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Most African-American Patients with Rheumatoid Arthritis Do Not Have the Rheumatoid Antigenic Determinant (Epitope)
- From the University of Alabama at Birmingham, Birmingham, Alabama, and the University of Texas Health Science Center, Houston, Texas. Requests for Reprints: John D. Reveille, MD, The University of Texas Health Science Center, Division of Rheumatology and Clinical Immunogenetics, PO Box 20708, Houston, TX 77225. Acknowledgments: The authors thank Dr. William J. Koopman of the University of Alabama at Birmingham and Dr. Kenneth J. Hardy of the University of Mississippi for their helpful comments; the Clinical Faculty and Associates of the Division of Rheumatology and Clinical Immunology of the University of Alabama at Birmingham and the Division of Rheumatology and Clinical Immunogenetics of the University of Texas Health Science Center at Houston for allowing us to study their patients; and Ms. Ella Henderson for expert secretarial assistance. Grant Support: By National Institutes of Health grants PO-AR 20614 and AR39325.
Abstract
Objective: To evaluate the relation between the presence of the “rheumatoid epitope,” defined by a sequence motif in the HLA-DRB1 alleles, and disease severity in African-American patients with rheumatoid arthritis.
Design: Cross-sectional study.
Setting: Rheumatology outpatient clinics at two university medical centers.
Patients: 86 African-American patients with rheumatoid arthritis (66 seropositive and 20 seronegative for the rheumatoid factor) attending the clinics and 88 healthy African-American persons.
Measurements: HLA-DRB1 alleles were determined by restriction fragment length polymorphism and by allele-specific oligonucleotide typing of polymerase chain reaction-amplified HLA-DRB1 second exons.
Results: With the exception of an increased frequency of HLA-DRB1*04 alleles in seropositive patients with rheumatoid arthritis (27.3%) compared with controls (13.1%) (P = 0.02), the frequencies of HLA-DRB1 alleles were similar in patients and controls. Most seropositive (48 of 66) and seronegative (15 of 20) patients were HLA-DR4 negative, but some (7 of 48 seropositive patients and 3 of 15 seronegative persons) inherited the rheumatoid epitope on a non-DR4 allele. Disease features, including severity, were similar for patients without the epitope and for those with either a single or a double dose of an epitope-positive allele. Positivity for rheumatoid factor, but not for the rheumatoid epitope, was weakly associated with severity in these patients.
Conclusion: Most African-American patients with rheumatoid arthritis did not express the rheumatoid epitope. The predisposition to and severity of rheumatoid arthritis in African-Americans appears to be independent of the presence and dose of the shared rheumatoid epitope.
Rheumatoid arthritis is a relatively common systemic inflammatory disorder characterized by symmetric joint involvement, a variable frequency of extraarticular features (such as rheumatoid nodules, vasculitis, and scleritis) and genetic predisposition conferred by the major histocompatibility complex class II specificity, HLA-DR4 (and others). An association between rheumatoid arthritis and HLA-DR4 was first reported by Stastny in 1976 [1] and was subsequently confirmed by investigators throughout the world. (See the Appendix for a glossary of terms.) It was subsequently found that in some populations, HLA-DR4 was not the HLA-DR specificity associated with rheumatoid arthritis; rather, HLA-DR1, DR10, or a DRw6 allele (HLA-DRB1*1402) was [2-16]. More recently, investigators have shown that alleles associated with rheumatoid arthritis, including HLA-DR1 (DRB1*0101 and *0102), DRw6 (DRB1*1402 and *1406), DR10 (DRB1*1001), and some DR4 alleles share a common sequence motif designated the “rheumatoid epitope,” which is characterized by the amino acid coding sequence QKRAA/QRRAA/RRRAA (see the Appendix for definitions of alphabetic abbreviations) in the third hypervariable region, encompassing amino acid residues 70 to 74 of the HLA-DRB1 chain of the major histocompatibility complex class II molecule [8, 10, 12, 14-17].
