Tissue Antigens (1976), 7, 239-242 Published by Munksgaard, Copenhagen, Denmark N o part may be reproduced by any process without written permission from the author@)

HLA Antigen (A & B Loci) Frequency in Addisonian Pernicious Anaemia M.A. Horton’ and R.T.D. Oliver’ ‘Department of Haematology; and ‘I.C.R.F. Department of Medical Oncology, St. Bartholomew’s Hospital and Medical College, London, England Sixty-six patients with Addisonian pernicious anaemia have been HLA typed and compared with 86 controls of the same ethnic group. N o deviation in antigen frequency was found to be specific for the disease group as a whole. T h e most significant deviation found was an increased frequency of HLA-B8 in those patients with coincident thyroid autoantibodies. Pernicious anaemia seems to be the exception to the demonstrated association between HLA-B8 and the organ specific autoimmune diseases studied u p to the present time. Received for publication 26 January, accepted 1 1 February 1976

Although the aetiology of Addisonian perni cious anaemia (PA) is not fully understood and involves both immunological and genetic factors, it is considered by many workers to be a member of the group of organ specific autoimmune diseases. Other conditions within this group, including autoimmune thyroiditis and adrenal failure, have all shown an associated, but variable, increase in frequency of the histocompatibili t y B locus antigen HLA-B8 (Svejgaard et al. 1975). It has been suggested that the association with this antigen may be the common denominator for the production of organ specific autoantibodies in certain autoimmune diseases (Nerup et al. 1974). With this in mind a series of 66 cases of PA have been HLA typed in order to see if this relationship holds true for a disease involving autoimmune reactivity towards gastric antigens and whether this might throw some light upon the pathogenesis of this disease.

Materials and Methods Patients T h e 66 cases of PA studied were selected retrospectively from the various medical clinics at St. Bartholomew’s Hospital. Altogether 24 were men and 42 were women, their ages at the time of initial presentation ranging from 31 to 82 years (mean 60.0 years). Diagnosis was based upon the usual criteria of vitamin B12 malabsorption corrected by intrinsic factor in the absence of previous gastric surgery, vitamin B12 deficiency leading to megaloblastic anaemia or neurological damage, characteristic gastric changes and, in the majority of cases, autoantibodies to gastric parietal cells or intrinsic factor. Serological Methods Autoantibodies to gastric parietal and thyroid antigens were detected in the serum by a standard sandwich immunofluoresc-

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ence technique (Roitt & Doniach 1969). Thyroglobulin antibody was detected by tanned red cell haemagglutination. Intrinsic factor vitamin Bi2 binding site blocking antibodies (Type I) were estimated by the zerconium gel method of Hansen et al. (1966). All sera were collected and stored at -20°C and tested within 1 month. HLA Typing HLA typing was performed using the platelet complement fixation (Colombani et al. 1970) and standard N.I.H. lymphocytotoxicity tests (Brand et al. 1970) with sera defining nine antigens of the A locus and 15 antigens of the B locus, as well as the complex specificities, 4a and 4b. T h e subjects in the control series for HLA antigen frequencies were all healthy staff members from the St. Bartholomew’s and Royal Marsden Hospitals. Results Of the 66 cases studied 51 (77%) had antibodies to gastric parietal cell antigens, 2 1

(37%) thyroid cytoplasmic or microsomal antibodies and 19 (29%) to gastric intrinsic factor. T h e frequencies of HLA antigens of the A and B loci in the patients with PA and controls are shown in Table 1. As can be seen there are no significant deviations in antigen frequency, particularly of HLA-B8, when controls and patients are compared. N o relationship was found to the antigens 4a and 4b. Table 2 shows the frequencies of antigens HLA-A1, 2 and 3 and HLA-B7,8 and € 2 in controls and cases of PA with and wothout thyroid and intrinsic factor antibodies. There is no significant association between any of the HLA antigens or phenotypes ( A l ; B8-A2; B12-A3; B7) and the presence o r absence of Type I intrinsic factor antibodies. A weak association between the presence of thyroid autoantibodies and HLA-B8 (x2 = 5.0, P = 0.025), but not with the phenotype A l ; B8, within the disease population studies has been demonstrated.

