假肥大型肌营养不良症(DMD/BMD)遗传学诊断及剪接突变的致病性分析（英文）
|
摘要
目的:针对100例无亲缘关系的假肥大型肌营养不良症(DMD/BMD)患者,联合应用多种检测技术进行遗传学诊断,并且建立个体化产前诊断及胚胎植入前遗传学诊断方案,以最大限度地降低DMD/BMD患儿的出生。创新点:通过minigene剪接实验分析DMD:c.1149+1G>A和c.1150-2A>G突变是否导致剪接异常,并确定剪接方式。方法:收集100例无亲缘关系DMD/BMD患者的临床资料,应用多重连接依赖式探针扩增技术(MLPA)、第二代测序(NGS)、minigene剪接实验(HMSA)进行遗传学诊断,并通过单体型分析及性别鉴定进行胚胎植入前遗传学诊断。结论:联合应用多种检测技术可以尽早地对患者进行遗传学诊断,为临床遗传咨询和产前诊断及胚胎植入前遗传学诊断提供了科学依据。
Duchenne muscular dystrophy(DMD) and Becker muscular dystrophy(BMD) are caused by mutations in the DMD gene. The aim of this study is to identify pathogenic DMD variants in probands and reduce the risk of recurrence of the disease in affected families. Variations in 100 unrelated DMD/BMD patients were detected by multiplex ligation-dependent probe amplification(MLPA) and next-generation sequencing(NGS). Pathogenic variants in DMD were successfully identified in all cases, and 11 of them were novel. The most common mutations were intragenic deletions(69%), with two hotspots located in the 5' end(exons 2–19) and the central of the DMD gene(exons 45–55), while point mutations were observed in 22% patients. Further, c.1149+1 G>A and c.1150-2 A>G were confirmed by hybrid minigene splicing assay(HMSA). This two splice site mutations would lead to two aberrant DMD isoforms which give rise to severely truncated protein. Therefore, the clinical use of MLPA, NGS, and HMSA is an effective strategy to identify variants. Importantly, eight embryos were terminated pregnancies according to prenatal diagnosis and a healthy boy was successfully delivered by preimplantation genetic diagnosis(PGD). Early and accurate genetic diagnosis is essential for prenatal diagnosis/PGD to reduce the risk of recurrence of DMD in affected families.
Duchenne muscular dystrophy(DMD) and Becker muscular dystrophy(BMD) are caused by mutations in the DMD gene. The aim of this study is to identify pathogenic DMD variants in probands and reduce the risk of recurrence of the disease in affected families. Variations in 100 unrelated DMD/BMD patients were detected by multiplex ligation-dependent probe amplification(MLPA) and next-generation sequencing(NGS). Pathogenic variants in DMD were successfully identified in all cases, and 11 of them were novel. The most common mutations were intragenic deletions(69%), with two hotspots located in the 5' end(exons 2–19) and the central of the DMD gene(exons 45–55), while point mutations were observed in 22% patients. Further, c.1149+1 G>A and c.1150-2 A>G were confirmed by hybrid minigene splicing assay(HMSA). This two splice site mutations would lead to two aberrant DMD isoforms which give rise to severely truncated protein. Therefore, the clinical use of MLPA, NGS, and HMSA is an effective strategy to identify variants. Importantly, eight embryos were terminated pregnancies according to prenatal diagnosis and a healthy boy was successfully delivered by preimplantation genetic diagnosis(PGD). Early and accurate genetic diagnosis is essential for prenatal diagnosis/PGD to reduce the risk of recurrence of DMD in affected families.
