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The OTF3 Gene Polymorphism Confers Susceptibility to Psoriasis Independent of the Association of HLA-Cw*0602

2000; Elsevier BV; Volume: 115; Issue: 5 Linguagem: Inglês

10.1046/j.1523-1747.2000.00133.x

1523-1747

Segundo González, Jesús Martínez‐Borra, Antonio López‐Vázquez, Miguel Ángel Blanco-Gelaz, Carlos López‐Larrea, J. Sánchez del Río, Jorge Santos‐Juanes,

Dermatology and Skin Diseases

Psoriasis has been strongly associated to HLA-Cw6, but it remains unclear whether Cw6 itself or a closely linked gene is associated with the disease. The aim of this study was to clarify whether the HLA-C itself determines disease susceptibility or whether it acts only as a marker for the susceptibility allele. We examined a sample of 95 type I psoriasis patients and 104 Spanish matched controls to investigate whether HLA-Cw*0602 or other closely related class I loci, such as HLA-B and MICA (which are centromeric to HLA-C), or corneodesmosin gene and octamer transcription factor-3 genes (which are telomeric to HLA-C), might play a part in disease development. DNA samples were genotyped by polymerase chain reaction/sequence-specific primers (HLA-C), polymerase chain reaction/sequence-specific primers (HLA-B), radioactive polymerase chain reaction (MICA-TM polymorphism in the transmembrane region), and polymerase chain reaction/restriction fragment length polymorphism (protein S and octamer transcription factor-3). Our results show a significant increase of Cw*0602 in psoriasis patients (odds ratio = 3.64; pc < 0.0006). A significant association between the β allele of octamer transcription factor-3 (HindIII) and psoriasis was also detected (odds ratio = 3.76; pc < 0.0003). The allele octamer transcription factor-3B (etiologic fraction = 0.62) was found to be more strongly associated to psoriasis vulgaris than Cw*0602 (etiologic fraction = 0.35) and the increase of octamer transcription factor-3 B allele is independent of the linkage disequilibrium with Cw*0602 as this was also found in Cw*0602 negative patients (odds ratio = 3.63; pc < 0.015, etiologic fraction = 0.55). We did not detect an association between the corneodesmosin gene and psoriasis. This fact suggests that the psoriasis susceptibility gene is located within a critical region of 147 kb, telomeric to HLA-C and centromeric to the corneodesmosin gene, and the association of Cw6 to psoriasis may be secondary to linkage disequilibrium. Psoriasis has been strongly associated to HLA-Cw6, but it remains unclear whether Cw6 itself or a closely linked gene is associated with the disease. The aim of this study was to clarify whether the HLA-C itself determines disease susceptibility or whether it acts only as a marker for the susceptibility allele. We examined a sample of 95 type I psoriasis patients and 104 Spanish matched controls to investigate whether HLA-Cw*0602 or other closely related class I loci, such as HLA-B and MICA (which are centromeric to HLA-C), or corneodesmosin gene and octamer transcription factor-3 genes (which are telomeric to HLA-C), might play a part in disease development. DNA samples were genotyped by polymerase chain reaction/sequence-specific primers (HLA-C), polymerase chain reaction/sequence-specific primers (HLA-B), radioactive polymerase chain reaction (MICA-TM polymorphism in the transmembrane region), and polymerase chain reaction/restriction fragment length polymorphism (protein S and octamer transcription factor-3). Our results show a significant increase of Cw*0602 in psoriasis patients (odds ratio = 3.64; pc < 0.0006). A significant association between the β allele of octamer transcription factor-3 (HindIII) and psoriasis was also detected (odds ratio = 3.76; pc < 0.0003). The allele octamer transcription factor-3B (etiologic fraction = 0.62) was found to be more strongly associated to psoriasis vulgaris than Cw*0602 (etiologic fraction = 0.35) and the increase of octamer transcription factor-3 B allele is independent of the linkage disequilibrium with Cw*0602 as this was also found in Cw*0602 negative patients (odds ratio = 3.63; pc < 0.015, etiologic fraction = 0.55). We did not detect an association between the corneodesmosin gene and psoriasis. This fact suggests that the psoriasis susceptibility gene is located within a critical region of 147 kb, telomeric to HLA-C and centromeric to the corneodesmosin gene, and the association of Cw6 to psoriasis may be secondary to linkage disequilibrium. psoriasis vulgaris MHC class I chain-related gene A octamer transcription factor corneodesmosin gene etiologic fraction Psoriasis vulgaris (PV) is a chronic cutaneous disease characterized by hyperproliferation of keratinocytes, and recruitment of T lymphocytes and mononuclear cells in the affected skin. PV affects 1–2% of the subjects in Caucasian populations (Bhalerao and Bowcock, 1998Bhalerao J. Bowcock A.M. The genetics of psoriasis: a complex disorder of skin and immune system.Hum Mol Genet. 