A Comprehensive Investigation on Potential Risk Factors for NSCL/P in a Rural District of Hebei Province, China.

Non syndromic cleft lip with or without palate (NSCL/P), one of the most common birth defects, is closely related to various risk factors. However, information regarding risk factors for NSCL/P in rural districts in China is very limited thus far. The objective of this study was designed to identify the potential risk factors for NSCL/P in rural districts.A comprehensive retrospective investigation including 435 NSCL/P patients and 402 healthy children was carried out in Hebei Province, China. Multiple logistic regression analysis and transmission disequilibrium test (TDT) were respectively used to identify non-genetic and genetic risk factors for NSCL/P, and then PLINK was used to explore the relationship between non-genetic and genetic risk factors.The results showed that maternal periconceptional exposure to pesticides and herbicides, as well as low parental education level were involved in the increased risk of NSCL/P, whereas maternal folic acid and multivitamin supplementation use during preconception period were associated with the reduced risk of NSCL/P. TDT analysis identified 2 single nucleotide polymorphisms (SNPs) (rs7078160 and rs4752028) in VAX1 and one SNP (rs17563) in BMP4 as the genetic risk factors for NSCL/P. Further analysis showed that the genetic risk factors were closely related with the negative non-genetic risk factors.Our study identified the potential risk factors for NSCL/P in rural districts, thus providing a theoretical basis for the prevention of NSCL/P occurrence.

Cleft palate morphology, genetic etiology, and risk of mortality in infants with Robin sequence.

Robin sequence (RS) has many genetic and nongenetic causes, including isolated Robin sequence (iRS), Stickler syndrome (SS), and other syndromes (SyndRS). The purpose of this study was to determine if the presence and type of cleft palate varies between etiologic groups. A secondary endpoint was to determine the relationship of etiologic group, cleft type, and mortality. Retrospective chart review of patients with RS at two high-volume craniofacial centers. 295 patients with RS identified. CP was identified in 97% with iRS, 95% with SS, and 70% of those with SyndRS (p < .0001). U-shaped CP was seen in 86% of iRS, 82% with SS, but only 27% with SyndRS (p < .0001). At one institution, 12 children (6%) with RS died, all from the SyndRS group (p < .0001). All died due to medical comorbidities related to their syndrome. Only 25% of children who died had a U-shaped CP. The most common palatal morphology among those who died was an intact palate. U-shaped CP was most strongly associated with iRS and SS, and with a lower risk of mortality. RS with submucous CP, cleft lip and palate or intact palate was strongly suggestive of an underlying genetic syndrome and higher risk of mortality.

A comprehensive analysis of AHRR gene as a candidate for cleft lip with or without cleft palate.

Cleft lip and palate (CL/P) is among the most common congenital malformations and affects 1 in 700 newborns. CL/P is caused by genetic and environmental factors (maternal smoking, alcohol or drug use and others). Many genes and loci were associated with cleft lip/palate but the amount of heterogeneity justifies identifying new causal genes and variants. AHRR (Aryl-Hydrocarbon Receptor Repressor) gene has recently been related to CL/P however, few functional studies analyze the genotypephenotype interaction of AHRR with CL/P. Several studies associate the molecular pathway of AHRR to CL/P which indicates this gene as a functional candidate in CL/P etiology. METHODS: Systematic Literature Review was performed using PUBMED database with the keywords cleft lip, cleft palate, orofacial cleft, AHRR and synonyms. SLR resulted in 37 included articles. RESULTS: AHRR is a positional and functional candidate gene for CL/P. In silico analysis detected interactions with other genes previously associated to CL/P like ARNT and CYP1A1. AHRR protein regulates cellular toxicity through TCDD mediated AHR pathway. Exposure to TCDD in animal embryos is AHR mediated and lead to cleft palate due to palate fusion failure and post fusion rupture. AHRR regulates cellular growth and differentiation, fundamental to lip and palatogenesis. AHRR decreases carcinogenesis and recently a higher tumor risk has been described in CL/P patients and families. AHRR is also a smoking biomarker due to changed methylation sites found in smokers DNA although folate intake may partially revert these methylation alterations. This corroborates the role of maternal smoking and lack of folate supplementation as risk factors for CL/P. CONCLUSION: This research identified the importance of AHRR in dioxin response and demonstrated an example of genetic and environmental interaction, indispensable in the development of many complex diseases.