引文
Bakker E,Veenema H,den Dunnen JT,et al.,1989.Germinal mosaicism increases the recurrence risk for‘new’Duchenne muscular dystrophy mutations.J Med Genet,26(9):553-559.https://doi.org/10.1136/jmg.26.9.553
Baralle D,Baralle M,2005.Splicing in action:assessing disease causing sequence changes.J Med Genet,42(10):737-748.https://doi.org/10.1136/jmg.2004.029538
Beggs AH,Koenig M,Boyce FM,et al.,1990.Detection of98%of DMD/BMD gene deletions by polymerase chain reaction.Hum Genet,86(1):45-48.https://doi.org/10.1007/BF00205170
Bovolenta M,Neri M,Fini S,et al.,2008.A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies.BMC Genomics,9:572.https://doi.org/10.1186/1471-2164-9-572
Buratti E,Baralle FE,2004.Influence of RNA secondary structure on the pre-m RNA splicing process.Mol Cell Biol,24(24):10505-10514.https://doi.org/10.1128/MCB.24.24.10505-10514.2004
Buratti E,Baralle M,Baralle FE,2006.Defective splicing,disease and therapy:searching for master checkpoints in exon definition.Nucleic Acids Res,34(12):3494-3510.https://doi.org/10.1093/nar/gkl498
Burset M,Seledtsov IA,Solovyev VV,2001.SpliceDB:database of canonical and non-canonical mammalian splice sites.Nucleic Acids Res,29(1):255-259.https://doi.org/10.1093/nar/29.1.255
Chen LJ,Diao ZY,Xu ZP,et al.,2016.The clinical application of preimplantation genetic diagnosis for the patient affected by congenital contractural arachnodactyly and spinal and bulbar muscular atrophy.J Assist Reprod Genet,33(11):1459-1466.https://doi.org/10.1007/s10815-016-0760-y
Echevarria L,Aupy P,Goyenvalle A,2018.Exon-skipping advances for Duchenne muscular dystrophy.Hum Mol Genet,27(R2):R163-R172.https://doi.org/10.1093/hmg/ddy171
Fairclough RJ,Wood MJ,Davies KE,2013.Therapy for Duchenne muscular dystrophy:renewed optimism from genetic approaches.Nat Rev Genet,14(6):373-378.https://doi.org/10.1038/nrg3460
Flanigan KM,von Niederhausern A,Dunn DM,et al.,2003.Rapid direct sequence analysis of the dystrophin gene.Am J Hum Genet,72(4):931-939.https://doi.org/10.1086/374176
Girardet A,Hamamah S,Dechaud H,et al.,2003.Specific detection of deleted and non-deleted dystrophin exons together with gender assignment in preimplantation genetic diagnosis of Duchenne muscular dystrophy.Mol Hum Reprod,9(7):421-427.https://doi.org/10.1093/molehr/gag050
Haas J,Katus HA,Meder B,2011.Next-generation sequencing entering the clinical arena.Mol Cell Probes,25(5-6):206-211.https://doi.org/10.1016/j.mcp.2011.08.005
Habara Y,Takeshima Y,Awano H,et al.,2009.In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by+1G→A mutations in introns of the dystrophin gene.J Med Genet,46(8):542-547.https://doi.org/10.1136/jmg.2008.061259
Hertel KJ,2008.Combinatorial control of exon recognition.JBiol Chem,283(3):1211-1215.https://doi.org/10.1074/jbc.R700035200
Jefferies JL,Eidem BW,Belmont JW,et al.,2005.Genetic predictors and remodeling of dilated cardiomyopathy in muscular dystrophy.Circulation,112(18):2799-2804.https://doi.org/10.1161/CIRCULATIONAHA.104.528281
Juan-Mateu J,González-Quereda L,Rodríguez MJ,et al.,2013.Interplay between DMD point mutations and splicing signals in dystrophinopathy phenotypes.PLo SONE,8(3):e59916.https://doi.org/10.1371/journal.pone.0059916
Kralovicova J,Hwang G,Asplund AC,et al.,2011.Compensatory signals associated with the activation of human GC5'splice sites.Nucleic Acids Res,39(16):7077-7091.https://doi.org/10.1093/nar/gkr306
Krawczak M,Thomas NST,Hundrieser B,et al.,2007.Single base-pair substitutions in exon-intron junctions of human genes:nature,distribution,and consequences for mRNAsplicing.Hum Mutat,28(2):150-158.https://doi.org/10.1002/humu.20400
Lalic T,Vossen RHAM,Coffa J,et al.,2005.Deletion and duplication screening in the DMD gene using MLPA.Eur J Hum Genet,13(11):1231-1234.https://doi.org/10.1038/sj.ejhg.5201465
Lee SH,Kwak IP,Cha KE,et al.,1998.Preimplantation diagnosis of non-deletion Duchenne muscular dystrophy(DMD)by linkage polymerase chain reaction analysis.Mol Hum Reprod,4(4):345-349.https://doi.org/10.1093/molehr/4.4.345
