【作者】 张玉；

【导师】 杜玉开；

【作者基本信息】 华中科技大学 ， 儿少卫生与妇幼保健学， 2013， 博士

【摘要】 目的：1.调查了解非综合征型唇腭裂发生的危险因素，分析各因素的危险程度；2.探索TGFα基因TaqI、BamHI和RsaI单核苷酸多态性位点突变与非综合征型唇腭裂易感性的关系；3.探索Wnt3基因rs142167(A/G)和rs3809857(G/T)单核苷酸多态性位点突变与非综合征型唇腭裂易感性的关系；4.揭示TGFα、Wnt3基因多态性和环境因素的交互作用与非综合征型唇腭裂易感性的关联性。方法：本研究采用1:1配对病例对照研究的方法，对广西柳州地区178例确诊的非综合征型唇腭裂患儿和178例正常对照儿童进行了非综合征型唇腭裂环境影响因素的流行病学调查，并采集病例组与对照组的外周静脉血2ml，提取DNA，应用限制性片段长度多态性聚合酶链反应(PCR-RFLP)技术对病例组与对照组TGFα基因TaqI、BamHI、RsaI位点以及Wnt3基因rs142167、rs3809857位点进行基因型分布频率的检测，分析了其多态性与非综合征型唇腭裂易感性的关系。采用配对资料χ2检验和单因素及多因素条件logistic回归分析筛选出与非综合征型唇腭裂易感性有关的环境因素及基因型，并分析其作用强度。应用相加模型分析基因与环境危险因素之前可能存在的交互作用对非综合征型唇腭裂易感性的影响。所有资料均使用SPSS13.0进行统计分析，以P＜0.05判定为差异有统计学意义。结果：1.本次研究共调查非综合征型唇腭裂儿童与正常健康对照儿童178对，其中男孩121对，女孩57对。非综合征型唇腭裂患儿中单纯唇裂53例，唇裂合并腭裂79例，单纯腭裂46例，单纯唇裂：唇裂合并腭裂：单纯腭裂=1：1.49：0.87。2.环境暴露因素分析多因素条件logistic回归分析结果显示：居住在农村(OR=2.35，95%CI=1.10-5.05)、母亲孕前6个月或孕早期接触宠物(OR=4.46，95%CI=1.46-13.66)、母亲孕前6个月或孕早期被动吸烟(OR=2.20，95%CI=1.28-3.77)、先兆流产(OR=20.84，95%CI=1.42-304.85)、母亲慢性病史(OR=31.84，95%CI=3.20-316.60)、母亲出生缺陷家族史(OR=11.14，95%CI=1.30-95.75)、父亲职业有害物理因素接触史(OR=4.62，95%CI=1.10-19.47)是非综合征型唇腭裂易感性的危险因素，而母亲孕前6个月至孕早期补充叶酸(OR=0.30，95%CI=0.10-0.91)、父亲文化程度高(OR=0.14，95%CI=0.03-0.62)是非综合征型唇腭裂易感性的保护因素。3.基因多态性分析3.1TGFα基因TaqI位点多态性与非综合征型唇腭裂易感性有统计学关联，携带C2等位基因的个体患非综合征型唇腭裂的风险是非携带者的1.82倍(OR=1.82，95%CI=1.13-2.91，P=0.013)；基因型为C1C2或C2C2的个体患NSCL/P的风险增加，是C1C1基因型个体的1.84倍(ORC1C2+C2C2vs C1C1=1.84，95%CI=1.10-3.10，P=0.020)。3.2TGFα基因BamHI位点多态性与非综合征型唇腭裂易感性有统计学关联，携带A1等位基因的个体其患非综合征型唇腭裂的风险是非携带者的2倍(OR=2.00，95%CI=1.30-3.06，P=0.002)；基因型为A1A1或A1A2的个体患非综合征型唇腭裂的风险是A2A2基因型个体的2.19倍(ORA1A1+A1A2vs A2A2=2.19，95%CI=1.37-3.50，P=0.001)。3.3TGFα基因RsaI位点多态性与非综合征型唇腭裂易感性有统计学关联，尽管携带B1突变等位基因的个体患NSCL/P的风险并未见增加(OR=1.38，95%CI=0.89-2.14，P=0.149)，但在隐性遗传模式下，B1B1基因型个体发生非综合征型唇腭裂的风险是B1B2+B2B2基因型的3.42倍(ORB1B1vs B1B2+B2B2=3.42，95%CI=1.29-9.06，P=0.014)。3.4Wnt3基因rs142167(A/G)位点多态性与非综合征型唇腭裂易感性有统计学关联，携带G等位基因的个体患非综合征型唇腭裂的风险是非携带者的1.65倍(OR=1.65，95%CI=1.15-2.34，P=0.006)，在隐性遗传模式下，携带GG基因型个体NSCL/P易感性是AG+AA基因型个体的2.64倍(ORGG vs AG+AA=2.64，95%CI=1.03-6.76，P=0.043)。3.5Wnt3基因rs3809857(A/G)位点多态性未见与非综合征型唇腭裂易感性存在统计学关联。4.基因-环境交互作用分析4.1TGFα基因TaqI多态性与孕母在孕前6个月至孕早期被动吸烟、孕前6个月至孕早期经常饮酒、先兆流产、母亲出生缺陷家族史及父亲职业有害物理因素接触史呈现正相加模型交互作用。TGFα BamHI多态性与与孕前或孕早期经常接触宠物、孕前6个月至孕早期不补充叶酸、父亲职业有害物理因素接触史存在正相加模型交互作用。TGFα RsaI多态性与孕前6个月至孕早期经常饮酒、先兆流产、父亲职业有害物理因素接触史存在正相加模型交互作用。4.2Wnt3基因rs142167多态性与孕前6个月至孕早期不补充叶酸、孕前6个月至孕早期被动吸烟、孕前6个月至孕早期经常饮酒、先兆流产和母亲出生缺陷家族史存在正相加模型交互作用。rs3809857位点多态性与孕前或孕早期经常接触宠物、孕前6个月至孕早期不补充叶酸、孕前6个月至孕早期被动吸烟、先兆流产和父亲职业有害物理因素接触史存在正相加模型交互作用。结论：1.居住在农村、母亲孕前6个月或孕早期接触宠物、母亲孕前6个月或孕早期被动吸烟、先兆流产、母亲慢性病史、母亲出生缺陷家族、父亲职业有害物理因素接触史是非综合征型唇腭裂易感性的危险因素，而母亲孕前6个月至孕早期补充叶酸、父亲文化程度高是非综合征型唇腭裂易感性的保护因素。2. TGFα基因TaqI、BamHI、RsaI位点及Wnt3基因rs142167(A/G)位点多态性与广西地区非综合征型唇腭裂的发生有关联；Wnt3基因rs3809857(A/G)位点未能发现与非综合征型唇腭裂易感性有关。3. TGFα基因的TaqI、BamHI与RsaI三个常见位点突变与孕母在孕前6个月至孕早期被动吸烟、孕前6个月至孕早期经常饮酒、孕前或孕早期经常接触宠物、母亲出生缺陷家族史及父亲职业有害物理因素接触史等对非综合征型唇腭裂的发生在不同程度上存在正相加模型交互作用。4. Wnt3基因rs142167与rs3809857位点突变与孕前6个月至孕早期不补充叶酸、孕前6个月至孕早期被动吸烟、孕前6个月至孕早期经常饮酒、先兆流产和母亲出生缺陷家族史、父亲职业有害物理因素接触史等对非综合征型唇腭裂的发生在不同程度上存在正相加模型交互作用。5.非综合征型唇腭裂是由遗传因素与环境因素共同作用的结果，研究它们之间的相互关系对阐明非综合征型唇腭裂的危险因素及发病机理有重要意义。更多还原

