Rare and common variants in CARD14, encoding an epidermal regulator of NF-kappaB, in psoriasis.

Psoriasis is a common inflammatory disorder of the skin and other organs. We have determined that mutations in CARD14, encoding a nuclear factor of kappa light chain enhancer in B cells (NF-kB) activator within skin epidermis, account for PSORS2. Here, we describe fifteen additional rare missense variants in CARD14, their distribution in seven psoriasis cohorts (>6,000 cases and >4,000 controls), and their effects on NF-kB activation and the transcriptome of keratinocytes. There were more CARD14 rare variants in cases than in controls (burden test p value = 0.0015). Some variants were only seen in a single case, and these included putative pathogenic mutations (c.424G>A [p.Glu142Lys] and c.425A>G [p.Glu142Gly]) and the generalized-pustular-psoriasis mutation, c.413A>C (p.Glu138Ala); these three mutations lie within the coiled-coil domain of CARD14. The c.349G>A (p.Gly117Ser) familial-psoriasis mutation was present at a frequency of 0.0005 in cases of European ancestry. CARD14 variants led to a range of NF-kB activities; in particular, putative pathogenic variants led to levels >2.5× higher than did wild-type CARD14. Two variants (c.511C>A [p.His171Asn] and c.536G>A [p.Arg179His]) required stimulation with tumor necrosis factor alpha (TNF-α) to achieve significant increases in NF-kB levels. Transcriptome profiling of wild-type and variant CARD14 transfectants in keratinocytes differentiated probably pathogenic mutations from neutral variants such as polymorphisms. Over 20 CARD14 polymorphisms were also genotyped, and meta-analysis revealed an association between psoriasis and rs11652075 (c.2458C>T [p.Arg820Trp]; p value = 2.1 × 10(-6)). In the two largest psoriasis cohorts, evidence for association increased when rs11652075 was conditioned on HLA-Cw*0602 (PSORS1). These studies contribute to our understanding of the genetic basis of psoriasis and illustrate the challenges faced in identifying pathogenic variants in common disease.

Magnitude and distribution of linkage disequilibrium in population isolates and implications for genome-wide association studies.

The genome-wide distribution of linkage disequilibrium (LD) determines the strategy for selecting markers for association studies, but it varies between populations. We assayed LD in large samples (200 individuals) from each of 11 well-described population isolates and an outbred European-derived sample, using SNP markers spaced across chromosome 22. Most isolates show substantially higher levels of LD than the outbred sample and many fewer regions of very low LD (termed 'holes'). Young isolates known to have had relatively few founders show particularly extensive LD with very few holes; these populations offer substantial advantages for genome-wide association mapping.

High resolution mapping in the major histocompatibility complex region identifies multiple independent novel loci for psoriatic arthritis.

OBJECTIVE: Psoriatic arthritis (PsA) has a clear familial predisposition, the major histocompatibility complex (MHC) region being the strongest genetic locus. The study primary objective was to identify single nucleotide polymorphisms (SNPs) independent of known human leucocyte antigen (HLA) alleles within the MHC region that are associated with PsA using a high-density SNP map. METHOD: In all, 914 samples were assessed, including 427 PsA cases from 2 well established PsA cohorts and 487 controls from Canada. The genotype data consisted of 2521 SNPs from 2 Illumina Goldengate MHC panels, spanning 4.9 Mb of chromosome 6 with an average spacing of 2 kb. Classical HLA alleles were genotyped in all subjects using sequence-specific oligonucleotide probes or sequence-specific primers. A conditioning approach was used to distinguish between new associations and those in linkage disequilibrium (LD) with known HLA alleles. RESULTS: Unconditional association analysis revealed 43 markers with p<7.26×10(-5) (calculated experiment-wide significance threshold). In the conditional analysis, 10 SNPs showed statistically significant association at a threshold of p<7.26×10(-5). Seven SNPs were in strong LD in the study data (pairwise r(2) >0.77 in the controls) reflecting one association signal. These SNPs spanned a 1.6 Mb region. SNP rs1150735 is 1.5 kb upstream from ring finger protein 39 (RNF39). RNF39 SNPs have been associated with HIV1 disease progression and set point CD4 T cell count. CONCLUSION: Four new loci for either psoriasis or PsA in the MHC region that are independent of known HLA alleles have been identified. The effect size of these variants is modest. Replication of these variants in multiple larger populations is necessary.

