To evaluate the implication of methymalonic acid (MMA) in the early diagnosis of neural tube defects (NTD), a quantitative assay for MMA was established by using gas chromatography-mass spectrometry with stable isotope of MMA as an internal standard. Amniotic fluid and maternal urine MMA concentration, maternal serum folate, red blood cell folate and vitamin B12 levels were measured in the middle term of NTD-affected and normal pregnancies. Amniotic fluid and maternal urine MMA concentrations in the middle term of NTD affected pregnancies (1.4 +/- 0.9 micromol/L, and 22.1 +/- 12.6 nmol/micromol creatinine) were significantly higher than that of normal pregnancies (1.0 +/- 0. 4 micromol/L, and 2.5 +/- 1.1 nmol/micromol creatinine). There was no significant difference between normal and NTD pregnancies for serum folate, red blood cell folate and vitamin B12 levels. The results suggested that MMAs in amniotic fluid and maternal urine are sensitive markers for early diagnosis of NTD. Vitamin B12 is an active cofactor involved in the remethylation of homocycteine and its deficiency is an independent risk factor for NTD. MMA is a specific and sensitive marker for intracellular vitamin B12 deficiency. This study suggests that it is necessary to monitor the vitamin B12 deficiency and advocates vitamin B12 supplementation with folate prevention program.
Amniotic Fluid/*chemistry ; Biological Markers/analysis ; Biological Markers/urine ; Folic Acid/blood ; Methylmalonic Acid/analysis ; Methylmalonic Acid/*urine ; Neural Tube Defects/*diagnosis ; Neural Tube Defects/metabolism ; Pregnancy Trimester, Second ; *Prenatal Diagnosis ; Vitamin B 12/blood

OBJECTIVEMethylmalonic acidemia complicated with homocysteinemia, cblC type, is the most common inborn error of cobalamin metabolism. The gene MMACHC (OMIM 277400) is located on chromosome 1p34.1 with four coding exons and a 5th non-coding exon. It encodes for a protein with 282 amino acid residues. So far, more than 40 mutations have been detected, in which 271dupA (R91KfsX14) is the hot spot of MMACHC gene. However, there have not been relevant reports in China. The present study aimed to identify the mutation types of MMACHC gene and analyze the genotype-phenotype correlations in Chinese patients.

METHODThe diagnosis of this disease mainly depends on the measurement of C3 propionylcarnitine, C3/C0 (free carnitine) and C3/C2 (acetylcarnitine) in the blood by tandem mass spectrometry, the detection of methylmalonic acid in the urine by gas-chromatography mass spectrometry, the determination of total homocysteine in the serum, and the loading test of vitamin B12. The entire coding region of MMACHC gene was screened by polymerase chain reaction (PCR) combined with DNA direct sequencing in 28 Chinese patients. Genomic DNA was extracted using phenol-chloroform method from the peripheral blood leukocytes of each patient. PCR amplification products were checked by 1.8% agarose gel electrophoresis and were subsequently sequenced with both the forward and reverse primers. Mutational analyses were performed using normal human genomic MMACHC sequence as a reference (GenBank ID: 25974).

RESULTIn this study, ten mutations were identified in 27 of 28 Chinese patients. Most of them were located in exons 3 and 4 (91.3%). We detected four mutations reported, which were 609G>A (W203X), 217C>T (R73X), 271dupA (R91KfsX14), and 394C>T (R132X), and six novel mutations, which were 1A>G, 365A>T, 658_660delAAG, 301-3_327del 30, 567_568insT, and 625_626insT. The 609G>A (W203X) is the most common mutation, which was detected in 30 of 56 alleles (53.6%), including 10 homozygote mutations and 10 heterozygote mutations. In addition, three gene polymorphisms were detected, namely, -302T>G (rs3748643), -234A>G (rs3728644), and 321G>A (rs2275276). These mutations include missense mutations, nonsense mutations, duplication, deletions, and insertions.

CONCLUSIONIn this study, we found a part of gene mutations spectrum in Chinese patients with methylmalonic acidemia and homocysteinemia, in which the 609G>A (W203X) may be the hotspot mutation of MMACHC gene. This would be helpful in the prenatal diagnosis and gene screening programs of methylmalonic acidemia and homocystinemia.

