Systematic Linkage Disequilibrium Analysis of SLC12A8 at PSORS5 Confirms a Role in Susceptibility to Psoriasis Vulgaris
2005; Elsevier BV; Volume: 125; Issue: 5 Linguagem: Inglês
10.1111/j.0022-202x.2005.23847.x
ISSN1523-1747
AutoresUlrike Hüffmeier, Jesús Lascorz, Heiko Traupe, Beate Böhm, Funda Schürmeier-Horst, Markward Ständer, Reinhard Kelsch, Claudia Baumann, Wolfgang Küster, Harald Burkhardt, André Reis,
Tópico(s)Mast cells and histamine
ResumoThe gene for solute carrier family 12 member A8 has recently been proposed as a candidate gene for psoriasis susceptibility (PSORS5) on chromosome 3q based on association of five single nucleotide polymorphisms (SNP) in Swedish patients. To investigate whether this locus is relevant for German psoriasis vulgaris (PsV) patients, we analyzed a group of 210 trios and a case–control group including 375 patients. Based on our investigation of the linkage disequilibrium (LD) structure of SLC12A8, we assayed 35 haplotype tag SNP and grouped them into nine LD-blocks. In the case–control study, we detected an association for six SNP and three LD-based haplotypes. Association was strongest for ss35527511 (χ2=11.224, p=0.0008) and haplotype E-2 (χ2=11.788, p=0.00059) and independent of the presence of an HLA-associated PSORS1 risk allele. Through extended haplotype analysis, we could show that two independent association signals exist in SLC12A8, suggesting allelic heterogeneity. None of the SNP showed association in trios, apart from a weak association of rs2228674 (transmission disequilibrium test statistics p=0.048), probably due to insufficient power. We conclude that SLC12A8 is a susceptibility locus for PsV. In order to establish the exact nature of this association, efforts to identify the disease-causing variants are ongoing. The gene for solute carrier family 12 member A8 has recently been proposed as a candidate gene for psoriasis susceptibility (PSORS5) on chromosome 3q based on association of five single nucleotide polymorphisms (SNP) in Swedish patients. To investigate whether this locus is relevant for German psoriasis vulgaris (PsV) patients, we analyzed a group of 210 trios and a case–control group including 375 patients. Based on our investigation of the linkage disequilibrium (LD) structure of SLC12A8, we assayed 35 haplotype tag SNP and grouped them into nine LD-blocks. In the case–control study, we detected an association for six SNP and three LD-based haplotypes. Association was strongest for ss35527511 (χ2=11.224, p=0.0008) and haplotype E-2 (χ2=11.788, p=0.00059) and independent of the presence of an HLA-associated PSORS1 risk allele. Through extended haplotype analysis, we could show that two independent association signals exist in SLC12A8, suggesting allelic heterogeneity. None of the SNP showed association in trios, apart from a weak association of rs2228674 (transmission disequilibrium test statistics p=0.048), probably due to insufficient power. We conclude that SLC12A8 is a susceptibility locus for PsV. In order to establish the exact nature of this association, efforts to identify the disease-causing variants are ongoing. alpha-helix coiled-coil rod homolog gene haplotype tag SNP linkage disequilibrium minor allele frequency gene for psoriasis susceptibility psoriasis vulgaris single nucleotide polymorphism transmission disequilibrium test At chromosome 3q21, (Enlund et al., 1999Enlund F. Samuelsson L. Enerback C. et al.Psoriasis susceptibility locus in chromosome region 3q21 identified in patients from southwest Sweden.Eur J Hum Genet. 1999; 7: 783-790Crossref PubMed Scopus (118) Google Scholar proposed a candidate region showing linkage and association of the microsatellite D3S1551 to psoriasis after stratification of their sample for parental origin (at least one parent originating from Southwest Sweden). The following linkage disequilibrium mapping of the candidate region on 3q21 identified SLC12A8 as a candidate gene based on association of psoriasis to several intronic single nucleotide polymorphisms (SNPs) as well as to haplotypes (Hewett et al., 2002Hewett D. Samuelsson L. Polding J. et al.Identification of a psoriasis susceptibility candidate gene by linkage disequilibrium mapping with a localized single nucleotide polymorphism map.Genomics. 