采用二代测序对脊髓性肌萎缩家系行胚胎植入前遗传学诊断
|
摘要
目的探讨二代测序技术对脊髓性肌萎缩症(SMA)家系行胚胎植入前遗传学诊断(PGD)的应用价值和优势。方法选取1对于苏州市立医院生殖与遗传中心就诊的SMA基因携带者夫妇,经家系调查后,抽取家系成员外周血,采用Real-time PCR法进行分析,明确基因突变位点。针对突变位点涉及基因及基因的上下游区域内选择高密度紧密连锁的单核苷酸多态(SNP)作为遗传标记。二代测序(NGS)后,选择若干有效位点进行单体型分析。最后使用单体型连锁分析技术进行单基因PGD。结果此对夫妇的SMN1基因exon7均为单拷贝,而先证者为SMN1基因exon7纯合缺失。通过单体型连锁分析之后进行单基因病PGD周期,形成4枚囊胚,NGS PGD检测结果为未携带突变的整倍体囊胚、单拷贝携带的整倍体囊胚、单拷贝携带但为非整倍体囊胚和致病的整倍体囊胚各1枚。将未携带突变的整倍体囊胚行冻融胚胎移植后获得临床妊娠,产前诊断结果显示未见异常,足月剖宫产分娩一正常男婴,体重3 150g,健康状况良好。结论采用NGS对SMA家系行PGD可以阻断此单基因病在该家系中的再发风险,还可以避免选择非整倍体胚胎而导致的流产问题。
Objective:To investigate the value and advantages of next generation sequencing technology(NGS)for pre-implantation genetic diagnosis(PGD)in the patients who are spinal muscular atrophy(SMA)carrier.Methods:One couple with SMA SMN1 carriers visited the Reproductive and Genetic Center of Suzhou Municipal Hospital for IVF treatment.The peripheral blood samples of the family members were taken and analyzed by Real-time PCR.High-density tightly-linked single nucleotide polymorphisms(SNPs)were selected as genetic markers for upstream and downstream regions of genes.After next-generation sequencing(NGS),several valid sites were selected for haplotype analysis.Monoclonal genetic disease PGD was performed by using haplogroup linkage analysis.Results:The examination results showed that the SMN1 gene exon7 was a single copy of the couple,while the proband was a homozygous deletion.Four blastocysts were formed after haplogroup linkage analysis.The results of NGS PGD showed that there were 1 non-carrying mutation euploid blastocyst,1 single copy mutation euploid blastocyst,1 single copy mutation but aneuploid blastocyst,and 1 pathogenic euploid blastocyst.The clinical pregnancy was obtained after the non-carrying mutation euploid blastocyst was transferred in a frozen-thawed cycle.Prenatal diagnosis showed no abnormalities,and a normal fullterm boy with weigh of 3 150 g was delivered after cesarean.Conclusions:The recurrence risk of SMA monogenic disease in a family can be blocked by adopting NGS for PGD.This technology can avoid miscarriage caused by selecting aneuploid embryos transfer.
Objective:To investigate the value and advantages of next generation sequencing technology(NGS)for pre-implantation genetic diagnosis(PGD)in the patients who are spinal muscular atrophy(SMA)carrier.Methods:One couple with SMA SMN1 carriers visited the Reproductive and Genetic Center of Suzhou Municipal Hospital for IVF treatment.The peripheral blood samples of the family members were taken and analyzed by Real-time PCR.High-density tightly-linked single nucleotide polymorphisms(SNPs)were selected as genetic markers for upstream and downstream regions of genes.After next-generation sequencing(NGS),several valid sites were selected for haplotype analysis.Monoclonal genetic disease PGD was performed by using haplogroup linkage analysis.Results:The examination results showed that the SMN1 gene exon7 was a single copy of the couple,while the proband was a homozygous deletion.Four blastocysts were formed after haplogroup linkage analysis.The results of NGS PGD showed that there were 1 non-carrying mutation euploid blastocyst,1 single copy mutation euploid blastocyst,1 single copy mutation but aneuploid blastocyst,and 1 pathogenic euploid blastocyst.The clinical pregnancy was obtained after the non-carrying mutation euploid blastocyst was transferred in a frozen-thawed cycle.Prenatal diagnosis showed no abnormalities,and a normal fullterm boy with weigh of 3 150 g was delivered after cesarean.Conclusions:The recurrence risk of SMA monogenic disease in a family can be blocked by adopting NGS for PGD.This technology can avoid miscarriage caused by selecting aneuploid embryos transfer.