In white patients, seropositive rheumatoid arthritis is strongly associated with the HLA-DRB1*0401, *0404, and *0408 alleles but not with the *0402 and *0403 alleles [6]. In Japanese, Greek, Polynesian, and Spanish populations with seropositive rheumatoid arthritis, the rheumatoid epitope is encoded by HLA-DRB1*0405 [7-9]; in Italians, by DRB1*0101 and *0102 [11]; in Israeli Jews, Asian-Indians, Basques, and some Spanish populations, by DRB1*0101 and *1001 [10, 12, 14]; and in Yakima Indians and mestizo Peruvians, by DRB1*1402 [15, 16].
It has been postulated that the severity of rheumatoid arthritis, particularly the presence of extraarticular features and the extent of destructive arthropathy, depends on the dose of the rheumatoid epitope and whether it is encoded by an HLA-DR4 allele or a non-DR4 allele [18-21]. These data raise the possibility of determining early in the course of the disease whether a person is at risk for having severe and destructive arthropathy and perhaps of formulating approaches directed toward early and aggressive treatment that may prevent the subsequent occurrence of joint damage [18-21]. Before such tests can be uniformly used and recommended for this purpose, these findings must be validated in other populations. We have previously studied many African-American patients with seropositive and seronegative rheumatoid arthritis by serologic typing [22] and found, as others have [23-27], that the association with HLA-DR4 was present but was not as strong as in white patients (22.2% of African-American patients with rheumatoid arthritis were DR4 positive compared with 56.3% of white patients); the relative risks were 3.6 and 8.0, respectively [22, 28].
We sought to investigate further the genetic basis of rheumatoid arthritis in a group of African-American patients with rheumatoid arthritis who had clinical and radiologic disease characteristics similar to those seen in white patients with rheumatoid arthritis [29]. We have examined the frequency distribution of the rheumatoid epitope in this well-characterized African-American population and compared the clinical features of those patients having none, one, or two doses of the rheumatoid epitope encoded by their respective HLA-DRB1 alleles.
Methods
Patients
We studied 86 African-American patients with rheumatoid arthritis from the University of Alabama at Birmingham, the University of Texas Medical Center at Houston, and affiliated hospitals and clinics. All patients met the 1987 American Rheumatism Association (now the American College of Rheumatology) revised criteria for the classification of rheumatoid arthritis [30]. The study rheumatologists obtained clinical and demographic data by chart review, interview, and physical examination. The American Rheumatism Association functional class, the presence of extra-articular manifestations (anemia, nodules, serositis, pulmonary involvement, and vasculitis), and the use of disease-modifying antirheumatic drugs were noted. Radiographs of the hand and wrist (obtained within 6 months of the typing) were graded on the basis of the actual films (or their descriptions, if films were not available) by one of the study rheumatologists using the method of Berens and Lin [31]. Rheumatoid factor was determined by the latex fixation test or by nephelometry. Seropositivity was defined as a titer ≥ 1:320 by the former method and as at least 30 IU by the latter.
Controls
The controls were healthy, unrelated African-American persons who had no personal or family history of a rheumatic disease and who were recruited in Alabama and Texas. The proportions of persons from Alabama and Texas were approximately equal (3:1) for both patients and controls.
DNA Typing
The HLA-DRB1 genotypes were determined first by restriction fragment length polymorphism using Taq I endonuclease and a HLA-DRB1 complementary DNA probe [32, 33]. The HLA-DRB1 alleles were further determined by amplification of the second exon of the DRB1 gene and by subsequent hybridization with Phosphorus-32-labeled, allele-specific oligonucleotide probes. Briefly, DNA amplification was done using the polymerase chain reaction with either the Perkin-Elmer DNA Thermal Cycler (Norwalk, Connecticut) or the MJ Research Thermal Cycler (Watertown, Massachusetts). One hundred to 500 ng of genomic DNA obtained from peripheral blood nucleated cells was used in each reaction. Target DNA was amplified using DRB1 group-specific primers (DR1, DR2, DR4, DR52, and DR53) as reported previously [34]. All reaction mixtures were done in a volume of 50 to 100 µL that contained 50 mM of KCl, 10 mM of Tris-HCl (pH, 8.3), 1.5 mM of MgCl2, 0.01% (weight in volume) gelatin, 200 mM each of deoxyribonucleotide triphosphate and DNA template, and 1.25 U of Taq DNA polymerase (GIBCO-BRL/Life Technologies, Gaithersburg, Maryland).