2002; 79: 305-314Crossref PubMed Scopus (91) Google Scholar). It has long been known that in general, SNPs show stronger linkage disequilibrium (LD) the closer they are located, although this relation can be erratic in many cases. Some distant SNPs can show strong LD, whereas close ones show almost none. Since the first publications ofReich et al., 2001Reich D.E. Cargill M. Bolk S. et al.Linkage disequilibrium in the human genome.Nature. 2001; 411: 199-204Crossref PubMed Scopus (1288) Google Scholar andDaly et al., 2001Daly M.J. Rioux J.D. Schaffner S.F. Hudson T.J. Lander E.S. High-resolution haplotype structure in the human genome.Nat Genet. 2001; 29: 229-232Crossref PubMed Scopus (1383) Google Scholar, it has been recognized that the genome consists of haplotype blocks characterized by few or no recombinations; therefore, SNPs within the same haplotype block are in strong LD. The haplotype blocks are confined by recombination hot spots with little or no LD. Only few different haplotypes within one block account for the majority of detected haplotypes. The haplotype diversity can be ascertained by genotyping haplotype tag SNPs (htSNPs) (Johnson et al., 2001Johnson G.C. Esposito L. Barratt B.J. et al.Haplotype tagging for the identification of common disease genes.Nat Genet. 2001; 29: 233-237Crossref PubMed Scopus (999) Google Scholar), whose combination represents the (majority of) detected haplotypes within a haplotype block. Due to the consistency of these haplotype blocks during the last millennia, it is being recognized that it might be a more advantageous approach to characterize the block structure of a candidate region and to use this information for association studies instead of single SNPs for defining genetic variants underlying common complex diseases. We set out to replicate the finding ofHewett et al., 2002Hewett D. Samuelsson L. Polding J. et al.Identification of a psoriasis susceptibility candidate gene by linkage disequilibrium mapping with a localized single nucleotide polymorphism map.Genomics. 2002; 79: 305-314Crossref PubMed Scopus (91) Google Scholar in our psoriasis samples of 375 psoriasis vulgaris (PsV) patients and of 210 trios of German origin. In order to systematically investigate this locus, we determined the LD-based haplotype block structure of SLC12A8 and tested single SNP as well as haplotypes within the block structure for allelic association in both study groups. In order to determine whether the association with SLC12A8 was independent of the major psoriasis susceptibility locus (PSORS1), which is linked to the MHC locus, we stratified for the risk allele at this locus and tested again for association. A total of 102 SNP with minor allele frequency (MAF)>0.1 were identified by direct sequencing of the 71 amplicons described above. From these, the 68 SNPs showing MAF>0.2 in the 24 patients sequenced were included in the study, leading to an average density of one SNPs every 3 kb (see Table S3). The only coding SNP identified is rs2981482, located in exon 14, and leading to an aminoacid change from arginine to glutamine (R664Q); the remainder were located in introns or in untranslated regions (UTR). After reconstruction of haplotypes in the whole region with PHASE (Stephens et al., 2001Stephens M. Smith N.J. Donnelly P. A new statistical method for haplotype reconstruction from population data.Am J Hum Genet. 2001; 68: 978-989Abstract Full Text Full Text PDF PubMed Scopus (6192) Google Scholar), SNPtagger (Ke and Cardon, 2003Ke X. Cardon L.R. Efficient selective screening of haplotype tag SNPs.Bioinformatics. 2003; 19: 287-288Crossref PubMed Scopus (173) Google Scholar) was used to determine the 35 tag SNPs that were subsequently genotyped in the cohorts. The average genotyping rate was 98.1%. Hardy–Weinberg equilibrium for all tag SNPs was confirmed in all cohorts. Download .doc (.1 MB) Help with doc files Table S3SNP positions The case–control study provided association with six SNPs, with ss35527511 showing the strongest association (χ2=11.224, p=0.0008, odds ratio (OR)=1.51 [1.19–1.92]) (Table I). Two of the tag SNPs, ss35527511 and rs651630, remained significant even after Bonferroni correction for multiple testing (e.g., ss35527511 pc=0.028; 35 SNPs tested), which is overly conservative and known to overcorrect data (Cardon and Bell, 2001Cardon L.R. Bell J.I. Association study designs for complex diseases.Nat Rev Genet. 