Li Y,Pan Q,Gu YS,2017.Phenotype-genotype correlation with Sanger sequencing identified retinol dehydrogenase12(RDH12)compound heterozygous variants in a Chinese family with Leber congenital amaurosis.J Zhejiang Univ-Sci B(Biomed&Biotechnol),18(5):421-429.https://doi.org/10.1631/jzus.B1600156
Ma PP,Zhang S,Zhang H,et al.,2018.Comprehensive genetic characteristics of dystrophinopathies in China.Orphanet J Rare Dis,13:109.https://doi.org/10.1186/s13023-018-0853-z
Muntoni F,Torelli S,Ferlini A,2003.Dystrophin and mutations:one gene,several proteins,multiple phenotypes.Lancet Neurol,2(12):731-740.https://doi.org/10.1016/S1474-4422(03)00585-4
Parada GE,Munita R,Cerda CA,et al.,2014.A comprehensive survey of non-canonical splice sites in the human transcriptome.Nucleic Acids Res,42(16):10564-10578.https://doi.org/10.1093/nar/gku744
Pico AR,Bader GD,Demchak B,et al.,2014.The Cytoscape app article collection.F1000Res,3:138.https://doi.org/10.12688/f1000research.4642.1
Rando TA,2001.The dystrophin-glycoprotein complex,cellular signaling,and the regulation of cell survival in the muscular dystrophies.Muscle Nerve,24(12):1575-1594.https://doi.org/10.1002/mus.1192
Roberts RG,Gardner RJ,Bobrow M,1994.Searching for the 1in 2,400,000:a review of dystrophin gene point mutations.Hum Mutat,4(1):1-11.https://doi.org/10.1002/humu.1380040102
Roca X,Sachidanandam R,Krainer AR,2003.Intrinsic differences between authentic and cryptic 5'splice sites.Nucleic Acids Res,31(21):6321-6333.https://doi.org/10.1093/nar/gkg830
Richards S,Aziz N,Bale S,et al.,2015.Standards and guidelines for the interpretation of sequence variants:a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet Med,17(5):405-424.https://doi.org/10.1038/gim.2015.30
Sakthivel Murugan SM,Arthi C,Thilothammal N,et al.,2013.Carrier detection in Duchenne muscular dystrophy using molecular methods.Indian J Med Res,137(6):1102-1110.
Schouten JP,McElgunn CJ,Waaijer R,et al.,2002.Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res,30(12):e57.https://doi.org/10.1093/nar/gnf056
Stockley TL,Akber S,Bulgin N,et al.,2006.Strategy for comprehensive molecular testing for Duchenne and Becker muscular dystrophies.Genet Test,10(4):229-243.https://doi.org/10.1089/gte.2006.10.229
Takeshima Y,Yagi M,Okizuka Y,et al.,2010.Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center.J Hum Genet,55(6):379-388.https://doi.org/10.1038/jhg.2010.49
Tuffery-Giraud S,Béroud C,Leturcq F,et al.,2009.Genotypephenotype analysis in 2,405 patients with a dystrophinopathy using the UMD-DMD database:a model of nationwide knowledgebase.Hum Mutat,30(6):934-945.https://doi.org/10.1002/humu.20976
van Essen AJ,Abbs S,Baiget M,et al.,1992.Parental origin and germline mosaicism of deletions and duplications of the dystrophin gene:a European study.Hum Genet,88(3):249-257.https://doi.org/10.1007/BF00197255
Wei XM,Dai Y,Yu P,et al.,2013.Targeted next-generation sequencing as a comprehensive test for patients with and female carriers of DMD/BMD:a multi-population diagnostic study.Eur J Hum Genet,22(1):110-118.https://doi.org/10.1038/ejhg.2013.82
Xia ZJ,Lin J,Lu LP,et al.,2018.An intronic mutation c.6430-3C>G in the F8 gene causes splicing efficiency and premature termination in hemophilia A.Blood Coagul Fibrinoly,29(4):381-386.https://doi.org/10.1097/MBC.0000000000000730
Ye Y,Yu P,Yong J,et al.,2014.Preimplantational genetic diagnosis and mutation detection in a family with duplication mutation of DMD gene.Gynecol Obstet Invest,78(4):272-278.https://doi.org/10.1159/000365083
Zhang XHF,Leslie CS,Chasin LA,2005.Dichotomous splicing signals in exon flanks.Genome Res,15(6):768-779.https://doi.org/10.1101/gr.3217705
Zimowski JG,Massalska D,Holding M,et al.,2014.MLPAbased detection of mutations in the dystrophin gene of 180Polish families with Duchenne/Becker muscular dystrophy.Neurol Neurochir Pol,48(6):416-422.https://doi.org/10.1016/j.pjnns.2014.10.004
