高通量测序在稽留流产绒毛组织染色体检测中的应用分析
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摘要
目的:应用新一代高通量测序技术对稽留流产患者绒毛组织染色体进行分析,为临床诊治提供依据。方法:选择本院行手术终止妊娠的稽留流产患者作为研究对象,收集绒毛组织标本-80℃保存,解冻检测DNA总量和浓度。构建并获得全基因组重测序文库行Illumina HiSeq平台测序。将高通量测序所得数据进行生物信息学分析,获得染色体拷贝数变异数据,分析稽留流产患者染色体特点。结果:共检测成功54例稽留流产患者绒毛组织,检测成功率88.5%,染色体异常检出率77.8%(42/54)。其中染色体数目异常31例(73.8%,31/42),主要为非整倍体数目异常,1例同时有15和18三体异常;染色体结构异常11例(26.2%,11/42)。染色体非整倍体检出率57.4%(31/54),其中以X染色体单体(22.6%)及16号染色体三体最为常见(22.6%)。染色体双三体1例(1/54)。结论:在稽留流产绒毛组织中染色体异常占较大比例,异常多为非整倍体,其中近50%是X染色体单体及16号染色体三体。高通量测序技术不仅可以检测染色体数目和结构异常,还可以检测100kb染色体片段的微缺失和微重复,具有较好的技术优势。
Objective:To analyze the new generation of high-throughput sequencing technology used to detect chromosomes of villus tissue of patients with missed abortion,and to provide the evidence for clinical diagnosis and treatment.Methods:The patients with missed abortion who underwent surgical abortion were selected as the research objects,and the villi specimens of all included women were collected and conserved in-80 degree refrigerator,and the total amount and concentration of DNA were detected in thawing the villus.The whole genome sequencing library was constructed and sequenced by Illumina HiSeq platform.Bioinformatic analysis of data by high-throughput sequencing was performed to obtain variation data of chromosome copy number,and then the chromosomal characteristics of villus tissue of patients with missed abortion were analyzed.Results:A total of 54 villi specimens of patients with missed abortion were detected successfully,and the success rate was 88.5%.The rate of chromosomal abnormalities was 77.8%(42/54).The number of abnormal chromosomes of patients was 31(73.8%,31/42),which included aneuploidy mainly,1 cases with 15 and 18 trisomy abnormalities,and 11 cases with chromosomal structural abnormalities(26.2%,11/42).The rate of chromosome aneuploidy was 57.4%(31/54),of which X chromosome(22.6%)and trisomy 16 were the most common(22.6%).1 cases of chromosome double trisomy(1/54).Conclusion:In the villi of patient with missed abortion,chromosome abnormality occupies a large proportion,most of which are aneuploidy,and nearly50% of which are X chromosome and trisomy 16.High throughput sequencing can not only detect chromosomal number or structural abnormalities,but also can detect microdeletion or microrepetition of 100 kb chromosome fragments,which have good technical advantage.
Objective:To analyze the new generation of high-throughput sequencing technology used to detect chromosomes of villus tissue of patients with missed abortion,and to provide the evidence for clinical diagnosis and treatment.Methods:The patients with missed abortion who underwent surgical abortion were selected as the research objects,and the villi specimens of all included women were collected and conserved in-80 degree refrigerator,and the total amount and concentration of DNA were detected in thawing the villus.The whole genome sequencing library was constructed and sequenced by Illumina HiSeq platform.Bioinformatic analysis of data by high-throughput sequencing was performed to obtain variation data of chromosome copy number,and then the chromosomal characteristics of villus tissue of patients with missed abortion were analyzed.Results:A total of 54 villi specimens of patients with missed abortion were detected successfully,and the success rate was 88.5%.The rate of chromosomal abnormalities was 77.8%(42/54).The number of abnormal chromosomes of patients was 31(73.8%,31/42),which included aneuploidy mainly,1 cases with 15 and 18 trisomy abnormalities,and 11 cases with chromosomal structural abnormalities(26.2%,11/42).The rate of chromosome aneuploidy was 57.4%(31/54),of which X chromosome(22.6%)and trisomy 16 were the most common(22.6%).1 cases of chromosome double trisomy(1/54).Conclusion:In the villi of patient with missed abortion,chromosome abnormality occupies a large proportion,most of which are aneuploidy,and nearly50% of which are X chromosome and trisomy 16.High throughput sequencing can not only detect chromosomal number or structural abnormalities,but also can detect microdeletion or microrepetition of 100 kb chromosome fragments,which have good technical advantage.
