VANGL基因和DACT1基因与神经管畸形的相关性研究

【作者】 雷云平；

【导师】 金力； 王红艳；

【作者基本信息】 复旦大学 ， 遗传学， 2010， 博士

【摘要】 神经管畸形是由于胚胎发育早期神经管不闭合或闭合不全所造成的一组严重出生缺陷,包括头部未闭合产生的无脑儿、脑膨出、颅脊柱裂和尾部未闭合产生的脊柱裂等。前者因头部神经管不闭合,表现为严重脑发育不全、颅骨损伤,一般在出生前或出生后一小段时间内死亡,而很多脊柱裂患儿可以存活,却造成终身残疾和生活质量低下,给社会、家庭和个人带来沉重的精神压力和经济负担。近年来越来越多的神经管畸形小鼠模型研究表明平面细胞极化信号通路中的基因对神经管的闭合起着重要作用,而Vangl2就是该通路中第一个被定位克隆的神经管畸形相关基因。本文采用病例-对照的研究方法,结合酵母双杂交和双荧光素酶报告基因系统的功能试验,对VANGL基因(包括VANGL2, VANGL1)和DACT1基因与人类神经管畸形发生的相关性进行研究。在对神经管畸形VANGL2基因编码区的突变筛查中,我们在头部相关的神经管畸形患者中发现了三个神经管畸形特有的VANGL2错义突变位点：S84F,R353C,F437S。酵母双杂交实验显示：R353C减弱了VANGL2与Dishevelleds原有的相互作用,而F437S则使VANGL2完全丧失了与Dishevelleds互作的能力。这些实验数据表明VANGL2基因突变与头部神经管畸形的发生存在相关性。VANGL2基因启动子区多态性与神经管畸形发生的相关性研究迄今尚无文献报道。本文通过对VANGL2基因启动子区测序,检测到11个变异位点,其中三个位点(SNP7:-992T>G,SNP8: -545T>G, SNP9: 42G>C)低频等位基因频率(MAF)在10%以上。在关联分析中,我们发现SNP9与颅脊柱裂相关(p=0.03),低频等位基因(42C)呈现出保护效应(OR=0.49,95%CI：0.21-0.96),这种相关性在显性模型下更为显著(p=0.019)。用双荧光素酶报告基因系统检测该位点的功能,发现低频等位基因(42C)的转录活性为高频等位基因(42G)的1.4倍,差异显著(p=0.002)。VANGL2启动子区的关联和功能分析也表明VANGL2基因与头部的神经管畸形相关,与我们编码区的研究结果一致。采用同一个体不同组织对VANGL2父源和母源等位基因表达差异进行研究发现,VANGL2基因存在等位基因表达不平衡现象。在神经管畸形患者的病理组织中,错义突变的等位基因VANGL2是高表达的,而非病理组织中,优势表达的是野生型的等位基因VANGL2该基因的不平衡表达的现象在健康人群的肝脏组织中被进一步证实。并且,研究发现这种不平衡表达现象与启动子区的甲基化有一定的相关关系,优势表达的等位基因与低的甲基化状态相对应,但是这种对应关系并不显著(p=0.313)。VANGL1基因突变与人类脊柱裂等尾部神经管畸形的相关关系已经明确,但是VANGL1基因是否与人类的头部神经管畸形相关并不清楚。我们通过对100余例头部相关神经管畸形患者的测序检测,发现了两个头部神经管畸形相关的VANGL1错义突变(G39S,N313S)。酵母双杂交实验显示,N313S减弱了VANGL1与Dishevelled1和Dishevelled2之间的相互作用。这些结果表明VANGL1基因与人类头部神经管畸形的发生也存在一定的相关性。小鼠模型中,Dactl基因纯合突变体中约有20%表现出脊柱裂的神经管畸形,并且Dact1基因纯合突变体能与Vangl2突变杂合子在表型上互相补救,说明两者存在遗传相互作用。本文首次尝试在神经管畸形中检测DACT1的基因突变。在163例神经管畸形患者中,我们一共检测到了5个错义突变位点,这些错义突变位点全部发生在女性中,以杂合子形式存在。其中三个位点(R45W,D142G,N356K)在进化上十分保守,突变后的氨基酸残基改变了原有氨基酸残基的性质,并且三个突变的携带者在临床表型上极为相似,都表现为无脑儿,枕颈脊柱裂。在正常胎儿女性对照组测序中,未发现DACT1的错义突变；而在正常成年女性对照组中检测到的三个错义突变位点进化上并不保守。这些数据表明DACT1与女性神经管畸形相关。更多还原

