Journal of Neuroimmunology, 27 (1990) 149-153 Elsevier
149
JNI 00916
Major histocompatibility complex class I and class II polymorphism in chronic idiopathic demyelinating polyradiculoneuropathy R.W, Vaughan
1 A.M. A d a m
2, I.A. G r a y 2, R.A.C. Hughes 2, E.A.C.M. Sanders M. van D a m a and K.I. Welsh a
2,
Departments of 1 Molecular Immunogenetics and 2 Neurology, United Medical and Dental Schools of Guy's and St Thomas's Hospitals, Guy's Campus, London SE1 9RT, UK. (Received 3 August 1989) (Revised, received 19 September 1989) (Accepted 20 September 1989)
Key words: Histocompatibility antigen; Chronic idiopathic demyelinating polyradiculoneuropathy; HLA-Cw7
Summa W
Thirty-one chronic idiopathic demyelinating polyradiculoneuropathy (CIDP) patients have been typed for HLA-A, -B and -C antigens serologically and for HLA-DR, -DQ and -DP class II genes by RFLP analysis. Our results confirm a previously reported slight association with HLA-B8 and identify a stronger association with HLA-Cw7.
Introduction
Chronic idiopathic demyelinating polyradiculoneuropathy (CIDP) is a slowly progressive disorder of the peripheral nervous system for which autoimmune mechanisms have been proposed (Dyck and Arnason, 1984). It resembles GuillainBarr6 syndrome (GBS) but pursues a more prolonged course. GBS is usually monophasic reaching its nadir within 4 weeks whereas CIDP progresses over 3 or more months and often pursues a relapsing and remitting course (McCombe et al., 1987). For epidemiological purposes it is convenient to set an arbitrary boundary for the duration
Address for correspondence: Prof. R.A.C. Hughes, Department of Neurology, Guy's Hospital, London SE1 9RT, U.K.
of the time from onset to nadir for GBS of 4 weeks (Hughes and Winer, 1984; Winer et al., 1988b). This definition leaves a small proportion of patients with progressive disease reaching a nadir in more than 4 but less than 12 weeks: we have included these patients in a subacute category of the CIDP group. CIDP has only been loosely defined on a combination of clinical features and electrophysiological evidence of non-uniform slowing of conduction and multifocal conduction block (Dyck and Arnason, 1984; Albers, 1987). The pathological substrate is a multifocal demyelinating process in which macrophages strip and phagocytose the myelin sheath. Lymphocytic infiltration is rarely observed in peripheral nerve biopsies but rare autopsy studies have revealed perivascular infiltration of cells in spinal roots (Dyck and Arnason,
0165-5728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
150 1984). This histological evidence of an immune response in the peripheral nerves and its resemblance to chronic experimental allergic neuritis (EAN) induced in rats by immunisation with myelin or P2 protein and in rabbits with myelin or galactocerebroside have led to the proposal that CIDP is an autoimmune condition (Craggs et al., 1986; Harvey et al., 1987; Adam et al., 1989). The major histocompatibility complex (MHC) on chromosome 6 contains HLA genes essential to the control of the immune response. Stewart et al. (1978) found a significant association with HLAA30 and -A31 and probable associations with HLA-B8 and -Dw3 in 16 Australian CIDP patients. Adams et al. (1979) did not find an increased frequency of HLA-A30 and A31 but did report an association with HLA-1, -B8, -DRW3 and -DW3 in 14 patients in the U.K. We report the HLA types of a further series of 31 Caucasoid CIDP patients.
Patients and methods
Consecutive Caucasoid patients, encountered by one of the authors, who fulfilled the following diagnostic criteria for CIDP were studied: (1) progressive weakness of two or more limbs due to peripheral neuropathy for more than 4 weeks; (2) diminution or loss of tendon reflexes; (3) absence of family history or clinical features of hereditary motor and sensory neuropathy, diabetes mellitus, paraproteinaemia, toxin exposure or other known cause of demyelinating neuropathy; (4) electrophysiological evidence of demyelinating neuropathy according to criteria similar to those of Albers (1987). The clinical course was considered subacute if the nadir was reached more than 4 but less than 12 weeks from the onset, chronic progressive if the nadir was reached after more than 12 weeks and chronic relapsing if the progressive course was interrupted by remissions followed by relapses. A remission was defined as improvement of one disability grade for at least one week, and a relapse as a worsening of one clinical grade for at least one week. The clinical grades were modified from those used by Hughes et al. (1978): 0 = healthy; 1 = minor symptoms or signs; 2 = able to
walk 5 metres without assistance; 3 = able to walk 5 metres with assistance; 4 = chair b o u n d / b e d bound; and 5 = requiring assisted ventilation. Patients who had recurrent attacks of acute neuropathy resembling GBS in reaching their nadir within 4 weeks were included as a separate recurrent acute group.
