上海地区遗传病基因变异高通量测序项目室间质量评价分析

doi:10.3969/j.issn.1673-8640.2025.02.010

摘要/Abstract

摘要：

目的通过开展遗传病基因变异高通量测序项目室间质量评价计划，评估参评实验室的检测能力，提高检测质量。方法 2024年室间质量评价样本盘包含普拉德-威利综合征（PWS）、杜氏进行性肌营养不良症（DMD）和亨廷顿舞蹈症（HD）3种遗传病的阳性样本，涉及拷贝数变异（CNV）和动态突变2种变异类型，共3支样本，类型为细胞培养物。要求参评实验室收到样本后在规定时间内检测样本，并网上上报检测结果。依据上报信息，汇总不同样本的总体符合率，并分析参评实验室检测基本情况、“湿实验”（实验室操作）检测相关信息、生物信息学分析流程、检测相关质控情况。结果有16家实验室参加遗传病基因变异高通量测序项目室间质量评价计划，共收到11份有效回报结果。CNV检测符合率为100.0%（22/22），动态突变符合率为81.8%（9/11），样本变异检出的总体符合率为93.9%（31/33）。11家回报有效结果的参评实验室中，有8家（72.7%）“湿实验”和“干实验”均自行完成，其他3家均存在不同程度的外送情况；有9家（81.8%）实验室采用WES进行检测，有2家（19.2%）采用全基因组测序（WGS）进行检测。11家实验室检测范围均能覆盖单核苷酸变异（SNV）、小的插入/缺失（InDel）和CNV类型，但CNV检测范围有一定差异。所有33个样本的测序数据质量整体较高，达到Q30标准的测序数据占91.60%~96.91%。采用WES检测的27个样本的探针捕获效率差异较大（49.20%~87.91%）。11家实验室均规定了测序文库最低浓度，但对其他环节的质控要求各异。结论上海地区部分实验室针对某些特定变异类型的检测水平有待提高。基于高通量测序的遗传病基因变异检测流程和关键环节质控还有待进一步改进。

关键词: 高通量测序, 基因变异, 遗传病, 室间质量评价, 上海

Abstract:

Objective To evaluate the performance of next-generation sequencing for genetic testing of inherited diseases in external quality assessment（EQA） program and improve the quality of detection results. Methods In 2024 EQA program，3 different neurogenetic disorders including Prader-Willi syndrome（PWS），Duchenne muscular dystrophy（DMD） and Huntington's disease（HD） were investigated. Totally，2 types of genetic variations of copy number variations（CNV） and dynamic mutations were involved. The sample panel consisted of 3 different cell cultures. Participating laboratories were asked to report the results before deadlines. The overall coincidence of detection and report of the pathogenic variations were calculated，and the basic next-generation sequencing conditions，wet-lab procedure，bioinformatics analysis workflow，quality control measures were also analyzed. Results A total of 16 laboratories enrolled in the EQA program and 11 valid laboratory results were received in the EQA program. The coincidence rate of CNV and dynamic mutations were 100.0%（22/22） and 81.8%（9/11），respectively. The overall coincidence rates were 93.9%（31/33）. Among the 11 laboratories that returned valid results，8（72.7%） completed both "wet lab" and "dry lab" procedure independently，while the other 3 laboratories had different levels of testing outsourcing. The 9（81.8%） employed whole-exome sequencing（WES），and 2（19.2%） used whole-genome sequencing（WGS）. The detection scope of all 11 laboratories covered single nucleotide variations（SNV），small insertions-deletion（InDel），as well as CNV，but varied in CNV detection range. The overall quality of sequencing data for all the 33 samples was high，with the proportion of sequencing data meeting the Q30 standard ranging from 91.60% to 96.91%. The probe capture efficiency of the 27 samples for WES varied（49.20%-87.91%）. All the 11 laboratories specified the minimum concentration of sequencing libraries，but the quality control requirements for other steps varied. Conclusions Some laboratories in Shanghai need to enhance their detection capabilities for specific mutation types. There is a need for additional improvements in the experimental procedure as well as quality control of NGS applications for detecting genetic variations.

Key words: Inherited disease, Genetic variation, Next-generation sequencing, External quality assessment, Shanghai

中图分类号:

R446.7

鲍芸, 郁婷婷, 权静, 尹刘翻, 张芃胤, 肖艳群. 上海地区遗传病基因变异高通量测序项目室间质量评价分析[J]. 检验医学, 2025, 40(2): 160-164.