引文
[1]谢幸,苟文丽,主编.妇产科学[M].北京:人民卫生出版社,2013:49.
[2]马钰,丘峻朝,方艾荪.育龄妇女稽留流产的相关因素研究[J].齐齐哈尔医学院学报,2015,36(19):2877-2878.
[3]Eiben B,Bartels I,Bahr-Porsch S,et al.Cytogenetic analysis of 750spontaneous abortions with the direct-preparation method of chorionic villi and its implications for studying genetic causes of pregnancy wastage[J].Am J Hum Genet,1990,47(4):656-663.
[4]周丽颖,余兰,梁毓,王树玉.自然流产遗传病因的高通量测序检测[J].中国优生与遗传杂志,2016,(8)35-36.
[5]胡浩梅,杨华,殷振惠,赵露.稽留流产患者染色体检查[J].中华医学杂志,2015,95(35)2837-2840.
[6]刘惠莲.海峰,吕发辉,等.应用高通量基因测序技术测定稽留流产胚胎染色体异常及临床意义[J]吉林医学,2014,35(10):2041-2043.
[7]乞艳华,余珊珊,李小鹏,麻妙艳,邬晋芳,周琦,雷小莹.孕早期流产物的高通量测序研究[J].山西医科大学学报,2017,48(3):271-275.
[8]Feldman B,Aviram-Goldring A,Evans MI.Interphase FISH for prenatal diagnosis of common aneuploidies[J].Methods Mol Biol,2002,204:219-241.
[9]罗岚蓉.自然流产的细胞遗传学研究进展[J].中国工程科学,2015,17(6):65-69.
[10]Wang Z,Andrews P,Kendall J,et al.SMASH,a fragmentation and sequencing method for genomic copy number analysis[J].Genome Res,ana,2016,26(6):844—851.
[11]Yuan Y,Jiang F,Hua S,et al.Feasibility Study of Semiconductor Sequencing for Noninvasive Prenatal Detection of Fetal Aneuploidy[J].Clin Chem,2013,Jan 30.
[12]Cohen K,Tzika A,wood H,et al.Diagnosis of fetal submicroscopic chromosomal abnormalities in failed array CGH samples:copy number by sequencing as an alternative to microarrays for invasive fetal testing[J].Ultrasound Obetet Gynecol,2015,45(4):394-401.
[13]Farr SL,Schieve LA,Jamieson DJ.Pregnancy loss among pregnancies conceived through assisted reproductive technology,United States,1999-2002[J].Am J Epidemiol,2007,165(12):1380-1388.
[14]Mastenbroek S,Twisk M,van Echten-Arends J,et al.In vetro fertilization with preimplantation genetic screening[J].N Engl J Med,2007,357(1):9-17.
[15]JacobsPA,Hassold TJ.Chromosome abnormalities:origin and etiology in abortions and live births.In:Vogal F and Sperling K,eds.Human genetics.Berlin:Springer-Verlag,1987:233-244.
[16]DiegoAlvarezl D,GarciaHoyos M,Trujillo MJ,et a1.Application of quantitative fluorescent PCR with short tandem repeat markers to the study of aneuploidies in spontaneous miscarriages[J].Hum Reprod,2005,20(5):1235-1237.
[17]杨锴,祝建疆,戚红,等.1389例稽留流产绒毛细胞染色体核型分析[J].中国优生与遗传杂志,2014,22(7):53-54.
