Macrophage Migration Inhibitory Factor Gene Polymorphism is Associated with Psoriasis
2004; Elsevier BV; Volume: 123; Issue: 3 Linguagem: Inglês
10.1111/j.0022-202x.2004.23314.x
ISSN1523-1747
AutoresRachelle Donn, Darren Plant, Francine Jury, Helen L. Richards, Jane Worthington, David Ray, C.E.M. Griffiths,
Tópico(s)Macrophage Migration Inhibitory Factor
ResumoMacrophage migration inhibitory factor (MIF), an important pro-inflammatory cytokine, is over-expressed in plaques of psoriasis and increased levels are found in the sera of patients with psoriasis. Promoter polymorphisms of the MIF gene are associated with increased production of MIF and have been found to confer increased risk of susceptibility to chronic inflammatory diseases. We investigated whether there is an association between promoter polymorphisms of the MIF gene and chronic plaque psoriasis. Two hundred and twenty-eight UK caucasian patients with chronic plaque psoriasis, and a control panel of 401 UK caucasian normal volunteers were studied. MIF promoter polymorphisms were genotyped by allelic discrimination, or by a fluorescently labeled primer method, and capillary gel electrophoresis. Carriage of either the MIF-173*C polymorphism or the MIF CATT7 polymorphism was positively correlated with psoriasis (odds ratios (OR) 1.52 95% confidence intervals (CI) 1.05–2.19 (p=0.024) and OR 1.67 95% CI 1.1–2.5 (p=0.013), respectively. The OR for presence of the CATT7–MIF-173*C haplotype versus all other haplotypes combined was 1.69 95% CI 1.2–2.5 (p=0.008). The results provide evidence for polymorphisms in the MIF gene, and in particular the CATT7–MIF-173*C haplotype, being of importance in susceptibility to psoriasis. Macrophage migration inhibitory factor (MIF), an important pro-inflammatory cytokine, is over-expressed in plaques of psoriasis and increased levels are found in the sera of patients with psoriasis. Promoter polymorphisms of the MIF gene are associated with increased production of MIF and have been found to confer increased risk of susceptibility to chronic inflammatory diseases. We investigated whether there is an association between promoter polymorphisms of the MIF gene and chronic plaque psoriasis. Two hundred and twenty-eight UK caucasian patients with chronic plaque psoriasis, and a control panel of 401 UK caucasian normal volunteers were studied. MIF promoter polymorphisms were genotyped by allelic discrimination, or by a fluorescently labeled primer method, and capillary gel electrophoresis. Carriage of either the MIF-173*C polymorphism or the MIF CATT7 polymorphism was positively correlated with psoriasis (odds ratios (OR) 1.52 95% confidence intervals (CI) 1.05–2.19 (p=0.024) and OR 1.67 95% CI 1.1–2.5 (p=0.013), respectively. The OR for presence of the CATT7–MIF-173*C haplotype versus all other haplotypes combined was 1.69 95% CI 1.2–2.5 (p=0.008). The results provide evidence for polymorphisms in the MIF gene, and in particular the CATT7–MIF-173*C haplotype, being of importance in susceptibility to psoriasis. confidence intervals juvenile idiopathic arthritis migration inhibitory factor odds ratios single nucleotide polymorphism tumor necrosis factor-α Psoriasis is a chronic, currently incurable, inflammatory dermatosis that affects approximately 2% of the western population. Skin lesions of the commonest form of psoriasis––chronic plaque––consist of heavily scaled, red plaques commonly situated on extensor aspects of elbows and knees, lower back, and scalp, although any skin surface may be affected. Early onset "type I" psoriasis (Henseler and Christophers, 1985Henseler T. Christophers E. Psoriasis of early and late onset: Characterization of two types of psoriasis vulgaris.J Am Acad Dermatol. 