Amino Acid Metabolism, Inborn Errors ; complications ; genetics ; Cysteine ; blood ; DNA ; DNA Mutational Analysis ; Exons ; Humans ; Hyperhomocysteinemia ; complications ; genetics ; Methylmalonic Acid ; blood ; Mutation

To evaluate the implication of methymalonic acid (MMA) in the early diagnosis of neural tube defects (NTD), a quantitative assay for MMA was established by using gas chromatography-mass spectrometry with stable isotope of MMA as an internal standard. Amniotic fluid and maternal urine MMA concentration, maternal serum folate, red blood cell folate and vitamin B12 levels were measured in the middle term of NTD-affected and normal pregnancies. Amniotic fluid and maternal urine MMA concentrations in the middle term of NTD affected pregnancies (1.4 +/- 0.9 micromol/L, and 22.1 +/- 12.6 nmol/micromol creatinine) were significantly higher than that of normal pregnancies (1.0 +/- 0. 4 micromol/L, and 2.5 +/- 1.1 nmol/micromol creatinine). There was no significant difference between normal and NTD pregnancies for serum folate, red blood cell folate and vitamin B12 levels. The results suggested that MMAs in amniotic fluid and maternal urine are sensitive markers for early diagnosis of NTD. Vitamin B12 is an active cofactor involved in the remethylation of homocycteine and its deficiency is an independent risk factor for NTD. MMA is a specific and sensitive marker for intracellular vitamin B12 deficiency. This study suggests that it is necessary to monitor the vitamin B12 deficiency and advocates vitamin B12 supplementation with folate prevention program.
Adult ; Amniotic Fluid ; chemistry ; Biomarkers ; analysis ; urine ; Female ; Folic Acid ; blood ; Humans ; Methylmalonic Acid ; analysis ; urine ; Neural Tube Defects ; diagnosis ; metabolism ; Pregnancy ; Pregnancy Trimester, Second ; Prenatal Diagnosis ; Vitamin B 12 ; blood

BACKGROUNDMethylmalonic aciduria (MMA) is the most frequent disease of organic aciduria in China. Various biochemical strategies are followed for the prenatal diagnosis of MMA. However, since fetuses affected by MMA have decreased excretion of methylmalonic acid, the difficulties of prenatal biochemical diagnosis are obvious. Gas chromatography mass spectrometry (GC/MS) and tandem mass spectrometry (ESI/MS/MS) have allowed us to identify the disease in affected fetuses. The aim of this study was to determine the value of analysis of organic acids and total homocysteine in amniotic fluid in prenatal diagnosis of MMA.

METHODSThe clinical diagnoses and outcomes of nine probands with MMA and the prenatal diagnoses based on biochemical analysis of nine fetuses at risk for MMA were investigated. Amniotic fluid samples from pregnancies at risk for MMA and metabolically normal pregnancies were obtained at 16 - 24 weeks of gestation. Methylmalonic acid and methylcitric acid were measured by GC/MS, propionylcarnitine was analyzed by ESI/MS/MS, and total homocysteine was determined by fluorescence polarization immunoassay.

RESULTSIn two pregnancies, high levels of methylmalonic acid, methylcitric acid, propionylcarnitine, and total homocysteine indicated combined MMA and homocysteinemia in the fetuses. One of the mothers continued pregnancy and received cobalamin supplement as prenatal treatment, and the other terminated her pregnancy. In one pregnancy, significantly elevated levels of methylmalonic acid, methylcitric acid, and propionylcarnitine, and normal level of total homocysteine was found indicating isolated MMA in the fetus; abortion was performed on this case. In the other six pregnancies, all the levels of the above mentioned metabolites were normal suggesting that the fetuses were not affected by MMA. The diagnoses were confirmed after delivery by testing urinary organic acids and plasma total homocysteine.

CONCLUSIONSThe metabolic abnormalities of MMA occur early in gestation. The level of total homocysteine in amniotic fluid may be an additional indicator of fetal combined MMA and homocysteinemia. Determination of total homocysteine level in amniotic fluid may become a convenient and reliable method for prenatal diagnosis of the disease.