2001; 2: 91-99Crossref PubMed Scopus (1139) Google Scholar). A complete overview of the association with single SNPs in the case–control study is presented in Figure 1. In the trios with PsV, single SNP analysis resulted in only a marginal significant transmission disequilibrium test (TDT) p-value of 0.048 for rs2228674 (EC2 (Hewett et al., 2002Hewett D. Samuelsson L. Polding J. et al.Identification of a psoriasis susceptibility candidate gene by linkage disequilibrium mapping with a localized single nucleotide polymorphism map.Genomics. 2002; 79: 305-314Crossref PubMed Scopus (91) Google Scholar)).Table IAllele frequencies of the associated tag SNP in patients and controls and results of χ2 statisticsPsoriasis vulgarisSNP rs/ss-numberControls n (%)n (%)χ2p-valueOR [95% confidence interval]ss35527513 Allele A605 (81)636 (86)8.1640.00431.50 [1.14–1.98] Allele T145 (19)102 (14)ss35527511 Allele A205 (27)149 (20)11.2240.00081.51 [1.19–1.92] Allele G543 (73)595 (80)rs9813946 Allele A489 (65)528 (71)6.5050.0111.33 [1.07–1.65] Allele C261 (35)212 (29)rs9831295 Allele A500 (69)551 (74)5.2670.0221.31 [1.04–1.64] Allele G224 (31)189 (26)rs1554241 Allele A265 (36)305 (41)3.8890.0491.24 [1.00–1.52] Allele T471 (64)439 (59)rs651630 Allele A423 (57)354 (48)10.970.00091.40 [1.14–1.72] Allele G325 (43)384 (52)SNP, single nucleotide polymorphism; OR, odds ratio. Open table in a new tab SNP, single nucleotide polymorphism; OR, odds ratio. The 68 SNPs with MAF>0.2 sequenced in 24 probands were used to determine the haplotype block structure of SLC12A8. A total of nine haplotype blocks between 3 and 20 kb long were identified (Figure 2). Using the 35 tag SNPs genotyped in the case–control samples, this haplotype block structure was confirmed in the two groups (375 PsV patients and 376 controls) (data not shown). Data for associated haplotypes in the case–control study are shown in Table II, and Figure 3 summarizes the results for all haplotype blocks in this study group. Strong association with haplotypes in block B and block E was observed. Haplotype B-2 (χ2=10.552, p=0.001, OR=1.41 [1.15–1.73]) as well as haplotype E-1 (χ2=8.821, p=0.003, OR=1.40 [1.130–1.75]) were the risk haplotypes. One further haplotype, A-5, was also strongly associated (χ2=10.202, p=0.001, OR=2.27 [1.36–3.80]), whereas it accounted for only a minor number of 6% of haplotypes in patients and 3% in controls. Two other adjacent haplotypes, A-3 and H-3, were also slightly associated. The PsV trios were not tested for association at the haplotype level.Table IIFrequencies of the associated haplotypes within a haplotype block in patients and controls and results of χ2 statisticsPsoriasis vulgarisBlock-haplotypeAllele combinationControls n (%)n (%)χ2p-valueOR [95% confidence interval]A-3A G G C A T A67 (9)45 (6)4.6070.032A-5A A A T G T G22 (3)48 (6)10.2020.0012.27 [1.36–3.80]B-2A A274 (36)335 (45)10.5520.0011.41 [1.15–1.73]B-3A A145 (19)102 (14)8.8230.003E-1T A C G G A A197 (26)249 (33)8.8210.0031.40 [1.13–1.75]E-2T A C A G C G180 (24)126 (17)11.7880.00059H-3T A G G192 (26)232 (31)5.4070.021.31 [1.04–1.64]OR, odds ratio. Open table in a new tab OR, odds ratio. We next investigated whether the haplotype blocks associated are part of the same long-range haplotype and whether we could clarify the localization of the association signal. We used Haploview (Barrett et al., 2005Barrett J.C. Fry B. Maller J. Daly M.J. Haploview: Analysis and visualization of LD and haplotype maps.Bioinformatics. 