The genetic basis for most patients with pustular skin disease remains elusive.

BACKGROUND: Rare variants in the genes IL36RN, CARD14 and AP1S3 have been identified to cause or contribute to pustular skin diseases, primarily generalized pustular psoriasis (GPP). OBJECTIVES: To better understand the disease relevance of these genes, we screened our cohorts of patients with pustular skin diseases [primarily GPP and palmoplantar pustular psoriasis (PPP)] for coding changes in these three genes. Carriers of single heterozygous IL36RN mutations were screened for a second mutation in IL36RN. METHODS: Coding exons of IL36RN, CARD14 and AP1S3 were sequenced in 67 patients - 61 with GPP, two with acute generalized exanthematous pustulosis and four with acrodermatitis continua of Hallopeau. We screened IL36RN and AP1S3 for intragenic copy-number variants and 258 patients with PPP for coding changes in AP1S3. Eleven heterozygous IL36RN mutations carriers were analysed for a second noncoding IL36RN mutation. Genotype-phenotype correlations in carriers/noncarriers of IL36RN mutations were assessed within the GPP cohort. RESULTS: The majority of patients (GPP, 64%) did not carry rare variants in any of the three genes. Biallelic and monoallelic IL36RN mutations were identified in 15 and five patients with GPP, respectively. Noncoding rare IL36RN variants were not identified in heterozygous carriers. The only significant genotype-phenotype correlation observed for IL36RN mutation carriers was early age at disease onset. Additional rare CARD14 or AP1S3 variants were identified in 15% of IL36RN mutation carriers. CONCLUSIONS: The identification of IL36RN mutation carriers harbouring additional rare variants in CARD14 or AP1S3 indicates a more complex mode of inheritance of pustular psoriasis. Our results suggest that, in heterozygous IL36RN mutation carriers, there are additional disease-causing genetic factors outside IL36RN.

Evidence for genetic overlap between adult onset Still's disease and hereditary periodic fever syndromes.

OBJECTIVE: Adult onset Still's disease (AOSD) is a severe, autoimmune disease that can be challenging to treat with conventional therapeutics and biologicals in a considerable number of cases. Therefore, there is a high need to understand its pathogenesis better. As major clinical symptoms overlap between AOSD and hereditary periodic fever syndromes (HPFS), we analysed four known HPFS genes in AOSD. METHODS: We performed Sanger sequencing and quantitative analysis of all coding regions of MEFV, TNFRSF1A, MVK and NLRP3 in 40 AOSD patients. All rare coding variants (n = 6) were evaluated for several aspects to classify them as benign to pathogenic variants. Statistical analysis was performed to analyse whether variants classified as (likely) pathogenic were associated with AOSD. RESULTS: We identified three rare variants in MEFV, one previously not described. Association to the three likely pathogenic MEFV variants was significant (p c = 2.34E- 03), and two of the three carriers had a severe course of disease. We observed strong evidence for significant association to mutations in TNFRSF1A (p c = 2.40E- 04), as 5% of patients (2/40) carried a (likely) pathogenic variant in this gene. Both of them received a biological for treatment. CONCLUSION: Our results indicate TNFRSF1A as a relevant gene in AOSD, especially in patients with a more challenging course of disease, while causal variants remain to be identified in the majority of patients.

Characterisation of psoriasis susceptibility locus 6 (PSORS6) in patients with early onset psoriasis and evidence for interaction with PSORS1.