HLA typing 30 ml of blood were taken from each patient or control. 20 ml aliquots were anticoagulated with 5% EDTA and stored at - 20 ° C before extraction of DNA. 10 ml were anticoagulated with 4% sodium citrate and used for serological HLA-A, -B and -Cw typing as described (Welsh and Batchelor, 1978), with cell viability assessed by phasecontrast microscopy. Controls for the HLA-A, -B and -Cw analysis consisted of 346 volunteer normal Caucasoid subjects. The class II study was conducted by analysis of the R F L P pattern of genomic DNA restricted with the endonucleases TaqI or MspI. D N A was isolated from blood, digested, electrophoresed and blotted as described (Demaine et al., 1987) onto Hybond N (Amersham International). Hybridisation was performed in glass tubes in Bachoffer rotisserie ovens. The following class II probes were used: DRB (pII-fl-4, SacI and HindIII fragment) (Gustafsson et al., 1984a), DQA (pII-a-5, ApaI fragment) (Schenning et al., 1984), DQB (pII-/~-I, AuaI fragment) (Larhammar et al., 1982), DPA (pDAdl3b, EcoRI fragment) (Trowsdale et al., 1985), DPB (pII-/~-7, RsaI and HpaII fragment) (Gustafson et al., 1984b). The DPB probe was hybridised with D N A digested with the restriction enzyme MspI. All the other probes were hybridised with TaqI-digested DNA. The autoradiographs were assessed and the typing scheme was based on our own observations and the work of others. The DRB definition follows Bidwell et al. (1987) and Bidwell (1988). The simplified DQ assignment defines DQw5, DQw6, DQw2, DQw7, DQw8 and DQw9. The DQwl splits are defined by the DQA probe where DQw5 corresponds to Bidwell's D Q l a and DQw6 refers to bands at 6 or 6.5 kb. The DQw2 and DQw7 and DQw8 specifities are defined using the DQB probe. DQw9 is defined by the DRB 7b pattern and the DQw8 DQB band at 1.9 kb. DR7 DQw9 and DR9 DQw9
151
cannot be separated in this way and are referred to as D R 7 / 9 . The DP definition is a short-hand nomenclature and is based on the work of Hyldig-Neilsen et al. (1987). The correlation between DP R F L P and D P alleles may be slightly less accurate than that for D R and DQ; the following simplified scheme has been adopted: D P w l has a MspI DPB band at 4.5 kb;
-
DPw2 has a TaqI D P A band at 13 kb and a band at 2.1 kb; D P w 3 / 6 has a TaqI D P A band at 6.3 kb and a MspI DPB band at 3 kb; DPw4 has a TaqI D P A band at 14 kb and a MspI DPB band at 1.1 kb; DPw5 has a TaqI D P A band at 6.5 kb; DP CP63 has a MspI DPB band at 1.8 kb. The significance of the results was tested with
MspI DPB -
-
-
-
-
TABLE1 C L I N I C A L F E A T U R E S A N D H L A P O L Y M O R P H I S M O F CIDP PATIENTS Code
Age
Sex
Duration
Worst
H L A class I serology
(years)
disability grade
HLA-A
Chronic progressive 1 65 M 2 61 M 3 51 M 4 38 M 5 49 F 6 39 M 7 66 M 8 35 M
0.5 1.0 1.0 3.0 5.0 6.0 7.0 9.0
3 3 4 4 2 2 3 3
29, 1, 1, 3, 1, 2, 1, 2,
Chronic relapsing 9 26 M 10 55 M 11 29 F 12 47 M 13 21 M 14 60 F 15 34 M 16 32 M 17 25 M 18 27 M 19 86 F 20 34 M 21 68 M 22 64 M 23 49 M 24 60 M
0.5 1.0 1.0 2.0 2.0 5.0 5.0 6.0 6.0 6.0 7.0 8.0 16.0 18.0 20.0 27.0
2 4 5 4 4 4 5 3 3 4 4 4 2 4 3 4
2, 31 1, 2 1, 2 3, 1, 2, 24 2, 29 1, 2 11, 25 1, 11 1, 2 2, 11 3, 1, 11 3, 24 1, 2
Recurrent acute 25 62 26 48 27 62 28 88
M M M M
13.0 15.0 29.0 45.0
4 5 5 4
2, 2, 1, 2,
Subacute 29 31 30 46 31 2l
F M M
0.5 0.5 1.5
2 4 4
1, 31 3, 30 2, 3
N.T., not tested.
