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TGF-β1 gene polymorphism in psoriasis vulgaris
www.elsevier.com/locate/issn/10434666 Cytokine 38 (2007) 8–11
TGF-b1 gene polymorphism in psoriasis vulgaris Wojciech Baran
a,*
, Jacek C. Szepietowski
a,b
, Grzegorz Mazur c, Eugeniusz Baran
a
a
c
Department of Dermatology, Venereology and Allergology, Wroclaw Medical University, Chalubinskiego 1, 50-368 Wroclaw, Poland b Institute of Immunology and Experimental Therapy, Polish Academy of Science, Rudolfa Weigla 12, 53-114 Wroclaw, Poland Department of Haematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical University, Pasteura 4, 50-367 Wroclaw, Poland Received 8 March 2007; received in revised form 29 March 2007; accepted 11 April 2007
Abstract Overexpression of TGF-b1 has been implicated in the pathology of many inflammatory diseases, including psoriasis. This study was performed to investigate the association between TGF-b1 single nucleotide polymorphism and susceptibility for psoriasis vulgaris. DNA from 78 patients with psoriasis vulgaris and 74 healthy volunteers was investigated. Polymorphism of TGF-b1 gene in codon 10 (T/C) and codon 25 (G/C) was evaluated by PCR-SSP and the results were compared between group of psoriatic patients, divided into early onset of psoriasis (type I) and late onset of psoriasis (type II) subgroups, and control healthy subjects. Frequencies in genotypes were similar between patients and control group (p > 0.7), but between type I and type II psoriasis patients highly significant difference was found (p < 0.0003). Higher frequency of CC/GG (intermediate producer) and TC/GG (high producer) was noted in the type I group, but the second high producer genotype (TT/GG) was more common in type II group. Also between type II psoriasis patients and healthy controls statistically significant difference was found (p < 0.000001). In analyzing frequencies of carriage and alleles no significant differences were found. TGF-b1 gene polymorphism in codon 10 and 25 is not associated with susceptibility to psoriasis vulgaris, but may be important for the type of the disease. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: TGF-b; Single nucleotide polymorphism; Psoriasis
1. Introduction Psoriasis is a hyperproliferative cutaneous disease of multifactorial etiology including: genetic background, environmental factors, vascular and immune system disturbances. It affects about 3% of the population and is characterized by an abnormal pattern of keratinocyte growth, alteration of dermal blood vessels and inflammation of both dermis and epidermis. Disorders of cell proliferation and differentiation observed in psoriatic epidermis may be explained by the imbalance of specific growth factors [1–3]. Altered levels of transforming growth factors alfa and beta (TGF-a, TGF-b), epidermal growth factor (EGF) and interleukin-6 (IL-6) have been reported in psoriatic patients’ skin and serum. These alterations may be *
Corresponding author. Fax: +48713270942. E-mail address: [email protected] (W. Baran).
1043-4666/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2007.04.004
responsible for disturbances in the keratinocytes growth and differentiation in psoriasis and many researches concerning this phenomenon disclosed interesting results [4–8]. Transforming growth factor beta (TGF-b) is a multifunctional regulator of both cell growth and differentiation. TGF-b has five isoforms TGF-b1–5, however only TGF-b1, TGF-b2 and TGF-b3 have been found in mammals. TGFb1 is produced by many cells including activated inflammatory infiltrate cells and keratinocytes. TGF-b1 and TGF-b2 were found in the human epidermis whereas TGF-b3 is distributed in the dermis, mainly in the upper dermis [9–11]. TGF-b1 inhibits proliferation of keratinocytes, activates angiogenesis and stimulates fibroblasts to proliferation and production of extracellular matrix elements. Moreover, TGF-b1 may increase production of Th1 type cytokines what is very important in contemporary understanding of psoriasis as a type 1 immune response disease [12,13]. TGF-b1 also antagonizes the acanthotic
W. Baran et al. / Cytokine 38 (2007) 8–11
