Genetic differences between b27 positive patients with ankylosing spondylitis and b27 positive healthy controls.
код для вставкиСкачать1460 GENETIC DIFFERENCES BETWEEN B27 POSITIVE PATIENTS WITH ANKYLOSING SPONDYLITIS AND B27 POSITIVE HEALTHY CONTROLS ANDRE1 CALIN, AMY MARDER, ELENA BECKS, and THOMAS BURNS In a controlled study of the 499 available first degree relatives of 79 consecutive HLA-B27 positive patients with ankylosing spondylitis and 69 HLA-B27 positive healthy blood donors, 19 cases of ankylosing spondylitis were found: 16 (15 B27 positive) among the 282 relatives of the patients with ankylosing spondylitis, and 3 (1 B27 positive, 1 B27 negative, 1 unknown) among the 217 relatives of healthy donors y (,: = 5.11; P < 0.025). However, if all cases of possible spondylarthritis are included, 48 cases of ankylosing spondylitis were found: 37 of 282 relatives of patients with ankylosing spondylitis and 11 of 217 relatives of healthy donors y ,(: = 8.29; P < 0.01). Assuming that 50% of relatives of B27 positive individuals carry this antigen, 15 of 142 (10.6%) B27 positive relatives of patients and 2 of 108 = (1.9%) B27 positive relatives of healthy subjects y(,: 5.91; P < 0.025) have ankylosing spondylitis. The relative risk of spondylarthropathy for B27 positive relatives of B27 positive patients compared with relatives of B27 positive healthy subjects is 5.6. Assuming all subjects were evaluated in a similar manner, analysis of these data suggests genetic differences between B27 positive diseased individuals and B27 positive healthy subjects. From the Division of Rheumatology, Veterans Administration Hospital, Palo Alto, CA, and Stanford University Medical Center, Stanford, CA. Supported by the Veterans Administration Research Service and the Northern California Chapter Arthritis Foundation. Andrei Calin, MD, MRCP; Amy Marder, BA; Elena Becks, BA; Thomas Burns, MD. Address reprint requests to Dr. Andrei Calin, Division of Immunology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305. Submitted for publication January 31, 1983; accepted in revised form July 22, 1983. Arthritis and Rheumatism, Vol. 26, No. 12 (December 1983) In spite of the recognized association between ankylosing spondylitis (AS) and HLA-B27 (1,2), the precise nature of the predisposition to this disorder remains an enigma. About 20% of whites with HLAB27 develop sacroiliitis (SI) (3-5) following an unknown inciting event. The figure reaches 50% for Pima Indian males (6). The environmental agent or agents that precipitate AS may be ubiquitous since the disease is prevalent with a worldwide distribution, the frequency of which mirrors that of B27 (7). It is also recognized that approximately 20% of subjects with HLA-B27 develop Reiter’s syndrome (RS) after nonspecific urethritis (8), shigellosis (9), or other triggering events. The explanation of why the remaining 80% of people with HLA-B27 remain healthy is unclear. One possibility is that B27 positive patients with AS have an additional susceptibility gene o r genes, that are lacking in healthy B27 positive subjects. Alternatively, B27 may not be homogeneic, that is, healthy B27 positive subjects may lack a hypothetical disease epitope present on B27 molecules of diseased individuals. To analyze these possibilities, studies can be performed both at epidemiologic and molecular levels. The former require in-depth family studies, whereas the latter rely on monoclonal antibody and biochemical investigations. We report a controlled study of the prevalence of AS among first degree relatives of B27 positive diseased individuals and among relatives of B27 positive healthy controls. A comparable prevalence of disease in both groups would provide evidence against genetic differences between the two B27 positive populations. Alternatively, if disease were found more frequently in relatives of B27 positive patients than in INHERITANCE OF AS relatives of B27 positive controls, one could assume that either additional gene or genes were operative, or that the B27 molecules themselves were distinct. Moreover, if a putative susceptibility gene in juxtaposition to B27 or a diseased B27 epitope were operative, the majority of B27 positive relatives of patients would be expected to have disease. By contrast, if the additional genes were distant from B27, or on a different chromosome, one would expect the frequency of AS among relatives of B27 positive patients t o range from that found in a random B27 positive population t o a higher prevalence. The actual figure would depend on number and location of genes, crossover frequency, and other unknown factors. PATIENTS AND METHODS Ankylosing spondylitis (AS) patients with symptornatic bilateral sacroiliitis (SI) grade I1 