Amniotic Fluid ; chemistry ; Carnitine ; analogs & derivatives ; analysis ; Citrates ; analysis ; Female ; Gas Chromatography-Mass Spectrometry ; Homocysteine ; analysis ; blood ; Humans ; Male ; Methylmalonic Acid ; urine ; Pregnancy ; Prenatal Diagnosis ; methods ; Spectrometry, Mass, Electrospray Ionization

BACKGROUND: Methylmalonic aciduria can be caused by inherited defects in the methylmalonyl-CoA mutase enzyme, Inherited defects in the metabolism of vitamin Bl2 and acquired or inherited vitamin Bl2 deficiency. Quantitation of urinary methylmalonic acid (MMA) is very useful In diagnosis of methylmalonic acidemia and cobalamin deficiency. We evaluated a quantitation method of urinary MMA and determined reference values. METHODS: The method involved stable isotope dilution gas chromatographymass spectrometry (GC-MS) with (methyl 2H3)-MMA as the internal standard. We determined the detection limit, linearity and periodic variations of the assay. Urinary MMA levels were measured in 70 individuals of ages newborn to 58 years with no metabolic disorders. RESULTS: The lower limit of detection calculated from blank runs (mean+/-3SD) was 2.62nmo1/m1. One control urine tramp)e analyzed 23 times within 3 weeks game results of 7.83+/-1.09 (mean+/-SD, CV=13.8%) nmol/mL. The linearity at four different concentrations of MMA was acceptable (R2=0.9992). The concentration of urinary MMA in 70 individuals was 2.33+/-2.19 mmol/mol creatinine (mean+/-SD). Age related reference values which decreased with age were also reported (p=1.23x10-9). CONCLUSIONS: The described method is sensitive, specific and noninvasive, which is considered the gold standard method for measuring MMA. The method could be used as a screening test for cobalamin deficiency and inherited methyl malonic acidemia. On the basis of the narrow range of normal concentration, it is expected that the method would readily detect mild cobalamin deficiency.
Chromatography, Gas* ; Creatinine ; Diagnosis ; Gas Chromatography-Mass Spectrometry* ; Humans ; Infant, Newborn ; Limit of Detection ; Mass Screening ; Metabolism ; Methylmalonic Acid* ; Methylmalonyl-CoA Mutase ; Reference Values ; Spectrum Analysis ; Vitamin B 12 ; Vitamins

OBJECTIVETo identify the mutation of the methylmalonic aciduria (cobalamin deficiency) CblC type, with homocystinuria (MMACHC) gene in a pedigree with methylmalonic aciduria.

METHODSThe MMACHC gene mutation was detected using polymerase chain reaction (PCR) and DNA sequencing. The MMACHC gene of 50 healthy people was also sequenced as control.

RESULTSA new mutation of 146_154 del CCTTCCTGG was found in the patient and his father, and was absent in the controls.

CONCLUSIONA new mutation (146_154 del CCTTCCTGG) in the MMACHC gene was detected in a Chinese family with methylmalonic aciduria.

Amino Acid Metabolism, Inborn Errors ; genetics ; metabolism ; Amino Acid Sequence ; Animals ; Base Sequence ; Carrier Proteins ; chemistry ; genetics ; Case-Control Studies ; Child, Preschool ; DNA Mutational Analysis ; Exons ; genetics ; Fathers ; Female ; Humans ; Male ; Methylmalonic Acid ; metabolism ; Molecular Sequence Data ; Mutation ; Pedigree ; Polymerase Chain Reaction ; Pregnancy ; Protein Structure, Secondary

Adult ; Age of Onset ; Amino Acid Metabolism, Inborn Errors ; complications ; diagnosis ; genetics ; therapy ; Betaine ; administration & dosage ; therapeutic use ; Carrier Proteins ; genetics ; metabolism ; Child ; China ; epidemiology ; DNA Mutational Analysis ; Gas Chromatography-Mass Spectrometry ; Genotype ; Homocysteine ; urine ; Humans ; Hydroxocobalamin ; administration & dosage ; therapeutic use ; Hyperhomocysteinemia ; complications ; diagnosis ; genetics ; therapy ; Infant ; Methylmalonic Acid ; blood ; urine ; Mutation ; Vitamin B 12 ; metabolism