Allele-Specific Oligonucleotide Hybridization
Deoxyribonucleic acid amplified with primers specific for each HLA-DRB1 group types was transferred to Nytran membranes (Schleicher and Schuell, Keene, New Hampshire) by dot blotting and was hybridized with Phosphorus-32-labeled, DRB1 allele-specific oligonucleotide probes. Ten pmol of each DRB1 eighteenmer allele-specific oligonucleotide probe was 5′ end-labeled with g Phosphorus-32-adenosine triphosphate (ATP) (7000 Ci/mmol) (ICN, Irvine, California) using T4 polynucleotide kinase (GIBCO-BRL/Life Technologies). Membranes were prehybridized for 1 hour at 54 °C in buffer containing 50 mM of Tris-HCl (pH, 8.0), 3.0 M of tetramethylammonium chloride, 2 mM of ethylenediaminetetraacetic acid (pH, 8.0), 5X Denhart solution, 0.1% sodium dodecyl sulfate (SDS), and 100 µg/mL of heat-denatured herring-sperm DNA. The labeled DRB1 probe was then added to the membrane and was further incubated for 2 to 8 hours. After hybridization, each membrane was washed twice in 2xSSPE/0.1% SDS for 10 minutes at room temperature, then once in tetramethylammonium chloride solution (50 mM of Tris-HCl [pH, 8.0], 3.0 M of tetramethylammonium chloride, 2 mM of ethylenediaminetetraacetic acid, and 0.1% SDS) at room temperature, and then once at 58 °C followed by 2xSSPE/0.1% SDS at room temperature. Membranes were exposed to XAR-5 radiograph film for 2 to 12 hours at 80 °C.
DNA Sequencing
In most cases, the DR4 subtypes were also determined by sequencing. The DNA sequence was determined by direct sequencing of the polymerase chain reaction product, which was amplified using the HLA-DR4 sequence-specific primers. In brief, the amplified product was spin-dialyzed against 10 mL of sterile distilled water through Centricon 10 filters (Amicon, Beverly, Massachusetts) and concentrated to a volume of 50 to 100 µL. One pmol of each amplification primer was end-labeled with γ Phosphorus-32-ATP (7000 Ci/mmol) and T4 polynucleotide kinase (GIBCO-BRL/Life Technologies).
Terminology
A glossary of immunogenetic terms relevant to our article is provided in the Appendix. The relation of various HLA-DR specificities and HLA-DRB1 alleles relative to the presence of the rheumatoid epitope (and the sequences thereof) is shown in Table 1.
Statistical Analyses
We first compared the frequency distribution of the HLA-DRB1 alleles in seropositive and seronegative patients and in controls using the chi-square distribution; we did not use a Bonferroni correction because the association of HLA-DR4 with rheumatoid arthritis in African-Americans has been well documented. We next examined the demographic and clinical characteristics of the seropositive and seronegative patients using analysis of variance for means and chi-square for proportions. For the next set of comparisons, we divided the seropositive patients into three groups according to the dose of the epitope (none, single, and double); we examined their demographic and clinical features either by chi-square (for the proportions) with Yates correction if appropriate or by analysis of variance (for the means). Finally, we examined seropositivity, the presence and the dose of the rheumatoid epitope, and erosive disease by analysis of covariance (with disease duration as the covariate). For all comparisons, a critical P value of ≤ 0.05 was established a priori.
