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Association between T-lymphocyte regulatory gene CTLA4 single nucleotide polymorphism at position 49 in exon 1 and HLA-DRB1*08 in Japanese patients with psoriasis vulgaris
Letters to the Editor / Journal of Dermatological Science 62 (2011) 64–71
70
Letter to the Editor Association between T-lymphocyte regulatory gene CTLA4 single nucleotide polymorphism at position 49 in exon 1 and HLA-DRB1*08 in Japanese patients with psoriasis vulgaris Psoriasis vulgaris (PV) is a multifactorial skin disease involving T lymphocyte-mediated inflammation. The major genetic determinant of psoriasis is PSORS1, which accounts for more than 30% of the heritability of PV. A recent genome-wide association study revealed that the human leukocyte antigen (HLA) complex, particularly HLA-Cw*0602, is the most plausible candidate gene among various ethnicities [1]. On the other hand, cytotoxic T-lymphocyte antigen 4 (CTLA4), which is located on chromosome 2q33, is a costimulatory molecule that is expressed on activated T lymphocytes and is important in the counter-regulation of CD28 T-cell activation via CD80/CD86 (B7-1/B7-2) on the antigen-presenting cells. Several polymorphisms of CTLA4 were found to be associated with some autoimmune diseases in humans [2]. However, there have been only a few studies on CTLA4 polymorphisms in psoriasis in Japanese [3], Korean [4], Polish [5] and Swedish [6] populations. Three known polymorphisms have been reported in CTLA4: the 318(rs5742909)C/T single nucleotide polymorphism (SNP) at position 318 (counting from the ATG start codon) in the promoter region; the 49(rs231775)A/G (A as the ancestral allele and G as the minor allele) SNP at position 49 in exon 1, resulting in an alanine for threonine amino acid substitution; and variations in the length of a dinucleotide (AT)n repeat in the 30 -untranslated region (30 -UTR). The above reports did not find any associations between either PV or psoriatic arthritis and the CTLA4 SNP sites. With respect to HLA, HLA-A2-B46-DR8 haplotype was found to be associated with psoriasis in a Japanese population [7]. We therefore investigated whether both HLA (HLA-C and -DRB1) and the 49A/G SNP in CTLA4 were associated with disease susceptibility in Japanese PV patients. Eighty-two unrelated Japanese psoriatics (male = 64, female = 18; age range, 16–81 years; mean age at onset of PV, 38.5 years) and 80 normal healthy controls (age range, 20–73 years; mean age, 36.2 years) were enrolled in the present study. The study was approved by the Ethics Committee for Genome Research of the Yamaguchi University School of Medicine. All patients and controls involved gave written informed consent for
genetic studies. Genomic DNA was isolated from peripheral venous blood using a QIAamp DNA Blood Maxi kit (QIAGEN, Hilden, Germany) and the 49 A/G SNP (rs231775) was typed using PCR, as described previously [5]. HLA class I and class II genotypes were determined using Micro SSPTM HLA typing trays (One Lambda Inc., Canoga Park, CA, USA). Statistical significance was determined by x2 test for differences in genotype and allele frequency. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using Woolf’s method. Differences between groups of data were analyzed by Student’s t-test. The frequencies of genotypes and alleles at the 49A/G SNP are shown in Table 1. The 49 A/G genotypes and alleles were similarly distributed in PV patients and controls. When we calculated the frequency of HLA-DRB1*08 in the presence of combined (G/G + G/A) genotypes at position 49 of CTLA4, the most significant difference between PV patients and controls was observed (38.1% in PV vs. 17.4% in controls; p < 0.01), indicating an additional requirement of CTLA4 and the HLA-DRB1*08 allele for psoriasis susceptibility. On the other hand, with respect to genotype A/A at position 49, a significant difference was not seen between the two groups. Patient demographic data in the PV-associated HLA-DRB1*08-positive group (n = 28) are as follows: mean age at onset SE = 33.9 17.3 years vs. 39.8 12.0 years in HLA-DRB1*08-negative