【作者】 傅夏洲；

【导师】 程汉华； 周荣家；

【作者基本信息】 武汉大学 ， 细胞生物学， 2014， 博士

【摘要】 唇腭裂是一种高发病率的先天性遗传疾病,在胚胎早期发育的过程中,颌面部正常结构的形成异常所致。唇腭裂通常分为综合征型唇腭裂和非综合征型唇腭裂两类,综合征型唇腭裂大多属于单基因遗传疾病,非综合征型唇腭裂属于多基因遗传疾病,并与环境因素的影响有关。最新的研究发现8q24.21区域在欧洲和美洲人群中与非综合征型唇腭裂有紧密的连锁关系,但在亚洲人群中这种关联并不明显。目前尚未报道这一区域与唇腭裂相关的致病基因。8q24.21区域在中国中部人群中是否也存在与非综合征型唇腭裂的紧密连锁关系?这一区域是否存在潜在的唇腭裂致病基因?本研究对这些科学问题进行了研究。1.中国中部人群的非综合征型唇腭裂与8q24.21区域存在紧密连锁以中国中部人群的三代以上大家系,两代核心家系以及散发病例的唇腭裂样本为研究对象,依据HapMap人种异质性数据选择了8q24.21区域中适合中国人群的新的SNP位点,并在210例唇腭裂患者和234例正常对照组中进行SNP位点分型分析。发现在8q24.21区域存在两个新的SNP位点rs17241323和rs12546523与唇腭裂存在紧密连锁关系。SNP位点rs17241323的C等位基因与唇腭裂存在连锁关系,P=0.02285,0.01<P=0.02285<0.05,0R(95%CI)=1.450(1.38-1.53),统计学分析显著差异；基因型C/C也与唇腭裂存在连锁关系,P=0.006934<0.01,0R(95%CI)=1.682(1.56～1.81),统计学分析极显著差异。SNP位点rs12546523的G等位基因与唇腭裂存在连锁关系,P=0.01329,0.01<P=0.01329<0.05,0R(95%CI)=1.526(1.44～1.62),统计学分析显著差异；基因型G/G也与唇腭裂存在连锁关系, P=0.004261<0.01,OR(95%CI)=1.752(1.62～1.89),统计学分析极显著差异。因此,非综合征型唇腭裂在中国中部人群中与8q24.21区域存在紧密连锁关系。2. EST BF691581与单纯腭裂的发生相关为了探寻8q24.21区域是否存在潜在的唇腭裂致病基因,我们用GENESCAN软件对SNP位点rs17241323和rs12546523附近以及欧美人群中报道的与唇腭裂紧密连锁程度最高的SNP位点rs987525、rsl7241253和rs1530300附近的基因组序列进行扫描,没有发现可能的编码基因。然后我们对这些区域的EST序列在不同组织和细胞系中进行半定量分析,结果显示BF573425和BF691581在唇部和腭部组织有表达,且BF691581的表达量较高。通过Northern blot的方法鉴定了BF691581分子的大小,并且结合RACE分析和cDNA文库菌落原位杂交的方法获得了BF691581EST序列。荧光定量PCR结果提示BF691581在单纯腭裂患者中表达量较高,组织原位杂交实验表明BF691581表达在腭板的外层和口腔侧的腭上皮组织,表达部位与一些已知的唇腭裂候选基因如成纤维生长因9(FGF9),声猥蛋白(SHH)以及干扰素调节因子6(IRF6)相似,提示BF691581可能在胚胎发育早期促进腭板中缝融合的过程中发挥作用。从细胞水平分析,BF691581表达于基底层细胞中；基底层细胞是唯一可以分裂的表皮细胞,可能与颅面部的早期形态发育有关。3.唇腭裂候选基因TBX225’端调控区域一个新的功能性突变构建了19个已知的唇腭裂易感基因的启动子的荧光素酶表达载体,并选择5个唇腭裂易感基因IRF6,TBX22,MSX1,BMP4以及FGF8鉴定其核心启动子。在7个三代以上大家系和22个两代核心家系样本中对这5个唇腭裂候选基因的核心启动子区域进行测序分析,在一个三代家系中发现TBX22的转录起始位点上游-73bp处存在一个G→A点突变；在一个核心家系中发现IRF6转录起始位点上游-32bp处存在一个A→C点突变。其中,TBX22转录起始位点上游-73bp的G→A点突变是一个有功能的点突变。通过生物信息学软件预测,这个G→A点突变位于转录因子c-ETS-1的结合序列上；荧光素酶报告基因分析,TBX22转录起始位点上游-73bp的G→A点突变可以使TBX22核心启动子的转录起始活性显著下降约三分之一：而TBX22的低表达曾被证实是导致X连锁腭裂综合征(CPX)的主要原因。因此,提示这个在一个三代单纯腭裂家系中发现的新的点突变可能是一个有功能的点突变。更多还原

