Frans J.M. Trijbels

A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects?

Recently, we showed that homozygosity for the common 677(C-->T) mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, causing thermolability of the enzyme, is a risk factor for neural-tube defects (NTDs). We now report on another mutation in the same gene, the 1298(A-->C) mutation, which changes a glutamate into an alanine residue. This mutation destroys an MboII recognition site and has an allele frequency of .33. This 1298(A-->C) mutation results in decreased MTHFR activity (one-way analysis of variance [ANOVA] P < .0001), which is more pronounced in the homozygous than heterozygous state. Neither the homozygous nor the heterozygous state is associated with higher plasma homocysteine (Hcy) or a lower plasma folate concentration-phenomena that are evident with homozygosity for the 677(C-->T) mutation. However, there appears to be an interaction between these two common mutations. When compared with heterozygosity for either the 677(C-->T) or 1298(A-->C) mutations, the combined heterozygosity for the 1298(A-->C) and 677(C-->T) mutations was associated with reduced MTHFR specific activity (ANOVA P < .0001), higher Hcy, and decreased plasma folate levels (ANOVA P <.03). Thus, combined heterozygosity for both MTHFR mutations results in similar features as observed in homozygotes for the 677(C-->T) mutation. This combined heterozygosity was observed in 28% (n =86) of the NTD patients compared with 20% (n =403) among controls, resulting in an odds ratio of 2.04 (95% confidence interval: .9-4.7). These data suggest that the combined heterozygosity for the two MTHFR common mutations accounts for a proportion of folate-related NTDs, which is not explained by homozygosity for the 677(C-->T) mutation, and can be an additional genetic risk factor for NTDs.

Heterozygosity for Homocystinuria in Premature Peripheral and Cerebral Occlusive Arterial Disease

Premature arteriosclerosis and thromboembolic events are well-known complications of homozygous homocystinuria due to cystathionine synthase deficiency. It is unknown whether heterozygosity for homocystinuria predisposes to premature vascular disease. We explored the frequency of excessive homocysteine accumulation after standardized methionine loading in 75 patients presenting with clinical signs of ischemic disease before the age of 50:25 with occlusive peripheral arterial disease, 25 with occlusive cerebrovascular disease, and 25 with myocardial infarction. In seven patients in each of the first two groups but in none of the patients in the third group, heterozygosity for homocystinuria was established on the basis of pathological homocysteinemia after methionine loading and cystathionine synthase deficiency in skin fibroblast cultures. Because the frequency of heterozygosity for homocystinuria in the normal population is 1 in 70 at the most, we conclude that this condition predisposes to the development of premature occlusive arterial disease, causing intermittent claudication, renovascular hypertension, and ischemic cerebrovascular disease.

Maternal hyperhomocysteinemia: A risk factor for neural-tube defects?

The maternal vitamin status, especially of folate, is involved in the pathogenesis of neural-tube defects (NTDs). Maternal folate administration can prevent these malformations. The precise metabolic mechanism of the beneficial effect of folate is unclear. In this study we focus on homocysteine accumulation, which may derive from abnormalities of metabolism of folate, vitamin B12, and vitamin B6. We studied nonpregnant women, 41 of whom had given birth to infants with NTDs and 50 control women who previously had normal offspring. The determinations included the plasma total homocysteine both in the fasting state and 6 hours after the ingestion of a methionine load. In addition, we measured the fasting blood levels of folate, vitamin B12, and vitamin B6. The mean values for both basal homocysteine and homocysteine following a methionine load were significantly increased in the group of women who previously had infants with NTDs. In nine of these subjects and two controls, the values after methionine ingestion exceeded the mean control by more than 2 standard deviations. Cystathionine synthase levels in skin fibroblasts derived from these methionine-intolerant women were within the normal range. Our findings suggest a disorder in the remethylation of homocysteine to methionine due to an acquired (ie, nutritional) or inherited derangement of folate or vitamin B12 metabolism. Increased homocysteine levels can be normalized by administration of vitamin B6 or folate. Therefore, we suggest that the prevention of NTDs by periconceptional folate administration may effectively correct a mild to moderate hyperhomocysteinemia.

