Association Study Of Environmental Interactions And Candidate Genes Of ABCA4 And MAFB With NSCL/P In A Northeast Chinese Population

Objective:To investigate the environmental factors of morbidity of non-syndromic cleft lip and palatte,analyze the degree of influence of the environmental factors;To explore the relationship of candidate gene ABCA4 loci rs481931 and rs560426 of single nucleotide polymorphisms and mutations in the pathogenesis of the non-syndromic cleft lip and palate in Northeast Han population;To explore the relationship of candidate gene MAFB loci rs17820943 of single nucleotide polymorphism and mutation in the pathogenesis of the non-syndromic cleft lip and palate in Northeast Han population;To reveal the correlationship of ABCA4,MAFB gene polymorphisms and environmental factors interaction with non-syndromic cleft lip and palate.Methods:It was a case-control study conducted in China Medical University,Hospital of Stomatology.206 hospitalized children of oral cleft lip and palate from Oral and Maxillofacial Surgery Department and 319 healthy controls from Liaoning Provincial Family Planning Research Institute took part in the questionnaire,which concerned the type of deformity,gender,urban and rural origin,parents’education level and possible risk factors.The information of the questionnaire was collected and logistic regression analysis was used to find out the environmental risk factors that affected the incidence of cleft lip and palate.5 ml peripheral blood were drawn form 210 patients and 184 healthy controls,including 98 nuclear families（children and parents）,a total of 590 blood samples.The DNAs in the blood sample were amplified by polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP）technique.The single nucleotide polymorphism analysis was carried for rs481931,rs560426,rs17820943 site.The Hardy-Weinberg equilibrium inspection was used to test whether the objects of this study were from the same group of Mendelian inheritance.Genotype and allele of the patients,their partents and control group were calculated.Genotype and allele frequencies and constitution were compared between case group and control group.The OR value and 95%confidence interval were used to express the results.FBAT was used for genetic pedigree relevance analysis of complex diseases.Logistic regression analysis of the interaction between genes and the environment was used to look for their connections.Different OR value calculated form regression equation under different exposure factors and additive model were used for qualitative judgments.While the relative excess risk of interaction（RERI）,attributable proportion due to interaction（AP）and the synergy index（S）were used for quantitative analysis,taken form the relevant formulas of relative risk interaction.In this study,all the statistical analysis was done by SPSS20.0 statistical software.And FBAT was done by FBAT5.0 software.Results:Among the 206 cases of non-syndromic cleft lip and palate patients,cleft lip had the biggest percentage with the number of 80 cases,accounting for 38.83%;followed by patients with cleft lip and palate of 76 cases,accounting for 36.90%;cleft palate only of50 cases,accounting for 24.27%.In patients with cleft lip only and cleft lip with palate,males were higher than females,male to female ratio was 1.11:1 and 2.04:1.In patients with cleft palate only,the male to female ratio was 0.79:1.Comparing the sex ratio of the three types of non-syndromic cleft lip and palate,theχ²=7.115,P value was 0.029<0.05,which showed statistical significant.Among the patients of cleft lip and palate,71 cases came from city,accounting for 34.47%.While 135 cases came from rural areas,accounting for 65.53%.Compared the sources of urban and rural areas with the control group,χ²=77.755,P<0.001,the difference had statistical significant.The cleft patients’parents with low educational background held a larger proportion of junior high school and below.The rate was 70.39%and 78.16%respectively.Mothers always had lower levels of education than fathers.Compared the parents’education background with the control group,χ_母²=64.415,χ_父²=114.181,both P<0.001,the differences had statistical significant.Mothers’age（≥35 years）of pregnancy,family history,master/passive smoking,previous birth malformations,toxic substance exposure during early pregnancy,early pregnancy sick,early pregnancy medication,the above seven risk factors were selected and taken into binary logistic regress model.Folic acid supplementation was put in multiple logistic regression analysis.This research found that four risk factors had a significant correlation（P<0.05）with the prevalence of cleft lip and palate.And the folic acid supplementation was a protective factor also had a significant correlation（P<0.05）with the prevalence of cleft lip and palate.Among all the cases of non-syndromic cleft lip and palate in this study,there were 116 males,with an average age of 11.8;and 94 females,mean age 11.3 years.The Hardy-Weinberg equilibrium test of the three loci in control groups showed p>0.05which proved the distribution of genotype and allele frequencies of this disease was in Hardy-Weinberg genetic equilibrium.The case group patients were stratified according to their malformation diagnosis.Compared with the control group,the research showed that the genotype and allele frequencies of rs481931,rs560624 had no statistical significant（P>0.05）.But the genotype frequencies of rs17820943 in cleft lip group（P=0.035）and the total number of cases group（P=0.010）had statistical significant.Case-control study of rs17820943 was separated to find out the influence of genotype.CT genotype[P=0.023,OR=0.446（0.219-0.906）]was much higher than the normal control group,which showed statistical significant.But the TT,CT+TT genotype frequencies of rs17820943 had no significant difference（p>0.05）,compared with the CC genotype.FBAT analysis showed,in C allele of rs481931,Z=2.063,P=0.039<0.05 which had statistical significant.It meant excessive transfer of C allele from parents to children and indicated the the C allele associated with the occurrence of cleft lip and palate.HBAT analysis showed that haplotype combinations was C allele（rs481931）and G allele（rs560426）,the Z=2.882,P=0.004<0.05 which showed statistical significant.The haplotype combinations of rs481931 C allele and rs560426 G allele were associated with the occurrence of cleft lip and palate.The mutant allele gene（A→C）of rs481931 of ABCA4 had negative additive model of interaction with folic acid supplementation（S=-0.763）,which showed adequate folic acid supplementation could reduce the morbidity of cleft Lip and palate caused by AC genotype.The allele mutation（C→T）of rs17820943 of MAFB was the risk factor of occurrence of cleft lip and palate.It had a positive additive model of interaction with family history（S=1.877）,and also with mothers’smoking-alcohol abuse during early pregnancy（S=1.581）.Two conditions occurred simultaneously and resulted in a synergistic effect.The family history and mothers’smoking-alcohol abuse during early pregnancy enhanced fetal risk of cleft lip and palate.For the SNP mutation of this loci,REPI _{family history}2.639>REPI _mothers’smoking_{-alcohol abuse} 1.299,it indicated that the family history had greater synergistic enhancement effect.Conclusions:1.The present study shows that parents’living environment,level of education will indirectly affect the occurrence of cleft lip and palate of their children.Family history,master/passive smoking and drinking habits in mothers’early pregnancy,illness and medication early pregnancy may increase the risk factors with cleft lip and palate;Folic acid supplements in early pregnancy can reduce the time of the occurrence of cleft lip and palate.2.CT genotype of rs17820943 allele MAFB gene is a protective factor in the incidence of cleft lip and palate,which carry the risk of people suffering from cleft lip CT genotype was 0.466 times the CC genotype.ABCA4 rs481931 sites C allele gene is passed over,and is therefore related to the occurrence of cleft lip and palate.5.Two loci rs481931 and rs560426 ABCA4 gene haplotype combinations when the C allele of rs481931 and rs560426 G allele and the incidence of cleft lip and palate related.3.MAFB gene rs17820943 site with smoking and drinking among mothers during early pregnancy,family history and a positive interaction between the sum of the risk of non-syndromic cleft lip enhancement of synergistic effects,greater enhancing effect of family history.4.ABCA4 rs481931 gene locus and folic acid supplementation had negative additive model and play antagonistic effect of interaction of the risk of non-syndromic cleft lip.