or greater (10) were selected for this study. One hundred twenty consecutive patients with AS (95% whites) who were seen at the Stanford University Medical Center and Palo Alto Veterans Administration Hospital were evaluated. Sixteen patients were excluded either because they were HLA-B27 negative (9 patients) or because their HLA type was unknown (7 patients). Of the remaining I04 patients, 25 were unsuitable for study because they were adopted, the only surviving member of the family, or unwilling to release names of their relatives. Therefore, of the original patient population, 79 (65.8%) were appropriate for study. Likewise, 99 consecutive HLA-B27 positive blood donors (97% whites) located through the Stanford University, Irwin Memorial, and Midpeninsula Blood Banks and the San Jose Red Cross agreed to take part in the study. Nineteen were excluded because at the initial survey they were found to have symptoms suggestive of spondylarthropathy or uveitis. Eleven olhps were excluded, again because they were adopted, the only member of the family, or unwilling to release names of relatives. Thus, of the original 99 donors, 69 (69.7%) were suitable for study. The 79 patients with AS provided us with names of 330 living first degree relatives (mother, father, brother, sister, son, or daughter); 282 (85.5%) of these relatives signed a consent form and provided personal data. These represented an average of 3.6 relatives per propositus. Comparable figures for the 69 controls were 275 first degree relatives of whom 217 (78.9%) consented to take part, which is an average of 3.1 relatives per propositus. The male to female ratio was 55:24 for the patients and 38:31 for the donors. The mean ages for the propositi were 40.0 years and 37.9 years, respectively. The male to female ratio of the relatives of the spondylitics was 129: 153 and for the relatives of the donors 102: 1 15, and mean ages of the relatives were 51.0 and 48.4 years, respectively. Screening questionnaires were sent to the 605 relatives, 79 spondylitics, and 69 controls. All individuals were evaluated without knowledge of their study status. Analysis included a standardized evaluation of questionnaires pertain- 1461 ing to inflammatory spinal disease (i.e., insidious onset, duration >3 months, age at onset <40 years, association with morning stiffness, improvement with exercise) (1 1,12), and the peripheral stigmata of spondylarthropathy (uveitis, keratodermia, mouth ulcers, urethritis, and peripheral joint disease) (13). Pelvic radiographs, at no cost to the subject, were sought from individuals who gave a history of back pain that included 4 or more of the 5 “inflammatory” symptoms. Those subjects who had less than 4 of the 5. but at least a total of 6 features, including those of the stigmata of spondylarthropathy, were also asked to have pelvic radiographic evaluations. All 79 spondylitis patients had a diagnosis of AS confirmed by radiograph. The 69 controls were considered disease free (i.e., the absence of the above criteria or normal pelvic radiograph results in symptomatic individuals). We chose not to request radiographs from individuals with fewer criteria since the sensitivity of the questionnaire was found to be accurate to 95% in a prior study (1 I , 12). Specificity was of less concern since a definitive diagnosis of AS would depend on the “blind” reading of the pelvic radiograph. Completed questionnaires were received from 282 (85.5%) of the 330 relatives of the spondylitic patients and from 217 (78.9%) of the 275 relatives of the 69 controls. The sacroiliac radiographs were read “blindly” according to the New York criteria for AS (14) (grade 0 = normal, I = suspicious change, I1 = minimal SI, I11 = moderate SI, IV = fused) by 2 independent observers. If the observers did not agree on individual readings, they reached a consensus. Statistical analysis was performed using chi-square with Yates correction. RESULTS One hundred seven (37.9%) of the 282 relatives of the patients with ankylosing spondylitis gave a positive response t o the questionnaire (i.e., 1 4 of 5 “inflammatory” back pain symptoms or a t least 6 features, including those suggesting peripheral stigmata of spondylarthritis), compared with 55 (25.3%) of 217 relatives of the controls (xc2 = 8.25; P < 0.01). Seventy (65.4%) of the 107 “symptomatic” relatives of the patients with AS agreed to have a pelvic radiograph evaluation as did 41 (74.5%) of the 55 relatives of the controls (not significant). Twenty (28.5%) of the 70 radiographs of the symptomatic relatives of t he patient group showed abnormal results (sacroiliac grade 211) compared with 4 (9.8%) of the 41 radiographs of the symptomatic relatives of the healthy controls (20 of 282 