儿童脊肌萎缩症的基因诊断
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摘要
目的对临床诊断疑似为脊肌萎缩症(SMA)的患儿进行运动神经元生存基因(SMN)缺失分析,通过基因诊断对SMA进行确诊。方法收集2015年1月至2016年12月上海市儿童医院新生儿科及神经内科就诊的疑似SMA患儿29例,患儿就诊年龄11 d～14岁。采用多重连接探针扩增(MLPA)技术对上述患儿外周血DNA样本进行基因诊断。结果 15例存在SMA致病突变,疑似患儿的SMA基因诊断率为51.7%(15/29)。其中8例为SMN1基因外显子7和外显子8纯合缺失;3例为SMN1基因外显子7和外显子8纯合缺失,SMN2基因外显子7拷贝数增加;4例为SMN1基因外显子7纯合缺失,外显子8杂合缺失。另外14例存在不直接致病的突变。按照SMA国际诊断标准,15例确诊患者中Ⅰ型5例,Ⅱ型5例,Ⅲ型5例,均占33.3%。其中Ⅰ型的5例患儿均为SMN1基因外显子7、8纯合缺失,仅有1例存在SMN2基因外显子7拷贝的轻度增加。结论 MLPA技术灵敏度较高,可作为SMA疑似患者首选的诊断方法,为SMA的明确诊断提供依据。
Objective To analyze the deletion of survival motor neuron(SMN) gene in children suspected of spinal muscular atrophy(SMA), and to diagnose SMA by gene diagnosis. Methods A total of 29 patients suspected of SMA were recruited from January 2015 to December 2016 in the Department of Neonatology or Neurology of Shanghai Children's Hospital. The age of these patients ranged from 11 days to 14 years old. Gene diagnosis of SMN1 and SMN2 were performed by Mutiplex Ligation-dependent Probe Amplification(MLPA) in these peripheral DNA samples.Results Pathological mutations were identified in 15 cases, and the diagnosis rate in suspected cases was 51.7%(15/29). Among them, 8 cases had homozygous deletion in exon 7 and exon 8 of SMN1 gene, 3 cases had homozygous deletion in exon 7 and exon 8 of SMN1 gene combined with copy number increase in exon 7 of SMN2 gene, and 4 cases had homozygous deletion in exon 7 and heterozygous deletion in exon 8 of SMN1 gene. In other 14 cases, some non-pathogenic mutations were identified. According to the international diagnostic criteria of SMA, 5 cases of type Ⅰ, 5 cases of type Ⅱ, and 5 cases of type Ⅲ were diagnosed, accounting for 33.3%, 33.3%, 33.3%, respectively. Homozygous deletion in exon 7 and exon 8 of SMN1 gene were identified in all of the cases of type Ⅰ, while slight copy number increase in exon 7 of SMN2 gene were only found in one case. Conclusion MLPA is a high sensitive method for SMA diagnosis, which can be considered as the preferred genetic test for the suspected SMA patients. It would provide basis for a definitive diagnosis of SMA.
Objective To analyze the deletion of survival motor neuron(SMN) gene in children suspected of spinal muscular atrophy(SMA), and to diagnose SMA by gene diagnosis. Methods A total of 29 patients suspected of SMA were recruited from January 2015 to December 2016 in the Department of Neonatology or Neurology of Shanghai Children's Hospital. The age of these patients ranged from 11 days to 14 years old. Gene diagnosis of SMN1 and SMN2 were performed by Mutiplex Ligation-dependent Probe Amplification(MLPA) in these peripheral DNA samples.Results Pathological mutations were identified in 15 cases, and the diagnosis rate in suspected cases was 51.7%(15/29). Among them, 8 cases had homozygous deletion in exon 7 and exon 8 of SMN1 gene, 3 cases had homozygous deletion in exon 7 and exon 8 of SMN1 gene combined with copy number increase in exon 7 of SMN2 gene, and 4 cases had homozygous deletion in exon 7 and heterozygous deletion in exon 8 of SMN1 gene. In other 14 cases, some non-pathogenic mutations were identified. According to the international diagnostic criteria of SMA, 5 cases of type Ⅰ, 5 cases of type Ⅱ, and 5 cases of type Ⅲ were diagnosed, accounting for 33.3%, 33.3%, 33.3%, respectively. Homozygous deletion in exon 7 and exon 8 of SMN1 gene were identified in all of the cases of type Ⅰ, while slight copy number increase in exon 7 of SMN2 gene were only found in one case. Conclusion MLPA is a high sensitive method for SMA diagnosis, which can be considered as the preferred genetic test for the suspected SMA patients. It would provide basis for a definitive diagnosis of SMA.