2005; 21: 263-265Crossref PubMed Scopus (11430) Google Scholar) to reconstruct longer-range haplotypes from single SNPs between blocks B and E and their adjacent regions in the gene (Figure 4). When we included the five haplotypes of block C in the analysis of the two associated haplotypes B-2 or E-1, we failed to observe any significant difference between patients and controls, indicating that the two signals observed in block B and block E are independent of each other. We next included rs1554241 and rs651630, which are also associated in the case–control study at a single SNP level, but that are not included in neither of their flanking blocks E and F nor G and H, respectively, into haplotypes with haplotype E1. All these haplotypes showed association, whereas a haplotype comprising E-1 and rs1712458, located immediately upstream and not contained in any block, showed a stronger association than haplotype E-1 alone. When also including rs684030, the tag SNP of haplotype block D, however, the association appeared to be weaker, indicating that the causative variant is located between both blocks in a region of low LD. In the proximal part of the gene, the longer-range haplotype comprising haplotypes A-5 and B-2 was associated (χ2=11.8, p=0.0006), even at a higher level than either of the two haplotypes alone. As expected, we found strong association with the risk alleles of the two single HCR-SNPs at PSORS1 (4.26 × 10−14 for HCR-325*T and 1.15 × 10−8 for HCR-2327*G). Deviation from Hardy–Weinberg equilibrium for the two HCR-SNPs was also detected due to significant excess of patients heterozygous for the risk alleles. In contrast, Hardy–Weinberg equilibrium was confirmed in the control group. We next investigated whether patients of the case–control study carrying the HCR risk haplotype at PSORS1 had a higher frequency of haplotypes A-5, B-2, E-1, or H-3. We found no significant differences in the two study groups, indicating that the risk alleles in LD with haplotypes A5, B-2, E-1, or H3 do not add any additional risk to that conferred by the PSORS1 risk haplotype, and thus act independently. Our systematic LD analysis provides the first replication of association with SLC12A8, a candidate gene for psoriasis at PSORS5 on 3q21 (Hewett et al., 2002Hewett D. Samuelsson L. Polding J. et al.Identification of a psoriasis susceptibility candidate gene by linkage disequilibrium mapping with a localized single nucleotide polymorphism map.Genomics. 2002; 79: 305-314Crossref PubMed Scopus (91) Google Scholar). Of five associated SNPs in the original study, only two were significant on their own in our study: rs1554241 (B1551S3, p=0.049 in the single cases) and rs2228674 (EC2, TDT p=0.048 in the trios). In addition, rs531740 (BES2) is part of haplotype E-1, which is strongly associated in our case–control study. SNP rs702045 (B1551S4), further reported to be associated in the Swedish group, was not included in our study due to its low MAF (0.05). In the initial report, the five associated variants were reported to form a haplotype that was highly associated with PsV (p=3.8 × 10−5). Constructing haplotypes without taking into account the underlying LD structure of a region can result in a false-positive association due to casual LD between these markers and the localization of the true causal SNP some distance away (Daly et al., 2001Daly M.J. Rioux J.D. Schaffner S.F. Hudson T.J. Lander E.S. High-resolution haplotype structure in the human genome.Nat Genet. 2001; 29: 229-232Crossref PubMed Scopus (1383) Google Scholar). Also, the resolution of the haplotype block structure of a gene and the use of the corresponding haplotypes in each block is much more powerful than marker-by-marker analysis (Zhang et al., 2002Zhang K. Calabrese P. Nordborg M. Sun F. Haplotype block structure and its applications to association studies: Power and study designs.Am J Hum Genet. 2002; 71: 1386-1394Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Therefore, we determined the haplotype structure of SLC12A8 and used these haplotypes to confirm and extend the association analysis based on single SNPs and to clarify the localization of the signal. Due to the current awareness that haplotypes can be better discerned when using ancestral SNPs, which are therefore more frequent, we set a cutoff for SNPs with MAF>0.2 based on the initial sample of probands. Thus, we obtained a block-like structure of regions with high LD (D′>0.8) sharply demarcated from regions of low LD (Figure 2). Blocks of high LD varied between 3 and 20 kb, whereas haplotype diversity varied between three and five different common haplotypes, consistent with data in the literature (Gabriel et al., 2002Gabriel S.B. Schaffner S.F. Nguyen H. et al.The structure of haplotype blocks in the human genome.Science. 