Article Text
Abstract
Objective A recent population-based study identified several HLA alleles as conferring a risk for psoriatic arthritis (PsA) among patients with psoriasis. The authors aimed to confirm these results using a family-based association study.
Methods PsA probands, psoriasis probands and their first-degree family members were included. All participants were evaluated for the presence of psoriasis and inflammatory arthritis. HLA-B and -C genotyping was performed. The family-based association test was used to test for differences between PsA and psoriasis patients in transmission of candidate alleles from parents to offspring.
Results A total of 178 PsA and 30 psoriasis probands and 561 first degree family members were analysed. The following HLA alleles were over-transmitted to PsA compared with psoriasis: HLA-C*12 (p=0.005), HLA-B*38 (p=0.04), HLA-B*39 (p=0.03), HLA-B*27 (p=0.002).
Conclusions HLA-B*27, HLA-B*38, HLA-B*39 and HLA-C*12 alleles are potential PsA-specific genetic markers among patients with psoriasis.
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Psoriasis is an immune-mediated skin disease affecting 2–3% of the population. Psoriatic arthritis (PsA) is an inflammatory arthritis that affects approximately one-third of people with psoriasis.1 PsA can be viewed as a ‘disease within a disease’ as most patients first develop psoriasis and only later develop PsA.2 One of the models that describes the relation between these diseases considers PsA as a more severe phenotype of cutaneous psoriasis (PsC) that occurs due to a greater number of susceptibility genes or environmental factors.3 Genes that differentiate patients with PsA from those with psoriasis alone serve as markers for the development of PsA in patients with psoriasis.
Epidemiological studies have implicated a strong genetic basis to PsA. A strong familial aggregation of PsA was found in several family studies. Moll and Wright4 were the first to report a recurrence risk ratio in first-degree relatives (λ1) of 55, compared with estimates ranging from five to 10 in PsC. Another study from the UK, reported a PsA prevalence of 14.3% among siblings and recurrence risk ratio (λs) of 47.5 Our group has found a PsA prevalence of 7.6% among first-degree relatives leading to a λ1 of 30.4.6 These findings support the strong heritability of PsA.
Studies have shown a strong association between PsC and the major histocompatibility complex region.7,–,9 Within this region the strongest and most consistently reported association is with human leucocyte antigen (HLA)-C*06.10 While HLA-C*06 is also increased in PsA patients compared with the general population, this association is stronger with PsC than with PsA.11 There is limited information about specific genetic markers for PsA among patients with PsC as only a few studies have directly compared these two groups of patients. We recently performed a large population-based association study that compared patients with PsA with those with PsC to identify specific genetic markers for PsA within the HLA loci. We found higher frequencies of HLA-B*27, HLA-B*38 and HLA-B*08 in PsA compared with PsC patients, while the HLA-C*06 allele was less frequent in PsA. The HLA-B*39 allele was associated with the development of axial PsA.12
In the present family-based association study we aimed to confirm the above-mentioned identified associations within the HLA-B and HLA-C loci. The advantage of this study design is that it avoids population stratification bias, a potential threat to the validity of any population-based genetic association study. To our knowledge, no such family-based association study has been published on PsA.
Methods
Study population
PsA and PsC probands and their first-degree family members (parents and siblings) were included in this analysis. Altogether, 178 PsA probands, 30 PsC probands and 561 first-degree family members belonging to 208 families were analysed.
The PsA probands were recruited from the University of Toronto PsA cohort. All patients were carefully phenotyped by a rheumatologist and satisfied the CASPAR criteria for the classification of PsA.13 Of these patients, 100 participants were recruited for a previous study that assessed familial aggregation of PsA.6 Consecutive patients with PsA were approached to participate in a family investigation. If a patient did not wish their family to participate or if there were no available family members, the next patient was approached until 100 PsA probands were recruited. The remaining patients participated in a linkage study and were selected due to a positive family history of psoriasis and/or PsA. These patients underwent assessment according to a standard protocol that included a complete history, physical examination and laboratory evaluation at 6–12-month intervals and radiological assessment at 2-year intervals irrespective of symptoms.2 The PsC probands were part of a recently established psoriasis cohort that consecutively recruits and follows psoriasis patients without arthritis at their first visit. All of these patients were assessed by a rheumatologist to exclude those with inflammatory arthritis. Subjects were interviewed and examined according to a standardised protocol as previously described.14 Psoriasis was confirmed in both cohorts by a dermatologist. We made thorough efforts to assess all family members and the great majority of them underwent physical examination for signs and symptoms of psoriasis and inflammatory arthritis. In only a few cases in which a family member was unable to attend the clinic for clinical assessment, the screening questionnaire was used to determine their phenotype. All assessed family members irrespective of their phenotype (PsA, psoriasis without arthritis or healthy individuals) were included in the study. Overall, three families were bilineal for PsC and one for PsA.