1998; 7: 1537-1545Crossref PubMed Scopus (230) Google Scholar). There is clear evidence from association and twin studies of a strong heritable component in this disease (Faber et al., 1974Faber E.M. Nall L. Watson W. Natural history of psoriasis in 61 twin pairs.Arch Dermatol. 1974; 109: 207-211Crossref PubMed Scopus (201) Google Scholar;Brandrup et al., 1978Brandrup F. Hauge M. Henningse J. Eriksen B. Psoriasis in unselected series of twins.Arch Dermatol. 1978; 114: 874-878Crossref PubMed Scopus (92) Google Scholar). Linkage analysis have confirmed the strong association of major histocompatibility complex (MHC) to psoriasis, and they also provide some evidence that other non-MHC genes influence disease susceptibility (Tomfohrde et al., 1994Tomfohrde J. Silverman A. Barnes R. et al.Gene for familial psoriasis susceptibility mapped to the distal end of human chromosome, 17q.Science. 1994; 264: 1141-1145Crossref PubMed Scopus (363) Google Scholar;Matthews et al., 1996Matthews D. Fry L. Powless A. et al.Evidence that a locus for familial psoriasis maps on chromosome 4q.Nature Genet. 1996; 14: 231-233Crossref PubMed Scopus (183) Google Scholar;Nair et al., 1997Nair R.P. Hensler T. Jenisch S. et al.Evidence for two psoriasis susceptibility loci (HLA and 17q) and two novel candidate regions (16q and 20p) by genome scans.Hum Mol Genet. 1997; 6: 1349-1356Crossref PubMed Scopus (357) Google Scholar;Trembath et al., 1997Trembath R.C. Clough R.L. Rosbotham J.L. et al.Identification of major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis.Hum Mol Genet. 1997; 6: 813-820Crossref PubMed Scopus (440) Google Scholar;Jenisch et al., 1998Jenisch S. Henseler T. Nair R.P. et al.Linkage analysis of human leucocyte antigen (HLA) marker in familial psoriasis: strong disequilibrium effects provide evidence for major determinant in the HLA-B/-C region.Am J Hum Genet. 1998; 63: 191-199Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar;Balendran et al., 1999Balendran N. Clough R.L. Arguello J.R. et al.Characterization of the major susceptibility region for psoriasis at chromosome 6p21.3.J Invest Dermatol. 1999; 113: 322-328Crossref PubMed Scopus (97) Google Scholar). The role of the MHC in the development of the disease is not clear, but it has been proposed that HLA molecules are directly involved in the etiology of PV (Menssen et al., 1995Menssen A. Trommler P. Vollmer S. et al.Evidence for an antigen-specific cellular immune response in skin lesions of patients with psoriasis vulgaris.J Immunol. 1995; 155: 4078-4093PubMed Google Scholar;Valdimarsson et al., 1995Valdimarsson H. Baker B.S. Jonsdottir I. Powles A. Fry L. Psoriasis. A. T-cell-mediated autoimmune disease induced by streptococcal superantigens?.Immunol Today. 1995; 16: 145-149Abstract Full Text PDF PubMed Scopus (329) Google Scholar). Classically, the HLA-Cw*0602 allele is the strongest association found in PV (Brenner et al., 1978Brenner W. Gschnait F. Mayr R.W. HLAs B13, B17, B37 and Cw6 in psoriasis vulgaris: association with age of onset.Arch Dermatol Res. 1978; 262: 337-339Crossref PubMed Scopus (74) Google Scholar;Roitberg-Tambur et al., 1994Roitberg-Tambur A. Friedman A. Tzfoni E.E. Ashina A. Brautbar C. Do specific pockets of HLA-C molecules predispose to psoriasis vulgaris.J Am Acad Dermatol. 1994; 31: 964-968Abstract Full Text PDF PubMed Scopus (44) Google Scholar). Other HLA alleles have also been associated with PV (B13, B47, B57). Nevertheless, it has been established that the primary association is with HLA-Cw*0602 and that other HLA-B alleles related to PV are due to linkage disequilibrium between these antigens and Cw6. Only 10% of HLA-Cw6 positive individuals develop PV, however, and this suggests a major role for additional genes and/or environmental factors. Other disease-associated alleles, such as Cw7, have also been observed in some populations, thus indicating that different haplotypes may be associated with disease in different racial groups (Jenisch et al., 1999aJenisch S. Koch S. Henseler T. Nair R.P. et al.Corneodesmosin gene polymorphism demonstrates strong linkage disequilibrium with HLA and association with psoriasis vulgaris.Tissue Antigens. 1999; 54: 439-449Crossref PubMed Scopus (73) Google Scholar). An association between a specific amino acid residue of HLA-C (Ala-73) was found increased in PV patients in some populations (Asahina et al., 1991Asahina A. Akazaki S. Nakanawa H. Kuwata S. Tokunaga K. Ishibashi Y. Juji T. Specific nucleotide sequence of HLA-C is strongly associated with psoriasis vulgaris.J Dermatol. 1991; 97: 254-258Google Scholar;Ikaheimo et al., 1994Ikaheimo I. Silvennoinen-Kassinen S. Karvonen J. Tiilikainen A. Alanine at position 73 of HLA-C is associated with psoriasis vulgaris in Finland.Br J Dermatol. 1994; 131: 257-259Crossref PubMed Scopus (33) Google Scholar;Roitberg-Tambur et al., 1994Roitberg-Tambur A. Friedman A. Tzfoni E.E. Ashina A. Brautbar C. Do specific pockets of HLA-C molecules predispose to psoriasis vulgaris.J Am Acad Dermatol. 