Patrizia De Marco

Reduced folate carrier polymorphism (80A-->G) and neural tube defects.

Transport of folates in mammalian cells occurs by a carrier-mediated mechanism. The human folate carrier (RFC-1) gene has been isolated and characterized. Within this gene, a common polymorphism, 80A→G, changing a histidine to an arginine in exon 2 (H27R), was recently identified. Defects in folate metabolism, such as defective carrier molecules, could be implicated in the etiology of neural tube defects (NTDs). In the present case–control study, we recruited 174 Italian probands with nonsyndromic NTD, 43 mothers, 53 fathers and 156 control individuals and evaluated the impact of RFC-1 variant on NTD risk. A statistically significant risk was calculated for the 80GG genotype of the NTD cases (OR=2.35; 95% CI 1.21–4.58) and mothers (OR=2.74; 95% CI 0.92–8.38). On the contrary, the heterozygous genotype of the mothers and both heterozygous and homozygous genotypes of the fathers did not seem to be significant NTD risk factors. Furthemore, according to the multifactorial inheritance of NTDs, we demonstrated that the combined genotypes for MTHFR 1298A→C and RFC-1 80A→G polymorphisms of cases resulted in greater NTD risk than heterozygosity or homozygosity for RFC-1 80A→G variant alone. Conversely, our data provide no evidence for an association between NTD phenotype and combined MTHFR C677T/RFC-1 A80G genotypes. Moreover, here we describe the combinations of the two MTHFR polymorphic sites (677CT and 1298AC) with RFC-1 genotypes. We found that both patients and controls could have at most quadruple-mutation combinations. Interestingly, 27% (7/26) of the mothers and 18.75% (30/160) of the cases genotyped presented four mutant alleles in comparison with 8.5% (11/129) of the controls. Finally, the frequency of NTD cases and mothers carrying combined heterozygosity for the two MTHFR polymorphisms and RFC-1 80GG homozygosity (677CT/1298AC/80GG) (cases=11.3%; mothers 11.5%) was increased compared with controls (1.6%). Altogether, our findings support the hypothesis that RFC-1 A80G variant may contribute to NTD susceptibility in the Italian population.

Novel Mutations in VANGL1 in Neural Tube Defects

Neural tube defects (NTDs) are severe congenital malformations caused by failure of the neural tube to close during neurulation. Their etiology is complex involving both environmental and genetic factors. We have recently reported three mutations in the planar cell polarity gene VANGL1 associated with NTDs. The aim of the present study was to define the role of VANGL1 genetic variants in the development of NTDs in a large cohort of various ethnic origins. We identified five novel missense variants in VANGL1, p.Ser83Leu, p.Phe153Ser, p.Arg181Gln, p.Leu202Phe and p.Ala404Ser, occurring in sporadic and familial cases of spinal dysraphisms. All five variants affect evolutionary conserved residues and are absent from all controls analyzed. This study provides further evidence supporting the role of VANGL1 as a risk factor in the development of spinal NTDs.

Evaluation of a methylenetetrahydrofolate-dehydrogenase 1958G>A polymorphism for neural tube defect risk

AbstractGenetic variants of enzymes involved in the folate pathway might be expected to have an impact on neural tube defect (NTD) risk. Given its key role in folate metabolism, the methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene could represent an attractive candidate in NTD aetiology. In this study, the impact of the MTHFD1 1958G>A polymorphism on NTD risk in the Italian population was examined both by hospital-based case-control and family-based studies. The MTHFD1 1958G>A polymorphism was genotyped in 142 NTD cases, 125 mothers, 108 fathers and 523 controls. An increased risk was found for the heterozygous 1958G/A (OR=1.69; P=0.04) and homozygous 1958A/A (OR=1.91; P=0.02) genotypes in the children. Significant association was also found when combined 1958G/A and 1958A/A genotypes of cases were compared with the 1958G/G genotype (OR=1.76; P=0.02). The risk of an NTD-affected pregnancy of the mothers was increased 1.67-fold (P=0.04) only when a dominant effect (1958G/A or 1958A/A vs 1958G/G) of the 1958A allele was analysed. The combined TDT/1-TDT (Z=2.11; P=0.03) and FBAT (Z=2.4; P=0.01) demonstrated a significant excess of transmission of the 1958A allele to affected individuals. In summary, our results indicate that heterozygosity and homozygosity for the MTHFD1 1958G>A polymorphism are genetic determinants of NTD risk in the cases examined.