versus 4 of 217; xc2 = 6.33; P < 0.025). When only bilateral disease was considered, the numbers were 16 (22.9%) and 3 (7.3%), respectively (xc’ = 5.11; P < 0.025). When questionable radiograph results were included (grades I and above), the total numbers were 37 (52.9%) and I 1 (26.8%), respectively, for unilateral CALIN ET AL 1462 and bilateral SI (37 of 282 versus I 1 of 217; xc2 = 8.29; P < 0.01). The abnormal radiograph results of relatives of the patients included 7 with grade IV :IV (bilateral fused joints), 3 with 111: 111 (moderate sacroiliitis), 4 with II:III, 2 with II:II, 2 with II:I, and 1 each with 1:III and 0:III. The 4 relatives of the controls with abnormal radiograph results included 1 each with II:III, III:IV, II:II, and 1I:O. The ma1e:female distribution of the 16 diseased relatives (AS, symptomatic bilateral SI) of the patient group was 10:6 compared with 2: 1 for the 3 patients in the control group. Together, these 12 men and 7 women were derived from 231 male and 268 female relatives: 12 of 231 (5.2%) and 7 of 268 (2.6%), a distribution of 2: 1 in favor of males. Of 282 relatives, 20 had grade I1 or more sacroiliitis, at least on one side. Assuming that spondylarthritis occurs predominantly in B27 positive subjects, and that 50% of our study relatives are B27 positive, 20 of 141 (14.2%) B27 positive relatives are affected. Comparable figures for symmetric disease and suspicious change are 16 of 241 ( I 1.3%) and 37 of 241 (26.2%) respectively. Since none of the propositi had more than 1 relative with symptomatic bilateral SI, 16 (20.3%) of 79 patients with AS had a diseased relative compared with 3 (4.3%) of the 69 controls. When the 79 propositi with AS were separated into male and female subgroups, 10 (18.2%) of the 55 men had a relative with AS compared with 6 (25%) of the 24 women (not significant). Fifteen (93.8%) of the 16 relatives of patients with AS who had bilateral SI were B27 positive. The single B27 negative individual had a 111:I1 reading for SI. Of the 3 diseased relatives of the healthy controls, 1 was B27 positive, 1 was B27 negative (sacroiliitis III:II), and 1 was HLA type unknown. DISCUSSION Analysis of the data suggests that there is a highly significant difference in the prevalence of ankylosing spondylitis among the relatives of B27 positive patients with AS compared with occurrence of AS among the relatives of B27 positive healthy controls ( P < 0.025). There are three potential explanations for these findings: 1) a systematic bias has occurred, 2) a difference in the environment between the families of healthy B27 positive subjects and the families of B27 positive patients, or 3) genetic differences between HLA-B27 positive diseased individuals and HLA-B27 positive healthy subjects. Inevitably, biases are inherent in a study of this nature. To some extent, these can be anticipated and avoided. However, many problems remain and their effect and direction can only be surmised. The ideal study would require that patients be selected randomly from B27 positive individuals who are found to have symptomatic SI in a population survey. Patients referred to clinics should not be used in these studies since such individuals may be more likely to have diseased relatives than a sick individual found during a routine survey. However, the perfect study could not be attempted for logistic and ethical reasons. The use of blood donors for “healthy” controls is suspect since it is recognized that donors are often not healthy individuals (3). Moreover, relatives of diseased individuals may be more willing to donate blood than subjects in the general population. We have defined AS as symptomatic bilateral SI (10). Ideally, all 605 relatives would have received a pelvic radiograph evaluation since asymptomatic SI can occur (15). From a pathogenic point of view, asymptomatic SI is as important as symptomatic disease. How many such individuals have been missed remains unknown, though we have previously shown that most patients with SI do have symptoms (15). However, symptoms, by definition, are subjective and our initial screening technique may have been too stringent. Apart from ethical considerations, cost precluded taking additional radiographs. Nevertheless, it seems likely that any under-defining of sacroiliac disease would have affected both the patient and control groups equally. Had we used the New York criteria for the definition of AS, 10 relatives of patients with AS would have had definite AS (grade 111-IV bilateral SI with any clinical criterion) compared with 1 relative of healthy controls. The remaining 6 relatives of patients with AS in whom the pelvic radiographs revealed bilateral SI equal to or greater than grade I1 and the 2 subjects