Results
Clinical and Demographic Features
The demographic and clinical characteristics of the 86 patients (66 patients seropositive for the rheumatoid factor and 20 patients seronegative for the rheumatoid factor), including age at disease onset, disease duration, American Rheumatism Association functional class, frequency of extra-articular manifestations, use of disease-modifying antirheumatic drugs, radiographic scores, and seropositivity for rheumatoid factor, are shown in Table 2. The seropositive and seronegative patients differed in disease duration (12.1 ±7.7 years compared with 8.1 ±6.9 years; P = 0.04), radiographic scores (3.6 ±1.7 compared with 2.5 ±2.1; P =0.02), and the use of disease-modifying antirheumatic drugs (96.4% of patients compared with 65.0%; P = 0.01).
Frequencies of HLA-DR4 and Non-DR4 Alleles Encoding the Rheumatoid Epitope in Patients with Rheumatoid Arthritis
The frequencies of DRB1 alleles were similar in both seropositive and seronegative patients with rheumatoid arthritis, with the exception of an increased frequency of HLA-DRB1*04 alleles in both groups compared with the controls (seropositive patients, 27.3%; seronegative patients, 25%; controls, 13.1%; P = 0.02 for seropositive patients compared with controls and P = 0.2 for seronegative patients compared with controls) (Table 3). Most African-American patients with rheumatoid arthritis were HLA-DR4 negative (48 of 66 seropositive patients [72.7%] Figure 1 and 15 of 20 seronegative patients [75%] Figure 2). Among the 18 HLA-DR4-positive, rheumatoid factor-positive patients (Figure 1), 14 (77.8%) had at least one rheumatoid epitope encoded from either HLA-DRB1*0401 or DRB1*0404. Of the remaining 4 HLA-DR4-positive seropositive patients with rheumatoid arthritis, 2 expressed the HLA-DRB1*0403 allele and the other 2 expressed the DRB1*0411 allele, both of which have an amino-acid substitution, ALA to GLU, at residue 74 that does not encode the rheumatoid epitope. Among the 48 HLA-DR4-negative, rheumatoid factor-positive patients with rheumatoid arthritis, 1 encoded the epitope in a DRB1*0101 allele and 6 encoded it in a DRB1*0102 allele. Among the 20 seronegative patients with rheumatoid arthritis (Figure 2), 5 (25%) were HLA-DR4 positive and all encoded the rheumatoid epitope in either HLA-DRB1*0401 (60%) or DRB1*0404 (40%). Three of the 15 HLA-DR4-negative patients with rheumatoid arthritis encoded the rheumatoid epitope: 1 in an HLA-DRB1*0101 allele and 2 in an HLA-DRB1*0102 allele.
Presence of the Rheumatoid Epitope and Its Association with Disease Severity
The frequencies with which patients had different doses of the epitope were similar in the seropositive and seronegative patients (double dose, 7.6% in the seropositive patients and 5% in the seronegative patients; single dose, 30.3% in the seropositive patients and 35% in the seronegative patients). The HLA-DRB1 alleles encoding the rheumatoid epitope were absent in 62.1% of seropositive patients and 60% of seronegative patients.
Disease features were also similar for seropositive patients with rheumatoid arthritis and either double, single, or no doses of the epitope; specifically, patients in the three groups had similar radiographic scores, extra-articular manifestations, and American Rheumatism Association functional class (Table 4). The small number of HLA-DR4-positive persons in each category precluded comparison between persons encoding the epitope by an HLA-DR4 allele and those encoding it by a non-DR4 allele. Disease characteristics were also similar for seronegative patients in the three epitope dose groups (data not shown).
When data for seropositive and seronegative patients were combined and examined by analysis of covariance, seropositivity (P = 0.07), but not the presence of the rheumatoid epitope, was found to be weakly associated with radiographically determined disease severity. This finding is similar to our previous findings [29].
Discussion
An overwhelming body of evidence supports the role of the similar sequence motif in residues 70 to 74 of the third hypervariable region of the HLA-DRB1 chain encoded by the HLA-DRB1*0401, *0404/0408, *0405, *0409, *0410, *0101, *0102, *1001, *1402, and *1406 alleles in the predisposition to rheumatoid arthritis. Recently, this sequence, also called the rheumatoid epitope, was implicated as a genetic factor that can influence the clinical presentation and severity of the disease in some white populations [18-21]. It has been postulated that having HLA-DR4 alleles encoding the epitope and being homozygous for these alleles is associated with more severe rheumatoid arthritis and that major histocompatibility complex typing may be useful in predicting the course of the disease in patients with early rheumatoid arthritis [18-21].