1994; 31: 964-968Abstract Full Text PDF PubMed Scopus (44) Google Scholar;Mallon et al., 1997Mallon E. Bunce M. Wojnarowska F. Welsh K. HLA-Cw*0602 is a susceptibility factor in type I psoriasis, and evidence Ala-73 is increased in male type I psoriatics.J Invest Dermatol. 1997; 109: 183-186Crossref PubMed Scopus (81) Google Scholar). Furthermore, population- and family-based studies, have suggested that psoriasis is associated with the class I side of the extended haplotypes EH57.1 and 13.1 rather than to a particular HLA locus (Schmitt-Egenolf et al., 1996Schmitt-Egenolf M. Eiermann T.H. Boehncke W.F. Stander M. Sterry W. Familial juvenile onset psoriasis is associated with the human leukocyte antigen (HLA) class I side of the extended haplotype Cw6–B57-DRB*0701-DQA1*0201-DQB*0303: a population and family based study.J Invest Dermatol. 1996; 106: 711-714Crossref PubMed Scopus (112) Google Scholar). Clearly, the possibility exists that the HLA-C itself is not the primary locus responsible for disease susceptibility and its association with psoriasis may be secondary to linkage disequilibrium with a nearby gene responsible for PV susceptibility. In accordance with this idea, several studies have recently associated psoriasis with nearby genes and polymorphic markers, located centromeric (Dawkins et al., 1999Dawkins R. Leelayuwat C. Gaudieri S. et al.Genomics of the major histocompatibility complex: haplotypes, duplication, retroviruses and disease.Immunol Rev. 1999; 167: 275-304Crossref PubMed Scopus (252) Google Scholar) or telomeric to HLA-C (Ishihara et al., 1996Ishihara N. Yamagata N. Ohno S. et al.Genetic polymorphism in the keratin-like S gene within the major histocompatibility complex and association analysis on the susceptibility of psoriasis vulgaris.Tissue Antigen. 1996; 48: 182-186Crossref PubMed Scopus (51) Google Scholar;Tazi Ahnini et al., 1999aTazi Ahnini R. Camp N.J. Cork M.J. Mee J.B. Keohane S.G. Duff G.W. Giovine F.S. Novel association between the corneodesmosin (MHC S) gene and susceptibility to psoriasis.Hum Mol Genet. 1999; 8: 1135-1140Crossref PubMed Scopus (116) Google Scholar,Tazi Ahnini et al., 1999bTazi Ahnini R. Giovine F.S. Cox A. Keohane S.G. Cork M.J. Corneodesmosin (MHC S) gene in guttate psoriasis.Lancet. 1999; 354: 597Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar;Oka et al., 1999Oka A. Tamiya G. Tomizawa M. et al.Association analysis using refined microsatellite markers localizes a susceptibility locus for psoriasis vulgaris within a 111 kb segment telomeric to HLA-C gene.Hum Mol Genet. 1999; 12: 2165-2170Crossref Scopus (149) Google Scholar); however, the exact location of this putative susceptible locus, and whether its association to PV is primary or secondary to HLA-C, has not been established. In this study we tested the hypothesis of whether the PV predisposition is controlled by Cw6 alone or other closely linked class I loci [MHC class I chain-related gene A (MICA), HLA-B, octamer transcription factor-3 (OTF3), MHC corneodesmosin gene (S gene)], which flank the HLA-C locus. MICA is a new polymorphic gene located 47 kb centromeric to HLA-B and is expressed mainly in fibroblasts and epithelial cells (Bahram et al., 1994Bahram S. Bresnahan M. Geraghty D.E. Spies T. A second lineage of mammalian major histocompatibility complex class I genes.Proc Natl Acad Sci USA. 1994; 91: 6259-6263Crossref PubMed Scopus (685) Google Scholar). Recently, it has been implicated as a candidate gene for psoriasis type I susceptibility (Dawkins et al., 1999Dawkins R. Leelayuwat C. Gaudieri S. et al.Genomics of the major histocompatibility complex: haplotypes, duplication, retroviruses and disease.Immunol Rev. 1999; 167: 275-304Crossref PubMed Scopus (252) Google Scholar). The HLA-C telomeric portion of the class I region may also contain putative genes involved in PV susceptibility (Figure 1). In this critical region of 147 kb between HLA-C locus and the S gene, three known genes: OTF3 (or POU5F1), TCF19 (SC1), and S have been described (Guillaudeux et al., 1998Guillaudeux T. Janer M. Wong G.K. Spies T. Geraghty E. The complete genomic sequence of 424,015 bp at the centromeric end of the HLA class I region: Gene content and polymorphism.Proc Natl Acad Sci USA. 1998; 95: 9494-9499Crossref PubMed Scopus (53) Google Scholar;Shiina et al., 1999aShiina T. Tamiya G. Oka A. Takishima N. Inoko H. Genome sequencing analysis of the 1.8 Mb entire human MHC class I region.Inmunol Rev. 1999; 167: 193-199Crossref Scopus (56) Google Scholar,Shiina et al., 1999bShiina T. Tamiya G. Oka A. et al.Molecular dynamics of MHC genesis unraveled by sequence analysis of the 1,796,938 bp HLA class I region.Proc Natl Acad Sci USA. 1999; 96: 13282-13287Crossref PubMed Scopus (156) Google Scholar). A new gene encoding an octamer binding factor, OTF3, is located 96 kb telomeric to the HLA-C locus (Takeda et al., 1992Takeda J. Seino S. Bell G.I. Human OTC3 gene family: cDNA sequence, alternative splicing, gene organization, chromosomal location, and expression at low levels in adult tissues.Nucleic Acid Res. 1992; 20: 4613-4620Crossref PubMed Scopus (249) Google Scholar). OTF3 is expressed in pluripotent stem cells and plays a part in the early development of embryonic cells (Marshall et al., 1993Marshall B. Leelayuwat C. Degli-Espositi M.A. Pinelli M. Abraham L.J. Dawkins R.L. New mayor histocompatibility complex genes.Hum Immunol. 