Clin. exp. Immunol. (1976) 25, 352-354.
BRIEF COMMUNICATION
HLA antigens in pernicious anaemia A. H. GOLDSTONE,* D. VOAK,t J. C. CAWLEY* & W. J. IRVINE:*Department of Haematology, Addenbrooke's Hospital, t Regional Transfusion and Immuno-Haematology Centre, Cambridge, and $ Clinical Immunology, Royal Infirmary, Edinburgh (Received 20 February 1976)
SUMMARY The increased frequency of HLA-B7 alone and HLA-A3/B7 together, in the same patient, has been confirmed in pernicious anaemia. There is no increased prevalence of HLA-A3 alone. No association has been found between the presence of serum intrinsic factor type 1 antibody and HLA-A3 or HLA-B7.
INTRODUCTION The investigation of possible associations between the HLA system and disease has recently turned towards the area of organ-specific autoimmune disorders (Grumet et al., 1973; Platz et al., 1974; Nerup et al., 1974). It seems likely that there is an association between the antigens HLA-A3 and HLA-B7 and gastric-autoimmune disease (Whittingham et al., 1975; Mawhinney et al., 1975). In pernicious anaemia (PA) itself there may well be an increased incidence of HLA-B7 and HLA-A3/B7 together (Mawhinney et al., 1975; Zittoun et al., 1975). In this paper we report a study of HLA antigens in a further forty-two patients with PA and our results confirm and extend previous reports. PATIENTS AND METHODS Patients and controls. (a) Pernicious anaemia. A total of forty-two patients was studied; all were receiving maintenance B12 and had normal haematology. Diagnosis had been originally made by the presence of a macrocytic anaemia, megaloblastic bone marrow, low serum B12, achlorhydria and an abnormal vitamin B12 absorption test corrected by the addition of intrinsic factor. (b) Controls. 216 Random group 0 blood donors were typed concurrently with patients. HLA typing. Lymphocytes were separated from fresh ACD blood samples by sedimentation over Ficoll-Triosil. Phagocytic mononuclear cells were removed by incubation with carbonyl iron and extraction with a magnet. Typing for HLA specificities was carried out on a Terasaki type plate by a microlymphocytotoxicity technique. 2 pul of lymphocyte suspension at a concentration of 106 cells/ml were added to the test wells of the typing plates containing 2Al of the known anti-HLA antisera, left at room temperature for 30 min. Then 4 ul of complement (3 parts rabbit serum to 1 part human-group AB serum) were added to each test well. After incubation at room temperature for 1 hr, 1 p1 of trypan blue (0-2%/ in isotonic saline) was added to each well to which after 15 min I ul of1% bovine albumin and then 1 ul of 5%O acetic acid were added and the tests read on an inverted Wild microscope. Lymphocytes from patients and controls were tested for the following twenty-two antigens HLA-A1, -A2, -A3, -A9, -A10, -A29 and -AW32 at the first locus and HLA-B5, -B7, -B8, -B12, -B13, -B14, -BW17, -B27, -BW35, -BW40, -BW15, -B18, -BW22, -BW37 at the second locus. Three antisera were used for each of the common HLA specificities including HLA-A3 and HLA-B7. Controls and patients were tested on at least two separate occasions. Autoantibody studies. Serum intrinsic factor (IF) type 1 antibody was demonstrated by the charcoal absorption method (Irvine 1966). Statistics. The hypothesis that, on the basis of their reactivity against the specific sera listed, the control group and the patient group were drawn from the same population was tested using thex2 (Chi2) test. Where the tested number reacting against an individual antigen was less than 5, results were pooled. AX2 value of 12-104 with 12 degrees of freedom showed that Correspondence: Dr A. H. Goldstone, Department of Haematology, University College Hospital, London.
352
HLA antigen in pernicious anaemia
353
there was no significant difference at the 95% level. The difference at each antigen specificity was further tested by determining the confidence limits for the difference in proportions of each.
