HLA class 11-associated genetic susceptibility in multiple sclerosis: A critical evaluation 0. Olerup, J. Hillert. HLA class 11-associated genetic susceptibility in multiple sclerosis: A critical evaluation. Tissue Antigens 1991: 38: 1-15. Abstract: Multiple sclerosis (MS) has, since the 1970s, been known to be associated with the HLA-Dw2 and -DR2 specificities in Caucasian Europeans and North Americans. By the use of genomic typing techniques, the association has been specified to be with the DRwlS,DQw6,Dw2, i.e. the DRBI* I.5OI-DQAI*0102-DQBl*O602 haplotype. A significant DPw4 association in Scandinavian MS patients has been described in one report. However, this association has not been confirmed in several subsequent studies with patients from the same and other ethnic groups. During the last few years several reports, based on serological, RFLP and PCR-SSO data, have suggested that the HLA class 11-associated MS susceptibility gene(s) may be more closely associated with the DQ than with the DR subregion. The observations that the HLA-DQBI genes of MS patients share long stretches of sequence motifs and also carry DQAl alleles encoding glutamine at position 34 of the DQ a chain have received considerable attention. It has been suggested that the susceptibility to develop MS might be determined by the corresponding DQ a-P heterodimers either encoded in cis or in trans. We have investigated these issues in a large group of Swedish MS patients (n = 179). We found that the associations with the suggested DQBl sequences and position 34 of the DQ a chain were due to linkage disequilibrium and secondary to the association with the DRw 15,DQw6,Dw2 haplotype (p< lo-' and p < respectively). No overrepresentation of the implicated DQ a-0 heterodimers was observed in DRw 1S,DQw6,Dw2-negative patients. We conclude that available data does not allow the localization of the HLA class IIassociated MS susceptibility genes within the DRw15,DQw6,I>w2 haplotype. We have previously described immunogenetic differences between different clinical forms of MS. In the present study of a new group of MS patients compared with a new control panel, these differences were partly confirmed. The DRw 17,DQw2 association in relapsing/remitting MS was affirmed. However, in patients with primarily chronic progressive MS a negative association with the DQw7 allele was not substantiated and a positive association with the DR4.DQw8 haplotype could neither be confirmed nor rejected.
Iutredrctlen
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disorder of the central nervous system often causing neurological deficits. The etiology and many aspects of the pathogenesis remain unclear. Most current hypotheses postulate that the disease is caused by a T cell-mediated autoimmune destruction of the myelin sheath in the genetically predisposed individual. The racial and ethnic differences in the incidence of MS, the familial occurrence of MS with a
Ollr Olenp12 and Jan Hlllert'~' 'Center for BioTechnology. Karolinska Institute, NOVUM. Huddinge. zDepartment of Clinical Immunology and 'Department of Neurology, Karolinska Institute at Huddinge Hospital, Huddinge, Sweden
Key words: genetic susceptibility - HLA-0 a n k gens - HLA-DP - HLA-W 4 L A - D R - multiple sclerosis - polymerase chain reaction - primarily chronic progressive multiple sclerosis rehpsinglremitting multiple sclerosis - restriction fragment length polymorphism Received 3 May, accepted lor publication 6 May 1991
tendency to increasing prevalence with increasing degree of kinship (reviewed in ref. l), as well as the significantly higher concordance rate in monozygotic twins compared to dizygotic twins (2) indicate a major genetic etiologic component. In most population studies of Caucasian MS patients strong associations with the HLA class I1 specificities Dw2 and DR2 have been observed (3, reviewed in ref. 4). In formal segregation and linkage analysis an excellent fit was obtained for a model with a dominant MS genetic factor tightly linked to the DR2 haplotype and a second unlinked determinant 1
Olerup & Hillert (5). However, as only limited data are available on the concordance rate in HLA-identical siblings, the influence of non-HLA linked genes is difficult to ascertain. During the past 5 years many reports on HLA class I1 associations in MS have suggested that the HLA-D region-associated MS susceptibility gene(s) are more closely associated with the DQ than the D R subregion. The existence of DQ-associated MS resistande genes has also been proposed (6, 7). Furthermore, it has been suggested that MS is one of the HLA-associated diseases related to specific amino acids or epitopes of the class I1 molecules (8,9). Analogous to the findings in celiac disease, the susceptibility to MS has been hypothesized to be jointly determined by DQAI and DQBl gene products forming a-P heterodimers encoded in cis or in trans (9). Since these findings have not been undisputed and as they are of great potential interest in the unravelling of the HLA-D region-associated MS susceptibility genetic factor(s), we have found it of interest to critically review them and also to investigate these issues in a large group of Swedish MS patients. A second aim of the present study was to test if our previously published finding of immunogenetic differences between primarily chronic progressive (PCP) MS and relapsing/remitting (R/R) MS (7, 10) could be verified in an independent study.