1985; 13: 450-456Abstract Full Text PDF PubMed Scopus (735) Google Scholar) presenting at or before the age of 40 y is familial and in >60% of subjects is associated with HLA-Cw6 (Enerback et al., 1997Enerback C. Martinsson T. Inerot A. Wahlstrom J. Enlund F. Yhr M. Swanbeck G. Evidence that HLA-Cw6 determines early onset of psoriasis, obtained using sequence-specific primers (PCR-SPP).Acta Derm Venereol. 1997; 77: 273-276PubMed Google Scholar). Considerable interest is invested in ascertaining the genetic basis of psoriasis. As yet no gene or gene product has been found as causative but at least seven psoriasis susceptibility loci have been identified on a number of chromosomes (Capon et al., 2002Capon F. Munro M. Barker J. Trembath R. Searching for the major histocompatibility complex psoriasis susceptibility gene.J Invest Dermatol. 2002; 118: 745-751Crossref PubMed Scopus (136) Google Scholar). The evidence, to date, indicates that psoriasis is a complex, polygenic disorder. Current understanding of the pathomechanisms integral to the psoriatic process is that it is a disease of abnormal epidermal keratinocyte hyperproliferation and differentiation driven by a T cell predominant infiltrate (Griffiths and Voorhees, 1996Griffiths C.E.M. Voorhees J.J. Psoriasis, T cells and autoimmunity.J R Soc Med. 1996; 89: 315-319PubMed Google Scholar). Tumor necrosis factor-α (TNF-α;Nickoloff et al., 1991Nickoloff B.J. Karabin G.D. Barker J.N.W.N. et al.Cellular localization of interleukin-γ and its inducer, tumor necrosis factor-8 in psoriasis.Am J Pathol. 1991; 138: 129-140PubMed Google Scholar) and Th1 cytokines including interferon-γ and interleukins (IL)-2 and -12 are key drivers of the psoriatic process (Austin et al., 1999Austin L.M. Ozawa M. Kikuchi T. Walters I.B. Krueger J.G. The majority of epidermal cells in psoriasis vulgaris lesions can produce type I cytokines, interferon-γ, defining TC1 (cytotoxic T lymphocyte) and TH1 effector populations: A type I differentiation bias is also measured in circulating blood T cells in psoriatic patients.J Invest Dermatol. 1999; 113: 752-759Crossref PubMed Scopus (417) Google Scholar). Carriage of the TNF-α A-238 allele 2 is associated with early onset psoriasis (Reich et al., 2002Reich K. Mossner R. Konig I.R. Westphal G. Ziegler A. Neumann C. Promoter polymorphisms of the genes encoding tumor necrosis factor-α and interleukin-1β are associated with different subtypes of psoriasis characterized by early and late disease onset.J Invest Dermatol. 2002; 118: 155-163Crossref PubMed Scopus (100) Google Scholar). Macrophage migration inhibitory factor (MIF) is a macrophage derived cytokine important in a number of pro-inflammatory events (Bernhagen et al., 1996Bernhagen J. Bacher M. Calandra T. Metz C.N. Doty S.B. Donnelly T. Bucala R. An essential role for macrophage migration inhibitory factor in the tuberculin delayed-type hypersensitivity reaction.J Exp Med. 1996; 183: 377-382Crossref Scopus (244) Google Scholar). These events include inducing secretion of TNF-α and promotion of T cell activation (Bernhagen et al., 1996Bernhagen J. Bacher M. Calandra T. Metz C.N. Doty S.B. Donnelly T. Bucala R. An essential role for macrophage migration inhibitory factor in the tuberculin delayed-type hypersensitivity reaction.J Exp Med. 1996; 183: 377-382Crossref Scopus (244) Google Scholar;Leech et al., 2000Leech M. Metz C. Bucala R. Morand E.F. Regulation of macrophage migration inhibitory factor by endogenous glucocorticoids in rad adjuvant-induced arthritis.Arthritis Rheum. 2000; 43: 827-833Crossref PubMed Scopus (86) Google Scholar). MIF has been identified as a key regulator of TNF-α production in rheumatoid arthritis (Leech et al., 1999Leech M. Metz C. Hall P. et al.Macrophage migration inhibitory factor in rheumatoid arthritis: Evidence of proinflammatory function and regulation by glucocorticoids.Arthritis Rheum. 