Abstracts

Rationale: In a genome-wide association study (GWAS) of symptomatic epilepsy (Two-stage genome-wide association study identifies variants in CAMSAP1L1 as susceptibility loci for epilepsy in Chinese. Hum Mol Genet. 2012;21:1184-9), the strongest association we identified was with a polymorphism in the CAMSAP1L1 gene: p=1.0×10^-8 and odds ratio=0.63 for the G allele of rs2292096. Little is known about CAMSAP1L1. However, a homolog of this gene is known to bind microtubules and to inhibit neurite outgrowth. With double immunostaining, we demonstrated partial colocalization of CAMSAP1L1 with β-tubulin. We hypothesize that the G allele of rs2292096 tags a haplotype expressing increased levels of CAMSAP1L1, which inhibits neurite growth after brain insults, thus reducing the risk of forming ectopic neural circuits that lead to epilepsy. Methods: To test the hypothesis, we performed quantitative real-time PCR to measure CAMSAP1L1 and GAPDH expression in RNA of brain samples from surgery on refractory epilepsy patients. We genotyped rs2292096 in the patients and compared expression among genotypes. To determine the effect of CAMSAP1L1 on neurite growth, we transfected SH-SY5Y human neuroblastoma cells, treated with 25 μM retinoic acid (RA), with siRNA to CAMSAP1L1 or negative control siRNA. Western blotting demonstrated gene knockdown. Cells were fixed 60 hours after transfection and were stained with β-tubulin antibody to label neurites and 4'-6-diamidino-2-phenylindole (DAPI) to label nuclei. Metamorph software was used to quantify total neurite outgrowth, mean outgrowth per cell (neurite length per cell), and neurite branches per cell. Results: In 28 individuals contributing brain samples, there were 20 AA, 6 AG, and 2 GG genotypes. CAMSAP1L1 expression, corrected for GAPDH expression, tended to differ by genotype (Kruskal-Wallis test, p=0.17 for 26 hippocampal samples and p=0.06 for 20 temporal lobe samples), with higher levels for GG. Transfection by siRNA reduced CAMSAP1L1 expression ~50%. Knockdown of CAMSAP1L1 increased neurite length per cell (p<0.05) for both undifferentiated and differentiated cells (Figure 1A). Knockdown also increased neurite branches per cell (p<0.05) for both undifferentiated (0 μM RA) and differentiated (25 μM RA) cells (Figure 1B). Double immunostaining (Figure 2) for CAMSAP1L1 (red) and β-tubulin (green) demonstrated partial colocalization of CAMSAP1L1 with microtubules (from our poster: CAMSAP1L1 is a potential epilepsy gene in the Chinese population. 9th Asian & Oceanian Epilepsy Congress, Manila, Philippines, 22-25 March 2012). Conclusions: CAMSAP1L1 inhibits neurite outgrowth, and the G allele of rs2292096, which is associated with decreased risk of symptomatic epilepsy, may also be associated with increased expression of CAMSAP1L1. These results are consistent with the hypothesis that CAMSAP1L1 expression inhibits neurite growth, thus reducing the risk of symptomatic epilepsy.