HLA typing
DNA was extracted from peripheral blood using a modified salting out procedure (Gentra Puregene blood kit; Qiagen, Germantown, Maryland, USA). Extracted genomic DNA was amplified by PCR using locus-specific primers for each of the HLA-A, HLA-B, HLA-C, HLA-DR and DQ loci. PCR amplicons were identified by sequence-specific oligonucleotide probes using the reverse line blot technique (RELI sequence-specific oligonucleotide HLA typing kits; Life Technologies, Grand Island, New York, USA).15 Ambiguous results were resolved using sequence-specific primers.
Definitions of traits
In our previous population-based study three groups were assessed: PsA, PsC and healthy individuals. As the family-based association test (FBAT) method allows direct comparison of only two phenotypic subgroups, the data were analysed in two ways:
To compare PsA and PsC, PsA patients were coded as affected and PsC as unaffected, excluding healthy siblings. As only the parental genotypes are relevant for calculation of the FBAT test statistic, parents were included in the analysis irrespective of their disease status.
In order to compare psoriatic disease (PsD; including PsC and PsA) with healthy controls, all available information was included. PsA and PsC probands and siblings were coded as affected and healthy siblings were coded as unaffected. In this analysis probands with PsD were compared with their healthy siblings.
The rationale of assessing the PsD phenotype was based on a genetic model that views PsA as a disease within a disease, with psoriasis as the parent disease. This model predicts that cases ascertained by psoriasis will be genetically homogenous and that cases ascertained by PsA will completely overlap those of psoriasis apart from several additional distinct genes.
Statistical analysis: FBAT
The FBAT software (version 2.0.3) uses a unified approach to family-based tests of association.16 ,17 Based on the original transmission disequilibrium test,18 in which alleles transmitted to affected offspring are compared with the expected distribution of alleles among offspring under Mendelian transmission, the FBAT test statistic examines the distribution of the offspring genotypes conditional on the offspring trait information and the parental genotypes. If one of the parental genotypes in a trio is not observed, the test statistic is conditioned on the sufficient statistics for the offspring trait distribution. An additive model was specified.
A family-based test statistic (Z value) greater than 0 indicates over-transmission of the allele, while a Z value of less than 0 indicates under-transmission. The test statistic was computed using the empirical variance (-e) option19 because we were testing for an association in an area of known linkage with multiple sibs in a family.
The power of a family-based study to detect an association is partly related to the number of informative families. FBAT requires at least one heterozygote parent per family, otherwise the family cannot be used, because it is not possible to determine which of the two alleles was transmitted. Such families are called non-informative families. In this study, the number of families that contributed to a specific allele test depended on whether the allele of interest was observed in the family. As each HLA locus is multiallelic, the frequency of each allele in the study population was relatively low resulting in a relatively low number of informative families for each allele. Our single-locus analysis focused on each one of several predefined HLA alleles that had been identified as potential PsA-specific genetic markers in our previous population-based study, so no adjustment for multiple testing was applied. The remaining HLA alleles did not show a statistically significant association with either PsA or PsD.
Haplotype analysis
The linkage phase of HLA-B and HLA-C alleles was determined using the pedigree data available for all participants. Due to the multiallelic nature of the investigated loci, 101 haplotypes were identified; however, as FBAT can only analyse up to 80 haplotypes at a time, we limited the analysis to those haplotypes that included alleles that were identified as being associated with PsA in the single-locus analysis.
Results
Of the 208 families available, four families (two PsA probands, two psoriasis probands and their 13 family members) were excluded due to Mendelian errors. The study population included 47 complete trios (two parents and a proband) and 157 families with affected–unaffected sib-pairs with or without their parents, as detailed in table 1. One hundred of 201 (49.7%) probands had a positive family history of psoriasis and 42 out of 201 (20.8%) probands had a family history of PsA. In three trios the disease status of the parents was unknown.
Several HLA-B and HLA-C alleles were found to be associated with PsA in the single-locus FBAT analysis (table 2). The association with HLA-B*27, the strongest genetic marker for PsA in the population-based analysis, was confirmed in the family-based study. HLA-B*27 was over-transmitted in PsA and PsD compared with PsC and healthy individuals (table 2, p=0.002) and healthy controls (table 3, p=0.04), respectively. Furthermore, this study confirmed several HLA associations that we reported previously. HLA-C*12 was over-transmitted in PsA patients compared with PsC (p=0.005). However, no significant association was found in PsD compared with healthy sibling controls (table 3, p=0.13). HLA-B*38, which is in strong linkage disequilibrium with HLA-C*12, was over-transmitted in PsA probands when compared with siblings with PsC (table 2, p=0.04) and there was no significant association with PsD (table 3, p=0.14). HLA-B*39 was over-transmitted to patients with PsA compared with PsC (table 3, p=0.03). There was no significant association between HLA-B*08 and either PsA or PsD. Interestingly, HLA-C*06, the strongest risk allele for psoriasis, was not significantly associated with either PsA or PsD. However, the test statistics (Z) indicate over-transmission of HLA-C*06 to PsD probands compared with healthy siblings, while under-transmission of the allele to PsA compared with siblings with PsC, corresponding to the direction of association of HLA-C*06 in the population-based study.