Material and Methods Patients
One-hundred-and-seventy-nineunrelated Swedish patients with clinically definite MS (1 1) were studied. Thirty-six patients had PCP MS, i.e. gradually progressive neurological symptoms from the onset of the disease without remissions. Onehundred-and-forty-three patients had R/R MS. This latter group included patients who experienced secondary progressive evolution of symptoms after initial relapses and remissions. One hundred of the patients, 26 with PCP MS and 74 with R/R MS, were included in a previous study on DR-DQ associations in MS (7). The DP typings of 110 of the patients, 28 with PCP MS and 82 with R/R MS, have been reported elsewhere (12).
Controls Two-hundred-and-fifty randomly selected, healthy Swedes were used as controls. The HLA class I1 typings of 100 of the controls have been reported previously; DR and DQ (7, 13), and DP (12). 2
Cell lines
Five DRZpositive (DO28915 and AMAL, Dw2; E4181324, Dw12; KASOll, Dw21; RML, Dw22) and seven DR6positive homozygous cell lines (DEU and WT51, Dw4; YAR, DwlO; JHAF, Dw13; PE117 and MT14B, Dw14; LKT3, Dw15) of the 10th International HistocompatibilityWorkshop, as well as 26 homozygous cell lines representing the 13 DQBI alleles recognized by the HLA Nomenclature Committee in 1989 (14) were used as controls in the DQB EcoRV RFLP, DR4 PCRSSO and DQB PCR-SSO analyses, respectively. Southern blot analysis
DNA was isolated from peripheral blood leukocytes and from the homozygous cell lines by phenol/chloroform extraction of proteinase-K treated nuclei in microscale. Aliquots of 8-10 pg DNA were digested with BurnHI, EcoRI, EcoRV, MspI and TuqI (Boehringer-Mannheim, Mannheim, Germany) according to the manufacturer’s recommendations. Agarose gel electrophoresis, capillary blotting of size-separated DNA fragments onto nylon membranes, labeling of purified probe inserts, prehybridization, hybridization, stringency washes and autoradiography were performed according to standard techniques with minor modifications (described in ref. 7). Bql DRB, DQA and DQB RFLP typing Allelic TaqI DRB, DQA and DQB restriction fragment patterns were analyzed as previously described (15-17). To avoid local RFLP nomenclature, the serologically defined DR and DQ specificities associated with the different allelic TaqI DRB-DQA-DQB haplotypes are given in ,the text and Tables (15-17). All MS patients and controls were DR and DQ typed by Tuql RFLP analysis. EcoRV DQB RFLP analysis
EcoRV-digestions were performed on samples from 30 DRwl5,DQw6,Dw2-positiveMS patients, 30 DRwl S7DQw6,Dw2-positivecontrols and the five DR2-positive cell lines. BamHI-, EcoRI-, EcoRV-, MspI- and TaqI-cleaved DNA from DRwl5,DQw6,Dw2 homozygous MS patients (n=7), controls (n=6) and cell lines (n=2) were hybridized with DRB, DQA and DQB cDNA probes. Mspl DPA and DPB RFLP typing
Allelic MspI DPA and DPB restriction fragment patterns were analyzed as described in refs. 18-20.
Multiple sclerosis and HLA class I1 genes
The cellularly defined DP specificities associated with the different allelic MspI patterns are given in the text and Tables (18-20). One-hundred-andseventy-two of the MS patients and all the controls were DP typed by MspI RFLP analysis.
given in ref. 21. The PCR amplified DQB genes were hybridized with the same SSOs that were used in the Norwegian study, i.e. p26.2 and p.26.3, complementary to codon 23-30 of DQBl *O6U2, 0302 and 0303, and to codons 24-31 of DQBl*0603 and 0604, respectively (8).
Primers for in vitro DNA amplification
For PCR amplification of DRBl genes in DR4 haplotypes, primers 337C and 336C were used (21). Initial group-specific amplifications were followed by a second amplification step with internal primers complementary to codons 3 9 4 5 and 78-84 for increased efficiency of amplification. Primers GLPDQPl and GAMPDQXj32 were used for amplification of DQB genes (22).
