遺傳性痙攣性截癱致病基因SLC33A1突變位點c.339T＞G（p.Ser113Arg）的確定及功能分析
發(fā)布時間：2018-08-21 07:59
【摘要】：遺傳性痙攣性截癱(Hereditary spastic paraplegia, HSP或SPG)是一種在遺傳學(xué)及臨床上很受關(guān)注的神經(jīng)系統(tǒng)遺傳疾病。HSP主要臨床特征為對稱性雙下肢進行性肌無力和肌張力增高,主要病理改變?yōu)殡p側(cè)皮質(zhì)脊髓束的軸索變性。到目前為止,已經(jīng)有76個HSP致病基因位點被定位,確定的HSP致病基因也己達54個。其中,SPG42致病基因SLC33A1是本實驗室利用一個來自山東省青島地區(qū)的HSP家系資源定位并確定的。我們發(fā)現(xiàn)該家系患者的SLC33A1基因均發(fā)生了c.339TG (p.Ser113Arg)錯義突變,而家系的正常人及群體中均未發(fā)現(xiàn)該突變。在斑馬魚模型中下調(diào)slc33a1的表達會導(dǎo)致斑馬魚出現(xiàn)尾部發(fā)育不良、運動神經(jīng)元軸突生長異常等表型。在此基礎(chǔ)上,本論文利用全外顯子測序進一步確認SPG42發(fā)生是由于 SLC33A1基因c.339TG(p.Ser113Arg)突變引起的,并進一步對SLC33A1的功能進行初步研究。首先,我們利用全外顯子組測序?qū)σ粋�SPG42患病家系所有的候選致病突變進行檢測,發(fā)現(xiàn)在該家系兩個患者中均存在5個點突變和4個堿基插入改變,而且這些改變都位于致病基因定位區(qū)域。SLC33A1中第113位絲氨酸突變?yōu)榫彼?p.Ser112Arg)和VEPH1中第433位谷氨酰胺突變?yōu)榻M氨酸(p.Gln433His),這兩個位點的改變都與疾病的表型共分離。但軟件預(yù)測結(jié)果顯示,VEPH1p.Gln433His位點改變后該蛋白仍能維持正常功能,因此,我們認為,SLC33A1 c.339TG (p.Ser113Arg)位點的改變最有可能是該SPG42家系的致病突變。為進一步研究SLC33A1基因c.339TG (p.Ser113Arg)突變的突變性質(zhì),我們利用斑馬魚模型,用人類野生型和/或c.3391G(p.Ser113Arg)突變型SLC33A1 mRNA與slc33al嗎啉代反義寡核苷酸(Morpholino, MO)單獨或以不同比例對斑馬魚胚胎進行共注射,并對其進行表型分析。結(jié)果發(fā)現(xiàn)人野生型SLC33A1 mRNA可以拯救由于注射slc33a1 MO下調(diào)slc33a1所引起的斑馬魚異常表型,而同時注射人突變型SLC33A1 mRNA則抵消了這種拯救效果。這些結(jié)果提示c.339TG (p.Ser113Arg)突變具有顯性負效應(yīng)。此外,為了進一步研究遺傳性痙攣性截癱致病基因SLC33A1的功能及其致病機制,我們在前期研究的基礎(chǔ)上,利用SPG42患者及正常人的皮膚成纖維細胞進一步確定,患者由于SLC33A1的錯義突變導(dǎo)致骨形態(tài)發(fā)生蛋白I型受體A(Bone morphogenetic protein receptor IA, BMPR1A)表達上調(diào),進而導(dǎo)致BMP信號通路的異常激活。另外,通過半衰期的檢測,確定BMPR1A的表達上調(diào)是由于BMPR1A在細胞內(nèi)的降解異常造成的。綜上所述,我們利用全外顯子測序進一步確定SPG42發(fā)生是SLC33A1基因c.339TG (p.Ser113Arg)突變引起的,此錯義突變具有顯性負效應(yīng)。SLC33A1的突變導(dǎo)致BMPR1A在細胞內(nèi)的降解異常,進而引起B(yǎng)MPR1A表達上調(diào),最終導(dǎo)致BMP信號通路的異常激活。這一結(jié)果將有利于更加深入地認識SLC33A1的功能及其導(dǎo)致HSP的分子機制。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R682.22
本文編號：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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R682.22
【相似文獻】
相關(guān)碩士學(xué)位論文 前2條
1 李昭輝;遺傳性痙攣性截癱致病基因SLC33A1突變位點c.339T＞G（p.Ser113Arg）的確定及功能分析[D];山東大學(xué);2015年
2 趙寶悅;對遺傳性痙攣性截癱致病基因SLC33A1功能及致病機制的初步探討[D];山東大學(xué);2014年
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