Table 1 H L A antiEen frequencyin battents with Addzsontan bernicious anaemia Locus A Locus B

HLA-A 1 A2

Controls*

Patients

(n = 86)

(n

.35 .43

66) .32 .42

Controls (n = 86)

=

HLA-B5

.08

B7

.22 .28 .35 .01

.2 1

.26(21**

B8

.20

B12

.I2 .20

B13

All

.20 .07 .14

A28

.08

.08

AW 30+31

.27 .2 I

.23

A3 A9 A10

Blank ‘4a’ ‘4b

.17

.52

.54

.8 1

.9 I

B14 B18 B27

Patients (n = 66)

.06

.27(3)** .21“’

.2 1

.I0

.05 .08

.04

.03

.08

BW35 BW40

.09

.15 .12

.08

.17

BW17 BW21

.I0 .06

.12 .06

.03 .03 .03 .33

24 1

HLA ANTIGENS I N PERNICIOUS- ANAEMIA

Table 2 H L A antigen frequency i n patients with thyroid and intrinsic factor antibodies Controls (n = 86)

HLA-A 1 A2

A3 HLA-B7 B8 B12 ~~~

.35 .43 .21 .22 .28 .35

Thyroid antibody positive

Thyroid antibody negative

( n = 21, 37%)

(n = 45)

.33* .33 .I4 .29 .381.* .19

.31 .47 .31 .24 .I3 .22

I.F,Ab, +ve ( n = 19;29%)

.21** .47 .16 .26 .I@.** .32

I,F,Ab. -ve (n = 47)

.36 .40 .30 .26 .23 .17

~

1 . B8, Thyroid Ab. pos. vs. neg. X2 = 5.0, P = 0.025. 2. B8, I.F. Ab. pos. vs. neg. X2 = 0.47, P = N.S. * Phenotype Al-B8. Thyroid Ab. pos. vs. neg. X2 = 1.72, P = N.S. ** Phenotype AI-B8. I.F. Ab. pos. vs. neg. X2 = 1.72, P = N.S.

Discussion No overall relationship has been found between any HLA antigen and the disease population studied. Unlike the other organ specific autoimmune diseases there is no association with the antigen HLA-B8, a possible explanation for this being that the group of patients studied is not homogenous, with different mechanisms producing the same resultant syndrome. However, if the results are re-analysed, with a consequent reduction in disease heterogeneity, for the presence or absence of serum intrinsic factor antibodies, there is no significant alteration in any HLA antigen or phenotype frequency. This is in contrast to the association between HLA-A3 and serum intrinsic factor antibody reported in a group of patients with pernicious anaemia (Workshop 1974). In this study no association was found with the HLA specificities A3 and B7 or the phenotype A3; B7, as has been previously reported by Whittingham et al. ( 1 9751, Mawhinney et al. (1975) and Zittoun et al. (1975) from studies in smaller groups of patients. However, when the data from these series, including our own, is combined there is a marginally statistically significant excess of HLA-B7 (Svejgaard, personal

communication), which justifies further investigation. Multiple sclerosis and paralytic polio are the only diseases to show an association with HLA-B7 in previous studies. As a proportion of patients with PA develop neurological complications (peripheral neuropathy o r subacute combined degeneration of the cord) it will be important to assess the neurological status of HLA-B7 positive and negative patients, though to date there is no clear cut association of neurological disease with HLA-B7 in our patients. There is no demonstrable age or sex difference in the frequency of HLA-B8, in our series, as has been shown to occur in both juvenile onset diabetes mellitus and cases of myasthenia gravis with thymic hyperplasia (Feltkamp et a]. 1974, Nerup et al. 1974), though few early onset PA’s were included in this series. A weak association between HLA-B8 and the presence of thyroid autoantibodies in the serum of the cases investigated has been demonstrated, but this correlation is not significant if a correction for the number of possible phenotypes analysed is made. It has been suggested that the presence of HLA-B8 is the common denominator for the production of organ specific autoantibodies in certain conditions with a probable

