Newborn screening for carnitine palmitoyltransferase II deficiency using (C16+C18:1)/C2: Evaluation of additional indices for adequate sensitivity and lower false-positivity.

BACKGROUND: Carnitine palmitoyltransferase (CPT) II deficiency is one of the most common forms of mitochondrial fatty acid oxidation disorder (FAOD). However, newborn screening (NBS) for this potentially fatal disease has not been established partly because reliable indices are not available. METHODS: We diagnosed CPT II deficiency in a 7-month-old boy presenting with hypoglycemic encephalopathy, which apparently had been missed in the NBS using C16 and C18:1 concentrations as indices. By referring to his acylcarnitine profile from the NBS, we adopted the (C16+C18:1)/C2 ratio (cutoff 0.62) and C16 concentration (cutoff 3.0nmol/mL) as alternative indices for CPT II deficiency such that an analysis of a dried blood specimen collected at postnatal day five retroactively yielded the correct diagnosis. Thereafter, positive cases were assessed by measuring (1) the fatty acid oxidation ability of intact lymphocytes and/or (2) CPT II activity in the lysates of lymphocytes. The diagnoses were then further confirmed by genetic analysis. RESULTS: The disease was diagnosed in seven of 21 newborns suspected of having CPT II deficiency based on NBS. We also analyzed the false-negative patient and five symptomatic patients for comparison. Values for the NBS indices of the false-negative, symptomatic patient were lower than those of the seven affected newborns. Although it was difficult to differentiate the false-negative patient from heterozygous carriers and false-positive subjects, the fatty acid oxidation ability of the lymphocytes and CPT II activity clearly confirmed the diagnosis. Among several other indices proposed previously, C14/C3 completely differentiated the seven NBS-positive patients and the false-negative patient from the heterozygous carriers and the false-positive subjects. Genetic analysis revealed 16 kinds of variant alleles. The most prevalent, detected in ten alleles in nine patients from eight families, was c.1148T>A (p.F383Y), a finding in line with those of several previous reports on Japanese patients. CONCLUSIONS: These findings suggested that CPT II deficiency can be screened by using (C16+C18:1)/C2 and C16 as indices. An appropriate cutoff level is required to achieve adequate sensitivity albeit at the cost of a considerable increase in the false-positive rate, which might be reduced by using additional indices such as C14/C3.

Screening of MCAD deficiency in Japan: 16years' experience of enzymatic and genetic evaluation.

BACKGROUND: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a representative disorder of fatty acid oxidation and is one of the most prevalent inborn errors of metabolism among Caucasian populations. In Japan, however, it was as late as 2000 when the first patient was found, and enzymatic and genetic evaluation of MCAD deficiency began. METHODS: We measured octanoyl-CoA dehydrogenase activity in lymphocytes of symptomatic children and newborn screening (NBS)-positive subjects who showed elevated levels of C8-acylcarnitine in blood. The results were further confirmed by direct sequencing of the ACADM gene. RESULTS: The disease was diagnosed in 9 out of 18 symptomatic children. The affected patients showed residual activities from 0% to 3% of the normal average value, except for one patient with 10% activity. Concerning 50 NBS-positive subjects, 18 with enzymatic activities around 10% or lower and 14 with activities ranging from 13% to 30% were judged to be affected patients, and biallelic variants were detected in most of the cases tested. Newborns with higher enzymatic activities were estimated to be heterozygous carriers or healthy subjects, though biallelic variants were detected in 5 of them. Genetic analysis detected 22 kinds of variant alleles. The most prevalent was c.449_452delCTGA (p.T150Rfs), which was followed by c.50G>A (p.R17H), c.1085G>A (p.G362E), c.157C>T (p.R53C), and c.843A>T (p.R281S); these five variants accounted for approximately 60% of all the alleles examined. CONCLUSION: Our study has revealed the unique genetic backgrounds of MCAD deficiency among Japanese, based on the largest series of non-Caucasian cases. A continuous spectrum of severity was also observed in our series of NBS-positive cases, suggesting that it is essential for every nation and ethnic group to accumulate its own information on gene variants, together with their enzymatic evaluation, in order to establish an efficient NBS system for MCAD deficiency.

Significance of ACADM mutations identified through newborn screening of MCAD deficiency in Japan.