Haplotype analysis
As FBAT is limited by the number of haplotypes that can be concurrently assessed, we analysed only haplotypes that included HLA-B or HLA-C alleles that were identified in the single-locus analysis as being associated with PsA (table 2). The following haplotypes were over-transmitted to probands with PsA compared with their siblings with PsC: HLA-B*38-C*12 (p=0.02), HLA-B*39-C*12 (p=0.005) and HLA-B*27-C*02 (p=0.04). The results are presented in table 4.
Discussion
The main aim of this study was to confirm the results of individual-level genetic association analyses we previously conducted in PsA and PsC patients who identified several genetic markers for PsA within the HLA-B and HLA-C loci. In this family-based association study we investigated the association between HLA alleles within these loci and PsA compared with PsC. We were able to confirm the association between several of the HLA risk alleles for PsA identified in our recent population-based study.20 HLA-B*27, the strongest genetic marker for PsA, was associated with both PsA and PsD. The associations between HLA-B*38, HLA-B*39, HLA-B*12 and PsA versus PsC were also confirmed.
In accordance with several population-based studies, including ours,20 HLA-B*27 was found to be a strong genetic marker for PsA compared with PsC. In this study HLA-B*27 was over-transmitted to PsA probands compared with their unaffected family members. HLA-B*27 is also part of a risk haplotype, along with HLA-C*02, which showed an association with PsA versus PsC. Previous studies in different ethnic groups have consistently shown that HLA-B*27 is an independent risk allele for PsA that is unrelated to the skin disease.21,–,24 However, unlike the situation in ankylosing spondylitis, the majority of PsA patients do not carry this allele and it can only account for a small proportion of the total genetic risk in PsA. Other HLA alleles, as well as genes outside the HLA region, probably account for the reminder of the genetic risk.
Three additional alleles were transmitted more often in PsA compared with PsC: HLA-B*38, HLA-B*39 and HLA-C*12. Our recent population-based association study showed that while HLA-B*38 and HLA-C*12 were associated with PsA irrespective of the arthritis pattern, HLA-B*39 was only associated with PsA patients who exhibited axial involvement.12 Furthermore, HLA-B*38 was more frequent in PsA compared with PsC in two additional studies in Caucasians from the USA and Jewish PsA patients from Israel.25 ,26 HLA-B*39 was more frequent in PsA patients with axial involvement in a small study from Spain,27 and was also identified as being associated with joint damage progression.28 HLA-B*38 and HLA-B*39 are part of common ancestral haplotypes along with HLA-C*12. A recent population-based association study from Ireland confirmed the association between HLA-B*39:01 and HLA-B*38:01 with PsA compared with PsC. The frequency of the haplotype HLA-B*39:01-C*12 was also higher among PsA patients compared with PsC.29 These data suggest that HLA-B*39 and HLA-B*38 can be considered as susceptibility alleles for PsA among PsC patients.
HLA-B*39 and HLA-B*38 are splits of the broad B16 antigen and share significant peptide sequence homology. Previous studies have indicated an increased frequency of HLA-B*39 among patients with HLA-B*27-negative spondylarthropathies (SpA).30 However, only a few functional studies that investigated the mechanism underlying this association have been conducted. Sobao et al31 found that HLA-B27 and HLA-B39 antigens recognise overlapping peptide repertoires, supporting the hypothesis that peptides presented by both of these class I antigens play a role in the pathogenesis of SpA. Further functional studies are needed to investigate the role of HLA-B*39 and HLA-B*38 in the pathogenesis of PsA.
The lack of association between the HLA-C*06 allele and either PsA or PsD is interesting, as this allele is the strongest genetic risk allele for psoriasis. The frequency of HLA-C*06 is increased in PsA patients compared with the general population; however, it is lower than among psoriasis patients. Two recent population-based studies that compared PsA with PsC patients have found that the frequency of HLA-C*06 was significantly lower in PsA patients (approximately 28%) compared with PsC patients (ranging from 44% to 57%).12 ,29 In the present study the frequency of HLA-C*06 among the PsA probands was similar to that reported among PsA cases in these population-based studies. Although it is possible that the lack of association is due to low power, an alternative explanation may be related to the genetic heterogeneity of PsA and PsC. Patients who carry HLA-C*06 develop more severe and early psoriasis, yet they are less likely to develop arthritis, while others who carry other HLA risk alleles such as HLA-C*12, HLA-B*39, HLA-B*38 tend to develop both psoriasis and inflammatory arthritis. Although not statistically significant, the over-transmission of HLA-C*06 to PsD probands compared with healthy siblings, while the under-transmission of the allele to PsA compared with siblings with PsC, corresponds to the direction of association of HLA-C*06 in the above-mentioned population-based studies.
In summary, in this family-based study, we investigated genetic markers for PsA among psoriasis patients, and confirmed associations of HLA-B*27, HLA-B*39, HLA-B*38 and HLA-C*12 with PsA. These alleles can be considered as conferring a risk for the development of PsA among PsC patients. Future studies should focus on the mechanism underlying these associations.
References
Footnotes
Funding The Psoriatic Arthritis Program is partly funded by the Arthritis Society, Canadian Institutes of Health Research and the Krembil Foundation.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.