2002; 296: 2225-2229Crossref PubMed Scopus (4531) Google Scholar). The number of SNPs required to reliably detect the underlying LD structure is dependent on the overall LD of the region. The average density in our study with one SNP every 3 kb is much denser than the current HapMap data available, and allowed us to detect boundaries of critical regions with association more reliably, as reported previously (Cardon and Abecasis, 2003Cardon L.R. Abecasis G.R. Using haplotype blocks to map human complex trait loci.Trends Genet. 2003; 19: 135-140Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). We chose tag SNPs based on the LD structure in order to ascertain the underlying haplotype variability using various computational tools. This choice of tag SNPs independent of exact block boundaries has the advantage of identifying disease association with either specific haplotypes or with clades of related haplotypes (Carlson et al., 2004Carlson C.S. Eberle M.A. Rieder M.J. Yi Q. Kruglyak L. Nickerson D.A. Selecting a maximally informative set of single-nucleotide polymorphisms for association analyses using linkage disequilibrium.Am J Hum Genet. 2004; 74: 106-120Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar), as neighboring blocks can be correlated (Cardon and Abecasis, 2003Cardon L.R. Abecasis G.R. Using haplotype blocks to map human complex trait loci.Trends Genet. 2003; 19: 135-140Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar) and an etiological variant may lie inside a recombination hot spot between two haplotype blocks (Kauppi et al., 2004Kauppi L. Jeffreys A.J. Keeney S. Where the crossovers are: Recombination distributions in mammals.Nat Rev Genet. 2004; 5: 413-424Crossref PubMed Scopus (241) Google Scholar). By using these tag SNPs for association studies, we detected a total of six SNPs showing significant association in the case–control study different from those SNPs originally reported. Two of them (ss35527511 and rs651630) remained significant even after the overly conservative Bonferroni correction for multiple testing, indicating that the association is a true finding. The association with single SNPs was confirmed at haplotype level: two of the three most significantly associated SNPs are contained in haplotype blocks B and E, respectively. The level of association for block E is similar to that of SNP 34, whereas association with blocks B and A is remarkably higher than for single SNP analysis. This effect is well known as haplotypes can be more informative than single bi-allelic SNPs, when they are only in LD with the causative variant. The corresponding OR for the highly associated ss35527511 (OR=1.51 [1.19–1.92]) and for haplotype B-2 (OR=1.41 [1.15–1.73]) are in the expected range for a complex disease such as PsV with its multiple susceptibility loci. Next, we tried to unify the association signals seen from blocks A, B, and E through reconstruction of long-range haplotypes between these blocks. Although we tried several combinations (Figure 4), we failed to observe any association with these longer-range haplotypes, suggesting that the association signals are independent of each other. If verified, this would demonstrate allelic heterogeneity in SLC12A8. This heterogeneity could account for the difficulty that we and Hewett and co-workers had in finding significant evidence for association. We assume that the association reported for the Swedish population corresponds to that observed for haplotype E-1 in the German patients. Once an association is found in a case–control study, it is desirable to confirm it in an independent sample and preferably by using a different study design, e.g., TDT, known to have less power (Morton and Collins, 1998Morton N.E. Collins A. Tests and estimates of allelic association in complex inheritance.Proc Natl Acad Sci USA. 1998; 95: 11389-11393Crossref PubMed Scopus (227) Google Scholar) but to be more resistant to possible population stratification (Martin et al., 2000Martin E.R. Lai E.H. Gilbert J.R. et al.SNPing away at complex diseases: Analysis of single-nucleotide polymorphisms around APOE in Alzheimer disease.Am J Hum Genet. 2000; 67: 383-394Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). No association in our group of 210 parent–offspring trios was detected, only a marginal significant p-value for SNP 68 (EC2 inHewett et al., 2002Hewett D. Samuelsson L. Polding J. et al.Identification of a psoriasis susceptibility candidate gene by linkage disequilibrium mapping with a localized single nucleotide polymorphism map.Genomics. 