BACKGROUND: Since the first case was detected in 2000, there has been a remarkable increase in Japanese patients diagnosed with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. Genetic analysis has revealed a spectrum of mutations that is quite different from those observed in Caucasian populations. In 2014, Japan initiated nationwide newborn screening (NBS) for MCAD using tandem mass spectrometry (MS/MS). It is an urgent issue to assess the risk of acute metabolic decompensation from the respective novel mutations found thus far. METHODS: To evaluate the pathogenic effect of each mutation, we established a eukaryotic cell expression system and prepared 11 mutant proteins identified in five symptomatic patients and eight MS/MS-NBS-positive newborns, as well as two common Caucasian mutations, p.K329E (c.985G>A) and p.Y67H (c.157C>T) for comparison. RESULTS: The expression of four mutant proteins (p.Q45R, p.P92L, p.P128X and p.Y397N) were severely impaired, whereas the others expressed normally, as did p.K329E and p.Y67H. Based on their dehydrogenase activities toward n-octanoyl-CoA, we determined three mutations (p.R53C, p.R281S and p.G362E) to be disease-causing, two mutations having (p.R17H and p.M274V) to be of marginal risk, and two mutations (p.K271E and p.I416T) as benign. Their allele-specific activities were as a whole in accordance with those estimated from the results of measurement in peripheral blood mononuclear cells. CONCLUSION: As most of the mutations detected in the Japanese population are unique, prudent genetic and enzymatic analysis is essential to precisely evaluate the latent risk of clinical onset for screening-positive newborns.

Effects of idursulfase enzyme replacement therapy for Mucopolysaccharidosis type II when started in early infancy: comparison in two siblings.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder that is progressive and involves multiple organs and tissues. While enzyme replacement therapy (ERT) with idursulfase has been shown to improve many somatic features of the disease, some such as dysostosis multiplex and cardiac valve disease appear irreversible once established, and little is known about the preventative effects of ERT in pre-symptomatic patients. We report on two siblings with severe MPS II caused by an inversion mutation with recombination breakpoints located within the IDS gene and its adjacent pseudogene, IDS-2. The siblings initiated treatment with idursulfase at 3.0 years (older brother) and 4 months (younger brother) of age, and we compared their outcomes following 2 years of treatment. At the start of treatment, the older brother showed typical features of MPS II, including intellectual disability. After 34 months of ERT, his somatic disease was stable or improved, but he continued to decline cognitively. By comparison, after 32 months of ERT his younger brother remained free from most of the somatic features that had already appeared in his brother at the same age, manifesting only exudative otitis media. Skeletal X-rays revealed characteristic signs of dysostosis multiplex in the older brother at the initiation of treatment that were unchanged two years later, whereas the younger brother showed only slight findings of dysostosis multiplex throughout the treatment period. The younger brother's developmental quotient trended downward over time to just below the normal range. These findings suggest that pre-symptomatic initiation of ERT may prevent or attenuate progression of the somatic features of MPS II. Follow-up in a larger number of patients is required to confirm the additive long-term benefits of ERT in pre-symptomatic patients.

Molecular pathogenesis of a novel mutation, G108D, in short-chain acyl-CoA dehydrogenase identified in subjects with short-chain acyl-CoA dehydrogenase deficiency.

Short-chain acyl-CoA dehydrogenase (SCAD) is a mitochondrial enzyme involved in the beta-oxidation of fatty acids. Genetic defect of SCAD was documented to cause clinical symptoms such as progressive psychomotor retardation, muscle hypotonia, and myopathy in early reports. However, clinical significance of SCAD deficiency (SCADD) has been getting ambiguous, for some variants in the ACADS gene, which encodes the SCAD protein, has turned out to be widely prevailed among general populations. Accordingly, the pathophysiology of SCADD has not been clarified thus far. The present report focuses on two suspected cases of SCADD detected through the screening of newborns by tandem mass spectrometry. In both subjects, compound heterozygous mutations in ACADS were detected. The mutated genes were expressed in a transient gene expression system, and the enzymatic activities of the obtained mutant SCAD proteins were measured. The activities of the mutant SCAD proteins were significantly lower than that of the wild-type enzyme, confirming the mechanism underlying the diagnosis of SCADD in both subjects. Moreover, the mutant SCAD proteins gave rise to mitochondrial fragmentation and autophagy, both of which were proportional to the decrease in SCAD activities. The association of autophagy with programmed cell death suggests that the mutant SCAD proteins are toxic to mitochondria and to the cells in which they are expressed. The expression of recombinant ACADS-encoded mutant proteins offers a technique to evaluate both the nature of the defective SCAD proteins and their toxicity. Moreover, our results provide insight into possible molecular pathophysiology of SCADD.