and degenerative effect of transforming growth factor-a which increases expression of epidermal growth factor receptors that may induce hyperplasia of psoriatic keratinocytes [14]. Experiments on animal model also showed very strong influence of TGF-b1 on proliferation of the epidermis—TGF-b1 knock-out mice showed hyperproliferation of the epidermis and transgenic mice with the overexpression of TGF-b1 presented inhibition of the skin development [15,16]. It has been proved that the ability of an individual to produce high or low amounts of TGF-b1 may be genetically predetermined and polymorphisms in the gene for TGF-b1 regulate its expression [17]. The human TGF-b1 gene is located on the long arm of chromosome 19. There are five described polymorphisms in this gene: two in the promoter region at positions 800 G/A and 509 C/T and three located in the coding sequence at positions 869 T/C, 915 G/C and 1628 C/A. The point mutation in position 869 at codon 10 involves alleles T (leucine) and C (proline) and the second one in position 915 at codon 25 G (arginine) and C (proline). The presence of leucine at codon 10 and arginine at codon 25 determines high producer genotype, while proline in both codons predisposes to low producer status [18]. There are some conflicting results concerning codon 10 polymorphism, Hutchinson et al. [19] proved relations described above, but other authors [20] reported opposite, with leucine, not proline associated with an increased production of TGF-b1. Despite this, there is no doubt that those polymorphisms at codon 10 and 25 influence the production of TGF-b1. This study was designed to identify whether polymorphism of TGF-b1 is a risk factor for the developing of psoriasis. To the best of our knowledge, no studies concerning TGF-b1 single nucleotide polymorphism in psoriasis have been published, but many papers proving importance of TGF-b1 in the pathogenesis of psoriasis are available. 2. Materials and methods 2.1. Patients and controls Seventy-eight patients with psoriasis vulgaris [37 females (47.43%) and 41 males (52.57%)] were included into the study. Two subsets of patients were established—early onset psoriasis (type I—onset not later than at the age of 40 years with positive family history of psoriasis) and type II psoriasis (onset after the age of 40 years with negative family history of the disease). Type I group included 28 females (51.85%) and 26 males (48.15%) with mean age of 44.12 ± 11.80 years (range 19–67 years). The type II group had a mean age of 61.37±11.21 years and consisted of 9 females (37.5%) and 15 males (62.5%). The healthy control group consisted of 74 unrelated subjects (33 females and 41 males) with no family history of psoriasis. Study was approved by the Commission of Bioethics at Wroclaw Medical University (KB 359/2003).
9
2.2. TGF-b1 genotyping DNA was isolated from the whole peripheral blood taken on EDTA with the use of Qiagen DNA Isolation Kit (Qiagen GmbH, Hilden, Germany). Biallelic polymorphism within the TGF-b1 gene in codon 10 (T/C) and codon 25 (G/C) was determined by PCR-SSP technique employing commercial primers (One Lambda, Inc., Canoga Park, CA, USA). The use of this kit (due to number of primer mix combination) allows assessing the presence of particular TGF-b1 genotypes (high, TT/GG, TC/GG; intermediate, TC/GC, CC/GG and low producers, CC/GC, CC/CC). For each polymorphic site one PCR was carried out on DNA template with a pair of specific primers, the additional control primers, reaction mix (provided by a manufacturer), and Taq polymerase (Invitrogen, USA) in a total volume of 10 ll. Amplifications were performed in MJ Research Apparatus (Watertown, MA, USA). PCR cycling conditions were as follows: 96 °C for 130 s, 63 °C for 60 s, followed by nine cycles of 96 °C for 10 s, 63 °C for 60 s, and followed by 20 cycles of 96 °C for 10 s, 59 °C for 50 s, 72 °C for 30 s, ending with 4 °C. PCR products were analyzed electrophoretically in 2% agarose gel in the presence of ethidium bromide. The DNA bands were visualized under UV light. 2.3. Evaluation and statistical analysis Genotype and allele frequencies were compared between the study groups by the v2 test with Yates correction or Fisher’s exact test when necessary. p values less than 0.05 were considered statistically significant. 3. Results In our study two polymorphic positions within the TGF-b codon 10 and 25 were analyzed (codon 10–869 T/C, codon 25–915 G/C). None of the following TGF-b1 codon 10 and 25 genotypes: TT/GC, TT/CC and TC/CC was detected in patients and controls, while the presence CC/CC was found only in two patients with psoriasis and one in the control group. In both groups dominant genotypes were TC/GG and TT/GG—high producers and CC/GG—intermediate producer. Frequencies in genotypes were similar between patients and control group (p > 0.7), but between type I and type II psoriasis patients highly significant difference was found (p < 0.0003) (Table 1). Higher frequency of CC/GG (intermediate producer) and TC/GG (high producer) was noted in the early onset group, but the second high producer genotype (TT/GG) was more common in late onset of psoriasis group. Also between type II psoriasis patients and healthy controls statistically significant difference was found (p < 0.000001). Analyzing frequencies of carriage and alleles no significant differences were found (Table 2).