with unilateral SI grade 111, may also have had definite AS, although this diagnosis would have required evaluation of spinal mobility. Patients with “limitation of back movement in all three planes” in addition to grade 111-IV unilateral SI or grade I1 bilateral SI are said to have definite disease (14). Our concern with this definition is that limitation of movement is not adequately defined. Regardless of the definition of AS, the pelvic radiographs were read “blindly” and therefore, over or under reading would have affected both groups equally. At each step data collection was less than INHERITANCE OF AS complete and the completion rate differed for the 2 groups. For example, 282 (85.5%) of first degree relatives of patients returned questionnaires while only 217 (78.9%) of control relatives did so. By contrast, radiographs were obtained from more of the selected control groups, 41 of 55 (74.5%) than from the patient group. 70 of 107 (65.4%). Although our screening questionnaire was developed in a controlled setting (11) and used in other studies (12,13), its precise sensitivity and specificity in other populations remain unknown. Nevertheless, it should be equally applicable to both patient and control groups. However, we are aware that relatives of “healthy” people may answer questions differently from those of patients. Evaluation of radiographs is notoriously unreliable (3,lO). But any systematic under or over reading would not bias the central issue. Yet, from the point of view of derived prevalence data, the numbers are probably underestimated since the completion rates were not 100%. There were more women than men among relatives of both groups (53.0% and 54.3%, respectively). Since the percentages are comparable, there appears to be no additional bias. The preponderance of women would be expected to decrease the number of diagnosed cases, since spondylarthritis is more common in men than women. No attempt was made to control for duration of familial cohabitation. Since the putative infective incident may occur at any time, such an exercise seemed unwarranted. Nevertheless, the probands with AS and their diseased relatives often developed illness at different times and places. Although an environmental factor must be operative, homozygotic twin studies have shown a concordance rate of no more than 70% (16). The possibility remains that the clustering of cases within family units related to diseased probands could be explained by a common infective or toxic etiology. However, we consider this unlikely since AS occurs in about 20% of random cases of B27 positive individuals worldwide, indicating a prevalent environmental trigger (3-5). The initial finding that 19 of the 99 B27 positive donors had to be excluded since they had underlying uveitis or signs of spondylarthropathy was expected and confirmed previous data (3,4). The male to female ratio of 2 : l for spondylarthritis among the relatives approaches the figure suggested by more recent studies (3,17). If the differences between B27 positive diseased individuals and B27 positive healthy subjects that were suggested by our study can be confirmed. 1463 two major hypotheses could explain the findings: 1) differences exist between the B27 molecules of healthy and diseased individuals (i.e., B27 is heterogeneic), or 2) there must be one or more additional genes present in those individuals who develop AS. The location of such genes could be (a) in juxtaposition to HLA-B27, or (b) distant from B27 on the same or a different chromosome. If we accept that about 70% of subjects encounter the infective trigger, a figure derived from the twin series (16), our family studies should reveal a similar prevalence of AS in the B27 positive relatives of B27 positive patients if hypothesis 1 or 2a was operative. In addition, a much lower figure among relatives of B27 positive healthy subjects would be expected. Alternatively, if 2b were the genetic explanation, the prevalence of disease among relatives of B27 positive patients would be greater than among relatives of random B27 positive individuals, but less than the hypothetical 70%. Our data are more consistent with the latter possibility. That B27 is the susceptibility gene has been suggested by the reported cross-reactivity between antibodies directed toward Klebsiella K43 and B27 positive lymphocytes of patients with AS but not of control subjects or B27 negative patients (18). Recent evidence that Yersinia enterocoliticn serotype 3 has an intimate relationship with HLA molecules in patients with Reiter’s syndrome also directs attention toward B27 (19). Moreover, the use of monoclonal antibodies in preliminary studies has differentiated two classes of B27 molecules designated