32 2 26 29 31 3 32
3 11 25 4
HLA-B
8, 7, 8, 7, 8, 7, 8, 7,
44 62 71 44 39 18 60
HLAclass IlRFLP HLA-C
HLA-DR
HLA-DQ
HLA-DP
5, 3, 7, 7, 7, 7, 7, 3,
4, 4, 2, 2 1, 3, 3 2,
w2, w6, w2, w6 w5, w2, w2 w6,
N.T. 2 4 3/6 4 4, CP63 2, CP63 4
7 7 7
3 w13 3 5 w13 4
w3 w7 w6 w7 w6 w7
7, 39 13, 58 8, 44 7, 14 55, 57 8, 60 7, 35 8, 51 18, 27 8, 27 8, 14 14, 35 27, 35 7, 8 7, 18 8, -
7, 6, 7 5, 7 7, 3, 6 3, 7 4, 7 7, 2, 1, 7 7, 4, 2, 3 7, 7, 7, -
2, w13 5, 7 4, 7 1, w13 4 3, w13 2, w13 2, 4 7 w13, w14 3, w13 1, 4 2 2, 3 5 3
w6 w7, w2, w5, w7, w2, w6 w6, w2, w5, w2, w5, w6 w2, w7 w2
7, 7, 8, 39,
5, 7, 7, 3,
7 7
4, w13 2, w14 2, 3 4
w6, w7 w5, w6 w6, w2 w7
4, 3 / 6 4, 3 4 4, 3 / 6
7, 7, 1, -
3 4, w13 4
w2 w3, w6 w7
N.T. 2, 1 / 3 / 6 CP63
44 51 18 62
8, 51 7, 44 51, 56
w2 w8 w6 w8 w6 w3 w9 w6 w6 w3 w6
2 4, CP63 3, 4 N.T. 4, 3 / 6 N.T. 2, CP63 2 4 4, 3 / 6 1, 5 2 4, 6 4 5 4, 1
152
the chi-square test and two-tailed probabilities are given. They have not been corrected for the number of antigens tested on the grounds that we are seeking to confirm an association with HLA-A1, -B8 and -DR3 (then -Dw3) which has been identified in two separate previous studies.
Results and discussion
Our results confirm the previously reported small increase in frequency of HLA-A1 and HLAB8 in CIDP patients (Stewart et al., 1978; Adams et al., 1979) and show a similar small increase in HLA-B7 (Tables 1 and 2). The most significant association in our series of CIDP patients was with HLA-Cw7. The significance of the association has to be interpreted with caution since the p value has not been corrected for the number of HLA class I allotypes tested (80). Since there is strong linkage disequilibrium between HLA-Cw7 and both the B locus antigens HLA-B7 and HLA-B8, an association with a gene close to these B locus antigens might cause the observed increased frequency of HLA-Cw7. However, our study of M H C class II genes at the D N A level, the first to be published in this disease, suggests that this is not the explanation. Linkage disequilibrium with HLA-B8 and HLA-B7 would usually be associated with an increase in HLADR3 and H L A - D R 2 respectively, but the frequency of these antigens was not increased. The
TABLE 2 THE FREQUENCY OF HLA ANTIGENS A1, B7, B8, Cw7 AND CLASS II RFLP FOR DR2, DR3 AND DQw6 IN CIDP PATIENTS AND CONTROLS CIDP
HLA-A1 HLA-B7 HLA-B8 HLA-Cw7 HLA-DR2 HLA-DR3 HLA-DQw6
Controls
p
Ratio a
Percent
Ratio a
Percent
14/31 12/31 13/31 26/31 10/31 10/31 18/31
45.2 38.7 41.9 83.8 32.2 32.2 58
118/346 74/346 85/346 184/346 18/60 21/60 26/60
34.1 21.4 24.6 53.2 36 33 43.3
0.29 0.05 0.06 0.002 0.98 0.97 0.27
a Number of patients positive/number of patients tested.
