利用靶向基因测序技术诊断遗传性骨髓衰竭综合征

Targeted Panel Sequencing for Clinical Diagnosis in Inherited Bone Marrow Failure Syndromes

目的 遗传性骨髓衰竭综合征（IBMFS）是一组以骨髓造血功能衰竭、先天多发畸形及易发肿瘤为主要特征的遗传异质性疾病,其临床表型复杂多样,给临床诊断带来困难.本研究旨在基于高通量靶向基因测序（TPS）技术,建立IBMFS的单管高通量基因诊断方法,并应用于临床诊断.方法 选择2009年8月至2013年12月,于上海交通大学医学院附属上海儿童医学中心就诊的21例临床疑似IBMFS患者为研究对象.采集其DNA样本,利用Agilent Haloplex方法对IBMFS已知致病基因及相关基因进行文库捕获,基于Illumina MiSeq技术平台进行DNA高通量TPS,测序数据经NextGENe软件匹配分析后,采用Ingenuity在线软件系统进行基因变异筛选及解释,最后采用Sanger法测序验证基因突变情况（本研究遵循的程序符合上海交通大学医学院附属上海儿童医学中心人体试验委员会制定的伦理学标准,得到该委员会批准,并征得受试对象监护人的知情同意）.结果 本组21例患者的测序数据中,总reads数为1 570 558～3 577 386个,95％以上的reads与人类基因组序列相匹配,85％以上的reads位于目标基因靶序列以内,平均测序深度为444x～1 092x,测序深度大于20x的区域达95％以上,覆盖度均一性达85％以上.本组21例患者中,12例患者有明确的基因诊断结果,包括6例范可尼贫血（FA）、2例先天性角化不良症（DC）、2例先天性中性粒细胞减少症（SCN）、1例先天性纯红细胞再生障碍性贫血（DBA）和1例Shwachman-Diamond综合征（SDS）.5例患者经造血干细胞移植（HSCT）后成功治愈,2个家庭以此诊断结果为产前诊断依据被批准生育二胎.结论 本研究建立了IBMFS的单管高通量TPS分子诊断方法,临床应用结果显示,该方法捕获效率高、测序质量可靠、生物信息学软件全面,可有效检测IBMFS致病基因突变,为产前诊断IBMFS及其及时根治性治疗提供了强有力的依据.

Objective Inherited bone marrow failure syndromes （IBMFS） is a group of genetically heterogeneous diseases characterized by bone marrow failure,congenital anomalies,and susceptibility to tumour.Because of its phenotypic variability,diagnosis can be challenging for clinicians.This study is aimed to develop a targeted panel sequencing pipeline to clinical diagnosis in IBMFS patients.Methods From August 2009 to December 2013,21 suspected IBMFS patients who received treatment in Shanghai Children＇s Medical Center,Affiliated Hospital of Shanghai Jiaotong University School of Medicine were selected into this study.Agilent Haloplex method was used to capture the IBMFS Library of known genes and related genes.Illumina platform was used for high-throughput sequencing,and the sequencing data were aligned by NextGENe software.The variants were filtered and interpreted by the online tool Ingenuity Variant Analysis,and Sanger sequencing was used to confirm the variations （The study protocol was approved by the Ethical Review Board of Investigation in Human Being of Shanghai Children＇s Medical Center,Affiliated Hospital of Shanghai Jiaotong University School of Medicine.Informed consent was obtained from the guardians of all patients）.Results According to the 21 patients＇ sequencing data,the total number of reads were 1 570 558-3 577 386.More than 95% of the sequencing reads were aligned to human genome reference sequence and more than 85 of the sequencing reads were in the target sequence.The average sequencing depth was 444x-1 092x.More than 95% of the coverage area that the sequencing depth was higher than 20 x and the uniformity was higher than 85 %.Among the 21 patients with suspected IBMFS,pathogenic mutations have been identified in 12 patients,including 6 cases with Fanconi anemia （FA）,2 cases with dyskeratosis congenital （DC）,2 cases with severe congenital neutropenia （SCN）,1 case with Diamond-Blackfan anemia （DBA）,and 1 case with Shwachman-Diamond syndrome （SDS）.Five patients were successfully cured through hematopoietic stem cell transplantation （HSCT）.Prenatal diagnosis had been successfully performed in 2 families.Conclusions The target panel sequencing method for molecular diagnosis of IBMFS is successfully established in this study.The clinical results show that the target panel sequencing method can effectively detect and identify pathogenic genes in monogenic disorders and to provide a strong basis for completely cure and prenatal diagnosis.Because the efficiency of captured target region is excellent,the quality of sequencing data is reliable,and the analysis by the bioinformatics software is very comprehensive.