RESULTS The frequencies of the HLA-antigen specificities in the control population and in the PA patients are shown in Table 1. The incidence of serum IF antibody type 1 in patients with HLA-A3, HLA-B7 and HLA-A3 and -B7 together in the same patient respectively are shown in Table 2. TABLE 1. Frequencies of HLA specificities in PA Controls HLA
PA = 42)
(n = 216)
(n
84 (39) 100 (46 2) 53 (24.5) 31 (14-3) 22 (10-2) 38 (17-6) 6 (2.8) 13 (6 0) 20 (9.25) 55 (25.5) 59 (27.3) 72 (33.3) 6 (2.7) 7 (3.2) 24 (11.1) 22 (10.2) 19 (8-8) 38 (17-6) 27 (12-5) 6 (2.8) 5 (2.3) 3 (1-3) 17 (7 9)
19 (45.2) 16 (38-1) 10 (23 8) 5 (11.9) 8 (19-0) 7 (16.7) 3 (7.1) 3 (7-1) 2 (4 8) 18 (43)* 13 (30-1) 9 (21-5)
Specificities HLA-A1 -A2 -A3 -A9 -A1O -All -A29 -AW32 -B5 -B7 -B8 -B12 -B13 -B14 -BW17 -B27 -BW35 -BW40 -BW15 -B18 -BW22 -BW37 HLA-A3, B7
0
(0)
2 (4.8) 2 (4 8) 3 (7-1) 2 (4.8) 9 (21-5) 3 (7-1) 0
(0)
2 (4 8) 1 (2.4) 9 (21-4)t
* For HLA-B7 statistically significant -0-327< P1 - P2< - 0*023 (P< 0-05 > 0-01). t For HLA-A3, B7 together in the same patient statistically significant -0-258< P1-P2 < 0-012
(P< 0-05> 0.01). TABLE 2. The incidence of IF antibody type 1 with HLA-A3, with HLA-B7 and with HLA-A3 plus -B7 PA patients
Total HLA-A3 HLA-B7
HLA-A3, B7
Number tested
Number positive
34 7 12 5
17 0 4 0
Age
(0%) 50 0 33-3 0
354
A. H. Goldstone et al.
The incidence of IF antibody corresponds to that normally found in PA (Irvine 1965) and no HL-A specificity was found to correlate with the presence of IF antibody.
DISCUSSION The finding of an increase in HLA-B7 and of HLA-A3/B7 together in PA confirms previous findings (Mawhinney et al., 1975; Zittoun et al., 1975), although disputed by others (Horton & Oliver, 1976) and is of considerable interest. There appears to be an association between gastric-autoimmune disease and HLA-A3 (Whittingham et al., 1975; Mawhinney et al., 1975) but our results confirm that this is not so for PA alone, although the HLA-A3, -B7 is increased in PA. We find, like others (Mawhinney et al., 1975), that a previous suggestion of a link between HLA specificity and the presence of IF antibody (Workshop, 1974) is not confirmed. Interestingly, a sibship has been reported in which the HLA-A3, B7 haplotype is associated with gastric autoimmune disease (McKay et al., 1974) and it would take detailed family studies to establish that HLA-A3 and B7 were together on the same chromosome in our own patients. The only other reports of an association between HLA-A3, B7 haplotype and disease appear to be with multiple sclerosis and high-titre measles antibody (Jersild et al., 1973) and with paralytic poliomyelitis (Pietsch & Morris, 1974). We wish to thank Dr J. Darnborough, Regional Transfusion and Immuno-Haematology Centre, Cambridge, for HLA typing facilities; Dr J. K. H. Rees for help in sample collection and Dr D. G. Chalmers for help with statistics. The patients studied were under the care of Professor F. G. J. Hayhoe and other physicians for Addenbrooke's Hospital, Cambridge. W.J.I. is supported by a grant from the Cancer Research Campaign.