1993; 38: 24-29Crossref PubMed Scopus (41) Google Scholar). The MHC S gene is located 147 kb telomeric to HLA-C. It is expressed specifically in keratinocytes in the terminal phases of differentiation (Zhou and Chaplin, 1993Zhou Y. Chaplin D.D. Identification in HLA class I region of a gene expressed late in keratinocyte differentiation.Proc Natl Acad Sci USA. 1993; 90: 9470-9473Crossref PubMed Scopus (83) Google Scholar;Simon et al., 1997Simon M. Montézin M. Guerrin M. Durieux J.J. Serre G. Characterization and purification of human corneodesmosin, an epidermal basic glycoprotein associated with corneocyte-specific modified desmosomes.J Biol Chem. 1997; 272: 31770-31776Crossref PubMed Scopus (75) Google Scholar;Guerrin et al., 1998Guerrin M. Simon M. Montézin M. Haftek M. Vincent C. Serre G. Expression cloning of human corneodesmosin proves its identity with the product of the S gene and allows improved characterization of its processing during keratinocyte differentiation.J Biol Chem. 1998; 28: 22640-22647Crossref Scopus (56) Google Scholar). The close proximity of the MHC S gene to HLA-C and the potential importance of the S protein in keratinocyte differentiation has defined the S gene as a possible candidate gene for PV. It has been reported that position +1243 in the MHC S gene could be involved in the susceptibility to psoriasis in UK patients (Tazi Ahnini et al., 1999aTazi Ahnini R. Camp N.J. Cork M.J. Mee J.B. Keohane S.G. Duff G.W. Giovine F.S. Novel association between the corneodesmosin (MHC S) gene and susceptibility to psoriasis.Hum Mol Genet. 1999; 8: 1135-1140Crossref PubMed Scopus (116) Google Scholar). The analysis of these polymorphic genes (MICA, HLA-B, HLA-C, OTF-3 and MHC S gene) in type I psoriasis patients, shows that HLA-C itself is not primarily responsible for psoriasis susceptibility and we define a susceptible locus in a region located between HLA-C and the S gene. Ninety-five unrelated Spanish patients (39 males) with early-onset chronic plaque psoriasis (type I) were examined in this study. None of the patients had arthritis or clinical signs of extra-epithelial manifestation of disease. The mean age at onset of type I psoriasis was 26.9 ± 15.1 y. Typing was also performed on a control population of 104 matched donors from the Spanish population. The differences between the frequencies in the populations were assessed using the chi square test with Yates' correction. The odds ratio (OR) was calculated by the cross-product ratio. The p-values were corrected (pc) by multiplying these by the number of comparisons at every locus. The potential impact for each marker was estimated by the etiologic fraction (EF), which indicates the proportion of disease cases among the total population that are attributable to one allele when OR > 1. The extent of linkage disequilibrium between two loci is expressed as the observed disequilibrium value (D) that is, a proportion of the theoretical maximum disequilibrium value (Dmax) achievable for this combination of alleles. The D standardized value (Ds) was calculated using the formula: Ds = Pab – (Pa·Pb)/Pa(1 - Pb) = D/Dmax. Analysis for typing class I antigens was performed by standard methods. DNA was isolated from lymphocytes using standard procedures. HLA-C alleles were specifically amplified with a combination of sense primer SV1 (exon 2, codon 45) and the anti-sense primer SV2 (exon 3, codon 182), described previously by us, spanning sequences containing the hypervariable regions of HLA-C exon 2/exon 3 (680 bp). HLA-C alleles were examined by sequence-specific probes (González et al., 1999González S. Martínez-Borra J. Torre-Alonso J.C. González-Roces S. Sanchez del Río Rodríguez Pérez A. Brautbar C. López-Larrea C. The MIC-A9 triplet repeat polymorphism in the transmembrane region confers additional susceptibility to develop psoriatic arthritis, and is independent of the association of Cw*0602 in psoriasis.Arthritis Rheum. 1999; 42: 1010-1016Crossref PubMed Scopus (140) Google Scholar). Amplification of DNA using sequence-specific primers was used to genotype B13, B38, B39, B47 and B57 alleles (Bunce et al., 1995Bunce M. Fanning G.C. Welsh K.I. Comprehensive, serologically equivalent DNA typing for HLA-B by PCR using sequence-specific primers (PCR-SSP).Tissue Antigens. 