10
W. Baran et al. / Cytokine 38 (2007) 8–11
Table 1 Distribution of TGF-b1 (codon 10 T/C and codon 25 G/C) genotypes among psoriasis patients and healthy controls Genotype
TGF-b1 genotypes
p
Psoriasis
CC/CC CC/GC CC/GG TC/GC TC/GG TT/GG
CC/CC CC/GC CC/GG TC/GC TC/GG TT/GG
Controls
n
f
n
f
1 7 11 5 33 21
0.013 0.090 0.141 0.064 0.423 0.269
2 5 11 6 28 22
0.027 0.068 0.149 0.081 0.378 0.297
Type I psoriasis
Type II psoriasis
1 5 10 2 25 11
0 2 1 3 8 10
0.019 0.093 0.185 0.037 0.463 0.204
>0.7
<0.0003
0 0.083 0.042 0.125 0.333 0.417
n, number; f, relative frequency; G, guanine; T, thymine; C, cytosine.
Table 2 Distribution of TGF-b1 (codon 10 T/C and codon 25 G/C) carriages and alleles among psoriasis patients, type I and type II subgroups and healthy controls Psoriasis
Controls
n
f
n
f
Carriage CC GC GG TC TT
19 12 65 38 22
0.122 0.077 0.417 0.244 0.141
18 11 61 34 22
0.123 0.075 0.418 0.233 0.151
Alleles CC GC GG TC TT
20 12 65 38 22
0.127 0.076 0.414 0.242 0.140
20 11 61 34 22
0.135 0.074 0.412 0.230 0.149
Type I psoriasis
Type II psoriasis
Carriage CC GC GG TC TT
16 7 46 27 22
0.136 0.059 0.390 0.229 0.186
3 5 19 11 22
0.050 0.083 0.317 0.183 0.367
Allele CC GC GG TC TT
17 7 46 27 22
0.143 0.059 0.387 0.226 0.185
3 5 19 11 22
0.050 0.083 0.317 0.183 0.367
4. Discussion The expression of many cytokines is thought to be influenced by polymorphism in their gene loci and this may contribute to the development of inflammatory diseases.
TGF-b1 polymorphisms are associated with asthma, Crohn’s disease, rheumatoid arthritis, systemic sclerosis, liver cirrhosis, renal dysfunction after heart transplantation, liver graft acceptance and many other disorders [18,21–25]. Single nucleotide polymorphism of the cytokine genes in psoriasis is the object of intensive researches and many of them disclosed interesting findings. In this study, we did not find significant differences in genotypes between patients and control group, but between type I and type II psoriasis patients highly significant difference was observed. Unfortunately, higher frequency of high and intermediate producer was noted in the early onset group (type I), but the second high producer genotype was more common in late onset of psoriasis group (type II), so it is difficult to asses genotype influence for the type of the disease. The second difficulty is that we could not compare these results with other studies because no other paper concerning TGF-b1 polymorphism in psoriasis vulgaris was found. In 1996 Bonifati et al. [7] found increased serum concentrations of TGF-b1 in psoriatic patients and strong correlation between TGF-b1 concentration and severity of the disease, similar results obtained Nockowski et al. [6] but contradictory data were published by Flisiak et al. [8]. Other authors checked for presence of TGF-b1 in psoriatic skin, but results were controversial, depending on antibodies used in immunohistochemical examination [9,26]. Leivo et al. [27] obtained very interesting results in immunohistochemical analysis of TGF-b receptors in the skin. Skin biopsies from healthy controls and non-lesional skin from psoriatic patients