International Journal of Rheumatic Diseases 2014; 17: 268–273
ORIGINAL ARTICLE
HLA-DR typing in polyarticular juvenile idiopathic arthritis: a study from a tertiary care hospital in northern India Behera DIBYA RANJAN,1 Singh NAND KUMAR,1 Singh USHA2 and Verma PRAMOD KUMAR2 1
Department of Medicine, Banaras Hindu University, and 2Department of Pathology and Immunopathology, Banaras Hindu University, Varanasi, India
Abstract Introduction: Many studies of human leukocyte antigen (HLA) association with juvenile idiopathic arthritis (JIA) have reported conflicting results, which were probably related to ethnic differences. Moreover, in India, studies on HLA-DR typing on JIA, particularly polyarticular JIA, is lacking. Objective: The aim of our study was to reveal the frequency of HLA DR types in a cohort of polyarticular JIA in northern India. Methods: Fifty-two polyarticular JIA patients were included as per the recent International League of Associations for Rheumatology classification, 2001. HLA-DR typing was performed in 21 patients (18 rheumatoid factor [RF]+ and three RF ) by a DNA-based polymerase chain reaction method for the determination of HLA alleles using sequence specific primers (SSP). The results were compared with that of 23 healthy controls of the same age and sex. Results: HLA-DR4 was present in five cases (23%) in the diseased group while only in one case (4.3%) in the control group with a relative risk of 5.47, but when compared with only RF+ polyarticular JIA, HLA-DR4 was found to be significantly high (27.7% vs. 4.43%; P < 0.05) with a relative risk of 6.3. Further, DR4, DR1, DR2, DR9, DR10 were also non-significantly high in these patients with relative risk of 3.2 for DR9 and 1.8 for DR10. In contrast, HLA-DR6 was seen only in 5.5% of polyarticular JIA cases, whereas it was present in 39% of controls (P < 0.05), a showing negative association. Conclusion: HLA-DR4 codes for susceptibility to RF+ polyarticular JIA with a six-fold risk, whereas HLA-DR6 offers protection. Key words: HLA-DR, juvenile idiopathic arthritis, polyarticular JIA.
INTRODUCTION Juvenile idiopathic arthritis (JIA) is not a single disease, but a term that encompasses all forms of arthritis that begin before the age of 16 years, persist for more than 6 weeks and are of unknown cause.1 Many studies of human leukocyte antigen (HLA) associations with juve-
Correspondence: Dr Behera Dibya Ranjan, Room no. 37, New PG Doctors Hostel, B.H.U., Varanasi – 221005, India. Email: [email protected]
nile rheumatoid arthritis (JRA) have reported conflicting results, which were probably related to ethnic or nationality differences or the possibility that the disease includes several disease entities.2,3 Several studies have reported association of JRA with different human HLA class II alleles by either serological or DNA typing techniques, but the majority of these findings came from studies performed in Whites.4 For polyarticular JRA, HLA-DR4, particularly the alleles HLA-DB1*0401, DRB1*01 and DRB1*08, have been found to be associated with susceptibility.5–14 However, many of such
© 2013 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
HLA-DR typing in polyarticular JIA
studies performed with limited numbers of patients and from different geographic as well as ethnic backgrounds. It is known that great geographic and ethnic variation occurs in JIA; thus, it is prudent to understand the HLA-DR association of Indian polyarticular JIA patients. We want to know whether it is the same type of HLA association prevalent outside India or something else. However, in India, studies on HLAtyping, particularly HLA-DR typing in JIA are lacking. (We have searched PubMed with terms HLA DR association in juvenile arthritis/ juvenile idiopathic arthritis/ juvenile rheumatoid arthritis/ JRA/ JIA in India). So, the aim of our study was to reveal the frequency of HLA-DR types in a cohort of polyarticular JIA in our country.
MATERIAL AND METHODS The study was carried out in Sir Sunderlal Hospital, Institute of Medical Sciences, Banaras Hindu University, Varanasi, which is a tertiary care hospital situated in northern India. Fifty-two children fulfilling the recent 2001 International League of Associations for Rheumatology (ILAR) classification criteria, Edmonton15 (Table 1) of polyarticular JIA were included in the study. The study group consisted of an Indo-Aryan ethnic group.
Investigation Besides routine investigations, rheumatoid factor (RF: immunoglobulin G [IgG], IgM, IgA), antinuclear anti-
body (ANA) were performed by enzyme-linked immunosorbent assay (ELISA) Omega Diagnostics (Misfisher Scientific India pvt. Limited, Mumbai, India).
