遗传性痉挛性截瘫致病基因SLC33A1突变位点c.339T＞G（p.Ser113Arg）的确定及功能分析
发布时间：2018-08-21 07:59
【摘要】：遗传性痉挛性截瘫(Hereditary spastic paraplegia, HSP或SPG)是一种在遗传学及临床上很受关注的神经系统遗传疾病。HSP主要临床特征为对称性双下肢进行性肌无力和肌张力增高,主要病理改变为双侧皮质脊髓束的轴索变性。到目前为止,已经有76个HSP致病基因位点被定位,确定的HSP致病基因也己达54个。其中,SPG42致病基因SLC33A1是本实验室利用一个来自山东省青岛地区的HSP家系资源定位并确定的。我们发现该家系患者的SLC33A1基因均发生了c.339TG (p.Ser113Arg)错义突变,而家系的正常人及群体中均未发现该突变。在斑马鱼模型中下调slc33a1的表达会导致斑马鱼出现尾部发育不良、运动神经元轴突生长异常等表型。在此基础上,本论文利用全外显子测序进一步确认SPG42发生是由于 SLC33A1基因c.339TG(p.Ser113Arg)突变引起的,并进一步对SLC33A1的功能进行初步研究。首先,我们利用全外显子组测序对一个SPG42患病家系所有的候选致病突变进行检测,发现在该家系两个患者中均存在5个点突变和4个碱基插入改变,而且这些改变都位于致病基因定位区域。SLC33A1中第113位丝氨酸突变为精氨酸(p.Ser112Arg)和VEPH1中第433位谷氨酰胺突变为组氨酸(p.Gln433His),这两个位点的改变都与疾病的表型共分离。但软件预测结果显示,VEPH1p.Gln433His位点改变后该蛋白仍能维持正常功能,因此,我们认为,SLC33A1 c.339TG (p.Ser113Arg)位点的改变最有可能是该SPG42家系的致病突变。为进一步研究SLC33A1基因c.339TG (p.Ser113Arg)突变的突变性质,我们利用斑马鱼模型,用人类野生型和/或c.3391G(p.Ser113Arg)突变型SLC33A1 mRNA与slc33al吗啉代反义寡核苷酸(Morpholino, MO)单独或以不同比例对斑马鱼胚胎进行共注射,并对其进行表型分析。结果发现人野生型SLC33A1 mRNA可以拯救由于注射slc33a1 MO下调slc33a1所引起的斑马鱼异常表型,而同时注射人突变型SLC33A1 mRNA则抵消了这种拯救效果。这些结果提示c.339TG (p.Ser113Arg)突变具有显性负效应。此外,为了进一步研究遗传性痉挛性截瘫致病基因SLC33A1的功能及其致病机制,我们在前期研究的基础上,利用SPG42患者及正常人的皮肤成纤维细胞进一步确定,患者由于SLC33A1的错义突变导致骨形态发生蛋白I型受体A(Bone morphogenetic protein receptor IA, BMPR1A)表达上调,进而导致BMP信号通路的异常激活。另外,通过半衰期的检测,确定BMPR1A的表达上调是由于BMPR1A在细胞内的降解异常造成的。综上所述,我们利用全外显子测序进一步确定SPG42发生是SLC33A1基因c.339TG (p.Ser113Arg)突变引起的,此错义突变具有显性负效应。SLC33A1的突变导致BMPR1A在细胞内的降解异常,进而引起BMPR1A表达上调,最终导致BMP信号通路的异常激活。这一结果将有利于更加深入地认识SLC33A1的功能及其导致HSP的分子机制。
[Abstract]:Hereditary spastic paraplegia (Hereditary spastic paraplegia, HSP or SPG) is a genetic and clinical genetic disease of nervous system. The main pathological changes were axonal degeneration of bilateral corticospinal tract. Up to now, 76 HSP pathogenicity genes have been located, and 54 HSP pathogenicity genes have been identified. The SLC33A1 gene was identified by using a HSP pedigree from Qingdao, Shandong Province. We found that c.339TG (p.Ser113Arg) missense mutation was found in the SLC33A1 gene of all the patients in this pedigree, but it was not found in the normal people and the population of the pedigree. The down-regulation of slc33a1 expression in zebrafish model may lead to hypoplasia of tail and abnormal axonal growth of motor neurons in zebrafish. On this basis, the whole exon sequencing was used to further confirm that SPG42 was caused by c.339TG (p.Ser113Arg) mutation in the SLC33A1 gene, and to further study the function of SLC33A1. First, we detected all candidate pathogenicity mutations in a SPG42 family by total exon sequencing, and found that there were 5 point mutations and 4 base insertion changes in both patients. These changes were located in the pathogenetic locus. SLC33A1, the 113th serine mutated to arginine (p.Ser112Arg) and the 433rd glutamine to histidine (p.Gln433His) in VEPH1, both of which were coisolated from the phenotype of the disease. However, the results of software prediction showed that the protein could maintain normal function after the alteration of the p.Ser113Arg site. Therefore, we believe that the change of the SLC33A1 c.339TG (p.Ser113Arg) locus is most likely to be the pathogenic mutation in the SPG42 pedigree. In order to further study the mutagenicity of c.339TG (p.Ser113Arg) mutation in SLC33A1 gene, we used zebrafish model to co-inject zebrafish embryos with slc33al morpholine antisense oligonucleotide (Morpholino, MO) and human wild-type and / or c.3391G (p.Ser113Arg) mutant SLC33A1 mRNA alone or in different proportions. Phenotypic analysis was carried out. The results showed that human wild-type SLC33A1 mRNA could save the abnormal phenotype of zebrafish caused by slc33a1 MO down-regulation of slc33a1, while injection of human mutant SLC33A1 mRNA could counteract the rescue effect. These results suggest that c.339TG (p.Ser113Arg) mutations have dominant negative effects. In addition, in order to further study the function and pathogenesis of hereditary spastic paraplegia gene SLC33A1, we further determined the function of skin fibroblasts in patients with SPG42 and normal people on the basis of previous studies. The missense mutation of SLC33A1 leads to the up-regulation of bone morphogenetic protein type I receptor (A (Bone morphogenetic protein receptor IA, BMPR1A) expression, which leads to abnormal activation of BMP signaling pathway. In addition, the detection of half-life indicated that the up-regulation of BMPR1A expression was due to the abnormal degradation of BMPR1A in cells. In conclusion, we further confirmed that SPG42 was caused by the c.339TG (p.Ser113Arg) mutation of the SLC33A1 gene by using total exon sequencing. The missense mutation had a dominant negative effect. SLC33A1 mutation led to the abnormal degradation of BMPR1A in cells, which led to the upregulation of BMPR1A expression. Finally, abnormal activation of BMP signaling pathway is caused. This result will be helpful to a deeper understanding of the function of SLC33A1 and its molecular mechanism leading to HSP.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R682.22
