Investigating the Relationship between CAG Repeat Length and HTT Alternative Transcript Processing

Huntington's disease (HD) is a neurodegenerative disorder resulting in motor, cognitive, and behavioral manifestations. Within the Huntingtin protein (HTT), exon 1 of HTT has an expanded CAG (Cytosine, Adenosine, Guanosine; trinucleotide RNA repeat) tract. Over time, expanded regions of CAG repeats in the HTT fragments result in somatic instability and polyQ generation, leading to neuronal atrophy. 

This study investigates the relationship between CAG repeat length and HTT1a, an N-terminal fragment produced from an alternative HTT transcript, in patient-derived Lymphoblastoid Cell Lines (LCLs). A methodological procedure using a sequential two-step model for HD to accurately characterize the HTT1a transcript was conducted by amplifying HTT1a's signal via a 3' RACE (Rapid Amplification of cDNA Ends) assay—quantification analysis of the relationship between full transcript HTT and HTT1a ratio utilized a multiplex ddPCR. To analyze the relationship between HTT1a and HTT exon 1-2 in LCLs, cell lines derived from different patients with varying lengths were grown to be propagated. The range was 17-150 repeats, with most repeat lengths being 40-60. By extracting DNA and RNA, cDNA was made with the primer handle from the previously conducted 3' RACE assay, and the HTT1a's signal was quantified with ddPCR. Quantitative analysis revealed a positive correlation between HTT1a and CAG repeats in LCLs. In relation to somatic repeat expansion, this study supports that longer repeats lead to greater somatic expansion, thereby increasing the production of HTT1a and causing more toxicity. These results have implications for further confirming how HTT1a could be a mechanism that drives toxicity.

For more information, please email: molivarescely@eckerd.edu