psoriatics (n = 54) (not significant); male:female = 19:9; presence of arthropathy = 7/28 (25.0%) vs. 2/54 (3.7%) in HLA-DRB1*08-negative psoriatics (p < 0.01); and type of skin lesion, plaque type:guttate type:erythrodermic type = 25:2:1. With regard to streptococcal infection, the results based on our previously described ELISA system [8] showed that serum IgG antibody levels against recombinant M protein originating from Streptococcus pyogenes were significantly elevated in HLADRB1*08-positive psoriatics when compared with HLA-DRB1*08negative psoriatics (7,211 815 U/ml vs. 3,420 485 U/ml; p < 0.01). Based on these data, HLA-DRB1*08 is relevant to arthropathy and elevated immune response to streptococcal infection. With regard to HLA-Cw*0602 and the 49A/G SNP in CTLA4, no associations were seen in the present study (data not shown) (Table 2). Tsunemi et al. [3] concluded that there were no statistically significant differences in the 49 A/G SNP of CTLA4 exon1 in Japanese patients with PV, but there was a slightly increased genotype frequency for 49 G/G in PV patients as compared to controls (41.8% in PV vs. 32.7% in controls; not significant at the 5%
Table 1 Genotype and allele frequencies of 49A/G of CTLA4 in psoriasis. CTLA4
Genotypes
PV (n = 82)
Controls (n = 80)
p value
49A/G (rs231775)
G/G G/A A/A
35/82 (42.7%) 28/82 (34.1%) 19/82 (23.2%)
34/80(42.5%) 35/80 (43.8%) 11/80 (13.7%)
p > 0.08 p > 0.10 p > 0.10
CTLA4
Alleles
PV (2n = 164)
Controls (2n = 160)
p value
49A/G (rs231775)
G A
98/164 (59.8%) 66/164 (40.2%)
103/160 (64.4%) 57/160 (35.6%)
p > 0.40 p > 0.40
Table 2 Frequencies of HLA-DRB1*08a with either (G/G + G/A)or A/A CTLA4 genotype.
49A/G (rs231775)
G/G + G/A A/A
a b
PV (n = 82)
Controls (n = 80)
ORb (95% CI)
p value
(n = 63) 24/63 (38.1%) (n = 19) 4/19 (21.1%)
(n = 69) 12/69 (17.4%) (n = 11) 3/11 (27.3%)
2.92 (1.31 6.53)
p < 0.01
Frequency of HLA-DRB1*08 was 34.1% (=28/82) in PV, and 18.8% (=15/80) in controls, respectively (OR = 2.25 (95%CI = 1.09 4.63), p < 0.03). OR: Odds ratios.
p > 0.50
Letters to the Editor / Journal of Dermatological Science 62 (2011) 64–71
level). They also found that 49 A/G SNP showed strong linkage disequilibrium with the 318 C/T SNP (x2 = 26.96; p = 2.1 107). Similarly, the 49 G variant allele of CTLA4 exon1 does not appear to be associated with PV in Koreans. However, the frequency of the 49 G and CT60 (rs3087243) G haplotypes was significantly lower in Polish PV patients with middle-age onset [9]. Based on a functional analysis for the CTLA4 protein in Graves’ disease, in which more individuals have the combined (G/G + G/A) genotypes at position 49 of CTLA exon 1 when compared to controls (82.2% vs. 65.1% in controls), the G allele was found to exert inhibitory effects on CTLA4 in T cells [10]. Taken together with clinical evidence of an association between the HLA-A2-B46-DR8(DRB1*08) haplotype and disease susceptibility to PV in Japanese, the present observations suggest that G variants at position 49 of CTLA4 exon 1 and HLA-DRB1*08 may contribute to the development of PV through mutual action. In conclusion, to our knowledge, this is the first report showing an association between CTLA4 SNP (49 G allele) and HLA-DRB1*08 with psoriasis in Japanese PV patients, although the number of subjects was limited. Larger population-based studies are essential in order to confirm these findings. Acknowledgements This work was supported in part by a grant from the Ministry of Health, Labour and Welfare (Research on Measures for Intractable Diseases), Japan and by a Grant-in Aid for Scientific Research (22591223) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References [1] Nestle FO, Kaplan DH, Barker J. Psoriasis mechanisms of disease. N Engl J Med 2009;361:496–509. [2] Kristiansen OP, Larsen ZM, Pociot F. CTLA-4 in autoimmune diseases—a general susceptibility gene to autoimmunity? Genes Immun 2000;1:170–84. [3] Tsunemi Y, Saeki H, Kishimoto M, Mitsui H, Tada Y, Torii H, et al. Cytotoxic T lymphocyte antigen-4 gene (CTLA4) polymorphisms in Japanese patients with psoriasis vulgaris. J Dermatol Sci 2003;32:163–5.