Baralle D,Baralle M,2005.Splicing in action:assessing disease causing sequence changes.J Med Genet,42(10):737-748.https://doi.org/10.1136/jmg.2004.029538
Beggs AH,Koenig M,Boyce FM,et al.,1990.Detection of98%of DMD/BMD gene deletions by polymerase chain reaction.Hum Genet,86(1):45-48.https://doi.org/10.1007/BF00205170
Bovolenta M,Neri M,Fini S,et al.,2008.A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies.BMC Genomics,9:572.https://doi.org/10.1186/1471-2164-9-572
Buratti E,Baralle FE,2004.Influence of RNA secondary structure on the pre-m RNA splicing process.Mol Cell Biol,24(24):10505-10514.https://doi.org/10.1128/MCB.24.24.10505-10514.2004
Buratti E,Baralle M,Baralle FE,2006.Defective splicing,disease and therapy:searching for master checkpoints in exon definition.Nucleic Acids Res,34(12):3494-3510.https://doi.org/10.1093/nar/gkl498
Burset M,Seledtsov IA,Solovyev VV,2001.SpliceDB:database of canonical and non-canonical mammalian splice sites.Nucleic Acids Res,29(1):255-259.https://doi.org/10.1093/nar/29.1.255
Chen LJ,Diao ZY,Xu ZP,et al.,2016.The clinical application of preimplantation genetic diagnosis for the patient affected by congenital contractural arachnodactyly and spinal and bulbar muscular atrophy.J Assist Reprod Genet,33(11):1459-1466.https://doi.org/10.1007/s10815-016-0760-y
Echevarria L,Aupy P,Goyenvalle A,2018.Exon-skipping advances for Duchenne muscular dystrophy.Hum Mol Genet,27(R2):R163-R172.https://doi.org/10.1093/hmg/ddy171
Fairclough RJ,Wood MJ,Davies KE,2013.Therapy for Duchenne muscular dystrophy:renewed optimism from genetic approaches.Nat Rev Genet,14(6):373-378.https://doi.org/10.1038/nrg3460
Flanigan KM,von Niederhausern A,Dunn DM,et al.,2003.Rapid direct sequence analysis of the dystrophin gene.Am J Hum Genet,72(4):931-939.https://doi.org/10.1086/374176
Girardet A,Hamamah S,Dechaud H,et al.,2003.Specific detection of deleted and non-deleted dystrophin exons together with gender assignment in preimplantation genetic diagnosis of Duchenne muscular dystrophy.Mol Hum Reprod,9(7):421-427.https://doi.org/10.1093/molehr/gag050
Haas J,Katus HA,Meder B,2011.Next-generation sequencing entering the clinical arena.Mol Cell Probes,25(5-6):206-211.https://doi.org/10.1016/j.mcp.2011.08.005
Habara Y,Takeshima Y,Awano H,et al.,2009.In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by+1G→A mutations in introns of the dystrophin gene.J Med Genet,46(8):542-547.https://doi.org/10.1136/jmg.2008.061259
Hertel KJ,2008.Combinatorial control of exon recognition.JBiol Chem,283(3):1211-1215.https://doi.org/10.1074/jbc.R700035200
Jefferies JL,Eidem BW,Belmont JW,et al.,2005.Genetic predictors and remodeling of dilated cardiomyopathy in muscular dystrophy.Circulation,112(18):2799-2804.https://doi.org/10.1161/CIRCULATIONAHA.104.528281
Juan-Mateu J,González-Quereda L,Rodríguez MJ,et al.,2013.Interplay between DMD point mutations and splicing signals in dystrophinopathy phenotypes.PLo SONE,8(3):e59916.https://doi.org/10.1371/journal.pone.0059916
Kralovicova J,Hwang G,Asplund AC,et al.,2011.Compensatory signals associated with the activation of human GC5'splice sites.Nucleic Acids Res,39(16):7077-7091.https://doi.org/10.1093/nar/gkr306
Krawczak M,Thomas NST,Hundrieser B,et al.,2007.Single base-pair substitutions in exon-intron junctions of human genes:nature,distribution,and consequences for mRNAsplicing.Hum Mutat,28(2):150-158.https://doi.org/10.1002/humu.20400
Lalic T,Vossen RHAM,Coffa J,et al.,2005.Deletion and duplication screening in the DMD gene using MLPA.Eur J Hum Genet,13(11):1231-1234.https://doi.org/10.1038/sj.ejhg.5201465
Lee SH,Kwak IP,Cha KE,et al.,1998.Preimplantation diagnosis of non-deletion Duchenne muscular dystrophy(DMD)by linkage polymerase chain reaction analysis.Mol Hum Reprod,4(4):345-349.https://doi.org/10.1093/molehr/4.4.345