association between CIDP and HLA decreases with genomic distance from HLA-C so that if there is a CIDP susceptibility gene on chromosome 6 it is likely to be closer to the C locus than the DR, A or B loci and in linkage dysequilibrium with Cw7. There was no significant distortion of the frequency of R F L P detected with the DP, DQ or D R probes in CIDP patients apart from a small but non-significant increase in DQw6 (Table 2). Our study did not confirm a weak association with DR3 reported in two earlier studies (Stewart et al., 1978; Adams et al., 1979). This may be due to the smaller numbers in those studies or improvements in class II definition in the last 10 years. The small numbers in clinical subgroups preclude drawing conclusions about differences in the frequency of class I or II antigens but the details are presented for comparison with future studies. Subdivision according to progressive or relapsing disease course, or disease severity did not strengthen the association with Cw7 and any other group, except that Cw7 was present in all seven patients with a chronic progressive course and all four with a recurrent acute course. The association of CIDP with a class I allele invites speculation about mechanisms. We propose that the class I allele is itself a disease susceptibility gene although since Cw7 is common and CIDP rare polygenic influence and environmental factors must also be important. Class I molecules usually contain endogenous peptides in their antigen binding cleft (Bjorkman, 1987). These endogenous peptides may be bound at the time of synthesis or assembly of the class I molecule. If the cell becomes infected, virally derived peptides may replace the usual endogenous peptide (Townsend, 1987). The binding of a viral peptide in the HLA-Cw7 cleft might trigger individuals who are predisposed by yet other genes to an aberrant autoimmune response presenting as CIDP. Alternatively minor myelin damage by viral infection, trauma or other factor might release myelin antigens which bind to the HLA-Cw7 cleft and stimulate an autoimmune response. A third possibility is that HLA-Cw7 or a linked gene is not responsible for the initiation of CIDP but for the failure to limit the disease to a monophasic illness. This possibility is supported by the finding
153
of a slightly decreased frequency of HLA-Cw7 in Guillain-Barr6 syndrome (Winer et al., 1988a).
References Adam, A.M., Atkinson, P.F., Hall, S.M., Hughes, R.A.C. and Taylor, W.A. (1989) Chronic experimental allergic neuritis in Lewis rats. Neuropathol. Appl. Neurobiol. 15, 249-264. Adams, D., Gibson, J.D., Thomas, P.K., Batchelor, J.R., Hughes, R.A.C., Kennedy, L., Festenstein, H. and Sachs, J. (1977) HLA antigens in Guillain-Barr~ syndrome. Lancet 2, 504-505. Adams, D., Festenstein, H., Gibson, J.D., Hughes, R.A.C., Jaraquemada, J., Papasteriadis, C., Sachs, J. and Thomas, P.K. (1979) HLA antigens in chronic relapsing idiopathic inflammatory polyneuropathy. J. Neurol. Neurosurg. Psychiatry 4, 184-186. Albers, J.W. (1987) Inflammatory demyelinating polyradiculoneuropathy. In: W.F. Brown and C.F. Bolton (Eds.), Clinical Electromyography, Butterworth, Boston, MA. Asbury, A.K. (1981) Diagnostic considerations in GuillainBarr6 syndrome. Ann. Neurol. (suppl. 9), 1-5. Bidwell, J. (1988) DNA-RFLP analysis and genotyping of HLA-DR and DQ antigens. Immunol. Today 9, 18-23. Bidwell, J.L., Bidwell, E.A., Lanndy, G.J., Klouda, P.T. and Bradley, B.A. (1987) Allogenotypes defined by short DQd and DQ beta cDNA probes correlate with and define splits of HLA-DQ serological specificities. Mol. Immunol. 24, 513-522. Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennet, W.S., Strominger, J.L. and Wiley, D.C. (1987) Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329, 506-511. Craggs, R., Brosnan, J.V., King, R.H.M. and Thomas, P.K. (1986) Chronic relapsing experimental allergic neuritis in Lewis rats: effects of thymectomy and spleenectomy. Acta Neuropathol. 70, 22-29. Demaine, A.G., Vaughan, R.W., Taube, D.H., Williams, D.G. and Welsh, K.I. (1987) T-cell receptor beta chain and immunoglobulin heavy chain switch region gene polymorphisms are associated with membranous nephropathy. Clin. Sci. 72 (Suppl. 16), 46-51. Dyck, P.J. and Arnason, B.G.W. (1984) Chronic inflammatory demyelinating polyradiculoneuropathy. In: P.J. Dyck, P.K. Thomas, E.H. Lambert and R. Bunge (Eds.), Peripheral Neuropathy, Saunders, Philadelphia, PA, pp. 2101-2114. Gorodezky, C., Varela, B., Castro-Escobar, L.E., ChavezNegrete, Escobar-Guitierrez, A. and Martinez-Mata, J. (1983) HLA-DR antigens in Mexican patients with Guillain-Barr~ syndrome. J. Neuroimmunol. 4, 1-7. Gustafsson, K.,. Wiman, K., Emmoth, E., Larhammar, D., Bohme, J., Hyldig-Nielsen, J.J., Ronne, H., Peterson, P.A. and Rask, L. (1984a) Mutations and selection in the generation of class II histocompatibility antigen polymorphism. EMBO J. 3, 1655-1661.