1995; 45: 81-90Crossref PubMed Scopus (129) Google Scholar), all of which have been described as susceptibility factors in psoriasis. For analysis of microsatellite repeat polymorphism in the MICA gene, polymerase chain reaction (PCR) was carried out by the same procedure described byMizuki et al., 1997Mizuki N. Ota M. Kimura M. et al.Triplet repeat polymorphism in the transmembrane region of the MICA gene: a strong association of six GCT repetitions with Behçet disease.Proc Natl Acad Sci USA. 1997; 94: 1298-1303Crossref PubMed Scopus (363) Google Scholar, except for the use of the primers flanking the TM region: sense MICA 5′-ACATTCCATGTTT CTGCTGTTG-3′ (MICA located 33 bp 3′-of exon 5) and the anti-sense primer 5′-TCACCTGGACCCTCTGCAG-3′ (MICA exon/intron 5 boundary region). Allele designation was based on the number of repeat units present in the PCR products. Four distinct alleles consisting of CGT repetitions were designated as A4 (104 bp), A5 (107 bp), A6 (110 bp), and A9 (119 bp). One additional A5 (A5.1) with one nucleotide insertion (G) was also detected (108 bp). Two reported polymorphisms were analyzed in the OTF3 gene by PCR/restriction fragment length polymorphism (Takeda et al., 1992Takeda J. Seino S. Bell G.I. Human OTC3 gene family: cDNA sequence, alternative splicing, gene organization, chromosomal location, and expression at low levels in adult tissues.Nucleic Acid Res. 1992; 20: 4613-4620Crossref PubMed Scopus (249) Google Scholar): a T/G replacement in the second position for the initiating ATG codon was characterized using the sense primer 5′-GTAGTCCTTTGTTA CATGCATGAGTCAGT-3′ and the anti-sense primer 5′-TGAATACCTTCCCAAA TAGAACCCC-3′. MnlI produced 276 + 78 bp for allele 1 (ATG) and 183 + 93 + 78 for allele 2 (AGG). A second set of a sense primer 5′-AGCTCATTGTCTAATGT-CAT-3′ [intron 4, located at position 5311 of the OTF3 sequence (EMBL accession number Z11900)] and an anti-sense primer 5′-CAGCTA-CATGGTGACTGAGT-3′ (exon 5, located at position 6112) were used to span the polymorphic HindIII polymorphic site (located at position 5856). HindIII digestion generated 546 + 256 fragments (allele B) and where it does not cut 802 bp (allele A). We analyzed the polymorphisms at amino acid positions 186 (Gly/Val), 393 (Gly/Val) and 394 (Ser/Leu). Genomic DNA was amplified by PCR with the S7/S8- and S15/S16-specific primer pairs described (Ishihara et al., 1996Ishihara N. Yamagata N. Ohno S. et al.Genetic polymorphism in the keratin-like S gene within the major histocompatibility complex and association analysis on the susceptibility of psoriasis vulgaris.Tissue Antigen. 1996; 48: 182-186Crossref PubMed Scopus (51) Google Scholar). The nucleotide differences at the polymorphic positions were distinguished by restriction fragment length polymorphism digestion using MnlI (C/T substitution at nucleotide position +619), MspI (G/T at position +1240) and HphI (C/T at position +1243) restriction enzymes. MnlI cleaved a PCR product (261 bp) into 66 + 12 (allele 2) or uncut (allele 1). MspI digestion generated a 126 + 60 (allele 1), and HphI produced 123 + 89 bp for allele 1, where it does not cut for allele 2 (212 bp). Allelic analysis was performed by electrophoresis on 6% polyacrylamide-urea gel (+619) or 2% agarose gel (+1240/1243). We analyzed the distribution of HLA-C antigens in a Spanish population of 95 patients with chronic plaque type I psoriasis and 104 matched controls by PCR/SSOP typing. The analysis showed a strong association between psoriasis and Cw*0602 (Table 1). This allele was found in 49% of the patients, but it was only carried by 21% of the control individuals (pc < 0.0006.; OR = 3.64, EF = 0.35). We investigated the role of Ala-73 in psoriasis which is present in Cw*04, Cw*0602, Cw*07, Cw*12, Cw*1503, and Cw*17. The overall frequency of Ala-73 was found to be similar in the psoriatic group (85%) compared with the controls (84%) (Table 1).Table IDistribution of HLA-B, HLA-C, MICA, OTF3, and S alleles in healthy controls and patients with psoriasisLocusControl (n=104)PV (n=95)B*57016 (5.7%)17 (17.8%)B*13010 (0%)7 (7.3%)Cw*0602aχ2=16.95; Corrected p (pc)<6·10-4; Odds ratio (OR)=3.64; Etiologic fraction (EF)=0.35.22 (21.1%)47 (49.4%)Cw-Ala 73bCw-Ala73 is present in Cw*04, Cw*0602, Cw*07, Cw*12, Cw*1503 and Cw*17.88 (84.6%)81 (85.2%)MICA-A932 (30.7%)29 (30.5%)OTF3-Bcχ2=14.53; pc<2.8·10-4; OR=3.76; EF=0.625.63 (60.5%)81 (85.2%)S(186)/Phe101 (97.1%)93 (97.8%)S(186)/Ser35 (33.6%)29 (30.5%)S(393)/Gly102 (98%)95 (100%)S(393)/Val2 (1.9%)0 (0%)S(394)/Ser90 (86.5%)92 (96.8%)S(394)/LeudSer394 (+1243) N.S.66 (63.4%)54 (56.8%)a χ2=16.95; Corrected p (pc)<6·10-4; Odds ratio (OR)=3.64; Etiologic fraction (EF)=0.35.b Cw-Ala73 is present in Cw*04, Cw*0602, Cw*07, Cw*12, Cw*1503 and Cw*17.c χ2=14.53; pc<2.8·10-4; OR=3.76; EF=0.625.d Ser394 (+1243) N.S. Open table in a new tab The contribution of the polymorphism of MICA and HLA-B genes to PV susceptibility was analyzed. A considerable incidence of the HLA-B*1301 (7.3% vs 0%) and B*5701 (17.8% vs 7%) in PV patients was found, but this was not statistically significant (Table 1). These alleles are known to take part in the B13.1 and B57.1 ancestral haplotypes and the associations of B*1301 and B*5701 are secondary to the stronger Cw*0602 allele. To analyze the association of MICA with psoriasis, microsatellite polymorphism in its transmembrane region was investigated. There were no significant differences in the distribution of MICA transmembrane polymorphism between the psoriasis and control groups. The triplet repeat A9, recently described by our group increased in psoriatic patients with arthritis (González et al., 1999González S. Martínez-Borra J. Torre-Alonso J.C. González-Roces S. Sanchez del Río Rodríguez Pérez A. Brautbar C. López-Larrea C. The MIC-A9 triplet repeat polymorphism in the transmembrane region confers additional susceptibility to develop psoriatic arthritis, and is independent of the association of Cw*0602 in psoriasis.Arthritis Rheum. 