【作者】 赵坤；

【导师】 吴志英； 王柠；

【作者基本信息】 福建医科大学 ， 神经病学， 2009， 博士

【摘要】 目的筛查并分析1996年至2008年我院神经内科所收集的来自全国的24例遗传性痉挛性截瘫(HSP)家系的先证者和32例散发性HSP患者spastin、atlastin、NIPA1和REEP1基因的突变,了解中国人这四个基因的突变特点和基因型-表型关系,为该病的基因诊断奠定基础。进一步对以上患者行MJD基因的筛查研究,明确是否存在MJD的痉挛性截瘫亚型。方法联合应用变性高效液相色谱分析(DHPLC)和DNA序列分析方法对以上HSP患者的gDNA进行spastin、atlastin和NIPA1基因的突变筛查,并对24例HSP家系的先证者进一步直接测序行以上3个基因的DNA序列分析;另外直接应用测序方法筛查以上全部患者的REEP1基因突变。采用PCR扩增后产物分别进行2%琼脂糖凝胶电泳和变性聚丙烯酰胺凝胶(PAGE)电泳的方法检测上述患者的MJD基因,条带异常的行测序证实。结果在1个AD-HSP家系中发现1个位于spastin基因上的1616+1g→t杂合突变,是一个新型突变;未发现atlastin基因突变;在1个AD-HSP家系中发现1个位于NIPA1基因上的c.316G＞A杂合突变,是一个已知突变;未发现SPG31基因突变。此外,我们还发现8种spastin基因多态、10种atlastin基因多态、1种NIPA1基因多态,还有5种REEP1基因多态。另外我们发现4个家系存在MJD基因变异,大片段CAG重复数分别为80/28、86/28、83/33和76/29。结论(1)首次在国内外报道spastin基因上的1616+1g→t杂合突变。(2)提示atlastin基因型在中国人中可能少见,其突变频率可能存在一定的种族差异。(3)报道NIPA1基因上的已知杂合突变c.316G＞A,提示为一突变热点;NIPA1基因型在中国人中可能少见,其突变频率与国外报道的基本一致。(4)首次在国内开展REEP1基因突变分析,提示REEP1基因型在中国人中可能少见,其突变频率可能存在一定的种族差异。(5)发现了以上4个基因的大量多态,丰富了其多态库;但突变在本组HSP患者中较少见,需要继续筛查HSP的其他基因。(6) 4例临床诊断HSP的患者通过基因诊断可以确诊为MJD,证实了HSP和SCAs临床表现的复杂性和基因诊断的意义。(7) DHPLC结合DNA序列分析是一种有效、经济的筛查突变的方法。更多还原

【Abstract】 ObjectivesTo detect the mutations of spastin,atlastin,NIPA1 and REEP1 Gene in Chinese patients with hereditary spastic paraplegia(HSP) and establish the base of the gene diagnosis of HSP.To screen the MJD gene changes for the patients above and ascertain the spastic paraplegia subtype of MJD.MethodsMutation analysis of spastin,atlastin and NIPA1 gene,carried out by polymerase chain reaction and DHPLC combined with sequencing in 56 unrelated HSP individuals consisting of the indexes from 24 HSP families and 32 sporadic cases.Then spastin, atlastin and NIPA1 genes of these 24 indexes,while REEP1 gene of all 56 individuals were screened by polymerase chain reaction combined with direct sequencing.MJD gene of individuals above were segregated by means of Agrose Gel Electrophoresis as well as Polyacrylamide Gel Electrophoresis after polymerase chain reaction proceeded, and then Changes of them detected were confirmed by sequencing.ResultsOne novel mutation of the spastin gene was identified(1616+1g＞t) in one autosomal dominant family;no disease causing mutation of the atlastin gene was detected in all cases;A reported mutation of the NIPA1 gene(c.316G＞A) was identified in an autosomal dominant family;no mutation was identified in REEP1 gene of all cases.8 polymorphisms in the spastin gene,10 polymorphisms in the atlastin gene,1 polymorphism in the NIPA1 gene and 5 polymorphisms in the REEP1 gene were identified.Conclusions(1) The mutation of spastin gene,1616+1g→t,have not been reported previously in the world. (2) The mutation of atlastin gene in Chinese HSP patients may be rare.The lower mutation rate may be correlated with racial diferentation.(3) We identified c.316G＞A,a mutation of NIPA1 gene in an autosomal dominant HSP family,which is a hotspot.(4) The REEP1 gene were first screened in Chinese patients,mutations of which may be rare.(5) We also found a lot of polymorphisms in the four genes.Polymorphisms are common in HSP patients,but mutations are less in the four genes.We should detect other genes of HSP.(6) We found changes of the MJD gene occure in 4 previously diagnosed HSP families, suggesting the importance of gene screening.(7) DHPLC together with DNA sequencing is an efficient and economical method for screening mutations.更多还原