BACKGROUND: Psoriasis is a genetically complex, chronic inflammatory skin disease. The authors have previously identified a susceptibility locus on chromosome 19p13 (PSORS6). METHODS AND RESULTS: In a follow-up linkage disequilibrium (LD) study in an independent family based cohort, the authors found evidence for association to a newly discovered microsatellite at this locus (D19SPS21, p<5.3x10(-5)). An LD based association scan in 300 trios revealed association to several single, single nucleotide polymorphisms (SNPs) in one LD block. When the authors stratified this cohort for carrying the PSORS1 risk allele at the HLA-C locus, evidence for association became much stronger at single SNP and haplotype levels (p values between 1.0x10(-4) and 8.0x10(-4)). In a replication study of 1114 patients and 937 control individuals, evidence for association was also observed after stratification to the PSORS1 risk allele. In both study groups, logistic regression showed evidence for interaction between the risk alleles at PSORS1 and PSORS6. Best p values for rs12459358 in both study groups remained significant after correction for multiple testing. The associated LD block did not comprise any known genes. Interestingly, an adjacent gene, MUC16, coding for a large glycosylated protein expressed in epithelia and of unknown function, could be shown to be also expressed in tissues relevant for pathogenesis of psoriasis such as skin and thymus. Immunohistochemical analyses of skin revealed focal staining for MUC16 in suprabasal epidermal cells. Further functional studies are required to clarify its potential role in psoriasis and identify the causal variant(s) at this locus. CONCLUSION: The data establish PSORS6 as a confirmed psoriasis susceptibility locus showing interaction with PSORS1.

Ichthyosis vulgaris: novel FLG mutations in the German population and high presence of CD1a+ cells in the epidermis of the atopic subgroup.

BACKGROUND: Ichthyosis vulgaris (IV) is a genetic disorder with a prevalence of 1:250-1000 caused by filaggrin (FLG) mutations, which also predispose to atopic diseases. OBJECTIVES: To study the genotype/phenotype relationship in IV and to analyse whether the suggested skin barrier defect is associated with differences of epidermal dendritic cells. PATIENTS/METHODS: We evaluated a cohort of 26 German patients with IV, established an IV severity score and analysed epidermal ultrastructure, histology, filaggrin and CD1a antigens. Mutations were screened by restriction enzyme analysis. Particular sequencing techniques allowed the complete FLG analysis to reveal novel mutations. RESULTS: The combined null allele frequency of R501X and 2282del4 was 67.3%. Patients also showed the mutations S3247X and R2447X as well as five novel FLG mutations: 424del17 and 621del4 (profilaggrin S100 domain), 2974delGA (repeat 2), R3766X (repeat 10(1)) and E4265X (repeat 10(2)). Their combined allele frequency in controls was <0.7%. No mutation was found in one IV patient, all in all approximately 27% were heterozygous, and the majority (approximately 69%) showed two null alleles. The IV severity score and ultrastructure showed a significant correlation with genotypes. Interestingly, CD1a cell counts showed a significant difference between nonatopic and atopic IV patients both with eczema and without eczema. CONCLUSIONS: We confirm that the mutations R501X and 2282del4 represent the most frequent genetic cause in German IV patients. The novel mutations are probably population and family specific. The observed differences of CD1a cells support the hypothesis that there is a barrier defect that predisposes to atopic manifestations, possibly independent of atopic eczema.

Loss-of-function mutations in the filaggrin gene: no contribution to disease susceptibility, but to autoantibody formation against citrullinated peptides in early rheumatoid arthritis.

OBJECTIVES: Autoantibody formation to citrullinated (pro)filaggrin has proven to be a highly specific serological marker for rheumatoid arthritis (RA). To test the potential relevance of mutations of the filaggrin (FLG) gene for disease susceptibility and elicitation of humoural autoimmunity in RA, a case-control association study of three loss-of-function FLG variants was performed. METHODS: DNA was obtained from 282 patients with early RA (mean disease duration: 6.5 months) and from 376 control individuals. Three loss-of-function variants of the FLG gene (*R501X, *2282del4 and *3702del1) were genotyped. RESULTS: No significant differences in genotype frequencies were observed between control probands and the population of RA patients. The FLG*3702del1 allele was not identified in any of the patients nor controls, and none of the probands was homozygous or compound heterozygous. In the RA cohort, heterozygous carriers of either of the FLG variants exhibited a significantly elevated prevalence of autoantibodies to citrullinated peptides (CCP-2) (80%) compared to non-carriers (51.9%) (p = 0.018, odds ratio: 3.71 (1.20-11.46)). CONCLUSIONS: The investigated FLG variants do not confer an overall risk for the development of RA. However, loss-of-function mutations in the FLG gene may contribute to the development of humoural autoimmunity, targeting citrullinated determinants in early RA.

