Valeria Capra

Study of MTHFR and MS polymorphisms as risk factors for NTD in the Italian population.

AbstractHomozygosity for the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene is a risk factor for neural tube defects (NTDs) in many populations, including Italians. Another common mutation on the MTHFR gene, A1298C, has also been described as a risk mutation. Furthermore, several studies have suggested that a defective methionine synthase (MS) enzyme could be a critical defect in folate-related NTDs. An A-to-G transition at bp 2756 on the MS gene has also been reported. In this case-control study, we studied the frequencies of these two polymorphisms in 203 Italian probands with non-syndromic NTDs: 98 mothers, 67 fathers, and 210 control individuals. Although the A1298C polymorphism is common in the Italian population (0.25), the allelic frequency was significantly higher among NTD cases and their parents. Heterozygous patients and mothers have an odds ratio (OR) of 1.98 and 2.11, respectively. The risk associated with the 1298CC genotype was higher for cases (OR = 3.67), for fathers (OR = 3.28), and, above all, for mothers (OR = 6.23). The prevalence of the A2756G polymorphism of the MS gene was determined (0.15). No increased prevalence of the mutated G allele was found in NTD families. This study shows that the MTHFR A1298C polymorphism is a genetic determinant for NTD risk in Italy. No association between the MS A2756G and NTD susceptibility was found.

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.

VACTERL/caudal regression/Currarino syndrome-like malformations in mice with mutation in the proprotein convertase Pcsk5

We have identified an ethylnitrosourea (ENU)-induced recessive mouse mutation (Vcc) with a pleiotropic phenotype that includes cardiac, tracheoesophageal, anorectal, anteroposterior patterning defects, exomphalos, hindlimb hypoplasia, a presacral mass, renal and palatal agenesis, and pulmonary hypoplasia. It results from a C470R mutation in the proprotein convertase PCSK5 (PC5/6). Compound mutants (Pcsk5(Vcc/null)) completely recapitulate the Pcsk5(Vcc/Vcc) phenotype, as does an epiblast-specific conditional deletion of Pcsk5. The C470R mutation ablates a disulfide bond in the P domain, and blocks export from the endoplasmic reticulum and proprotein convertase activity. We show that GDF11 is cleaved and activated by PCSK5A, but not by PCSK5A-C470R, and that Gdf11-deficient embryos, in addition to having anteroposterior patterning defects and renal and palatal agenesis, also have a presacral mass, anorectal malformation, and exomphalos. Pcsk5 mutation results in abnormal expression of several paralogous Hox genes (Hoxa, Hoxc, and Hoxd), and of Mnx1 (Hlxb9). These include known Gdf11 targets, and are necessary for caudal embryo development. We identified nonsynonymous mutations in PCSK5 in patients with VACTERL (vertebral, anorectal, cardiac, tracheoesophageal, renal, limb malformation OMIM 192350) and caudal regression syndrome, the phenotypic features of which resemble the mouse mutation. We propose that Pcsk5, at least in part via GDF11, coordinately regulates caudal Hox paralogs, to control anteroposterior patterning, nephrogenesis, skeletal, and anorectal development.

Toward understanding the genetic basis of neural tube defects

Neural tube defects (NTDs) represent a common group of severe congenital malformations that result from failure of neural tube closure during early development. Their etiology is quite complex involving environmental and genetic factors and their underlying molecular and cellular pathogenic mechanisms remain poorly understood. Animal studies have recently demonstrated an essential role for the planar cell polarity pathway (PCP) in mediating a morphogenetic process called convergent extension during neural tube formation. Alterations in members of this pathway lead to NTDs in vertebrate models, representing novel and exciting candidates for human NTDs. Genetic studies in NTDs have focused mainly on folate‐related genes based on the finding that perinatal folic acid supplementation reduces the risk of NTDs by 60–70%. A few variants in these genes have been found to be significantly associated with an increased risk for NTDs. The candidate gene approach investigating genes involved in neurulation has failed to identify major causative genes in the etiology of NTDs. Despite this history of generally negative findings, we are achieving a rapid and impressive progress in understanding the genetic basis of NTDs, based mainly on the powerful tool of animal models.