showed intense immunoreactivity for receptors in the epidermis, but lesional psoriatic skin lacked detectable immunoreactivity of them. This phenomenon suggests down-regulation of these structures in psoriasis. Recently, it has been documented that yeast— Malassezia furfur up-regulate TGF-b1 in the psoriatic skin [28]. In conclusion, TGF-b1 gene polymorphism in codon 10 and 25 is not associated with susceptibility to psoriasis vulgaris, but may be important for the type of disease. Further studies concerning TGF-b1 polymorphisms seem to be required to determine completely the molecular basis of the susceptibility to psoriasis in the context of increased levels of TGF-b1 itself, down-regulated receptors for TGF-b1 in the psoriatic lesions epidermis and To the best of our knowledge this study presents for the first time the difference in genotyped TGF-b1 polymorphisms between type I and type II psoriasis. References [1] Bos JD, De Rie MA. The pathogenesis of psoriasis: immunological facts and speculations. Immunol Today 1999;20:40–6. [2] Krueger G, Ellis CN. Psoriasis – recent advances in understanding its pathogenesis and treatment. J Am Acad Dermatol 2005;53:94–100. [3] Ozawa M, Aiba S. Immunopathogenesis of psoriasis. Curr Drug Targets Inflamm Allergy 2004;3:137–44.
W. Baran et al. / Cytokine 38 (2007) 8–11 [4] Boyce ST. Epidermis as a secretory tissue. J Invest Dermatol 1994;102:8–10. [5] Grossman RM, Krueger J, Yourish D, Granelli-Piperno A, Murphy DP, May LT, et al. Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes. Proc Natl Acad Sci USA 1989;86:6367–71. [6] Nockowski P, Szepietowski JC, Ziarkiewicz M, Baran E. Serum concentrations of transforming growth factor beta 1 in patients with psoriasis vulgaris. Acta Dermatovenerol Croat 2004;12:2–6. [7] Bonifati C, Carducci M, Mussi A, Pitarello A, D’Agosto G, Fazio M. The levels of transforming growth factor-b1 are increased in the serum of patients with psoriasis and correlate with disease severity. Eur J Dermatol 1996;6:486–90. [8] Flisiak I, Chodynicka B, Porebski P, Flisiak R. Association between psoriasis severity and transforming growth factor beta (1) and beta (2) in plasma and scales from psoriatic lesions. Cytokine 2002;19:121–5. [9] Kane CJ, Knapp AM, Mansbridge JN, Hanawalt PC. Transforming growth factor-beta 1 localization in normal and psoriatic epidermal keratinocytes in situ. J Cell Physiol 1990;144:144–50. [10] Wang XJ, Greenhalgh DA, Bickenbach JR, Jiang A, Bundman DS, Krieg T, et al. Expression of a dominant-negative type II transforming growth factor beta (TGF-beta) receptor in the epidermis of transgenic mice blocks TGF-beta-mediated growth inhibition. Proc Natl Acad Sci USA 1997;94:2386–91. [11] Wataya-Kaneda M, Hashimoto K, Kato M, Miyazono K, Yoshikawa K. Differential localization of TGF-beta-precursor isotypes in normal human skin. J Dermatol Sci 1994;8:38–44. [12] Massague J. The transforming growth factor-b family. Annu Rev Cell Biol 1990;6:597–641. [13] Lawrence DA. Transforming growth factor-beta: a general review. Eur Cytokine Netw 1996;7:363–74. [14] Kondo S, Hozumi Y, Maejima H, Aso K. Organ culture of