基于外显子组测序的Axenfeld-Rieger综合征致病突变筛查

Screening of pathogenic mutation in a family with Axenfeld-Rieger syndrome by whole exome sequencing

目的对Axenfeld-Rieger综合征(ARS)一家系进行临床检查和基因检测,寻找致病突变。方法采用家系调查研究方法,纳入2018年于哈尔滨医科大学附属第二医院就诊的中国汉族ARS一家系,该家系3代共15人,其中患者3例。详细记录家族史,进行眼科及全身一般检查。采集受检者外周静脉血2~5 ml并提取淋巴细胞基因组DNA和RNA。对先证者DNA进行外显子测序,利用人群数据库及生物信息学分析筛选可疑突变,Sanger测序和实时荧光定量PCR进行验证,对可疑突变进行保守性和有害性预测,参照美国医学遗传学与基因组学学会(ACMG)遗传变异分类标准与指南对候选罕见变异进行致病性评估。结果3例患者均存在ARS典型的眼部、牙齿和肚脐发育异常,且均携带PITX2基因c.525delC(p.Asp175Glufs^(*))杂合突变,家系中其他人员无相应临床表型且未检测到该变异位点,符合家系共分离。3例患者PITX2 mRNA相对表达量为0.672±0.063,明显低于健康对照的1.015±0.179,差异有统计学意义(t=8.847,P<0.001)。dbSNP、1000G、gnomeAD、ExAC、Korea1K、EVS数据库中均未收录该变异,MutationTaster评为有害,受影响的氨基酸序列在9种动物中保守存在。ACMG遗传变异分类标准和指南评价该变异位点为致病变异。结论PITX2基因c.525delC(p.Asp175Glufs*)变异为该家系患者的致病变异,首次在中国ARS家系中报道。

Objective To identify disease-causing variation in a Chinese family with Axenfeld-Rieger syndrome(ARS)through the analysis of clinical symptoms and hereditary information.Methods The method of pedigree investigation was adopted.A Chinese ARS family including 15 family members of 3 generations was recruited in the Second Affiliated Hospital of Harbin Medical University in 2018.There were 3 patients in the family.The family history and clinical data were collected.Ophthalmic and general examinations were carried out in all the members included.DNA and RNA were extracted from collected peripheral venous blood samples of 2-5 ml from each member.Whole exome sequencing was used to screen the variations in the proband.Suspected variations screened through searching population databases and bioinformatics analysis were verified by Sanger sequencing and real-time quantitative PCR.Conservation analysis and deleteriousness prediction of suspected variations were conducted.The pathogenecity of candidate rare variations were evaluated according to the American College of Medical Genetics and Genomics(ACMG)standards and guidelines.This study followed the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University(No.KY2019-231).Written informed consent was obtained from each subject or custodian prior to entering the study cohort.Results The 3 patients all had typical ARS clinical features in eyes,teeth and umbilicus,and carried the same heterozygous variant,c.525delC(p.Asp175Glufs^(*))in the PITX2 gene,which were not found in other members,indicating co-segregation.The relative expression of PITX2 mRNA was 0.672±0.063 in the patients,which was significantly lower than 1.015±0.179 in the healthy controls(t=8.847,P<0.001).This variant was not recorded in dbSNP,1000G,gnomeAD,ExAC,Korea1K and EVS databases,and it was labelled as deleterious by MutationTaster.The affected conservative amino acid sequences were found in 9 species.The variant was determined as pathogenic according to the ACMG standards and guidelines.Conclusions The c.525delC(p.Asp175Glufs*)mutation of PITX2 gene is pathogenic in the pedigree.This is the first time that this mutation has been reported in Chinese family with ARS.