[18]彩霞,张月萍.1437例早孕期自然流产胚胎核型分析[J].生殖与避孕,2014,34(4):328-331.
[19]张丽,谢岚,谢红敏.基于高通量测序技术的78例稽留流产绒毛染色体分析[J].山西医药杂志,2017,46(12):1467-1468.
[20]李亚丽,余小平,王方娜,等.复发性流产患者流产组织物的遗传学分析[J].中国妇幼保健,2016,31(3):536-538.
[21]Yakut S,Toru HS,cetin Z,et al.Chromosome abnormalities i-dentified in 457spontaneous abortions and their histopathological findings[J].Turk Patoloji Derg,2015,31(2):111-118.
[22]赵佳,毕川,高雅,等.NGS技术检测自然流产胚胎或绒毛染色体非整倍体及拷贝数变异的研究[J].中国优生与遗传杂志.2015,23(2):12-15.
[23]Creasy MR,Crolla JA,Alberman ED.A Cytogenetic Study of Human Spontaneous Abortions Using Banding Techniques.Hum Genet,1976,31:177-196.
[24]Stephenson MD,Awartani KA,Robinson WP.Cytogenetic analysis of miscarriages from couples with recurrent miscarriage:a casecontrol study.Hum Reprod,2002,17:446-51.
[25]Kalousek DK,Pantzar T,Tsai M,Paradice B.Early spontaneous abortion:morphologic and karyotypic findings in 3912cases.Birth Defects Orig Artic Ser,1993.29:53-61.
[26]Benirschke K,Burton GJ,Baergen RN.Pathology of the human Pla-centa[J].Mayo Clinic Proceedings,2000,66(5):674-675.
[27]Huijsdens-van AK,Barge-Schaapveld DQ,Mathijssen IB,etal.Prenatal diagnosiso fatrisomy7/trisomy13 mosaicism[J].Mol Cytogenet,2012,5(1):8.
[28]Kearney HM,Thorland EC,Brown KK,et al.American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants[J].Genet Med,2011,13(7):680-685.
[29]戚红,蔡莉蓉,祝建疆,杨锴,闻小慧,曾雯,陈佳靓.基于高通量测序的染色体组拷贝数分析技术在流产胚胎组织遗传学诊断中的应用价值[J].中华妇产科杂志,2016,(2):92-97.
[2]马钰,丘峻朝,方艾荪.育龄妇女稽留流产的相关因素研究[J].齐齐哈尔医学院学报,2015,36(19):2877-2878.
[3]Eiben B,Bartels I,Bahr-Porsch S,et al.Cytogenetic analysis of 750spontaneous abortions with the direct-preparation method of chorionic villi and its implications for studying genetic causes of pregnancy wastage[J].Am J Hum Genet,1990,47(4):656-663.
[4]周丽颖,余兰,梁毓,王树玉.自然流产遗传病因的高通量测序检测[J].中国优生与遗传杂志,2016,(8)35-36.
[5]胡浩梅,杨华,殷振惠,赵露.稽留流产患者染色体检查[J].中华医学杂志,2015,95(35)2837-2840.
[6]刘惠莲.海峰,吕发辉,等.应用高通量基因测序技术测定稽留流产胚胎染色体异常及临床意义[J]吉林医学,2014,35(10):2041-2043.
[7]乞艳华,余珊珊,李小鹏,麻妙艳,邬晋芳,周琦,雷小莹.孕早期流产物的高通量测序研究[J].山西医科大学学报,2017,48(3):271-275.
[8]Feldman B,Aviram-Goldring A,Evans MI.Interphase FISH for prenatal diagnosis of common aneuploidies[J].Methods Mol Biol,2002,204:219-241.
[9]罗岚蓉.自然流产的细胞遗传学研究进展[J].中国工程科学,2015,17(6):65-69.
[10]Wang Z,Andrews P,Kendall J,et al.SMASH,a fragmentation and sequencing method for genomic copy number analysis[J].Genome Res,ana,2016,26(6):844—851.