1999; 42: 1601-1608Crossref PubMed Scopus (282) Google Scholar). The MIF gene maps to chromosome 22q11.2 and a single nucleotide polymorphism (SNP; G to C transition) in the 5′-flanking region at position -173 of the MIF gene has been associated with susceptibility to adult inflammatory arthritis (Barton et al., 2003Barton A. Lamb R. Symmons D. Silman A. Thomson W. Worthington J. Donn R. Macrophage migration inhibitory factor (MIF) gene polymorphism is associated with susceptibility to but not severity of inflammatory polyarthritis.Genes Immun. 2003; 3: 170-176Google Scholar) and juvenile idiopathic arthritis (Donn et al., 2001Donn R.P. Shelley E. Oliver W.E. Thomson W. A novel 5′-flanking region polymorphism of macrophage migration inhibitory factor is associated with systemic-onset juvenile idiopathic arthritis.Arthritis Rheum. 2001; 44: 1782-1785Crossref PubMed Scopus (180) Google Scholar, Donn et al., 2004Donn R.P. Alourfi Z. Zeggini E. et al.A functional promoter haplotype of macrophage migration inhibitory factor (MIF) is linked and associated with juvenile idiopathic arthritis.Arthritis Rheum. 2004; 50: 1604-1610Crossref PubMed Scopus (117) Google Scholar). This polymorphism also has the potential to be associated with increased expression of MIF via production of an activator protein-4 response element in the MIF promoter. A CATT tetranucleotide repeat element beginning at -794, within the MIF promoter (Donn et al., 2004Donn R.P. Alourfi Z. Zeggini E. et al.A functional promoter haplotype of macrophage migration inhibitory factor (MIF) is linked and associated with juvenile idiopathic arthritis.Arthritis Rheum. 2004; 50: 1604-1610Crossref PubMed Scopus (117) Google Scholar) also appears to play a role in determining susceptibility to rheumatoid arthritis (Barton et al., 2003Barton A. Lamb R. Symmons D. Silman A. Thomson W. Worthington J. Donn R. Macrophage migration inhibitory factor (MIF) gene polymorphism is associated with susceptibility to but not severity of inflammatory polyarthritis.Genes Immun. 2003; 3: 170-176Google Scholar). Given the observation that serum levels of MIF are elevated in psoriasis (Shimizu et al., 2001Shimizu T. Nishihira J. Mizue Y. et al.High macrophage migration inhibitory factor (MIF) serum levels associated with extended psoriasis.J Invest Dermatol. 2001; 116: 989-990Crossref PubMed Google Scholar) and the central importance of TNF-α in the cutaneous inflammatory process we investigated whether promoter polymorphisms of the MIF gene are implicated in susceptibility to psoriasis. No deviation from Hardy–Weinberg equilibrium was seen for the patients with psoriasis or the controls for either MIF-173 SNP (p=1.0; 0.30) or the CATT repeat element (p=0.58; 0.13). MIF-173 genotype frequencies were compared between patients with psoriasis and controls (p=0.080; Table I). But comparing carriage of the MIF-173*C polymorphism (GC+CC vs GG) in patients with psoriasis to that seen in controls revealed a positive association with disease odds ratios (OR) 1.52, 95% confidence intervals (CI) 1.05–2.19, p=0.024.Table IMIF-173 genotype (% frequencies) in psoriasis patients and controlsMIF-173 genotypePsoriasis (n=225)Controls (n=375)MIF-173*GG152 (67.5)285 (76.0)MIF-173GC67 (29.8)83 (22.1)MIF-173CC6.0 (2.7)7 (1.9) Open table in a new tab Similarly, genotype frequencies of the CATT repeat element were initially compared between the patients with psoriasis and the control panel (p=0.090; Table II). When carriage of the MIF CATT7 (5/7+6/7+7/7 vs 5/5+5/6+6/6) was compared between patients with psoriasis and controls a significant association with disease was observed OR 1.67, 95% CI 1.1–2.5, p=0.013.Table IIMIF CATT repeat genotype (% frequencies) in psoriasis patients and controlsMIF CATT genotypePsoriasis (n=218)Controls (n=378)5/59 (4.1)22 (5.8)5/658 (26.7)132 (34.9)5/716 (7.3)16 (4.2)6/697 (44.5)162 (42.9)6/734 (15.6)39 (10.3)7/74 (1.8)7 (1.9) Open table in a new tab Frequencies of estimated haplotypes (inferred using the Markov chain-Monte Carlo algorithm, Phase 1.0; Table III) in cases with psoriasis were compared with controls (Table III). The OR for the presence of the CATT7–MIF-173*C haplotype versus all other haplotypes combined was 1.69, 95% CI 1.2–2.5, p=0.008. This provides evidence for this haplotype being of importance in susceptibility to psoriasis.Table IIIFrequencies (%) of estimated CATT–MIF-173 haplotypes in psoriasis patients and controlsHaplotypePsoriasis (2n=456) (%)Controls (2n=802) (%)CATT5–MIF-173*G92 (20.2)198 (24.7)CATT6–MIF-173*G276 (60.5)487 (60.7)CATT7–MIF-173*G9 (2.0)16 (2.0)CATT6–MIF-173*C26 (5.7)43 (5.4)CATT7–MIF-173*C53 (11.6)58 (7.2) Open table in a new tab One hundred and twenty-two (66%) of the 184 patients genotyped were HLA-Cw6 positive. When the frequency of the MIF polymorphisms was considered in just the Cw6 positive patient group compared to controls (n=401) no significant associations were seen (carriage of MIF-173*C OR 1.22, 95% CI 0.7–2.0, p=0.4; carriage of MIF CATT7 OR 1.62, 95% CI 0.9–2.7, p=0.06; presence of the CATT7–MIF-173*C haplotype OR 0.7 95% CI 0.3–1.6, p=0.4). Comparison of the MIF polymorphisms between Cw6 negative patients with psoriasis (n=62) and the controls (n=401) was borderline significant for carriage of the MIF-173*C allele (OR 1.83 95% CI 0.9–3.3, p=0.05) but non-significant for carriage of the MIF CATT7 repeat (OR 1.71, 95% CI 0.8–3.4, p=0.09) and for the presence of the CATT7–MIF-173*C haplotype OR 2.1 95% CI 0.8–4.9, p=0.09). This study has shown significant association between carriage of the MIF-173*C SNP, and also with carriage of the MIF CATT7 allele, and chronic plaque psoriasis. Furthermore, the CATT7–MIF-173*C haplotype conferred an OR of 1.7 (p=0.008) for increased risk of susceptibility to chronic psoriasis. This is the same MIF haplotype that has been found to confer increased risk of susceptibility to JIA (Donn et al, 2003) and to adult inflammatory polyarthritis (Barton et al., 2003Barton A. Lamb R. Symmons D. Silman A. Thomson W. Worthington J. Donn R. Macrophage migration inhibitory factor (MIF) gene polymorphism is associated with susceptibility to but not severity of inflammatory polyarthritis.Genes Immun. 2003; 3: 170-176Google Scholar). When the sample set was divided based on age of onset or HLA-Cw6 genotype the effect of MIF was not enhanced in either subset. Although the complex immunogenetics of chronic plaque psoriasis are, at present, poorly understood psoriasis is a chronic inflammatory disease whose expression is dependent on pro-inflammatory cytokines, such as TNF-α, and Th1 cytokines. Plaques of psoriasis contain high levels of TNF-α activity (Nickoloff et al., 1991Nickoloff B.J. Karabin G.D. Barker J.N.W.N. et al.Cellular localization of interleukin-γ and its inducer, tumor necrosis factor-8 in psoriasis.Am J Pathol. 1991; 138: 129-140PubMed Google Scholar) and are characterized by a predominance of Th1 cytokines (Austin et al., 1999Austin L.M. Ozawa M. Kikuchi T. Walters I.B. Krueger J.G. The majority of epidermal cells in psoriasis vulgaris lesions can produce type I cytokines, interferon-γ, defining TC1 (cytotoxic T lymphocyte) and TH1 effector populations: A type I differentiation bias is also measured in circulating blood T cells in psoriatic patients.J Invest Dermatol. 1999; 113: 752-759Crossref PubMed Scopus (417) Google Scholar). The importance of these cytokines to the psoriatic process is underscored by the ability of TNF-α blocking agents (Chaudhari et al., 2001Chaudhari U. Romano P. Mulcahy L.D. Dooley L.T. Baker D.G. Gottlieb A.B. Efficacy and safety of infliximab monotherapy for plaque-type psoriasis: Randomised trial.Lancet. 