Statistical analysis
The polymorphic second exon of the DRBl genes was group-specifically PCR-amplified by nested PCR in 44 of the 54 DRCpositive patients, 69 of the 93 DR4-positive controls and the seven DR4positive cell lines. The latter were used as amplification and hybridization controls. PCR amplification was performed in a programmable thermal cycler (Perkin Elmer, Cetus) under conditions recommended by the manufacturer. In vitro amplification were performed as follows; 20 cycles with primers 337C and 336C, 1 rnin denaturation at 94"C, 1 min annealing at 56°C and 1 rnin extension at 72"C, followed by 20 cycles with the internal primers, 1 min denaturation at 94"C, 1 min annealing at 57°C and 1 rnin extension at 72°C.
Data were analyzed by the chi-square test or Fisher's exact test when the criteria of the chi-square test were not fulfilled. When looking for DR-DQ associations other than the well-established DRw 15,DQw6,Dw2 association, gene frequencies in patients and controls were compared after subtraction of DwZpositive haplotypes (24). When appropriate, probability (p) values were corrected for multiple comparisons with a factor of 32 for the number of DR-DQ haplotypes.(n = 24), DQA2 RFLPs (n=2) and DP alleles (n=6) determined (pC32)and with a factor of 2 for dividing the patients into two groups, i.e. PCP MS and R/R MS (pc2).p values were not corrected for multiple comparisons when associations with previously published RFLPs or DQAl and DQBl sequences were investigated, or when our previously described immunogenetic differences between PCP MS and R/R MS were investigated in a new group of patients compared to a new group of controls. Strength of associations are given as relative risks (RR), calculated according to Woolf (25). Two-locus linkage analyses were performed as described in ref. 26. The mode of inheritance was investigated by the method of antigen genotype frequencies (27).
In vitro amplification of DQB genes
Statistical study design
The polymorphic second exon of the DQB genes was amplified in 115 MS patients, 100 controls and 26 homozygous cell lines representing 13 DQBl alleles. The latter were used as amplification and hybridization controls. PCR amplification was performed in a programmable thermal cycler (Perkin Elmer, Cetus) under conditions recommended by the manufacturer. Thirty cycles of amplification were performed as follows; 1 min denaturation at 94"C, 1 rnin annealing at 55°C and 1 rnin extension at 72°C.
The findings of our previously published studies on DR-DQ associations in clinical subgroups of MS (n= 100) and the distribution of DP alleles (n = 110) in MS (7, 12), were investigated in independent groups of patients (n = 79 for DR-DQ typings and n=62 for DP typings) and controls (n= 150). When the findings of other researchers were investigated, the two groups of MS patients (n= 179) and the two groups of controls (n = 250) were combined.
In vitro amplification of DRB7 genes in DR4 haplotypes
Hybridization with oligonucleotide probes
Results and Discussion DR-DQ associations in MS patients
The PCR-amplified product was manually dot blotted onto nylon membranes. 3'end-labelling of synthetic oligonucleotide probes with 32P,prehybridization, hybridization and stringency washes was performed as described in ref. 23. The sequences of the SSOs used for DR4 subtyping are
An association in MS with HLA class I1 determinants was first described by Jersild et al. in 1973 (3). In most population studies of Caucasians MS patients from Europe and North America, consistently strong associations with the Dw2 and DR2 specificities have been obtained (reviewed in ref. 3
Olerup & Hillert
4). By the use of RFLP analysis and PCR-SSO typings, the associations have been further delineated, by us and by others, to be with the DRwlS,DQw6 haplotype or with genomic nomenclature DRBl* 1501-DQAl *0102-DQB1*0602 (7, 9, 28-30). In our large group of genotyped MS patients the well-established DRw 15,DQw6 association (DRwl5 is a split of DR2) in MS was confirmed (p< lo-”; RR=3.9) (Table 1). All DRwlS,DQw6positive MS patients and all but one of the DRw 1S7DQw6-positivecontrols carried the Dw2associated TuqI DRB-DQA-DQB allelic RFLP patterns (15-17). A significant increase of the DRwl7,DQw2 haplotype was observed in all MS patients (p < 0.005, pc32< 0.05; RR = 1.4) (Table 1). When the patients were stratified according to clinical form of MS, it was found that only R/R MS was positively associated with DRwl7,DQw2 (p < lod4, pew< 0.005; R R = 1.7) (Table 1). An increase of DRwl7 (DRw17 is the DR3-split seen in Caucasians) can be seen in several earlier studies, but has not always been commented upon (29, 31-33). A second HLA association will be concealed by a Table 1. Frequencies (%) of DR-DQ phenotypes in patients with R MS compared to healthy controls DR