Identification and characterization of novel rare mutations in the planar cell polarity gene PRICKLE1 in human neural tube defects

The planar cell polarity (PCP) pathway controls the process of convergent extension (CE) during gastrulation and neural tube closure, and has been implicated in the pathogenesis of neural tube defects (NTDs) in animal models and human cohorts. In this study, we analyzed the role of one core PCP gene PRICKLE1 in these malformations. We screened this gene in 810 unrelated NTD patients and identified seven rare missense heterozygous mutations that were absent in all controls analyzed and predicted to be functionally deleterious using bioinformatics. Functional validation of five PRICKLE1 variants in a zebrafish model demonstrated that one variant, p.Arg682Cys, antagonized the CE phenotype induced by the wild‐type zebrafish prickle1a (zpk1a) in a dominant fashion. Our study demonstrates that PRICKLE1 could act as a predisposing factor to human NTDs and further expands our knowledge of the role of PCP genes in the pathogenesis of these malformations. 32:1371–1375, 2011. ©2011 Wiley Periodicals, Inc.

FZD6 is a novel gene for human neural tube defects

Neural tube defects (NTDs) are severe malformations of the central nervous system, affecting 1 of 1,000 live births. Mouse models were instrumental in defining the signaling pathways defective in NTDs, including the planar cell polarity (PCP), also called noncanonical Frizzled/Disheveled pathway. Based on the highly penetrant occurrence of NTDs in double Fzd3/Fzd6−/− mutant mice, we investigated the role of the human orthologues, FZD3 and FZD6, by resequencing a cohort of 473 NTDs patients and 639 ethnically matched controls. While we could not demonstrate a significant contribution of FZD3 gene, we identified five rare FZD6 variants that were absent in all controls and predicted to have a functional effect by computational analysis: one de novo frameshift mutation (c.1843_1844insA), three missense changes (p.Arg405Gln, p.Arg511Cys p.Arg511His), and one substitution (c.*20C>T) affecting the 3′‐untranslated region (UTR) of the gene. The overall rate of predicted deleterious variants of FZD6 was 5.1‐fold higher in cases compared to controls, resulting in a significantly increased NTDs mutation burden. This study demonstrates that rare nonsynonymous variants in FZD6 may contribute to NTDs in humans and enlarges the spectrum of mutations that link PCP pathway to NTDs. Hum Mutat 33:384–390, 2012.

Mutations in the planar cell polarity gene, Fuzzy, are associated with neural tube defects in humans

Neural tube defects (NTDs) are a heterogeneous group of common severe congenital anomalies which affect 1-2 infants per 1000 births. Most genetic and/or environmental factors that contribute to the pathogenesis of human NTDs are unknown. Recently, however, pathogenic mutations of VANGL1 and VANGL2 genes have been associated with some cases of human NTDs. Vangl genes encode proteins of the planar cell polarity (PCP) pathway that regulates cell behavior during early stages of neural tube formation. Homozygous disruption of PCP genes in mice results in a spectrum of NTDs, including defects that affect the entire neural axis (craniorachischisis), cranial NTDs (exencephaly) and spina bifida. In this paper, we report the dynamic expression of another PCP gene, Fuzzy, during neural tube formation in mice. We also identify non-synonymous Fuzzy amino acid substitutions in some patients with NTDs and demonstrate that several of these Fuzzy mutations affect formation of primary cilia and ciliary length or affect directional cell movement. Since Fuzzy knockout mice exhibit both NTDs and defective primary cilia and Fuzzy is expressed in the emerging neural tube, we propose that mutations in Fuzzy may account for a subset of NTDs in humans.

Current perspectives on the genetic causes of neural tube defects

Neural tube defects (NTDs) are a group of severe congenital abnormalities resulting from the failure of neurulation. The pattern of inheritance of these complex defects is multifactorial, making it difficult to identify the underlying causes. Scientific research has rapidly progressed in experimental embryology and molecular genetics to elucidate the basis of neurulation. Crucial mechanisms of neurulation include the planar cell polarity pathway, which is essential for the initiation of neural tube closure, and the sonic hedgehog signaling pathway, which regulates neural plate bending. Genes influencing neurulation have been investigated for their contribution to human neural tube defects, but only genes with well-established role in convergent extension provide an exciting new set of candidate genes. Biochemical factors such as folic acid appear to be the greatest modifiers of NTDs risk in the human population. Consequently, much research has focused on genes of folate-related metabolic pathways. Variants of several such genes have been found to be significantly associated with the risk of neural tube defects in more studies. In this manuscript, we reviewed the current perspectives on the causes of neural tube defects and highlighted that we are still a long way from understanding the etiology of these complex defects.

