全基因组测序鉴定3对单卵双胞胎中眶距增宽症致病基因突变及其验证研究

Whole genome sequencing for the identification and verification of causative genes involved in orbital hypertelorism patients—3 monozygotic twins

目的在3对表型存在差异(眶距增宽/眶距正常)的单卵双胞胎中鉴定面裂相关眶距增宽症的致病基因突变并进行验证和机制研究。方法纳入2014年5月至2019年5月就诊于上海交通大学医学院附属第九人民医院的3对单卵双胞胎,男2例,女4例,年龄5~18岁,双胞胎中均为1例眶距增宽症患者,1例眶距正常者,且眶距增宽均为面裂所致。对3对双胞胎进行全基因组测序,筛选眶距增宽症的突变基因。纳入该院同期33例面裂相关眶距增宽症患者和50例健康人作为验证样本,采用Sanger法进行外显子测序,验证全基因组测序筛选出的致病基因。在整形外科手术中获取患者和健康人颅面部骨膜组织,进行细胞培养,测定两组的碱性磷酸酶(ALP)活性,并进行茜素红染色鉴定细胞成骨分化情况;分别采用实时定量PCR和蛋白质印迹法检测两组骨膜细胞Notch和Wnt信号传导通路mRNA、蛋白表达水平。正态分布计量数据以均数±标准差表示,多组之间比较采用单因素方差分析,多组之间两两比较采用Bonferroni’s检验。结果全基因组测序分析显示,3对双胞胎中的眶距增宽症患者均在MAML3基因发现1个新的同义突变(c.1479 G>A,p.Q493Q)。Sanger法外显子测序结果中,33例眶距增宽症患者中有17例(51.5%)携带该突变;而在50例正常对照者中没有检测到该突变。骨膜来源细胞学实验结果显示:患者来源细胞中MAML3的mRNA和蛋白表达水平均低于健康者来源细胞;成骨诱导后3、7、14 d,患者来源细胞中ALP活性均高于健康者来源细胞(8.540±1.450、20.740±2.514、24.090±3.213 vs.5.268±0.482、11.680±1.527、13.200±0.592;P值均<0.05);成骨诱导后14 d,茜素红染色结果显示患者来源细胞内红斑形成多于健康者来源细胞,提示MAML3突变可能导致人骨膜来源细胞过度成骨分化;成骨诱导后14 d,与健康者来源细胞相比,Notch信号途径下游的靶转录因子hes1、hes5的mRNA和蛋白表达水平在患者骨膜来源细胞中均下调,Wnt信号途径的Wnt3a、β-catenin mRNA和蛋白表达水平上调。结论MAML3基因(c.1479G>A,p.Q493Q)突变是面裂相关眶距增宽症的一种致病基因,该基因突变使患者颅面部骨膜组织中MAML3蛋白表达水平下调。Notch和Wnt/β-catenin信号途径在眶距增宽症发病中起重要作用。

Objective To identify the gene mutations associated with facial cleft-related orbital hypertelorism in 3 pairs of monozygotic twins with different phenotypes(with/without hypertelorism)and to investigate their mechanisms.Methods From May 2014 to May 2019,3 pairs of monozygotic twins,2 males and 4 females,aged 5-18 years,were treated in Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,one with normal orbital distance and widening of orbital distance was caused by facial fissure.Among the twins,there was 1 case of orbital hypertelorism and the other case of without orbital hypertelorism,and the hypertelorism was caused by facial cleft.To screen for mutations in hypertelorism,whole genome sequencing was performed on 3 pairs of twins.The Sanger method was used to sequence the exons of 33 patients with facial fissure associated hypertelorism and 50 healthy individuals in the same period to identify the genes selected by the whole genome sequencing.The periosteal tissues were obtained from patients and healthy people during plastic surgery.The cells were cultured,the activity of alkaline phosphatase was measured,and the osteogenic differentiation was identified by alizarin red staining,real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of signal transduction pathways in periosteal cells.Results Whole genome sequencing analysis showed that in all three sets of twins,a new synonymous mutation(c.1479G>A,p.Q493Q)was found in the MAML3.In Sanger exon sequencing,17(51.5%)of 33 patients with hypertelorism carried the mutation,while no mutation was detected in 50 normal controls.The result of periosteum-derived cytology showed that the expression of MAML3 mRNA and protein in the patient-derived cells was lower than that in the healthy-derived cells.Three,7,14 days after osteoinduction,the ALP activity in the cells from the patients was higher than that from the healthy subjects(8.540±1.450,20.740±2.514,24.090±3.213 vs.5.268±0.482,11.680±1.527,13.200±0.592;all P<0.05).Fourteen days after osteoinduction,the result of alizarin red staining showed that there were more erythema formation in the cells from the patients than those from the healthy subjects,these result suggest that MAML3 mutation may lead to over-differentiation of human periosteal-derived cells.The mRNA and protein expression levels of hes1 and hes5 downstream of the Notch signal pathway were down-regulated in the periosteal cells of the patients,while Wnt3a andβ-catenin mRNA and protein expression levels were up-regulated in the Wnt signal pathway.Conclusions The MAML3 gene(c.1479G>A,p.Q493Q)mutation is one of the causative genes of facial cleft-related hypertelorism.Notch and Wnt/β-catenin signaling pathway play an important role in the pathogenesis of hypertelorism.