HLADR typing Because of financial constraints, we had performed HLA-DR typing in 46 cases, 23 polyarticular JIA patients and 23 age- and sex-matched controls. Two of the polyarticular JIA patients were excluded from the study as both were subsequently diagnosed as systemic onset JIA on further follow-up visits. Thus, HLA-DR typing was performed on 21 polyarticular JIA patients, of which 18 were RF+ and three were RF . In all these cases the age of onset was more than 6 years. DR typing was performed by the Morgan HLA SSP DRB Typing Kit (Texas Biogene, Richardson, TX, USA) as a DNA-based method for the determination of HLA alleles using sequence specific primers (SSP). This kit produces the HL-DRB typing results with a low to medium resolution.
Principles The Morgan HLA SSP DRB Typing Kit is for determining HLA alleles using polymerase chain reaction (PCR) techniques with SSPs (PCR-SSP). Allele SSP pairs are designed to selectively amplify target sequences that are specific to a single allele or group of alleles. This PCRSSP method is based on the principle that only primers with completely matched sequences to the target sequences result in amplified products under controlled PCR conditions. The presence of amplified DNA fragment is a positive indication of the existence of allelespecific sequences within the genomic DNA. On the
Table 1 ILAR classification criteria, 2001 for polyarticular JIA Category
Definition
Exclusion
Polyarthritis (RF negative)
Arthritis affecting five or more joints during the first 6 months of disease; a test for RF is negative
(A) Psoriasis or a history of psoriasis in the patient or a first-degree relative (B) Arthritis in an HLA-B27 positive male beginning after the sixth birthday (C) Ankylosing spondylitis, enthesitis related arthritis, sacroiliitis with inflammatory bowel disease, Reiter’s syndrome, or acute anterior uveitis, or a history of one of these disorders in a first-degree relative (D) The presence of lgM RF on at least two occasions at least 3 months apart (E) The presence of systematic JIA in the patient
Polyarthritis (RF positive)
Arthritis affecting five or more joints during the first 6 months of disease; two or more test for RF at least 3 months apart during the first 6 months of disease are positive
A,B,C,E
HLA, human leukocyte antigen; JIA, juvenile idiopathic arthritis; RF, rheumatoid factor.
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other hand, mismatched primers do not generate amplicons. In addition to SSPs, an internal control primer pair, which amplifies a conserved region of the housekeeping gene, cystic fibrosis gene, is included in every PCR reaction mix, and the PCR product of the internal control primer pair serves as an indication of the integrity of PCR reaction. When the DNA fragment amplified by SSP is present, the product of the internal control primer pair is sometimes weak or absent due to the differences in amplifying efficiency between the specific primer pair and then the internal control primer pair. The PCR reaction products were examined by agarose gel electrophoresis which separated the DNA fragments by size. The amplified fragments were visualized as bands through ethidium bromide staining followed by ultraviolet light exposure. The Morgan HLA SSP DRB Typing Kit supplied a panel of primer mixes that was designed to provide positive identification of HLA alleles. A worksheet was provided to assist the determination of HLA types by analyzing the resulting PCR reactions.
Statistical analysis Comparisons of frequencies were accomplished by chisquare analysis. Statistical significance (P-values) for the various associations in patients versus controls were calculated by the chi-square test based on a 2 9 2 table
(Mantel–Haenszel). In cases where the 2 9 2 table contained a value < 5, Fisher’s exact test was performed. Relative risk (RR) was also calculated to understand the strength of association.
RESULTS Polyarticular JIA A total number of 52 patients satisfying the ILAR classification, 2001, for polyarticular JIA were included in the study. Among them 16 (30.7%) were male and 36 (69.7%) were female. Of these 39 (75%) were RF+ and 13 (25%) were RF . In the majority of patients age of onset was between 11–16 years. The clinical features are described in Table 3. The clinical features of the RF+ group was very much similar to the RF group. In the RF+ group the proximal interphalangeal joints (PIP) were involved most commonly (95%), followed by metacarpophalangeal joints (MCP) (91%), wrist (91%), elbow (87%), knee (87%), and metatarsophalangeal joints (MTP) (78%). No case of uveitis was reported in either group. ANA was non-significantly high in the RF+ group(12%) rather than the RF group (9%) (Table 2).
Frequency of HLA-DR types in polyarticular JIA HLA DR typing of 21 polyarticular JIA patients was carried out as mentioned earlier. Of them, 18 were of RF+
Table 2 Distribution and comparison of clinical features between RF+ and RF Clinical feature
Joint involvement wrist PIP MCP DIP Elbow Shoulder Hip Knee Ankle MTP Cervical spine TMJ Symmetry Uveitis
polyarticular JIA
Polyarticular (RF+) (n = 39)
Polyarticular (RF ) (n = 13)
P-value*
No.
