Sandrine Barbaux

The methionine synthase reductase (MTRR) A66G polymorphism is a novel genetic determinant of plasma homocysteine concentrations.

Epidemiological evidence has revealed that an elevated plasma homocysteine level (hyperhomocysteinemia) confers an increased risk of cardiovascular disease and neural tube defects. Hyperhomocysteinemia is caused by both nutritional (e.g. folate, vitamins B(6) and B(12)) and genetic factors, including functional polymorphisms of key enzymes involved in homocysteine metabolism. One such enzyme, methionine synthase reductase (MTRR), maintains adequate levels of methylcob(III)alamin, the activated cofactor for methionine synthase, which catalyzes the remethylation of homocysteine to methionine. A common MTRR polymorphism, i.e. a 66 A-->G substitution specifying an isoleucine to methionine substitution (I22M), was recently identified. To assess the influence of this polymorphism on total plasma homocysteine (tHcy), we undertook a genotype/phenotype analysis in a study population of 601 Northern-Irish men, aged 30--49, for which biochemical and genetic data relevant to folate/homocysteine metabolism had already been acquired. The 66AA genotype has a frequency of 29% in this population. We established that there was a significant influence of MTRR genotype on tHcy ranking (P=0.004) and that the 66AA genotype contributes to a moderate increase in tHcy levels across the distribution [OR 1.59 (95% CI: 1.10--2.25) for the 66AA genotype to be in the upper half of the tHcy distribution, P=0.03]. The homocysteine-elevating effect of the 66AA genotype is independent of serum folate, vitamin B(12) and vitamin B(6) levels. Based on published estimates of the enhanced cardiovascular disease risk conferred by defined increments of plasma tHcy, we estimate that 66AA homozygotes have, on average, an approximately 4% increase in cardiovascular disease risk compared to 66GG homozygotes. This study provides the first evidence that the MTRR A66G polymorphism significantly influences the circulating tHcy concentration.

Maternal Genetic Effects, Exerted by Genes Involved in Homocysteine Remethylation, Influence the Risk of Spina Bifida

There is currently considerable interest in the relationship between variation in genes that are involved in the folate-homocysteine metabolic axis and the risk of spina bifida. The evaluation of this relationship is, however, complicated by the potential involvement of both the maternal and the embryonic genotype in determination of disease risk. The present study was designed to address questions regarding both maternal and embryonic genetic risk factors for spina bifida by use of the two-step transmission/disequilibrium test. Analysis of data on variants of two genes involved in homocysteine remethylation/methionine biosynthesis--methionine synthase (MTR) A2756G and methionine synthase reductase (MTRR) A66G--provided evidence that both variants influence the risk of spina bifida via the maternal rather than the embryonic genotype. For both variants, the risk of having a child with spina bifida appears to increase with the number of high-risk alleles in the maternal genotype: MTR (R1=2.16, 95% CI 0.92-5.06; R2=6.58, 95% CI 0.87-49.67) and MTRR (R1=2.05, 95% CI 1.05-3.99; R2=3.15, 95% CI 0.92-10.85). These findings highlight the importance of considering both the maternal and embryonic genotype when evaluating putative spina bifida susceptibility loci.

The human and mouse methylenetetrahydrofolate reductase (MTHFR) genes: genomic organization, mRNA structure and linkage to the CLCN6 gene.

Methylenetetrahydrofolate reductase (MTHFR), a pivotal enzyme in folate metabolism, regulates the proportional distribution of one-carbon moieties between cellular methylation reactions and nucleic acid synthesis. The organization of the MTHFR gene and the structure of its mRNA were characterized in human and mouse. There are three mRNA transcripts of 2.8, 7.2 and 9.8 kb in human and two of 3.2 and 7.5 kb in mouse. Northern blot analysis revealed that human MTHFR MRNA is only present at low abundance in most tissues tested. Five kilobases of sequence flanking the 3 end of the human gene were isolated, and polyadenylation sites were defined by 3 RACE. The shorter 2.8 kb transcript and the two larger 7.2 and 9.8 kb transcripts utilize different polyadenylation signal sequences, 629 and 4937 bp downstream of the stop codon, respectively. The two mRNA species in mouse also result from differential polyadenylation. Approximately 7 and 3.5 kb upstream of the human and mouse genes, respectively, were isolated and sequenced. Transcription start sites in human MTHFR were mapped using 5 RACE. The 2.8 and 7.2 kb mRNAs originate from one of two transcription start sites that are 206 and 243 bp upstream of the ATG initiation codon, whereas transcription of the 9.8 kb mRNA is initiated at a start site located 2.8 kb upstream of the translation start codon. The putative MTHFR promoter does not have a TATA box but contains CpG islands and multiple potential Sp1 binding sites. The MTHFR gene was finely mapped to interval 16 of chromosome 1p36.3, a region deleted in many tumors, by establishing a close linkage to CLCN6, a putative chloride channel gene. A novel CA-repeat polymorphism identified within intron 2 of the CLCN6 gene may be useful in assessing loss of heterozygosity in such tumors. The multiple MTHFR mRNA species identified in this report may reflect an underlying complex set of gene regulatory mechanisms acting through an alternative transcription start site and/or polyadenylation signal sequence utilization.

Polymorphisms of genes controlling homocysteine/folate metabolism and cognitive function

Elevated concentrations of the amino acid homocysteine and/or folate deficiency have been reported to affect neural development/function in both human patients and animal models. We have investigated the distribution of functional polymorphisms in genes involved in homocysteine/folate metabolism in children with high IQ and in children with average IQ. No differences in the frequencies of genetic variants in the methionine synthase or methylenetetrahydrofolate reductase genes were found. However, the cystathionine β-synthase (CBS) 844ins68 allele was significantly underrepresented in children with high IQ. The mechanism by which a functional genetic variant in the CBS gene may influence cognitive function remains to be determined.