DQ
All MS n=179
PCP MS n=36
MS, PCP MS and RI
RIR MS n=143
Controls n=250
1, ’Br‘
w5
11
14
11
18
wl5 wl6
w6 w5
63’ 2
58‘ 0
643 2
30 1
wl7 wl8
w2 w4
2z4 0
6 0
275 0
17 0
4 4
w7 w8
9 22
8 396
9 18
12 26
wll w12
w7 w7
5
3
6
5
0
6
14 6
wl3 w13 wl4 wl4
w6 w7 w5 w7
14 1 3 0
28 0 3 0
10 1 3
0
29 2 6 0
7 7
w2 w9
7 5
3 6
8 4
7 5
w8 w8
w4 w7
12 0
14 0
12 0
9 1
9
w9
4
0
5
4
WlO
w5
1
3
0
2
’ p < lo-’’; 3
6
4
RR 3.9. p < 0.005; p& < 0.005; RR 3.3. p
Iutredrctlen
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disorder of the central nervous system often causing neurological deficits. The etiology and many aspects of the pathogenesis remain unclear. Most current hypotheses postulate that the disease is caused by a T cell-mediated autoimmune destruction of the myelin sheath in the genetically predisposed individual. The racial and ethnic differences in the incidence of MS, the familial occurrence of MS with a
Ollr Olenp12 and Jan Hlllert'~' 'Center for BioTechnology. Karolinska Institute, NOVUM. Huddinge. zDepartment of Clinical Immunology and 'Department of Neurology, Karolinska Institute at Huddinge Hospital, Huddinge, Sweden
Key words: genetic susceptibility - HLA-0 a n k gens - HLA-DP - HLA-W 4 L A - D R - multiple sclerosis - polymerase chain reaction - primarily chronic progressive multiple sclerosis rehpsinglremitting multiple sclerosis - restriction fragment length polymorphism Received 3 May, accepted lor publication 6 May 1991
tendency to increasing prevalence with increasing degree of kinship (reviewed in ref. l), as well as the significantly higher concordance rate in monozygotic twins compared to dizygotic twins (2) indicate a major genetic etiologic component. In most population studies of Caucasian MS patients strong associations with the HLA class I1 specificities Dw2 and DR2 have been observed (3, reviewed in ref. 4). In formal segregation and linkage analysis an excellent fit was obtained for a model with a dominant MS genetic factor tightly linked to the DR2 haplotype and a second unlinked determinant 1
Olerup & Hillert (5). However, as only limited data are available on the concordance rate in HLA-identical siblings, the influence of non-HLA linked genes is difficult to ascertain. During the past 5 years many reports on HLA class I1 associations in MS have suggested that the HLA-D region-associated MS susceptibility gene(s) are more closely associated with the DQ than the D R subregion. The existence of DQ-associated MS resistande genes has also been proposed (6, 7). Furthermore, it has been suggested that MS is one of the HLA-associated diseases related to specific amino acids or epitopes of the class I1 molecules (8,9). Analogous to the findings in celiac disease, the susceptibility to MS has been hypothesized to be jointly determined by DQAI and DQBl gene products forming a-P heterodimers encoded in cis or in trans (9). Since these findings have not been undisputed and as they are of great potential interest in the unravelling of the HLA-D region-associated MS susceptibility genetic factor(s), we have found it of interest to critically review them and also to investigate these issues in a large group of Swedish MS patients. A second aim of the present study was to test if our previously published finding of immunogenetic differences between primarily chronic progressive (PCP) MS and relapsing/remitting (R/R) MS (7, 10) could be verified in an independent study.
Material and Methods Patients
One-hundred-and-seventy-nineunrelated Swedish patients with clinically definite MS (1 1) were studied. Thirty-six patients had PCP MS, i.e. gradually progressive neurological symptoms from the onset of the disease without remissions. Onehundred-and-forty-three patients had R/R MS. This latter group included patients who experienced secondary progressive evolution of symptoms after initial relapses and remissions. One hundred of the patients, 26 with PCP MS and 74 with R/R MS, were included in a previous study on DR-DQ associations in MS (7). The DP typings of 110 of the patients, 28 with PCP MS and 82 with R/R MS, have been reported elsewhere (12).