B27M2 positive and B27M2 negative (20). However, a contrasting biochemical study found no differences between B27 molecules of healthy and diseased individuals (21). AS and Reiter’s syndrome (RS) are closely related spondylarthropathies. Our own controlled data and previously reported studies suggest that the nature of the diseases AS and RS is comparable within families to the substantial exclusion of the other disorders (22,23). This would suggest that phenotypic expression is modified by an additional gene or genes. In each group one relative with AS was found to be B27 negative. The explanation for familial disease in the absence of B27 is unknown. One possibility is that a laboratory “error” has been made (24). Alternatively, genes related to clinically silent psoriasis or inflammatory bowel disease may be relevant (16). Assuming that the one diseased relative of unknown HLA type was B27 positive, and that 50% of relatives of B27 positive individuals carry this antigen, 15 of 142 (10.6%) B27 positive relatives of patients will have AS CALIN ET AL compared with 2 of 108 (1.9%)B27 positive relatives of healthy subjects (x: = 5.91; P < 0.025). T h e relative risk of AS for relatives of B27 positive patients compared with relatives of B27 positive healthy subjects is 5.6. In summary, our findings suggest that genetic differences exist between B27 positive healthy subjects and B27 positive patients with AS. Taken togethe r , the molecular a n d epidemiologic data have not yet defined these putative differences. ACKNOWLEDGMENTS We thank the Stanford University Blood Bank, Irwin Memorial Blood Bank, Midpeninsula Blood Bank, and the San Jose Red Cross for allowing us to contact their B27 positive blood donors. We also wish to thank Doni Saunders who was instrumental in preparing this manuscript and Dr. G. Thomson for her gracious assistance. REFERENCES I. Brewerton DA, Caffrey M, Hart FD, James DCO, Sturrock RD: Ankylosing spondylitis and HL-A27. Lancet 1:904-906, 1973 2. Schlosstein L, Terasaki PI, Bluestone R, Pearson C: High association of an HL-A antigen W27, with ankylosing spondylitis. N Engl J Med 288:704-705, 1973 3. Calin A, Fries JF: Striking prevalence of ankylosing spondylitis in “healthy” W27 positive males and females: a controlled study. N Engl J Med 2932335-839, 1975 4. Cohen LM, Mittal KK, Schmid FR, Rogers LF. Cohen K: Increased risk for spondylitis stigmata in apparently healthy HLA W27 men. Ann Intern Med 84:l-7, 1976 5 . HLA-B27 and risk of ankylosing spondylitis (editorial). Br Med J 2:650-65 I , 1978 6. Lisse JR, Kuberski TT, Bennett PH, Knowler WC, Gofton JP, Calin A, Mann DL: High risk of sacroiliitis in HLA-B27-positive Pima Indian men (letter). Arthritis Rheum 25:236-238, 1982 7. Calin A: The relationship between genetics and environment in the pathogenesis of rheumatic diseases. West J Med 131:205-218, 1979 8, Keat AC, Maini RN, Nkwazi GC, Pegrum GD, Ridgeway GL, Scott JT: Role of chlamydia trachomatis and HLA B27 in sexually acquired reactive arthritis. Br Med J 1:605-607, 1978 9. Calin A, Fries JF: An “experimental” epidemic of Reiter’s syndrome revisited: follow-up evidence on genetic and environmental factors. Ann Intern Med 84~564-566, 1976 10. Calin A: Ankylosing spondylitis, Textbook of Rheumatology. Vol. 11. Edited by WN Kelly, ED Harris, S Ruddy, CB Sledge. Philadelphia, WB Saunders, 1981, pp 1017-1032 11. Calin A, Porta J, Fries JF, Schurman DJ: The clinical history as a screening test in ankylosing spondylitis. JAMA 237:2613-2614, 1977 12. Calin A, Kaye B, Sternberg M, Antell B, Chan M: The prevalence and nature of back pain in an industrial complex. Spine 5:201-205, 1980 13. 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Wiesenhutter CW, Brenner MB, Kobayashi S, Huberman A, Yu DTY: Participation of HLA molecules in the interaction between human lymphocytes and a Reiter’sdisease-causing-bacterium (abstract). Arthritis Rheum (supp1)25:S38, I982 20, Grumet FC, Fendly BM, Fish L, Foung S, Engleman EG: Monoclonal antibody (B27M2) subdividing HLAB27. Hum Immunol 5:61-64. 1982 21. Karr RW, Hahn Y, Schwartz BD: Structural identity of human histocompatibility leukocyte antigen-B27 molecules from patients with ankylosing spondylitis and normal individuals. J Clin Invest 69:443-450, 1982 22 Calin A, Barnett M. Marder A, Marks S: The nature and prevalence of spondylarthritis among relatives of probands with ankylosing spondylitis and Reiter’s syndrome (abstract). Arthritis Rheum (supp1)24:S78, 1981 23. Hochberg M, Bias W. Arnett FC: Family studies in HLA-B27 associated arthritis. Medicine 57:463-475, 1978 24. Calin A, Barnett M: HLA-27 positive, negative or both? N Engl J Med 302:1366-1367, 1980
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