Non-syndromic cleft palate: Association analysis on three gene polymorphisms of the folate pathway in Asian and Italian populations.

Periconceptional folic acid supplementation can reduce the risk of inborn malformations, including orofacial clefts. Polymorphisms of MTHFR, TCN2, and CBS folate-related genes seem to modulate the risk of cleft lip with or without cleft palate (CL/P) in some populations. CL/P and cleft palate only (CPO) are different malformations that share several features and possibly etiological causes. In the present investigation, we conducted a family-based, candidate gene association study of non-syndromic CPO. Three single nucleotide polymorphisms, namely, rs1801133 of MTHFR, rs1801198 of TCN2, and rs4920037 of CBS, were investigated in a sample that included 129 Italian and 65 Asian families. No evidence of association between the three genotyped polymorphisms and CPO was found in the Italian and Asian cases, indeed the transmission disequilibrium test did not detect any asymmetry of transmission of alleles. This investigation, although with some limitation, further supports that CL/P and CPO diverge in their genetic background.

A genome-wide scan of cleft lip triads identifies parent-of-origin interaction effects between ANK3 and maternal smoking, and between ARHGEF10 and alcohol consumption.

Background: Although both genetic and environmental factors have been reported to influence the risk of isolated cleft lip with or without cleft palate (CL/P), the exact mechanisms behind CL/P are still largely unaccounted for. We recently developed new methods to identify parent-of-origin (PoO) interactions with environmental exposures (PoOxE) and applied them to families with children born with isolated cleft palate only. Here, we used the same genome-wide association study (GWAS) dataset and methodology to screen for PoOxE effects in the larger sample of CL/P triads. Methods: Genotypes from 1594 complete triads and 314 dyads (1908 nuclear families in total) with CL/P were available for the current analyses. Of these families, 1024 were Asian, 825 were European and 59 had other ancestries. After quality control, 341,191 SNPs remained from the original 569,244. The exposures were maternal cigarette smoking, use of alcohol, and use of vitamin supplements in the periconceptional period. The methodology applied in the analyses is implemented in the R-package Haplin. Results: Among Europeans, there was evidence of a PoOxSmoke effect for ANK3 with three SNPs (rs3793861, q=0.20, p=2.6e-6; rs7087489, q=0.20, p=3.1e-6; rs4310561, q=0.67, p=4.0e-5) and a PoOxAlcohol effect for ARHGEF10 with two SNPs (rs2294035, q=0.32, p=2.9e-6; rs4876274, q=0.76, p=1.3e-5). Conclusion: Our results indicate that the detected PoOxE effects have a plausible biological basis, and thus warrant replication in other independent cleft samples. Our demonstration of the feasibility of identifying complex interactions between relevant environmental exposures and PoO effects offers new avenues for future research aimed at unravelling  the complex etiology of cleft lip defects.

Epigenomic profiling of newborns with isolated orofacial clefts reveals widespread DNA methylation changes and implicates metastable epiallele regions in disease risk.

Cleft lip with or without cleft palate (CL/P) is a common human birth defect whose etiologies remain largely unknown. Several studies have demonstrated that periconceptional supplementation of folic acid can reduce risk of CL/P in offspring. In this study, we tested the hypothesis that the preventive effect of folic acid is manifested through epigenetic modifications by determining whether DNA methylation changes are associated with CL/P. To more readily observe the potential effects of maternal folate on the offspring epigenome, we focused on births prior to mandatory dietary folate fortification in the United States (i.e. birth year 1997 or earlier). Genomic DNA methylation levels were assessed from archived newborn bloodspots in a 182-member case-control study using the Illumina® Human Beadchip 450K array. CL/P cases displayed striking epigenome-wide hypomethylation relative to controls: 63% of CpGs interrogated had lower methylation levels in case newborns, a trend which held up in racially stratified sub-groups. 28 CpG sites reached epigenome-wide significance and all were case-hypomethylated. The most significant CL/P-associated differentially methylated region encompassed the VTRNA2-1 gene, which was also hypomethylated in cases (FWER p = 0.014). This region has been previously characterized as a nutritionally-responsive, metastable epiallele and CL/P-associated methylation changes, in general, were greater at or near putative metastable epiallelic regions. Gene Set Enrichment Analysis of CL/P-associated DMRs showed an over-representation of genes involved in palate development such as WNT9B, MIR140 and LHX8. CL/P-associated DNA methylation changes may partly explain the mechanism by which orofacial clefts are responsive to maternal folate levels.