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[4] Kim Y-K, Pyo C-W, Hur S-S, Kim T-Y, Kim T-G. No associations of CTLA-4 and ICAM-1 polymorphisms with psoriasis in the Korean population. J Dermatol Sci 2003;33:75–7. [5] Łuszczek W, Kubicka W, Jasek M, Barant E, Cisło M, Nockowski P, et al. CTLA-4 gene polymorphisms and natural soluble CTLA-4 protein in psoriasis vulgaris. Int J Immunogenet 2006;33:217–24. [6] Alenius G-M, Friberg C, Nilsson S, Wahlstro¨m J, Dahlqvist SR, Samuelsson L. Analysis of 6 genetic loci for disease susceptibility in psoriatic arthritis. J Rheumatol 2004;31:2230–5. [7] Nakagawa H, Akazaki S, Asahina A, Tokunaga K, Matsuki K, Kuwata S, et al. Study of HLA class I, class II and complement genes (C2, C4A, C4B and BF) in Japanese psoriatics and analysis of a newly-found high-risk haplotype by pulsed field gel electrophoresis. Arch Dermatol Res 1991;283:281–4. [8] Muto M, Date Y, Ichimiya M, Moriwaki Y, Mori K, Kamikawaji N, et al. Significance of antibodies to streptococcal M protein in psoriatic arthritis and their association with HLA-A*0207. Tissue Antigens 1996;48:645–50. [9] Łuszczek W, Majorczyk E, Nockowski P, Plucins´ki P, Jasek M, Nowak I, et al. Distribution of the CTLA-4 single nucleotide polymorphism CT60G > A and +49A > G in psoriasis vulgaris patients and control individuals from a Polish Caucasian population. Int J Immunogenet 2007;35:51–5. [10] Kouki T, Sawai Y, Gardine CA, Fisfalen M-E, Alegre M-L, DeGroot LJ. CTLA-4 gene polymorphism at position 49 in exon1 reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of Graves’ disease. J Immunol 2000;165:6606–11.
Masahiko Muto* Hirotaka Deguchi Akemi Tanaka Takayuki Inoue Makoto Ichimiya Department of Dermatology, Yamaguchi University Graduate School of Medicine, 1-1-1, Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan *Corresponding author. Tel.: +81 836 22 2269; fax: +81 836 22 2269 E-mail address: [email protected] (M. Muto). 15 March 2010 doi:10.1016/j.jdermsci.2010.10.012
70
Letter to the Editor Association between T-lymphocyte regulatory gene CTLA4 single nucleotide polymorphism at position 49 in exon 1 and HLA-DRB1*08 in Japanese patients with psoriasis vulgaris Psoriasis vulgaris (PV) is a multifactorial skin disease involving T lymphocyte-mediated inflammation. The major genetic determinant of psoriasis is PSORS1, which accounts for more than 30% of the heritability of PV. A recent genome-wide association study revealed that the human leukocyte antigen (HLA) complex, particularly HLA-Cw*0602, is the most plausible candidate gene among various ethnicities [1]. On the other hand, cytotoxic T-lymphocyte antigen 4 (CTLA4), which is located on chromosome 2q33, is a costimulatory molecule that is expressed on activated T lymphocytes and is important in the counter-regulation of CD28 T-cell activation via CD80/CD86 (B7-1/B7-2) on the antigen-presenting cells. Several polymorphisms of CTLA4 were found to be associated with some autoimmune diseases in humans [2]. However, there have been only a few studies on CTLA4 polymorphisms in psoriasis in Japanese [3], Korean [4], Polish [5] and Swedish [6] populations. Three known polymorphisms have been reported in CTLA4: the 318(rs5742909)C/T single nucleotide polymorphism (SNP) at position 318 (counting from the ATG start codon) in the promoter region; the 49(rs231775)A/G (A as the ancestral allele and G as the minor allele) SNP at position 49 in exon 1, resulting in an alanine for threonine amino acid substitution; and variations in the length of a dinucleotide (AT)n repeat in the 30 -untranslated region (30 -UTR). The above reports did not find any associations between either PV or psoriatic arthritis and the CTLA4 SNP sites. With respect to HLA, HLA-A2-B46-DR8 haplotype was found to be associated with psoriasis in a Japanese population [7]. We therefore investigated whether both HLA (HLA-C and -DRB1) and the 49A/G SNP in CTLA4 were associated with disease susceptibility in Japanese PV patients. Eighty-two unrelated Japanese psoriatics (male = 64, female = 18; age range, 16–81 years; mean age at onset of PV, 38.5 years) and 80 normal healthy controls (age range, 20–73 years; mean age, 36.2 years) were enrolled in the present study. The study was approved by the Ethics Committee for Genome Research of the Yamaguchi University School of Medicine. All patients and controls involved gave written informed consent for