Dermatan sulfate and heparan sulfate as a biomarker for mucopolysaccharidosis I.

Mucopolysaccharidosis I (MPS I) is an autosomal recessive disorder caused by deficiency of alpha-L-iduronidase leading to accumulation of its catabolic substrates, dermatan sulfate (DS) and heparan sulfate (HS), in lysosomes. This results in progressive multiorgan dysfunction and death in early childhood. The recent success of enzyme replacement therapy (ERT) for MPS I highlights the need for biomarkers that reflect response to such therapy. To determine which biochemical markers are better, we determined serum and urine DS and HS levels by liquid chromatography tandem mass spectrometry in ERT-treated MPS I patients. The group included one Hurler, 11 Hurler/Scheie, and two Scheie patients. Seven patients were treated from week 1, whereas the other seven were treated from week 26. Serum and urine DS (DeltaDi-4S/6S) and HS (DeltaDiHS-0S, DeltaDiHS-NS) were measured at baseline, week 26, and week 72. Serum DeltaDi-4S/6S, DeltaDiHS-0S, and DeltaDiHS-NS levels decreased by 72%, 56%, and 56%, respectively, from baseline at week 72. Urinary glycosaminoglycan level decreased by 61.2%, whereas urine DeltaDi-4S/6S, DeltaDiHS-0S, and DeltaDiHS-NS decreased by 66.8%, 71.8%, and 71%, respectively. Regardless of age and clinical severity, all patients showed marked decrease of DS and HS in blood and urine samples. We also evaluated serum DS and HS from dried blood-spot samples of three MPS I newborn patients, showing marked elevation of DS and HS levels compared with those in control newborns. In conclusion, blood and urine levels of DS and HS provide an intrinsic monitoring and screening tool for MPS I patients.

Surgical intervention for patent ductus venosus.

Patent ductus venosus (PDV) is a rare condition, which usually presents secondary to hepatic atrophy and hepatic failure. We have treated eight cases of PDV, all with hypergalactosemia and hyperbilirubinemia. Ultrasonography and three-dimensional computed tomography demonstrated communication between the portal vein and the inferior vena cava. Of the eight PDV cases, three from the older age group (ages 9, 11, and 14 years) had high-density lesions in their brain nucleus, and one case (age 19 years) had undergone prior Kasai portoenterostomy for biliary atresia. Six PDV patients underwent ligation of PDV and the remaining two cases underwent partial banding of PDV with intraoperative monitoring to maintain portal vein pressure (PVP) under 30 cm H(2)O. Improvement of the intrahepatic portal vein flow was achieved by ligation or banding of PDV. Postoperatively, serum galactose and bilirubin fell to normal ranges, but portal thrombus occurred postoperatively in the first case. We subsequently administered postoperative anticoagulation in the remaining cases and experienced no major complications. These results suggest that PDV ligation and banding are effective surgical approaches for patients with PDV. Close postoperative monitoring to avoid portal thrombus is imperative in these cases.

Development of a new enzymatic diagnosis method for very-long-chain Acyl-CoA dehydrogenase deficiency by detecting 2-hexadecenoyl-CoA production and its application in tandem mass spectrometry-based selective screening and newborn screening in Japan.

The introduction of tandem mass spectrometry (MS/MS) has made it possible to screen for very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. To confirm the diagnosis in cases with an abnormal profile of blood acylcarnitines, we developed a new enzymatic assay method for determining dehydrogenase activity toward palmitoyl-CoA (C16:0) in lymphocytes. Using this method, the production of 2-hexadecenoyl-CoA (C16:1) by crude cell lysates can be directly quantified using high performance liquid chromatography (HPLC). We applied the assay to 7 myopathic patients, 7 hypoglycemic patients, and 2 presymptomatic newborns with elevated levels of tetradecenoylcarnitine (C14:1 AC) in blood, and found impaired VLCAD activity in all of the 7 myopathic patients and both of the 2 newborns. All of the 7 hypoglycemic patients had normal level of the enzyme activity. Results of the ACADVL gene analysis were in consistent with the enzymatic diagnosis. These results suggest that MS/MS-based screening for VLCAD deficiency using blood C14:1 AC as the indicator may show a considerably high false-positive rate in selective screening of symptomatic patients. Our practical enzymatic assay can be a useful test for the accurate diagnosis of VLCAD deficiency cases screened by MS/MS.