Differential haplotypic expression of the interleukin-18 gene

Interleukin 18 (IL-18) is suspected to play an important role in atherosclerosis and plaque vulnerability. We had previously shown that haplotypes combining two IL18 gene polymorphisms in complete linkage disequilibrium, C-105T (rs360717) in 5′-untranslated region (UTR) and A+183G (rs5744292) in 3′-UTR, were related to IL-18 circulating levels and cardiovascular outcome, the C−105G+183 haplotype being associated with lower IL-18 levels and lower cardiovascular risk. This study was aimed at investigating the functional role of the two polymorphisms and their haplotypes on IL18 expression levels. Allelic imbalance experiments conducted in 24 and 20 subjects heterozygous for the C-105T and the A+183G polymorphisms did not detect any difference when subjects were considered as a whole (−0.009±0.044, P=0.85 and +0.114±0.082, P=0.18, respectively). However, when splitting individuals according to their haplo-genotype, the haplotype C−105G+183 was associated with a lower expression level than C−105A+183 (−0.287±0.076, P=0.005), but did not differ from T−105A+183 (−0.138±0.083, P=0.13). The lower expression associated with C−105G+183 was confirmed by real-time reverse transcription-PCR. Transfection of different haplotypic versions of the 3′-UTR did not show any difference in the expression of an upstream reporter gene. A 10-h study of the mRNA degradation kinetics by allelic imbalance with the A+183G polymorphism did not show any differential allelic degradation. In conclusion, the haplotype associated with lower IL-18 circulating concentrations and a lower cardiovascular risk was consistently associated with decreased IL18 expression levels, although the exact functional mechanisms remain to be elucidated.

Haplotypes of the Caspase-1 Gene, Plasma Caspase-1 Levels, and Cardiovascular Risk

Caspase-1 processes the interleukin (IL)-1&bgr; and IL-18 inactive precursors to the biologically active cytokines that are known to have proatherogenic effects. The present study investigated the genetic variability of the CASP1 gene and plasma levels of caspase-1 in relation to cardiovascular risk. In Europeans, 3 tag SNPs captured 4 common haplotypes of the CASP1 gene. Among these, the Ain6 allele of the G+7/in6A polymorphism was less frequent in 246 cases with myocardial infarction and a parental history of disease than in 253 controls free of familial history of disease (0.13±0.02 versus 0.20±0.02; P=0.005). However, in a larger case/control study (n=1774), these effects are borderline restricted to the UK population. In a prospective cohort of 1168 patients with coronary artery disease followed up during a median period of 6.0 years, the Ain6 allele exhibited a borderline association with future cardiovascular death (hazard ratio [HR]: 0.64, 0.41 to 1.01; P=0.053) and was associated with lower serum IL-18 levels (P=0.014). Baseline caspase-1 levels in the top quartile of the distribution were predictive of cardiovascular deaths (HR=3.62, 1.81 to 7.27; P=0.0003 compared with the bottom quartile). Finally, in vitro assays of allelic imbalance showed that the CASP1 haplotype carrying the Ain6 allele was associated with a lower mRNA expression. These results indicate that caspase-1 levels are predictive of future cardiovascular death in patients with coronary artery disease. The role of CASP1 genetic variations in the susceptibility to myocardial infarction requires further investigation.

The human T locus and spina bifida risk

The transcription factor T is essential for mesoderm formation and axial development during embryogenesis. Embryonic genotype for a single-nucleotide polymorphism in intron 7 of T (TIVS7 T/C) has been associated with the risk of spina bifida in some but not all studies. We developed a novel genotyping assay for the TIVS7 polymorphism using heteroduplex generator methology. This assay was used to genotype spina bifida case—parent trios and the resulting data were analyzed using the transmission disequilibrium test and log-linear analyses. Analyses of these data demonstrated that heterozygous parents transmit the TIVS7-C allele to their offspring with spina bifida significantly more frequently than expected under the assumption of Mendelian inheritance (63 vs 50%, P=0.02). Moreover, these analyses suggest that the TIVS7-C allele acts in a dominant fashion, such that individuals carrying one or more copies of this allele have a 1.6-fold increased risk of spina bifida compared with individuals with zero copies. In silico analysis of the sequence surrounding this polymorphism revealed a potential target site for olfactory neuron-specific factor-1, a transcription factor expressed in the neural tube during development, spanning the polymorphic site. Several other putative, developmentally important and/or environmentally responsive transcription factor-binding sites were also identified close to the TIVS7 polymorphism. The TIVS7 polymorphism or a variant that is in linkage disequilibrium with the TIVS7 polymorphism may, therefore, play a role in T gene expression and influence the risk of spina bifida.

A genome-wide search for new imprinted genes in the human placenta identifies DSCAM as the first imprinted gene on chromosome 21

We identified, through a genome-wide search for new imprinted genes in the human placenta, DSCAM (Down Syndrome Cellular Adhesion Molecule) as a paternally expressed imprinted gene. Our work revealed the presence of a Differentially Methylated Region (DMR), located within intron 1 that might regulate the imprinting in the region. This DMR showed a maternal allele methylation, compatible with its paternal expression. We showed that DSCAM is present in endothelial cells and the syncytiotrophoblast layer of the human placenta. In mouse, Dscam expression is biallelic in foetal brain and placenta excluding any possible imprinting in these tissues. This gene encodes a cellular adhesion molecule mainly known for its role in neurone development but its function in the placenta remains unclear. We report here the first imprinted gene located on human chromosome 21 with potential clinical implications.