引文
[1]张云茜,章印红,王建林.脊髓性肌萎缩症的研究进展[J].重庆医学,2014,43:1521-1523.
[2]王柠,何瑾,陈万金.脊髓性肌萎缩症临床诊断研究进展[J].中国现代神经疾病杂志,2012,3:252-256.
[3]Ar Rochmah M,Awano H,Awaya T,et al.Spinal muscular atrophy carriers with two SMN1copies[J].Brain Dev,2017,39:851-860.
[4]冯春,郑芳,彭剑虹,等.脊髓性肌萎缩症产前诊断病例分析并遗传咨询指导[J].中国优生与遗传杂志,2017,25:94-96.
[5]Li W.How do SMA-linked mutations of SMN1lead to structural/functional deficiency of the SMA protein?[J/OL].PLoS One,2017,12:e0178519.
[6]徐晨阳,项延包,卢金芳,等.温州地区14个脊髓性肌萎缩症家系临床表型与基因分析及产前诊断[J].中国优生与遗传杂志,2017,25:21-24.
[7]Lefebvre S,Bürglen L,Reboullet S,et al.Identification and characterization of a spinal muscular atrophy-determining gene[J].Cell,1995,80:155-165.
[8]Lefebvre S,Burlet P,Liu Q,et al.Correlation between severity and SMN protein level inspinal muscular atrophy[J].Nat Genet,1997,16:265-269.
[9]Ou J,Wang W,Feng T,et al.Identification of small segmental translocations in patients with repeated implantation failure and recurrent miscarriage using next generation sequencing after in vitro fertilization/intracytoplasmic sperm injection[J].Mol Cytogenet,2015,8:105.
[10]Iannaccone ST.Spinal muscular atrophy[J].Semin Neurol.1998,18:19-26.
[11]张蕾,颉小玲,李娟,等.脊髓性肌萎缩症遗传学及治疗研究进展[J].临床儿科杂志,2017,35:632-635.
[12]卢丽萍.脊髓性肌萎缩症SMN基因检测及其表达的研究[D].中国医科大学,2005:38-47.
[13]邓坤仪,彭建明,范汉恭,等.荧光定量PCR快速筛查脊髓性肌肉萎缩症smn1致病基因携带者[J].国际检验医学杂志,2016,37:2983-2984.
[14]曾光群,杨季云,张丁丁,等.脊髓性肌萎缩症2个核心家系SMN1基因分析[J].中国实用神经疾病杂志,2015,18:1-2.
[15]王柠,吴志英,慕容慎行,等.脊髓性肌萎缩症的快速基因诊断研究[J].中国神经免疫学和神经病学杂志,1999,6:124-127.
[16]李晓光,郭玉璞,刘天慈.脊髓性肌萎缩症临床与肌肉病理学研究[J].中华神经科杂志,2000,33:32-35.
[17]常迈利,李玥.脊髓性肌萎缩症的基因诊断[J].中国优生与遗传杂志,2003,11:29.
[18]李文磊,吴婷,丁新生.脊髓性肌萎缩症的基因诊断[J].国际神经病学神经外科学杂志,2004,31:346-349.
[19]苏峻峰,陈万金,吴志英,等.短串联重复序列位点的优选及其在脊髓性肌萎缩症产前诊断中的应用[J].中华神经科杂志,2007,40:460-464.
[20]马素参,袁丽芳,刘天慈,等.进行性脊髓性肌萎缩症患者神经元存活基因及神经元凋亡抑制蛋白基因的缺失[J].中国医学科学院学报,2000,22:551-554.
