脊髓小腦運動失調症：SCA2、SCA14的遺傳檢測及擴增SCA17 TBP蛋白與HMGB1蛋白的交互作用分析

Abstract

脊髓小腦運動失調症(Spinocerebellar ataxia；簡稱SCA)，為一種異質性的神經退化性疾病，大部分起因為特定基因上三核苷重複擴增的結果，患者在小腦、腦幹、脊髓以及週邊神經系統會出現漸進式的退化現象。現在已知的脊髓小腦運動失調症約有28種亞型，本研究第一部份將重點放在屬於三核苷重複擴增的第二型(SCA2)以及不屬於三核苷重複擴增的第十四型(SCA14)的遺傳分析。這兩型的致病原因分別為Ataxin-2基因的CAG三核苷重複擴增和protein kinase Cγ (PRKCG)基因的突變。首先會對長庚醫院神經內科所提供的正常人(179位)、運動失調症(ataxia)患者(15位)、帕金森氏症(Parkinson's disease)患者(137位)、失智症(dementia)患者(124位)、震顫(tremor)患者(84位)及其他神經疾病患者(舞蹈症、肌張力異常症等，41位)進行SCA2 CAG三核苷重複擴增的分子檢測，結果並未發現任何Ataxin-2 CAG重複擴增的等位基因。在SCA14的遺傳分析方面，利用PCR增幅及DNA定序，檢視了30位帕金森氏症徵候群(Parkinsonism)患者PRKCG基因最常被報導發生突變的表現子(exon) 4及5，結果亦未發現任何突變。本研究的第二部份著重在分析SCA17 TBP蛋白與HMGB1蛋白的交互作用。首先製備包含20、45、61個麩醯胺的N端TBP (nTBP-Q20/Q45/Q61)及全長TBP (fTBP-Q20/Q45/Q61)的GST融合蛋白，再利用石英晶體微量天平(Quartz Crystal Microbalance)來量測TBP重複擴增對其與HMG1分子間交互作用的影響，結果發現N端TBP或全長TBP與HMG1之間的交互作用相同，但不同polyQ長度之TBP蛋白與HMG1之間的交互作用不同，其中以nTBP-Q45的結合力最強(ΔHz = 13.0)，nTBP-Q61的結合力最弱(ΔHz = 4.3)。故推測HMGB1可能和TBP的polyQ部位結合。

Spinocerebellar ataxias (SCAs) are a group of neurodegenerative disorders characterized by cerebellar dysfunction alone or in combination with other neurological abnormalities. More than 28 SCA types have been described. Among them, SCA2 and SCA17 were caused by the expansions of coded CAG trinucleotide repeats and SCA14 cause by mutations in the protein kinase Cγ. We examined the CAG repeat range of SCA2 Ataxin-2 gene in 179 normal controls and in patients with various neurodegenerative diseases, including 15 ataxia, 137 Parkinson's disease, 124 dementia, 84 essential tremor, and 41 chorea and dystonia. No SCA2 expanded allele was found. In the screening of SCA14 PRKCG gene mutation, we sequenced the exons 4 and 5-containing DNA fragment from 30 patients with parkinsonism and again no mutation was found. To study the protein-protein interaction between HMGB1 and TBP, GST fused HMGB1, nTBP-Q20/Q45/Q61 (N-terminal TBP) and fTBP-Q20/Q45/Q61 (full-length TBP) were overexpressed in E. coli BL21 cells and purified by affinity chromatography. Quartz Crystal Microbalance (QCM) was used to study the interactions between HMGB1 and TBP protein carrying 20, 45 or 61 polyQ track. While the interaction between HMGB1 and full-length or N-terminal TBP was similar, the length of polyQ track affects the binding of HMGB1 to TBP, with strongest binding of nTBP-Q45 (ΔHz = 13.0) and weakest binding of nTBP-Q61 (ΔHz = 4.3).