CNV-seq应用于产前诊断及流产物检测意外发现DMD基因变异临床分析

Clinical analysis of accidental discovery of DMD gene variations in prenatal diagnosis and detection of abortus by CNV-seq

摘要

目的 探讨全基因组拷贝数变异测序技术(CNV-seq)应用在产前诊断及流产物检测中发现Duchenne型肌营养不良症(DMD)的重要价值。方法 本研究使用CNV-seq技术对2019年9月至2020年7月来西北妇女儿童医院进行产前诊断的羊水样本和流产胎儿样本共784例进行检测,对DMD基因变异样本进一步采用多重连接依赖性探针扩增(MLPA)方法进行验证。结果 本研究意外发现3例样本存在DMD基因的缺失或重复,同时MLPA验证了这些突变.3例均为女胎,胎儿1为DMD基因64-79号外显子重复,胎儿2为DMD基因1-7号外显子重复,这两例羊水样本的变异为新发突变;流产胎儿3的变异遗传自母亲,母亲检测为DMD基因45-51号外显子缺失。结论 CNV-seq技术应用于产前诊断及流产物不仅能检测染色体片段缺失或重复,还有助于发现基因重复或缺失所致的单基因疾病,提高了染色体及基因异常疾病的检出率,从而为临床诊断和遗传咨询提供更全面的理论依据.

Abstract

Objective To explore important value of copy number variation sequencing (CNV-seq) technology in identifying Duchenne muscular dystrophy (DMD) in prenatal diagnosis and abortus detection. Methods The amniotic fluid and abortus samples of 784 pregnant women who received prenatal diagnosis in our hospital from September 2019 to July 2020 were examined using CNV-seq technology, and those samples with DMD gene variations were validated further by multiplex ligation-dependent probe amplification (MLPA). Results DMD gene deletions or duplication in three samples were accidentally discovered, and they were all validated by MLPA. All three samples were female fetuses. The Fetus 1 had duplication of exons 64-79 of the DMD gene and the fetus 2 had duplication of exons 1-7 of the DMD gene. The variations of the DMD gene in the two amniotic fluid samples were De novo (novel) mutations. The variation of the DMD gene in aborted fetus 3 was deletion of exons 45-51 of the DMD gene which inherited from the mother. Conclusion Application of CNV-seq technology in prenatal diagnosis and abortus examination cannot only detect deletion or duplication of chromosome segments, but also contribute to identification of monogenic diseases caused by gene duplication or deletions, which improves detection rate of chromosome or gene abnormalities, and thus provides more comprehensive theoretical basis for clinical diagnosis and genetic consultation.

关键词

全基因组拷贝数变异测序技术 / Duchenne型肌营养不良症 / 产前诊断 / 流产物

Key words

copy number variation sequencing / Duchenne muscular dystrophy / prenatal diagnosis / abortus

参考文献

[1] Fortunato F, Farnè M, Ferlini A. The DMD gene and therapeutic approaches to restore dystrophin[J]. Neuromuscul Disord, 2021, 31(10): 1013-1020.

[2] Keegan NP. Pseudoexons of the DMD Gene[J]. J Neuromuscul Dis, 2020, 7(2): 77-95.

[3] Nascimento O A, Medina C J, Camacho S A, et al. Consensus on the diagnosis, treatment and follow-up of patients with Duchenne muscular dystrophy[J]. Neurologia (Engl Ed), 2019, 34(7): 469-481.

[4] 杨李, 许晓燕, 朱静, 等. Duchenne肌营养不良家系临床表型及遗传学分析[J]. 中国当代儿科杂志, 2020, 22(8): 867-873.

[5] 马韵翼. 孕早期自然流产组织基因组拷贝数变异分析[D]. 郑州: 郑州大学, 2019.

[6] 徐晓昕, 徐志勇, 王勤, 等. 结合多重连接探针扩增技术与第二代测序技术建立假性肥大型肌营养不良产前诊断技术平台[J]. 中华检验医学杂志, 2018, 41(1): 70-72.

[7] Elangkovan N, Dickson G. Gene therapy for Duchenne muscular dystrophy[J]. J Neuromuscul Dis, 2021, 8(s2): S303-S316.

[8] Hanson B, Wood M J A, Roberts T C. Molecular correction of Duchenne muscular dystrophy by splice modulation and gene editing[J]. RNA Biol, 2021, 18(7): 1048-1062.

[9] 朱若男, 时盼来, 赵军红, 等. 20038例羊水染色体核型与不同产前诊断指征关系的分析[J]. 中华医学遗传学杂志, 2021, 38(3): 300-302.

[10] 向萍霞, 刘翎, 胡晞江, 等. CNV-seq在高危孕妇产前诊断中的应用价值[J]. 中华医学遗传学杂志, 2023, 40(1): 17-20.

[11] 高明雅, 柴玉琼, 王亚男. 核型分析联合CNV-seq技术在羊水染色体嵌合体检测中的应用[J]. 检验医学与临床, 2022, 19(15): 2082-2085, 2090.

[12] 屈素真, 时盼来, 张田园, 等. CNV-seq与染色体核型分析在平衡易位携带者产前诊断中的应用[J]. 中华医学遗传学杂志, 2022, 39(4): 366-369.

[13] Luo S, Chen X, Yan T, et al. Application of copy number variation sequencing in genetic analysis of miscarriages in early and middle pregnancy[J]. Cytogenet Genome Res, 2020, 160(11-12): 634-642.

[14] Silva T H D, Anequini I P, Fávero F M, et al. Functional performance and muscular strength in symptomatic female carriers of Duchenne muscular dystrophy[J]. Arq Neuropsiquiatr, 2020, 78(3): 143-148.

[15] Takeshita E, Minami N, Minami K, et al. Duchenne muscular dystrophy in a female with compound heterozygous contiguous exon deletions[J]. Neuromuscul Disord, 2017, 27(6): 569-573.

[16] Chen J, Zheng H, Wang Z, et al. A female carrier of a novel DMD mutation with slightly skewed X-chromosome inactivation shows a suspected case of Becker muscular dystrophy in a Chinese family[J]. Mol Genet Genomics, 2021, 296(3): 541-549.

[17] 高值, 刘莉娜, 王艳丽, 等. 中原地区67个DMD家系产前诊断结果的分析[J]. 中华医学遗传学杂志, 2022, 39(7): 669-673.

基金资助

国家自然科学基金(82201865)

陕西省重点研发计划项目(2023GYBSFG305)

西北妇女儿童医院院内科研项目(2020ZD03)