Genetic epidemiology of complex phenotypes.

Genetic factors play a substantive role in the susceptibility to common diseases. Due to recent advancements in the characterization of genetic variants and large-scale genotyping platforms, multiple genes have now been identified for common, complex diseases. As a result, there is an immense interest in elucidating genetic factors in complex traits. Therefore, it is important to understand the design as well as the technical and statistical issues of the two most common approaches for gene identification: linkage and association.

Association of TNF-alpha polymorphisms in Crohn disease.

Clinical and molecular studies implicate tumor necrosis factor alpha (TNF-alpha) as a key mediator in the initiation and propagation of Crohn disease (CD). Genetic associations have been documented between promoter polymorphisms of TNF-alpha and CD; however, these associations have not been universally replicated. In this study, we set out to examine the association of five promoter TNF-alpha polymorphisms in CD subjects from a founder population. In total, 128 CD subjects and 103 ethnically matched healthy controls were genotyped with time-of-flight mass spectrometry for the following five single nucleotide polymorphisms (SNPs) in the 5' flanking region of TNF-alpha gene: -1031 (T-->C), -863 (C-->A), -857 (C-->T), -308 (G-->A), and -238 (G-->A). Primer sequences, termination mixes, and multiplexing were determined with Sequenom SpectroDESIGNER software v1.3.4. The minor allele frequency for the TNF-alpha SNPs in subjects with CD and healthy controls, respectively, were -238 (5.5% vs. 5.3%); -308 (17.6% vs. 18.9%); -857 (5.1% vs. 7.8%); -863 (19.1% vs. 17.5%), and -1031 (24.6% vs. 22.8%). Thus, none of the TNF-alpha variants was associated with CD. Furthermore, no genotype/phenotype correlations were observed for the mutant allele of the TNF-alpha variants and selected clinical outcomes. In conclusion, there was no significant association for any of the TNF-alpha promoter polymorphism tested and CD in the Newfoundland population.

Association of SEEK1 polymorphisms in Crohn's disease.

Crohn's disease (CD) is an inflammatory bowel disease that is likely the result of an interplay of genetic and environmental factors. Recent studies have postulated similarities in genetic susceptibility of CD and psoriasis. Because the +39604 SEEK1 polymorphism in chromosome 6p has recently been associated with psoriasis, the prevalence of this polymorphism, as well as two additional single nucleotide polymorphisms in the SEEK1 gene, in patients with CD from the Newfoundland population were determined. In total, 126 CD and 105 ethnically matched healthy controls were genotyped via time-of-flight mass spectrometry for the following SEEK1 polymorphisms: +39604 and +39709 in exon 2 and +26680 in exon 5. The allele frequency for the mutant allele for +39604, +39709, and +26680 was 16.3%, 44.5%, and 0%, respectively, in subjects with CD as compared with 17.6%, 42.0%, and 0%, respectively, in controls. There was no statistical difference in any of the variants between patients with CD and controls. The SEEK1polymorphisms +39604, +39709, and +26680 are not associated with CD in the Newfoundland population.

Genome-wide signatures of 'rearrangement hotspots' within segmental duplications in humans.

The primary objective of this study was to create a genome-wide high resolution map (i.e., >100 bp) of 'rearrangement hotspots' which can facilitate the identification of regions capable of mediating de novo deletions or duplications in humans. A hierarchical method was employed to fragment segmental duplications (SDs) into multiple smaller SD units. Combining an end space free pairwise alignment algorithm with a 'seed and extend' approach, we have exhaustively searched 409 million alignments to detect complex structural rearrangements within the reference-guided assembly of the NA18507 human genome (18× coverage), including the previously identified novel 4.8 Mb sequence from de novo assembly within this genome. We have identified 1,963 rearrangement hotspots within SDs which encompass 166 genes and display an enrichment of duplicated gene nucleotide variants (DNVs). These regions are correlated with increased non-allelic homologous recombination (NAHR) event frequency which presumably represents the origin of copy number variations (CNVs) and pathogenic duplications/deletions. Analysis revealed that 20% of the detected hotspots are clustered within the proximal and distal SD breakpoints flanked by the pathogenic deletions/duplications that have been mapped for 24 NAHR-mediated genomic disorders. FISH Validation of selected complex regions revealed 94% concordance with in silico localization of the highly homologous derivatives. Other results from this study indicate that intra-chromosomal recombination is enhanced in genic compared with agenic duplicated regions, and that gene desert regions comprising SDs may represent reservoirs for creation of novel genes. The generation of genome-wide signatures of 'rearrangement hotspots', which likely serve as templates for NAHR, may provide a powerful approach towards understanding the underlying mutational mechanism(s) for development of constitutional and acquired diseases.

