Targeted 3'-end RNA sequencing uncovers cryptic polyadenylation in Huntington's disease linked to somatic instability and CAG repeat purity

Huntingtons disease (HD) is a progressive neurodegenerative disorder, caused by a CAG trinucleotide expansion in the first exon of the HTT gene. The full-penetrance threshold for HD is established at 40 CAG repeats, which are translated into a polyglutamine (polyQ) tract within the huntingtin (HTT) protein. Beyond this threshold, somatic repeat instability in the brain and CAG repeat purity can further modulate disease onset and severity. HTT can be processed into multiple N-terminal fragments. Among these, the exon 1 HTT fragment containing an expanded polyQ tract is considered the most pathogenic. While some studies propose this fragment is generated by HTT proteolytic cleavage, others indicate it is primarily generated by the choice of a premature, cryptic polyadenylation site within HTT intron 1. Here, we apply a targeted RNA sequencing approach to systematically characterize HTT cryptic polyadenylation in several HD mouse models, human cell lines, and HD postmortem brain tissue. This method, known as 3-end targeted RNA sequencing or 3TRS, enables an accurate, quantitative, and cost-effective measurement of alternative polyadenylation. We describe an experimental and computational protocol that can also be adapted to additional transcripts and disease models. We show that activation of HTT cryptic polyadenylation is highly selective for the distal polyadenylation site located 7.3kb downstream intron 1 and requires very long, uninterrupted CAG repeat expansions. In humanized YAC128 mice, cryptic HTT expression was detected across all inspected brain regions, whereas in knock-in mice it correlated strongly with tissue-specific somatic repeat instability, being most prominent in the striatum. In human cell lines, pure CAG tracts triggered cryptic polyadenylation, while CAA interruptions within the repeats completely suppressed HTT1a expression. In HD patient samples, cryptic HTT transcripts were detected only in a juvenile-onset fibroblast line carrying an ultralong CAG expansion and in brain regions exhibiting high somatic instability. Together, these data support a model in which long and unstable CAG repeat expansions drive HD pathogenesis by activating HTT cryptic polyadenylation. By enabling quantitative comparison of cryptic and canonical HTT isoforms, 3TRS provides a robust framework to investigate HTT1a biogenesis and to support the development and evaluation of HTT-lowering therapeutic strategies.