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Evidence-based Laboratory Medicine and Test Utilization |
1 Department of Neurology, Columbia University College of Physicians and Surgeons, New York, NY.
2 Centre d’Investigacions en Bioquímica i Biologia Molecular, Hospital Universitari Vall d’Hebron, Barcelona, Spain.
3 Division of Molecular Neurogenetics, National Neurological Institute "Carlo Besta", Milan, Italy.
4 Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.
aAddress correspondence to this author at: Department of Neurology, Columbia University College of Physicians and Surgeons, P&S 4-443, 630 West 168th St., New York, NY 10032. Fax 212-305-3986; e-mail mh29{at}columbia.edu.
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Abstract |
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Methods: We studied 180 patients with clinical features suggestive of MNGIE, 14 asymptomatic TP mutation carriers, and 20 controls. TP enzyme activity in the buffy coat was determined by a fixed-time method, and the plasma nucleosides thymidine (dThd) and deoxyuridine (dUrd) were assessed by a gradient-elution reversed phase HPLC method. TP was sequenced through standard procedures in patients who met the clinical criteria for MNGIE.
Results:Twenty-five of the 180 patients fulfilled the clinical criteria for MNGIE and had homozygous or compound heterozygous TP mutations. All had drastically decreased TP activity [mean (SD), 10 (15) nmol thymine formed · h-1 · (mg protein)-1 vs 634 (217) nmol thymine formed · h-1 · (mg protein)-1 for the controls]. Relative to the control mean, TP activities were reduced to 35% in mutation carriers and 65% in MNGIE-like patients. All 25 MNGIE patients had detectable plasma dThd [8.6 (3.4) µmol/L] and dUrd [14.2 (4.4) µmol/L]. Controls, carriers, and MNGIE-like patients showed no detectable plasma dThd and dUrd.
Conclusions:We propose a diagnostic algorithm based on the determination of plasma dThd and dUrd, TP activity in buffy coat, or both to make a definitive diagnosis of MNGIE. Increased concentrations of dThd (>3 µmol/L) and dUrd (>5 µmol/L) in plasma or a decrease in buffy coat TP activity to 
8% relative to controls is sufficient to diagnose MNGIE.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Although the clinical manifestations are homogeneous and recognizable, MNGIE is often misdiagnosed, particularly early in the course of the disease before all of the clinical manifestations are apparent. Consequently, patients presenting with progressive external ophthalmoplegia and ptosis have been given the diagnosis of another mitochondrial disease or myasthenia gravis. By contrast, individuals presenting with demyelinating peripheral neuropathy have been misdiagnosed as having chronic inflammatory demyelinating polyneuropathy (7). Gastrointestinal dysmotility is the most common presenting feature and has been misattributed to inflammatory bowel disease, celiac disease, superior mesenteric artery syndrome, and anorexia nervosa.