2002; 79: 305-314Crossref PubMed Scopus (91) Google Scholar). The power of TDT-based studies requires significantly more patients to find the same level of association as case–control studies (Cardon and Bell, 2001Cardon L.R. Bell J.I. Association study designs for complex diseases.Nat Rev Genet. 2001; 2: 91-99Crossref PubMed Scopus (1139) Google Scholar). In order to estimate the sample size of a TDT study that would have allowed us to find the same level of association as in our case–control study, we applied the model ofMitchell, 2000Mitchell L.E. Relationship between case–control studies and the transmission/disequilibrium test.Genet Epidemiol. 2000; 19: 193-201Crossref PubMed Scopus (20) Google Scholar. In this model, a T value is calculated with the results of the case–control study, and is used to estimate Nmin, the minimum number of informative transmissions required in the TDT study to reject the null hypothesis of no linkage. A requirement of a number of informative trios between 151 and 613 was calculated to reach a significance level of 0.05 with 80% power (Table S4). In our study, this figure was only attained for rs651630. Interestingly, this SNP is highly associated in the single cases, and is the only one that is close to the significance level in the TDT study (p=0.064). We thus conclude, that our group of trios was underpowered to detect the association. Download .doc (.03 MB) Help with doc files Table S4Estimation of sample sizes for TDT It has been previously reported that other psoriasis susceptibility loci show epistasis to the major psoriasis susceptibility locus at PSORS1 (Veal et al., 2001Veal C.D. Clough R.L. Barber R.C. et al.Identification of a novel psoriasis susceptibility locus at 1p and evidence of epistasis between PSORS1 and candidate loci.J Med Genet. 2001; 38: 7-13Crossref PubMed Scopus (122) Google Scholar). Therefore, we investigated whether the association with SLC12A8 was contingent to the presence of the risk allele at PSORS1. We determined this allele indirectly based on an HCR risk haplotype, which is in very high LD with the common PSORS1 risk allele, a procedure that has been used before (Asumalahti et al., 2003Asumalahti K. Laitinen T. Lahermo P. et al.Psoriasis susceptibility locus on 18p revealed by genome scan in Finnish families not associated with PSORS1.J Invest Dermatol. 2003; 121: 735-740Crossref PubMed Scopus (46) Google Scholar; Huffmeier et al., 2005Huffmeier U. Traupe H. Burkhardt H. et al.Lack of evidence for genetic association to RUNX1 binding site at PSORS2 in different German psoriasis cohorts.J Invest Dermatol. 2005; 124: 107-110Crossref PubMed Scopus (23) Google Scholar). Interestingly, we detected no difference in allele frequencies independent of the presence or absence of the risk allele. Thus, we found no evidence for interaction of this SLC12A8 with PSORS1 and conclude that their effects in the pathogenesis of PsV are independent of each other. The exact nature of the disease-causing variants in SLC12A8 yet remains to be elucidated. The only coding SNP that we identified (R664Q, rs2981482) did not show any association and is also in a region with comparably low association at haplotype level. In the regions showing stronger association, no further coding variant was identified. We thus assume that variants affecting regulatory or splicing processes will be the risk factors at PSORS5. Since these processes are only partially understood and since the density of SNP in intronic sequences is much higher than in coding ones, it will require extensive sequencing efforts in large numbers of patients as well as functional studies in order to pinpoint the exact variants. New questions arise from the still unknown function of SLC12A8 and its potential role in the pathogenesis of PsV. Thus, the gene SLC12A8 encodes a protein-sharing homology with a family of cation-chloride-coupled cotransporters that are responsible for the electroneutral transport of ions in a variety of different cell types (Delpire and Mount, 2002Delpire E. Mount D.B. Human and murine phenotypes associated with defects in cation-chloride cotransport.Annu Rev Physiol. 