REFERENCES LYNGSOE, J., PoULSEN, J.A., RYDER, L.P., NIELSEN, L.S., THOMSEN, M. & SVEJGAARD, A. (1974) HL-A antigens and diabetes mellitus. Lancet, ii, 864. 493. HORTON, M.A. & OLIVER, R.T.D. (1976) HL-A and perni- PIETSCH, M.C. & MORRIS, P.J. (1974) Association of HL-A3 and HL-A7 with paralytic poliomyelitis. Tissue Antigens, cious anaemia. New Engl. 7. Med. 294, 396 (letter). 4, 50. IRVINE, W.J. (1965) Immunological aspects of pernicious PLATZ, P., RYDER, L., STAUB NIELSEN, L., SVEJGAARD, A., anaemia. New Engl. J. Med. 273, 432. THOMSEN, M., NERUP, J. & CHRISTY, M. (1974) HL-A IRVINE, W.J. (1966) Immunoassay of gastric intrinsic factor and idiopathic Addison's disease. Lancet, ii, 289. and the titration of antibody to intrinsic factor. Clin. exp. WHITTINGHAM, S., YOUNGCHAIYUD, U., MACKAY, I.R., Immunol. 1, 99. BUCKLEY, J.D. & MORRIS, P.J. (1975) Thyrogastric JERSILD, C., AMMITZBOLL, T., CALUSEN, J. & FOG, T. (1973) autoimmune disease. Studies on the cell-mediated immune Association between HLA antigens and measles antibody system and histocompatibility antigens. Clin. exp. Immunol. in multiple sclerosis. Lancet, i, 151. 19, 289. MACKAY, I.R., WHITTINGHAM, S., MORRIS, P. & KING, A. (1974) Familial autoimmunity-immunological and WORKSHOP ON PERNICIOUS ANAEMIA, DIABETES MELLITUS AND ADDISON'S DISEASE (1974) Progress in Immunology, genetic analysis. Aust. N.Z. J. Med. 4, 105 (abstract). Volume II, Proceedings on the 2nd International Congress MAWHINNEY, H., LAWTON, J.M.W., WHITE, A.G. & IRVINE, of Immunology, p. 301. North-Holland, Amsterdam. W.J. (1975) HL-A3 and HL-A7 in Pernicious Anaemia and autoimmune atrophic gastritis. Clin. exp. Immunol. 22, ZITTOUN, R., ZITTOUN, J., SEIGNALET, J. & DAUSSET, J. (1975) HL-A and pernicious anaemia. New Engl. 5. Med. 293, 47. 1324 (letter). NERup, J., PLATZ, P., ANDERSON, 0.0., CHRISTY, M., GRUMET, C., KONISHI, J., PAYNE, R. & KRIss, J.P. (1973) Association of Graves disease with HLA-B8. Clin. Res. 21,
BRIEF COMMUNICATION
HLA antigens in pernicious anaemia A. H. GOLDSTONE,* D. VOAK,t J. C. CAWLEY* & W. J. IRVINE:*Department of Haematology, Addenbrooke's Hospital, t Regional Transfusion and Immuno-Haematology Centre, Cambridge, and $ Clinical Immunology, Royal Infirmary, Edinburgh (Received 20 February 1976)
SUMMARY The increased frequency of HLA-B7 alone and HLA-A3/B7 together, in the same patient, has been confirmed in pernicious anaemia. There is no increased prevalence of HLA-A3 alone. No association has been found between the presence of serum intrinsic factor type 1 antibody and HLA-A3 or HLA-B7.
INTRODUCTION The investigation of possible associations between the HLA system and disease has recently turned towards the area of organ-specific autoimmune disorders (Grumet et al., 1973; Platz et al., 1974; Nerup et al., 1974). It seems likely that there is an association between the antigens HLA-A3 and HLA-B7 and gastric-autoimmune disease (Whittingham et al., 1975; Mawhinney et al., 1975). In pernicious anaemia (PA) itself there may well be an increased incidence of HLA-B7 and HLA-A3/B7 together (Mawhinney et al., 1975; Zittoun et al., 1975). In this paper we report a study of HLA antigens in a further forty-two patients with PA and our results confirm and extend previous reports. PATIENTS AND METHODS Patients and controls. (a) Pernicious anaemia. A total of forty-two patients was studied; all were receiving maintenance B12 and had normal haematology. Diagnosis had been originally made by the presence of a macrocytic anaemia, megaloblastic bone marrow, low serum B12, achlorhydria and an abnormal vitamin B12 absorption test corrected by the addition of intrinsic factor. (b) Controls. 