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

Abstract

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 444×-1 092×.More than 95％ of the coverage area that the sequencing depth was higher than 20 × 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. Key words: Bone marrow failure syndromes; Diagnosis; Target panel sequencing