Poster abstracts

Poster number 78 submitted by Megan Pino

Variants in the ALS-associated gene KIF5A affect RNA splicing and structure

Megan G. Pino, MS (Neuroscience Graduate Program), Anton Blatnik, PhD (Biological Chemistry and Pharmacology, Molecular Genetics), Vicki McGovern, PhD (Biological Chemistry and Pharmacology, Molecular Genetics), Arthur Burghes, PhD (Biological Chemistry and Pharmacology, Molecular Genetics), Stephen J. Kolb, MD, PhD (Neurology, Biological Chemistry and Pharmacology)

Abstract:
Approximately one-third of all disease-associated genetic mutations exhibit disruption of the correct pattern of pre-mRNA splicing.1,2 Aberrant splicing is thought to be a mechanism by which variants in the neuron-specific cytoskeletal motor protein, Kinesin Family Member 5A (KIF5A), cause amyotrophic lateral sclerosis (ALS)—a rapidly progressive, fatal neurodegenerative disease with no cure.3,4 Variants in the KIF5A C-terminal cargo binding domain were recently identified through whole genome/exome sequencing and genome-wide association studies.5-7 8 of the 12 KIF5A variants are located near exon 27 splice-site junctions and are predicted to cause missplicing. Exon 27 is the penultimate exon and encodes part of the cargo binding domain, therefore changes in exon 27 splicing likely result in a novel C-terminus and altered KIF5A cargo binding. However, RNA splicing is heterogeneous, and the extent of each KIF5A variant’s effect on splicing—and thus the clinical relevance—have yet to be determined.8 Furthermore, disease presentation varies widely among patients with reported ALS-associated KIF5A variants, with age of disease onset occurring as early as 29 years and survival time ranging from one to 22 years—a stark contrast to the 3-5 year average survival of ALS patients.3,9,10 We hypothesize that KIF5A variants alter the incorporation of exon 27, resulting in a novel C-terminus that gains a toxic function causing ALS. We have identified over 20 consensus splice factor and enhancer motifs that overlap with C-terminal KIF5A variants. To investigate the biochemical consequences of variant-specific splice alterations in KIF5A, we performed minigene splicing assays and droplet digital PCR to quantify the extent of exon 27 inclusion at the RNA level. Our results show that KIF5A variants exhibit a wide range of exon 27 inclusion, and there does not appear to be a correlation between percent exon 27 inclusion and ALS survival. We will assess correlations with other ALS clinical outcomes. To further investigate the RNA, we generated structural predictions of variant KIF5A RNAs and demonstrated alterations in base pairing and secondary structure. We are performing additional experiments to confirm these predictions in vitro.

References:
1. Cáceres JF & Kornblihtt AR 2002. https://doi.org/10.1016/s0168-9525(01)02626-9
2. Montes M et al. 2019. https://doi.org/10.1016/j.tig.2018.10.002
3. NINDS ALS Fact Sheet https://www.ninds.nih.gov/Disorders/Patient-Caregiver-Education/Fact-Sheets/Amyotrophic-Lateral-Sclerosis-ALS-Fact-Sheet
4. Kiernan MC et al. 2011. https://doi.org/10.1016/S0140-6736(10)61156-7
5. Brenner D et al. 2018. https://doi.org/10.1093/brain/awx370
6. Nicolas A et al. 2018. https://doi.org/10.1016/j.neuron.2018.02.027
7. He J et al. 2020. https://doi.org/10.1136/jnnp-2019-320483
8. Roggenbuck J et al. 2020. https://doi.org/10.1212/NXG.0000000000000390
9. Tandan R & Bradley WG 1985. https://doi.org/10.1002/ana.410180302
10. Mulder DW et al. 1986. https://doi.org/10.1212/wnl.36.4.511

Keywords: Kinesin Family Member 5A (KIF5A), Amyotrophic lateral sclerosis (ALS), RNA splicing and processing mechanisms