%
No.
%
35 37 35 0 34 6 1 34 25 30 3 1 39 0
91 95 91 0 87 17 2.5 87 65 78 7.6 2.5 100 0
11 13 13 0 7 5 0 11 11 9 1 0 13 0
84 100 100 0 76 38.5 0 87.5 87.5 75 7.6 0 100 0
1.000 1.000 1.000 1.000 1.000 0.334 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
*Chi-square test. DIP, distal interphalangeal joints; JIA, juvenile idiopathic arthritis; MCP, metacarpophalangeal joints; MTP, metatarsophalangeal joints; PIP, proximal interphalangeal joints; RF, rheumatoid factor; TMJ, temporomandibular joint.
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Table 3 Frequency of HLA-DR types in polyarticular JIA HLA-DR type
DR1 DR2, DR15(2), DR16(2) DR3, DR17(3) DR18(3) DR4 DR5, DR11(5) DR12(5) DR6, DR13(6) DR14(6) DR7 DR8 DR9 DR10
(A) Polyarticular JIA (n = 21)
(B) Healthy control (n = 23)
No.
%
No.
%
2 13 0 5 1 2 6 0 1 5
9.5 62 0 23 4.7 9.5 28.5 0 4.7 23
0 11 1 1 5 9 10 2 0 3
0 47.8 4.3 4.3 21.7 39 43.5 8.7 0 13
Relative risk
Chi square A versus B
P-value
5.45 1.29 1.33 5.47 0.21 0.24 0.65 0.21 3.2 1.8
2.295 0.878 0.934 3.53 2.68 5.13 0.545 1.91 1.12 0.851
Relative risk
Chi square A versus B
P-value
3.7 1.39 0.42 6.3 0.25 0.14 0.51 0.25 1.26 2.12
1.31 1.45 0.802 4.43 2.11 6.17 2.01 1.65 1.396
0.252 0.227 0.370 0.035 0.145 0.013 0.154 0.199 0.905 0.237
0.129 0.348 0.333 0.060 0.101 0.023 0.460 0.166 0.289 0.355
HLA, human leukocyte antigen; JIA, juvenile idiopathic arthritis.
Table 4 Frequency of HLA DR types in RF+ve Polyarticular JIA HLA DR type
DR 1 DR 2, DR 15(2), DR16(2) DR3, DR17(3) DR 18(3) DR 4 DR 5, DR11(5) DR 12(5) DR 6, DR13(6) DR14(6) DR 7 DR 8 DR 9 DR 10
(A) RF+ve Polyarticular JIA (n = 18)
(B) Healthy control (n = 23)
No.
%
No.
%
1 12 0 5 1 1 4 0 0 5
5.5 66.6 0 27.7 5.5 5.5 22.2 0 0 27.7
0 11 1 1 5 9 10 2 0 3
0 47.8 4.3 4.3 21.7 39 43.5 8.7 0 13
and three were of RF . The result was compared with that of 23 healthy controls of the same age and sex (Table 3). HLA-DR1 was present two cases (9.5%) of the polyarticular group whereas no patients in the control group had this, with a RR of 5.45. Similarly, HLA-DR4 was present in five cases (23%) in the diseased group but only in one case (2.3%) in the control group, with a RR of 5.47, which was borderline significant. DR9 (4.7% vs. 0%), DR10 (23% vs. 13%) were also non-significantly high in patients with RR of 3.2 and 1.8, respectively. RR was highest for DR4 (5.47). In contrast, HLA-DR5 (21.7% vs. 4.7%), DR6 (39% vs. 9.5%, P < 0.05), DR7 (43.5% vs. 28.5%) were detected more in controls, suggesting that HLA-DR5,
International Journal of Rheumatic Diseases 2014; 17: 268–273
DR6, DR7 may have protective roles against polyarticular JIA in this population. As the majority of patients were in the RF+ group, we also compared only RF+ polyarticular JIA with the controls (Table 4). Analyzing frequency of HLA-DR antigens in RF+ polyarticular JIA, this showed significant association with HLA-DR4 (27.7% vs. 4.3%; P < 0.05; RR = 6.3). Similarly as seen in Table 4, HLA-DR6 was negatively associated with RF+ polyarticular JIA (P = 0.013).