Hyperhomocysteinemia: a risk factor in women with unexplained recurrent early pregnancy loss * †

OBJECTIVE To establish the prevalence of hyperhomocysteinemia in women with unexplained recurrent early pregnancy loss. DESIGN In a patient-control study, the methionine-homocysteine metabolism was investigated by a standardized oral methionine-loading test. SETTING Gynecologic outpatient department of university hospital. PATIENTS One-hundred and two women who had been referred to the hospital because they suffered from at least two consecutive unexplained spontaneous abortions (study group) as well as 41 controls who were recruited by public advertisement were selected. INTERVENTIONS Blood samples were collected just before and 6 hours after oral methionine administration to determine plasma total homocysteine concentrations. MAIN OUTCOME MEASURE Plasma total homocysteine concentrations 6 hours after methionine loading. Hyperhomocysteinemia was defined as total homocysteine concentration at 6 hours exceeding the 97.5 percentile level of the controls. RESULTS Hyperhomocysteinemia was diagnosed in 21 women of the study group (21%). In the parous women of the study group, the prevalence of hyperhomocysteinemia was more than two times greater compared with the nulliparous subjects (33% and 14%, respectively). CONCLUSION Hyperhomocysteinemia is a risk factor in women with unexplained recurrent early pregnancy loss.

Folate, Homocysteine and Neural Tube Defects: An Overview

Folate administration substantially reduces the risk on neural tube defects (NTD). The interest for studying a disturbed homocysteine (Hcy) metabolism in relation to NTD was raised by the observation of elevated blood Hcy levels in mothers of a NTD child. This observation resulted in the examination of enzymes involved in the folate-dependent Hcy metabolism. Thus far, this has led to the identification of the first and likely a second genetic risk factor for NTD. The C677T and A1298C mutations in the methylenetetrahydrofolate reductase (MTHFR) gene are associated with an increased risk of NTD and cause elevated Hcy concentrations. These levels can be normalized by additional folate intake. Thus, a dysfunctional MTHFR partly explains the observed elevated Hcy levels in women with NTD pregnancies and also, in part, the protective effect of folate on NTD. Although the MTHFR polymorphisms are only moderate risk factors, population-wide they may account for an important part of the observed NTD prevalence.

The First Nuclear-Encoded Complex I Mutation in a Patient with Leigh Syndrome

Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest multiprotein enzyme complex of the respiratory chain. The nuclear-encoded NDUFS8 (TYKY) subunit of complex I is highly conserved among eukaryotes and prokaryotes and contains two 4Fe4S ferredoxin consensus patterns, which have long been thought to provide the binding site for the iron-sulfur cluster N-2. The NDUFS8 cDNA contains an open reading frame of 633 bp, coding for 210 amino acids. Cycle sequencing of amplified NDUFS8 cDNA of 20 patients with isolated enzymatic complex I deficiency revealed two compound heterozygous transitions in a patient with neuropathologically proven Leigh syndrome. The first mutation was a C236T (P79L), and the second mutation was a G305A (R102H). Both mutations were absent in 70 control alleles and cosegregated within the family. A progressive clinical phenotype proceeding to death in the first months of life was expressed in the patient. In the 19 other patients with enzymatic complex I deficiency, no mutations were found in the NDUFS8 cDNA. This article describes the first molecular genetic link between a nuclear-encoded subunit of complex I and Leigh syndrome.

Demonstration of a New Pathogenic Mutation in Human Complex I Deficiency: A 5-bp Duplication in the Nuclear Gene Encoding the 18-kD (AQDQ) Subunit

We report the cDNA cloning, chromosomal localization, and a mutation in the human nuclear gene encoding the 18-kD (AQDQ) subunit of the mitochondrial respiratory chain complex I. The cDNA has an open reading frame of 175 amino acids and codes for a protein with a molecular mass of 23.2 kD. Its gene was mapped to chromosome 5. A homozygous 5-bp duplication, destroying a consensus phosphorylation site, in the 18-kD cDNA was found in a complex I-deficient patient. The patient showed normal muscle morphology and a remarkably nonspecific fatal progressive phenotype without increased lactate concentrations in body fluids. The childs parents were heterozygous for the mutation. In 19 other complex I-deficient patients, no mutations were found in the 18-kD gene.