Association of interleukin 23 receptor variants with psoriatic arthritis.

OBJECTIVE: A recent genome-wide pooling study noted a significant association of interleukin 23 receptor (IL-23R) and psoriasis. Overxpression of IL-23 has been detected in lesional psoriatic skin, and induces epidermal proliferation. Given the interplay between psoriasis and PsA, we examined the association of IL-23R variants in 2 independent Canadian Caucasian cohorts of patients with psoriatic arthritis (PsA). METHODS: We examined 496 PsA probands and 476 controls. Cases and controls were genotyped for a panel of 11 single-nucleotide polymorphisms (SNP) in IL-23R. Allele and haplotype associations were calculated using WHAP software. P values for haplotype associations were calculated using a permutation test. RESULTS: The 381Gln allele of the coding SNP Arg381Gln (rs11209026) was found to be protective in the Canadian population (p=0.004; corrected p=0.044). A 2-marker haplotype from SNP rs7530511 and rs11209026 was associated with PsA (p=0.011). All 3-marker sliding windows containing SNP rs11209026 were associated with PsA (p=0.02 for all 3 windows). The magnitude of effect of IL-23R association in PsA appears to be similar to that reported in uncomplicated psoriasis. CONCLUSION: Significant associations between Arg381Gln SNP and haplotypes encoding this variant were noted in PsA. It remains to be determined what contribution of this association, if any, is specifically due to the inflammatory arthritis (PsA) rather than psoriasis.

IL-23R polymorphisms in patients with ankylosing spondylitis in Korea.

OBJECTIVE: IL23R polymorphisms have been shown to have a significant association with ankylosing spondylitis (AS). To date, these studies have been restricted to Caucasian patients with AS. Our study addresses this relationship in Korean patients with AS. METHODS: A total of 451 patients with AS and 392 ethnically matched healthy controls were enrolled. All patients were native Koreans with AS satisfying the modified New York criteria. In total, 10 single nucleotide polymorphisms (SNP) within the IL-23R gene cluster were genotyped. RESULTS: No IL-23R SNP were found to be associated with AS in Koreans. CONCLUSION: The association of IL23R and AS that is seen in Caucasian patients with AS is not present in Korean patients with AS.

Association of interleukin-23 receptor variants with ankylosing spondylitis.

OBJECTIVE: Recent studies have shown that a nonsynonymous single-nucleotide polymorphism (SNP) (Arg381Gln; rs11209026) in the interleukin-23 receptor (IL-23R) gene on chromosome 1p31 is associated with Crohn's disease and psoriasis. Given the clinical and immunologic overlap between ankylosing spondylitis (AS) and these diseases, and the potential function of this candidate SNP, this study was undertaken to examine the association of IL-23R variants with AS in multiple Canadian populations. METHODS: We examined 3 cohorts of AS patients from established rheumatic disease centers in Canada. The majority of AS patients were Caucasians of northern European descent, and all patients satisfied the modified New York classification criteria for AS or for juvenile spondylarthritis. We examined 424 AS probands and 401 controls from Alberta, 251 AS probands and 122 controls from Toronto, and 121 AS probands and 219 controls from Newfoundland. Ten IL-23R SNPs were genotyped, 9 of which were incorporated in the haplotype analysis. Allele and haplotype associations were calculated using the WHAP software package. P values for haplotype associations were calculated using a permutation test. RESULTS: The primary SNP of interest in a previous study of inflammatory bowel disease (IBD) (Arg381Gln; rs11209026) was found to be protective against AS in the Newfoundland population (P=0.04) and in the Toronto population (P=0.04) in single-marker univariate analysis. The strongest association, however, was with SNP rs11465804 (P=0.007 for the Newfoundland population and P=0.0007 for the Toronto population). A 3-marker sliding window omnibus test revealed a significant association with markers rs10489629, rs2201841, and rs11465804 in both the Newfoundland population (P=0.04) and the Alberta population (P=0.034). Our results were independent of the IBD and psoriasis status of the AS patients. CONCLUSION: This concurrent analysis of 3 distinct AS populations and their regional controls demonstrates a disease association with the IL-23R locus and implicates the same polymorphisms associated with IBD and psoriasis.