Controls Two-hundred-and-fifty randomly selected, healthy Swedes were used as controls. The HLA class I1 typings of 100 of the controls have been reported previously; DR and DQ (7, 13), and DP (12). 2
Cell lines
Five DRZpositive (DO28915 and AMAL, Dw2; E4181324, Dw12; KASOll, Dw21; RML, Dw22) and seven DR6positive homozygous cell lines (DEU and WT51, Dw4; YAR, DwlO; JHAF, Dw13; PE117 and MT14B, Dw14; LKT3, Dw15) of the 10th International HistocompatibilityWorkshop, as well as 26 homozygous cell lines representing the 13 DQBI alleles recognized by the HLA Nomenclature Committee in 1989 (14) were used as controls in the DQB EcoRV RFLP, DR4 PCRSSO and DQB PCR-SSO analyses, respectively. Southern blot analysis
DNA was isolated from peripheral blood leukocytes and from the homozygous cell lines by phenol/chloroform extraction of proteinase-K treated nuclei in microscale. Aliquots of 8-10 pg DNA were digested with BurnHI, EcoRI, EcoRV, MspI and TuqI (Boehringer-Mannheim, Mannheim, Germany) according to the manufacturer’s recommendations. Agarose gel electrophoresis, capillary blotting of size-separated DNA fragments onto nylon membranes, labeling of purified probe inserts, prehybridization, hybridization, stringency washes and autoradiography were performed according to standard techniques with minor modifications (described in ref. 7). Bql DRB, DQA and DQB RFLP typing Allelic TaqI DRB, DQA and DQB restriction fragment patterns were analyzed as previously described (15-17). To avoid local RFLP nomenclature, the serologically defined DR and DQ specificities associated with the different allelic TaqI DRB-DQA-DQB haplotypes are given in ,the text and Tables (15-17). All MS patients and controls were DR and DQ typed by Tuql RFLP analysis. EcoRV DQB RFLP analysis
EcoRV-digestions were performed on samples from 30 DRwl5,DQw6,Dw2-positiveMS patients, 30 DRwl S7DQw6,Dw2-positivecontrols and the five DR2-positive cell lines. BamHI-, EcoRI-, EcoRV-, MspI- and TaqI-cleaved DNA from DRwl5,DQw6,Dw2 homozygous MS patients (n=7), controls (n=6) and cell lines (n=2) were hybridized with DRB, DQA and DQB cDNA probes. Mspl DPA and DPB RFLP typing
Allelic MspI DPA and DPB restriction fragment patterns were analyzed as described in refs. 18-20.
Multiple sclerosis and HLA class I1 genes
The cellularly defined DP specificities associated with the different allelic MspI patterns are given in the text and Tables (18-20). One-hundred-andseventy-two of the MS patients and all the controls were DP typed by MspI RFLP analysis.
given in ref. 21. The PCR amplified DQB genes were hybridized with the same SSOs that were used in the Norwegian study, i.e. p26.2 and p.26.3, complementary to codon 23-30 of DQBl *O6U2, 0302 and 0303, and to codons 24-31 of DQBl*0603 and 0604, respectively (8).
Primers for in vitro DNA amplification
For PCR amplification of DRBl genes in DR4 haplotypes, primers 337C and 336C were used (21). Initial group-specific amplifications were followed by a second amplification step with internal primers complementary to codons 3 9 4 5 and 78-84 for increased efficiency of amplification. Primers GLPDQPl and GAMPDQXj32 were used for amplification of DQB genes (22).
Statistical analysis
The polymorphic second exon of the DRBl genes was group-specifically PCR-amplified by nested PCR in 44 of the 54 DRCpositive patients, 69 of the 93 DR4-positive controls and the seven DR4positive cell lines. The latter were used as amplification and hybridization controls. PCR amplification was performed in a programmable thermal cycler (Perkin Elmer, Cetus) under conditions recommended by the manufacturer. In vitro amplification were performed as follows; 20 cycles with primers 337C and 336C, 1 rnin denaturation at 94"C, 1 min annealing at 56°C and 1 rnin extension at 72"C, followed by 20 cycles with the internal primers, 1 min denaturation at 94"C, 1 min annealing at 57°C and 1 rnin extension at 72°C.