genetic studies. Genomic DNA was isolated from peripheral venous blood using a QIAamp DNA Blood Maxi kit (QIAGEN, Hilden, Germany) and the 49 A/G SNP (rs231775) was typed using PCR, as described previously [5]. HLA class I and class II genotypes were determined using Micro SSPTM HLA typing trays (One Lambda Inc., Canoga Park, CA, USA). Statistical significance was determined by x2 test for differences in genotype and allele frequency. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using Woolf’s method. Differences between groups of data were analyzed by Student’s t-test. The frequencies of genotypes and alleles at the 49A/G SNP are shown in Table 1. The 49 A/G genotypes and alleles were similarly distributed in PV patients and controls. When we calculated the frequency of HLA-DRB1*08 in the presence of combined (G/G + G/A) genotypes at position 49 of CTLA4, the most significant difference between PV patients and controls was observed (38.1% in PV vs. 17.4% in controls; p < 0.01), indicating an additional requirement of CTLA4 and the HLA-DRB1*08 allele for psoriasis susceptibility. On the other hand, with respect to genotype A/A at position 49, a significant difference was not seen between the two groups. Patient demographic data in the PV-associated HLA-DRB1*08-positive group (n = 28) are as follows: mean age at onset SE = 33.9 17.3 years vs. 39.8 12.0 years in HLA-DRB1*08-negative psoriatics (n = 54) (not significant); male:female = 19:9; presence of arthropathy = 7/28 (25.0%) vs. 2/54 (3.7%) in HLA-DRB1*08-negative psoriatics (p < 0.01); and type of skin lesion, plaque type:guttate type:erythrodermic type = 25:2:1. With regard to streptococcal infection, the results based on our previously described ELISA system [8] showed that serum IgG antibody levels against recombinant M protein originating from Streptococcus pyogenes were significantly elevated in HLADRB1*08-positive psoriatics when compared with HLA-DRB1*08negative psoriatics (7,211 815 U/ml vs. 3,420 485 U/ml; p < 0.01). Based on these data, HLA-DRB1*08 is relevant to arthropathy and elevated immune response to streptococcal infection. With regard to HLA-Cw*0602 and the 49A/G SNP in CTLA4, no associations were seen in the present study (data not shown) (Table 2). Tsunemi et al. [3] concluded that there were no statistically significant differences in the 49 A/G SNP of CTLA4 exon1 in Japanese patients with PV, but there was a slightly increased genotype frequency for 49 G/G in PV patients as compared to controls (41.8% in PV vs. 32.7% in controls; not significant at the 5%
Table 1 Genotype and allele frequencies of 49A/G of CTLA4 in psoriasis. CTLA4
Genotypes
PV (n = 82)
Controls (n = 80)
p value
49A/G (rs231775)
G/G G/A A/A
35/82 (42.7%) 28/82 (34.1%) 19/82 (23.2%)
34/80(42.5%) 35/80 (43.8%) 11/80 (13.7%)
p > 0.08 p > 0.10 p > 0.10
CTLA4
Alleles
PV (2n = 164)
Controls (2n = 160)
p value
49A/G (rs231775)
G A
98/164 (59.8%) 66/164 (40.2%)
103/160 (64.4%) 57/160 (35.6%)
p > 0.40 p > 0.40
Table 2 Frequencies of HLA-DRB1*08a with either (G/G + G/A)or A/A CTLA4 genotype.
49A/G (rs231775)
G/G + G/A A/A
a b
PV (n = 82)
Controls (n = 80)
ORb (95% CI)
p value
(n = 63) 24/63 (38.1%) (n = 19) 4/19 (21.1%)
(n = 69) 12/69 (17.4%) (n = 11) 3/11 (27.3%)
2.92 (1.31 6.53)
p < 0.01
Frequency of HLA-DRB1*08 was 34.1% (=28/82) in PV, and 18.8% (=15/80) in controls, respectively (OR = 2.25 (95%CI = 1.09 4.63), p < 0.03). OR: Odds ratios.
