摘要
遗传性痉挛性截瘫(Hereditary spastic paraplegia,HSP)是一组以双下肢进行性肌张力增高和无力、痉挛步态为特征的具有明显遗传异质性的综合征,有常染色体显性、隐性和X连锁隐性三种遗传方式。患病率为2/10万~10/10万。发病机制尚不详。HSP至少有43个致病基因相关位点,按发现时间顺序依次命名为SPG1～SPG43。其中SPG2为X连锁隐性遗传,致病基因位于Xq21～22,编码蛋白脂蛋白1(PLP1),主要表现为单纯型,即仅有痉挛性截瘫,无脊髓外系的症状。本研究对一遗传性痉挛性截瘫家系进行研究,根据临床症状及遗传方式,对照孟德尔在线已公布的资料定位于SPG2,应用PCR体外克隆方法获得SPG2基因的片段,对获得序列进行测序。发现先证者exon6上的第844位碱基存在点突变,由T突变为C,引起了氨基酸的变化,由苯丙氨酸突变为亮氨酸。另一发病者exon6上的第844位碱基为C,但从基因测序的信号来分析为T和C的混杂信号,不排除此点也存在突变。遗传性痉挛性截瘫目前尚无有效的根治手段,及早诊断出遗传病是防治的第一步。防止患儿的出生是根治的重要途径,也是提高人群生活质量的主要因素。
Objective: Hereditary spastic paraplegia is a kind of inherited and neurodegenerative diseases, with significant genetic heterogeneity. The patients have slowly progressive lower extremity spastic paraplegia, scissors gait which lately occurred in the upper limbs . The diseases had high disability and seriously affected quality of life. Now there is no special treatment , so the only way to prevent sick children from born is prenatal diagnosis .At the same time ,this method is the most important factor which can improve the quality of people’s life . DNA sequences of a hereditary spastic paraplegia pedigree were detected in order to explore the family's gene mutation sites and further the value of clinical application.
Methods: Genomic DNA was extracted from blood samples of a hereditary spastic paraplegia pedigree , SPG2 was posited through the clinical symptoms and genetic mode. PLP1 was disease-causing genes. First cloning primers were designed , PLP1 exon3, exon5 were amplified by PCR, when the amount reached a certain level, products were recycled. Second PLP1 exon6, exon7 amplified by PCR were connected with T vector after recycling ,then transformed . Bacteria were shook and plasmid was extracted. PLP1 exon3, exon5 and exon6,exon7 were sent to sequence. Results of the sequencing was detected by nucleic acid and protein sequence comparison used DNAMAN Version5.2.
RESULTS: After PCR amplification and sequencing, exon3、exon5 and exon7 fragments obtained were the same to PLP1 sequence published by the NCBI; Point mutation occurred in the 844th base in exon6 of the 10th sample. The change that T mutated to C caused the change of the amino acids that phenylalanine changed to leucine. Although the 844th base is C exon6 on the 5 samples found in pairs for the C, gene sequencing analysis showed the mixed-signal of T signal and C signal, so the possibility of this point mutation was not temporarily excluded. Until now this mutation point has not been reported, remains to be further explored.
Conclusion:
1. Detection method of SPG2 gene of hereditary spastic paraplegia was successfully established .This method can be used early genetic diagnosis and prenatal diagnosis of X-recessive hereditary spastic paraplegia.
2. A new gene mutation sites has been founded by sequencing. The gene mutation sites has not been reported at home and abroad and remains to be further studied.
3. The 844th base of exon6 in PLP1 gene of the proband of the pedigree existed mutation that T instead of C, so far, this mutation has not been reported. A new genetic mutation site was added in the genetics. the 844th base is C exon6 on the 5 samples found in pairs for the C, gene sequencing analysis showed the mixed-signal of T signal and C signal, so the possibility of this point mutation was not temporarily excluded
4 .The changes that phenylalanine changed to leucine may lead to degeneration of the corticospinal tract axons and demyelination and cause a series of clinical symptoms including muscle tone increased, walking laborious.