psoriatic skin: effect of TGF-alpha and TGF-beta on epidermal structure in vitro. Arch Dermatol Res 1992;284:150–3. [15] Glick AB, Kulkarni AB, Tennenbaum T, Hennings H, Flanders KC, O’Reilly M, et al. Loss of expression of transforming growth factor beta in skin and skin tumors is associated with hyperproliferation and a high risk for malignant conversion. Proc Natl Acad Sci USA 1993;90:6076–80. [16] Sellheyer K, Bickenbach JR, Rothnagel JA, Bundman D, Longley MA, Krieg T, et al. Inhibition of skin development by overexpres-
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[28]
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sion of transforming growth factor beta 1 in the epidermis of transgenic mice. Proc Natl Acad Sci USA 1993;90:5237–41. Grainger DJ, Heathcote K, Chiano M, Snieder H, Kemp PR, Metcalfe JC, et al. Genetic control of the circulating concentration of transforming growth factor type beta1. Hum Mol Genet 1999;8:93–7. Kim SY, Han SW, Kim GW, Lee JM, Kang YM. TGF-beta1 polymorphism determines the progression of joint damage in rheumatoid arthritis. Scand J Rheumatol 2004;33:389–94. Hutchinson IV, Turner D, Sankaran D, Awad M, Pravica V, Sinnott P. Cytokine genotypes in allograft rejection: guidelines for immunosuppression. Transplant Proc 1998;30:3991–2. Suthanthiran M, Li B, Song JO, Ding R, Sharma VK, Schwartz JE, et al. Transforming growth factor-beta 1 hyperexpression in African–American hypertensives: a novel mediator of hypertension and/or target organ damage. Proc Natl Acad Sci USA 2000;97:3479–84. Baan CC, Balk AH, Holweg CT, van Riemsdijk IC, Maat LP, Vantrimpont PJ, et al. Renal failure after clinical heart transplantation is associated with the TGF-beta 1 codon 10 gene polymorphism. J Heart Lung Transplant 2000;19:866–72. Gomez-Mateo J, Marin L, Lopez-Alvarez MR, Moya-Quiles MR, Miras M, Marin-Moreno I, et al. TGF-beta1 gene polymorphism in liver graft recipients. Transpl Immunol 2006;17:55–7. Osterreicher CH, Datz C, Stickel F, Hellerbrand C, Penz M, Hofer H, et al. TGF-beta1 codon 25 gene polymorphism is associated with cirrhosis in patients with hereditary hemochromatosis. Cytokine 2005;31:142–8. Schulte CM, Goebell H, Roher HD, Schulte KM. C-509T polymorphism in the TGFB1 gene promoter: impact on Crohn’s disease susceptibility and clinical course?. Immunogenetics 2001;53:178–82. Pulleyn LJ, Newton R, Adcock IM, Barnes PJ. TGFbeta1 allele association with asthma severity. Hum Genet 2001;109:623–7. Wataya-Kaneda M, Hashimoto K, Kato M, Miyazono K, Yoshikawa K. Differential localization of TGF-beta-precursor isotypes in psoriatic human skin. J Dermatol Sci 1996;11:183–8. Leivo T, Leivo I, Kariniemi AL, Keski-Oja J, Virtanen I. Down-regulation of transforming growth factor-beta receptors I and II is seen in lesional but not non-lesional psoriatic epidermis. Br J Dermatol 1998;138:57–62. Baroni A, Paoletti I, Ruocco E, Agozzino M, Tufano MA, Donnarumma G. Possible role of Malassezia furfur in psoriasis: modulation of TGF-beta1, integrin, and HSP70 expression in human keratinocytes and in the skin of psoriasis-affected patients. J Cutan Pathol 2004;31:35–42.