2001; 357: 1842-1847Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar) and Th2 cytokines (Ghoreschi et al., 2003Ghoreschi K. Thomas P. Breit S. et al.Interleukin-4 therapy of psoriasis induces Th2 responses and improves human autoimmune disease.Nat Med. 2003; 9: 40-46Crossref PubMed Scopus (365) Google Scholar) to produce significant clinical improvement in patients with this disease. It is increasingly recognized that the innate immune response is involved in several components of the pathology of psoriasis, which include roles for natural killer cells (Cameron et al., 2002Cameron A.L. Kirby B. Fei W. Griffiths C.E.M. Natural killer and natural killer-T cells in psoriasis.Arch Dermatol Res. 2002; 294: 863-869Google Scholar) and IL-15 (Ruckert et al., 2000Ruckert R. Asadullah K. Seifert M. et al.Inhibition of keratinocyte apoptosis by IL-15: A new parameter in the pathogenesis of psoriasis?.J Immunol. 2000; 165: 2240-2250Crossref PubMed Scopus (177) Google Scholar) and response to infection such as β-hemolytic streptococcus––a trigger of guttate psoriasis (Telfer et al., 1992Telfer N.R. Chalmer R.J. Whale K. Colman G. The role of streptococcal infection in the initiation of guttate psoriasis.Arch Dermatol. 1992; 128: 39-42Crossref PubMed Scopus (261) Google Scholar). A number of investigators have linked cytokine polymorphisms with early onset psoriasis particularly carriage of TNF-α A-238 allele 2 (Reich et al., 2002Reich K. Mossner R. Konig I.R. Westphal G. Ziegler A. Neumann C. Promoter polymorphisms of the genes encoding tumor necrosis factor-α and interleukin-1β are associated with different subtypes of psoriasis characterized by early and late disease onset.J Invest Dermatol. 2002; 118: 155-163Crossref PubMed Scopus (100) Google Scholar), vascular endothelial growth factor +405 GC (Young et al., 2004Young H.S. Summers A.S. Bhushan M. Brenchley P.E. Griffiths C.E.M. Single nucleotide polymorphisms of vascular endothelial growth factor in psoriasis of early onset.J Invest Dermatol. 2004; 122: 209-215Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) and IL-1β (Reich et al., 2002Reich K. Mossner R. Konig I.R. Westphal G. Ziegler A. Neumann C. Promoter polymorphisms of the genes encoding tumor necrosis factor-α and interleukin-1β are associated with different subtypes of psoriasis characterized by early and late disease onset.J Invest Dermatol. 2002; 118: 155-163Crossref PubMed Scopus (100) Google Scholar) polymorphism. It is likely that although carriage of these particular SNP is significantly associated with susceptibility to psoriasis as compared with normal controls they are perhaps indicative of susceptibility to inflammation in general. A similar paradigm is the commonality of chromosomal susceptibility loci between psoriasis and atopic dermatitis, which infers common genes associated with skin inflammation and immunity (Cookson et al., 2001Cookson W.O. Ubhi B. Lawrence R. et al.Genetic linkage of childhood atopic dermatitis and psoriasis susceptibility loci.Nat Genet. 2001; 27: 372-373Crossref PubMed Scopus (326) Google Scholar). In common with cytokines such as TNF-α the actions of MIF involve both the innate and adaptive immune response to bacterial infections (Calandra et al., 1998Calandra T. Spiegel L.A. Metz C.N. Bucala R. Macrophage migration inhibitory factor is a critical mediator of the activation of immune cells by exotoxins of gram-positive bacteria.Proc Natl Acad Sci USA. 1998; 95: 11383-11388Crossref PubMed Scopus (217) Google Scholar;Bozza et al., 1999Bozza M. Satoskar A.R. Lin G. Lu B. Humbles A.A. Gerard C. David J.R. Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis.J Exp Med. 1999; 189: 341-346Crossref PubMed Scopus (480) Google Scholar) in part due to the ability of MIF to antagonize the effects of glucocorticoids perhaps by preferential utilization of mitogen-activated protein kinase pathways (Donnelly and Bucala, 1997Donnelly S.C. Bucala R. Macrophage migration inhibitory factor: A regulator of glucocorticoid activity with a critical role in inflammatory disease.Mol Med Today. 