Molecular Genetic Analysis of Spinocerebellar Ataxia Type 7 and a Further Study on the RNA Interference Analysis

Spinocerebellar ataxia type 7 (SCA7), autosomal dominant cerebellar ataxia type II (ADCA II), is a progressive autosomal dominant neurodegenerative disorder characterized clinically by cerebellar ataxia and blindness resulting from dysfunction and degeneration mainly of the cerebellum and retina. The prevalence of SCA7 is about 1-4 in 1,000,000 worldwide. SCA7 is caused by expansion of an unstable trinucleotide CAG repeat in ATXN7 gene which encodes Ataxin-7 protein. Normal ATXN7 alleles contain 4-35 CAG repeats. Expansion of CAG repeat in the mutant allele ranges from 36 to 460, with repeat length inversely correlated to the age of disease onset. However intermediate size alleles (having 28-35 CAG repeats) can be converted into mutational expanded alleles in offspring. Ataxin-7 exists in many CNS and non-CNS tissues but pathological symptoms are confined to a few types of neuronal cells (e.g. mainly Purkinje and cone-rod cells). The function of Ataxin-7 is not fully understood but currently supposed a subunit of a transcriptional coactivator complex (called TFTC or STAGA) that has histone acetyltransferase activity. So far effective treatmentsof polyglutamine diseases containing SCA7 have not yet been developed. Inhibition of mutated allele expression provides a direct approach to SCA7 therapy. More recently, gene silencing through RNA interference (RNAi) by double-stranded RNA (dsRNA) has emerged as a powerful method to inhibit expression of targeted gene. This new method provides a unique tool for sequence-specific gene suppression and may hold great promise for a potential therapy of gain-of-function diseases like polyglutamine diseases with a minimal side effect. In this study, we aimed establishing two RNAi systems for treatment of SCA7, a mutant allele-specific and a mutant allele-nonspecific system. For mutant allele-specific RNAi system, we used two known single nucleotide polymorphisms (SNPs) in the ATXN7 gene, a G/A SNP in the exon 12 (ORF region) and a G/A SNP in the exon 13 (3'-UTR region). At first, we have analyzed frequencies of two SNP types in the 16 Korean SCA7 patients. We found that in the exon 12 SNP A was 84% and SNP G was 16%, and in the exon 13 SNP G was 84% and SNP A was 16%. And heterozygosity is 31.3% in both of them. These all data are different from that of Japanese and Chinese. We first had assumed that combination of these two SNP typeswould be beneficialfor RNAi treatment of SCA7. But this assumption is not correct because two SNP types (G in the exon 12 and A in the exon 13) were related. To use the G/A SNP in the exon 12, we have engineered two short hairpin RNA (shRNA) expressing vector constructs targeting SNP A and G.. They suppressed the expression of mutant Ataxin-7 (55Q) with allele-specific manner by approximately 80% and 50%, respectively, in HeLa cells, indicating that theRNAi system for SNP A is more efficient than G.. Also we partially found that staurosporine-induced apoptotic progress decreased by this RNAi system in SK-N-SH (neuroblastoma cell line) cells. We have also engineered four shRNA constructs targeting ATXN directly by mutant allele-nonspecific manner. One of them decreased the expression of whole Ataxin-7 by approximately 80%. Furthermore, these two effective shRNAs significantly decreased the nuclear inclusion body (aggregation) generated by over-expression of YFP-Ataxin-7 (55Q) in HeLa cells. Our results suggest that a combination of two shRNA systems for allele-specific and non-specific silencing of mutant Ataxin-7 may produce an additive effect on a new RNAi-based therapy for SCA7.

Author dc:creator

• 장, 석훈

Contributors dc:contributor

• 정, 선용
• 김, 현주
• 대학원 의학과
• 200624182

• 척수소뇌성 운동실조증 제7형
• 상염색체우성 소뇌성운동실조증 제II형
• 폴리글루타민 질환
• Ataxin-7
• RNA 간섭
• 대립유전자 특이적 RNAi
• 단일염기변이
• spinocerebellar ataxia type 7(SCA7)
• autosomal dominant cerebellarataxia type II(ADCA II)
• polyglutamine disease
• RNA interference(RNAi)
• allele-specific RNAi
• single nucleotide polymorphism(SNP)

Language dc:language

ko

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000006968
000000006968

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/1834