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autoimmune pathogenesis. It is possible that any association between HLA-B8 and these diseases, for instance autoimmune adrenalitis, may be fortuitous and only related to the increased frequency of thyroid antibodies. However, from the literature it is clear that HLA-B8 is only associated with Graves’ disease and not with the other thyroid diseases with a n autoimmune pathogenesis (Bode et al. 1973, Grumet et al. 1973, Nelson & Pollet 1975, Richiardi et al. 1975). Furthermore, it must be remembered that, in the age group studied (the majority of patients being over 50 years old), serum thyroid and parietal cell autoantibodies are frequently present in “normals” with no evidence of PA or thyroid disease (Doniach & Roitt 1964). PA would thus seem to be an exception to the association between HLA-B8 and the organ specific autoimmune disease, with no increased frequency of this antigen in the disease group studied and only a slight increase in those patients with thyroid antibodies. Acknowledgements We thank Professor D.L. Mollin for helpful discussions and the physicians of St. Bartholomew’s Hospital for allowing us to study patients under their care. Financial support for this work was provided by I.C.R.F. (RDTO), a Lawrence Research Scholarship and the Welcome Trust (M.A.H.). References Bode, H., Dorf, M.E. & Forbes, A.P. (1973) Familial lymphocytic thyroiditis, analysis of linkage with histocompatibility and blood groups. J. clin. Endoc. 37, 692-697. Brand, D.L., Ray, J.G., Hare, D.B., Kayhoe, D. & McClelland, J.D. (1970) Preliminary trials towards standardisation of leucocyte typing. Histocompatibility Testing 1970, p. 357-367. Munksgaard, Copenhagen. Colombani, M., Colombani, J , Dehay, C. & Dausset, J. (1970) A microtechnique of platelet complement fixation. Histocompatibility Testing 1970, p. 553-557. Munksgaard, Copenhagen. Doniach, D. & Roitt, M. (1964) An evaluation of

gastric and thyroid autoimmunity in relation haematologic disorders. Sem. Haemat. 1, 313. Feltkamp. T.E.W., Van den Berg-Loonen, P.M., Nijenhuis, L.E., Engelfriet, C.P., Van Rossum, A.L, Van Loghem, J.J. & Oosterhuis, H.J.G.H. (1974) Myasthenia gravis, autoantibodies and HLA antigens. Brit. med. J. 1, 131-133. Grumet, C., Konishi, T., Payne, R.D. & Kriss, J.P. (1973) Association of Graves’ disease with HLA-8. Clin. Res. 21, 493. Hansen, H.J., Miller, O.N. & Tan, C.H. (1966) Assay of the autohumoral antibody that neutralises the vitamin B,, combining site of intrinsic factor in serum from patients with pernicious anaemia. Amer. J . Clin. Nutr. 19, 10-16. Mawhinney, H., Lawton, J.W.M., White, A.G. & Irvine, W.J. (1975) HL-A3 and HL-A7 in pernicious anaemia and autoimmune atrophic gastritis. Clin. exp. Immunol. 22, 47-53. Nelson, S.D. & Pollet, J.E. (1975) HLA antigens and thyrotoxicosis. Tissue Antigens 5 , 38-40. Nerup, J., Platz, P., Ortved Anderson, O., Christy, M., Lyngs@e,J., Poulsen, J.E., Ryder, L.P., Staub Nielsen, L., Thomsen, M. & Svejgaard, A. (1974) HLA antigens and diabetes mellitus. Lancet ii, 864-866. Richiardi, P., Salabe, G.B., Lucertinii, L. & Curtoni, E.S. (1975) Absence of linkage between the HLA system and production of antithyroglobulin antibodies. Tissue Antigens 5, 213-215. Roitt, M. & Doniach, D. (1969) Manual of Autoimmiine Serology. World Health Organisation, Geneva. Svejgaard, A,, Platz, P., Ryder, L.P., Staub Nielsen, L. & Thomsen, M. (1975) HLA and disease associations - a survey. Transplant. Rev. 22, 3-43. Whittingham, S., Youngchamud, U., Mackay, I.R., Buckley, J.D. & Morris, P.J. (1975)Thyrogastric autoimmune disease. Studies on the cell-mediated immune system and histocompatibility antigens. Clin. exp. Immunol. 19, 289-299 Workshop on pernicious anaemia, diabetes rnellitus and Addison’s disease (1974) Progress in Immunology, vol. 2, Proceedings of the 2nd International Congress of Immunology, p. 301. North-Holland, Amsterdam. Zittoun, R., Zittoun, J., Seignalet, J. & Dausset, J. (1975) Letter, HLA and pernicious anaemia. New Engl. f . Med. 293, 1324. Address: M . A . Horton Department of Haematology St. Bartholomew’s Hospital London, EClA 7BE, England