多重扩增子高通量测序对高苯丙氨酸血症基因变异实验室检测效率的研究  

An Evaluation of Laboratory Test Efficiency of Multiplex Amplicon Next Generation Sequencing for Genetic Variation of Hyperphenylalaninemia

作　　者：吉栩[1] 李锦[2] 潘亭亭 陈宁 沈鸣 田亚平[2] JI Xu;LI Jin;PAN Tingting;CHEN Ning;SHEN Ming;TIAN Yaping(Research Center of Translational Medicine,Medical Innovation Research Division of Chinese PLA GeneralHospital,Beijing 100853,China;Research Center of Birth Defect Prevention Technology,MedicalInnovation Research Division of Chinese PLA General Hospital,Beijing 100853,China;984^(th) Hospital of PLA Joint Logistic Support Force,Beijing 100094,China)

机构地区：[1]中国人民解放军总医院医学创新研究部转化医学研究中心,北京100853 [2]中国人民解放军总医院医学创新研究部出生缺陷防控技术研究中心,北京100853 [3]中国人民解放军联勤保障部队第九八四医院,北京100094

出　　处：《标记免疫分析与临床》2021年第10期1653-1658,共6页Labeled Immunoassays and Clinical Medicine

基　　金：国家重点研发项目(编号:2017YFC1001700);科技助力经济2020重点专项(编号:SQ2020YFF0426571);军队课题(编号:18-163-12-ZT-002-015-01)。

摘　　要：目的对高苯丙氨酸血症(hyperphenylalaninemia,HPA)干血片DNA样本进行多重扩增子高通量测序检测,研究该方法对HPA潜在病因的实验室诊断价值。方法选取既往70例生化筛查为HPA的患儿干血片,提取DNA后使用覆盖126种遗传病130个基因的扩增子建库试剂盒建库,进行高通量测序和数据分析,获得样本基因变异信息。经一代测序验证位点准确性后,分析对样本具有诊断价值的变异位点信息进行疑似病因判定,评价高通量测序的检测和诊断效能。结果高通量测序结果和既往部分样品的一代测序结果完全吻合。70例样本中,5例为PAH和PTPS纯合突变,53例检测出PAH或PTPS单个基因2~3个杂合突变位点,还有2例检出HPA信号通路相关的SPR基因或GCH1基因各1个突变。检测结果中c.158G>A、c.728G>A、c.1068C>A、c.1238G>C、c.611A>G位点为PAH基因频率最高的5个突变。结论通过高通量测序技术,可准确检测基因突变,提供病因解释,为后续召回和临床诊断提供重要参考。Objective To study the contribution of next generation sequencing(NGS)for candidate diagnosis of HPA by using DNA samples from HPA dry blood spots(DBS)for multi-amplicon based sequencing.Methods Seventy HPA DBS screened by previous biochemical test were selected for DNA extraction,and library preparation was performed with the amplicon-based library preparation kit,which covered 130 genes for 126 types of genetic disorders.Next NGS and data analysis were conducted to produce genetic variants information.After the sequence accuracy was verified by Sanger sequencing,variants potentially contributing to the suspected etiology were evaluated for the detection and diagnostic efficiency of NGS.Results Mutation results from high-throughput sequencing were completely consistent with former Sanger sequencing results.In all 70 samples,5 cases carried homozygous mutations of PAH(phenylalanine hydroxylase)and PTPS(6-pyruvoyl tetrahydropterinsynthase)gene.53 cases were detected with 2 to 3 heterozygous mutations of PAH or PTPS.And 2 cases each were found with one mutation of SPR or GCH1 gene related to HPA signaling pathway.The five most frequent mutations of PAH were c.158G>A,c.728G>A,c.1068C>A,c.1238G>C,and c.611A>G.Conclusion NGS detects genetic variants with high accuracy and provides explanation for disease cause,which is an important reference for subsequent recall and clinical diagnosis.

关 键 词：高苯丙氨酸血症 苯丙氨酸羟化酶 四氢生物喋呤(BH4)缺乏 高通量测序 遗传诊断 

分 类 号：R722.11[医药卫生—儿科]