1997; 3: 502-507Abstract Full Text PDF PubMed Scopus (115) Google Scholar). The role of MIF in the adaptive, T cell driven, immune response is exemplified by the observation that immunoneutralization of MIF inhibits cutaneous delayed-type hypersensitivity reactions in vivo (Bernhagen et al., 1996Bernhagen J. Bacher M. Calandra T. Metz C.N. Doty S.B. Donnelly T. Bucala R. An essential role for macrophage migration inhibitory factor in the tuberculin delayed-type hypersensitivity reaction.J Exp Med. 1996; 183: 377-382Crossref Scopus (244) Google Scholar). Furthermore, MIF induces macrophage production of IL-12 a component of the Th1 cytokine response (De Jung et al., 2001De Jung Y.P. Badia-Molina A.C. Satoskar A.R. et al.Development of chronic colitis is dependent on the cytokine MIF.Nat Immunol. 2001; 2: 1061-1066Crossref PubMed Scopus (266) Google Scholar). Increased expression of MIF occurs in plaques of psoriasis (Steinhoff et al., 1999Steinhoff M. Meinhardt A. Steinhoff A. Gemsa D. Bucala R. Bacher M. Evidence for a role of macrophage migration inhibitory factor in psoriatic skin disease.Br J Dermatol. 1999; 141: 1061-1066Crossref PubMed Scopus (47) Google Scholar) and sera from patients with psoriasis contain high levels of MIF (Shimizu et al., 2001Shimizu T. Nishihira J. Mizue Y. et al.High macrophage migration inhibitory factor (MIF) serum levels associated with extended psoriasis.J Invest Dermatol. 2001; 116: 989-990Crossref PubMed Google Scholar). MIF is also associated with other inflammatory conditions such as sarcoidosis, transplant rejection, asthma, and inflammatory eye disease (Morand et al., 2003Morand E.F. Bucala R. Leech M. Macrophage migration inhibitory factor. An emerging therapeutic target in rheumatoid arthritis.Arthritis Rheum. 2003; 48: 291-299Crossref PubMed Scopus (62) Google Scholar). Although MIF is involved predominantly in immune responses there is evidence that it also promotes angiogenesis, a key histological component of psoriasis (Yang et al., 2000Yang Y. Degranpre P. Kharfi A. Akoum A. Identification of macrophage migration inhibitory factor as a potent endothelial cell-growth promoting agent released by ectopic human endometrial cells.J Clin Endocrinol Metab. 2000; 85: 4721-4727Crossref PubMed Scopus (87) Google Scholar). Taken together these observations implicate MIF as an important pro-inflammatory cytokine in psoriasis. Our study provides evidence for the importance of MIF in psoriasis pathogenesis. It is likely, however, that the CATT7–MIF-173*C haplotype contributes to the expression of skin inflammation rather than specifically to the development of psoriasis, as this is the same haplotype associated with the chronic inflammatory diseases adult polyarthritis and juvenile idiopathic arthritis. Strategies aimed at therapeutic antagonism of MIF would to some extent remove an antagonist of glucocorticoid activity––a hypothesis proved by the observation that administration of an anti-MIF antibody reverses the effects of adrenalectomy in rat adjuvant arthritis (Leech et al., 2000Leech M. Metz C. Bucala R. Morand E.F. Regulation of macrophage migration inhibitory factor by endogenous glucocorticoids in rad adjuvant-induced arthritis.Arthritis Rheum. 