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Mannose-binding lectin polymorphisms in patients with Behçet's disease
Journal of Dermatological Science (2004) 36, 115—117
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR Mannose-binding lectin polymorphisms in patients with Behc¸et’s disease To the Editor, Behc¸et’s disease (BD) is a chronic inflammatory disorder with recurrent oral and genital ulcers, uveitis, and vasculitis, along with mucocutaneous, arthritic, and neurologic manifestations. The main microscopic finding at most sites of active BD is immune-mediated occlusive vasculitis. HLA-B51 is the most strongly associated genetic marker in BD patients. BD is probably not a simple hereditary disease, and its onset may be triggered by bacterial, viral, or other microorganism antigens [1]. Mannose-binding lectin (MBL) is a C-type serum lectin, secreted by the liver. It binds to mannose and N-acetyl-glucosamine oligosaccharides on the surfaces of yeast, bacteria and viruses and serves as the initiator of the third pathway of the complement system independent of antibodies [2]. Three polymorphic sites have been identified in exon 1 of the MBL gene, at codon 52 (CGT to TGT), codon 54 (GGC to GAC) and codon 57 (GGA to GAA) [2]. It has been reported that serum MBL levels and MBL activation are regulated by MBL gene polymorphisms. A low serum concentration of MBL in humans is the basis for a common opsonic defect and is associated with recurrent infections in adults and children, and possibly immune diseases [2]. Recent studies showed that MBL gene mutations are additive risk factors for susceptibility to systemic lupus erythematosus, Sjogren’s syndrome and rheumatic diseases [3,4]. Our hypothesis is that MBL genetic polymorphisms that are involved in immune responses to infections may influence the onset of BD. Therefore, we investigated MBL gene polymorphisms to examine the relationship between MBL gene mutations and microbiological factors in BD. Thirty Japanese patients with primary BD were randomly selected from patients followed up at
Fukushima Medical University Hospital and Yokohama City University Hospital. Thirty patients (16 men and 14 women), who fulfilled the Japanese diagnostic criteria for Behc¸et’s disease based on interviews and clinical findings using a controlled protocol, were included. Thirty healthy control subjects (21 males and 9 females) were also included [5]. The ages of the BD patients ranged from 30 to 70 years (mean, 36.5 10.8 years), while those of the control subjects ranged from 22 to 68 years (mean, 35.5 11.6 years). According to Shimizu’s classification, the clinical appearance of BD was classified into two types, incomplete type and complete type [5]. There were 10 BD patients (33.3%) with the complete type, and 20 patients (66.7%) with the incomplete type. Sixty percent of the BD patients (18/30) were HLA-B51 positive. DNA was extracted from peripheral blood mononuclear cells by a standard procedure. Codon 54 genotypes of MBL exon 1 were identified by PCR using the primers as described previously [6]. The sequences of the primers for MBL exon 1 were: 50 AGTCGACCCAGATTGTAGGACAGAG-30 (forward primer) and 50 -AGGATCCAGGCAGTTTCCTCTGGAAGG30 (reverse primer). Sample DNA was amplified in the presence of 1.5 mM MgCl2, 0.2 mM each dNTP, and 2.5 U/ml Ampli Taq DNA polymerase (TaKara, Tokyo, Japan). Polymerase chain reaction (PCR) conditions, using an automated PCR thermal cycler (Takara PCR Thermal Cycler), were an initial denaturation at 94 8C for 2 min, 35 cycles of denaturation at 94 8C for 1 min, annealing at 58 8C for 1 min, extension at 72 8C for 2 min and a final elongation at 72 8C for 4 min. The PCR products were digested at 50 8C for 2 h with the restriction enzyme BanI (5 U; Promega Corporation, USA), which permits identification of the mutation because of its unique cleavage site [6]. The genotypes were determined by electrophoresis on 2% agarose gels followed by staining with ethidium bromide. The wild-type (A) fragment is cleaved into two bands (260 bp and
0923-1811/$30.00 # 2004 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2004.07.011
116
Letter to the Editor
Fig. 1 PCR-restriction fragment length polymorphism analysis for codon 54 allele of MBL. The genotype is as follows: A/A, wild/wild; A/B, wild/mutant. M: marker.