【Abstract】 Objectives:1. To explore the risk factors of susceptibility of non-syndromic cleft lipwith or without cleft palate (NSCL/P) in Liuzhou City, Guangxi.2. To explore the variable contribution of the TaqI、BamHI and RsaI polymorphisms inTGFα gene to NSCL/P.3. To explore the variable contribution of rs142167(A/G) and rs3809857(G/T)polymorphisms in Wnt3gene to NSCL/P.4. To assess the gene-environment interaction on risk of NSCL/P.Methods: A matched case-control study was performed in this study. The studypopulation consisted of178cases with NSCL/P and178controls without any birth defectsborn in Guangxi between2007and2012. A questionnaire survey was carried out to detectthe information on maternal and paternal exposure before and during pregnancy. Thesamples of DNA were extracted from the peripheral blood of all children. The Genotypes ofTaqI, BamHI, RsaI in TGFα gene and rs142167, rs3809857in Wnt3gene of178pairssubjects were determined by polymerase chain reaction-based restriction fragment lengthpolymorphism (PCR-RFLP). McNemar test, univariate and multiple conditional logisticregression models were used to explore the association between environmental factors,different genotypes and the risk of NSCL/P. Additionally, the gene-environment interactionwas assessed by conditional logistic regression models. The odds ratio values (ORs) was calculated by using regression model to determine the addition effects among differentfactors and measure the interaction. All data analyses were performed using the statisticalsoftware package SPSS13.0(SPSS, Chicago, IL). A2-tailed P value <0.05was consideredstatistically significant.Results:1. In total,178pairs subjects with NSCL/P children as case and healthychildren as control were enrolled in this study. Among them,121pairs were boys and57pairs were girls. The cleft subtype ratio was1:1.49for cleft lip only with cleft lip and palate,and1:0.87for cleft lip only with cleft palate only.2. Risk factors in environmentThe results of multiple conditional logistic regression analysis indicated that living inrural area(OR:2.35;95%CI:1.10,5.05), keeping pets before six months to the early stagesof pregnancy (OR:4.46;95%CI:1.46,13.66), passive smoking before six months to theearly stages of pregnancy (OR:2.20;95%CI:1.28,3.77), threatened abortion (OR:20.84;95%CI:1.42,304.85), chronic disease history of mother (OR:31.84;95%CI:3.20,316.60), family history of birth defects (OR:11.14;95%CI:1.30,95.75) and father’soccupational exposure to physical adverse factors of father (OR:4.62;95%CI:1.10,19.47)were risk environment factors of NSCL/P. However, if supplement folic acid before sixmonths to the early stages of pregnancy (OR:0.30;95%CI:0.10,0.91) and the highereducational level of father (OR:0.14;95%CI:0.03,0.62), the risk of NSCL/P of theiroffsprings would decrease.3. Gene polymorphisms3.1The results suggested that TGFα gene TaqI polymorphism could be used as agenetic susceptibility marker for NSCL/P. Infants carrying the rarer C2allele showed a1.82-fold increase in risk for NSCL/P (OR:1.82;95%CI:1.13,2.91, P=0.013). C1C2orC2C2genotype was significantly associated with increased risk of NSCL/P compared withC1C1genotype (ORC1C2+C2C2vs C1C1:1.84;95%CI:1.10,3.10, P=0.020).3.2The results suggested that TGFα gene BamHI polymorphism could be used as a genetic susceptibility marker for NSCL/P. Infants carrying the rarer A1allele showed atwo-fold increase in risk for NSCL/P (OR:2.00;95%CI:1.30,3.06, P=0.002). A1A2orA1A1genotype was significantly associated with increased risk of NSCL/P compared withA2A2genotype (ORA1A1+A1A2vs A2A2:2.19;95%CI:1.37,3.50, P=0.001).3.3The results suggested that TGFα gene RsaI polymorphism could be used as agenetic susceptibility marker for NSCL/P. No association was found between the rarer A1allele and risk for NSCL/P in our data (OR:1.38;95%CI:0.89,2.14, P=0.149).Nevertheless, under a recessive genetic model, B1B1genotype was significantly associatedwith increased risk of NSCL/P compared with B1B2+B2B2genotype (ORB1B1vs B1B2+B2B2:3.42,95%CI:1.29,9.06, P=0.014).3.4The results suggested that Wnt3gene rs142167polymorphism could be used as agenetic susceptibility marker for NSCL/P. Infants carrying the rarer G allele showed a1.65-fold increase in risk for NSCL/P (OR:1.65;95%CI:1.15,2.34, P=0.006). Under arecessive genetic model, GG genotype was significantly associated with increased risk ofNSCL/P compared with AG or AA genotype (ORGG vs AG+AA:2.64;95%CI:1.03,6.76,P=0.043).3.5No significant association was detected when cases and controls were comparedwith the genotype for Wnt3gene rs3809857polymorphism in risk of NSCL/P.4. Gene-environment interaction between TGFα, Wnt3gene polymorphisms andenvironmental factors4.1The positive additive interactions were found between TGFα gene TaqIpolymorphism and passive smoking and alcohol use before six months to the early stages ofpregnancy, threatened abortion, family history of birth defects and father’s occupationalexposure to physical adverse factors. Similarly, a positive additive interaction was foundbetween TGFα gene BamHI polymorphism and keeping pets and no folic acid supplementbefore six months to the