In 1999, we identified loss-of-function mutations in TP as the cause of MNGIE (8). TP usually catalyzes the first step in the degradation of the pyrimidine nucleosides thymidine (dThd) and deoxyuridine (dUrd) (9). Over the last 4 years, our understanding of the pathomechanism of MNGIE has advanced dramatically (3)(8)(10)(11)(12). Measurement of TP activity in buffy coats from MNGIE patients has revealed total or nearly complete loss of TP activity (10), as well as dramatic accumulations of both dThd and dUrd in plasma (10)(11). Given the clear biochemical alterations in this disease, we have attempted to provide an algorithm to reliably diagnose MNGIE, based on measurements of TP activity and nucleosides in blood. The results are presented here.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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plasma DTHD and DURD assessments
dThd and dUrd were measured by a gradient-elution HPLC method as described previously (11). Plasma samples were deproteinized through addition of perchloric acid (final concentration, 0.5 mol/L), and 50 µL of the supernatant was injected into an Alliance HPLC apparatus (Waters Corporation) and eluted by use of two buffers, eluent A (20 mmol/L potassium phosphate, pH 5.6), and eluent B (20 mmol/L potassium phosphate–600 mL/L methanol, pH 5.6) at a constant flow rate of 1.5 mL/min. Samples were eluted over 110 min as follows: 0 to 5 min, 100% eluent A; 5 to 92 min, 100% to 0% eluent A; 92 to 93 min, 0% to 100% eluent A; 93 to 110 min, constant 100% eluent A. The column used was an Alltima C18 NUC reversed-phase column [100 Å pore size; 5 µm bead size; 250 x 4.6 mm (i.d.); Alltech], preceded by a guard column [Alltima C18; 5 µm bead size; 7.5x 4.6 mm (i.d.); Alltech]. For each sample, a second aliquot was treated with purified TP (Sigma-Aldrich) to specifically eliminate dThd and dUrd, and chromatographed to identify dThd and dUrd peaks definitively. The eluate was monitored at 267 nm, and the dThd and dUrd peaks were quantified by comparing their peak areas with a calibration curve obtained with aqueous calibrators. The detection limit of the method was 0.05 µmol/L for both dThd and dUrd.
tp activity
TP activity in buffy coat was determined as described previously (10). Buffy coats from anticoagulated blood (citrated or EDTA) were homogenized in lysis buffer (50 mmol/L Tris-HCl, pH 7.2, containing 10 mL/L Triton X-100, 2 mmol/L phenylmethylsulfonyl fluoride, and 0.2 mL/L 2-mercaptoethanol) and subjected to brief sonication. Samples were centrifuged at 20 000g for 30 min at 4 °C. The protein content of the supernatant was assessed by the bicinchoninic method (13), and 100–250 µg of supernatant protein was incubated with 10 mmol/L dThd in reaction buffer (0.1 mol/L Tris-arsenate buffer, pH 6.5) in a total volume of 0.1 mL. After incubation for 1 h at 37 °C, the reaction was terminated by the addition of 1 mL of 0.3 mol/L NaOH. In parallel with each sample, a blank was also processed, to which dThd was added after the addition of NaOH. The absorbance at 300 nm was measured for both mixtures and, after the value of the blank was subtracted, the amount of thymine formed was determined based on the 3.4 x 103 L-1 · mol · cm-1 difference in molar absorptivity between dThd and thymine at alkaline pH. Enzyme activity was expressed as nanomoles of thymine formed per hour and mg of protein.
The day-to-day imprecision of our spectrophotometric assay for TP activity in buffy coat was determined with use of 20 aliquots of buffy coat obtained from a single 50-mL blood sample from a healthy control. The aliquots were frozen at -80 °C until analysis.
The TP gene was sequenced as described (8) in those patients with increased plasma concentrations of dThd and dUrd, markedly decreased TP activity, or both.
TP activity, dThd and dUrd measurements, and TP gene sequencing were performed by two postdoctoral researchers or a technician, all with extensive experience in laboratory management. They were blinded to the clinical features and other biochemical results for the patients. As stated above, the patients were evaluated by clinicians worldwide or by a neurologist with 15 years of clinical experience; all were blinded to the results of the above-mentioned biochemical tests.
Because most of the samples from the patients were shipped to our laboratory, sometimes from overseas, we assessed the stability of TP enzyme activity in whole blood at room temperature. Two samples of blood from one healthy donor were collected into tubes containing EDTA or sodium citrate. The blood was stored at room temperature, and aliquots were taken for TP determination over time.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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) (11).
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The distribution of TP activities in buffy coats is shown in Table 2
. TP activity was virtually absent in all MNGIE patients (1.5% of controls on average), whereas carriers had substantially reduced activities (35% of controls), and MNGIE-like patients had partially reduced activities (65% of controls). All groups were statistically different from each other (P <0.01 in all cases, t-test with Bonferroni correction). The day-to-day imprecision, determined by serial measurements of 20 frozen aliquoted buffy coat samples from a single blood collection from a healthy individual, revealed a mean (SD) TP activity of 747 (78) nmol thymine · h-1 · (mg protein)-1 [n = 20; range, 638–889 nmol thymine · h-1 · (mg protein)-1; CV = 10%].
