lon Torrent半导体测序联合MLPA技术检测神经纤维瘤患者的致病突变

摘要:目的建 立临床适用的神经纤维瘤病(NF)的分子遗传分析方法,对NF进行分子遺传诊断和遗传咨询,并对NF家庭进行产前诊断。方法以南京医科大学附属妇产医院遗传咨询门诊收治的18 例NF患者为研究对象,收集患者及其家属的外周血标本并提取基因组DNA,应用lon Torrent半导体测序技术进行NF1/NF2基因测序并结合多重连接探针扩增反应(ML-PA)检测,筛选致病突变并用Sanger测序验证,结合亲子二代的分子遗传检测结果,制定适当的临床干预和随访计划。在NF家系母亲再次妊娠时,对胎儿进行产前诊断。结果应用 lon Torrent半导体测序技术检测出9例致病突变和1例可疑致病突变,其中包括3个未报道过的新突变;应用MLPA技术检测出1例拷贝数大片段缺失。对其中5个家系的孕妇行羊膜腔穿刺术抽取羊水进行产前诊断,其胎儿均不携带家系的致病突变。结论基于 lon Torent半导体测序及MLPA技术建立的基因诊断方法可为NF临床诊断及遗传咨询提供分子遗传学依据,具有较高的临床应用价值。

Detection of pathogenic mutations in patients with neurofibromatosis by Ion Torrent semiconductor sequencing combined with MLPA technology

Abstract: Objective To establish a dlinically applicable molecular genetic method for neurofibromatosis(NF), perform molecular genetic diagnosis and genetic counseling for NF, and carry out prenatal testing for NF families. Methods Eighteen NF patients admitted to the Genetic Counseling Clinic of Nanjing Matemity and Child Health Care Hospital were selected as the study subjects. The peripher-al blood samples of patients and their families were collected, and genomic DNA was extracted. The sequence of NFI/NF2 gene was determined by the lon Torrent semiconductor sequencing combined with multiplex ligation-dependent probe amplification ( MLPA )technology. Then, the pathogenic mutations were screened and validated by Sanger sequencing. Based on the molecular genetic testing results of the second generation of parents and children, the appropriate dlinical intervention and fllow -up plans were developed. When the mother in NF family was pregnant again, the prenatal testing was performed on the fetus. Results Nine pathogenic mutations and one suspected pathogenic mutation were detected using the lon Torrent semiconductor sequencing technology, among which included three unreported new mutations. One case of large copy number fragnent deletion was detected by the MLP A technology. Amniocentesis was performed on pregnant women from 5 families to extract amniotic fluid for prenatal testing, and their fetuses did not carry pathogenic mutations of the family. Conclusion The established gene diagnosis method based on lon Torrent semiconductor sequencing and MLPA technology may provide molecular genetics basis for the clinical diagnosis and genetic counseling of NF, which has high clinical application value.