Haplotype and Haplotype-Environment Interaction Analysis Revealed Roles of SPRY2 for NSCL/P among Chinese Populations.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is one of common birth defects in China, with genetic and environmental components contributing to the etiology. Genome wide association studies (GWASs) have identified SPRY1 and SPRY2 to be associated with NSCL/P among Chinese populations. This study aimed to further explore potential genetic effect and gene-environment interaction among SPRY genes based on haplotype analysis, using 806 Chinese case-parent NSCL/P trios drawn from an international consortium which conducted a genome-wide association study. After the process of quality control, 190 single nucleotide polymorphisms (SNPs) of SPRY genes were included for analyses. Haplotype and haplotype-environment interaction analyses were conducted in Population-Based Association Test (PBAT) software. A 2-SNP haplotype and three 3-SNP haplotypes showed a significant association with the risk of NSCL/P after Bonferroni correction (corrected significance level = 2.6 × 10-4). Moreover, haplotype-environment interaction analysis identified these haplotypes respectively showing statistically significant interactions with maternal multivitamin supplementation or maternal environmental tobacco smoke. This study showed SPRY2 to be associated with NSCL/P among the Chinese population through not only gene effects, but also a gene-environment interaction, highlighting the importance of considering environmental exposures in the genetic etiological study of NSCL/P.

Machine Learning Models for Genetic Risk Assessment of Infants with Non-syndromic Orofacial Cleft.

The isolated type of orofacial cleft, termed non-syndromic cleft lip with or without cleft palate (NSCL/P), is the second most common birth defect in China, with Asians having the highest incidence in the world. NSCL/P involves multiple genes and complex interactions between genetic and environmental factors, imposing difficulty for the genetic assessment of the unborn fetus carrying multiple NSCL/P-susceptible variants. Although genome-wide association studies (GWAS) have uncovered dozens of single nucleotide polymorphism (SNP) loci in different ethnic populations, the genetic diagnostic effectiveness of these SNPs requires further experimental validation in Chinese populations before a diagnostic panel or a predictive model covering multiple SNPs can be built. In this study, we collected blood samples from control and NSCL/P infants in Han and Uyghur Chinese populations to validate the diagnostic effectiveness of 43 candidate SNPs previously detected using GWAS. We then built predictive models with the validated SNPs using different machine learning algorithms and evaluated their prediction performance. Our results showed that logistic regression had the best performance for risk assessment according to the area under curve. Notably, defective variants in MTHFR and RBP4, two genes involved in folic acid and vitamin A biosynthesis, were found to have high contributions to NSCL/P incidence based on feature importance evaluation with logistic regression. This is consistent with the notion that folic acid and vitamin A are both essential nutritional supplements for pregnant women to reduce the risk of conceiving an NSCL/P baby. Moreover, we observed a lower predictive power in Uyghur than in Han cases, likely due to differences in genetic background between these two ethnic populations. Thus, our study highlights the urgency to generate the HapMap for Uyghur population and perform resequencing-based screening of Uyghur-specific NSCL/P markers.

Evaluating LINE-1 methylation in cleft lip tissues and its association with early pregnancy exposures.

AIM: To pilot investigation of methylation of long interspersed nucleotide element-1 in lip tissues from infants with nonsyndromic cleft lip, and its association with maternal periconceptional exposures. METHODS: The lateral and medial sides of the cleft lips of 23 affected infants were analyzed for long interspersed nucleotide element-1 methylation by bisulfite conversion and pyrosequencing. RESULTS: The medial side showed 1.8% higher methylation compared with the lateral side; p = 0.031, particularly in male infants (2.7% difference; p = 0.011) or when the mothers did not take folic acid during periconceptional period (2.4% difference; p = 0.011). These results were not statistically significant when Bonferroni adjustment was used. CONCLUSION: The observed differences in DNA methylation, although nonsignificant after correction for multiple comparisons, suggest that differential regulation of the two sides may impact lip fusion and warrant larger-scale replication.