本文编号：2195064
[Abstract]:Hereditary spastic paraplegia (Hereditary spastic paraplegia, HSP or SPG) is a genetic and clinical genetic disease of nervous system. The main pathological changes were axonal degeneration of bilateral corticospinal tract. Up to now, 76 HSP pathogenicity genes have been located, and 54 HSP pathogenicity genes have been identified. The SLC33A1 gene was identified by using a HSP pedigree from Qingdao, Shandong Province. We found that c.339TG (p.Ser113Arg) missense mutation was found in the SLC33A1 gene of all the patients in this pedigree, but it was not found in the normal people and the population of the pedigree. The down-regulation of slc33a1 expression in zebrafish model may lead to hypoplasia of tail and abnormal axonal growth of motor neurons in zebrafish. On this basis, the whole exon sequencing was used to further confirm that SPG42 was caused by c.339TG (p.Ser113Arg) mutation in the SLC33A1 gene, and to further study the function of SLC33A1. First, we detected all candidate pathogenicity mutations in a SPG42 family by total exon sequencing, and found that there were 5 point mutations and 4 base insertion changes in both patients. These changes were located in the pathogenetic locus. SLC33A1, the 113th serine mutated to arginine (p.Ser112Arg) and the 433rd glutamine to histidine (p.Gln433His) in VEPH1, both of which were coisolated from the phenotype of the disease. However, the results of software prediction showed that the protein could maintain normal function after the alteration of the p.Ser113Arg site. Therefore, we believe that the change of the SLC33A1 c.339TG (p.Ser113Arg) locus is most likely to be the pathogenic mutation in the SPG42 pedigree. In order to further study the mutagenicity of c.339TG (p.Ser113Arg) mutation in SLC33A1 gene, we used zebrafish model to co-inject zebrafish embryos with slc33al morpholine antisense oligonucleotide (Morpholino, MO) and human wild-type and / or c.3391G (p.Ser113Arg) mutant SLC33A1 mRNA alone or in different proportions. Phenotypic analysis was carried out. The results showed that human wild-type SLC33A1 mRNA could save the abnormal phenotype of zebrafish caused by slc33a1 MO down-regulation of slc33a1, while injection of human mutant SLC33A1 mRNA could counteract the rescue effect. These results suggest that c.339TG (p.Ser113Arg) mutations have dominant negative effects. In addition, in order to further study the function and pathogenesis of hereditary spastic paraplegia gene SLC33A1, we further determined the function of skin fibroblasts in patients with SPG42 and normal people on the basis of previous studies. The missense mutation of SLC33A1 leads to the up-regulation of bone morphogenetic protein type I receptor (A (Bone morphogenetic protein receptor IA, BMPR1A) expression, which leads to abnormal activation of BMP signaling pathway. In addition, the detection of half-life indicated that the up-regulation of BMPR1A expression was due to the abnormal degradation of BMPR1A in cells. In conclusion, we further confirmed that SPG42 was caused by the c.339TG (p.Ser113Arg) mutation of the SLC33A1 gene by using total exon sequencing. The missense mutation had a dominant negative effect. SLC33A1 mutation led to the abnormal degradation of BMPR1A in cells, which led to the upregulation of BMPR1A expression. Finally, abnormal activation of BMP signaling pathway is caused. This result will be helpful to a deeper understanding of the function of SLC33A1 and its molecular mechanism leading to HSP.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R682.22
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相关硕士学位论文 前2条
1 李昭辉;遗传性痉挛性截瘫致病基因SLC33A1突变位点c.339T＞G（p.Ser113Arg）的确定及功能分析[D];山东大学;2015年
2 赵宝悦;对遗传性痉挛性截瘫致病基因SLC33A1功能及致病机制的初步探讨[D];山东大学;2014年
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