216 Random group 0 blood donors were typed concurrently with patients. HLA typing. Lymphocytes were separated from fresh ACD blood samples by sedimentation over Ficoll-Triosil. Phagocytic mononuclear cells were removed by incubation with carbonyl iron and extraction with a magnet. Typing for HLA specificities was carried out on a Terasaki type plate by a microlymphocytotoxicity technique. 2 pul of lymphocyte suspension at a concentration of 106 cells/ml were added to the test wells of the typing plates containing 2Al of the known anti-HLA antisera, left at room temperature for 30 min. Then 4 ul of complement (3 parts rabbit serum to 1 part human-group AB serum) were added to each test well. After incubation at room temperature for 1 hr, 1 p1 of trypan blue (0-2%/ in isotonic saline) was added to each well to which after 15 min I ul of1% bovine albumin and then 1 ul of 5%O acetic acid were added and the tests read on an inverted Wild microscope. Lymphocytes from patients and controls were tested for the following twenty-two antigens HLA-A1, -A2, -A3, -A9, -A10, -A29 and -AW32 at the first locus and HLA-B5, -B7, -B8, -B12, -B13, -B14, -BW17, -B27, -BW35, -BW40, -BW15, -B18, -BW22, -BW37 at the second locus. Three antisera were used for each of the common HLA specificities including HLA-A3 and HLA-B7. Controls and patients were tested on at least two separate occasions. Autoantibody studies. Serum intrinsic factor (IF) type 1 antibody was demonstrated by the charcoal absorption method (Irvine 1966). Statistics. The hypothesis that, on the basis of their reactivity against the specific sera listed, the control group and the patient group were drawn from the same population was tested using thex2 (Chi2) test. Where the tested number reacting against an individual antigen was less than 5, results were pooled. AX2 value of 12-104 with 12 degrees of freedom showed that Correspondence: Dr A. H. Goldstone, Department of Haematology, University College Hospital, London.
352
HLA antigen in pernicious anaemia
353
there was no significant difference at the 95% level. The difference at each antigen specificity was further tested by determining the confidence limits for the difference in proportions of each.
RESULTS The frequencies of the HLA-antigen specificities in the control population and in the PA patients are shown in Table 1. The incidence of serum IF antibody type 1 in patients with HLA-A3, HLA-B7 and HLA-A3 and -B7 together in the same patient respectively are shown in Table 2. TABLE 1. Frequencies of HLA specificities in PA Controls HLA
PA = 42)
(n = 216)
(n
84 (39) 100 (46 2) 53 (24.5) 31 (14-3) 22 (10-2) 38 (17-6) 6 (2.8) 13 (6 0) 20 (9.25) 55 (25.5) 59 (27.3) 72 (33.3) 6 (2.7) 7 (3.2) 24 (11.1) 22 (10.2) 19 (8-8) 38 (17-6) 27 (12-5) 6 (2.8) 5 (2.3) 3 (1-3) 17 (7 9)
19 (45.2) 16 (38-1) 10 (23 8) 5 (11.9) 8 (19-0) 7 (16.7) 3 (7.1) 3 (7-1) 2 (4 8) 18 (43)* 13 (30-1) 9 (21-5)
Specificities HLA-A1 -A2 -A3 -A9 -A1O -All -A29 -AW32 -B5 -B7 -B8 -B12 -B13 -B14 -BW17 -B27 -BW35 -BW40 -BW15 -B18 -BW22 -BW37 HLA-A3, B7
0
(0)
2 (4.8) 2 (4 8) 3 (7-1) 2 (4.8) 9 (21-5) 3 (7-1) 0
(0)
2 (4 8) 1 (2.4) 9 (21-4)t
* For HLA-B7 statistically significant -0-327< P1 - P2< - 0*023 (P< 0-05 > 0-01). t For HLA-A3, B7 together in the same patient statistically significant -0-258< P1-P2 < 0-012
(P< 0-05> 0.01). TABLE 2. The incidence of IF antibody type 1 with HLA-A3, with HLA-B7 and with HLA-A3 plus -B7 PA patients
Total HLA-A3 HLA-B7
HLA-A3, B7
Number tested
Number positive
34 7 12 5
17 0 4 0
Age
(0%) 50 0 33-3 0
354
A. H. Goldstone et al.
The incidence of IF antibody corresponds to that normally found in PA (Irvine 1965) and no HL-A specificity was found to correlate with the presence of IF antibody.