【Abstract】 Neural tube defects (NTDs) are caused by a partial or complete failure of the neural tube to close during embryogenesis at any level of the rostrocaudal axis. They include anencephaly, craniorachischisis, encephalocele, iniencephaly and spina bifida that result from the failure of fusion in the cranial and spinal region of the neural tube, respectively. All infants with anencephaly are stillborn or die shortly after birth, whereas many infants with spina bifida now survive but with severe and life-long physical and developmental disabilities and are at risk for psychosocial maladjustment. In addition to the emotional cost of spina bifida, the estimated monetary cost is also staggering.Recently, more and more mouse models of neural-tube defects indicated that nearly all of the genes involved in planar cell polarity (PCP) pathway play an important role in neural tube closure. Among them, VANGL2 is the first reported gene associated with craniorachisis, a serve craninal neural-tube defects. However, no research groups nowadays succeeded to find VANGL2 mutations in human neural-tube defects.This study aimed to investigate whether mutations and polymorphisms in VANGL2, VANGL1, DACT1 are associated with human neural tube defects. We conducted a case-control study in the subjects containing a large number of cranial neural-tube defects, such as anencephaly, craniorachischisis and encephalocele. This study also evaluated the functions of missense mutations and polymorphisms in the promoter by yeast two hybrdization assay (Y2H) and dual-luciferase assay, respectively.Three NTD-specific missense mutations (i. e. S84F, R353C, F437S) in VANGL2 gene were detected in three patients with cranial neural tube defects, respectively. Y2H assays showed that R353C diminished the physical interactions between VANGL2 and Dishevelleds, and F437S abrogated these interactions. These data indicated VANGL2 was associated with human cranial neural tube defects.Eleven variants in VANGL2 promoter were detected in this study. Of them, the major allele frequencies (MAFs) of three (i. e. SNP7:-992T>G, SNP8:-545T>G, SNP9:42G>C) are higher than 10%. Case-control association analysis indicated that the minor allele (42C) of SNP9 was significantly associated with decreased risk for craniorachischisis (OR=0.49,95%CI: 0.21-0.96, p=0.03). Dual-luciferase assays indicated that the minor allele (42C) had a higher transcription activity (1.4 fold of the major allele) (p=0.002)Using cDNA from different tissues of a same individual, we found that the paternal and maternal VANGL2 expression levels were different at the same tissue. The mutated allele (VANGL2c.1057C) was predominant expressed in the pathogenic tissue and the wild-type allele (VANGL2 c.1057T) was predominant in the normal tissue. These allele imbalance expressions were relation to the promoter methylation status. Higher expression level of VANGL2 was correlated with Lower promoter methylation status, but this relation was nonsignificant (p=0.313).Although the associations between VANGL1 mutations and human spinal bifida have already been reported by others, the relationship between VANGL1 mutation and cranial neural-tube defects is still unknown. In this study, two cranial NTD-specific missense mutations (i.e. G39S, N313S) were detected in VANGL1. Y2H assays showed that N313S diminished the interaction between VANGL1 and Dishevelledl and Dishevelled2. These results suggested that VANLG1 was associated with human cranial NTDs.In mouse, about 20% of Dactl mutation homozygouses had spina bifida, and heterozygous mutation of Vangl2rescued recessive Dactl phenotypes, whereas loss of Dactl reciprocally rescued semidominant Vangl2 phenotypes, which is interesting. To date, no study reported that DACT1 was associated with human birth defects. In this study, we sequenced 163 NTDs and 480 controls, and detected five NTD-specific missense mutations. Multiple sequence alignment analysis suggested three of them (i. e. R45W, D142G, N356K) were conserved in evolution. And all the conserved mutations extremely changed the property of the wildtype amino acid residue. It was surprised that all the five mutations were uniformly found in female cases and were completely absent in all of female fetus controls. Although three missense mutations were detected in 192 control women, none of them was conserved through the protein blast analysis. These data demonstrated that DACT1 might be associated with female neural-tube defects.更多还原