Sandra G. Heil

Molecular genetic analysis of the gene encoding the trifunctional enzyme MTHFD (methylenetetrahydrofolate-dehydrogenase, methenyltetrahydrofolate-cyclohydrolase, formyltetrahydrofolate synthetase) in patients with neural tube defects

It is now well recognized that periconceptional folic acid or folic acid containing multivitamin supplementation reduces the risk of neural tube defects (NTDs). Recently we were able to show that homozygosity for a thermolabile variant of the enzyme methylenetetrahydrofolate reductase is associated with an increased risk for spina bifida in patients recruited from the Dutch population. However, this genetic risk factor could not account for all folic acid preventable NTDs. In an attempt to identify additional folate related enzymes that contribute to NTD etiology we now studied the methylenetetrahydrofolate dehydrogenase gene on chromosome 14q24 which encodes a single protein with three catalytic properties important in the folate metabolism. The cDNA sequence of 38 familial and 79 sporadic patients was screened for the presence of mutations by single strand conformation polymorphism (SSCP) analysis followed by sequencing. Two amino acid substitutions were identified. The first one (R293H) was detected in a patient with familial spina bifida and not in 300 control individuals. The mutation was inherited from the unaffected maternal grandmother and was also present in two younger brothers of the index patient, one of them displaying spina bifida occulta and the other being unaffected. The second change turned out to be an amino acid polymorphism (R653Q) that was present in both patients and controls with similar frequencies. Our results so far provide no evidence for a major role of the methylenetetrahydrofolate‐dehydrogenase (MTHFD) gene in NTD etiology. However, the identification of a mutation in one family suggests that this gene can act as a risk factor for human NTD.

Genetic variation in genes of folate metabolism and neural-tube defect risk

Neural-tube defects (NTD) are common congenital malformations that can lead to severe disability or even death. Periconceptional supplementation with the B-vitamin folic acid has been demonstrated to prevent 50-70% of NTD cases. Since the identification of the first genetic risk factor of NTD, the C677T single-nucleotide polymorphism (SNP) in the methylenetetrahydrofolate reductase (MTHFR) gene, and the observation that elevated plasma homocysteine levels are associated with NTD, research has focused on genetic variation in genes encoding for enzymes of folate metabolism and the closely-related homocysteine metabolism. In the present review relevant SNP in genes that code for enzymes involved in folate transport and uptake, the folate cycles and homocysteine metabolism are summarised and the importance of these SNP discussed in relation to NTD risk.

Endothelium-Derived Hyperpolarizing Factor–Mediated Renal Vasodilatory Response Is Impaired During Acute and Chronic Hyperhomocysteinemia

Background—Endothelial dysfunction is an early event in the development of vascular complications in hyperhomocysteinemia. Endothelial cells release a number of vasodilators, including NO and prostacyclin. Several lines of evidence have indicated the existence of a third vasodilator pathway, mediated by endothelium-derived hyperpolarizing factor (EDHF). EDHF is a major determinant of vascular tone in small resistance vessels. The influence of hyperhomocysteinemia on EDHF is unknown. The present in vivo study evaluates the integrity of the EDHF pathway in the renal microcirculation of rats with acute and chronic hyperhomocysteinemia. Methods and Results—EDHF-mediated vasodilation was evaluated as the renal blood flow (RBF) response to intrarenal acetylcholine during systemic NO synthase and cyclooxygenase inhibition. Acute hyperhomocysteinemia induced by intravenous homocysteine did not affect EDHF-mediated vasodilation. In contrast, intravenous methionine with subsequent hyperhomocysteinemia impaired the EDHF-mediated RBF response. When the methionine infusion was preceded by adenosine periodate oxidized to prevent the cleavage of S-adenosylhomocysteine to homocysteine and adenosine, a similar impairment of EDHF was observed, but with normal homocysteine levels. Animals with chronic hyperhomocysteinemia induced by a high-methionine, low–B vitamin diet during 8 weeks had a severely depressed EDHF-mediated vasodilation compared with those on a standard diet. Endothelium-independent vasodilation to deta-NONOate and pinacidil was not affected in acute and chronic hyperhomocysteinemia, demonstrating intact vascular smooth muscle reactivity. Conclusions—EDHF-dependent responses are impaired in the kidney of hyperhomocysteinemic rats. Because EDHF is a major regulator of vascular function in small vessels, these findings have important implications for the development of microangiopathy in hyperhomocysteinemia.

A 31 bp VNTR in the cystathionine beta-synthase (CBS) gene is associated with reduced CBS activity and elevated post-load homocysteine levels.

Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinaemia, an independent and graded risk factor for cardiovascular disease (CVD). Although heterozygosity for cystathionine β-synthase (CBS) deficiency has been excluded as a major genetic cause of mild hyperhomocysteinaemia in vascular disease, mutations in (non-)coding DNA sequences may lead to a mildly decreased CBS expression and, consequently, to elevated plasma homocysteine levels. We assessed the association between a 31 bp VNTR, that spans the exon 13-intron 13 boundary of the CBS gene, and fasting, post-methionine load and increase upon methionine load plasma homocysteine levels in 190 patients with arterial occlusive disease, and in 381 controls. The 31u2009bp VNTR consists of 16, 17, 18, 19 or 21 repeat units and shows a significant increase in plasma homocysteine concentrations with an increasing number of repeat elements, in particular after methionine loading. In 26 vascular disease patients the relationship between this 31u2009bp VNTR and CBS enzyme activity in cultured fibroblasts was studied. The CBS enzyme activity decreased with increasing number of repeat units of the 31 bp VNTR. RT–PCR experiments showed evidence of alternative splicing at the exon 13-intron 13 splice junction site. The 31u2009bp VNTR in the CBS gene is associated with post-methionine load hyperhomocysteinaemia that may predispose individuals to an increased risk of cardiovascular diseases.

Molecular genetic analysis of human folate receptors in neural tube defects.

Neural tube defects (NTDs) are the most common congenital malformations and are considered to have a multifactorial origin, having both genetic and environmental components. Periconceptional folate administration reduces the recurrence and occurrence risk by 70–100%. Recently we discovered the first genetic risk factors for NTDs: the 677 C→T and the 1298 A→C mutations in the methylenetetrahydrofolate reductase gene explaining at the most 35–50% of the protective effect of folate. In this study we further explored the genetic component of NTDs by analysing the coding region, including the intron–exon boundaries and signal sequences of the folate receptor genes by SSCP analysis. Among 39xa0patients with spina bifida (SB), 47xa0mothers with a child with SB, and 10xa0controls, no polymorphism was present in the folate receptor alpha (FR-α) gene or in the folate receptor beta (FR-β) gene.

METHYLENETETRAHYDROFOLATE REDUCTASE POLYMORPHISMS IN PREECLAMPSIA AND THE HELLP SYNDROME

Objective: To investigate the prevalence of the 677 (C → T) and 1298 (A → C) polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene in our preeclamptic population. For a summary estimation of the risk of the 677 (C → T) polymorphism for preeclampsia, we also performed a meta-analysis on four previously published case-control studies to which our results were added. Methods: Genotypes were analyzed by polymerase chain reaction followed by restriction enzyme analysis. The results of 176 nonpregnant women, previously hospitalized for preeclampsia in a tertiary care center, were compared with 403 Dutch population-based controls.Results were statistically analyzed with a chi-square test. Mean Outcome Measures: The incidence of the 677 (C → T) and 1298 (A → C) polymorphisms in the MTHFR gene. Results: The incidence of both MTHFR missense polymorphisms was not significantly different between cases and controls. We found an odds ratio (OR) of 1.5 [95% confidence interval (CI) 0.8–0.6, p = 0.17] and an OR of 1.0 (95% CI 0.6–1.9, p = 0.23) for the 677 (C → T) and the 1298 (A → C) polymorphism, respectively, in cases comparing the prevalence of the homozygous genotype versus the other two genotypes. The meta-analysis resulted in a significant OR of 2.0 (95% CI 1.4–2.9). Conclusions: In contrast to four previous studies, we were neither able to confirm an increased risk for preeclampsia to the 677 (C → T) polymorphism nor did we find an increased risk for preeclampsia to the 1298 (A → C) polymorphism. From the meta-analysis, however, we conclude that it cannot be ruled out that the homozygous 677TT genotype is a modest but significant risk factor for preeclampsia.

Automated extraction and amplification of DNA from whole blood using a robotic workstation and an integrated thermocycler.

Growing knowledge of the genetic basis of inheritable diseases has resulted in a rapidly increasing demand for DNA mutation analysis. Current methods are reliable and suitable for low‐throughput mutation analyses, but are unable to cope with the increasing demand for genetic analyses, necessitating the development of new, fully automated and reliable methods. We developed a semi‐automated method for DNA mutation analysis by integrating a thermocycler into a robotic pipetting workstation. DNA was extracted from 84 samples of 10 μl of EDTA‐treated whole blood using magnetic beads within 2 h. Directly after isolation, the DNA was automatically transferred to an integrated thermocycler for amplification. Our semi‐automated method proved to be reliable and robust, showing unambiguously interpretable PCR signals without occurrence of contamination. It is also faster than conventional manual methods. Only a brief manual intervention is required to remove and refit the seal of the PCR plate. This semi‐automated assay is a step forward in the development of fully automated assays for DNA mutation analysis.