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The TP activities in buffy coat samples isolated from blood stored at room temperature in sodium citrate or in ETDA for variable lengths of time are shown in Fig. 1
. The enzyme activity remained stable for at least for 10 days when blood was collected into sodium citrate. In contrast, when the anticoagulant was EDTA, the enzyme was less stable, with a >50% loss of activity after 5 days at room temperature.
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Sequencing of the TP gene in the MNGIE group confirmed the presence of homozygous or compound heterozygous mutations in all (5). We found no correlations between the genotypes, clinical phenotypes, or biochemical abnormalities (5).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Analysis of the TP gene sequence in suspected MNGIE patients is a valuable tool to confirm or rule out the diagnosis, but this strategy has limitations. One limitation is that, given the diverse nucleotide changes in the TP gene that have been identified in MNGIE patients, at a minimum the nine protein-coding exons with flanking intronic regions must be sequenced (8), but mutations in the promoter or nonflanking intronic sequences may be missed. Another limitation is that when a new polymorphism is identified, additional tests are needed to confirm that pathogenicity (i.e., screening of healthy controls and assays to assess the TP activity). Consequently, a reliable functional assay for TP has advantages over genetic screening.
Consistent with previous reports (1)(2)(3)(4)(5), all of our MNGIE patients had TP mutations causing total or almost complete loss of TP enzyme function. The total or near-complete loss of function accounts for the homogeneous clinical phenotype. The notion that almost total absence of TP enzyme function is necessary to cause MNGIE is supported by the observation that asymptomatic mutation carriers have partial TP activity (35% of the activity in healthy individuals). This value approximates the expected 25% enzyme activity in a homodimeric enzyme (such as TP) with a single mutant allele that obliterates enzyme activity in heterodimeric (mutant plus wild-type) or homodimeric mutant proteins.
In our series of patients, the mean TP activity in the MNGIE-like group was lower than the mean activity in the controls. Rather than a true difference in TP activity between healthy controls and MNGIE-like patients, this result is probably an artifact related to the shipment of blood samples to our laboratory over 1–7 days. In contrast, the controls and many of the heterozygotes were processed on the day of sample collection. Partial inactivation and/or degradation of the enzyme over this time might account for the reduced activity. Our assessment of TP stability in blood stored at room temperature supports this explanation because many of the samples received for TP measurements in suspected MNGIE patients were collected with EDTA. With this anticoagulant, partial inactivation or degradation of TP was noted over few days. Although this difference in sample processing provides an explanation for the TP differences in these two groups of patients, it does not undermine the value of the TP test to diagnose the disease. MNGIE patients have undetectable or barely detectable TP activities, which are clearly different from the other groups, including carriers and samples from MNGIE-like patients.
Our analysis of the substrates for TP has also produced consistent results. Only MNGIE patients have increased plasma concentrations of both dUrd (>5 µmol/L) and dThd (>3 µmol/L), whereas mutation carriers with partially reduced TP activity have no detectable pyrimidine nucleosides (<0.05 µmol/L) in plasma. Therefore, despite the partial reduction of TP activity, pyrimidines were effectively catabolized in all mutation carriers analyzed, including those whose measured TP activities were as low as 90 nmol thymine · h-1 · (mg protein)-1 (
15% of the control mean). In addition, patients with MNGIE-like disorders, but not fulfilling the clinical diagnostic criteria, have shown no detectable pyrimidine nucleosides. These biochemical features of MNGIE patients are clearly distinguishable from those of healthy controls, MNGIE-like patients, and carriers of the TP mutation. On the basis of these findings, we have concluded that measurement of dThd and dUrd concentrations in plasma or TP activity in buffy coat can definitively confirm the diagnosis of MNGIE. The consistent increases in plasma dThd and dUrd in patients with MNGIE also support the hypothesis that increased circulating nucleosides are important in the pathogenesis of the disease (10)(11).
