Analysis of CNTNAP2 polymorphisms in Polish population with pseudoexfoliation syndrome

Editor, T he incidence of pseudoexfoliation (PEX) syndrome varies among ethnic groups – from 0% in Greenland Eskimos to 25% in the Scandinavian countries (Challa et al. 2008; Arnarsson 2009). Three single-nucleotide polymorphisms (SNPs) in the lysyl oxidase-like 1 gene (LOXL1) have been confirmed to be associated with PEX in our previous study (Malukiewicz et al. 2011). In a recent investigation, an association between PEX and two SNPs, rs2107856 and rs2141388 of the contactin-associated protein-like 2 encoded by the CNTNAP2 gene located in intron 11 was found in Germans but not in Italians (Krumbiegel et al. 2011). We investigated the association between the SNPs rs2107856 and rs2141388 and PEX in Polish population. We studied 48 patients (16 men and 32 women), mean age 75 (SD = 7) with PEX and no other ocular diseases, for example, glaucoma. Control group comprised 30 healthy subjects (11 men and 19 women), mean age 76 (SD = 8). Pseudoexfoliation changes were identified as the presence of typical PEX material on the anterior lens surface, iris or corneal endothelium in either eye. Genomic DNA was extracted from blood as described previously (Malukiewicz et al. 2011). Genotypes of the SNPs rs2107856 and rs2141388 were determined using a commercially available TaqMan genotyping assays with the ABI Prism Sequence Detector 7000 (Applied Biosystems, Foster City, CA, USA). Correctness of genotyping was evaluated by direct sequencing for randomly selected samples. The Arlequin software version 3.1. was used to determine the Hardy– Weinberg equilibrium and estimate haplotype frequencies. The Fisher exact test was performed to compare patient and control groups for possible associations between allele frequency and disease state. Odds ratios were also calculated. The significance level for all statistical tests was 0.05. Statistical analysis was performed using Statistica software (version 8). The genotype distribution of rs2107856 and rs214138 was found in Hardy–Weinberg equilibrium in both groups. The allele frequencies in patients with PEX sample were not significantly different from those in the control group, for either rs2107856 (p = 0.38) or rs214138 (p = 0.38) (Table 1). Linkage disequilibrium analysis gave the value of r = 1 which proved that both SNPs are perfectly correlated to each other. There were no significant differences in haplotype frequencies between PEX and control samples. The haplotype (GC), present in 72% of cases and 65% of controls, confers a 1.38-fold of increased disease risk (95% CI, 0.685–2.765; p = 0.367). Krumbiegel et al. (2011) detected a significant association of both SNPs and their haplotype TT with PEX ⁄ PEXG in German but not the Italian cohort. They reported that the TT haplotype confers risk of the disease of 1.42 for German population. In our study, the minor T allele frequency is 0.28 in cases and 0.35 in controls. While the T frequency in our cases is the same as in Krumbiegel et al. (2011), the frequency in controls is higher (0.35 compared to 0.224), and this difference is statistically significant (p = 0.0094). The main cause of these conflicting observations can be either relatively small sizes of Polish samples or genetic heterogeneity of European populations. It was shown that the pronounced population differentiation at the level of haploid markers exists between the two geographically neighbouring countries, Poland and Germany (Kayser et al. 2005). In this respect, it should be emphasized that our study has only 18% power to detect the association, because of the small sample size. These findings strongly support the need for both replication of the CNTNAP2 SNPs association studies with PEX in Polish and other groups of larger sizes and further research into hidden population substructure which may affect the case–control data.