5. Clinical symptoms of hereditary spastic paraplegia were varied. The gold diagnosis standard is genetic testing.
6.This study provided basic research for further gene therapy. Abnormal gene was suppressed through gene regulation in order to achieve therapeutic purposes.
引文
[1]罗纯、章伟.医学遗传学.华中科技大学出版社.2007.第一版.6.
[2]Vogel-Motulsky(美).罗会元(译).人类遗传学—问题与方法.人民卫生出版社.1999.第三版.
[3]黄诒森、张光毅.生物化学与分子生物学.科学出版社.2003.第二版.361.
[4]FinkJK.et01.N.urolo.1995.45:325.
[5]Giovanni Stevanin. Recent Advances in the Genetics of Spastic Paraplegias.Genetics, 2008,8(3):198-210.
[6]吴江.神经病学.人民卫生出版社.2005.第一版.321.
[7]刘小民,赵国华,遗传性痉挛性截瘫与转运通路,国外医学神经病学神经外科学分册,2004,31(1):81.
[8]杜鹃,唐北沙,遗传性痉挛性截瘫分子发病机制研究进展,卒中与神经疾病,2009,16(3):192
[9].Hazan J, Lamy C, Melki J, et al.Autosomal dominant familial spastic paraplegia is genetically heterogeneous and one locus maps to chromosome 14q.Nat Genet, 1993, 5:163-167.
[10].Zhao X, Alvarado D, Rainier S, et al·Mutation in a newly identified GTPase gene cause autosomal dominant hereditary spastic paraplegia. Nat Genet, 2001, 29:326-331.
[11].Zhang Y, Moheban DB, Conway BR, et al.Cell surface Trk receptors mediate NGF-induced survival while internalized receptors regulate HGF-induced differentiation. Neurosci, 2000, 20: 5671-5678.
[12].Zhao X,Alvarado D,Rainier S,et al.Mutations in a newly identified GTPase gene cause autosomal dominant hereditary spatic paraplegia.Nat Genet,2001,29(3):326-331
[13]Sara Salinas,Christos Proukakis.Hereditary spastic paraplegia:clinical features and pathogenetic mechanisms. Lancet Neurol,2008,7(12):1127-1138.
[14]. Muglia M, Magariello A, Nicoletti G, et al·Further evidence that SPG3A gene mutations cause autosomal dominant hereditary spastic.
[15].Hazan J, Fonknechten N, Mavel D, et al.Spastin, a new AAA protein, is altered in themost frequent form of autosomal dominant spastic paraplegia. Nat Genet, 1999, 23: 296-303.
[16].Giovanni Stevanin. Recent Advances in the Genetics of Spastic Paraplegias. Genetics,2008,8(3):198-210.
[17].Higgins JJ, Loveless JM, Goswami S, et al. An atypical intronic deletion widens the spectrum of mutations in hereditary spastic paraplegia[J]. Neurology,2001, 56(11): 1482-1485.
[18].Yip AG, Durr A, Marchuk DA, et al. Meta-analysis of age at on set in spastin-associated hereditary spastic paraplegia provides no evideoce for a correlation with mutational class[J]. J Med Genet,2003,40(9):e106.
[19].Yabe I, Sasaki H, Tashiro K, et al. Spastin gene mutation inJapanese with hereditary spastic paraplegia[J]. J Med Genet,2002,39(8):e46.
[20].Proukakis C, Auer-Grumbach M, Wagner K, et al. Screening of patients with hereditary spastic paraplegia reveals seven novel mutations in the SPG4(Spastin) gene[J]. HumMutat,2003,21(2):170.
[21].Zhao GH, Tang BS, Luo W, et al. Spastin gene mutation in Chinese patientswith hereditary spastic paraplegia[J]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi,2003, 20(3):177-180.
[22].Sauter S, Miterski B, klimpe S, et al. Mutation analysis of the spastin gene(SPG4) in patients in Germanywith autosomal dominant hereditary spastic paraplegia[J]. Hum Mutat,2002,20(2):127-132.
[23].Errico A,Ballabio A,Rugarli EI.Spastin,the peotein mutated in autosomal dominant hereditary spastic paraplegia,is involved in mictotubule dynamics.Hum Mol Genet, 2002,11(2)153-163.