On the basis of our results, we propose an algorithm using measurements of dThd and dUrd in plasma and TP activity in buffy coat to diagnose MNGIE. Fig. 2
contains flow charts diagramming the specific guidelines. We did not identify any individuals with plasma dThd between 0.05 and 3 µmol/L or dUrd between 0.05 and 5µmol/L in either the present work or in previous reports (10)(11); we therefore recommend that threshold concentrations of nucleosides (dThd >3 µmol/L and dUrd >5 µmol/L) and TP activity (<8% of controls) be considered abnormal. In our experience, either the measurement of plasma dThd and dUrd (Fig. 2A
) or the assessment of TP activity in buffy coat (Fig. 2B
) is sufficient to make a definitive diagnosis because there is complete concordance between virtually complete loss of function of the TP enzyme and the increased concentrations of pyrimidine deoxynucleosides. We previously demonstrated a direct concordance between dThd and dUrd concentrations (11). All MNGIE patients were positive for the test index (TP <8% of controls), and all other groups were negative (TP >8% of controls), indicating that this test is 100% sensitive and 100% specific. dThd and dUrd measurements were also 100% sensitive and specific because all MNGIE patients, and only MNGIE patients, had detectable plasma dThd and dUrd.
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In cases with moderately increased plasma nucleoside concentrations, we propose measurement of buffy coat TP activity to confirm the diagnosis of MNGIE. In this situation, a severe loss of TP function is considered diagnostic. On the other hand, our data indicate that TP activity values between 15% and 60% of controls are compatible with a heterozygous TP mutation. This degree of TP deficiency does not alter plasma nucleosides because carriers, like controls, have undetectable amounts of dThd and dUrd. However, because of the overlap of TP activities in carriers and controls, this test does not reliably distinguish heterozygotes. To determine whether one member of a family is a carrier of a TP mutation or mutation free, it is necessary to look directly for the mutation, because measurements of dThd and dUrd in plasma are not informative and assessment of TP activity is ambiguous.
Once TP deficiency has been confirmed through the direct measurement of the enzyme activity or by detection of its substrates accumulated in plasma, the TP gene may be sequenced to identify the specific mutation(s).
In conclusion, the algorithm for biochemical screening of possible MNGIE patients proposed here provides a simple and practical method to definitively confirm the diagnosis. This approach avoids the more laborious and expensive DNA sequencing of TP and unambiguously detects patients with TP deficiency. Our experience indicates that the prevalence of this rare but devastating disease may have been underestimated in the past, but identification of the underlying molecular cause and a better understanding of its biochemical features have led to the proper diagnosis of many patients. We believe that implementation of biochemical screening for TP and pyrimidine nucleosides in clinical laboratories could lead to the detection of additional cases and avoid the misdiagnosis of MNGIE in patients with clinically similar disorders.
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Acknowledgments |
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Footnotes |
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1 Nonstandard abbreviations: MNGIE, mitochondrial neurogastrointestinal encephalomyopathy; TP, thymidine phosphorylase; dThd, thymidine; and dUrd, deoxyuridine. 
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  C. Rampazzo, S. Fabris, E. Franzolin, K. Crovatto, M. Frangini, and V. Bianchi Mitochondrial Thymidine Kinase and the Enzymatic Network Regulating Thymidine Triphosphate Pools in Cultured Human Cells J. Biol. Chem., November 30, 2007; 282(48): 34758 - 34769. [Abstract] [Full Text] [PDF]
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 P. F. Chinnery and J. Vissing Treating MNGIE: Is reducing blood nucleosides the first cure for a mitochondrial disorder? Neurology, October 24, 2006; 67(8): 1330 - 1332. [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or EDTA (•) for variable lengths of time.