1999; 42: 1010-1016Crossref PubMed Scopus (140) Google Scholar), was present in 30.5% of the patients and 30.7% of the controls (Table 1). Interestingly, MICA is in strong linkage disequilibrium with HLA-B locus, and each of both ancestral haplotypes described to be associated to PV in Caucasians (AH13.1 and AH57.1) carried different MICA transmembrane polymorphism. HLA-B*1301 is in linkage disequilibrium with MICA A5.1 (Ds = 0.74) and HLA-B*5701 is in linkage disequilibrium with MICA A9 (Ds = 0.90) (data not shown). We analyzed the MHC S polymorphism described to be a candidate PV gene in Caucasians. Three polymorphic changes resulting in amino acid change have been studied (at +619, +1240, and +1243). No significant differences of MHC S gene polymorphism were found between PV patients and healthy controls (Table 1). The distribution of MHC S genotypes was similar in both populations despite their close proximity to HLA-C. The MHC S (+1243) allele frequency corresponding with Leu-394 shows a similar frequency in patients and controls in our study (54% vs 63%). Cw*0602 was found in carriers the Phe-186/Gly-393/Ser-194 genotype and Cw*0701 was found to be predominantly associated with Phe-186/Gly-393/Leu-194 genotype (data not shown). Our results suggest that the S gene does not have an independent effect to HLA-Cw6 on the susceptibility to PV. The OTF3 polymorphism was also analyzed in PV patients and controls by PCR/restriction fragment length polymorphism. No differences were found in the frequency of the MnlI polymorphism in the patients compared with the controls; however, we detected a greater incidence of OTF3-B allele (HindIII) in psoriatic patients (Table 1). This allele was present in 85% of the PV patients (32% homozygous) and in 60% of the controls, 13% of whom carried the BB homozygous genotype (pc < 0.0003; OR = 3.76). It is important to emphasize that the allele OTF3-B (EF = 0.62) was found to be more strongly associated to PV than Cw*0602 (EF = 0.35). As we detected a strong linkage disequilibrium between Cw*0602 and OTF3-B (88% of Cw*0602 individuals carried the OTF3-B allele; Ds = 0.64), we investigated the OTF3 polymorphism in a group of positive and Cw*0602-negative patients in order to establish whether the association of allele OTF3-B (HindIII) is due to linkage disequilibrium with Cw6 (Table 2). The analysis of OTF3 in 48 psoriatic patients and 82 controls, all of whom were negative for Cw*0602, also showed a significant increase of OTF3-B polymorphism in the patient's group (81% in PV vs 57% in controls; pc < 0.015, OR = 3.63, EF = 0.55). These data indicate that the significant increase of OTF3-B in PV patients is independent of the Cw*0602 association.Table IIDistribution of OTF3-B allele and HLA-Cw6 in PV patients and healthy controlsnOTF3-BCw*0602 positiveaDesequilibrium standardized value (Ds)=0.64. PV4742 (89.3%) Control2219 (86.3%)Cw*0602 negativebOR=3.63; EF=0.55; pc<0.015 PV vs Controls. PV4839 (81%) Control8247 (57.3%)a Desequilibrium standardized value (Ds)=0.64.b OR=3.63; EF=0.55; pc<0.015 PV vs Controls. Open table in a new tab The association of MHC complex with psoriasis was established in the early 1970s (Russell et al., 1972Russell T.J. Schultes L.M. Kuban D.J. Histocompatibility (HL-A) antigen associated with psoriasis.N Engl J Med. 1972; 287: 738-743Crossref PubMed Scopus (270) Google Scholar). Recently, genome screenings have confirmed the strong linkage of the MHC region with this disease (Trembath et al., 1997Trembath R.C. Clough R.L. Rosbotham J.L. et al.Identification of major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis.Hum Mol Genet. 1997; 6: 813-820Crossref PubMed Scopus (440) Google Scholar). HLA-Cw6 is the major genetic determinant known at the present time, but its association with PV varies markedly among different ethnic populations. HLA-C typing has confirmed the association between Cw*0602 and psoriasis in our population. It is not, however, clear at present whether Cw6 itself is primarily associated with psoriasis or not. If Cw6 itself were a causative disease allele, then all the risk haplotypes described for Cw6 carriers could be equally associated with disease, independently of their ethnic group. Obviously, this is not the case, and there is no unanimity regarding the different studies of the association of EH13.1 and EH57.1 haplotypes in different populations. Therefore, the possibility clearly exists that the HLA-C gene itself is not the primary locus responsible for PV. To clarify this problem we studied, by association analysis the relation between particular polymorphic markers located around the HLA-C locus and the disease phenotype in a case–control study. This approach has proven to be more powerful than linkage analysis for identifying causative genes in a highly compact region, such as MHC (Risch and Merikangas, 1996Risch N. Merikangas K. The future of genetic studies of complex human diseases.Science. 