Steroid sulfatase deficiency with bilateral periventricular nodular heterotopia.

This report presents a case of steroid sulfatase deficiency with bilateral periventricular nodular heterotopia. A 13-year-old male was diagnosed as having steroid sulfatase deficiency because steroid sulfatase activity was not detected in his leukocytes. In deoxyribonucleic acid studies, steroid sulfatase locus and adjacent loci were found to be deleted in his deoxyribonucleic acid. Cranial magnetic resonance imaging revealed periventricular nodular heterotopia, disclosing an irregular contour of the lateral walls of the lateral ventricles due to small nodular masses that were isointense as to the gray matter. In steroid sulfatase deficiency patients, bilateral periventricular nodular heterotopia must be considered.

The peak height ratio of S-sulfonated transthyretin and other oxidized isoforms as a marker for molybdenum cofactor deficiency, measured by electrospray ionization mass spectrometry.

Molybdenum cofactor deficiency is a fatal neurological disorder, which follows an autosomal-recessive trait and is characterized by combined deficiency of the enzyme, sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. Early detection of molybdenum cofactor-deficient patients is essential for their proper care and genetic counseling of families at risk. We demonstrate the use of S-sulfonated transthyretin (TTR) as a marker for molybdenum cofactor deficiency. Plasma or sera obtained from 4 patients with molybdenum cofactor deficiency and 57 controls were studied by electrospray ionization mass spectrometry (ESIMS) following selective enrichment of TTR by immunoprecipitation using protein G/A agarose. The data obtained from molybdenum cofactor deficiency samples indicated a strong increase in the peak height of S-sulfonated TTR. A more significant difference was revealed if the peak height ratio of S-sulfonated TTR and the sum of the other oxidized TTR were determined. By accurate determination of the ratio, the samples of molybdenum cofactor deficiency patients could clearly be distinguished from controls without molybdenum cofactor deficiency.

Establishment of a practical enzymatic assay method for determination of isovaleryl-CoA dehydrogenase activity using high-performance liquid chromatography.

BACKGROUND: Isovaleric acidemia (IVA) is one of the various target disorders for tandem mass spectrometry (MS/MS) newborn screening. In the diagnosis of IVA, no enzymatic assay method for isovaleryl-CoA dehydrogenase (IVD) activity has been reported whereby the production of enoyl-CoA species was directly detected. We established a direct assay method to detect 3-methylcrotonyl-CoA (MC-CoA) production using high-performance liquid chromatography (HPLC). METHODS: Isovaleryl-CoA dehydrogenase crude enzyme was prepared by sonicating lymphocytes in peripheral blood. Aliquots were incubated with isovaleryl-CoA, flavin adenine dinucleotide, and phenazine methosulfate. 3-Methylcrotonyl-CoA produced in the samples was separated by HPLC and detected using an ultraviolet spectrophotometer. RESULTS: The detection of MC-CoA was reproducible depending upon the concentration of the substrates, the incubation time, and the number of cells contained in the crude enzyme solution. We applied this assay to three patients diagnosed with IVA and showed that neither of them had detectable residual activity. Only a few hours were required from the initial blood sampling to the end of the assay. CONCLUSIONS: These results demonstrate that this method for detecting MC-CoA production, using HPLC, is a practical assay for determining IVD activity. It can be a useful confirmatory test for IVA cases detected through MS/MS screening of newborns.

Stable-isotope dilution gas chromatography-mass spectrometric measurement of 3-hydroxyglutaric acid, glutaric acid and related metabolites in body fluids of patients with glutaric aciduria type 1 found in newborn screening.

We developed a simple and sensitive stable-isotope dilution method for the quantification of 3-hydroxyglutaric acid (3HGA) and glutaric acid (GA) in body fluids. In our method, tert-butyldimethylsilyl (tBDMS) derivatives of 3HGA and GA were measured with a conventional electron-impact ionization (EI) mode in gas chromatography-mass spectrometry (GC-MS). The control values for 3HGA in nmol/ml were 0.15+/-0.08 (serum; n=10) and 0.07+/-0.03 (CSF; n=10). In addition, glutarylcarnitine and free carnitine were quantified by electrospray tandem mass spectrometry. Using these methods, we monitored 3HGA, GA, and glutarylcarnitine in the body fluids of three patients with glutaric aciduria type 1 found during newborn screening. None of the patients had experienced neurological strokes, which are possibly caused by the accumulation of 3HGA, at 15-24 months of age under a disease-specific treatment, including carnitine supplementation. Our data showed that 3HGA levels were relatively high in some serum samples with lower glutarylcarnitine and carnitine levels, suggesting that carnitine supplementation may play a role in preventing the accumulation of 3HGA in patients with this disease.