early stages of pregnancy and father’s occupational exposure tophysical adverse factors. Aslo, a positive additive interaction was found between TGFα gene RsaI polymorphism and alcohol use before six months to the early stages of pregnancy,threatened abortion and father’s occupational exposure to physical adverse factors.4.2The positive additive interactions were found between Wnt3gene rs142167polymorphism and no folic acid supplement before six months to the early stages ofpregnancy, alcohol use before six months to the early stages of pregnancy, threatenedabortion, family history of birth defects of mother and father’s occupational exposure tophysical adverse factors. Similarly, the positive additive interactions were found betweenWnt3gene rs3809857polymorphism and keeping pets, no folic acid supplement, passivesmoking before six months to the early stages of pregnancy, threatened abortion andfather’s occupational exposure to physical adverse factors.Conclusions:1. Our findings demonstrated that living in rural area, keeping petsbefore six months to the early stages of pregnancy, passive smoking before six months tothe early stages of pregnancy, threatened abortion, chronic disease history of mother, familyhistory of birth defects and father’s occupational exposure to physical adverse factors offather were risk environment factors of NSCL/P. However, supplement folic acid before sixmonths to the early stages of pregnancy and the higher educational level of father wereprotective factors of NSCL/P.2. Our findings support the contribution of TGFα gene TaqI, BamHI, RsaIpolymorphism and Wnt3gene rs142167polymorphism in the etiology of NSCL/P in thepopulation of Liuzhou City, Guangxi. And no association was found between the Wnt3gene rs3809857polymorphism and the risk of NSCL/P in our data. But these resultswarrant further investigation.3. There are obvious interactions between TGFα gene TaqI, BamHI and RsaIpolymorphisms and environmental factors such as passive smoking, alcohol use andkeeping pets before six months to the early stages of pregnancy, family history of birthdefects and father’s occupational exposure to physical adverse factors.4. There are obvious interactions between Wnt3gene rs142167and rs3809857 polymorphisms and environmental factors such as no folic acid supplement, passivesmoking, alcohol use, and keeping pets before six months to the early stages of pregnancy,threatened abortion, family history of birth defects and father’s occupational exposure tophysical adverse factors.5. NSCL/P is caused by the interactions between many minor genes andenvironmental risk factors. It is very important to study their correlation to classify the riskfactors and pathogenesis of NSCL/P.更多还原