2002; 64: 803-843Crossref PubMed Scopus (176) Google Scholar). Our study provides evidence for involvement of the member A8 in the pathogenesis of psoriasis. Primarily functional studies are essential to investigate the regular role of SLC12A8, e.g., the nature of transported substrate(s) as well as further aspects also regarding the compromised function in PsV. In summary, our study replicates the previously described association with SLC12A8 at PSORS5 to PsV, and it further localizes the signals of association with two haplotype blocks at non-coding regions. This locus seems to act independent of PSORS1 at the HLA locus. In order to determine the exact nature of SLC12A8 in the pathogenesis of psoriasis, however, identification of the disease-causing variants and functional studies are required. PsV patients. For a detailed description of the sample of 210 trios, we refer toHensen et al., 2003Hensen P. Windemuth C. Huffmeier U. et al.Association scan of the novel psoriasis susceptibility region on chromosome 19: Evidence for both susceptible and protective loci.Exp Dermatol. 2003; 12: 490-496Crossref PubMed Scopus (23) Google Scholar. The 375 single patients with PsV were of German origin and were recruited through dermatology clinics at two psoriasis rehabilitation hospitals. An early onset form of psoriasis—mainly plaque type of PsV—was diagnosed in all but three patients, with an average age of onset of 23±11 y. The medium age at the time of recruitment was 50±12 y. Sixty-two percent of patients were male. The study, including recruitment of controls, was approved by the ethical committee of the University of Münster. The 376 controls had no PsV at the time of recruitment, when the average age was 32±10 y. All of them were healthy blood donors and were recruited in Northern Germany. Fifty-nine percent of probands were male. The individuals gave their written informed consent. The investigations were conducted according to the Declaration of Helsinki principles. Seventy-one amplicons extending 44 kb of the gene SLC12A8 were sequenced in a total of 24 samples (for primer sequences, see Table S1). Amplicons included the 14 exons of SLC12A8 fromHewett et al., 2002Hewett D. Samuelsson L. Polding J. et al.Identification of a psoriasis susceptibility candidate gene by linkage disequilibrium mapping with a localized single nucleotide polymorphism map.Genomics. 2002; 79: 305-314Crossref PubMed Scopus (91) Google Scholar and another 58, chosen for equal coverage of the gene and for evolutionary sequence conservation. After PCR reactions in an MJ Research thermocycler (Biozym, Hess Oldendorf, Germany), the PCR products were purified with the PCR Purification Kit (Qiagen, Hilden, Germany) on a robotic system (Tecan Miniprep 75-2 with vacuum station, Tecan, Crailsheim, Germany) with Millipore Montage PCR Cleanup Filter Plates (Millipore, Schwalbach, Germany). Sequencing reactions were performed using the Big Dye Terminator Cycle Sequencing Kit Version 3 or 3.1 (Applied Biosystems, Foster City, California) according to the manufacturer's instructions, and were purified on the same robotic system with the Millipore Montage SEQ Sequencing Reaction Cleanup Kit or with the Dye-Ex Purification Kit (Qiagen). They were analyzed in an ABI Prism Model 3100 or 3730 Sequencer (Applied Biosystems), whereas sequencing analysis was performed with the software SeqMan II (DNA-Star, Madison, Wisconsin). Download .doc (.08 MB) Help with doc files Table S1Sequences of PCR primers Genotyping of the 35 tag SNPs was performed with either Taqman SNP Genotyping Assays from Applied Biosystems or with self-designed primers and TaqMan mgb-probes (for assay information, see Table S2). All Taqman reactions were prepared from a PCR master mix consisting of 2 × Taqman Mastermix (Eurogentec, Seraing, Belgium) and of 20 × or 40 × prepared Genotyping Assay Mix of primers and probes or single primers and probes. They were carried out in an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) using standard thermal cycling conditions. Hardy–Weinberg equilibrium was tested in the two samples of single individuals for all SNPs with Finetti (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). Taqman genotypes for all polymorphisms were verified by direct sequencing of 24 randomly chosen samples. For stratification to PSORS1, two previously described htSNPs in the HCR gene at positions 325 and 2327 that cover the haplotype diversity within this gene were chosen (Asumalahti et al., 2003Asumalahti K. Laitinen T. Lahermo P. et al.Psoriasis susceptibility locus on 18p revealed by genome scan in Finnish families not associated with PSORS1.J Invest Dermatol. 