Analysis of single nucleotide polymorphisms in Toll-like receptor 4 shows no association with ankylosing spondylitis in a Korean population.

The role of microbial triggers in the pathogenesis of the ankylosing spondylitis (AS) has remained an active area of clinical and basic research but remains unresolved. We have recently found evidence of an important role for TLR4 in experimental reactive arthritis, raising the question to be addressed whether genetic polymorphisms in TLR4 affects susceptibility to AS. Innate immune responses to Gram-negative bacteria involve in a central role the binding of lipopolysaccharide to TLR4. Two commonly occurring SNPs in the human TLR4 gene (Asp299Gly and Thr399Ile) have been shown to be associated with increased risk of Gram-negative bacteremia in sepsis patients and with susceptibility to inflammatory bowel disease. It remains unresolved whether these SNPs are associated with AS and we have addressed this in a relatively genetically homogeneous population in Korea. A cohort of 200 Korean AS patients and 197 ethnically matched controls were studied. All patients were native Koreans with AS satisfying the modified New York criteria. Korean controls were examined and confirmed to be unaffected by AS. All subjects were genotyped for two functional SNPs in the TLR4 gene: Asp299Gly (A/G polymorphism) and TLR-4 (Thr399Ile) (C/T polymorphism) The Sequenom MassARRAY system was used for genotyping (Sequenom Inc., San Diego, CA, USA). All cases and controls were homozygous for the (A) allele for 299 variant and similarly for the 399 variant all cases and controls were homozygous for the (C) allele. Genetic-environmental interactions figure prominently in current concepts of the pathogenesis of AS. Our findings indicate that the polymorphisms in the TLR4 gene cannot be regarded as major contributors to AS susceptibility in the Korean population.

Interleukin 1 polymorphisms in patients with ankylosing spondylitis in Korea.

OBJECTIVE: Studies in ankylosing spondylitis (AS) and psoriatic arthritis (PsA) cohorts have demonstrated that the interleukin 1 (IL-1) gene cluster contains a major susceptibility locus for AS and PsA. We examined the association between the IL-1 gene cluster and susceptibility to AS in Korea. METHODS: In total, 451 patients with AS and 402 ethnically matched healthy controls were genotyped with 51 single-nucleotide polymorphisms (SNP) within the IL-1 gene cluster (specifically located within the IL1A, IL1B, IL1RN, and IL1F5-10 genes based on findings of previous association studies). Samples were genotyped by MALDI-TOF mass spectrometry using standard Sequenom iPLEX conditions. Genotyping assays were designed using AssayDESIGNER 2.0 and all SNP designed as 4 multiplex reactions. The resulting product was analyzed using the MassArray Compact Analyzer, and genotype results were determined. Univariate SNP marker distributions in case-control populations were tested by chi-square tests. RESULTS: No SNP showed association with p value < 0.05. Haplotype analysis revealed an association with the 6 markers (empirical p < or = 2 x 10(-4), corresponding to Bonferroni corrected p = 0.05). Analysis of each 2, 3, 4, 5-marker sliding window revealed association with the IL1A locus (especially haplotype rs12622683-rs11123148-rs10165537). CONCLUSION: Single SNP associations noted in outbred Caucasian populations were not found in the Korean AS cohort. Haplotype analysis revealed associations with IL1 locus. These results support the notion that the IL1 locus is associated with susceptibility to AS.

Association of toll-like receptor 4 variants and ankylosing spondylitis: a case-control study.