The 894 G > T variant of endothelial nitric oxide synthase (eNOS) increases the risk of recurrent venous thrombosis through interaction with elevated homocysteine levels

Summary.u2002 Background:u2003Venous thrombosis is a multicausal disease involving both genetic as well as acquired risk factors. Hyperhomocysteinemia is associated with a 2‐fold increased risk of recurrent venous thrombosis (RVT). Recently, the 894 Gu2003>u2003T variant of endothelial nitric oxide synthase (eNOS) was postulated to be associated with hyperhomocysteinemia. Objectives:u2003We hypothesized an interrelation of hyperhomocysteinemia, the eNOS 894 Gu2003>u2003T variant and RVT risk. Methods:u2003The eNOS 894 Gu2003>u2003T variant was studied in 170 cases with a history of RVT and 433 controls from the general population. Results:u2003The eNOS 894 TT genotype may increase RVT risk [odds ratio (OR) 1.3 (0.7–2.6)], but no association of the eNOS 894 Gu2003>u2003T variant with elevated homocysteine was found in controls. Interestingly, in RVT cases the coexistence of both the 894 TT genotype and elevated tHcy levels (>u200390th percentile) was more frequently present than in controls, which led to a substantially increased risk of recurrent venous thrombosis [fasting tHcy OR 5.3 (1.1–24.1), postload tHcy OR 6.5 (1.6–29.5)]. Conclusion:u2003The results of the present study demonstrate that the eNOS 894 Gu2003>u2003T variation interacts with elevated tHcy levels, leading to an increased risk of recurrent thrombotic events. This interaction points in the direction of S‐nitrosation as a mechanism by which homocysteine exerts its detrimental effects on the hemostatic system.

The Molecular Basis of Dutch Infantile Nephropathic Cystinosis

Infantile nephropathic cystinosis, an inborn error of metabolism with an autosomal recessive inheritance pattern, is characterized by lysosomal storage of the amino acid cystine due to an impaired transport of cystine out of the lysosomes. Initial clinical features consist of the renal Fanconi syndrome and crystals in the cornea. Oral therapy with cysteamine lowers the intracellular cystine content. Recently, the gene coding for the integral membrane protein cystinosin, which is responsible for membrane transport of cystine (CTNS), was cloned. Mutation analysis of the CTNS gene of Caucasian patients revealed a common 57-kb deletion, and several other mutations spread throughout the entire gene. In the present study, we developed an improved screening method for the detection of the common 57-kb deletion. By use of this method we detected the 57-kb deletion in 59% of the examined Dutch alleles. The remaining alleles were screened for other mutations by genomic sequencing of the different exons, revealing three previously described mutations. Furthermore, we studied a possible genotype-phenotype relation of the homozygous deleted patients, which could not be demonstrated in our study population. Next to biochemical determination of cystine in leukocytes or fibroblasts, molecular genetic analysis enables prenatal diagnosis and facilitates identification of carriers.

Inhibition of methylation and changes in gene expression in relation to neural tube defects.

BACKGROUNDnAn impaired DNA methylation has been suggested to underlie the complex etiology of neural tube defects (NTDs). Previously, we have demonstrated that inhibition of methylation by periodate oxidized adenosine (Adox) results in a widening of the anterior neuropore (ANP) in our in vitro chick embryo model. Since DNA methylation is the chief regulator of gene expression, we hypothesize that inhibition of methylation by Adox in our in vitro chick embryo model will affect the expression of genes that may be involved in neurulation. In the present study, we therefore examined differential gene expression between Adox-treated and control chick embryos, using the Affymetrix Genechip Chicken Genome Array.nnnMETHODSnChick embryos of 4/5 somites were cultured in vitro with saline (control) or Adox and cranial parts were excised. Gene expression profiling was determined using the Affymetrix Genechip Chicken Genome Array on RNA isolated from two pools of Adox-treated cranial parts (n = 12) and two pools of saline-treated cranial parts (n = 12). Microarray data were validated by QPCR analysis.nnnRESULTSnIn the Adox-treated chick embryos, 45 probesets were up-regulated (fold > or = 2.0, p < 0.05) and 32 probesets were down-regulated (fold < or = 0.5, p < 0.05). Of the 15 genes selected for QPCR analysis, the up-regulation of phosphoserine phosphatase (PSPH), unc-51-like kinase 1 (ULK1), and chemokine (C-X-C motif) ligand 12/stromal cell-derived factor 1 (CXCL12/SDF-1) was confirmed.nnnCONCLUSIONSnInhibition of methylation by Adox affects gene expression in our in vitro chick embryo model. Further research will focus on the gene-specific methylation patterns of PSPH, ULK1, and CXCL12/SDF-1 and the role of the products of these genes in neurulation.