PRKCA and multiple sclerosis: association in two independent populations

Abstract

Multiple sclerosis (MS) is a chronic disease of the central nervous system responsible for a large portion of neurological disabilities in young adults. Similar to what occurs in numerous complex diseases, both unknown environmental factors and genetic predisposition are required to generate MS. We ascertained a set of 63 Finnish MS families, originating from a high-risk region of the country, to identify a susceptibility gene within the previously established 3.4-Mb region on 17q24. Initial single nucleotide polymorphism (SNP)-based association implicated PRKCA (protein kinase C alpha) gene, and this association was replicated in an independent set of 148 Finnish MS families (p = 0.0004; remaining significant after correction for multiple testing). Further, a dense set of 211 SNPs evenly covering the PRKCA gene and the flanking regions was selected from the dbSNP database and analyzed in two large, independent MS cohorts: in 211 Finnish and 554 Canadian MS families. A multipoint SNP analysis indicated linkage to PRKCA and its telomeric flanking region in both populations, and SNP haplotype and genotype combination analyses revealed an allelic variant of PRKCA, which covers the region between introns 3 and 8, to be over-represented in Finnish MS cases (odds ratio = 1.34, 95% confidence interval 1.07-1.68). A second allelic variant, covering the same region of the PRKCA gene, showed somewhat stronger evidence for association in the Canadian families (odds ratio = 1.64, 95% confidence interval 1.39-1.94). Initial functional relevance for disease predisposition was suggested by the expression analysis: The transcript levels of PRKCA showed correlation with the copy number of the Finnish and Canadian "risk" haplotypes in CD4-negative mononuclear cells of five Finnish multiplex families and in lymphoblast cell lines of 11 Centre d'Etude du Polymorphisme Humain (CEPH) individuals of European origin.

Figure 1. Schematic Representation of the MS Locus on Chromosome 17q24

Positions of the SNPs used in stages I, II, and III are shown with arrowheads. Grey arrowheads indicate SNPs showing evidence for association: stage I, p < 0.05; stage II, p < 0.01. SNP rs887797 showing evidence for association in the two independent Finnish study sets (Study sets I and II) is shown with a black arrow. The horizontal bar indicates the position of the critical MS region identified by haplotype analysis in our previous study [21]. The positions of the genes mapping to the locus are shown as boxes below.

Figure 2. Haplotype Block Structure of the PRKCA Gene in Finnish and Canadian Populations

(A) shows the block structures of the PRKCA gene (between SNPs rs3764402 and rs4791037) in 211 Finnish MS families; (B), in the 554 Canadian MS families. The haplotype blocks were created using the solid line of LD of the Haploview program, version 3.2 [27]. The SNPs providing strongest evidence for association to MS in PRKCA in Finns and Canadians (rs887797, rs2361491 and rs2078153, rs1860984, respectively) are marked with black arrows, SNPs used in Haploview version 3.2 [27] haplotype analysis in 211 Finnish families are indicated with boxes, and those used in the two SNP genotype combinations are indicated with a white arrowhead with the letter H and the rs-numbers given above.

Figure 3. Expression of the PRKCA Gene and Overview of the Gene Region

(A) RT-PCR analysis of expression of the PRKCA gene in isolated human cells. (B) Relative levels of the PRKCA gene expression in CD4⁻ blood mononuclear cells in individuals with one (n[controls] = 4, n[MS] = 3) or two copies (n[controls] = 4, n[MS] = 5) of the “risk” MS haplotype. Ct(PRKCA) values are normalized against mean of Ct(HMBS) and Ct(β-actin) and are shown on the y-axis and the number of putative disease haplotypes (rs887797–rs1010544: C-A) in the x-axis. (Ct = number of amplification cycles required to reach the selected signal threshold, thus higher Ct values mean lower relative expression levels). Expression in MS cases is shown with black diamonds and in healthy controls with white squares (average ± SD are given for all individuals as horizontal lines). (C) UCSC Human Genome Browser view of the critical region of the PRKCA gene (chr17:61,980,000–62,158,000). The genomic DNA region covered by haplotype blocks 4–7 (in Canadians) and blocks 4–9 (in Finns) is indicated on top. The ruler shows map positions on chromosome 17 (UCSC Genome Browser, April, 2004). Names and positions of the SNPs used in the haplotype analysis are shown above the ruler. The names of the mRNA clones corresponding to the PRKCA gene variants are shown on the left and to the three other mRNA clones beside them in the middle. The spliced and unspliced ESTs, respectively, are shown in the middle of the figure. Conservation between species and the repeat masker are indicated below.