【作者】 刘鑫；

【导师】 刘珊玲；

【作者基本信息】 四川大学 ， 母婴医学， 2023， 硕士

【摘要】 目的:罗伯茨综合症(Roberts-SC Phocomelia Syndrome,RBS)是一种罕见的常染色体隐性遗传疾病,其临床特征为生长发育迟缓、身材矮小、四肢畸形等。产前生长发育迟缓的程度可以从轻微到严重不等,严重的患者可出现海豹肢畸形,甚至可能在孕期或新生儿期死亡,而轻度受影响的患者可以存活到成年。RBS由姐妹染色单体N-乙酰转移酶2(Establishment of Sisiter Chromatid Cohesion N-Acetyltransferase 2,ESCO2)基因变异所致,目前已报道的致病性变异有无义突变、错义突变、剪切突变、缺失突变和插入突变等。本研究中,临床一对夫妇两次妊娠胎儿均出现海豹肢畸形,第二次双胎妊娠,两个胎儿经家系全外显子测序发现均有ESCO2基因未经报道的复合杂合变异:c.754A>T及c.1276G>T,这两个变异均为无义突变。我们通过对这两个位点变异可能导致的蛋白结构及功能改变,以及相关致病机制进行初步研究,以期为ESCO2基因新变异位点在疾病的诊断及相关遗传咨询提供临床和实验室证据。方法:通过总结目前已发表的罗伯茨综合症患者病例,了解罗伯茨综合症患者的临床和分子特征。用Alpha Fold2在线分析软件对基因变异对蛋白可能造成的影响进行初步预测。以质粒pcDNA3.1和带荧光的质粒pIRES2-EGFP为骨架,克隆野生型ESCO2基因(pcDNA3.1-ESCO2-WT,pIRES2-EGFP-ESCO2-WT),以此为模板通过聚合酶链式反应(Polymerase Chain Reaction,PCR)介导的定点突变方法引入c.754A>T和c.1276G>T特定突变,得到4个重组质粒分别命名为pcDNA3.1-ESCO2-754,pcDNA3.1-ESCO2-1276,pIRES2-EGFP-ESCO2-754,pIRES2-EGFP-ESCO2-1276。将上述质粒分别转染入人胚胎肾细胞HEK293T进行ESCO2基因过表达,提取细胞总蛋白后通过蛋白免疫印迹实验检测c.754A>T和c.1276G>T两个变异对ESCO2蛋白表达的影响。将pIRES2-EGFP-ESCO2-WT,pIRES2-EGFP-ESCO2-754,pIRES2-EGFP-ESCO2-1276质粒转染HEK293T细胞,使用免疫荧光技术检测c.754A>T和c.1276G>T两个变异对ESCO2蛋白在细胞内表达定位的影响。将pcDNA3.1-ESCO2-WT,pcDNA3.1-ESCO2-754,pcDNA3.1-ESCO2-1276质粒转染HEK293T细胞,使用免疫荧光实验检测变异型ESCO2蛋白与增殖细胞核抗原(Proliferating Cell Nuclear Antigen,PCNA)在细胞内亚定位情况,从而分析c.754A>T和c.1276G>T两个变异可能对ESCO2蛋白功能的影响。将pcDNA3.1-ESCO2-WT和pcDNA3.1-ESCO2-1276质粒转染HEK293T细胞,提取细胞总蛋白,并在体外构建乙酰化反应体系,检测变异型ESCO2蛋白自身的赖氨酸乙酰化程度,从而判断变异型ESCO2蛋白的自乙酰化能力是否受影响。结果:1、目前已报道的罗伯茨综合症患者均由ESCO2基因变异引起,表现为生长迟缓、小头畸形、四肢畸形等症状。2、Alpha Fold2预测结果显示c.754A>T和c.1276G>T两个变异蛋白与野生型ESCO2蛋白三级结构吻合度差,提示可能影响该蛋白的生物学功能。3、蛋白免疫印迹结果显示c.754A>T变异没有出现可能的截短蛋白条带,提示变异可能导致蛋白表达下降或被降解。c.1276G>T变异出现分子量小于野生型ESCO2蛋白的条带,提示变异导致蛋白被截短。4、免疫荧光结果表明转染pIRES2-EGFP-ESCO2-754的HEK293T细胞中仅有染色后产生的微弱红色荧光信号,提示该变异可能导致ESCO2蛋白在细胞内表达下降或被降解。转染pIRES2-EGFP-ESCO2-1276质粒的HEK293T细胞中可检测出的ESCO2蛋白荧光信号,提示变异后的截短蛋白在细胞中仍可检测出表达。5、免疫荧光共定位结果显示转染pcDNA3.1-ESCO2-754、pcDNA3.1-ESCO2-1276质粒的细胞中,两种变异型ESCO2蛋白与PCNA在细胞内的共定位程度相较于转染pcDNA3.1-ESCO2-WT的细胞显著降低,提示两个变异可能影响ESCO2蛋白与PCNA的结合。6、体外乙酰化实验结果说明c.1276G>T变异型ESCO2蛋白相较于野生型ESCO2蛋白乙酰化自身赖氨酸程度降低,提示c.1276G>T变异可能导致蛋白的乙酰化能力下降。结论:ESCO2基因c.754A>T变异会可能导致ESCO2蛋白在细胞内表达下降或被降解,与野生型相比变异蛋白与PCNA在细胞内共定位程度下降,提示蛋白功能受损。c.1276G>T变异会造成ESCO2蛋白形成分子量较野生型小的截短蛋白,这段蛋白在细胞内仍然表达,但是与PCNA的共定位程度下降,体外乙酰化能力相较于野生型也下降,说明蛋白功能可能受损。结合病例随访结果,本病例夫妇选择未带有这两个变异位点的胚胎进行妊娠后,胎儿彩超未见异常。ESCO2基因c.754A>T和c.1276G>T是两个致病性无义突变,这两个位点的复合杂合突变可导致罗伯茨综合症。更多还原

【Abstract】 Objective:Roberts-SC Phocomelia Syndrome（RBS）is a rare autosomal recessive genetic disorder characterized by growth retardation,dwarfism,and limb malformations.The degree of prenatal growth retardation can range from mild to severe,with severe cases presenting with seal-like limb deformities and potential prenatal or neonatal death,while mildly affected individuals can survive into adulthood.RBS is caused by mutations in the Establishment of Sister