89 bp), while the mutant type (B) shows one band (349 bp) and A/B shows three bands (349 bp, 260 bp and 89 bp). The three patterns of MBL codon 54 are shown in Fig. 1: A/A (wild/wild), 54A/B (wild/ mutant), and 54B/B (mutant/mutant). Table 1 shows the genotypes and allele frequencies of MBL codon 54 in the BD patients and controls. The distribution of genotypes was significantly different in the patients and controls (x2 = 4.800, p < 0.05). The frequency of 54A/B was significantly higher in BD patients than in controls (0.467 versus 0.200). Nine BD patients of 14 BD cases with MBL A/B mutant were HLA-B51 positive and one control of six controls with MBL A/B mutant was HLA-B51 positive. Statistical analysis of these results revealed that there was no association between MBL mutant A/B and HLA-B51 (OR = 9, Fisher’s exact P = 0.1409). MBL mutant A/B was found in five cases (5/10) of the complete type and nine (9/20) of the incomplete type of BD. Statistical analysis of these results revealed that there was no association between MBL mutant A/B and BD clinical type (Fisher’s exact P = 1.000). MBL binds to clinical isolates and other sources of various species of bacteria. It has been reported that Staphylococcus aureus and b-haemolytic group A streptococci have strong MBL-binding capacity [8] and that BD patients have a high incidence of chronic streptococcal infections such as tonsillitis and dental caries, and hyperreactivity to streptococcal antigen might be related to these foci of chronic infection [7]. Recent studies showed that there are low serum levels of MBL in BD patients [9]. Thus, we suggest that MBL gene mutation at codon 54 may lead to susceptibility to bacterial infections such as streptococcal infection and affect the innate immune system in BD. In addition, lectins and Table 1 Genotype frequencies of MBL codon 54 in patients with BD and healthy controls Genotype
Patients (n = 30)
Controls (n = 30)
A/A A/B B/B
16 (0.533) 14 (0.467) 0
24 (0.800) 6 (0.200) 0
x2 = 4.8000, P = 0.028.
inflammation are related to coagulation activation [10]. There may be an interaction between increased coagulation and infection on the basis of distinct MBL polymorphisms in BD. However, because of the limited number of cases, further examination is needed to confirm our findings and the specificity of the function of MBL in BD.
References [1] Kaneko S, Suzuki N, Yamashita N, Nagafuchi H, Nakajima T, Wakisaka S, et al. Characterization of T cells specific for an epitope of human 60-kDa heat shock protein (hsp) in patients with Behc¸et’s disease (BD) in Japan. Clin Exp Immunol 1997;108:204—12. [2] Kilpatrick DC. Mannan-binding lectin: clinical significance and applications. Biochim Biophys Acta 2002;1572:401—13. [3] Garred P, Madsen HO, Halberg P, Petersen J, Kronborg G, Svejgaard A, et al. Mannose-binding lectin polymorphisms and susceptibility to infection in systemic lupus erythematosus. Arthritis Rheum 1999;42:2145—52. [4] Stanworth SJ, Donn RP, Hassall A, Dawes P, Ollier W, Snowden N. Absence of an association between mannose-binding lectin polymorphism and rheumatoid arthritis. Br J Rheumatol 1998;37:186—8. [5] Sungnack L. In: Sungnack L, Dongsik B, Eun-So L, Seonghang S, editors. Diagnostic criteria of Behcet Disease: problems and suggestions. Berlin: Springer; 2001. p. 115—8. [6] Madsen HO, Garred P, Thiel S, Kurtzhals JA, Lamm LU, Ryder LP, et al. Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein. J Immunol 1995;155:3013—20. [7] Kaneko F, Takahashi Y, Muramatsu Y, Miura Y. Immunological studies on aphthous ulcer and erythema nodosum-like eruptions in Behcet’s disease. Br J Dermatol 1985;113:303—12. [8] Neth O, Jack DL, Dodds AW, Holzel H, Klein NJ, Turner MW. Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition. Infect Immun 2000;68:688—93. [9] Inanc N, Birtas E, Yavuz S, Ergun T, Direskeneli H. Low serum mannose-binding lectin levels in Behcet’s disease. Adv Exp Med Biol 2003;528:287—9. [10] Kiraz S, Ertenli I, Ozturk MA, Haznedaroglu IC, Celik I, Calguneri M. Pathological haemostasis and prothrombotic state in Behcet’s disease. Thromb Res 2002;105:125—33.
