Reduced expression of the Kinesin-Associated Protein 3 (KIFAP3) gene increases survival in sporadic amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis is a degenerative disorder of motor neurons that typically develops in the 6th decade and is uniformly fatal, usually within 5 years. To identify genetic variants associated with susceptibility and phenotypes in sporadic ALS, we performed a genome-wide SNP analysis in sporadic ALS cases and controls. A total of 288,357 SNPs were screened in a set of 1,821 sporadic ALS cases and 2,258 controls from the U.S. and Europe. Survival analysis was performed using 1,014 deceased sporadic cases. Top results for susceptibility were further screened in an independent sample set of 538 ALS cases and 556 controls. SNP rs1541160 within the KIFAP3 gene (encoding a kinesin-associated protein) yielded a genome-wide significant result (P = 1.84 x 10(-8)) that withstood Bonferroni correction for association with survival. Homozygosity for the favorable allele (CC) conferred a 14.0 months survival advantage. Sequence, genotypic and functional analyses revealed that there is linkage disequilibrium between rs1541160 and SNP rs522444 within the KIFAP3 promoter and that the favorable alleles of rs1541160 and rs522444 correlate with reduced KIFAP3 expression. No SNPs were associated with risk of sporadic ALS, site of onset, or age of onset. We have identified a variant within the KIFAP3 gene that is associated with decreased KIFAP3 expression and increased survival in sporadic ALS. These findings support the view that genetic factors modify phenotypes in this disease and that cellular motor proteins are determinants of motor neuron viability.

Fig. 1.

Plot of −log₁₀(P) for survival, age of onset, site of onset and susceptibility of sporadic ALS. Analysis for survival, age of onset, site of onset and susceptibility was performed for 288,357 SNPs and the results for the entire genome were plotted as shown in A and (C–E). The x axis represents the chromosomal position and the y axis represents the −log₁₀ of the P value for each SNP. The dotted line represents the cutoff for Bonferroni significance. (A) P values from linear regression analysis of survival. SNPs rs1541160 (circled) and rs855913 were significant after Bonferroni correction. (B) A closer view of the rs1541160 region is shown. Dark points represent SNPs typed in the study, and light points represent SNPs whose genotypes were imputed. (Lower) Imputation certainty for each imputed SNP, defined as the average maximum posterior genotype call probability. The chromosomal region spans 5 Mb on either side of SNP rs1541160. Positions are in National Center for Biotechnology Information build-35 coordinates.

Fig. 2.

Influence of alleles of rs1541160 in KIFAP3 on survival in sporadic ALS. Survival curves were generated from sporadic ALS cases with different rs1541160 genotypes and analyzed using the Peto-Prentice generalized Wilcoxon test; each curve plots percentage survival versus duration for each of the 3 genotypes observed for rs1541160. To better visualize the differences between the genotypes, the curves were truncated at 10 years. As shown, individuals harboring the CC genotype (red) display an increased survival as compared with individuals with the CT (green) and TT (blue) genotypes. (A) uncensored data; (B) censored data. In B, small vertical marks superimposed on the survival curve show censored points at which individuals were lost to further assessment.

Fig. 3.

Association of rs1541160/rs522444 with expression of KIFAP3. (A and B) Total RNA was isolated from lymphoblastoid cell lines (A) or occipital cortex brain tissue harboring (B) either a CC or TT genotype for rs1541160. Relative expression of KIFAP3 was determined by real-time PCR. As shown, individuals harboring the CC genotype display decreased expression compared with individuals with the TT genotype. Error bars represent the 95% C.I. (C) The sequence of the KIFAP3 promoter region is shown. The arrow indicates the transcriptional start site. SNP rs522444 is indicated at the −25 position. The box represents the location of the putative Sp1 binding site. (D) The KIFAP3 promoter region and 5′ UTR (633 bp) were amplified from individuals harboring either the CC or TT genotype and subcloned upstream of the firefly luciferase gene. The schematic (not drawn to scale) represents the resultant constructs, which differ only at a single base pair located at rs522444. (E) The resultant constructs were transfected into SKN-AS cells and relative luciferase activity was measured. The error bars represent the 95% C.I. A promoterless vector yielded <1% relative activity. The construct containing the G allele displays higher luciferase activity relative to the C allele. *, P < 0.05; **, P < 0.01.