DISCUSSION The finding of an increase in HLA-B7 and of HLA-A3/B7 together in PA confirms previous findings (Mawhinney et al., 1975; Zittoun et al., 1975), although disputed by others (Horton & Oliver, 1976) and is of considerable interest. There appears to be an association between gastric-autoimmune disease and HLA-A3 (Whittingham et al., 1975; Mawhinney et al., 1975) but our results confirm that this is not so for PA alone, although the HLA-A3, -B7 is increased in PA. We find, like others (Mawhinney et al., 1975), that a previous suggestion of a link between HLA specificity and the presence of IF antibody (Workshop, 1974) is not confirmed. Interestingly, a sibship has been reported in which the HLA-A3, B7 haplotype is associated with gastric autoimmune disease (McKay et al., 1974) and it would take detailed family studies to establish that HLA-A3 and B7 were together on the same chromosome in our own patients. The only other reports of an association between HLA-A3, B7 haplotype and disease appear to be with multiple sclerosis and high-titre measles antibody (Jersild et al., 1973) and with paralytic poliomyelitis (Pietsch & Morris, 1974). We wish to thank Dr J. Darnborough, Regional Transfusion and Immuno-Haematology Centre, Cambridge, for HLA typing facilities; Dr J. K. H. Rees for help in sample collection and Dr D. G. Chalmers for help with statistics. The patients studied were under the care of Professor F. G. J. Hayhoe and other physicians for Addenbrooke's Hospital, Cambridge. W.J.I. is supported by a grant from the Cancer Research Campaign.
REFERENCES LYNGSOE, J., PoULSEN, J.A., RYDER, L.P., NIELSEN, L.S., THOMSEN, M. & SVEJGAARD, A. (1974) HL-A antigens and diabetes mellitus. Lancet, ii, 864. 493. HORTON, M.A. & OLIVER, R.T.D. (1976) HL-A and perni- PIETSCH, M.C. & MORRIS, P.J. (1974) Association of HL-A3 and HL-A7 with paralytic poliomyelitis. Tissue Antigens, cious anaemia. New Engl. 7. Med. 294, 396 (letter). 4, 50. IRVINE, W.J. (1965) Immunological aspects of pernicious PLATZ, P., RYDER, L., STAUB NIELSEN, L., SVEJGAARD, A., anaemia. New Engl. J. Med. 273, 432. THOMSEN, M., NERUP, J. & CHRISTY, M. (1974) HL-A IRVINE, W.J. (1966) Immunoassay of gastric intrinsic factor and idiopathic Addison's disease. Lancet, ii, 289. and the titration of antibody to intrinsic factor. Clin. exp. WHITTINGHAM, S., YOUNGCHAIYUD, U., MACKAY, I.R., Immunol. 1, 99. BUCKLEY, J.D. & MORRIS, P.J. (1975) Thyrogastric JERSILD, C., AMMITZBOLL, T., CALUSEN, J. & FOG, T. (1973) autoimmune disease. Studies on the cell-mediated immune Association between HLA antigens and measles antibody system and histocompatibility antigens. Clin. exp. Immunol. in multiple sclerosis. Lancet, i, 151. 19, 289. MACKAY, I.R., WHITTINGHAM, S., MORRIS, P. & KING, A. (1974) Familial autoimmunity-immunological and WORKSHOP ON PERNICIOUS ANAEMIA, DIABETES MELLITUS AND ADDISON'S DISEASE (1974) Progress in Immunology, genetic analysis. Aust. N.Z. J. Med. 4, 105 (abstract). Volume II, Proceedings on the 2nd International Congress MAWHINNEY, H., LAWTON, J.M.W., WHITE, A.G. & IRVINE, of Immunology, p. 301. North-Holland, Amsterdam. W.J. (1975) HL-A3 and HL-A7 in Pernicious Anaemia and autoimmune atrophic gastritis. Clin. exp. Immunol. 22, ZITTOUN, R., ZITTOUN, J., SEIGNALET, J. & DAUSSET, J. (1975) HL-A and pernicious anaemia. New Engl. 5. Med. 293, 47. 1324 (letter). NERup, J., PLATZ, P., ANDERSON, 0.0., CHRISTY, M., GRUMET, C., KONISHI, J., PAYNE, R. & KRIss, J.P. (1973) Association of Graves disease with HLA-B8. Clin. Res. 21,