Evidence for susceptibility determinant(s) to psoriasis vulgaris in or near PTPN22 in German patients.

INTRODUCTION: Variant R620W of protein tyrosine phosphatase non-receptor type 22 (PTPN22) has consistently been reported as a susceptibility factor for several autoimmune diseases. We investigated its role in susceptibility to psoriasis, the relevance of possibly other disease-causing variants, and interdependency of the major risk factor for psoriasis at PSORS1. METHODS: R620W was tested in a case-control study initially with 375 German patients and then with an enlarged sample of an additional 418 patients. Analyses were extended to linkage disequilibrium (LD) based haplotypes. Potential interaction between risk haplotypes of PTPN22 and the PSORS1 associated risk allele was tested by regression analysis. PTPN22 coding sequence was determined in 20 patients carrying the risk haplotype. Association and regression analysis were also performed in the extended case-control study. RESULTS: R620W was not associated in either case-control study, while significant association (corrected for multiple testing) with one haplotype (C-4) of the LD block encompassing PTPN22 as well with another haplotype (B-3) within an adjacent telomeric LD block was detected. No evidence for interaction between risk haplotype C-4 and the PSORS1 associated risk allele was found. Sequencing excluded other coding variants within PTPN22 as a basis for association findings. Analysis of the extended study group confirmed association for haplotypes B-3 and C-4 and independence of risk haplotypes C-4 and PSORS1. DISCUSSION: We exclude a major role of *620W in German psoriasis patients but suggest that other susceptibility determinant(s) within non-coding regions of PTPN22 or its proximity might exist acting independently of the major PSORS1 risk factor.

Systematic assessment of atypical deletions reveals genotype-phenotype correlation in 22q11.2.

INTRODUCTION: Clinical variability associated with the common 22q11.2 microdeletion is well known, and has led to a broad application of FISH diagnostics with probes for loci TUPLE1 or D22S75 (N25), although, rarely reported atypical deletions associated with the same phenotypic spectrum would not be discovered by these probes. As most types of 22q11.2 deletions occur between low copy repeats within the region (LCR22), we assumed that atypical deletions should be more common than has been reported. To address this question and the possibility of a deletion size related genotype-phenotype correlation, we systematically assessed the frequency of typical and atypical 22q11.2 deletions in a large cohort of patients. METHODS: We used a set of 10 fluorescent in situ hybridisation (FISH) DNA probes, capable of detecting all reported and hypothetical deletions between the LCR22, and analysed 350 patients. Deletion sizes in atypical deletions were established by use of further FISH probes. Frequency of certain atypical deletions was analysed in controls by FISH and quantitative PCR. RESULTS: Patients with conotruncal heart defects (ctCHD) and with typical VCFS phenotype showed the common 3 Mb or nested 1.5 Mb deletions (in 18.5% and 78.6%, respectively), but no atypical deletion, while 5% (3/63) of patients with a mildly suggestive, atypical phenotype showed atypical distal deletions, which were not detected in patients with mental retardation of unknown origin or in healthy controls. DISCUSSION: These statistically significant differences demonstrate that atypical distal 22q11.2 deletions are very uncommon in patients with ctCHDs, while atypical congenital heart defects and mild dysmorphism are recognisable feature of atypical distal deletions. Further phenotype-genotype analysis disclosed association of significant developmental delay with the distal part of the common deletion region, and choanal atresia and atypical CHDs with the adjacent distal deletion region.

Lack of genetic association of the three more common polymorphisms of CARD15 with psoriatic arthritis and psoriasis in a German cohort.