OBJECTIVE: Functional single nucleotide polymorphisms within the ectoplasmic domain of the Toll-like receptor 4 (TLR4) gene have been shown to result in an endotoxin-hyporesponsive phenotype and aberrant signal transduction for bacterial agonists. TLR4 is in proximity to a genome-wide linkage peak in 9q32-33. Given the proposed function and location of TLR4, we examined the association of 2 functional variants of TLR4 in patients with ankylosing spondylitis (AS) in Newfoundland. METHODS: In total, 101 AS patients and 100 ethnically matched controls were genotyped, using the Sequenom MassArray platform, for 2 functional variants in the TLR4 gene: Asp299Gly (A/G polymorphism) and Thr399Ile (C/T polymorphism). RESULTS: The minor allele frequency for the Asp299Gly variant (G) was significantly higher in AS cases compared to controls (7.5% vs 2.6%, respectively; OR 3.10, p = 0.037). The minor allele frequency for the Thr399Ile variant (T) for cases and controls was 7.4% vs 3.0% (OR 2.59, p = 0.071). Haplotype analysis using Haploview noted a higher proportion of GT in the cases (for GT, chi-squared p = 0.023). CONCLUSION: Given the functional role of TLR4 variants in the innate immune system, larger studies are now warranted to elucidate the association of TLR4 variants in AS.

Association between the interleukin-1 family gene cluster and psoriatic arthritis.

OBJECTIVE: The interleukin-1 (IL-1) cytokine elicits a wide variety of biologic activities that initiate and promote an inflammatory response. The loci in the IL1 gene cluster have recently been associated with ankylosing spondylitis (AS). Since there is clinical and immunologic overlap between psoriatic arthritis (PsA) and AS, we wanted to examine the association between a panel of single-nucleotide polymorphisms (SNPs) in the IL1 gene family cluster and chromosome 2q12-13 in a PsA cohort. METHODS: Two hundred twelve PsA patients and 150 ethnically matched controls were genotyped with 11 SNPs in IL1A, 9 SNPs in IL1B, and 9 SNPs in IL1F5-10. Univariate analyses of the 29 single markers and short intragenic haplotypes identified several associated regions. Seventeen markers of interest were noted and further investigated to determine which markers or short haplotypes independently predict case-control status, using a stepwise logistic model. RESULTS; Two regions contributing independently to risk of disease in PsA were noted: a region spanned by markers rs3783547, rs3783543, and rs17561 in IL1A, and a region near the end of IL1B, through IL1F7, IL1F8, and into IL1F10. The best model contained markers rs3811047, rs1562304, and rs3811058, and 1 haplotype constructed from the 3 markers in region 1, with a likelihood ratio of 25.34 (4 degrees of freedom). CONCLUSION: The IL1 locus appears to be a high-priority susceptibility locus in PsA, with at least 2 independent regions that confer increased risk.

Association of the IL1 gene cluster with susceptibility to ankylosing spondylitis: an analysis of three Canadian populations.

OBJECTIVE: To examine the association between the IL1 gene cluster and susceptibility to ankylosing spondylitis (AS) in 3 independent case-control cohorts. METHODS: We analyzed 394 patients and 446 controls from Alberta, Newfoundland, and Toronto, Canada. Samples were genotyped using a panel of 38 single-nucleotide polymorphism (SNP) markers within the IL1 gene cluster. Data from 20 informative and nonredundant SNP markers were analyzed using several association test strategies. First, we used the program WHAP to identify single-marker associations. Second, we used WHAP to analyze "sliding windows" of 3 contiguous markers along the entire extent of the IL1 gene cluster in order to identify haplotypic associations. Third, we used the linkage disequilibrium mapping program DMLE to estimate the posterior probability distribution of a disease locus. RESULTS: A total of 14 SNP markers showed significant single-locus disease associations, the most significant being rs3783526 (IL1A) (P = 0.0009 in the Alberta cohort, P = 0.04 in the Newfoundland cohort) and rs1143627 (IL1B) (P = 0.0005 in the Alberta cohort, P = 0.02 in the Newfoundland cohort). Analysis of 3-marker sliding windows revealed significant and consistent associations with all of the haplotypes in the IL1A and IL1B loci in the Alberta cohort and with IL1B in the Newfoundland cohort, especially haplotypes rs1143634/rs1143630/rs3917356 and rs1143630/rs3917356/rs3917354 (P = 0.006-0.0001). With DMLE, a strong peak in the probability distribution was estimated near IL1A in both the Alberta and the Newfoundland populations. CONCLUSION: These results indicate that the IL1 locus, or a locus close to IL1, is associated with susceptibility to AS.