DISCUSSION This is the first HLA-DR association study in polyarticular JIA in India. The study was done in Varanasi,
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situated in the bank of the river Ganga in the northern part of India. The study population mainly consisted of an Indo-Aryan ethnic group. Analysis of 52 polyarticular JIA patients showed that most features are the same in both RF+ and RF groups. The RF variety is described in the literature16,17 as a heterogeneous group. At least two subsets are identified, one with a ANA subset that resembles adult-onset RF rheumatoid arthritis, and the other ANA+ who share many features with ANA+ oligoarticular JIA and ANA+ psoriatic JIA. However, such heterogeneity was not seen in this study group. HLA-DR typing performed in 21 polyarticular JIA patients showed that some genes have a predisposing property. DR2, DR9, DR10 were non-significantly high in polyarticular JIA. However, HLA-DR4 was significantly high (P < 0.05) in RF+ polyarticular JIA as compared to the control group, suggesting susceptibility. Contrary to this, DR6 was significantly low (P < 0.05) in this group, suggesting its protective role against polyarticular JIA. It is to be noted that all HLA-DR4+ patients are RF+ and no patient from the RF group had HLA-DR4 positivity. So, there is a sharp contrast between RF+ and RF polyarticular JIA as far as HLA-DR4 association is concerned. This is very similar to seropositive rheumatoid arthritis in adults where HLA-DR4 association is well established. Barbara et al.,18 found DR4 in 77% of polyarticular JRA patients compared with 32% of controls with a RR of 7.2. (P < 0.001). Gloria et al.,19 also found significant association of the polyarticular JRA with HLA-DRB1*04, 01, and DRB1*1601, 1602, 1101 and 1104. However, there are no studies regarding HLA-DR association with JIA in India. Our study is perhaps the first in India indicating association between HLA-DR4 and RF+ polyarticular JIA. Our study found similar HLA association in RF+ polyarticular JIA (HLA-DR4) as in studies from other countries, reflecting uniformity regardless of geographic and/or ethnic variation. Another important observation is that HLA-DR6 seems to have protective value against polyarticular JIA (both RF+ and RF variety). No such data is available both from India or abroad. Thus, this may be a new area of research. Although the number of patients in our study was small, still we could find some important observations. However, we need larger studies from different parts of India to explore the hidden facts in this field.
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ACKNOWLEDGEMENTS We thank the staff of the Immunopathology laboratory, Banaras Hindu University and also our patients for their cooperation.
CONFLICT OF INTEREST We declare no conflict of interest.
REFERENCES 1 Cassidy JT, Petty RE (2005) Chronic arthritis in childhood. In: Cassidy JT, Petty RE (eds) Text Book of Pediatric Rheumatology, 5th edn, pp 206–60. Elsevier Saunders, Philadelphia. 2 Fernandez-Vina MA, Fink CW, Stastny P (1990) HLA antigens in juvenile arthritis. Arthritis Rheum 33, 1787–94. 3 Fernandez Vina MA, Fink CW, Stasny P (1994) HLA associations in juvenile arthritis. Clin Exp Rheumatol 12, 205– 14. 4 Reveilli JD, Spencer CH, Rivas-Chacon R, Myones B (1997) HLA-DRB1, DQA1, DQB1 and DPB1 alleles in children with juvenile arthritis from three ethnic groups. Arthritis Rheum 40, S241. 5 Melin-Aldana H, Giannini EH, Taylor J et al. (1992) Human leukocyte antigen-DRB1*1104 in the chronic iridocyclitis of pauciarticular juvenile rheumatoid arthritis. J Pediatr 121 (1), 56–60. 6 Ploski R, Vinje O, Ronningen KS et al. (1993) HLA class II alleles and heterogeneity of juvenile rheumatoid arthritis. DRB1*0101 may define a novel subset of the disease. Arthritis Rheum 36, 465–72. 7 Haas JP, Truckenbrodt H, Paul C, Hoza J, Scholz S, Albert ED (1994) Subtypes of Gloria Garavito de Egea Tesis Doctoral 2003 47 HLA-DRB1*03, *08, *11, *12, *13 and *14 in early onset pauciarticular juvenile chronic arthritis (EOPA) with and without iridocyclitis. Clin Exp Rheumatol 12 (10), S7–14. 8 Nepom B (1991) The immunogenetics of juvenile rheumatoid arthritis. Rheum Dis Clin North Am 17 (4), 825–42. 9 Brunner HI, Ivaskova E, Haas JP et al. (1993) Class I associations and frequencies of class II HLA-DRB alleles by RFLP analysis in children with rheumatoid-factor-negative juvenile chronic arthritis. Rheumatol Int 13, 83–8. 10 Scholz S, Albert ED (1993) Immunogenetic aspects of juvenile chronic arthritis. Clin Exp Rheumatol 11 (9), S37– 41. 11 Paul C, Schoenwald U, Truckenbrodt H et al. (1993) HLADP/DR interaction in early onset pauciarticular juvenile chronic arthritis. Immunogenetics 37 (6), 442–8. 12 Paul C, Hass JP, Truckenbrodt H (1993) HLA class I/class II interaction in EOPA juvenile chronic arthritis. Immunogenetics 39, 61.