The presence of HLA-DR4 has been found to be increased in black persons (Americans and Africans), but not as frequently as it is in white persons [23-27]. However, African-American and white patients with rheumatoid arthritis have similar disease severity, clinical and radiographic characteristics, and responses and toxicity patterns to different therapeutic agents [29].
We sought to investigate whether the rheumatoid epitope might be encoded by HLA-DRB1 alleles other than DR4 and whether it might be associated with disease severity in a group of well-characterized African-American patients from Alabama and southeast Texas. The low frequency of HLA-DR4-positive patients carrying the epitope was not surprising, but noteworthy findings were the absence of enrichment of any other HLA-DRB1 alleles and the fact that nearly two thirds of the seropositive and seronegative patients lacked the DRB1 alleles encoding the rheumatoid epitope. Furthermore, the presence and dose of these HLA-DRB1 alleles did not correlate with radiographically determined disease severity in either the seropositive or the seronegative patients. Rheumatoid factor positivity, but not the presence or the dose of the rheumatoid epitope, explained disease severity as determined solely by the presence or absence of erosions after adjustment for disease duration. In fact, of 66 seropositive patients, only 5 had a double dose of HLA-DRB1 alleles encoding the epitope, and none were homozygous for HLA-DRB1*0401 or DRB1*0404. Thus, the putatively worse disease in patients with early rheumatoid arthritis who are homozygous for HLA-DRB1*0401 described recently by Weyand and colleagues [18] could not be examined because none of our patients were in this category. In a smaller group of African-American patients with rheumatoid arthritis participating in a multicenter clinical trial, we also found no patients homozygous for this allele [35]. One patient who had moderately severe disease and was homozygous for HLA-DR4 carried HLA-DRB1*0403, an allele that does not encode the rheumatoid epitope and that has not been associated with rheumatoid arthritis. The low frequency of the rheumatoid epitope has been also noted in a small group of African blacks from Nigeria attending a general medicine clinic; such rarity has been considered to be the main reason for the low prevalence of rheumatoid arthritis in this and other African populations [36].
Whether the epitope itself predicts disease severity or whether severity is determined by the allele encoding for the epitope has not yet been determined [5, 37-41]. In fact, Nelson and colleagues [41] found an association between seropositivity and the epitope encoded by HLA-DR4 alleles but not by HLA-DR1 alleles. Gough and associates [19] found the epitope in approximately 45% of their controls and in a large proportion of patients with early polyarthritis who developed destructive arthropathy; they also found the epitope in a large proportion of patients with only transient polyarthritis. When considered with the data from our African-American patients, these data clearly indicate that the epitope itself cannot be used to predict seropositivity, severe destructive arthropathy, or extra-articular disease. However, when the allele encoding for the epitope, the dose of the epitope, seropositivity, and other clinical features (such as persistent synovitis or elevated C-reactive protein levels) are considered together, predicting disease course and outcome may be possible [42].