Association of functional variants of PTPN22 and tp53 in psoriatic arthritis: a case-control study.

Recent studies have implicated PTPN22 and tp53 in susceptibility to several autoimmune diseases, including rheumatoid arthritis, suggesting that these genes are important in maintaining immune homeostasis. Because autoimmune diseases may share similar susceptibility loci, investigation of these genes in psoriatic arthritis (PsA) is of potential relevance. As a result we investigated known coding polymorphisms in PTPN22 and tp53 in a homogenous Caucasian PsA cohort from Newfoundland, Canada and an admixed Caucasian PsA cohort from Toronto, Canada. We observed a moderate association of the R620W variant of PTPN22 with PsA in the Toronto population only. Because of the conflicting findings reported regarding the association of PTPN22 with PsA, further studies in other PsA populations are warranted.

Association of nuclear factor-kappaB in psoriatic arthritis.

OBJECTIVE: To examine the association of single nucleotide polymorphisms (SNP) in the NFKB1 gene, as well as 2 genes in the nuclear factor (NF)-kappaB functional complex (RelA and NFKBIA), in patients with psoriatic arthritis (PsA) from Newfoundland. METHODS: Patients with PsA and controls were genotyped for one 4-base insertion/deletion and 5 SNP in NFKB1, 4 SNP in RelA, and 7 SNP in NFKBIA by time-of-flight mass spectrometry, using the Sequenom platform. Chi-square analysis was used to test the single locus associations between SNP in the NF-kappaB complex and PsA. Associations between multi-locus haplotypes and case or control status were tested using the software PHASE. RESULTS: Two hundred and twenty-four patients with PsA (52% male) and 88 ethnically matched controls (64% male) were genotyped. No association was noted with any of the SNP tested for the single locus associations in NFKB1, RelA, and NFKBIA or with multi-locus haplotypes. In particular, the allele frequency for the NFKB1 -94delATTG was 41.7% in cases and 41.6% in the controls (p = 0.97). CONCLUSION: No association between the NFKB1 -94 ins/delATTG promoter polymorphism or with other NF-kappaB complex SNP in patients with PsA from Newfoundland was observed.

The Newfoundland population: a unique resource for genetic investigation of complex diseases.

The population of the province of Newfoundland and Labrador is genetically isolated. This isolation is evidenced by an overabundance of several monogenic disorders. The Newfoundland population, like that of other isolates, is now the focus of interest for identification of genes implicated in common diseases. However, the utility of such populations for this purpose remains unproven. In this paper, we review the current genetic architecture of the province, with respect to geographic isolation, homogeneity, founder effect, genetic drift and extended linkage disequilibrium. Based on these factors, we propose that the population of Newfoundland offers many advantages for genetic mapping of common diseases, compared with admixed populations, and even compared with other isolates.

Analysis of CARD15 polymorphisms in Korean patients with ankylosing spondylitis reveals absence of common variants seen in western populations.

OBJECTIVE: Substantial epidemiological and genetic evidence suggests that ankylosing spondylitis (AS) is likely due to an interplay of genetic and environmental factors. Recently, CARD15, located in chromosome 16q12, has been established as a disease susceptibility gene for Crohn's disease, Blau syndrome, and possibly psoriatic arthritis. Association studies in admixed populations from Northern European ancestry noted no such association between CARD15 mutations and AS. However, a homogenous population has yet to be studied. We investigated the prevalence of the 3 common CARD15 variants in a homogenous Korean population with AS. METHODS: All subjects were native Koreans with AS satisfying the modified New York criteria. Korean controls were examined and confirmed to be unaffected by AS. Subjects with AS were genotyped for the R702W, G908R, and Leu1007fsinsC variants of CARD15 using mass array MALDI-TOF mass spectrometry. RESULTS: A total of 205 AS subjects and 200 controls were genotyped. No subject with AS had any variants at the 702 and 1007 sites of CARD15. Only one subject was heterozygous for the 908 variant. The overall genotype frequency in AS for any CARD15 variant was 0.5%. No control had any of the 3 CARD15 variants. CONCLUSION: Our findings indicate that the CARD15 gene is not a major contributor to AS susceptibility in the Korean population.