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13 Van Kerckhove C, Luyrink L, Elma MS et al. (1990) HLADP/ DR interaction in children with juvenile rheumatoid arthritis. Immunogenetics 32 (5), 364–8. 14 Paul C, Yao Z, Nevinny-Stickel C et al. (1995) Immunogenetics of juvenile chronic arthritis. I. HLA interaction between A2, DR5/8-DR/DQ, and DPB1*0201 is a general feature of all subsets of early onset pauciarticular juvenile chronic arthritis II: DPB1 polymorphism plays a role in systemic juvenile chronic arthritis. Tissue Antigens 45, 280–3. 15 Petty RE, Southwood TR, Manners P et al. (2004) International League of Associations for Rheumatology classification of juvenile idiopathic arthritis: second revision, Edmonton, 2001. J Rheumatol 31, 390–2.
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16 Prakken B, Albani A, Martini A (2011) Juvenile idiopathic arthritis. Lancet 377, 2138–49. 17 Singh S, Jat KR (2008) Juvenile idiopathic arthritis: an update. Indian J Rheum 3 (3), 110–9. 18 Nepom BS, Nepom GT, Mickelson E, Schaller JG, Antonelli P, Hansen JA (1984) Specific HLA-DR4-associated histocompatibility molecules characterize patients with seropositive juvenile rheumatoid arthritis. J Clin Invest 74, 287–91. 19 Garavito G, Yunis EJ, Egea E et al. (2004) HLA-DRB1 alleles and HLA-DRB1 shared epitopes are markers for juvenile rheumatoid arthritis subgroups in colombian mestizos. Hum Immunol 65, 359–65.
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ORIGINAL ARTICLE
HLA-DR typing in polyarticular juvenile idiopathic arthritis: a study from a tertiary care hospital in northern India Behera DIBYA RANJAN,1 Singh NAND KUMAR,1 Singh USHA2 and Verma PRAMOD KUMAR2 1
Department of Medicine, Banaras Hindu University, and 2Department of Pathology and Immunopathology, Banaras Hindu University, Varanasi, India
Abstract Introduction: Many studies of human leukocyte antigen (HLA) association with juvenile idiopathic arthritis (JIA) have reported conflicting results, which were probably related to ethnic differences. Moreover, in India, studies on HLA-DR typing on JIA, particularly polyarticular JIA, is lacking. Objective: The aim of our study was to reveal the frequency of HLA DR types in a cohort of polyarticular JIA in northern India. Methods: Fifty-two polyarticular JIA patients were included as per the recent International League of Associations for Rheumatology classification, 2001. HLA-DR typing was performed in 21 patients (18 rheumatoid factor [RF]+ and three RF ) by a DNA-based polymerase chain reaction method for the determination of HLA alleles using sequence specific primers (SSP). The results were compared with that of 23 healthy controls of the same age and sex. Results: HLA-DR4 was present in five cases (23%) in the diseased group while only in one case (4.3%) in the control group with a relative risk of 5.47, but when compared with only RF+ polyarticular JIA, HLA-DR4 was found to be significantly high (27.7% vs. 4.43%; P < 0.05) with a relative risk of 6.3. Further, DR4, DR1, DR2, DR9, DR10 were also non-significantly high in these patients with relative risk of 3.2 for DR9 and 1.8 for DR10. In contrast, HLA-DR6 was seen only in 5.5% of polyarticular JIA cases, whereas it was present in 39% of controls (P < 0.05), a showing negative association. Conclusion: HLA-DR4 codes for susceptibility to RF+ polyarticular JIA with a six-fold risk, whereas HLA-DR6 offers protection. Key words: HLA-DR, juvenile idiopathic arthritis, polyarticular JIA.