Enzymatic diagnosis of medium-chain acyl-CoA dehydrogenase deficiency by detecting 2-octenoyl-CoA production using high-performance liquid chromatography: a practical confirmatory test for tandem mass spectrometry newborn screening in Japan.

Many of the previously described enzymatic assay methods for the diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency have been dependent upon the measurement of radioisotope-labeled co-products or reduction of electron acceptors. We have developed a direct assay method to detect 2-enoyl-CoA production using high-performance liquid chromatography (HPLC). Crude cell lysate prepared from lymphocytes were incubated with n-octanoyl-CoA and ferrocenium hexafluorophosphate. The detection of 2-octenoyl-CoA was significantly reproducible. We applied the assay to samples from four infants suspected to have MCAD deficiency by tandem mass spectrometry (MS/MS) newborn screening conducted in the Hiroshima area of Japan. Three of them were proved to have pathologically reduced residual enzyme activities, although they were associated with various clinical and biochemical phenotypes. In addition, another symptomatic Japanese patient and her presymptomatic sibling who were detected by MS/MS selective screening were successfully diagnosed by our enzymatic assay. These results indicate that the method can be a useful confirmatory test for MS/MS screening of MCAD deficiency.

Monitoring of urinary acrolein concentration in patients receiving cyclophosphamide and ifosphamide.

Acrolein, the metabolite of cyclophosphamide and ifosphamide, irritates mucous membranes and is considered pathogenetically important in hemorrhagic cystitis. Increasing fluid intake or administering sodium 2-mercaptoethanesulfonate (mesna), a thiol compound, can reduce the risk of this complication. We measured urinary acrolein concentrations using headspace-solid-phase microextraction gas chromatography and mass spectrometry (headspace-SPME-GC-MS) in 19 patients receiving cyclophosphamide and ifosphamide (36 occasions). Peak acrolein concentrations occurred at 1-12h (mean +/- S.D., 5.0+/-2.7) after starting therapy, ranging from 0.3 to 406.8 nM (39.7+/-76.7), with varying patterns over time. Maintaining high urine volume was important for preventing increases in urinary acrolein concentration, as urinary acrolein concentration tended to rise as urine volume decreased. Urinalysis detected occult blood in three cases, but the patients had no clinical symptoms of hemorrhagic cystitis. In clinical trials involving cyclophosphamide and ifosphamide, monitoring of urinary acrolein concentration could indicate when to take heightened preventive measures against hemorrhagic cystitis.

Inactivation of acrolein by sodium 2-mercaptoethanesulfonate using headspace-solid-phase microextraction gas chromatography and mass spectrometry.

Acrolein, the metabolite of cyclophosphamide and ifosphamide, is an irritant of mucous membranes and seems to play an important role in hemorrhagic cystitis. Several methods are available to reduce the risk of hemorrhagic cystitis. Mesna is a regional detoxificant which inactivates acrolein. However, the interaction of mesna and acrolein has never been reported because no available method can detect acrolein. In this study, we measured acrolein to evaluate the effect of mesna in urine or phosphate-buffered saline using a headspace-solid-phase microextraction gas chromatography and mass spectrometry method which we had previously established. We also investigated the effect of mesna at different conditions of pH. Mesna was effective in a dose-dependent (10 microM to 20 mM) fashion in both urine and phosphate-buffered saline and completely inactivated acrolein at concentrations over 10 mM. Furthermore, mesna was more effective in alkaline conditions than in acid.

Application of delayed extraction-matrix-assisted laser desorption ionization time-of-flight mass spectrometry for analysis of sphingolipids in pericardial fluid, peritoneal fluid and serum from Gaucher disease patients.