An alternative hypothesis is that other genes linked to the HLA-DRB1 locus are more important in the susceptibility to or modulation of rheumatoid arthritis, which may be linked to rheumatoid arthritis-associated HLA-DRB1 alleles in whites but not in Africans. The HLA-DQB1*0301, DQB1*0302, and DQB1*0501 alleles, linked with DR4 or DR1 alleles in many populations, have also been associated with rheumatoid arthritis in some studies [43, 44]. However, the role of HLA-DQ genes in rheumatoid arthritis has not been as well established as that of HLA-DRB1 genes. For instance, HLA-DRB1*0401 is in linkage disequilibrium with DQB1*0301 and with DQB1*0302 [37, 43], whereas HLA-DRB1*0405 (previously called HLA-Dw15), which is associated with rheumatoid arthritis in Japanese persons, is linked to DQB1*0401 [17]. Given that only a small proportion of African-American patients with rheumatoid arthritis have HLA-DR4 alleles and that there was no difference in the frequency of other HLA-DRB1 alleles between patients and controls, we would not expect to see an association between HLA-DQ alleles and rheumatoid arthritis in these African-American patients with rheumatoid arthritis. Our studies of HLA-DQB1 alleles and rheumatoid arthritis in another population of African-Americans, in which we found the same frequency of HLA-DRB1 alleles encoding the rheumatoid epitope, have further confirmed this assertion [35]. Furthermore, in the latter study, the same HLA-DQB1 alleles that are linked to the HLA-DRB1*01 and *04 alleles in whites were found in the African Americans. The HLA-DPB1*0301 allele was found to be associated with seronegative rheumatoid arthritis in white patients, and DPB1*0401 was found to be associated with seropositive rheumatoid arthritis [45, 46]. These associations were found to be independent of HLA-DR4 alleles in one study [45] but not in the other [46].
Thus, implication of HLA-DPB1 alleles in susceptibility and severity of rheumatoid arthritis seems to be premature. The TAP (transporter associated with antigen processing) genes are also located within the major histocompatibility complex class II region, between the HLA-DQ and DP loci, and their involvement in antigen processing makes them attractive candidates for disease-susceptibility genes. However, they appear to be in linkage disequilibrium with certain DR4 types, and no independent association with rheumatoid arthritis has yet been shown [47, 48].
The reason why the presence and gene dosage of HLA-DRB1 alleles, which may affect the severity of rheumatoid arthritis in whites, do not appear to do so in African Americans is still unclear. An attractive hypothesis is that other genes exist that interact with the products of HLA-DRB1 genes to affect disease outcome and that such interactive genes are present in whites but not in African Americans. If so, identification of these as-yet unidentified genes will clearly be important to a better understanding of the pathogenesis of rheumatoid arthritis.
In summary, we have shown that nearly two thirds of seropositive and seronegative African-American patients with rheumatoid arthritis do not have the rheumatoid epitope. Moreover, patients lacking the epitope had clinical features similar to those with one or two doses of the rheumatoid epitope. On the basis of these data, we believe that HLA-DRB1 typing cannot be used alone to predict the occurrence of destructive arthropathy among African-American patients with rheumatoid arthritis.
Appendix: Glossary
Amino acids: Standard scientific abbreviations are given; the most characteristic biochemical property for each amino acid is given in parentheses: A = alanine (hydrophobic); Q = glutamine and G = glycine (both polar but uncharged); R = arginine and K = lysine (both positively charged); D = aspartic acid and E = glutamic acid (both negatively charged).
HLA specificity: the HLA protein as expressed on the cell surface, a heterodimer composed (in the case of HLA-DR and other HLA class II specificities) of an α chain and a β chain. HLA specificities are commonly detected by serologic typing and are designated by terms such as HLA-DR1 or DR4.
Epitope: an antigenic determinant on a complex antigenic molecule.
Locus: a site on a chromosome occupied by a specific gene.
HLA allele: one of the potentially many forms of the HLA gene itself. Because this term refers to differences in the DNA sequence of the gene, different alleles (or forms) of an HLA gene are usually detected by examination of the DNA sequence, which is now possible through various techniques. Because DNA typing examines the alleles at a given locus, the name of the locus precedes the designation of the specific allele (with the two terms separated by an asterisk); for example, HLA-DRB1*0401 refers to the 0401 allele of the HLA-DRB1 locus. One HLA-DR specificity is encoded by several HLA-DRB1 alleles in conjunction with the product of the HLA-DRA1 locus; for example, more than 11 HLA-DRB1 alleles (HLA-DRB1*0401 to *0411) can encode the β chain of the HLA-DR4 specificity.
Linkage: two genes that are physically spaced close together on the same chromosome.
Linkage disequilibrium: alleles of two linked genes that tend to be passed from generation to generation en bloc and thus occur together more frequently than would be expected from the frequencies of the individual genes.
- Copyright ©2004 by the American College of Physicians
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