TGF-b1 gene polymorphism in psoriasis vulgaris Wojciech Baran
a,*
, Jacek C. Szepietowski
a,b
, Grzegorz Mazur c, Eugeniusz Baran
a
a
c
Department of Dermatology, Venereology and Allergology, Wroclaw Medical University, Chalubinskiego 1, 50-368 Wroclaw, Poland b Institute of Immunology and Experimental Therapy, Polish Academy of Science, Rudolfa Weigla 12, 53-114 Wroclaw, Poland Department of Haematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical University, Pasteura 4, 50-367 Wroclaw, Poland Received 8 March 2007; received in revised form 29 March 2007; accepted 11 April 2007
Abstract Overexpression of TGF-b1 has been implicated in the pathology of many inflammatory diseases, including psoriasis. This study was performed to investigate the association between TGF-b1 single nucleotide polymorphism and susceptibility for psoriasis vulgaris. DNA from 78 patients with psoriasis vulgaris and 74 healthy volunteers was investigated. Polymorphism of TGF-b1 gene in codon 10 (T/C) and codon 25 (G/C) was evaluated by PCR-SSP and the results were compared between group of psoriatic patients, divided into early onset of psoriasis (type I) and late onset of psoriasis (type II) subgroups, and control healthy subjects. Frequencies in genotypes were similar between patients and control group (p > 0.7), but between type I and type II psoriasis patients highly significant difference was found (p < 0.0003). Higher frequency of CC/GG (intermediate producer) and TC/GG (high producer) was noted in the type I group, but the second high producer genotype (TT/GG) was more common in type II group. Also between type II psoriasis patients and healthy controls statistically significant difference was found (p < 0.000001). In analyzing frequencies of carriage and alleles no significant differences were found. TGF-b1 gene polymorphism in codon 10 and 25 is not associated with susceptibility to psoriasis vulgaris, but may be important for the type of the disease. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: TGF-b; Single nucleotide polymorphism; Psoriasis
1. Introduction Psoriasis is a hyperproliferative cutaneous disease of multifactorial etiology including: genetic background, environmental factors, vascular and immune system disturbances. It affects about 3% of the population and is characterized by an abnormal pattern of keratinocyte growth, alteration of dermal blood vessels and inflammation of both dermis and epidermis. Disorders of cell proliferation and differentiation observed in psoriatic epidermis may be explained by the imbalance of specific growth factors [1–3]. Altered levels of transforming growth factors alfa and beta (TGF-a, TGF-b), epidermal growth factor (EGF) and interleukin-6 (IL-6) have been reported in psoriatic patients’ skin and serum. These alterations may be *
Corresponding author. Fax: +48713270942. E-mail address: [email protected] (W. Baran).
1043-4666/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2007.04.004
responsible for disturbances in the keratinocytes growth and differentiation in psoriasis and many researches concerning this phenomenon disclosed interesting results [4–8]. Transforming growth factor beta (TGF-b) is a multifunctional regulator of both cell growth and differentiation. TGF-b has five isoforms TGF-b1–5, however only TGF-b1, TGF-b2 and TGF-b3 have been found in mammals. TGFb1 is produced by many cells including activated inflammatory infiltrate cells and keratinocytes. TGF-b1 and TGF-b2 were found in the human epidermis whereas TGF-b3 is distributed in the dermis, mainly in the upper dermis [9–11]. TGF-b1 inhibits proliferation of keratinocytes, activates angiogenesis and stimulates fibroblasts to proliferation and production of extracellular matrix elements. Moreover, TGF-b1 may increase production of Th1 type cytokines what is very important in contemporary understanding of psoriasis as a type 1 immune response disease [12,13]. TGF-b1 also antagonizes the acanthotic
W. Baran et al. / Cytokine 38 (2007) 8–11
and degenerative effect of transforming growth factor-a which increases expression of epidermal growth factor receptors that may induce hyperplasia of psoriatic keratinocytes [14]. Experiments on animal model also showed very strong influence of TGF-b1 on proliferation of the epidermis—TGF-b1 knock-out mice showed hyperproliferation of the epidermis and transgenic mice with the overexpression of TGF-b1 presented inhibition of the skin development [15,16]. It has been proved that the ability of an individual to produce high or low amounts of TGF-b1 may be genetically predetermined and polymorphisms in the gene for TGF-b1 regulate its expression [17]. The human TGF-b1 gene is located on the long arm of chromosome 19. There are five described polymorphisms in this gene: two in the promoter region at positions 800 G/A and 509 C/T and three located in the coding sequence at positions 869 T/C, 915 G/C and 1628 C/A. The point mutation in position 869 at codon 10 involves alleles T (leucine) and C (proline) and the second one in position 915 at codon 25 G (arginine) and C (proline). The presence of leucine at codon 10 and arginine at codon 25 determines high producer genotype, while proline in both codons predisposes to low producer status [18]. There are some conflicting results concerning codon 10 polymorphism, Hutchinson et al. [19] proved relations described above, but other authors [20] reported opposite, with leucine, not proline associated with an increased production of TGF-b1. Despite this, there is no doubt that those polymorphisms at codon 10 and 25 influence the production of TGF-b1. This study was designed to identify whether polymorphism of TGF-b1 is a risk factor for the developing of psoriasis. To the best of our knowledge, no studies concerning TGF-b1 single nucleotide polymorphism in psoriasis have been published, but many papers proving importance of TGF-b1 in the pathogenesis of psoriasis are available. 2. Materials and methods 2.1. Patients and controls Seventy-eight patients with psoriasis vulgaris [37 females (47.43%) and 41 males (52.57%)] were included into the study. Two subsets of patients were established—early onset psoriasis (type I—onset not later than at the age of 40 years with positive family history of psoriasis) and type II psoriasis (onset after the age of 40 years with negative family history of the disease). Type I group included 28 females (51.85%) and 26 males (48.15%) with mean age of 44.12 ± 11.80 years (range 19–67 years). The type II group had a mean age of 61.37±11.21 years and consisted of 9 females (37.5%) and 15 males (62.5%). The healthy control group consisted of 74 unrelated subjects (33 females and 41 males) with no family history of psoriasis. Study was approved by the Commission of Bioethics at Wroclaw Medical University (KB 359/2003).