OBJECTIVE: To determine whether the three common independent sequence variants of the putative pleiotropic non-MHC autoimmune gene CARD15 influence disease susceptibility in large German cohorts of patients with psoriatic arthritis and psoriasis vulgaris, before and after stratification to HLA-C. METHODS: DNA was obtained from 375 patients with psoriatic arthritis, 281 patients with psoriasis vulgaris without joint involvement, and 376 controls. The three variants of the CARD15 gene (R702W, G908R, leu1007fsinsC), and two single nucleotide polymorphisms of the HCR gene (HCR-325, HCR-2327) for HLA-C stratification were genotyped using allelic discrimination Taqman assays. RESULTS: No significant differences in genotype frequencies were observed between controls and either the psoriatic arthritis or the psoriasis vulgaris patient population, even after stratification to HLA-C in both patient cohorts, or to the type of joint involvement within the psoriatic arthritis group. CONCLUSIONS: The lack of genetic association between the most common Crohn's disease alleles of the CARD15 gene and psoriatic joint disease on large cohorts of white patients does not support a recently claimed role for CARD15 as the first non-MHC susceptibility gene in the pathogenesis of psoriatic arthritis, but confirms and extends previous studies in the case of psoriasis vulgaris.

Interleukin-10 promoter polymorphism IL10.G and familial early onset psoriasis.

BACKGROUND: The anti-inflammatory cytokine interleukin (IL)-10 is considered to play a major role in the pathophysiology of psoriasis, which is characterized by an IL-10 deficiency. Systemic administration of IL-10 has been shown to be an effective therapy for psoriasis. The IL-10 promoter region contains a highly polymorphic microsatellite (IL10.G) and in a recent case-control study the IL10.G13 (144 bp) allele was found to be associated with familial early onset psoriasis (type 1 psoriasis) having a susceptible effect. OBJECTIVES: As it is essential in multifactorial diseases to replicate findings before definite conclusions can be drawn, we decided to perform a follow-up study and to follow a genetic approach analysing allele transmission in families with a positive family history of psoriasis. METHODS: We studied 137 nuclear families (trio-design) comprising 456 individuals and genotyped the IL10.G marker. For comparison we also genotyped the microsatellite tn62 as a reference marker of the major psoriasis susceptibility locus on chromosome 6p21 (PSORS1). In the present study allele transmission was evaluated using the family-based association test (FBAT) and GENEHUNTER 2.0 based on the transmission/disequilibrium test. RESULTS: The G13 allele (144 bp) had a frequency of 24%, was present in 88 families and clearly showed an even transmission (FBAT, P = 0.753). In contrast, allele 3 (IL10.G9) (136 bp) had a frequency of 39%, was present in 110 families and was transmitted in 43 trios and remained untransmitted in 67 trios (FBAT, P = 0.026), thus showing preferential nontransmission. For the HLA-linked tn62-marker we obtained a P-value of 0.00027 for allele 4 in the same study group. CONCLUSIONS: In conclusion, we failed to confirm the susceptible effect of the G13 allele, but provide the first data for a protective effect of allele 3 (IL10.G9) for familial psoriasis. Our results suggest that the IL10.G polymorphism is not a major locus, but acts as a minor locus.

Association scan of the novel psoriasis susceptibility region on chromosome 19: evidence for both susceptible and protective loci.

To follow up the novel psoriasis susceptibility region on chromosome 19 (PSORS6), we performed an association scan for psoriasis vulgaris using 45 evenly spaced DNA microsatellite markers. For this study, a new independent sample of 210 nuclear psoriasis families (trio design) from Northern Germany was recruited. We used the family based association test (FBAT) for an association scan over the chromosome 19 region encompassing 50.8 cM. We obtained a positive association for the markers D19S922 (allele 5, P = 0.008) and D19S916 (allele 13, P = 0.016), which correspond to the peak of the region identified in a previously performed scan. We identified two novel regions by a single marker, each showing negative association at D19S917 on 19p13.1 (allele 8, P = 0.0034) and at D19S425 (allele 9, P = 0.0005), compatible with the hypothesis of protective loci. These two novel regions were explored in more detail using novel microsatellite markers at an average distance of 100 kb. A separate analysis distinguishing between familial (n = 137) and sporadic (n = 73) psoriasis families showed that the familial trios contribute strongly in the region around D19S425 (P = 0.004), while the comparably small subset of 73 sporadic trios has a stronger effect at the locus around D19S917 (P = 0.026). These studies confirm the existence of a psoriasis susceptibility locus on chromosome 19 and give first evidence for the existence of both susceptible and protective loci in this region. Analysis of a dense marker set from these refined regions will eventually allow identification of the underlying susceptibility alleles.