Mutations in the complex I NDUFS2 gene of patients with cardiomyopathy and encephalomyopathy.

Human complex I is built up and regulated by genes encoded by the mitochondrial DNA (mtDNA) as well as the nuclear DNA (nDNA). In recent years, attention mainly focused on the relation between complex I deficiency and mtDNA mutations. However, a high percentage of consanguinity and an autosomal‐recessive mode of inheritance observed within our patient group as well as the absence of common mtDNA mutations make a nuclear genetic cause likely. The NDUFS2 protein is part of complex I of many pro‐ and eukaryotes. The nuclear gene coding for this protein is therefore an important candidate for mutational detection studies in enzymatic complex I deficient patients. Screening of patient NDUFS2 cDNA by reverse transcriptase–polymerase chain reaction (RT‐PCR) in combination with direct DNA sequencing revealed three missense mutations resulting in the substitution of conserved amino acids in three families. Ann Neurol 2001;49:195–201

A second common variant in the methylenetetrahydrofolate reductase (MTHFR) gene and its relationship to MTHFR enzyme activity, homocysteine, and cardiovascular disease risk

Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinemia, an independent and graded risk factor for cardiovascular disease (CVD). We examined the relationship of two polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, the 677C→T and 1298A→C variants, to MTHFR activity, homocysteine concentrations, and risk of CVD in a population of 190 vascular disease patients and 601 apparently healthy controls. The mean specific and residual MTHFR activities were significantly lower in 677CT and 677TT individuals (both P<0.001). The 1298A→C mutation alone showed no effect on MTHFR activities. However, when the 677C→T genotype was taken into account, the 1298A→C mutation also caused a significant decrease in MTHFR activities, which was observed in both the homozygous 1298CC (P<0.001) and the heterozygous 1298AC states (P=0.005). Both the 677TT as the 677CT genotypes were associated with significantly higher fasting and postload homocysteine levels than 677CC (P<0.001 and P=0.003, respectively). The 1298A→C mutation had no effect on fasting or postload homocysteine levels. Since homocysteine itself is considered to be positively associated with the risk of CVD, these findings indicate that the 1298A→C mutation cannot be considered a major risk factor for CVD.

Unique efficiency of methionine metabolism in premenopausal women may protect against vascular disease in the reproductive years.

Premenopausal women develop occlusive artery disease less frequently than postmenopausal women. In coronary heart disease, higher blood levels of homocysteine-cysteine mixed disulphide have been reported. Therefore, in healthy subjects, we studied the role of menopausal status in the transsulphuration of methionine in 10 premenopausal and 10 postmenopausal women. To exclude the role of aging, we compared these results with those in 10 younger and 10 older men of comparable age groups. An oral methionine load (0.1 g/kg of body weight) was administered after overnight fasting. Before and during 8 h, thereafter, serum levels of methionine, homocystine, and homocysteine-cysteine mixed disulphide were measured. In the fasting state, serum methionine levels were similar in the premenopausal women and both groups of men. Postmenopausal women had significantly lower fasting levels. Peak levels and clearances of methionine after loading did not differ between the groups. In the fasting state, homocystine was never detectable; yet, after methionine loading, slight homocystinemia was present in 12 out of 20 men, and was more pronounced in all postmenopausal women. However, homocystinemia did not occur in any of the premenopausal women after loading. Fasting serum homocysteine-cysteine mixed disulphide levels did not differ between both groups of men and postmenopausal women. In premenopausal women, both fasting and postloading disulphide levels were significantly lower than in any other group. We conclude that premenopausal women have a unique efficiency of methionine handling, and thereby are preserved against the accumulation of homocysteine after methionine loading. We speculate that this phenomenon might account for the lower incidence of vascular disease in women in the reproductive life cycle.