[21]岳佳,李永刚.胚胎植入前遗传学筛查技术的应用进展[J].生殖医学杂志,2015,24:676-681.
[22]Staessen C,Tournaye H,Van Assche E,et al.PGD in 47,XXY Klinefelter’s syndrome patients[J].Hum Reprod Update,2003,9:319-330.
[2]王柠,何瑾,陈万金.脊髓性肌萎缩症临床诊断研究进展[J].中国现代神经疾病杂志,2012,3:252-256.
[3]Ar Rochmah M,Awano H,Awaya T,et al.Spinal muscular atrophy carriers with two SMN1copies[J].Brain Dev,2017,39:851-860.
[4]冯春,郑芳,彭剑虹,等.脊髓性肌萎缩症产前诊断病例分析并遗传咨询指导[J].中国优生与遗传杂志,2017,25:94-96.
[5]Li W.How do SMA-linked mutations of SMN1lead to structural/functional deficiency of the SMA protein?[J/OL].PLoS One,2017,12:e0178519.
[6]徐晨阳,项延包,卢金芳,等.温州地区14个脊髓性肌萎缩症家系临床表型与基因分析及产前诊断[J].中国优生与遗传杂志,2017,25:21-24.
[7]Lefebvre S,Bürglen L,Reboullet S,et al.Identification and characterization of a spinal muscular atrophy-determining gene[J].Cell,1995,80:155-165.
[8]Lefebvre S,Burlet P,Liu Q,et al.Correlation between severity and SMN protein level inspinal muscular atrophy[J].Nat Genet,1997,16:265-269.
[9]Ou J,Wang W,Feng T,et al.Identification of small segmental translocations in patients with repeated implantation failure and recurrent miscarriage using next generation sequencing after in vitro fertilization/intracytoplasmic sperm injection[J].Mol Cytogenet,2015,8:105.
[10]Iannaccone ST.Spinal muscular atrophy[J].Semin Neurol.1998,18:19-26.
[11]张蕾,颉小玲,李娟,等.脊髓性肌萎缩症遗传学及治疗研究进展[J].临床儿科杂志,2017,35:632-635.
[12]卢丽萍.脊髓性肌萎缩症SMN基因检测及其表达的研究[D].中国医科大学,2005:38-47.
[13]邓坤仪,彭建明,范汉恭,等.荧光定量PCR快速筛查脊髓性肌肉萎缩症smn1致病基因携带者[J].国际检验医学杂志,2016,37:2983-2984.
[14]曾光群,杨季云,张丁丁,等.脊髓性肌萎缩症2个核心家系SMN1基因分析[J].中国实用神经疾病杂志,2015,18:1-2.
[15]王柠,吴志英,慕容慎行,等.脊髓性肌萎缩症的快速基因诊断研究[J].中国神经免疫学和神经病学杂志,1999,6:124-127.
[16]李晓光,郭玉璞,刘天慈.脊髓性肌萎缩症临床与肌肉病理学研究[J].中华神经科杂志,2000,33:32-35.
[17]常迈利,李玥.脊髓性肌萎缩症的基因诊断[J].中国优生与遗传杂志,2003,11:29.
[18]李文磊,吴婷,丁新生.脊髓性肌萎缩症的基因诊断[J].国际神经病学神经外科学杂志,2004,31:346-349.
[19]苏峻峰,陈万金,吴志英,等.短串联重复序列位点的优选及其在脊髓性肌萎缩症产前诊断中的应用[J].中华神经科杂志,2007,40:460-464.
[20]马素参,袁丽芳,刘天慈,等.进行性脊髓性肌萎缩症患者神经元存活基因及神经元凋亡抑制蛋白基因的缺失[J].中国医学科学院学报,2000,22:551-554.
[21]岳佳,李永刚.胚胎植入前遗传学筛查技术的应用进展[J].生殖医学杂志,2015,24:676-681.
[22]Staessen C,Tournaye H,Van Assche E,et al.PGD in 47,XXY Klinefelter’s syndrome patients[J].Hum Reprod Update,2003,9:319-330.