Gaucher disease is a glycolipid storage disorder characterized by the accumulation of glucosylceramide. Using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE-MALDI-TOF-MS), we analyzed sphingolipids in pericardial fluid, peritoneal fluid, and serum from two patients with Gaucher disease. Crude lipids were extracted from 1 ml each of pericardial fluid, peritoneal fluid, and serum with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by DE-MALDI-TOF-MS. The results were as follows: (a) in all the specimens, peaks of ceramide monohexoside and sphingomyelin were detected in both the controls and Gaucher disease patients; (b) in pericardial fluid, peritoneal fluid, and serum, the ceramide monohexoside/sphingomyelin ratio was increased in the Gaucher disease patients compared with in the controls. It was indicated that the accumulation of ceramide monohexoside in such samples from Gaucher disease patients can be easily detected with this DE-MALDI-TOF-MS method.

Newborn mass screening and selective screening using electrospray tandem mass spectrometry in Japan.

Electrospray tandem mass spectrometry was applied to detect a series of inherited metabolic disorders during a newborn-screening pilot study and a selective screening in Japan. In our mass screening of 102,200 newborns, five patients with propionic acidemia, two with methylmalonic acidemia, two with medium-chain acyl-CoA dehydrogenase deficiency, three with citrullinemia type II, and one with phenylketonuria were identified. In a selective screening of 164 patients with symptoms mainly related to hypoglycemia and/or hyperammonemia, 12 with fatty acid oxidation disorders and six with other disorders were found. The results indicated the importance of newborn screening using this technology in Japan.

Selective screening for fatty acid oxidation disorders by tandem mass spectrometry: difficulties in practical discrimination.

In a selective screening for fatty acid oxidation disorders by tandem mass spectrometry, we tested the diagnostic ratios and acylcarnitine concentrations in sera or blood spots, which were reported to be specific to very long-chain acyl CoA dehydrogenase deficiency, carnitine palmitoyltransferase I deficiency, and carnitine palmitoyltransferase II deficiency. While the acylcarnitine profiles in the majority of these patients were typical in the respective disorders, some overlapping of the indices was observed between these patients and the infants, who showed symptoms mainly related to hypoglycemia but did not have the disorders mentioned above. Although the diagnostic ratio of tetradecenoylcarnitine to dodecanoylcarnitine for very long-chain acyl CoA dehydrogenase deficiency seemed to minimize the overlapping in this study, additional measures including careful assessment of clinical data and enzyme assays may be necessary for the diagnosis in atypical cases.

The C677T mutation in the methylenetetrahydrofolate reductase gene contributes to hyperhomocysteinemia in patients taking anticonvulsants.

Hyperhomocysteinemia, a possible risk factor for vascular disease can result from folate deficiency due to anticonvulsant therapy. A reaction catalyzed by 5,10-methylenetetrahydrofolate reductase (MTHFR) supplies 5-methyltetrahydrofolate, needed to remethylate homocysteine to methionine. MTHFR gene mutation (C677T) also can lead to hyperhomocysteinemia. We examined interaction between anticonvulsant therapy, C677T homozygosity, serum folate concentration, and plasma total homocysteine (tHcy) concentration in 81 epileptic patients. Patients receiving monotherapy showed no difference in occurrence of hyperhomocysteinemia (tHcy>90th percentile for controls) between homozygotes for C677T and heterozygotes or patients with no mutant MTHFR. No monotherapy patient was folate deficient (<3 ng/ml). Among patients receiving multidrug therapy, hyperhomocysteinemia in homozygotes for C677T occured significantly more often than in heterozygotes or patients with no mutant enzyme (88.9 vs. 21.1%). The same was true for folate deficiency (44.4 vs. 0%). The C677T mutation is closely related to hyperhomocysteinemia and folate deficiency in epileptic patients taking multiple anticonvulsants.

Novel nonsense mutation (R194X) in the PMM2 gene in a Japanese patient with congenital disorder of glycosylation type Ia.

A Japanese boy had clinical features of congenital disorder of glycosylation type Ia (CDG Ia, also known as carbohydrate-deficient-glycoprotein syndrome, previously), and enzymatic and molecular assay of phosphomannomutase confirmed this diagnosis. During infancy, the patient showed delayed mental and motor development, hypotonia, ataxia, hepatomegaly, liver dysfunction, abnormal coagulation system and cerebellar hypoplasia. At present, though he is 3 years and 8 months old, he cannot utter meaningful words or sit by himself. These findings suggested that he had one of the severe phenotypes of Japanese CDG Ia. Mutational analysis demonstrated heterozygosity for the missense mutation in exon 4 (P113L) and a novel nonsense mutation in exon 7 (R194X). We report his clinical course and the results of molecular assay, and discuss correlation between clinical severity and genotype.