Gustafsson, K., Emmoth, E., Widmark, E., Bohme, J., Peterson, P. and Rask, L. (1984b) Isolation of a cDNA clone coding for an SB beta-chain. Nature 309, 76-78. Harvey, G.K., Pollard, J.D., Schindhelm, K. and Antony, J. (1987) Chronic experimental allergic neuritis. An electrophysiological and histological study in the rabbit. J. Neurol. Sci. 81,215-226. Hughes, R.A.C., Newsom Davis, J.M., Perkin, G.D. and Pierce, J.M. (1978) Controlled trial of prednisolone in acute polyneuropathy. Lancet 2, 750-753. Hughes, R.A.C., Winer, J.B. (1984) Guillain-Barr6 syndrome. In: W.B. Matthews and G.H. Glaser (Eds.)0 Recent Advances in Clinical Neurology, Vol. 4, Churchill-Livingstone, Edinburgh, pp. 19-49. Hyldig-Nielsen, J.J., Morling, N., Odum, N., Ryder, L.R., Platz, P., Jakobsen, B. and Svejgaard, A. (1987) Rectriction fragment length polymorphism of the HLA-DP subregion and correlations to HLA-DP phenotypes. Proc. Natl. Acad. Sci. U.S.A. 84, 1644-1648. Larhammar, D., Schenning, L., Gustafsson, K., Wiman, K., Claesson, L. and Rask, L. (1982) Complete amino acid sequence of an HLA-DR antigen-like beta chain as predicted from the nucleotide sequence: similarities with immunoglobulins and HLA-A, -B, and -C antigens. Proc. Natl. Acad. Sci. U.S.A. 799, 3687-3691. McCombe, P.A., Pollard, T.D. and McLeod, J.G. (1987) Chronic inflammatory demyelinating polyradiculoneuropathy. Brain 110, 1617-1630. Scherming, L., Larhammar, D., Bill, P., Wiman, K., Jonsson, A.-K., Rask, L. and Peterson, P.A. (1984) Both alpha and beta chains of HLA-DC class II histocompatibility antigens display extensive polymorphism in their amino-terminal domains. EMBO J. 3, 447-452. Stewart, G.J., Pollard, J.D., McLeod, J.G. and Wolnizer, C.M. (1978) HLA antigens in the Landry-Guillain-Barr6 syndrome and chronic relapsing polyneuritis. Ann. Neurol. 4, 285-289. Townsend, A.R.M. (1987) Recognition of influenza virus proteins by cytotoxic T lymphocytes. Immunol. Res. 6, 80-100. Trowsdale, J., Young, J.A.T., Kelly, A.P., Austin, P.J., Carson, S., Meunier, H., So, A., Ehrlich, H.A., Spielman, R.S., Bodmer, J. and Bodmer, W.F. (1985) Structure, sequence and polymorphism in the HLA-D region. Immunol. Rev. 85, 5-43. Welsh, K.I. and Batchelor, J.R. (1978) Assays for antibodies against histocompatability antigens. In: D. Weir (Ed.), Handbook of Experimental Immunology, Blackwell Scientific Publications, Oxford, pp. 1-20. Winer, J.B., Briggs, D., Welsh, K. and Hughes, R.A.C. (1988a) HLA antigens in the Guillain-Barr6 syndrome. J. Neuroimmunol. 18, 13-16. Winer, J.B., Hughes, R.A.C. and Osmond, C. (1988b) A prospective study of acute idiopathic neuropathy. I. Clinical features and their prognostic value. J. Neurol. Neurosurg. Psychiatry 51,605-612.
149
JNI 00916
Major histocompatibility complex class I and class II polymorphism in chronic idiopathic demyelinating polyradiculoneuropathy R.W, Vaughan
1 A.M. A d a m
2, I.A. G r a y 2, R.A.C. Hughes 2, E.A.C.M. Sanders M. van D a m a and K.I. Welsh a
2,
Departments of 1 Molecular Immunogenetics and 2 Neurology, United Medical and Dental Schools of Guy's and St Thomas's Hospitals, Guy's Campus, London SE1 9RT, UK. (Received 3 August 1989) (Revised, received 19 September 1989) (Accepted 20 September 1989)
Key words: Histocompatibility antigen; Chronic idiopathic demyelinating polyradiculoneuropathy; HLA-Cw7
Summa W
Thirty-one chronic idiopathic demyelinating polyradiculoneuropathy (CIDP) patients have been typed for HLA-A, -B and -C antigens serologically and for HLA-DR, -DQ and -DP class II genes by RFLP analysis. Our results confirm a previously reported slight association with HLA-B8 and identify a stronger association with HLA-Cw7.