Medulloblastoma Variants: Age-Dependent Occurrence and Relation to Gorlin Syndrome—A New Clinical Perspective

Purpose: We aimed to test the hypothesis that medulloblastoma (MB) variants show a different age distribution and clinical behavior reflecting their specific biology, and that MB occurring at very young age is associated with cancer predisposition syndromes such as Gorlin syndrome (GS). Experimental Design: We investigated the frequency, age distribution, location, response to treatment, outcome, and association with familial cancer predisposition syndromes in a series of 82 cases of MB in patients ages <14 years diagnosed at the Giannina Gaslini Childrens Hospital, Genoa, between 1987 and 2004. Results: Desmoplastic MB and MB with extensive nodularity (MBEN), were present in 22 of 82 cases (27%) and were more frequent in children ages ≤3 years (13 of 25; 52%). In this age group, MBEN was significantly more frequent than desmoplastic MB and classic MB (P < 0.001) and had a good prognosis. MBEN was associated with GS in 5 of 12 cases. Overall, 8 cases occurred in the context of familial tumor predisposition syndromes (5 GS, 1 each NF1, Li-Fraumeni, and Fragile X) and 7 of these patients were ages ≤3 years at diagnosis. Desmoplastic histology and a more intensive treatment represented independent favorable prognostic factors in multivariate analysis (P = 0.003 and P = 0.0139, respectively). Metastasis was a predictor of bad outcome (P = 0.0001). Conclusions: Our data indicate that biologically different MB entities warrant risk-adapted treatment and that MBEN is strongly associated with GS. Patients, ages ≤3 years, with MB and their families should be investigated for tumor predisposition syndromes such as GS.

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.

Contribution of VANGL2 mutations to isolated neural tube defects

Kibar Z, Salem S, Bosoi CM, Pauwels E, De Marco P, Merello E, Bassuk AG, Capra V, Gros P. Contribution of VANGL2 mutations to isolated neural tube defects.

The C677T mutation of the 5,10-methylenetetrahydrofolate reductase gene is a moderate risk factor for spina bifida in Italy.

OBJECTIVE: To estimate the risk for spina bifida associated with the common mutation C677T of the MTHFR gene in a country with a relatively low prevalence of NTDs. DESIGN: Case-control study. SUBJECTS: Cases: 203 living patients affected with spina bifida (173 myelomeningocele and 30 lipomeningocele); controls: 583 subjects (306 young adults and 277 unselected newborns) from northern and central-southern Italy. SETTING: Cases: three spina bifida centres; young adult controls: DNA banks; newborn controls: regional neonatal screening centres. MAIN OUTCOME MEASURES: Prevalence of the C677T genotypes in cases and controls by place of birth; odds ratios for spina bifida and estimated attributable fraction. RESULTS: The prevalence of T/T, T/C, and C/C genotype was 16.6%, 53.7%, and 29.7% in controls and 25.6%, 43.8%, and 30.6% in cases, respectively. We found no differences between type of defect or place of birth. The odds ratio for spina bifida associated with the T/T genotype v C/C plus T/C was 1.73 (95% CI 1.15, 2.59) and the corresponding attributable fraction was 10.8%. No increased risk was found for heterozygous patients (OR=0.79, 95% CI 0.53-1.18). CONCLUSION: This study, as well as the meta-analysis we updated, shows that homozygosity for the MTHFR C677T mutation is a moderate risk factor in Europe, and even in Italy where there is a relatively low prevalence of spina bifida. The estimated attributable fraction associated with this risk factor explains only a small proportion of cases preventable by periconceptional folic acid supplementation. Thus, other genes involved in folate-homocysteine metabolism, their interaction, and the interaction between genetic and environmental factors should be investigated further.

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.