[11]Yuan Y,Jiang F,Hua S,et al.Feasibility Study of Semiconductor Sequencing for Noninvasive Prenatal Detection of Fetal Aneuploidy[J].Clin Chem,2013,Jan 30.
[12]Cohen K,Tzika A,wood H,et al.Diagnosis of fetal submicroscopic chromosomal abnormalities in failed array CGH samples:copy number by sequencing as an alternative to microarrays for invasive fetal testing[J].Ultrasound Obetet Gynecol,2015,45(4):394-401.
[13]Farr SL,Schieve LA,Jamieson DJ.Pregnancy loss among pregnancies conceived through assisted reproductive technology,United States,1999-2002[J].Am J Epidemiol,2007,165(12):1380-1388.
[14]Mastenbroek S,Twisk M,van Echten-Arends J,et al.In vetro fertilization with preimplantation genetic screening[J].N Engl J Med,2007,357(1):9-17.
[15]JacobsPA,Hassold TJ.Chromosome abnormalities:origin and etiology in abortions and live births.In:Vogal F and Sperling K,eds.Human genetics.Berlin:Springer-Verlag,1987:233-244.
[16]DiegoAlvarezl D,GarciaHoyos M,Trujillo MJ,et a1.Application of quantitative fluorescent PCR with short tandem repeat markers to the study of aneuploidies in spontaneous miscarriages[J].Hum Reprod,2005,20(5):1235-1237.
[17]杨锴,祝建疆,戚红,等.1389例稽留流产绒毛细胞染色体核型分析[J].中国优生与遗传杂志,2014,22(7):53-54.
[18]彩霞,张月萍.1437例早孕期自然流产胚胎核型分析[J].生殖与避孕,2014,34(4):328-331.
[19]张丽,谢岚,谢红敏.基于高通量测序技术的78例稽留流产绒毛染色体分析[J].山西医药杂志,2017,46(12):1467-1468.
[20]李亚丽,余小平,王方娜,等.复发性流产患者流产组织物的遗传学分析[J].中国妇幼保健,2016,31(3):536-538.
[21]Yakut S,Toru HS,cetin Z,et al.Chromosome abnormalities i-dentified in 457spontaneous abortions and their histopathological findings[J].Turk Patoloji Derg,2015,31(2):111-118.
[22]赵佳,毕川,高雅,等.NGS技术检测自然流产胚胎或绒毛染色体非整倍体及拷贝数变异的研究[J].中国优生与遗传杂志.2015,23(2):12-15.
[23]Creasy MR,Crolla JA,Alberman ED.A Cytogenetic Study of Human Spontaneous Abortions Using Banding Techniques.Hum Genet,1976,31:177-196.
[24]Stephenson MD,Awartani KA,Robinson WP.Cytogenetic analysis of miscarriages from couples with recurrent miscarriage:a casecontrol study.Hum Reprod,2002,17:446-51.
[25]Kalousek DK,Pantzar T,Tsai M,Paradice B.Early spontaneous abortion:morphologic and karyotypic findings in 3912cases.Birth Defects Orig Artic Ser,1993.29:53-61.
[26]Benirschke K,Burton GJ,Baergen RN.Pathology of the human Pla-centa[J].Mayo Clinic Proceedings,2000,66(5):674-675.
[27]Huijsdens-van AK,Barge-Schaapveld DQ,Mathijssen IB,etal.Prenatal diagnosiso fatrisomy7/trisomy13 mosaicism[J].Mol Cytogenet,2012,5(1):8.
[28]Kearney HM,Thorland EC,Brown KK,et al.American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants[J].Genet Med,2011,13(7):680-685.
[29]戚红,蔡莉蓉,祝建疆,杨锴,闻小慧,曾雯,陈佳靓.基于高通量测序的染色体组拷贝数分析技术在流产胚胎组织遗传学诊断中的应用价值[J].中华妇产科杂志,2016,(2):92-97.