Oral vitamin B1-substitution does not decrease genetically determined cleft rate in mice (A/WySn).

PURPOSE: Cleft lip and palate (CL/P) are one of the most common human birth defects. Animal experiments and clinical investigations show a clear reduction of teratogenic clefts by a high-dose vitamin B supplementation during early pregnancy, especially in families at risk (reduction of recurrence). The aim of this work was to examine the influence of thiamine (vitamin B1) on CL/P appearance in genetically determined A/WySn mice within different supplementation starting points. MATERIALS AND METHODS: A total of 24 A/WySn female mice were orally supplemented with high doses (80 mg/kg) of thiamine at different times of pregnancy (5 groups, n = 90). The influence of thiamine on the abortion rate and CL/P appearance in the offspring was analyzed with respect to the concentration of thiamine in the serum and amniotic fluid (HPLC-chromatography). Immunochemical analyses of the ThTr-1 und ThTr-2 receptor-status were performed in midface sections of A/WySn-fetuses and the corresponding placenta, with and without CL/P. RESULTS: High doses of orally supplemented thiamine did not reduce the CL/P appearance in A/WySn mice. However, the different starting points of vitamin B1 substitution had some influence. Additionally, an obvious decrease in aborted fetuses was noticed in all supplemented groups. The oral substitution caused a clear increase of the serum concentration in all mothers, but showed no increase of the amniotic fluid concentration. Then immunohistochemistry detected an overexpression of ThTr-1 in the midface and an irregular localization of ThTr-2 in the placenta of fetuses with clefts. CONCLUSION: Our results suggest a time-dependent influence of thiamine on CL/P appearance in female mice. The prophylactic/periconceptional, but not the therapeutic supplementation, starting point can be proposed as a crucial step for regular facial and palatal fusion in embryonic development. The absolute rate of CL/P was not reduced, and the concentration of the water-soluble thiamine could not increase in the amniotic fluid. Thus the proposed local effect of thiamine failed in the development of genetically determined mice.

Risk factors for orofacial clefts in India: A case-control study.

BACKGROUND: Orofacial clefts (OFC) are linked with several genetic and environmental factors. The aim of this study was to explore the association of potential risk factors with OFCs in India. METHODS: This was a hospital-based, matched case-control (1:4 ratio; matching done for parity) study conducted in Hyderabad, Bengaluru, and Delhi-National Capital Region. Cases (nonsyndromic clefts) were recruited from treatment centers, while controls (live births) were recruited from maternity centers. Information on exposures was collected during personal interviews. Exposures of interest included folic acid supplementation during the peri-conceptional period, consanguineous marriage, exposure to drugs, infections during pregnancy, family history of OFC, and dietary factors. RESULTS: A total of 785 participants were included in the study: 157 cases and 628 controls. A family history of cleft lip/palate (adjusted odds ratio [AOR], 15.48; 95% confidence interval [CI], 4.36-54.96; p value = 0.001), exclusive vegetarianism (AOR, 4.47; 95% CI, 1.83-10.98; p value = 0.001), and delayed first conception (AOR, 2.55, 95% CI, 1.25-5.21, p = 0.01) were found to be strongly associated with higher risk of OFCs. Supplementation with folic acid during first 3 months of pregnancy was not found to be protective against OFCs (AOR, 1.24; 95% CI, 0.59-2.58; p value = 0.56). CONCLUSION: Our study confirmed the importance of family history as a risk factor for OFC. Our study did not show an association with folic acid supplementation but was underpowered to detect small effects. Our finding of higher risk among vegetarians requires replication. Birth Defects Research 109:1284-1291, 2017. © 2017 The Authors. Birth Defects Research Published by Wiley Periodicals, Inc.

