疑似脊髓性肌萎缩症患儿338例的运动神经元存活基因分析

Molecular analysis of survival motor neuron gene in 338 suspicious children patients with spinal muscular atrophy

目的研究儿童脊髓性肌萎缩症（SMA）运动神经元存活基因SMN1缺失和诊断的意义。方法根据国际诊断标准、病例随访和基因分析结果对338例疑似SMA的患儿进行诊断和分型。应用PCR-酶切方法分析患儿SMN1基因外显子7和外显子8的纯合缺失。应用等位基因特异PCR结合变性高效液相色谱分析（DHPLC）方法分析患儿的SMN1基因拷贝数，确定杂合缺失。结果（1）确诊SMA267例，其中Ⅰ型143例，Ⅱ型82例，Ⅲ型42例，分别占53．6％、30．7％和15．7％。（2）267例SMA患儿的SMN1基因缺失分析显示：SMN1基因外显子7和8均纯合缺失为183例，占68．5％（183／267），仅外显子7纯合缺失，外显子8不缺失为34例，占12．7％（34／267），外显子7杂合缺失为33例，占12．4％（33／267），非缺失为17例，占6．4％（17／267），未见SMN1基因外显子8的单独缺失。（3）Ⅰ型和Ⅱ型SMN1基因缺失率相近。Ⅲ型SMN1基因纯合缺失率较低于Ⅰ型和Ⅱ型，杂合缺失率较高于Ⅰ型和Ⅱ型。结论（1）我国儿童SMA的SMN1基因纯合缺失和杂合缺失频率提示，SMN1基因突变存在种族异质性，SMN1基因内微小突变需要研究。（2）SMN1基因诊断具有特异性和无创性，80％SMA患儿通过SMN1基因纯合缺失分析得到诊断。（3）Ⅲ型SMA的临床诊断和基因分析需要进一步研究。

Objective Spinal muscular atrophy （SMA） is an autosomal recessive disorder that results in symmetrical muscle weakness and wasting due to degeneration of the anterior horns of the spinal cord. The clinical picture of SMA is variable and childhood SMA has been classified into 3 types on the basis of the age of onset and clinical course. The survival motor neuron （SMN） gene was mapped to chromosome 5q13. The SMN1 gene has been recognized to be responsible for SMA because of homozygouse deletions or intragenic mutations in SMN1 results in childhood onset of SMA. The main objective of this study was to determine the deletion frequency of SMN1 gene and to apply gene analysis in children patients with SMA. Methods The SMA patients were diagnosed and clinically typed according to the international diagnostic criteria, following up cases, and gene analysis. The PCR enzyme assay was used to detect the homozygous deletion of SMN1 gene in SMA patients. A dosage assay that combined multiplexed allele-specific PCR and DHPLC was used to determine the copy numbers of the SMN1 and SMN2 and detect SMN1 heterozygous deletion. Results （ 1 ） A total of 267 patients with SMA were diagnosed from 338 suspicious cases and 143, 82, and 42 cases were typed as types Ⅰ ,Ⅱ, and Ⅲ with the percentages of 53.6% （ 143/267）, 30.7% （82/267） and 15.7% （42/267）, respectively. （2） Results of the present study showed that 68.5% （ 183/267 ） of SMA patients had homozygous deletions of exons 7 and 8 of SMN1 gene and 12.7% （34/267） had homozygous deletions of only exon 7 of SMN1 gene. The SMN1 heterozygous deletion was confirmed in 12.4% （33/267） of SMA patients. Non-deletion SMA patients accounted for 6.4% （17/267）.The homozygous deletions of only exon 8 of SMN1 gene could not be detected. （3） The rates of homozygous or heterozygous deletion in types Ⅰand Ⅱwere very similar. The rate of homozygous deletion was lower in type Ⅲ than that in type Ⅰ or Ⅱ and rate of heterozygous deletion of type Ⅲ was higher than that in types Ⅰ or Ⅱ. Condusion （ 1 ） The frequency and pattern of deletions in the Chinese children patients with SMA are significantly different from that observed in Caucasians populations. Further gene characterization and subtle mutations within the SMN1 gene need to be studied in order to define the molecular basis of SMA in the Chinese population. （2） The gene diagnosis is a special and non invasive method as compared with other methods. A total of 80% patients can be diagnosed through the analysis of the homozygous deletion of SMN1 gene. （3） The clinical diagnosis and gene detection need to be studied in future for the SMA patients with type Ⅲ.