INTRODUCTION Juvenile idiopathic arthritis (JIA) is not a single disease, but a term that encompasses all forms of arthritis that begin before the age of 16 years, persist for more than 6 weeks and are of unknown cause.1 Many studies of human leukocyte antigen (HLA) associations with juve-
Correspondence: Dr Behera Dibya Ranjan, Room no. 37, New PG Doctors Hostel, B.H.U., Varanasi – 221005, India. Email: [email protected]
nile rheumatoid arthritis (JRA) have reported conflicting results, which were probably related to ethnic or nationality differences or the possibility that the disease includes several disease entities.2,3 Several studies have reported association of JRA with different human HLA class II alleles by either serological or DNA typing techniques, but the majority of these findings came from studies performed in Whites.4 For polyarticular JRA, HLA-DR4, particularly the alleles HLA-DB1*0401, DRB1*01 and DRB1*08, have been found to be associated with susceptibility.5–14 However, many of such
© 2013 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
HLA-DR typing in polyarticular JIA
studies performed with limited numbers of patients and from different geographic as well as ethnic backgrounds. It is known that great geographic and ethnic variation occurs in JIA; thus, it is prudent to understand the HLA-DR association of Indian polyarticular JIA patients. We want to know whether it is the same type of HLA association prevalent outside India or something else. However, in India, studies on HLAtyping, particularly HLA-DR typing in JIA are lacking. (We have searched PubMed with terms HLA DR association in juvenile arthritis/ juvenile idiopathic arthritis/ juvenile rheumatoid arthritis/ JRA/ JIA in India). So, the aim of our study was to reveal the frequency of HLA-DR types in a cohort of polyarticular JIA in our country.
MATERIAL AND METHODS The study was carried out in Sir Sunderlal Hospital, Institute of Medical Sciences, Banaras Hindu University, Varanasi, which is a tertiary care hospital situated in northern India. Fifty-two children fulfilling the recent 2001 International League of Associations for Rheumatology (ILAR) classification criteria, Edmonton15 (Table 1) of polyarticular JIA were included in the study. The study group consisted of an Indo-Aryan ethnic group.
Investigation Besides routine investigations, rheumatoid factor (RF: immunoglobulin G [IgG], IgM, IgA), antinuclear anti-
body (ANA) were performed by enzyme-linked immunosorbent assay (ELISA) Omega Diagnostics (Misfisher Scientific India pvt. Limited, Mumbai, India).
HLADR typing Because of financial constraints, we had performed HLA-DR typing in 46 cases, 23 polyarticular JIA patients and 23 age- and sex-matched controls. Two of the polyarticular JIA patients were excluded from the study as both were subsequently diagnosed as systemic onset JIA on further follow-up visits. Thus, HLA-DR typing was performed on 21 polyarticular JIA patients, of which 18 were RF+ and three were RF . In all these cases the age of onset was more than 6 years. DR typing was performed by the Morgan HLA SSP DRB Typing Kit (Texas Biogene, Richardson, TX, USA) as a DNA-based method for the determination of HLA alleles using sequence specific primers (SSP). This kit produces the HL-DRB typing results with a low to medium resolution.
Principles The Morgan HLA SSP DRB Typing Kit is for determining HLA alleles using polymerase chain reaction (PCR) techniques with SSPs (PCR-SSP). Allele SSP pairs are designed to selectively amplify target sequences that are specific to a single allele or group of alleles. This PCRSSP method is based on the principle that only primers with completely matched sequences to the target sequences result in amplified products under controlled PCR conditions. The presence of amplified DNA fragment is a positive indication of the existence of allelespecific sequences within the genomic DNA. On the
Table 1 ILAR classification criteria, 2001 for polyarticular JIA Category
Definition
Exclusion
Polyarthritis (RF negative)
Arthritis affecting five or more joints during the first 6 months of disease; a test for RF is negative
(A) Psoriasis or a history of psoriasis in the patient or a first-degree relative (B) Arthritis in an HLA-B27 positive male beginning after the sixth birthday (C) Ankylosing spondylitis, enthesitis related arthritis, sacroiliitis with inflammatory bowel disease, Reiter’s syndrome, or acute anterior uveitis, or a history of one of these disorders in a first-degree relative (D) The presence of lgM RF on at least two occasions at least 3 months apart (E) The presence of systematic JIA in the patient
Polyarthritis (RF positive)
Arthritis affecting five or more joints during the first 6 months of disease; two or more test for RF at least 3 months apart during the first 6 months of disease are positive
A,B,C,E
HLA, human leukocyte antigen; JIA, juvenile idiopathic arthritis; RF, rheumatoid factor.