Genetic Interactions in Nonsyndromic Orofacial Clefts in Europe-EUROCRAN Study.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (nsCL±P) and nonsyndromic cleft palate (nsCP) are caused by a combination of genetic and environmental risk factors. We investigated gene-environment and gene-gene joint effects in a large multicenter study of case-parent triads. METHODS: The nsCL±P or nsCP triads were recruited in 11 European countries between 2001 and 2005. We collected DNA samples from infants and from their mothers and fathers, and mothers completed a questionnaire on exposures, including smoking and folic acid supplement use during pregnancy. We used log-linear regression to estimate relative risks (RRs) and 95% confidence intervals (CIs) for associations between nsCL±P or nsCP and variants in MTHFR, MTHFD1, TGFA, SATB2, and MSX1, stratifying by environmental or genetic factors. RESULTS: We obtained genotype and exposure data for 728 nsCL±P triads and 292 nsCP triads. In male infants, there was no association between the mother's homozygous MSX1 p(CA) *4/*4 genotype and nsCL±P (RR, 0.98; 95% CI, 0.63-1.54), but this maternal genotype resulted in a doubling of risk for female infants (RR, 2.21; 95% CI, 1.13-4.34). There was evidence suggestive of gene-gene joint-effects between MTHFR-TGFA for nsCP but not for nsCL±P. CONCLUSION: Although we chose the genes and their variants and putative joint effects based on associations previously reported in the literature, we replicated few associations. These results do not provide evidence supporting associations between these genes and oral clefts in European populations, although gene-environment and gene-gene interactions could play a role in oral cleft etiology.

[Association study between candidate genes on transforming growth factor-ß signaling pathway and the risk of non-syndromic cleft lip with or without cleft palate in Chinese populations].

OBJECTIVE: To explore the association between 10 candidate genes on transforming growth factor-ß (TGFB) signaling pathway and non-syndromic cleft lip with or without cleft palate (NSCL/P) among Chinese populations, and to study the gene-environment interaction. METHODS: A total of 806 Chinese Han NSCL/P trios were ascertained from an international consortium, which conducted a genome-wide association study using a case-parent trio design to investigate the genes affecting risk to NSCL/P. The transmission disequilibrium test (TDT) was used to test for effects of 343 single nucleotide polymorphisms (SNPs) in 10 genes on TGFB signaling pathway including DCN, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, BAMBI, SMAD2, SMAD3 and SMAD4. The conditional regression models were used to test for gene-environment interaction. RESULTS: For TDT, although 19 SNPs showed nominal significant association with NSCL/P, no significant evidence of association was seen for all SNPs in 806 NSCL/P trios after Bonferroni correction. The interactions between genes and maternal smoking, environmental tobacco smoke, alcohol consumption and multi-vitamin supplementation during pregnancy did not attain statistical significance after correction for multiple comparisons. CONCLUSION: No evidence for SNP effect of genes on TGFB signaling pathway and significant gene-environment interaction was seen in our data.

Evaluation of the association of polymorphisms in EYA1, environmental factors, and non-syndromic orofacial clefts in Western Han Chinese.

BACKGROUND: Non-syndromic orofacial clefts (NSOCs) are complex disease involving genetic triggers, environmental factors, and their interplay. Recent studies demonstrated that EYA1, a member of eye absent gene family, might contribute to NSOCs. METHODS: We investigated three single nucleotide polymorphisms (SNPs) and eight environmental factors (multivitamin, folic acid and calcium supplementation history, maternal alcohol consumption, common cold history, maternal smoking and environmental tobacco smoke in the first trimester, and paternal smoking in the 3 months before pregnancy) among 294 case-parent trios and 183 individual controls in western Han Chinese to evaluate the relationship between EYA1, environmental factors, and NSOCs. To be better known the gene's role in the etiology of NSOCs, we performed statistical analysis in different aspects including the linkage disequilibrium test, transmission disequilibrium test, haplotype analysis, multiple logistic regression analysis, and conditional logistic regression analysis. RESULTS: Allele C at rs3779748 showed an over-transmission in NSCL/P trios (P = 0.03), and genotype A/A at rs10094908 was under-transmitted among NSCL/P trios (P = 0.03), whereas over-transmitted among NSCPO trios (P = 0.02). The haplotype GC of rs10094908-rs3779748 was over-transmitted among NSCL/P trios (P = 0.05) and NSCPO trios (P = 0.05), respectively. Maternal common cold history, environmental tobacco smoke, and maternal alcohol consumption during the first trimester of pregnancy were risk factors for NSOCs, while calcium supplementation during the first trimester showed a protective effect. No evidence of interactions between EYA1 and environmental factors was found. CONCLUSIONS: These results revealed an association between EYA1, some environmental factors, and NSOCs in western Han Chinese.