2003; 121: 735-740Crossref PubMed Scopus (46) Google Scholar; Huffmeier et al., 2005Huffmeier U. Traupe H. Burkhardt H. et al.Lack of evidence for genetic association to RUNX1 binding site at PSORS2 in different German psoriasis cohorts.J Invest Dermatol. 2005; 124: 107-110Crossref PubMed Scopus (23) Google Scholar) and genotyped as described previously (Huffmeier et al., 2005Huffmeier U. Traupe H. Burkhardt H. et al.Lack of evidence for genetic association to RUNX1 binding site at PSORS2 in different German psoriasis cohorts.J Invest Dermatol. 2005; 124: 107-110Crossref PubMed Scopus (23) Google Scholar). Download .doc (.07 MB) Help with doc files Table S2Assay information Pairwise LD was estimated using Ldmax, as implementation of the program GOLD (graphical overview of LD) (Abecasis and Cookson, 2000Abecasis G.R. Cookson W.O. GOLD–graphical overview of linkage disequilibrium.Bioinformatics. 2000; 16: 182-183Crossref PubMed Scopus (673) Google Scholar). LD blocks were inferred from the data of the 24 samples sequenced. The same procedure was used for confirmation of the haplotype block structure in the groups of 375 PsV patients and of 376 unrelated controls. Haplotypes for the whole sequenced region and in every haplotype block were calculated with PHASE in the set of 24 probands (Stephens et al., 2001Stephens M. Smith N.J. Donnelly P. A new statistical method for haplotype reconstruction from population data.Am J Hum Genet. 2001; 68: 978-989Abstract Full Text Full Text PDF PubMed Scopus (6192) Google Scholar). SNPtagger (Ke and Cardon, 2003Ke X. Cardon L.R. Efficient selective screening of haplotype tag SNPs.Bioinformatics. 2003; 19: 287-288Crossref PubMed Scopus (173) Google Scholar) was used to determine a total of 35 tag SNPs. To test for allelic association of single SNPs in the trios, we used the family-based association test (FBAT) (Laird et al., 2000Laird N.M. Horvath S. Xu X. Implementing a unified approach to family-based tests of association.Genet Epidemiol. 2000; 19: S36-S42Crossref PubMed Scopus (734) Google Scholar). In the case–control study, association with single SNPs was tested using a χ2-statistics, or in the case of very few expected observations, with Fisher's exact test. In a first step we calculated a χ2-test based on the number of alleles in the case–control samples; for alleles yielding a significant p-value (p<0.05), we calculated an additional χ2-test based on the number of different genotypes to exclude that homozygosity for the risk allele falsified our results (Sasieni, 1997Sasieni P.D. From genotypes to genes: Doubling the sample size.Biometrics. 1997; 53: 1253-1261Crossref PubMed Scopus (738) Google Scholar). OR and their confidence intervals were determined for single SNP alleles with final p-values of <0.05. In the control and patient samples, haplotypes within the blocks were calculated with PHASE (Stephens et al., 2001Stephens M. Smith N.J. Donnelly P. A new statistical method for haplotype reconstruction from population data.Am J Hum Genet. 2001; 68: 978-989Abstract Full Text Full Text PDF PubMed Scopus (6192) Google Scholar) and Haploview (Barrett et al, 2004). Supported by grants from the Deutsche Forschungsgemeinschaft (DFG, Tr 228/5-4 and Re 679/10-4) and from the German Ministry for Education and Research (grant 001 KS 0002) within the Interdisciplinary Centre for Clinical Research at the University of Erlangen-Nuremberg (project A8). We thank all patients and other probands for participation in this study and Olga Zwenger and Verena Popp for excellent technical assistance. The following material is available online for this article. Table S1.Sequences of PCR primers Table S2.Assay information Table S3.SNP positions Table S4.Estimation of sample sizes for TDT
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