CCG•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•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•CGG interruptions at a higher frequency than sporadic SCA8 cases and that the number of CCG•CGG interruptions correlates with age at onset. At the molecular level, CCG•CGG interruptions increase RNA hairpin stability, and in cell culture experiments, increase p‐eIF2α and polyAla and polySer RAN protein levels. Additionally, CCG•CGG interruptions, which encode arginine interruptions in the polyGln frame, increase toxicity of the resulting proteins. In summary, SCA8 CCG•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. SYNOPSIS This study shows CCG•CGG interruptions within the ATXN8OS/ATXN8 CTG•CAG repeat are an important genetic modifier of disease penetrance in spinocerebellar ataxia type 8 (SCA8). CCG CGG interruptions in the ATXN8OS/ATXN8 CTG•CAG repeat expansion are more frequent in families with multiple affected individuals. The number of CCG•CGG interruptions, and not repeat length, correlates with age of onset in SCA8. CGG interrupted ATXN8 repeat expansions are more toxic to cells than pure ATXN8 repeat expansions. CGG interruptions increase RNA stability, p‐eIF2α levels and the levels of toxic polyAla and polySer repeat‐associated non‐AUG (RAN) proteins. Arginine‐interrupted polyGln expansion proteins expressed from CGG interrupted expansions are more toxic than pure polyGln expansion proteins. Graphical Abstract This study shows CCG•CGG interruptions within the ATXN8OS/ATXN8 CTG•CAG repeat are an important genetic modifier of disease penetrance in spinocerebellar ataxia type 8 (SCA8).