Data were analyzed by the chi-square test or Fisher's exact test when the criteria of the chi-square test were not fulfilled. When looking for DR-DQ associations other than the well-established DRw 15,DQw6,Dw2 association, gene frequencies in patients and controls were compared after subtraction of DwZpositive haplotypes (24). When appropriate, probability (p) values were corrected for multiple comparisons with a factor of 32 for the number of DR-DQ haplotypes.(n = 24), DQA2 RFLPs (n=2) and DP alleles (n=6) determined (pC32)and with a factor of 2 for dividing the patients into two groups, i.e. PCP MS and R/R MS (pc2).p values were not corrected for multiple comparisons when associations with previously published RFLPs or DQAl and DQBl sequences were investigated, or when our previously described immunogenetic differences between PCP MS and R/R MS were investigated in a new group of patients compared to a new group of controls. Strength of associations are given as relative risks (RR), calculated according to Woolf (25). Two-locus linkage analyses were performed as described in ref. 26. The mode of inheritance was investigated by the method of antigen genotype frequencies (27).
In vitro amplification of DQB genes
Statistical study design
The polymorphic second exon of the DQB genes was amplified in 115 MS patients, 100 controls and 26 homozygous cell lines representing 13 DQBl alleles. The latter were used as amplification and hybridization controls. PCR amplification was performed in a programmable thermal cycler (Perkin Elmer, Cetus) under conditions recommended by the manufacturer. Thirty cycles of amplification were performed as follows; 1 min denaturation at 94"C, 1 rnin annealing at 55°C and 1 rnin extension at 72°C.
The findings of our previously published studies on DR-DQ associations in clinical subgroups of MS (n= 100) and the distribution of DP alleles (n = 110) in MS (7, 12), were investigated in independent groups of patients (n = 79 for DR-DQ typings and n=62 for DP typings) and controls (n= 150). When the findings of other researchers were investigated, the two groups of MS patients (n= 179) and the two groups of controls (n = 250) were combined.
In vitro amplification of DRB7 genes in DR4 haplotypes
Hybridization with oligonucleotide probes
Results and Discussion DR-DQ associations in MS patients
The PCR-amplified product was manually dot blotted onto nylon membranes. 3'end-labelling of synthetic oligonucleotide probes with 32P,prehybridization, hybridization and stringency washes was performed as described in ref. 23. The sequences of the SSOs used for DR4 subtyping are
An association in MS with HLA class I1 determinants was first described by Jersild et al. in 1973 (3). In most population studies of Caucasians MS patients from Europe and North America, consistently strong associations with the Dw2 and DR2 specificities have been obtained (reviewed in ref. 3
Olerup & Hillert
4). By the use of RFLP analysis and PCR-SSO typings, the associations have been further delineated, by us and by others, to be with the DRwlS,DQw6 haplotype or with genomic nomenclature DRBl* 1501-DQAl *0102-DQB1*0602 (7, 9, 28-30). In our large group of genotyped MS patients the well-established DRw 15,DQw6 association (DRwl5 is a split of DR2) in MS was confirmed (p< lo-”; RR=3.9) (Table 1). All DRwlS,DQw6positive MS patients and all but one of the DRw 1S7DQw6-positivecontrols carried the Dw2associated TuqI DRB-DQA-DQB allelic RFLP patterns (15-17). A significant increase of the DRwl7,DQw2 haplotype was observed in all MS patients (p < 0.005, pc32< 0.05; RR = 1.4) (Table 1). When the patients were stratified according to clinical form of MS, it was found that only R/R MS was positively associated with DRwl7,DQw2 (p < lod4, pew< 0.005; R R = 1.7) (Table 1). An increase of DRwl7 (DRw17 is the DR3-split seen in Caucasians) can be seen in several earlier studies, but has not always been commented upon (29, 31-33). A second HLA association will be concealed by a Table 1. Frequencies (%) of DR-DQ phenotypes in patients with R MS compared to healthy controls DR
DQ
All MS n=179
PCP MS n=36
MS, PCP MS and RI
RIR MS n=143
Controls n=250
1, ’Br‘
w5
11
14
11
18
wl5 wl6
w6 w5
63’ 2
58‘ 0
643 2
30 1
wl7 wl8
w2 w4
2z4 0
6 0
275 0
17 0
4 4
w7 w8
9 22
8 396
9 18
12 26
wll w12
w7 w7
5
3
6
5
0
6
14 6
wl3 w13 wl4 wl4
w6 w7 w5 w7
14 1 3 0
28 0 3 0
10 1 3
0
29 2 6 0
7 7
w2 w9
7 5
3 6
8 4
7 5
w8 w8
w4 w7
12 0
14 0
12 0
9 1
9
w9
4
0
5
4
WlO
w5
1
3
0
2
’ p < lo-’’; 3
6
4
RR 3.9. p < 0.005; p& < 0.005; RR 3.3. p