引文
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[4]BURLET P,BüRGLEN L,CLERMONT O,et al.Large scale deletions of the 5q13 region are specific to Werdnig-Hoffmann disease[J].J Med Genet,1996,33(4):281-283.
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[6]D'AMICO A,MERCURI E,TIZIANO FD,et al.Spinal muscular atrophy[J].Orphanet J Rare Dis,2011,6:71.
[7]ROWLAND LP,MCLEOD JG.Classification of neuromuscular disorders[J].J Neurol Sci,1994,124(Suppl):109-130.
[8]LEFEBVRE S,BüRGLEN L,REBOULLET S,et al.Identification and characterization of a spinal muscular atrophy-determining gene[J].Cell,1995,80(1):155-165.
[9]FARRAR MA,KIERNAN MC.The Genetics of Spinal Muscular Atrophy:Progress and Challenges[J].Neurotherapeutics,2015,12(2):290-302.
[10]SCHOUTEN JP,MCELGUNN CJ,WAAIJER R,et al.Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification[J].Nucleic Acids Res,2002,30(12):e57.
[11]章印红,贺静,张云茜,等.154例脊肌萎缩症疑诊患者的基因诊断及其临床价值[J].中华检验医学杂志,2015,38(12):833-837.
[12]CARTEGNI L,KRAINER AR.Correction of disease-associated exon skipping by synthetic exon-specific activators[J].Nat Struct Biol,2003,10(2):120-125.
[13]FINKEL RS,CHIRIBOGA CA,VAJSAR J,et al.Treatment of infantile-onset spinal muscular atrophy with nusinersen:a phase 2,open-label,dose-escalation study[J].Lancet,2016,388(10063):3017-3026.r atrophy[J].Orphanet J Rare Dis,2011,6:71.
[2]MUNSAT TL,DAVIES KE.International SMA consortium meeting.(26-28 June 1992,Bonn,Germany)[J].Neuromuscul Disord,1992,2(5-6):423-428.
[3]DARRAS BT.Spinal muscular atrophies[J].Pediatr Clin North Am,2015,62(3):743-766.
[4]BURLET P,BüRGLEN L,CLERMONT O,et al.Large scale deletions of the 5q13 region are specific to Werdnig-Hoffmann disease[J].J Med Genet,1996,33(4):281-283.
[5]MONANI UR,LORSON CL,PARSONS DW,et al.A single nucleotide difference that alters splicing patterns distinguishes the SMAgene SMN1 from the copy gene SMN2[J].Hum Mol Genet,1999,8(7):1177-1183.
[6]D'AMICO A,MERCURI E,TIZIANO FD,et al.Spinal muscular atrophy[J].Orphanet J Rare Dis,2011,6:71.
[7]ROWLAND LP,MCLEOD JG.Classification of neuromuscular disorders[J].J Neurol Sci,1994,124(Suppl):109-130.
[8]LEFEBVRE S,BüRGLEN L,REBOULLET S,et al.Identification and characterization of a spinal muscular atrophy-determining gene[J].Cell,1995,80(1):155-165.
[9]FARRAR MA,KIERNAN MC.The Genetics of Spinal Muscular Atrophy:Progress and Challenges[J].Neurotherapeutics,2015,12(2):290-302.
[10]SCHOUTEN JP,MCELGUNN CJ,WAAIJER R,et al.Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification[J].Nucleic Acids Res,2002,30(12):e57.
[11]章印红,贺静,张云茜,等.154例脊肌萎缩症疑诊患者的基因诊断及其临床价值[J].中华检验医学杂志,2015,38(12):833-837.
[12]CARTEGNI L,KRAINER AR.Correction of disease-associated exon skipping by synthetic exon-specific activators[J].Nat Struct Biol,2003,10(2):120-125.
[13]FINKEL RS,CHIRIBOGA CA,VAJSAR J,et al.Treatment of infantile-onset spinal muscular atrophy with nusinersen:a phase 2,open-label,dose-escalation study[J].Lancet,2016,388(10063):3017-3026.r atrophy[J].Orphanet J Rare Dis,2011,6:71.