低深度全基因组拷贝数变异测序产前诊断发现DMD基因变异胎儿的遗传学分析

Genetic analysis of fetuses with DMD gene variations by low-depth whole-genome copy number variation sequencing

目的探讨低深度全基因组拷贝数变异测序(copy number variation sequencing,CNV-seq)在产前诊断中发现的无遗传病家族史胎儿DMD基因变异及家系检测结果,探索CNV-seq在DMD基因变异检测中的意义和结果解读。方法回顾性收集2019年12月至2023年8月就诊于郑州大学第一附属医院经低深度全基因组CNV-seq检测出的16例胎儿DMD基因缺失或重复的病例资料。16例抽取羊水或绒毛样本及家系成员外周血,提取基因组DNA。在应用CNV-seq技术检测胎儿染色体拷贝数变异情况同时通过多重连接探针扩增(multiplex ligation dependent probe amplification,MLPA)技术验证DMD基因缺失或重复情况,并经家系验证追溯变异来源。参考在线人类孟德尔遗传数据库等数据库以及家系验证结果分析DMD基因缺失或重复片段的致病性。结果16例均否认单基因遗传病家族史,CNV-seq产前诊断指征分别为唐氏综合征筛查高风险9例、高龄2例、胎儿超声检查异常3例、无创DNA产前检测提示X染色体异常2例。CNV-seq结果提示9例胎儿为DMD基因重复变异,7例为DMD基因缺失变异;通过MLPA方法验证,结果与CNV-seq结果一致。经家系分析,其中3例为新发变异,12例遗传自母亲,1例母亲外周血检测正常,但有一携带相同变异的姐姐,推测母亲为生殖腺嵌合体的可能性大。7例缺失变异为致病性变异的可能性大;9例为重复变异,其中4例变异位于DMD基因内部,可能导致DMD基因被打断,从而导致疾病的发生,另5例变异位于DMD基因5’端非编码区或3’端非编码区,为良性变异。结论低深度全基因组CNV-seq可检测出无DMD家族史胎儿DMD基因大片段缺失重复变异,避免新发变异患儿的出生。但对于检测出DMD基因大片段重复变异的胎儿应根据家系验证结果评估其致病性;重复区域包含DMD基因5’端非编码区或3’端非编码区时,为多态性变异的可能性大。

ObjectiveTo explore the significance and interpretation of low-depth whole-genome copy number variation sequencing(CNV-seq)in prenatal diagnosis in detecting DMD gene variations in fetuses without a family history of genetic diseases,and to investigate the results of family testing.MethodsRetrospectively collected case data of 16 fetuses with DMD gene deletions or duplications detected by low-depth whole-genome CNV-seq from December 2019 to August 2023 at the First Affiliated Hospital of Zhengzhou University.Amniotic fluid or chorionic villus samples and peripheral blood from family members were collected for all 16 cases,and genomic DNA was extracted.The fetal chromosomal copy number variations were detected using CNV-seq technology and the DMD gene deletions or duplications were verified by multiplex ligation-dependent probe amplification(MLPA),followed by family validation to trace the source of variation.The pathogenicity of the DMD gene deletion or duplication fragments was analyzed based on online Mendelian genetics databases and family validation results.ResultsAll 16 cases denied a family history of monogenic diseases.The indications for CNV-seq prenatal diagnosis were high-risk Down syndrome screening in nine cases,advanced maternal age in two cases,abnormal fetal ultrasound in three cases,and non-invasive prenatal DNA testing suggesting X chromosome abnormalities in two cases.CNV-seq results indicated nine cases of DMD gene duplication variations and seven cases of DMD gene deletion variations.MLPA validation confirmed results consistent with CNV-seq detection.Family analysis showed that three cases were de novo variations,12 cases were inherited from the mother,one case had a mother with normal peripheral blood testing but a sister carrying the same variation,suggesting a high possibility of the mother being a carrier of gonadal mosaic.The likelihood of pathogenic variation was high in seven cases of deletion;nine cases were duplication variations,four of which were located within the DMD gene and could potentially disrupt the gene,leading to disease,while the other five variations were located in the 5'untranslated region or 3'untranslated region,considered benign variations.ConclusionsLow-depth whole-genome CNV-seq can effectively detect large deletion and duplication variations of the DMD gene in fetuses without a family history,preventing the birth of children with de novo variations.However,the pathogenicity of fetuses with large DMD gene duplications should be assessed based on family validation.When the duplication region includes the 5'untranslated region or 3'untranslated region of the DMD gene,it is more likely to be a polymorphic variation.