECGF) or endothelial cell growth factor 1 (ECGF1), because of its angiogenic properties (16), or gliostatin, to denote its inhibitory effects on glial cell proliferation. TP is also present in prokaryotes, and its sequence is highly conserved (16). Human TP has been investigated extensively by cancer researchers, because TP expression and activity are increased in some tumors, presumably reflecting their neovascularization (9). Our MNGIE patients did not have vascular abnormalities, suggesting that the absence of TP activity does not interfere with normal angiogenesis.

TP is widely expressed in human tissues, including the gastrointestinal system, brain, peripheral nerves, spleen, bladder, and lung, but is not expressed in muscle, kidney, gall bladder, aorta, and fat (17). This expression pattern is consistent with the major clinical features of MNGIE: neuropathy, gastrointestinal dysmotility, and leukoencephalopathy. Because TP is also expressed in autonomic nerves, gastrointestinal symptoms may be partly due to autonomic neuropathy. TP has neurotrophic effects on cortical neurons and inhibits glial-cell proliferation (18). Therefore, encephalopathy in MNGIE may be caused by the loss of gliostatin function, rather than by decreased enzymatic activity. Paradoxically, skeletal muscle has no TP activity, although it is usually affected in MNGIE and harbors multiple mtDNA deletions. This paradox suggests that the mtDNA abnormalities in MNGIE might be an epi-phenomenon. A possible explanation for muscle involvement is that low levels (that is, undetectable levels) of TP might be vital for mtDNA maintenance (19). A second and perhaps more plausible possibility is that muscle mtDNA may be adversely affected by abnormal extracellular thymidine pools due to TP dysfunction (20). Therefore, although TP is not expressed in some aerobic tissues, such as muscle and kidney, TP activity may be indirectly essential for mtDNA maintenance in these tissues.

Thymidine is either degraded to thymine by TP catabolism or salvaged to deoxythymidine monophosphate (dTMP) by thymidine kinase (TK). Because mtDNA is constantly replicating, even in quiescent cells, a constant supply of thymidine and other nucleotides is likely to be vital for the maintenance of the mitochondrial genome. Mitochondria have physically separate deoxynucleotide triphosphate (dNTP) pools and may also have an independent thymidine salvage pathway (21). In support of this notion, at least two different forms of TK exist in eukaryotic cells. One (cytosolic, TK1) is highly active only in dividing cells, whereas the other (mitochondrial, TK2) is constitutively expressed (22), suggesting that the thymidine salvage pathway is important for mtDNA maintenance. Conceivably, the imbalance of dNTP pools caused by elevated thymidine levels might affect mtDNA more adversely than nuclear DNA, resulting in pathogenic multiple mtDNA deletion or partial mtDNA depletion, or both (23).

Finally, our results may have clinical implications for diagnosis and therapy of patients with MNGIE for which there is no effective treatment. For example, administration of parenteral TP or restoration of the normal nucleotide pools, or both, warrant investigation as potential therapies.

  • * To whom correspondence should be addressed. E-mail: mh29{at}


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