No major role for the EMX2 gene in schizencephaly

Schizencephaly (SCH) is a rare disorder of cerebral cortical development, characterized by full thickness clefts spanning the wall of the cerebral hemispheres that are lined and surrounded by polymicrogyric cortex. Based on pathological analysis, SCH was originally considered to have multiple causes including infectious and vascular injuries, and toxic agents. However, a few reports of familial SCH have suggested a possible genetic etiology. Ten years ago two articles identified EMX2 as the first causative gene for human SCH in 13 of 18 patients, although for several putative mutations no pathogenic role was demonstrated. Here, we reinterpret the original articles as showing a significantly lower mutational rate (17%) than originally reported (72%), and provide results of EMX2 sequencing in 39 new SCH patients, detecting no pathogenic mutations. We conclude that the reported association between SCH and EMX2 mutations is not adequately supported by current data, and that diagnostic testing of EMX2 is not justified, as any results would be uninterpretable.

Maternal periconceptional factors affect the risk of spina bifida-affected pregnancies: an Italian case–control study

PurposeNeural tube defects, including spina bifida and anencephaly, are the second most common birth defects with an incidence in Italy of 0.4–1/1,000. Information on factors playing a role in the pathogenesis of spina bifida is based on populations with different exposures, lifestyle, social and cultural habits compared to Italian people. Our objective was to fill this gap by using data from a case–control interview study carried out at the G. Gaslini Children’s Hospital, Genoa, from 2000 to 2008.MethodsWe surveyed questionnaires from 133 case mothers and 273 control women providing information on periconceptional risk factors. Univariate and multivariate logistic regression analyses were used to estimate risks by odds ratios (ORs) and 95% confidence intervals (95% CIs).ResultsUnivariate results suggest that birth order, low maternal educational level, age, smoking habits, alcohol consumption, high caffeine intake, lack of folate supplementation, low and high calorie diet, occasional consumption of fruit and vegetables, high emotional stress, and environmental pollution are associated with an increased spina bifida risk. Nevertheless, high caffeine intake (OR = 10.82; 95% CI, 3.78–31), low calorie diet (OR = 5.15; 95%CI, 1.79–14), occasional consumption of fruit and vegetables (OR = 3.38; 95% CI, 1.67–6.82), alcohol consumption (OR = 3.05; 95% CI, 1.24–7.50) and, above all, lack of folate supplementation at any time of pregnancy (OR = 20.54; 95% CI, 5.41–77) mainly determined spina bifida risk in the multivariate analysis.ConclusionOur findings point out that a common underlying mechanism, a disturbed folate/homocysteine metabolism, may be causative for the burden of spina bifida in the Italian population.

Genetic Analysis of Disheveled 2 and Disheveled 3 in Human Neural Tube Defects

Neural tube defects are severe malformations affecting 1/1,000 live births. The planar cell polarity pathway controls the neural tube closure and has been implicated in the pathogenesis of neural tube defects both in animal models and human cohorts. In mouse disruption of Dvl2 alone (Dvl2−/−) or Dvl2 and Dvl3 (Dvl2−/−; Dvl3+/−, Dvl2+/−; Dvl3−/−) results in incomplete neurulation, suggesting a role for Disheveled in neural tube closure. Disheveled is a multifunctional protein that is involved in both the canonical Wnt signaling and the noncanonical planar cell polarity pathway. In this study, we analyzed the role of the human orthologs DVL2 and DVL3 in a cohort of 473 patients with neural tube defects. Rare variants were genotyped in 639 ethnically matched controls. We identified seven rare missense mutations that were absent in all controls analyzed. Two of these mutations, p.Tyr667Cys and p.Ala53Val, identified in DVL2 were predicted to be detrimental in silico. Significantly, a 1-bp insertion (c.1801_1802insG) in exon 15 of DVL2 predicted to lead to the truncation of the protein was identified in a patient with a complex form of caudal agenesis. In summary, we demonstrate a possible role for rare variants in DVL2 gene as risk factors for neural tube defects.