产前诊断中采用拷贝数变异测序技术检测胎儿DMD基因缺失或重复的效能

Efficiency of CNV-seq in detecting fetal DMD gene deletion or duplication in prenatal diagnosis

目的评估产前诊断中采用低深度高通量全基因组拷贝数变异测序(CNV-seq)技术对胎儿DMD基因缺失或重复的诊断效能。方法对2018年1月至2023年7月于云南省第一人民医院行产前诊断的34544例胎儿的CNV-seq检测结果进行回顾性分析,共纳入156例胎儿,包括:有假性肥大型进行性肌营养不良[DMD;包括表型较轻的贝氏肌营养不良(BMD)]生育史或家族史的胎儿125例;无DMD/BMD生育史或家族史但因其他指征行产前诊断检出DMD基因缺失或重复的胎儿31例。采用多重连接依赖性探针扩增(MLPA)技术对胎儿样本进行检测验证,对CNV-seq和MLPA结果进行一致性检验,获得CNV-seq对胎儿DMD基因缺失或重复的检测效能。结果与MLPA相比,CNV-seq对胎儿DMD基因缺失或重复的检出总符合率为92.3%(144/156),敏感度和阳性预测值均为88.2%,特异度和阴性预测值均为94.3%,漏检率为3.8%,Kappa系数为0.839。有DMD/BMD生育史或家族史的胎儿中,MLPA共检出20例缺失和6例重复,其中4例缺失和2例重复因变异片段<100 Kb被CNV-seq漏检。无DMD/BMD生育史或家族史的胎儿中,CNV-seq检出缺失占42%(13/31),重复占58%(18/31),重复占比高于有DMD/BMD生育史或家族史的胎儿,其中3例位于第42~67号外显子(可能致病),9例覆盖DMD基因5’或3’末端,均包含第1或第79号外显子(临床意义未明),其余6例CNV-seq检出重复片段位于chrX:32650635_32910000(临床意义未明),但MLPA检测结果均为阴性。结论CNV-seq可有效检出胎儿DMD基因缺失或重复,但存在少量漏检风险,结果需MLPA验证。CNV-seq检测到chrX:32650635_32910000区域重复及检出覆盖DMD基因5′端或3′端重复的变异可能并不致病。

Objective To evaluate the diagnostic efficiency of copy number variation sequencing(CNV-seq)to detect the deletion or duplication of DMD gene in prenatal diagnosis.Methods A retrospective analysis was carried out on the CNV-seq results of 34544 fetuses diagnosed in the First People′s Hospital of Yunnan Province from January 2018 to July 2023.A total of 156 cases of fetuses were collected,including Group 1:125 cases with family history of Duchenne muscular dystrophy or Becker muscular dystrophy(DMD/BMD),and Group 2:31 cases with no family history but a DMD gene deletion or duplication was detected unexpectedly by CNV-seq.Multiplex ligation-dependent probe amplification(MLPA)was used as a standard method to detect the deletion or duplication.Consistency test was carried out basing on the results of CNV-seq and MLPA of all 156 cases.Results Comparing to MLPA,CNV-seq had a coincidence rate of 92.3%(144/156)for DMD gene deletion or duplication,with a sensitivity and positive predictive value of 88.2%,with a specificity and negative predictive value of 94.3%,a missed detection rate of 3.8%,and a Kappa value of 0.839.CNV-seq missed 4 cases with deletions and 2 with duplications due to involved fragments less than 100 Kb,among 20 cases of deletions and 6 cases of duplications detected by MLPA in Group 1.In Group 2,the deletions and duplications detected by CNV-seq were 42%(13/31)and 58%(18/31),respectively,in which the percentage of duplication was higher than that in Group 1.Among those 18 cases with duplications,3 cases with duplication locating in exon 42~67 were likely pathogenic;while 9 cases with duplication covering the 5′or 3′end of the DMD gene,containing exon 1 or 79 and with only one breakpoint within the gene,along with the last 6 cases with duplications locating at chrX:32650635_32910000 detected only by CNV-seq,which might be judged as variants of uncertain significance.Conclusions CNV-seq has a good efficiency to detect fetal DMD gene deletion or duplication in prenatal diagnosis,while a further verification test by MLPA is recommended.The duplications on chrX:32650635_32910000,5′or 3′end of DMD gene detected by CNV-seq should be carefully verified and assessed because those variants appear to be nonpathogenic polymorphisms.