1996; 273: 1516-1517Crossref PubMed Scopus (4160) Google Scholar). The analysis of further polymorphic markers (or genes) within this region and linkage disequilibrium mapping of different ethnic populations with different ancestral haplotypes may lead to a better definition of the psoriasis susceptibility region. The MHC region (class I) contains genes of both immune and nonimmune importance. Polymorphic analysis of several loci, especially MHC S, OTF3, and MICA loci around the HLA-C/B loci, will enable us to localize the susceptibility genes of psoriasis. Our data and previously reported data suggest that the susceptible gene for psoriasis is not located centromeric to HLA-C. MICA is located 47 kb centromeric to HLA-B and it has been implicated in PV susceptibility (Dawkins et al., 1999Dawkins R. Leelayuwat C. Gaudieri S. et al.Genomics of the major histocompatibility complex: haplotypes, duplication, retroviruses and disease.Immunol Rev. 1999; 167: 275-304Crossref PubMed Scopus (252) Google Scholar); however, in this study the analysis of MICA-TM polymorphism did not reach statistical significance in psoriatic patients. Moreover, our data do not clearly support this possible association, as each of the extended haplotypes classically associated with PV found in Caucasians (EH13.1 and EH57.1) carry different MICA polymorphism. HLA-B13 is in linkage disequilibrium with MICA A5.1 and HLA-B57 in linkage disequilibrium with MICA A9. We have recently reported that a trinucleotide repeat polymorphism in the TM region of the MICA gene (A9) is associated to psoriatic arthritis independently of Cw6 (González et al., 1999González S. Martínez-Borra J. Torre-Alonso J.C. González-Roces S. Sanchez del Río Rodríguez Pérez A. Brautbar C. López-Larrea C. The MIC-A9 triplet repeat polymorphism in the transmembrane region confers additional susceptibility to develop psoriatic arthritis, and is independent of the association of Cw*0602 in psoriasis.Arthritis Rheum. 1999; 42: 1010-1016Crossref PubMed Scopus (140) Google Scholar), but not with psoriasis without arthritis. These results suggest that MICA is not primarily associated with psoriasis susceptibility and its association with such might be secondary to the arthritis. The MHC S gene (corneodesmosin gene) is located 147 kb telomeric to HLA-C and is expressed specifically in keratinocyte differentiation. Corneodesmosin is processed during keratinocyte differentiation by cleavage in its carboxyl terminal domain, which is thought to be a prerequisite for desquamation (Simon et al., 1997Simon M. Montézin M. Guerrin M. Durieux J.J. Serre G. Characterization and purification of human corneodesmosin, an epidermal basic glycoprotein associated with corneocyte-specific modified desmosomes.J Biol Chem. 1997; 272: 31770-31776Crossref PubMed Scopus (75) Google Scholar). Three polymorphisms in the MHC S gene which give amino acid change at positions +619 (Ser/Phe), +1240 (Gly/Val), and +1243 (Ser/Leu) have been described (Ishihara et al., 1996Ishihara N. Yamagata N. Ohno S. et al.Genetic polymorphism in the keratin-like S gene within the major histocompatibility complex and association analysis on the susceptibility of psoriasis vulgaris.Tissue Antigen. 1996; 48: 182-186Crossref PubMed Scopus (51) Google Scholar). These substitutions might interfere with the processing of the S protein and thus contribute to the disruption of epidermal differentiation, which is a feature of psoriasis. All these data suggest that the S gene is an obvious candidate to psoriasis susceptibility; however, our study failed to detect any significant association between S gene polymorphisms and psoriasis. This is in accordance with the results published in Japanese patients (Ishihara et al., 1996Ishihara N. Yamagata N. Ohno S. et al.Genetic polymorphism in the keratin-like S gene within the major histocompatibility complex and association analysis on the susceptibility of psoriasis vulgaris.Tissue Antigen. 1996; 48: 182-186Crossref PubMed Scopus (51) Google Scholar) and contrasts with the reported association of MHC S gene at position +1243 (Leu) described in UK patients (Tazi Ahnini et al., 1999aTazi Ahnini R. Camp N.J. Cork M.J. Mee J.B. Keohane S.G. Duff G.W. Giovine F.S. Novel association between the corneodesmosin (MHC S) gene and susceptibility to psoriasis.Hum Mol Genet. 1999; 8: 1135-1140Crossref PubMed Scopus (116) Google Scholar). This probably indicates that the MHC S gene is not the disease-causing allele and this discrepancy only reflects the extended linkage disequilibrium between the causative gene and this allele in UK patients. Independently,Jenisch et al., 1999aJenisch S. Koch S. Henseler T. Nair R.P. et al.Corneodesmosin gene polymorphism demonstrates strong linkage disequilibrium with HLA and association with psoriasis vulgaris.Tissue Antigens. 