9
2.2. TGF-b1 genotyping DNA was isolated from the whole peripheral blood taken on EDTA with the use of Qiagen DNA Isolation Kit (Qiagen GmbH, Hilden, Germany). Biallelic polymorphism within the TGF-b1 gene in codon 10 (T/C) and codon 25 (G/C) was determined by PCR-SSP technique employing commercial primers (One Lambda, Inc., Canoga Park, CA, USA). The use of this kit (due to number of primer mix combination) allows assessing the presence of particular TGF-b1 genotypes (high, TT/GG, TC/GG; intermediate, TC/GC, CC/GG and low producers, CC/GC, CC/CC). For each polymorphic site one PCR was carried out on DNA template with a pair of specific primers, the additional control primers, reaction mix (provided by a manufacturer), and Taq polymerase (Invitrogen, USA) in a total volume of 10 ll. Amplifications were performed in MJ Research Apparatus (Watertown, MA, USA). PCR cycling conditions were as follows: 96 °C for 130 s, 63 °C for 60 s, followed by nine cycles of 96 °C for 10 s, 63 °C for 60 s, and followed by 20 cycles of 96 °C for 10 s, 59 °C for 50 s, 72 °C for 30 s, ending with 4 °C. PCR products were analyzed electrophoretically in 2% agarose gel in the presence of ethidium bromide. The DNA bands were visualized under UV light. 2.3. Evaluation and statistical analysis Genotype and allele frequencies were compared between the study groups by the v2 test with Yates correction or Fisher’s exact test when necessary. p values less than 0.05 were considered statistically significant. 3. Results In our study two polymorphic positions within the TGF-b codon 10 and 25 were analyzed (codon 10–869 T/C, codon 25–915 G/C). None of the following TGF-b1 codon 10 and 25 genotypes: TT/GC, TT/CC and TC/CC was detected in patients and controls, while the presence CC/CC was found only in two patients with psoriasis and one in the control group. In both groups dominant genotypes were TC/GG and TT/GG—high producers and CC/GG—intermediate producer. Frequencies in genotypes were similar between patients and control group (p > 0.7), but between type I and type II psoriasis patients highly significant difference was found (p < 0.0003) (Table 1). Higher frequency of CC/GG (intermediate producer) and TC/GG (high producer) was noted in the early onset group, but the second high producer genotype (TT/GG) was more common in late onset of psoriasis group. Also between type II psoriasis patients and healthy controls statistically significant difference was found (p < 0.000001). Analyzing frequencies of carriage and alleles no significant differences were found (Table 2).
10
W. Baran et al. / Cytokine 38 (2007) 8–11
Table 1 Distribution of TGF-b1 (codon 10 T/C and codon 25 G/C) genotypes among psoriasis patients and healthy controls Genotype
TGF-b1 genotypes
p
Psoriasis
CC/CC CC/GC CC/GG TC/GC TC/GG TT/GG
CC/CC CC/GC CC/GG TC/GC TC/GG TT/GG
Controls
n
f
n
f
1 7 11 5 33 21
0.013 0.090 0.141 0.064 0.423 0.269
2 5 11 6 28 22
0.027 0.068 0.149 0.081 0.378 0.297
Type I psoriasis
Type II psoriasis
1 5 10 2 25 11
0 2 1 3 8 10
0.019 0.093 0.185 0.037 0.463 0.204
>0.7
<0.0003
0 0.083 0.042 0.125 0.333 0.417
n, number; f, relative frequency; G, guanine; T, thymine; C, cytosine.