Introduction
Chronic idiopathic demyelinating polyradiculoneuropathy (CIDP) is a slowly progressive disorder of the peripheral nervous system for which autoimmune mechanisms have been proposed (Dyck and Arnason, 1984). It resembles GuillainBarr6 syndrome (GBS) but pursues a more prolonged course. GBS is usually monophasic reaching its nadir within 4 weeks whereas CIDP progresses over 3 or more months and often pursues a relapsing and remitting course (McCombe et al., 1987). For epidemiological purposes it is convenient to set an arbitrary boundary for the duration
Address for correspondence: Prof. R.A.C. Hughes, Department of Neurology, Guy's Hospital, London SE1 9RT, U.K.
of the time from onset to nadir for GBS of 4 weeks (Hughes and Winer, 1984; Winer et al., 1988b). This definition leaves a small proportion of patients with progressive disease reaching a nadir in more than 4 but less than 12 weeks: we have included these patients in a subacute category of the CIDP group. CIDP has only been loosely defined on a combination of clinical features and electrophysiological evidence of non-uniform slowing of conduction and multifocal conduction block (Dyck and Arnason, 1984; Albers, 1987). The pathological substrate is a multifocal demyelinating process in which macrophages strip and phagocytose the myelin sheath. Lymphocytic infiltration is rarely observed in peripheral nerve biopsies but rare autopsy studies have revealed perivascular infiltration of cells in spinal roots (Dyck and Arnason,
0165-5728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
150 1984). This histological evidence of an immune response in the peripheral nerves and its resemblance to chronic experimental allergic neuritis (EAN) induced in rats by immunisation with myelin or P2 protein and in rabbits with myelin or galactocerebroside have led to the proposal that CIDP is an autoimmune condition (Craggs et al., 1986; Harvey et al., 1987; Adam et al., 1989). The major histocompatibility complex (MHC) on chromosome 6 contains HLA genes essential to the control of the immune response. Stewart et al. (1978) found a significant association with HLAA30 and -A31 and probable associations with HLA-B8 and -Dw3 in 16 Australian CIDP patients. Adams et al. (1979) did not find an increased frequency of HLA-A30 and A31 but did report an association with HLA-1, -B8, -DRW3 and -DW3 in 14 patients in the U.K. We report the HLA types of a further series of 31 Caucasoid CIDP patients.
Patients and methods
Consecutive Caucasoid patients, encountered by one of the authors, who fulfilled the following diagnostic criteria for CIDP were studied: (1) progressive weakness of two or more limbs due to peripheral neuropathy for more than 4 weeks; (2) diminution or loss of tendon reflexes; (3) absence of family history or clinical features of hereditary motor and sensory neuropathy, diabetes mellitus, paraproteinaemia, toxin exposure or other known cause of demyelinating neuropathy; (4) electrophysiological evidence of demyelinating neuropathy according to criteria similar to those of Albers (1987). The clinical course was considered subacute if the nadir was reached more than 4 but less than 12 weeks from the onset, chronic progressive if the nadir was reached after more than 12 weeks and chronic relapsing if the progressive course was interrupted by remissions followed by relapses. A remission was defined as improvement of one disability grade for at least one week, and a relapse as a worsening of one clinical grade for at least one week. The clinical grades were modified from those used by Hughes et al. (1978): 0 = healthy; 1 = minor symptoms or signs; 2 = able to
walk 5 metres without assistance; 3 = able to walk 5 metres with assistance; 4 = chair b o u n d / b e d bound; and 5 = requiring assisted ventilation. Patients who had recurrent attacks of acute neuropathy resembling GBS in reaching their nadir within 4 weeks were included as a separate recurrent acute group.