【Abstract】 Cleft lip with or without cleft palate (CLP) is a common birth defect with complex aetiology, which is recognized as disruptions of normal facial structure. CLP affects approximately1in700live births, with wide variability across geographic origin, race and environment. CLP has been divided into nonsyndromic cleft lip with or without cleft palate (NSCLP) and syndromic cleft lip with or without cleft palate. Syndromic cleft lip with or without cleft palate is mostly caused by the single susceptibility gene, whereas the nonsyndromic cleft lip with or without cleft palate has a complex aetiology with both genetic and environmental contributions.The latest research has identified a new region on chromosome8q24.21in human that gave extremely strong evidence of association in their European and American case-control samples. However, this association is not evident in the Asia case-control samples (Philippines). No any candidate gene of cleft lip with or without palate has been identified in this8q24.21region. It remains to be studied that whether there is association between cleft lip with or without palate and the8q24.21region in the central Chinese population, and whether there is candidate gene in this8q24.21region of cleft lip with or without palate.1. There is strong evidence of association between8q24.21region SNPs and nonsyndromic cleft lip with or without cleft palate (NSCLP) in the central Chinese population.Eight nonsyndromic cleft lip with or without cleft palate (NSCLP) families of more than three generations, twenty two core nonsyndromic cleft lip with or without cleft palate (NSCLP) families of two generations and some separate case were screened.210cases and234controls were genotyped by the SNPs chosen from HapMap. Two new SNPs, rs17241323and rs12546523in the8q24.21region were associated with nonsyndromic cleft lip with or without cleft palate (NSCLP) in the central Chinese population. Linkage disequilibrium showed that the C allele of rs17241323was associated with nonsyndromic cleft lip with or without cleft palate (NSCLP). P=0.02285,0.01<P=0.02285<0.05, OR(95%CI)=1.450(1.38～1.53); C/C heterozygous genotype P=0.006934<0.01, OR(95%CI)=1.682(1.56～1.81). The G allele of rs12546523was also associated with nonsyndromic cleft lip with or without cleft palate (NSCLP), P=0.01329,0.01<P=0.01329<0.05OR(95%CI)=1.526(1.44-1.62); G/G heterozygous genotype P=0.004261<0.01, OR(95%CI)=1.752(1.62-1.89). These results showed that there is an association between8q24.21region SNPs and nonsyndromic cleft lip with or without cleft palate (NSCLP) in the central Chinese population..2. The EST BF691581is associated with cleft palate only.To investigate the candidate genes in the8q24.21region, the genome sequence near the SNPs were screened in the centre Chinese population for rs17241323, rs12546523, rs987525. rs17241253and rsl530300by the GENESCAN software. None coding candidate genes were observed in this region. RT-PCR was used to investigate expression of ESTs in the8q24.21region in adult tissues and cell lines. The ESTs BF573425and BF691581were expressed in lip and palate tissues. Northern blot and rapid amplification of cDNA ends (RACE) were used to determine length of BF691581. The real time fluorescence quantitative PCR (Q-PCR) showed that the BF691581was highly expressed in the cleft palate only (CPO) cases. In situ hybridization showed that BF691581was expressed in the epidermal basal layer cells, which were the exclusive epidermal cells, in the palatal outer shelves and the palate epithelium of middle oral. The expressive region of the BF691581was similar with the candidate genes, fibroblast growth factor9(FGF9), interferon regulatory factor6(IRF6) and sonic hedgehog (SHH). These results suggested that BF691581may be associated with the palate morphogenesis.3. A new functional mutation in the TBX22core promoter is associated with cleft palate only.Nineteen candidate gene promoters of non-syndromic cleft lip with or without cleft palate (NSCLP) including IRF6.TBX22,MSX1,BMP4and FGF8were investigated for possible mutations. The core promoters of these candidate genes in7nonsyndromic cleft lip with or without cleft palate (NSCLP) families of more than three generations.22core nonsyndromic cleft lip with or without cleft palate (NSCLP) families of two generations and some separate case, totally210cases and234controls were screened. A G→A mutation at the-73bp upstream of TBX22transcription start site (TSS) in a3generation family, and a A→C mutation at the-32bp upstream of IRF6transcription start site (TSS) in a2generation core family were identified. The G→A mutation at the-73bp upstream of TBX22transcription start site (TSS) was a functional mutation. Important transcription factors and their binding sites in the core promoter region were tested to elucidate possible mechanisms of TBX22gene in the palate development. A potential binding site of transcriptional factor c-ETS-1in the G→A mutation was observed. The luciferase reporter assays showed this G→A mutation at the-73bp upstream of TBX22resulted in a decreased activity of up to40%. These results showed that the new functional mutation of the TBX22gene promoter may be associated with the cleft palate only.更多还原