Table 2 Distribution of TGF-b1 (codon 10 T/C and codon 25 G/C) carriages and alleles among psoriasis patients, type I and type II subgroups and healthy controls Psoriasis
Controls
n
f
n
f
Carriage CC GC GG TC TT
19 12 65 38 22
0.122 0.077 0.417 0.244 0.141
18 11 61 34 22
0.123 0.075 0.418 0.233 0.151
Alleles CC GC GG TC TT
20 12 65 38 22
0.127 0.076 0.414 0.242 0.140
20 11 61 34 22
0.135 0.074 0.412 0.230 0.149
Type I psoriasis
Type II psoriasis
Carriage CC GC GG TC TT
16 7 46 27 22
0.136 0.059 0.390 0.229 0.186
3 5 19 11 22
0.050 0.083 0.317 0.183 0.367
Allele CC GC GG TC TT
17 7 46 27 22
0.143 0.059 0.387 0.226 0.185
3 5 19 11 22
0.050 0.083 0.317 0.183 0.367
4. Discussion The expression of many cytokines is thought to be influenced by polymorphism in their gene loci and this may contribute to the development of inflammatory diseases.
TGF-b1 polymorphisms are associated with asthma, Crohn’s disease, rheumatoid arthritis, systemic sclerosis, liver cirrhosis, renal dysfunction after heart transplantation, liver graft acceptance and many other disorders [18,21–25]. Single nucleotide polymorphism of the cytokine genes in psoriasis is the object of intensive researches and many of them disclosed interesting findings. In this study, we did not find significant differences in genotypes between patients and control group, but between type I and type II psoriasis patients highly significant difference was observed. Unfortunately, higher frequency of high and intermediate producer was noted in the early onset group (type I), but the second high producer genotype was more common in late onset of psoriasis group (type II), so it is difficult to asses genotype influence for the type of the disease. The second difficulty is that we could not compare these results with other studies because no other paper concerning TGF-b1 polymorphism in psoriasis vulgaris was found. In 1996 Bonifati et al. [7] found increased serum concentrations of TGF-b1 in psoriatic patients and strong correlation between TGF-b1 concentration and severity of the disease, similar results obtained Nockowski et al. [6] but contradictory data were published by Flisiak et al. [8]. Other authors checked for presence of TGF-b1 in psoriatic skin, but results were controversial, depending on antibodies used in immunohistochemical examination [9,26]. Leivo et al. [27] obtained very interesting results in immunohistochemical analysis of TGF-b receptors in the skin. Skin biopsies from healthy controls and non-lesional skin from psoriatic patients showed intense immunoreactivity for receptors in the epidermis, but lesional psoriatic skin lacked detectable immunoreactivity of them. This phenomenon suggests down-regulation of these structures in psoriasis. Recently, it has been documented that yeast— Malassezia furfur up-regulate TGF-b1 in the psoriatic skin [28]. In conclusion, TGF-b1 gene polymorphism in codon 10 and 25 is not associated with susceptibility to psoriasis vulgaris, but may be important for the type of disease. Further studies concerning TGF-b1 polymorphisms seem to be required to determine completely the molecular basis of the susceptibility to psoriasis in the context of increased levels of TGF-b1 itself, down-regulated receptors for TGF-b1 in the psoriatic lesions epidermis and To the best of our knowledge this study presents for the first time the difference in genotyped TGF-b1 polymorphisms between type I and type II psoriasis. References [1] Bos JD, De Rie MA. The pathogenesis of psoriasis: immunological facts and speculations. Immunol Today 1999;20:40–6. [2] Krueger G, Ellis CN. Psoriasis – recent advances in understanding its pathogenesis and treatment. J Am Acad Dermatol 2005;53:94–100. [3] Ozawa M, Aiba S. Immunopathogenesis of psoriasis. Curr Drug Targets Inflamm Allergy 2004;3:137–44.
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