Modeling anterior development in mice: diet as modulator of risk for neural tube defects.

Head morphogenesis is a complex process that is controlled by multiple signaling centers. The most common defects of cranial development are craniofacial defects, such as cleft lip and cleft palate, and neural tube defects, such as anencephaly and encephalocoele in humans. More than 400 genes that contribute to proper neural tube closure have been identified in experimental animals, but only very few causative gene mutations have been identified in humans, supporting the notion that environmental influences are critical. The intrauterine environment is influenced by maternal nutrition, and hence, maternal diet can modulate the risk for cranial and neural tube defects. This article reviews recent progress toward a better understanding of nutrients during pregnancy, with particular focus on mouse models for defective neural tube closure. At least four major patterns of nutrient responses are apparent, suggesting that multiple pathways are involved in the response, and likely in the underlying pathogenesis of the defects. Folic acid has been the most widely studied nutrient, and the diverse responses of the mouse models to folic acid supplementation indicate that folic acid is not universally beneficial, but that the effect is dependent on genetic configuration. If this is the case for other nutrients as well, efforts to prevent neural tube defects with nutritional supplementation may need to become more specifically targeted than previously appreciated. Mouse models are indispensable for a better understanding of nutrient-gene interactions in normal pregnancies, as well as in those affected by metabolic diseases, such as diabetes and obesity.

[Association study for gene polymorphism of folic acid/homocysteine metabolic pathway and nonsyndromic cleft lip with or without cleft palate in Chinese populations].

OBJECTIVE: To explore the association between 18 candidate genes encoding enzymes on the folate/homocysteine metabolism pathway and non-syndromic cleft lip with or without cleft palate (NSCL/P) in Chinese populations. METHODS: A total of 806 NSCL/P trios were drawn by an international consortium, which conducted a genome-wide association study using a case-parent trio design to investigate genes affecting risks to NSCL/P. The transmission disequilibrium test (TDT) was used for deviation from Mendelian expectations for 257 SNPs in 18 folate/homocysteine metabolism-related genes. The interactions between markers in these gene and environmental risk factors were also tested using conditional Logistic regressions. RESULTS: Although four SNPs (rs6428977, rs12060264, rs7730643 and rs4920037) showed nominal significant association with NSCL/P in the TDT on 806 NSCL/P trios (P<0.05), no significant evidence of linkage and association remained in all the SNPs after Bonferroni correction. Similar tests for interactions between genes and maternal smoking, environmental tobacco smoke, alcohol consumption and multi-vitamin supplementation during pregnancy did not attain statistical significance after correction for multiple comparisons. CONCLUSION: Folate/homocysteine metabolism-related genes could not influence the risk of NSCL/P.

Folic acid supplementation use and the MTHFR C677T polymorphism in orofacial clefts etiology: An individual participant data pooled-analysis.

BACKGROUND: This study examines gene-environment interaction between the MTHFR C667T polymorphism and folic acid in the etiology of orofacial clefts (OFC). We used a pooled-analytical approach on four studies that used similar methods. METHODS: We used logistic regression to analyze the pooled sample of 1149 isolated cases and 1161 controls. Fetal and maternal MTHFR C677T genotypes, and maternal periconceptional exposure to smoking, alcohol, vitamin containing folic acid and folic acid supplements were contrasted between the cleft types [non-syndromic clefts lip or without cleft palate (CL(P)) and non-syndromic cleft palate (CP)] and control groups. RESULTS: There was a reduced risk of CL(P) with maternal folic acid use (p = 0.008; OR = 0.70, 95% CI: 0.65-0.94) and with supplements containing folic acid (p = 0.028, OR = 0.80, 95% CI: 0.65-0.94). Maternal smoking increased the risk of both CL(P) (p < 10 e-3; OR = 1.62, 95% CI: 1.35-1.95) and CP (p = 0.028; OR = 1.38, 95% CI: 1.04-1.83). No significant risk was observed with either maternal or fetal MTHFR C677T genotypes. CONCLUSION: This individual participant data (IPD) meta-analysis affords greater statistical power and can help alleviate the problems associated with aggregate-level data-sharing. The result of this IPD meta-analysis is consistent with previous reports suggesting that folic acid and smoking influence OFC outcomes.