Hongwei Wang, Koichiro Nakamura* Tomoko Inoue, Hirokatsu Yanagihori Yoshio Kawakami, Shinichi Hashimoto Noritaka Oyama, Fumio Kaneko Department of Dermatology School of Medicine Fukushima Medical University, Hikarigaoka 1 Fukushima, Fukushima 960-1295, Japan Teizo Fujita Department of Biochemistry, School of Medicine Fukushima Medical University, Hikarigaoka 1 Fukushima, Fukushima 960-1295 Japan
Letter to the Editor
Tomomi Nishida, Nobuhisa Mizuki Department of Ophthalmology School of Medicine Yokohama City University, Fukuura 3-9 Kanazawaku, Yokohama, Kanagawa 236-0004 Japan
117 *
Corresponding author. Tel.: +81 24 548 2111 fax: +81 24 548 5412 E-mail address: [email protected] (K. Nakamura) 6 May 2004
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR Mannose-binding lectin polymorphisms in patients with Behc¸et’s disease To the Editor, Behc¸et’s disease (BD) is a chronic inflammatory disorder with recurrent oral and genital ulcers, uveitis, and vasculitis, along with mucocutaneous, arthritic, and neurologic manifestations. The main microscopic finding at most sites of active BD is immune-mediated occlusive vasculitis. HLA-B51 is the most strongly associated genetic marker in BD patients. BD is probably not a simple hereditary disease, and its onset may be triggered by bacterial, viral, or other microorganism antigens [1]. Mannose-binding lectin (MBL) is a C-type serum lectin, secreted by the liver. It binds to mannose and N-acetyl-glucosamine oligosaccharides on the surfaces of yeast, bacteria and viruses and serves as the initiator of the third pathway of the complement system independent of antibodies [2]. Three polymorphic sites have been identified in exon 1 of the MBL gene, at codon 52 (CGT to TGT), codon 54 (GGC to GAC) and codon 57 (GGA to GAA) [2]. It has been reported that serum MBL levels and MBL activation are regulated by MBL gene polymorphisms. A low serum concentration of MBL in humans is the basis for a common opsonic defect and is associated with recurrent infections in adults and children, and possibly immune diseases [2]. Recent studies showed that MBL gene mutations are additive risk factors for susceptibility to systemic lupus erythematosus, Sjogren’s syndrome and rheumatic diseases [3,4]. Our hypothesis is that MBL genetic polymorphisms that are involved in immune responses to infections may influence the onset of BD. Therefore, we investigated MBL gene polymorphisms to examine the relationship between MBL gene mutations and microbiological factors in BD. Thirty Japanese patients with primary BD were randomly selected from patients followed up at
Fukushima Medical University Hospital and Yokohama City University Hospital. Thirty patients (16 men and 14 women), who fulfilled the Japanese diagnostic criteria for Behc¸et’s disease based on interviews and clinical findings using a controlled protocol, were included. Thirty healthy control subjects (21 males and 9 females) were also included [5]. The ages of the BD patients ranged from 30 to 70 years (mean, 36.5 10.8 years), while those of the control subjects ranged from 22 to 68 years (mean, 35.5 11.6 years). According to Shimizu’s classification, the clinical appearance of BD was classified into two types, incomplete type and complete type [5]. There were 10 BD patients (33.3%) with the complete type, and 20 patients (66.7%) with the incomplete type. Sixty percent of the BD patients (18/30) were HLA-B51 positive. DNA was extracted from peripheral blood mononuclear cells by a standard procedure. Codon 54 genotypes of MBL exon 1 were identified by PCR using the primers as described previously [6]. The sequences of the primers for MBL exon 1 were: 50 AGTCGACCCAGATTGTAGGACAGAG-30 (forward primer) and 50 -AGGATCCAGGCAGTTTCCTCTGGAAGG30 (reverse primer). Sample DNA was amplified in the presence of 1.5 mM MgCl2, 0.2 mM each dNTP, and 2.5 U/ml Ampli Taq DNA polymerase (TaKara, Tokyo, Japan). Polymerase chain reaction (PCR) conditions, using an automated PCR thermal cycler (Takara PCR Thermal Cycler), were an initial denaturation at 94 8C for 2 min, 35 cycles of denaturation at 94 8C for 1 min, annealing at 58 8C for 1 min, extension at 72 8C for 2 min and a final elongation at 72 8C for 4 min. The PCR products were digested at 50 8C for 2 h with the restriction enzyme BanI (5 U; Promega Corporation, USA), which permits identification of the mutation because of its unique cleavage site [6]. The genotypes were determined by electrophoresis on 2% agarose gels followed by staining with ethidium bromide. The wild-type (A) fragment is cleaved into two bands (260 bp and
0923-1811/$30.00 # 2004 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2004.07.011
116
Letter to the Editor
Fig. 1 PCR-restriction fragment length polymorphism analysis for codon 54 allele of MBL. The genotype is as follows: A/A, wild/wild; A/B, wild/mutant. M: marker.