[24].Tsaousidou MK,Ouahchi K,Warner TT,et al.Sequence altera-tions within CYP7B1implicate defective cholcsterol homeostasis in motor-neuron degeneration.Am d Hum Genet,2008,82(2):510-515.
[25]Fink JK et al.Neurology,1996,46:835.
[26]Zhao J,Matthies DS,Botzolakis EJ,et al.Hereditary spastic paraplegia-associated mutationsin the NIPA1gene and its Caenorhabditis elegans homolog trigger neural degeneration in vitro and in vivo through a gain-of-function mechanism. J Neurosci, 2008,28(51):13938-13951.
[27] de Michele G, de Fusco M, Cavalcanti F, et al.A new locus for autosomal recessive hereditary spastic paraplegia maps to chromosome 16q24.3.Am J Hum Genet, 1998, 63: 135-139.
[28]Casari G, de Fusco M, Ciarmatori S, et al.Spastic paraplegia and OXPHOS impairment caused by mutation in paraplegin, a nuclearencoded mitochondrial metalloprotease. Cell, 1998, 93: 973-983.
[29]Settasatian C, Whitmore SA, Crawford J, et al.Genomic structure and expression analysis of the spastic paraplegia gene, SPG7.Hum Genet, 1999, 105: 139-144.
[30]Arnoldi A,Tonelli A,Crippa F,et al.A clinical, genetic, and biochemical characterization of SPG7mutations in a large cohort of patients with hereditary spastic paraplegia. Hum Mutah2008,29(4):522-531.
[31]Hedera et al.Am J Hum Genet,1999,64:563.
[32].Valdmanis PN,Meijer IA,Reynolds A, et al. Mutations in the KIAA0196Gene at the SPG8Locus Cause Hereditary Spastic Paraplegia.Am J Hum Genet,2007,80(1):152-161.
[33].Settasatian C, Whitmore SA, Crawford J, et al.Genomic structure and expression analysis of the spastic paraplegia gene, SPG7.Hum Genet, 1999, 105: 139-144.
[34].Reid E,Kloos M,Ashley-KochA,et al.Akinesin heavy chain(KIFSA)mutationinhe in hereditary spastic paraplegia (SPG10).AmJ Hum Genet,2002,71(5):l189-1194.
[35].Stevanin G, Santorelli FM, Azzedine H, et al. Mutations in SPG11,encoding spatacsin, are a major cause of spastic paraplegia with thin corpus callosum.Nat Genel, 2007,39(3):366-372.
[36].Hansen JJ, Durr A, Cournu-Rebeix I, et al.Hereditary spastic paraplegia SPG13 is associated with a mutation in the gene encoding the mitochondrial chaperonin Hsp60·Am J Hum Genet, 2002, 70:1328-1332.
[37]Ghosh JC,Dohi T,Kang BH,et al.Hsp60regulation of tumor cell apoptosis.J Biol Chem,2008,283(8):5188-5194.
[38]Hanein S,Martin E,Boukhris A,et al.Identification of the SPG15Gene,Encoding Spastizin, as a Frequent Cause of Complicated Autosomal-Recessive Spastic Paraplegia, Including Kjellin Syndrome. Am J Hum Genet,2008,82(4):992-1002.
[39]Ito D, Suzuki N. Seipinopathy: a novel endoplasmic reticulum stress-associated disease. Brain,2008,[Epub ahead of print].
[40]Sara Salinas,Christos Proukakis.Hereditary spastic paraplegia:clinical features and pathogenetic mechanisms. Lancet Neurol,2008,7(12):1127-1138.
[41].Beetz C,Schüle R,Deconinck T,et al.REEPI mutation spectrum and genotype/phenotype correlation in hereditary spastic paraplegia type31.Brain,2008,131(4):1078-1086.
[42].Lin P, Li J, Liu Q, Mao F, et al. A missense mutation in SLC33A1, which encodes the acetyl-CoA transporter, causes autosomal-dominant spastic paraplegia (SPG42). Am J Hum Gen-et,2008,83(6):752-759.
[43].遗传性痉挛性截瘫,脑与神经疾病杂志,1996,4(4):252。