Chromatid Cohesion N-Acetyltransferase 2（ESCO2）gene^[1,2],with reported pathogenic variations including nonsense mutations,missense mutations,splicing mutations,deletions,and insertions.In this study,a couple with two consecutive pregnancies resulting in infants with seal-like limb deformities was examined.In the second twin pregnancy,both fetuses were found to carry compound heterozygous variations in the ESCO2 gene,namely c.754A>T and c.1276G>T,both of which are nonsense mutations that have not been reported previously.We conducted preliminary research on the potential structural and functional changes caused by these mutations at the protein level and explored the underlying pathogenic mechanisms,aiming to provide clinical and laboratory evidence for the diagnosis and genetic counseling of novel ESCO2 gene variations associated with the disorder.Methods:By summarizing the currently published cases of Roberts syndrome,we aim to understand the clinical and molecular characteristics of patients with Roberts syndrome.The potential impact of gene mutations on protein structure is preliminarily predicted using the Alpha Fold2 online analysis tool.The wild-type ESCO2 gene was cloned using plasmids pcDNA3.1 and pIRES2-EGFP as backbones（named pcDNA3.1-ESCO2-WT and pIRES2-EGFP-ESCO2-WT）.Specific mutations c.754A>T and c.1276G>T were introduced using site-directed mutagenesis via polymerase chain reaction（PCR）,resulting in four recombinant plasmids named pcDNA3.1-ESCO2-754,pcDNA3.1-ESCO2-1276,pIRES2-EGFP-ESCO2-754,and pIRES2-EGFP-ESCO2-1276.These plasmids were transfected into human embryonic kidney cells（HEK293T）for ESCO2 gene overexpression.The impact of the c.754A>T and c.1276G>T mutations on ESCO2 protein expression was assessed through protein immunoblotting using total cellular protein extracts.HEK293T cells were transfected with pIRES2-EGFP-ESCO2-WT,pIRES2-EGFP-ESCO2-754,and pIRES2-EGFP-ESCO2-1276 plasmids,and immunofluorescence techniques were used to examine the effects of the two mutations on ESCO2 protein expression and localization in cells.HEK293T cells were transfected with pcDNA3.1-ESCO2-WT,pcDNA3.1-ESCO2-754,and pcDNA3.1-ESCO2-1276 plasmids,and immunofluorescence experiments were performed to analyze the subcellular localization of the mutant ESCO2 protein in relation to proliferating cell nuclear antigen（PCNA）,thereby assessing the potential impact of the c.754A>T and c.1276G>T mutations on ESCO2 protein function.HEK293T cells were transfected with pcDNA3.1-ESCO2-WT and pcDNA3.1-ESCO2-1276 plasmids,and total cellular protein was extracted for the construction of an in vitro acetylation reaction system to evaluate the acetylation level of the mutant ESCO2 protein,thereby determining if the acetylation capacity of the mutant ESCO2 protein is