1999; 54: 439-449Crossref PubMed Scopus (73) Google Scholar have recently reported new polymorphisms in the exon 2 of MHC S that could be grouped in six different alleles (which they termed CD1 to CD6). One allele (CD2) corresponding with Ser at position +1243, which has displayed strong disequilibrium with Cw6, was found to be more strongly associated with the disease than HLA-C locus in German patients. Assuming direct involvement of MHC S polymorphism, linkage of this allele (CD2) would be expected; however, an association with CD alleles independent of Cw6 has not been demonstrated. This indicates that the association of the CD alleles with psoriasis might only be due to linkage disequilibrium. Furthermore, Cw*0602 and Cw7 described to be associated to PV in different populations are linked with different MHC S alleles (CD2-Cw*0602, CD5-Cw*0701 and CD6-Cw*0702 haplotypes). These correspond with different MHC S polymorphism at +1243 position. These observations make it unlikely that MHC S gene polymorphism contributes to psoriasis independently to HLA-C linkage disequilibrium. OTF3 (POU5F1) has been previously localized to a region between the HLA-C and S genes by physical mapping. We found that the association of the allele of OTF3 gene (B) with PV is stronger than C*0602. PV patients carrying OTF3 B allele but lacking HLA-Cw*0602, were also significantly overrepresented compared with the controls. Thus, the association found between OTF3-B and PV cannot be attributed exclusively to the linkage disequilibrium with HLA-Cw6 in our population. OTF3 is a member of a transcription factor containing octamer-binding domains. It is expressed in pluripotent stem cells and plays a part in the early development of embryonic cells (Takeda et al., 1992Takeda J. Seino S. Bell G.I. Human OTC3 gene family: cDNA sequence, alternative splicing, gene organization, chromosomal location, and expression at low levels in adult tissues.Nucleic Acid Res. 1992; 20: 4613-4620Crossref PubMed Scopus (249) Google Scholar;Marshall et al., 1993Marshall B. Leelayuwat C. Degli-Espositi M.A. Pinelli M. Abraham L.J. Dawkins R.L. New mayor histocompatibility complex genes.Hum Immunol. 1993; 38: 24-29Crossref PubMed Scopus (41) Google Scholar). Based in its pattern of expression and basic functions, OTF3 does not appear to be an obvious candidate for the putative MHC-linked PV gene. Our results, however, allow us to define a critical region for psoriasis susceptibility within 147 kb segment, telomeric to HLA-C locus and centromeric to the S gene. This is consistent with the results recently published by using polymorphic microsatellite markers in the region of the HLA-C gene that revealed the pathogenic gene for psoriasis is located within a reduced interval spanning 111 kb telomeric to HLA-C (Oka et al., 1999Oka A. Tamiya G. Tomizawa M. et al.Association analysis using refined microsatellite markers localizes a susceptibility locus for psoriasis vulgaris within a 111 kb segment telomeric to HLA-C gene.Hum Mol Genet. 1999; 12: 2165-2170Crossref Scopus (149) Google Scholar). Recently, the MHC class I region has been completely sequenced (Shiina et al., 1999bShiina T. Tamiya G. Oka A. et al.Molecular dynamics of MHC genesis unraveled by sequence analysis of the 1,796,938 bp HLA class I region.Proc Natl Acad Sci USA. 1999; 96: 13282-13287Crossref PubMed Scopus (156) Google Scholar) Within the critical region for PV (between HLA-C locus and S gene), one additional known gene TCF19 (SC1), four putative new genes (NOB4, NOB5, HCGII-2, and HCGII3) and four potential new coding sequences (EST) have been identified. Some of these genes and ETS are expressed in most of the tissues examined, including keratinocytes, and are obvious candidates for playing a part in the disease development. Dysregulation and functional expression of these genes in lesional psoriatic skin could be important to identify the putative MHC-linked psoriasis gene. It is now necessary to study disease-associated polymorphism in these new genes in order to determine the definite position of the causative gene. In conclusion, this study shows an association between the OTF3-B allele and psoriasis. We found that most of the HLA-Cw*0602 genotypes carry the OTF3-B allele. The contribution of Cw*0602 to disease susceptibility appears to be weaker than OTF3 and secondary to HLA-Cw*0602/OTF3-B linkage disequilibrium. Analysis concerning the allelic association of this region supports the hypothesis that the Cw*0602/OTF3-B haplotype, which contains or is in linkage disequilibrium with the candidate gene involved in psoriasis, could be a common ancestor haplotype in Caucasians. Information on the haplotypic distribution of this critical region in other populations where the PV has not been found to be associated with HLA-Cw*0602, could help to identify the causative gene. We wish to thank David H. Wallace (Member of the Council of Science Editors and the European Association of Science Editors) for critical revision of the manuscript. Supported by a grant from the Spanish grants PM98-0004 and FIS 00/0208.