p > 0.50
Letters to the Editor / Journal of Dermatological Science 62 (2011) 64–71
level). They also found that 49 A/G SNP showed strong linkage disequilibrium with the 318 C/T SNP (x2 = 26.96; p = 2.1 107). Similarly, the 49 G variant allele of CTLA4 exon1 does not appear to be associated with PV in Koreans. However, the frequency of the 49 G and CT60 (rs3087243) G haplotypes was significantly lower in Polish PV patients with middle-age onset [9]. Based on a functional analysis for the CTLA4 protein in Graves’ disease, in which more individuals have the combined (G/G + G/A) genotypes at position 49 of CTLA exon 1 when compared to controls (82.2% vs. 65.1% in controls), the G allele was found to exert inhibitory effects on CTLA4 in T cells [10]. Taken together with clinical evidence of an association between the HLA-A2-B46-DR8(DRB1*08) haplotype and disease susceptibility to PV in Japanese, the present observations suggest that G variants at position 49 of CTLA4 exon 1 and HLA-DRB1*08 may contribute to the development of PV through mutual action. In conclusion, to our knowledge, this is the first report showing an association between CTLA4 SNP (49 G allele) and HLA-DRB1*08 with psoriasis in Japanese PV patients, although the number of subjects was limited. Larger population-based studies are essential in order to confirm these findings. Acknowledgements This work was supported in part by a grant from the Ministry of Health, Labour and Welfare (Research on Measures for Intractable Diseases), Japan and by a Grant-in Aid for Scientific Research (22591223) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References [1] Nestle FO, Kaplan DH, Barker J. Psoriasis mechanisms of disease. N Engl J Med 2009;361:496–509. [2] Kristiansen OP, Larsen ZM, Pociot F. CTLA-4 in autoimmune diseases—a general susceptibility gene to autoimmunity? Genes Immun 2000;1:170–84. [3] Tsunemi Y, Saeki H, Kishimoto M, Mitsui H, Tada Y, Torii H, et al. Cytotoxic T lymphocyte antigen-4 gene (CTLA4) polymorphisms in Japanese patients with psoriasis vulgaris. J Dermatol Sci 2003;32:163–5.
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[4] Kim Y-K, Pyo C-W, Hur S-S, Kim T-Y, Kim T-G. No associations of CTLA-4 and ICAM-1 polymorphisms with psoriasis in the Korean population. J Dermatol Sci 2003;33:75–7. [5] Łuszczek W, Kubicka W, Jasek M, Barant E, Cisło M, Nockowski P, et al. CTLA-4 gene polymorphisms and natural soluble CTLA-4 protein in psoriasis vulgaris. Int J Immunogenet 2006;33:217–24. [6] Alenius G-M, Friberg C, Nilsson S, Wahlstro¨m J, Dahlqvist SR, Samuelsson L. Analysis of 6 genetic loci for disease susceptibility in psoriatic arthritis. J Rheumatol 2004;31:2230–5. [7] Nakagawa H, Akazaki S, Asahina A, Tokunaga K, Matsuki K, Kuwata S, et al. Study of HLA class I, class II and complement genes (C2, C4A, C4B and BF) in Japanese psoriatics and analysis of a newly-found high-risk haplotype by pulsed field gel electrophoresis. Arch Dermatol Res 1991;283:281–4. [8] Muto M, Date Y, Ichimiya M, Moriwaki Y, Mori K, Kamikawaji N, et al. Significance of antibodies to streptococcal M protein in psoriatic arthritis and their association with HLA-A*0207. Tissue Antigens 1996;48:645–50. [9] Łuszczek W, Majorczyk E, Nockowski P, Plucins´ki P, Jasek M, Nowak I, et al. Distribution of the CTLA-4 single nucleotide polymorphism CT60G > A and +49A > G in psoriasis vulgaris patients and control individuals from a Polish Caucasian population. Int J Immunogenet 2007;35:51–5. [10] Kouki T, Sawai Y, Gardine CA, Fisfalen M-E, Alegre M-L, DeGroot LJ. CTLA-4 gene polymorphism at position 49 in exon1 reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of Graves’ disease. J Immunol 2000;165:6606–11.
Masahiko Muto* Hirotaka Deguchi Akemi Tanaka Takayuki Inoue Makoto Ichimiya Department of Dermatology, Yamaguchi University Graduate School of Medicine, 1-1-1, Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan *Corresponding author. Tel.: +81 836 22 2269; fax: +81 836 22 2269 E-mail address: [email protected] (M. Muto). 15 March 2010 doi:10.1016/j.jdermsci.2010.10.012