BAO Yun, YU Tingting, QUAN Jing, YIN Liufan, ZHANG Pengyin, XIAO Yanqun. Result analysis of external quality assessment of next-generation sequencing for genetic testing of inherited diseases in Shanghai[J]. Laboratory Medicine, 2025, 40(2): 160-164.

图/表 3

参考文献 11

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样本编号	临床表现	预期结果	预期疾病	变异类型	回报结果
样本编号	临床表现	预期结果	预期疾病	变异类型	回报数/份	符合/[家（%）]
2421	男；新生儿期喂养困难，生长迟缓；肥胖；性腺激素分泌不足，功能减退，隐睾症；轻度智力障碍	46，XY，del（15q11.2q13.1）.seq[GRCh37/hg19]（22693710-28818254）X 1	PWS	CNV	11	11（100）
2422	男；小腿肥大；进行性肌无力；9岁时出现肌肉无力、轻度脊柱侧弯和踝关节挛缩，需使用轮椅；10岁时诊断出心肌病	46，XY，del（Xp21.1）.seq[GRCh37/hg19]（32256952-32866852）X 0	DMD	CNV	11	11（100）
2423	女；短暂不能控制的装鬼脸、点头、手指跳动；肌张力障碍、不协调；认知能力下降；行为困难	NM_001388492.1（HTT）：c.54_110GCA[43]（p.Gln18[43]）	HD	动态突变	11	9（81.8）

样本编号	临床表现	预期结果	预期疾病	变异类型	回报结果
样本编号	临床表现	预期结果	预期疾病	变异类型	回报数/份	符合/[家（%）]
2421	男；新生儿期喂养困难，生长迟缓；肥胖；性腺激素分泌不足，功能减退，隐睾症；轻度智力障碍	46，XY，del（15q11.2q13.1）.seq[GRCh37/hg19]（22693710-28818254）X 1	PWS	CNV	11	11（100）
2422	男；小腿肥大；进行性肌无力；9岁时出现肌肉无力、轻度脊柱侧弯和踝关节挛缩，需使用轮椅；10岁时诊断出心肌病	46，XY，del（Xp21.1）.seq[GRCh37/hg19]（32256952-32866852）X 0	DMD	CNV	11	11（100）
2423	女；短暂不能控制的装鬼脸、点头、手指跳动；肌张力障碍、不协调；认知能力下降；行为困难	NM_001388492.1（HTT）：c.54_110GCA[43]（p.Gln18[43]）	HD	动态突变	11	9（81.8）

方法	实验室数/家	测序数据量/Gb	碱基质量Q30/%	平均测序长度/bp	平均测序深度	GC含量/%	中靶率/%	20×覆盖度/%
WES	9
2421		9.78~29.70	92.16~96.08	100~150	108×~341×	46.50~55.00	49.38~87.91	98.05~99.83
2422		9.99~37.29	92.00~96.21	100~150	114×~429×	46.40~55.00	49.20~87.29	98.01~99.85
2423		10.36~41.05	91.60~96.91	100~150	122×~494×	46.50~56.00	49.67~87.57	97.89~99.74
WGS	2
2421		164.78~181.27	95.65~96.48	150	55.44×~61.17×	40.32
2422		150.61~168.31	95.52~96.21	150	50.66×~56.78×	40.20~40.42
2423		176.51~186.24	95.98~96.14	150	55.44×~58.68×	40.37~40.41

方法	实验室数/家	测序数据量/Gb	碱基质量Q30/%	平均测序长度/bp	平均测序深度	GC含量/%	中靶率/%	20×覆盖度/%
WES	9
2421		9.78~29.70	92.16~96.08	100~150	108×~341×	46.50~55.00	49.38~87.91	98.05~99.83
2422		9.99~37.29	92.00~96.21	100~150	114×~429×	46.40~55.00	49.20~87.29	98.01~99.85
2423		10.36~41.05	91.60~96.91	100~150	122×~494×	46.50~56.00	49.67~87.57	97.89~99.74
WGS	2
2421		164.78~181.27	95.65~96.48	150	55.44×~61.17×	40.32
2422		150.61~168.31	95.52~96.21	150	50.66×~56.78×	40.20~40.42
2423		176.51~186.24	95.98~96.14	150	55.44×~58.68×	40.37~40.41

数据库	实验室数/家	更新时间
ClinVar	10	2024年1月—2024年9月
gonmAD	10	2018年10月—2023年11月
dbNSFP	7	2015年4月—2023年12月
OMIM	6	2022年7月—2024年8月