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other hand, mismatched primers do not generate amplicons. In addition to SSPs, an internal control primer pair, which amplifies a conserved region of the housekeeping gene, cystic fibrosis gene, is included in every PCR reaction mix, and the PCR product of the internal control primer pair serves as an indication of the integrity of PCR reaction. When the DNA fragment amplified by SSP is present, the product of the internal control primer pair is sometimes weak or absent due to the differences in amplifying efficiency between the specific primer pair and then the internal control primer pair. The PCR reaction products were examined by agarose gel electrophoresis which separated the DNA fragments by size. The amplified fragments were visualized as bands through ethidium bromide staining followed by ultraviolet light exposure. The Morgan HLA SSP DRB Typing Kit supplied a panel of primer mixes that was designed to provide positive identification of HLA alleles. A worksheet was provided to assist the determination of HLA types by analyzing the resulting PCR reactions.
Statistical analysis Comparisons of frequencies were accomplished by chisquare analysis. Statistical significance (P-values) for the various associations in patients versus controls were calculated by the chi-square test based on a 2 9 2 table
(Mantel–Haenszel). In cases where the 2 9 2 table contained a value < 5, Fisher’s exact test was performed. Relative risk (RR) was also calculated to understand the strength of association.
RESULTS Polyarticular JIA A total number of 52 patients satisfying the ILAR classification, 2001, for polyarticular JIA were included in the study. Among them 16 (30.7%) were male and 36 (69.7%) were female. Of these 39 (75%) were RF+ and 13 (25%) were RF . In the majority of patients age of onset was between 11–16 years. The clinical features are described in Table 3. The clinical features of the RF+ group was very much similar to the RF group. In the RF+ group the proximal interphalangeal joints (PIP) were involved most commonly (95%), followed by metacarpophalangeal joints (MCP) (91%), wrist (91%), elbow (87%), knee (87%), and metatarsophalangeal joints (MTP) (78%). No case of uveitis was reported in either group. ANA was non-significantly high in the RF+ group(12%) rather than the RF group (9%) (Table 2).
Frequency of HLA-DR types in polyarticular JIA HLA DR typing of 21 polyarticular JIA patients was carried out as mentioned earlier. Of them, 18 were of RF+
Table 2 Distribution and comparison of clinical features between RF+ and RF Clinical feature
Joint involvement wrist PIP MCP DIP Elbow Shoulder Hip Knee Ankle MTP Cervical spine TMJ Symmetry Uveitis
polyarticular JIA
Polyarticular (RF+) (n = 39)
Polyarticular (RF ) (n = 13)
P-value*
No.
%
No.
%
35 37 35 0 34 6 1 34 25 30 3 1 39 0
91 95 91 0 87 17 2.5 87 65 78 7.6 2.5 100 0
11 13 13 0 7 5 0 11 11 9 1 0 13 0
84 100 100 0 76 38.5 0 87.5 87.5 75 7.6 0 100 0
1.000 1.000 1.000 1.000 1.000 0.334 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
*Chi-square test. DIP, distal interphalangeal joints; JIA, juvenile idiopathic arthritis; MCP, metacarpophalangeal joints; MTP, metatarsophalangeal joints; PIP, proximal interphalangeal joints; RF, rheumatoid factor; TMJ, temporomandibular joint.
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Table 3 Frequency of HLA-DR types in polyarticular JIA HLA-DR type
DR1 DR2, DR15(2), DR16(2) DR3, DR17(3) DR18(3) DR4 DR5, DR11(5) DR12(5) DR6, DR13(6) DR14(6) DR7 DR8 DR9 DR10
(A) Polyarticular JIA (n = 21)
(B) Healthy control (n = 23)
No.
%
No.
%
2 13 0 5 1 2 6 0 1 5
9.5 62 0 23 4.7 9.5 28.5 0 4.7 23
0 11 1 1 5 9 10 2 0 3
0 47.8 4.3 4.3 21.7 39 43.5 8.7 0 13
Relative risk
Chi square A versus B
P-value
5.45 1.29 1.33 5.47 0.21 0.24 0.65 0.21 3.2 1.8
2.295 0.878 0.934 3.53 2.68 5.13 0.545 1.91 1.12 0.851
Relative risk
Chi square A versus B
P-value
3.7 1.39 0.42 6.3 0.25 0.14 0.51 0.25 1.26 2.12
1.31 1.45 0.802 4.43 2.11 6.17 2.01 1.65 1.396
0.252 0.227 0.370 0.035 0.145 0.013 0.154 0.199 0.905 0.237
0.129 0.348 0.333 0.060 0.101 0.023 0.460 0.166 0.289 0.355
HLA, human leukocyte antigen; JIA, juvenile idiopathic arthritis.