affected.Results:1、Roberts syndrome patients reported so far have mutations in the ESCO2 gene,exhibiting symptoms such as growth retardation,microcephaly,and limb malformations.2、Alpha Fold2 predictions indicate that the c.754A>T and c.1276G>T mutations show a lower structural similarity to the wild-type ESCO2 protein,suggesting a potential impact on the protein’s biological function.3、Protein immunoblotting results showed no corresponding protein band for the c.754A>T mutation,indicating a possible reduction in protein expression.The c.1276G>T mutation showed a protein band with a lower molecular weight than the wild-type ESCO2 protein,suggesting protein truncation due to the mutation.4、Immunofluorescence results showed only weak red fluorescence signals produced after staining in HEK293T cells transfected with pIRES2-EGFP-ESCO2-754,indicating that the mutation may lead to decreased expression of ESCO2 protein in cells.ESCO2 protein fluorescence signals could be detected in HEK293T cells transfected with pIRES2-EGFP-ESCO2-1276 plasmid,suggesting that the truncated protein after mutation can still be detected in cells.5、The immunofluorescence co-localization results showed that the co-localization of the two mutant ESCO2 proteins,pcDNA3.1-ESCO2-754 and pcDNA3.1-ESCO2-1276,with PCNA in the transfected cells was significantly reduced compared to cells transfected with pcDNA3.1-ESCO2-WT.This suggests that the two mutations may affect the binding between ESCO2 protein and PCNA.6、Results from in vitro acetylation experiments showed that the acetylation level of the c.1276G>T mutant ESCO2 protein on its own lysine residues was lower compared to the wild-type ESCO2 protein,indicating that the c.1276G>T mutation may lead to decreased acetylation ability of the protein.Conclusion:The c.754A>T mutation in the ESCO2 gene may lead to a decrease in ESCO2 protein expression or degradation within the cells.Compared to the wild-type,the mutated protein shows a decreased co-localization with PCNA in the cells,suggesting impaired protein function.The c.1276G>T mutation results in the formation of a truncated ESCO2 protein with a smaller molecular weight compared to the wild-type.Although this truncated protein is still expressed within the cells,its co-localization with PCNA is reduced,and its acetylation ability is decreased compared to the wild-type,indicating potential functional impairment.Based on follow-up results of the case,the couple chose to proceed with pregnancy using embryos that did not carry these two variant sites,and fetal ultrasound did not reveal any abnormalities.The c.754A>T and c.1276G>T mutations in the ESCO2 gene are two pathogenic nonsense mutations.The compound heterozygous mutations at these two sites can result in Roberts syndrome.更多还原