CCG center dot CGG interruptions in high-penetrance SCA8 families increase RAN translation and protein toxicity

Spinocerebellar ataxia type 8 (SCA8), a dominantly inherited neurodegenerative disorder caused by a CTG center dot CAG expansion, is unusual because most individuals that carry the mutation do not develop ataxia. To understand the variable penetrance of SCA8, we studied the molecular differences between highly penetrant families and more common sporadic cases (82%) using a large cohort of SCA8 families (n = 77). We show that repeat expansion mutations from individuals with multiple affected family members have CCG center dot CGG interruptions at a higher frequency than sporadic SCA8 cases and that the number of CCG center dot CGG interruptions correlates with age at onset. At the molecular level, CCG center dot CGG interruptions increase RNA hairpin stability, and in cell culture experiments, increase p-eIF2 alpha and polyAla and polySer RAN protein levels. Additionally, CCG center dot CGG interruptions, which encode arginine interruptions in the polyGln frame, increase toxicity of the resulting proteins. In summary, SCA8 CCG center dot CGG interruptions increase polyAla and polySer RAN protein levels, polyGln protein toxicity, and disease penetrance and provide novel insight into the molecular differences between SCA8 families with high vs. low disease penetrance.