Li Y,Pan Q,Gu YS,2017.Phenotype-genotype correlation with Sanger sequencing identified retinol dehydrogenase12(RDH12)compound heterozygous variants in a Chinese family with Leber congenital amaurosis.J Zhejiang Univ-Sci B(Biomed&Biotechnol),18(5):421-429.https://doi.org/10.1631/jzus.B1600156
Ma PP,Zhang S,Zhang H,et al.,2018.Comprehensive genetic characteristics of dystrophinopathies in China.Orphanet J Rare Dis,13:109.https://doi.org/10.1186/s13023-018-0853-z
Muntoni F,Torelli S,Ferlini A,2003.Dystrophin and mutations:one gene,several proteins,multiple phenotypes.Lancet Neurol,2(12):731-740.https://doi.org/10.1016/S1474-4422(03)00585-4
Parada GE,Munita R,Cerda CA,et al.,2014.A comprehensive survey of non-canonical splice sites in the human transcriptome.Nucleic Acids Res,42(16):10564-10578.https://doi.org/10.1093/nar/gku744
Pico AR,Bader GD,Demchak B,et al.,2014.The Cytoscape app article collection.F1000Res,3:138.https://doi.org/10.12688/f1000research.4642.1
Rando TA,2001.The dystrophin-glycoprotein complex,cellular signaling,and the regulation of cell survival in the muscular dystrophies.Muscle Nerve,24(12):1575-1594.https://doi.org/10.1002/mus.1192
Roberts RG,Gardner RJ,Bobrow M,1994.Searching for the 1in 2,400,000:a review of dystrophin gene point mutations.Hum Mutat,4(1):1-11.https://doi.org/10.1002/humu.1380040102
Roca X,Sachidanandam R,Krainer AR,2003.Intrinsic differences between authentic and cryptic 5'splice sites.Nucleic Acids Res,31(21):6321-6333.https://doi.org/10.1093/nar/gkg830
Richards S,Aziz N,Bale S,et al.,2015.Standards and guidelines for the interpretation of sequence variants:a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet Med,17(5):405-424.https://doi.org/10.1038/gim.2015.30
Sakthivel Murugan SM,Arthi C,Thilothammal N,et al.,2013.Carrier detection in Duchenne muscular dystrophy using molecular methods.Indian J Med Res,137(6):1102-1110.
Schouten JP,McElgunn CJ,Waaijer R,et al.,2002.Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res,30(12):e57.https://doi.org/10.1093/nar/gnf056
Stockley TL,Akber S,Bulgin N,et al.,2006.Strategy for comprehensive molecular testing for Duchenne and Becker muscular dystrophies.Genet Test,10(4):229-243.https://doi.org/10.1089/gte.2006.10.229
Takeshima Y,Yagi M,Okizuka Y,et al.,2010.Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center.J Hum Genet,55(6):379-388.https://doi.org/10.1038/jhg.2010.49
Tuffery-Giraud S,Béroud C,Leturcq F,et al.,2009.Genotypephenotype analysis in 2,405 patients with a dystrophinopathy using the UMD-DMD database:a model of nationwide knowledgebase.Hum Mutat,30(6):934-945.https://doi.org/10.1002/humu.20976
van Essen AJ,Abbs S,Baiget M,et al.,1992.Parental origin and germline mosaicism of deletions and duplications of the dystrophin gene:a European study.Hum Genet,88(3):249-257.https://doi.org/10.1007/BF00197255
Wei XM,Dai Y,Yu P,et al.,2013.Targeted next-generation sequencing as a comprehensive test for patients with and female carriers of DMD/BMD:a multi-population diagnostic study.Eur J Hum Genet,22(1):110-118.https://doi.org/10.1038/ejhg.2013.82
Xia ZJ,Lin J,Lu LP,et al.,2018.An intronic mutation c.6430-3C>G in the F8 gene causes splicing efficiency and premature termination in hemophilia A.Blood Coagul Fibrinoly,29(4):381-386.https://doi.org/10.1097/MBC.0000000000000730
Ye Y,Yu P,Yong J,et al.,2014.Preimplantational genetic diagnosis and mutation detection in a family with duplication mutation of DMD gene.Gynecol Obstet Invest,78(4):272-278.https://doi.org/10.1159/000365083
Zhang XHF,Leslie CS,Chasin LA,2005.Dichotomous splicing signals in exon flanks.Genome Res,15(6):768-779.https://doi.org/10.1101/gr.3217705
Zimowski JG,Massalska D,Holding M,et al.,2014.MLPAbased detection of mutations in the dystrophin gene of 180Polish families with Duchenne/Becker muscular dystrophy.Neurol Neurochir Pol,48(6):416-422.https://doi.org/10.1016/j.pjnns.2014.10.004
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |