No association with HLA-DR, -DQ or -DP alleles in Guillain-Barré syndrome

Journal of Neuroimmunology, 31 (1991) 67-72

67

Elsevier JNI 01039

No association with HLA-DR, -DQ or -DP alleles in Guillain-Barr6 syndrome Jan Hillert 1,2, P.O, O s t e r m a n 4 a n d Olle O l e r u p 1,3 s Center for BioTechnology, Karolinska Institute, Novum, Huddinge, Sweden, 2 Department of Neurology and s Department of Clinical Immunology, Karolinska Institute at Huddinge Hospital, Huddinge, Sweden, and 4 Department of Neurology, University of Uppsala, Uppsala, Sweden

(Received25 June 1990) (Revised, received24 August 1990) (Accepted 27 August 1990)

Key words: Genetic susceptibility; Guillain-Barr6 syndrome; HLA-D; Plasmapheresis treatment; Restriction fragment length poly-

morphism Summary The distribution of H L A class II alleles in Guillain-Barr6 syndrome (GBS) has previously been reported only for HLA-DR. We report here the results of genomic typing for HLA-DR, -DQ and -DP allelic variability by restriction fragment length polymorphism analysis in 49 patients with a history of GBS, No association was found to HLA-DR, -DQ or -DP alleles or H L A - D R - D Q haplotypes. Subgrouping of patients according to severity of disease, as measured by disability or muscular weakness, or response to plasmapheresis treatment, also failed to reveal significant associations. These data suggest that HLA class II genes do not confer susceptibility to GBS.

Introduction Guillain-Barr6 syndrome (GBS) or acute polyradiculoneuritis is an inflammatory disorder of unknown etiology; however, several features suggest an autoimmune component of the pathogenesis. Both humoral and cell-mediated immune response have been suspected of playing a role (for review, see Arnason et al., 1984). This concept is further supported by the close resemblance between GBS and experimental allergic neuritis

Address for correspondence:Jan Hillert, M.D., Center for BioTechnology,Karolinska Institute, NOVUM, S-141 52 Huddinge, Sweden.

(EAN), a well-characterized model for autoimmune disease. Since susceptibility to EAN varies between animal strains of different major histocompatibility complex (MHC) phenotype (Steinman et al., 1980), we considered it to be of interest to look for an association with HLA class II alleles in patients with GBS. Several diseases with autoimmune features have been found to be associated with different HLA antigens, for instance rheumatoid arthritis and multiple sclerosis. Associations of these disorders were first described with alleles of the HLA-A and -B loci of the HLA class I region. Later it has become clear that these associations, as the case has been in several other diseases studied, might be secondary to stronger associations with alleles

0165-5728/91/$03.50 © 1991 ElsevierSciencePublishers B.V. (BiomedicalDivision)

68 of the HLA class II region (for review, see Tiwari and Terasaki, 1985). The HLA class II region contains three expressed subregions, HLA-DR, -DQ and -DP. Typing for HLA class II allelic variability has until recently been performed by serologic (for HLADR and -DQ) and cellular (for HLA-Dw and -DP) methods. In recent years the use of molecular DNA techniques has allowed HLA class II typing at the genomic level which has revealed a much greater polymorphism than was previously recognized. Using a variety of techniques, including gene sequencing, it has been possible in a few diseases such as insulin-dependent diabetes mellitus and pemphigus vulgaris, to delineate the HLA association down to a single amino acid position in one of the protein chains (Todd et al., 1987; Erlich et al., 1988; Scharf et al., 1988). Plasmapheresis treatment has become an established therapeutic measure in GBS (Consensus Conference, 1986), but, though clearly effective in many patients, others do not respond. A predictive marker for selection of patients likely to improve from plasmapheresis would be of clinical value and has been sought, without convincing results (Osterman et al., 1988b). The aims of the present study were to study the distribution of HLA class II alleles in GBS, and to look for influence of HLA class II alleles on severity of the disease as well as response to plasmapheresis treatment.

Materials and methods

Patients Forty-nine unrelated adult patients with a history of severe GBS were included in the study. Forty-six of the patients came from a cohort of 77 patients included in earlier studies on the effect of plasmapheresis treatment in GBS (Osterman et al., 1984, 1988a). Of these 77 patients, two had died in the acute stage of their disease and one during the follow-up period of up to 8 years. Sixty-five patients could be traced to a recent address and 52 agreed to participate. Eventually, blood samples were recieved from 46 patients. Another three GBS patients were included prospectively during the study. All patients fulfilled

diagnostic criteria for GBS (NIH, 1978) and reached a disability score of at least 3 (Osterman et al., 1984). Forty-six patients were scored for muscular weakness (MWS) and disability grade in a standardized way at several times during their disease (Osterman et al., 1984). In short, disability was measured on a scale from 1 to 7, with higher scores indicating increasing incapacity; from score 5, patients could not stand or walk even with assistance. MWS is the sum of scores (0-4, where 0 indicates normal function and 4 indicates paralysis) from six muscle groups plus cranial function (score 0-3), giving a maximal score of 27 for total paralysis. Thus, it was possible to stratify the patients with regard to severity of symptoms as well as identify responders and non-responders among the 31 patients treated with plasmapheresis. A patient was identified as a responder if a reduction in MWS of at least 3 grades occurred within 14 days of the first plasmapheresis (Osterman et al., 1988a). A group of 100 healthy, randomly selected persons was used for comparison.

Probes The following almost full-length cDNA probes were used: DRB, a 790 base pair (bp) HindIIlSacI fragment of clone plI-fl-3 (Gustafsson et al., 1984a); DQA, a 584 bp RsaI-StuI fragment of clone pII-a-5 (Schenning et al., 1982); DQB, a 627 bp AvaI-AvaI fragment of clone pII-fl-1 (Larhammar et al., 1982); DPA, a 600 bp AvaI-EcoRI fragment of clone pDAa13b (Trowsdale and Kelly, 1985); DPB, a 601 bp HpaI-RsaI fragment of clone pII-~-7 (Gustafsson et al., 1984b). After purification, inserts were labeled by random hexanucleotide priming to a specific activity of 1-2 X 10 9 cpm//~g. Southern blot analysis Extraction of D N A from peripheral blood leukocytes, restriction enzyme digestions (TaqI and MspI), agarose gel electrophoresis, capillary blotting to nylon membranes, hybridization, stringency washes and autoradiography were performed by standard techniques. TaqI HLA-DR and -DQ RFLP analysis Genomic DR- and DQ-typing was performed by hybridizing TaqI cleaved DNA with DRB,

69

DQA and DQB probes. Allelic restriction frag-

TABLE 1

ment patterns were designated by the associated serologically defined D R and D Q specificities according to previous reports (Bidwell et al., 1987; Carlsson et al., 1987; reviewed by Bidwell, 1988).

FREQUENCIES (%) OF HLA-DR-DQ PHENOTYPES IN GBS PATIENTS AND CONTROLS

Msp I HLA-DP RFLP analysis DP-typing was performed by hybridizing MspI cleaved D N A with DPA and DPB c D N A probes. Allelic fragment patterns were designated by the associated cellularly defined DP specificities according to Hyldig-Nielsen et al. (1987, 1988).

Statistics Data were analysed by the chi-square test or by Fisher's exact test in the case of small expected numbers. P values were corrected for multiple comparisons by a factor of 25 for the 19 TaqI HLA-DRB-DQA-DQB haplotypes observed in patients and controls and for the six distinguishable H L A - D P allelic patterns.

HLA-DR-DQ haplotypes DR1, DQw5 DR4, DQw7 DR4, DQw8 DR7, DQw2 DR7, DQw9 DR8, DQw4 DR9, DQw9 DRwl0, DQw5 DRwll, DQw7 DRw12, DQw7 DRwl3, DQw6 DRwl3, DQw7 DRwl4, DQw5 DRwl4, DQw7 DRwl5, DQw6 DRwl6, DQw5 DRwl7, DQw2

GBS (n = 49) 8 18 29 8 4 8 2 4 18 4 22 0 4 2 43 6 18

Controls (n = 100) 21 11 24 7 4 9 4 3 12 6 33 1 5 0 30 2 15

the distribution of H L A class II alleles was similar in the two groups.

Results

HLA-DR, -DQ and -DP The frequencies of H L A - D R - D Q haplotypes in patients and controls are shown in Table 1. Only minor, insignificant differences were noted between the groups. Likewise, frequencies of H L A D P alleles were similar in patients and controls (Table 2).

Stratification according to severity of symptoms No clear tendency towards over- or under-representation of H L A class II alleles in severe or mild cases of GBS was noted, regardless if disability grade or muscle weakness score were assessed (Table 3).

Plasmapheresis Thirty-one patients received treatment with plasmapheresis, all of whom had considerable weakness. Eighteen were classified as responders on grounds of clear improvement within 14 days after onset o f treatment (see above) and 13 were classified as non-responders. As shown in Table 4,

Discussion In this study of H L A class II alleles in GBS the new strategy of genornic typing has been applied to all expressed H L A class II subregions, i.e. not only H L A - D R , but also H L A - D Q and -DP. Genomic H L A class II typing has important advantages over serologic and cellular techniques. Firstly, b y genomic typing all individuals are possible to type, whereas earlier methods have considTABLE 2 FREQUENCIES (%) OF HLA-DP ALLELES IN GBS PATIENTS AND CONTROLS DP allele DPwl DPw2 DPw3/DPw6 DPw4 DPw5 CDP HEI

GBS (n = 48) 8 21 52 81 2 0

Controls (n = 100) 9 20 37 82 6 1

70 TABLE 3 FREQUENCIES (%) OF THE MOST COMMON HLA-DRDQ HAPLOTYPES AND HLA-DP ALLELES IN GBS PATIENTS GROUPED ACCORDING TO SEVERITY OF SYMPTOMS AS MEASURED BY DISABILITY SCORE AND MUSCLE WEAKNESS SCORE HLA-DR-DQ Disability score haplotypes or <4 >5 HLA-DP alleles (n=18) (n=28)

Muscleweakness score < 12 > 19 (n=14) (n=13)

DRI, DQw5 DR4, DQw7 DR4, DQw8 DRwl 1, DQw7 DRw15, DQw6 DRwI7, DQw2

11 17 22 0 50 17

7 14 32 29 a 29 10

14 14 21 7 50 14

0 31 38 23 38 8

DPw2 DPw3/6 DPw4

17 67 72

18 43 79

7 64 79

31 54 77

a Pu .....

= 0.012;

Pcorr =

n.s.

erable frequencies of failures. Secondly, homozygosity is easily identified. This leads to m u c h safer figures of allelic frequencies. Thirdly, with genomic methods it is possible to identify a greater part of the biologically relevant p o l y m o r p h i s m than is feasible by serology. Only a few earlier reports have described the distribution of H L A - D R alleles in GBS. Stewart and co-workers (1978) and Latovitzki and coworkers (1979) reported negative findings in 22 TABLE 4 DISTRIBUTION OF THE MOST FREQUENT HLA-DRDQ HAPLOTYPES A N D HLA-DP ALLELES IN GBS PATIENTS GROUPED ACCORDING TO RESPONSE TO PLASMAPHERESIS TREATMENT HLA-DR-DQ haplotypes or HLA-DP alleles

% Responders (n = 18)

% Non-responders ( n = 13)

DR4, DQw7 DR4, DQw8 DRwll, DQw7 DRw15, DQw5 DRwl7, DQw2

22 22 22 39 6

23 46 8 31 31

DPwl DPw2 DPw3/6 DPw4

0 17 50 83

25 33 42 75

and 18 patients, respectively. In contrast, Gorodetzki and co-workers (1983), in a study on 38 Mexican patients with GBS, described an overrepresentation of DR3. However, since the frequency of this allele in the G B S group was only approximately 35%, this association could only account for the genetically determined susceptibility to G B S in a subgroup of cases. In a more recent study, Winer and co-workers (1988) investigated 79 G B S patients without finding any H L A - D R association. A n interesting finding in this study was the tendency towards an association between D R 2 and severe m o t o r s y m p t o m s as well as p o o r prognosis. The results of the present study show neither an increased frequency of H L A - D R 3 in the whole material, nor an over-representation of D R 2 in more severe cases. The distribution of H L A class II alleles was very similar in patients and controls, regardless of subgrouping of patients with regard to severity of disease and response to plasmapheresis. This makes it unlikely that a significant association is overlooked because of the limited size of the patient group. A possible disadvantage of the retrospective character of the study is that cases of p o o r o u t c o m e might be under-represented. The fact that the vast majority of GBS patients recover completely or almost completely (Arnason et al., 1984) could indicate that this p r o b l e m is of limited importance. Furthermore, as mentioned above, all patients included in this study presented with considerable weakness, so that a deviation towards cases with milder symptoms seems unlikely. It has been argued (Kaslow et al., 1984; Wirier et al., 1988) that one reason behind the inconclusive results in studies o n a possible H L A association in G B S is that the disease can be triggered by m a n y different events, and that associations can only be expected to be f o u n d in groups of patients with the same etiologic factor. The only good example of this is the increased occurrence of GBS following vaccinaton for swine flue in the U.S.A., 1976-1977. Kaslow and co-workers (1984), however, reported H L A class I . t y p i n g of 38 such G B S patients, decribing only a weak association to the H L A - B locus; the distribution of H L A - D R alleles was not reported. The patient group of Winer and co-workers (1988) included nine pa-

71

tients each with antecedent cytomegalovirus or Campylobacter jejuni infection, without strong tendencies towards association with H L A - D R alleles. In the present study no attempt was made to define triggering events. The observed differences in HLA allelic distribution between responders and non-responders to plasmapheresis were not statistically significant, but the numbers are small and it cannot be excluded that differences could be shown if more patients were analyzed. However, the results strongly suggest that HLA class II phenotype cannot be used as a marker to predict the outcome of plasmapheresis treatment in individual patients with GBS. In conclusion, the present study supports most previous results in showing a lack of HLA class II association in GBS, extending this to the full HLA class II region. If the susceptibility to GBS, in parallel to what has been found in EAN, is genetically influenced, it seems unlikely that the HLA class II region contributes significantly.

Acknowledgements This study was supported by grants from the Swedish Medical Research Council (project number 8709), the Karolinska Institute and the Swedish Society of Medicine.

References Arnason, B. (1984) Acute inflammatory demyelinating polyradiculoneuropathies. In: P. Dyck, P. Thomas, E. Lambert and R. Bunge (Eds.), Peripheral Neuropathy, Vol. 2, W.B. Saunders, Philadelphia, PA, pp. 2050-2100. Bidwell, J.L. (1988) DNA-RFLP-analysis and genotyping of HLA-DR and -DQ antigens. Immunol. Today 9, 18-23. Bidwell, J.L., Bidwel|, E.A., Laundy, C.J., Klouda, P.T. and Bradley, B.A. (1987) Allogenotypes defined by short DQa and DQfl cDNA probes correlate with, and define splits of HLA-DQ serological specificities. Mol. Immunol. 24, 513522. Consensus Conference (1986) The utility of therapeutic plasmapheresis for neurological disorders. J. Am. Med. Assoc. 256, 1333-1337. Gorodezki, C., Varela, B., Castro-Escobar, L.E., Ch~vezNegrete, A., Escobar-Guti~rrez, A. and Matinez-Mata, J.

(1983) HLA-DR antigens in Mexican patients with Guillain-Barr6 syndrome. J. Neuroimmunol. 4, 1-7. Gustafsson, K., Wiman, K., Emmoth, E., Larhammar, D., Bt~hme, 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.A. and Rask, L. (1984b) Isolation of a cDNA clone coding for a SB/~-chain. Nature 309, 76. Hyldig-Nielsen, J.J., Morling, N., Odum, N., Ryder, L., Platz, P., Jakobsen, B. and Svejgaard, A. (1987) Restriction fragment length polymorphism of the HLA-DP subregion and correlations to HLA-DP phenotypes. Proc. Natl. Acad. Sci. U.S.A. 84, 1644-1648. Hyldig-Nielsen, J.J., Odum, N., Morling, N. and Svejgaard, A. (1988) Restriction fragment length polymorphism (RFLP) of a 'new' HLA-DP specificity, CDP-HEI. Tissue Antigens 31, 161. Kaslow, R., Sullivan-Bolyai, J., Hafkin, B., Schonberger, L., Kraus, L., Moore, M., Yunis, E. and Williams, M. (1984) HLA antigens in Guillain-Barr~ syndrome. Neurology 34, 240-242. Larhammar, D., Schenning, L., Gustafsson, K., Wiman, K., Claesson, L., Rask, L. and Peterson, P.A. (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. Proc. Natl. Acad. Sci. U.S.A. 79, 3687-3691. Latovitzki, N., Suciu-Foca, N., Penn, A., Olarte, M. and Chutorian, A. (1979) HLA typing and Guillain-Barr6 syndrome. Neurology 29, 743-745. NIH (1978) Diagnostic criterions for Guillain-Barrr. J. Am. Med. Assoc. 240, 1709-1710. Osterman, P.O., Fagius, J., Lundemo, C., Pihlstedt, P., Pirskanen, R., Sid6n, .~. and Safwenberg, J. (1984) Beneficial effects of plasma exchange in acute inflammatory polyradiculoneuropathy. Lancet ii, 1296-1298. Osterman, P.O., Fagius, J., S~ifwenberg, J. and Wikstr6m, B. (1988a) Early relapse of acute inflammatory polyradiculoneuropathy after successful treatment with plasma exchange. Acta Neurol. Scand. 77, 273-277. Osterman, P.O., Vedeler, C.A., Ryberg, B., Fagius, J. and Nyland, H. (1988b) Serum antibodies to peripheral nerve tissue in acute Guillain-Barr6 syndrome in relation to outcome of plasma exchange. J. Neurol. 235, 285-289. Scharf, S., Friedman, A., Brautbar, C., Szafer, F., Steinman, L., Horn, C., Gyllensten, U. and Erlich, H. (1988) HLA class II allelic variation and susceptibility to pemphigus vulgaris. Proc. Natl. Acad. Sci. U.S.A. 85, 3504-3508. Schenning, L., Larhammar, D., Bill, P., Wiman, K., Jonsson, A.-K., Rask, L. and Peterson, P.A. (1984) Both a and fl chains of HLA-DC class I1 histocompatibility antigens display extensive polymorphism in their aminoterminal domains. EMBO J. 3, 447-452. Steinman, L., Smith, M. and Forno, L. (1980) Genetic susceptibility to experimental allergic neuritis. Neurology 30, 363 (abstract).

72 Stewart, G., Pollard, J., McLeod, J. and Wolnizer, C. (1978) HLA antigens in the Landry-Guillain-Barrd syndrome and chronic relapsing neuritis. Ann. Neurol. 4, 285-289. Todd, J,, Bell, J. and McDevitt, H. (1987) HLA-DQ/~ gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus, Nature 328, 599-604.

Trowsdale, J. and Kelly, A. (1985) The human class I1 c~ chain gene DZc~ is distinct from genes in the DP, DQ and DR subregions. EMBO J. 4, 2231. Winer, J., Briggs, D., Welsh, K. and Hughes, R. (1988) HLA antigens in the Guillain-Barr6 syndrome. J. Neuroimmunol. 18, 13-16.