HLA typing 30 ml of blood were taken from each patient or control. 20 ml aliquots were anticoagulated with 5% EDTA and stored at - 20 ° C before extraction of DNA. 10 ml were anticoagulated with 4% sodium citrate and used for serological HLA-A, -B and -Cw typing as described (Welsh and Batchelor, 1978), with cell viability assessed by phasecontrast microscopy. Controls for the HLA-A, -B and -Cw analysis consisted of 346 volunteer normal Caucasoid subjects. The class II study was conducted by analysis of the R F L P pattern of genomic DNA restricted with the endonucleases TaqI or MspI. D N A was isolated from blood, digested, electrophoresed and blotted as described (Demaine et al., 1987) onto Hybond N (Amersham International). Hybridisation was performed in glass tubes in Bachoffer rotisserie ovens. The following class II probes were used: DRB (pII-fl-4, SacI and HindIII fragment) (Gustafsson et al., 1984a), DQA (pII-a-5, ApaI fragment) (Schenning et al., 1984), DQB (pII-/~-I, AuaI fragment) (Larhammar et al., 1982), DPA (pDAdl3b, EcoRI fragment) (Trowsdale et al., 1985), DPB (pII-/~-7, RsaI and HpaII fragment) (Gustafson et al., 1984b). The DPB probe was hybridised with D N A digested with the restriction enzyme MspI. All the other probes were hybridised with TaqI-digested DNA. The autoradiographs were assessed and the typing scheme was based on our own observations and the work of others. The DRB definition follows Bidwell et al. (1987) and Bidwell (1988). The simplified DQ assignment defines DQw5, DQw6, DQw2, DQw7, DQw8 and DQw9. The DQwl splits are defined by the DQA probe where DQw5 corresponds to Bidwell's D Q l a and DQw6 refers to bands at 6 or 6.5 kb. The DQw2 and DQw7 and DQw8 specifities are defined using the DQB probe. DQw9 is defined by the DRB 7b pattern and the DQw8 DQB band at 1.9 kb. DR7 DQw9 and DR9 DQw9
151
cannot be separated in this way and are referred to as D R 7 / 9 . The DP definition is a short-hand nomenclature and is based on the work of Hyldig-Neilsen et al. (1987). The correlation between DP R F L P and D P alleles may be slightly less accurate than that for D R and DQ; the following simplified scheme has been adopted: D P w l has a MspI DPB band at 4.5 kb;
-
DPw2 has a TaqI D P A band at 13 kb and a band at 2.1 kb; D P w 3 / 6 has a TaqI D P A band at 6.3 kb and a MspI DPB band at 3 kb; DPw4 has a TaqI D P A band at 14 kb and a MspI DPB band at 1.1 kb; DPw5 has a TaqI D P A band at 6.5 kb; DP CP63 has a MspI DPB band at 1.8 kb. The significance of the results was tested with
MspI DPB -
-
-
-
-
TABLE1 C L I N I C A L F E A T U R E S A N D H L A P O L Y M O R P H I S M O F CIDP PATIENTS Code
Age
Sex
Duration
Worst
H L A class I serology
(years)
disability grade
HLA-A
Chronic progressive 1 65 M 2 61 M 3 51 M 4 38 M 5 49 F 6 39 M 7 66 M 8 35 M
0.5 1.0 1.0 3.0 5.0 6.0 7.0 9.0
3 3 4 4 2 2 3 3
29, 1, 1, 3, 1, 2, 1, 2,
Chronic relapsing 9 26 M 10 55 M 11 29 F 12 47 M 13 21 M 14 60 F 15 34 M 16 32 M 17 25 M 18 27 M 19 86 F 20 34 M 21 68 M 22 64 M 23 49 M 24 60 M
0.5 1.0 1.0 2.0 2.0 5.0 5.0 6.0 6.0 6.0 7.0 8.0 16.0 18.0 20.0 27.0
2 4 5 4 4 4 5 3 3 4 4 4 2 4 3 4
2, 31 1, 2 1, 2 3, 1, 2, 24 2, 29 1, 2 11, 25 1, 11 1, 2 2, 11 3, 1, 11 3, 24 1, 2
Recurrent acute 25 62 26 48 27 62 28 88
M M M M
13.0 15.0 29.0 45.0
4 5 5 4
2, 2, 1, 2,
Subacute 29 31 30 46 31 2l
F M M
0.5 0.5 1.5
2 4 4
1, 31 3, 30 2, 3
N.T., not tested.
32 2 26 29 31 3 32
3 11 25 4
HLA-B
8, 7, 8, 7, 8, 7, 8, 7,
44 62 71 44 39 18 60
HLAclass IlRFLP HLA-C
HLA-DR
HLA-DQ
HLA-DP
5, 3, 7, 7, 7, 7, 7, 3,
4, 4, 2, 2 1, 3, 3 2,
w2, w6, w2, w6 w5, w2, w2 w6,
N.T. 2 4 3/6 4 4, CP63 2, CP63 4
7 7 7
3 w13 3 5 w13 4
w3 w7 w6 w7 w6 w7
7, 39 13, 58 8, 44 7, 14 55, 57 8, 60 7, 35 8, 51 18, 27 8, 27 8, 14 14, 35 27, 35 7, 8 7, 18 8, -
7, 6, 7 5, 7 7, 3, 6 3, 7 4, 7 7, 2, 1, 7 7, 4, 2, 3 7, 7, 7, -
2, w13 5, 7 4, 7 1, w13 4 3, w13 2, w13 2, 4 7 w13, w14 3, w13 1, 4 2 2, 3 5 3
w6 w7, w2, w5, w7, w2, w6 w6, w2, w5, w2, w5, w6 w2, w7 w2
7, 7, 8, 39,
5, 7, 7, 3,
7 7
4, w13 2, w14 2, 3 4
w6, w7 w5, w6 w6, w2 w7
4, 3 / 6 4, 3 4 4, 3 / 6
7, 7, 1, -
3 4, w13 4
w2 w3, w6 w7
N.T. 2, 1 / 3 / 6 CP63
44 51 18 62
8, 51 7, 44 51, 56
w2 w8 w6 w8 w6 w3 w9 w6 w6 w3 w6
2 4, CP63 3, 4 N.T. 4, 3 / 6 N.T. 2, CP63 2 4 4, 3 / 6 1, 5 2 4, 6 4 5 4, 1
152
the chi-square test and two-tailed probabilities are given. They have not been corrected for the number of antigens tested on the grounds that we are seeking to confirm an association with HLA-A1, -B8 and -DR3 (then -Dw3) which has been identified in two separate previous studies.
Results and discussion
Our results confirm the previously reported small increase in frequency of HLA-A1 and HLAB8 in CIDP patients (Stewart et al., 1978; Adams et al., 1979) and show a similar small increase in HLA-B7 (Tables 1 and 2). The most significant association in our series of CIDP patients was with HLA-Cw7. The significance of the association has to be interpreted with caution since the p value has not been corrected for the number of HLA class I allotypes tested (80). Since there is strong linkage disequilibrium between HLA-Cw7 and both the B locus antigens HLA-B7 and HLA-B8, an association with a gene close to these B locus antigens might cause the observed increased frequency of HLA-Cw7. However, our study of M H C class II genes at the D N A level, the first to be published in this disease, suggests that this is not the explanation. Linkage disequilibrium with HLA-B8 and HLA-B7 would usually be associated with an increase in HLADR3 and H L A - D R 2 respectively, but the frequency of these antigens was not increased. The
TABLE 2 THE FREQUENCY OF HLA ANTIGENS A1, B7, B8, Cw7 AND CLASS II RFLP FOR DR2, DR3 AND DQw6 IN CIDP PATIENTS AND CONTROLS CIDP
HLA-A1 HLA-B7 HLA-B8 HLA-Cw7 HLA-DR2 HLA-DR3 HLA-DQw6
Controls
p
Ratio a
Percent
Ratio a
Percent
14/31 12/31 13/31 26/31 10/31 10/31 18/31
45.2 38.7 41.9 83.8 32.2 32.2 58
118/346 74/346 85/346 184/346 18/60 21/60 26/60
34.1 21.4 24.6 53.2 36 33 43.3
0.29 0.05 0.06 0.002 0.98 0.97 0.27
a Number of patients positive/number of patients tested.
association between CIDP and HLA decreases with genomic distance from HLA-C so that if there is a CIDP susceptibility gene on chromosome 6 it is likely to be closer to the C locus than the DR, A or B loci and in linkage dysequilibrium with Cw7. There was no significant distortion of the frequency of R F L P detected with the DP, DQ or D R probes in CIDP patients apart from a small but non-significant increase in DQw6 (Table 2). Our study did not confirm a weak association with DR3 reported in two earlier studies (Stewart et al., 1978; Adams et al., 1979). This may be due to the smaller numbers in those studies or improvements in class II definition in the last 10 years. The small numbers in clinical subgroups preclude drawing conclusions about differences in the frequency of class I or II antigens but the details are presented for comparison with future studies. Subdivision according to progressive or relapsing disease course, or disease severity did not strengthen the association with Cw7 and any other group, except that Cw7 was present in all seven patients with a chronic progressive course and all four with a recurrent acute course. The association of CIDP with a class I allele invites speculation about mechanisms. We propose that the class I allele is itself a disease susceptibility gene although since Cw7 is common and CIDP rare polygenic influence and environmental factors must also be important. Class I molecules usually contain endogenous peptides in their antigen binding cleft (Bjorkman, 1987). These endogenous peptides may be bound at the time of synthesis or assembly of the class I molecule. If the cell becomes infected, virally derived peptides may replace the usual endogenous peptide (Townsend, 1987). The binding of a viral peptide in the HLA-Cw7 cleft might trigger individuals who are predisposed by yet other genes to an aberrant autoimmune response presenting as CIDP. Alternatively minor myelin damage by viral infection, trauma or other factor might release myelin antigens which bind to the HLA-Cw7 cleft and stimulate an autoimmune response. A third possibility is that HLA-Cw7 or a linked gene is not responsible for the initiation of CIDP but for the failure to limit the disease to a monophasic illness. This possibility is supported by the finding
153
of a slightly decreased frequency of HLA-Cw7 in Guillain-Barr6 syndrome (Winer et al., 1988a).
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