Table 4 Frequency of HLA DR types in RF+ve Polyarticular JIA HLA DR type
DR 1 DR 2, DR 15(2), DR16(2) DR3, DR17(3) DR 18(3) DR 4 DR 5, DR11(5) DR 12(5) DR 6, DR13(6) DR14(6) DR 7 DR 8 DR 9 DR 10
(A) RF+ve Polyarticular JIA (n = 18)
(B) Healthy control (n = 23)
No.
%
No.
%
1 12 0 5 1 1 4 0 0 5
5.5 66.6 0 27.7 5.5 5.5 22.2 0 0 27.7
0 11 1 1 5 9 10 2 0 3
0 47.8 4.3 4.3 21.7 39 43.5 8.7 0 13
and three were of RF . The result was compared with that of 23 healthy controls of the same age and sex (Table 3). HLA-DR1 was present two cases (9.5%) of the polyarticular group whereas no patients in the control group had this, with a RR of 5.45. Similarly, HLA-DR4 was present in five cases (23%) in the diseased group but only in one case (2.3%) in the control group, with a RR of 5.47, which was borderline significant. DR9 (4.7% vs. 0%), DR10 (23% vs. 13%) were also non-significantly high in patients with RR of 3.2 and 1.8, respectively. RR was highest for DR4 (5.47). In contrast, HLA-DR5 (21.7% vs. 4.7%), DR6 (39% vs. 9.5%, P < 0.05), DR7 (43.5% vs. 28.5%) were detected more in controls, suggesting that HLA-DR5,
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DR6, DR7 may have protective roles against polyarticular JIA in this population. As the majority of patients were in the RF+ group, we also compared only RF+ polyarticular JIA with the controls (Table 4). Analyzing frequency of HLA-DR antigens in RF+ polyarticular JIA, this showed significant association with HLA-DR4 (27.7% vs. 4.3%; P < 0.05; RR = 6.3). Similarly as seen in Table 4, HLA-DR6 was negatively associated with RF+ polyarticular JIA (P = 0.013).
DISCUSSION This is the first HLA-DR association study in polyarticular JIA in India. The study was done in Varanasi,
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situated in the bank of the river Ganga in the northern part of India. The study population mainly consisted of an Indo-Aryan ethnic group. Analysis of 52 polyarticular JIA patients showed that most features are the same in both RF+ and RF groups. The RF variety is described in the literature16,17 as a heterogeneous group. At least two subsets are identified, one with a ANA subset that resembles adult-onset RF rheumatoid arthritis, and the other ANA+ who share many features with ANA+ oligoarticular JIA and ANA+ psoriatic JIA. However, such heterogeneity was not seen in this study group. HLA-DR typing performed in 21 polyarticular JIA patients showed that some genes have a predisposing property. DR2, DR9, DR10 were non-significantly high in polyarticular JIA. However, HLA-DR4 was significantly high (P < 0.05) in RF+ polyarticular JIA as compared to the control group, suggesting susceptibility. Contrary to this, DR6 was significantly low (P < 0.05) in this group, suggesting its protective role against polyarticular JIA. It is to be noted that all HLA-DR4+ patients are RF+ and no patient from the RF group had HLA-DR4 positivity. So, there is a sharp contrast between RF+ and RF polyarticular JIA as far as HLA-DR4 association is concerned. This is very similar to seropositive rheumatoid arthritis in adults where HLA-DR4 association is well established. Barbara et al.,18 found DR4 in 77% of polyarticular JRA patients compared with 32% of controls with a RR of 7.2. (P < 0.001). Gloria et al.,19 also found significant association of the polyarticular JRA with HLA-DRB1*04, 01, and DRB1*1601, 1602, 1101 and 1104. However, there are no studies regarding HLA-DR association with JIA in India. Our study is perhaps the first in India indicating association between HLA-DR4 and RF+ polyarticular JIA. Our study found similar HLA association in RF+ polyarticular JIA (HLA-DR4) as in studies from other countries, reflecting uniformity regardless of geographic and/or ethnic variation. Another important observation is that HLA-DR6 seems to have protective value against polyarticular JIA (both RF+ and RF variety). No such data is available both from India or abroad. Thus, this may be a new area of research. Although the number of patients in our study was small, still we could find some important observations. However, we need larger studies from different parts of India to explore the hidden facts in this field.
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ACKNOWLEDGEMENTS We thank the staff of the Immunopathology laboratory, Banaras Hindu University and also our patients for their cooperation.
CONFLICT OF INTEREST We declare no conflict of interest.
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