2000; 43: 827-833Crossref PubMed Scopus (86) Google Scholar). The development of a humanized anti-MIF would be a logical approach to treatment of psoriasis and in accord with other therapeutic strategies directed against pro-inflammatory cytokines. Peripheral blood samples were obtained, with informed consent, from 228 UK Caucasian patients with chronic plaque psoriasis. The male:female ratio was 1.3:1, and the mean age of disease onset 27 y (range 1–66 y) for this patient cohort. The majority, 188, of these patients were known to be of early onset (≤40 y) type I psoriasis. Similarly, blood was taken from 401 normal healthy, unrelated UK Caucasian controls. Genomic DNA was isolated by phenol chloroform extraction. The study was approved by the Salford and Trafford Local Research Ethics Committee and all patients gave written, informed consent. Genotyping was performed using an Assays-on-Demand (SNP ID hCV2213785; AB Biosystems, Warrington, UK) allelic discrimination assay on a Taqman 7700 platform (AB Biosystems) according to manufacturers' instructions except that a 5 μL rather than a 25 μL reaction volume was used (http://www.appliedbiosystems.com). Briefly, the PCR reaction contained 10 ng genomic DNA, 2.5 μL Taqman master mix and 0.125 μL of 40 × assay mix. PCR was performed using 384 well plates on an ABI 9700 thermal cycler (reaction conditions 50°C for 2 min 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min). The Taqman 7700 was used to perform end plate reading using the allelic discrimination option. Alleles of the CATT repeat element were identified using a fluorescently labeled PCR primer and capillary electrophoresis. Genomic DNA (50 ng) was amplified by PCR in a total reaction volume of 10 μL containing 5 pmol of both the forward and reverse primers: (forward primer 5′ TTG CAC CTA TCA GAG ACC 3′; Reverse primers 5′ TCC ACT AAT GGT AAA CTC G 3′). The forward primer was pre-labeled with a FAM fluorescent dye. Four nmol of each of the four deoxynucleotide triphosphates (dNTP), 0.2 U Taq polymerase (Bioline, London, UK), 1.5 mM MgCl2 buffer 1 × KCl buffer and 1 mM betaine were included in the PCR mix. The PCR were performed in 96-well microtiter plates on a Tetrad thermal cycler. Forty PCR cycles were carried out each with denaturation (1 min) 95°C, primer annealing 54°C (1 min) and extension 45 s at 72°C. A final extension step was conducted at 72°C for 5 min. Amplified product was pooled with the Tamra 350 size standard (PE Biosystems). Gel electrophoresis was performed on a 0.4 mm 6% polyacrylamide gel on a PE Biosystems 377 DNA sequencer (PE Biosystems, Warrington, UK). Gels were run at 1200 V for 2 h. Semi-automated genotyping was carried out using Genscan analysis and Genotyper 3.6 software, with all the genotyping being manually checked. All results were analyzed using Genescan analysis and manually checked using Genotyper 3.6 software. One hundred and eighty-four of the 188 patients known to have early onset psoriasis were genotyped for HLA-Cw6 using the Dynal auto REL1 48 (Dynal, Bromborough, UK) fully automated strip-processing system (Mackay et al., 2002Mackay K. Eyre S. Myerscough A. et al.Whole-genome linkage analysis of rheumatoid arthritis susceptibility loci in 252 affected sibling pairs in the United Kingdom.Arthritis Rheum. 2002; 46: 582-584Crossref PubMed Scopus (179) Google Scholar). Hardy–Weinberg equilibrium was checked using the χ2 test. Comparison of genotype and phenotype frequencies for each MIF promoter polymorphism was by χ2 analysis. OR with 95% CI were calculated in Stata (Stata Corporation, College Station, Texas). Haplotypes were inferred using the Markov chain-Monte Carlo algorithm (PHASE ver 1.0) (Stephens et al., 2001Stephens M. Smith N.J. Donnelly P. A new statistical method for haplotype reconstruction from population data.Am J Human Genet. 2001; 68: 978-989Abstract Full Text Full Text PDF PubMed Scopus (6195) Google Scholar). Haplotype frequencies were then compared using χ2 analysis (Stata). p<0.05 was taken as significant throughout. This work was supported in part by the Medical Research Council of the UK––Grant no. GG9901171 and an unrestricted grant from Stiefel UK to DP.
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