Association between the ACCN1 gene and multiple sclerosis in Central East Sardinia

Abstract

Multiple genome screens have been performed to identify regions in linkage or association with Multiple Sclerosis (MS, OMIM 126200), but little overlap has been found among them. This may be, in part, due to a low statistical power to detect small genetic effects and to genetic heterogeneity within and among the studied populations. Motivated by these considerations, we studied a very special population, namely that of Nuoro, Sardinia, Italy. This is an isolated, old, and genetically homogeneous population with high prevalence of MS. Our study sample includes both nuclear families and unrelated cases and controls. A multi-stage study design was adopted. In the first stage, microsatellites were typed in the 17q11.2 region, previously independently found to be in linkage with MS. One significant association was found at microsatellite D17S798. Next, a bioinformatic screening of the region surrounding this marker highlighted an interesting candidate MS susceptibility gene: the Amiloride-sensitive Cation Channel Neuronal 1 (ACCN1) gene. In the second stage of the study, we resequenced the exons and the 3' untranslated (UTR) region of ACCN1, and investigated the MS association of Single Nucleotide Polymorphisms (SNPs) identified in that region. For this purpose, we developed a method of analysis where complete, phase-solved, posterior-weighted haplotype assignments are imputed for each study individual from incomplete, multi-locus, genotyping data. The imputed assignments provide an input to a number of proposed procedures for testing association at a microsatellite level or of a sequence of SNPs. These include a Mantel-Haenszel type test based on expected frequencies of pseudocase/pseudocontrol haplotypes, as well as permutation based tests, including a combination of permutation and weighted logistic regression analysis. Application of these methods allowed us to find a significant association between MS and the SNP rs28936 located in the 3' UTR segment of ACCN1 with p = 0.0004 (p = 0.002, after adjusting for multiple testing). This result is in tune with several recent experimental findings which suggest that ACCN1 may play an important role in the pathogenesis of MS.

Figure 1. Studied genomic region.

This figure consists of three panels, (A,B,C). Panel A shows the location of the 17q11.2 region on Chromosome 17. Panel B maps the position of four known genes (blue rectangles) in the region around D17S798, where a horizontal line represents DNA sequence, and a pink bar marks the location of microsatellites D17S798 and D17S1800. The diagram conveys the fact that TMEM98 and SPACA3 (represented above the horizontal line) lie on the forward helix, whereas Myo1D and ACCN1 (represented below the horizontal line) lie on the reverse helix. Panel C zooms on gene ACCN1, showing the locations of the genotyped markers relative to D17S798, in Kb. In this latter panel, the horizontal line represents genomic DNA, the pink dots represent the SNPs we have genotyped, the pink vertical bar represents microsatellite D17S1850, the blue bars represent ACCN1 exons and the wavy solid line between exons corresponds to the ACCN1 introns. The ACCN1 gene is located on the reverse strand, so the portion of ACCN1 near D17S798, where SNP rs28936 and SNP rs28933 are located, is the 3′ UTR-exon10 region of the gene. The width of the introns and exons in panel C is proportional to the actual length, with the exception of the first three very long introns (right portion of the panel) which have been shortened. Gaps are represented by diagonal bars: three bars (intron1-2) = 1,043,911 base pairs; two bars (intron 3–4) = 60,470 base pairs; one bar (intron 2–3) = 22,926 base pairs