89 bp), while the mutant type (B) shows one band (349 bp) and A/B shows three bands (349 bp, 260 bp and 89 bp). The three patterns of MBL codon 54 are shown in Fig. 1: A/A (wild/wild), 54A/B (wild/ mutant), and 54B/B (mutant/mutant). Table 1 shows the genotypes and allele frequencies of MBL codon 54 in the BD patients and controls. The distribution of genotypes was significantly different in the patients and controls (x2 = 4.800, p < 0.05). The frequency of 54A/B was significantly higher in BD patients than in controls (0.467 versus 0.200). Nine BD patients of 14 BD cases with MBL A/B mutant were HLA-B51 positive and one control of six controls with MBL A/B mutant was HLA-B51 positive. Statistical analysis of these results revealed that there was no association between MBL mutant A/B and HLA-B51 (OR = 9, Fisher’s exact P = 0.1409). MBL mutant A/B was found in five cases (5/10) of the complete type and nine (9/20) of the incomplete type of BD. Statistical analysis of these results revealed that there was no association between MBL mutant A/B and BD clinical type (Fisher’s exact P = 1.000). MBL binds to clinical isolates and other sources of various species of bacteria. It has been reported that Staphylococcus aureus and b-haemolytic group A streptococci have strong MBL-binding capacity [8] and that BD patients have a high incidence of chronic streptococcal infections such as tonsillitis and dental caries, and hyperreactivity to streptococcal antigen might be related to these foci of chronic infection [7]. Recent studies showed that there are low serum levels of MBL in BD patients [9]. Thus, we suggest that MBL gene mutation at codon 54 may lead to susceptibility to bacterial infections such as streptococcal infection and affect the innate immune system in BD. In addition, lectins and Table 1 Genotype frequencies of MBL codon 54 in patients with BD and healthy controls Genotype
Patients (n = 30)
Controls (n = 30)
A/A A/B B/B
16 (0.533) 14 (0.467) 0
24 (0.800) 6 (0.200) 0
x2 = 4.8000, P = 0.028.
inflammation are related to coagulation activation [10]. There may be an interaction between increased coagulation and infection on the basis of distinct MBL polymorphisms in BD. However, because of the limited number of cases, further examination is needed to confirm our findings and the specificity of the function of MBL in BD.
References [1] Kaneko S, Suzuki N, Yamashita N, Nagafuchi H, Nakajima T, Wakisaka S, et al. Characterization of T cells specific for an epitope of human 60-kDa heat shock protein (hsp) in patients with Behc¸et’s disease (BD) in Japan. Clin Exp Immunol 1997;108:204—12. [2] Kilpatrick DC. Mannan-binding lectin: clinical significance and applications. Biochim Biophys Acta 2002;1572:401—13. [3] Garred P, Madsen HO, Halberg P, Petersen J, Kronborg G, Svejgaard A, et al. Mannose-binding lectin polymorphisms and susceptibility to infection in systemic lupus erythematosus. Arthritis Rheum 1999;42:2145—52. [4] Stanworth SJ, Donn RP, Hassall A, Dawes P, Ollier W, Snowden N. Absence of an association between mannose-binding lectin polymorphism and rheumatoid arthritis. Br J Rheumatol 1998;37:186—8. [5] Sungnack L. In: Sungnack L, Dongsik B, Eun-So L, Seonghang S, editors. Diagnostic criteria of Behcet Disease: problems and suggestions. Berlin: Springer; 2001. p. 115—8. [6] Madsen HO, Garred P, Thiel S, Kurtzhals JA, Lamm LU, Ryder LP, et al. Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein. J Immunol 1995;155:3013—20. [7] Kaneko F, Takahashi Y, Muramatsu Y, Miura Y. Immunological studies on aphthous ulcer and erythema nodosum-like eruptions in Behcet’s disease. Br J Dermatol 1985;113:303—12. [8] Neth O, Jack DL, Dodds AW, Holzel H, Klein NJ, Turner MW. Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition. Infect Immun 2000;68:688—93. [9] Inanc N, Birtas E, Yavuz S, Ergun T, Direskeneli H. Low serum mannose-binding lectin levels in Behcet’s disease. Adv Exp Med Biol 2003;528:287—9. [10] Kiraz S, Ertenli I, Ozturk MA, Haznedaroglu IC, Celik I, Calguneri M. Pathological haemostasis and prothrombotic state in Behcet’s disease. Thromb Res 2002;105:125—33.
Hongwei Wang, Koichiro Nakamura* Tomoko Inoue, Hirokatsu Yanagihori Yoshio Kawakami, Shinichi Hashimoto Noritaka Oyama, Fumio Kaneko Department of Dermatology School of Medicine Fukushima Medical University, Hikarigaoka 1 Fukushima, Fukushima 960-1295, Japan Teizo Fujita Department of Biochemistry, School of Medicine Fukushima Medical University, Hikarigaoka 1 Fukushima, Fukushima 960-1295 Japan
Letter to the Editor
Tomomi Nishida, Nobuhisa Mizuki Department of Ophthalmology School of Medicine Yokohama City University, Fukuura 3-9 Kanazawaku, Yokohama, Kanagawa 236-0004 Japan
117 *
Corresponding author. Tel.: +81 24 548 2111 fax: +81 24 548 5412 E-mail address: [email protected] (K. Nakamura) 6 May 2004