Oral folic acid supplementation decreases palate and/or lip cleft occurrence in Pug and Chihuahua puppies and elevates folic acid blood levels in pregnant bitches.

The aim of this study was to compare the frequency of the occurrence of lip and/or palate cleft (CL/CP) in new-borns of two breeds, Pugs and Chihuahuas, and to measure the folic acid blood levels in bitches during gestations both with and without folic acid oral supplementation. Bitches of 13 Pugs and 17 Chihuahuas with CL/CP cases were used in the study. In trial 1, the animals of the experimental group (n=25) were given additional folic acid from the onset of heat till the 40th day of gestation. The females of the control group (n=12) were fed a traditional diet. From all the animals blood was collected at the onset of heat, 14 days later and on the 30th day of the gestation to estimate folic acid concentration. In trial 2, the prevalence of CP/CL cases in litters from pregnancies before and after supplementation was compared. The percentage of puppies with CL/CP after supplementation decreased in both Pugs and Chihuahua puppies (10.86% and 15.78% vs. 4.76% and 4.8% respectively). On Day 0, the concentrations of folic acid were at a low physiological level (around 8 ng/ml) in all the animals. In bitches of the experimental group the blood level of folic acid on day 14th and 30th of the treatment showed an increase in both breeds (13.65 +/- 4.27 ng/ml in Pugs, 10.79 +/- 2.84 ng/ml in Chihuahuas, and 14.94 +/- 3.22 ng/ml in Pugs, 12.95 +/- 3.58 in Chihuahuas, respectively) while in the control group, this level decreased with time of gestation both in Pugs and in Chihuahuas (around 6 ng/ml). Folic acid supplementation seems to be a simple, effective preventive method to reduce the risk of CL/CP, especially in the predisposed breeds.

Application of a novel hybrid study design to explore gene-environment interactions in orofacial clefts.

Orofacial clefts are common birth defects with strong evidence for both genetic and environmental causal factors. Candidate gene studies combined with exposures known to influence the outcome provide a highly targeted approach to detecting GxE interactions. We developed a new statistical approach that combines the case-control and offspring-parent triad designs into a "hybrid design" to search for GxE interactions among 334 autosomal cleft candidate genes and maternal first-trimester exposure to smoking, alcohol, coffee, folic acid supplements, dietary folate and vitamin A. The study population comprised 425 case-parent triads of isolated clefts and 562 control-parent triads derived from a nationwide study of orofacial clefts in Norway (1996-2001). A full maximum-likelihood model was used in combination with a Wald test statistic to screen for statistically significant GxE interaction between strata of exposed and unexposed mothers. In addition, we performed pathway-based analyses on 28 detoxification genes and 21 genes involved in folic acid metabolism. With the possible exception of the T-box 4 gene (TBX4) and dietary folate interaction in isolated CPO, there was little evidence overall of GxE interaction in our data. This study is the largest to date aimed at detecting interactions between orofacial clefts candidate genes and well-established risk exposures.

Association between C677T and A1298C MTHFR gene polymorphism and nonsyndromic orofacial clefts in the Turkish population: a case-parent study.

Two common MTHFR gene polymorphisms (C677T and A1298C) have been implicated in the etiology of nonsyndromic cleft lip/palate (nsCL/P). To investigate the genotype association among nsCL/P in the Turkish population, 56 case-parent trios were recruited into the study. Genotype frequencies were compared to two groups of controls from the same population. A total of 46 case-parent trios were included in transmission disequilibrium test (TDT) analysis. The mothers of the study group had a higher frequency of 677TT genotype, with a three-fold increased risk of having nsCL/P offspring (odds ratio [OR]: 3.14, p=0.03). The combined 677CT/1298AC genotype was also common among these mothers (28%), but it did not reach statistical significance (OR: 2.27, p=0.07). TDT analysis for (C677T) T allele transmission did not reveal a significant association. In conclusion, mothers carrying 677TT genotype or with 677CT/1298AC combined genotype have increased risk of having nsCL/P offspring; therefore, higher periconceptional folic acid supplementation should be advised for decreasing the recurrence risk.