Restriction fragment length polymorphism analysis of HLA-DR- and DQ-linked alleles in multiple sclerosis in Spain

HLA-DR allele subtypes in multiple sclerosis (MS) individuals in Spain were determined by restriction fragment length polymorphism (RFLP) analysis. The well-established association of DRw15, DQw6, Dw2 alleles and MS susceptibility was confirmed. The strength of its association, however, was relatively weak in our MS population and no involvement with any other DR allele was observed. DQw6 increase correlated with the elevation of the involved DR2 subtype. The hypothesis that MS-associated susceptibility genes may be more closely associated with the DQ than the DR region was not supported by our findings which, on the other hand, could be in concordance with the concept that multiple genes contribute to MS susceptibility.     

HLA-DR beta, -DQ alpha, and -DQ beta restriction fragment length polymorphisms in multiple sclerosis

Restriction fragment length polymorphism (RFLP) studies were performed on DNA from unrelated Caucasian patients with multiple sclerosis (MS) using cDNA probes to the HLA class II genes DR beta, DQ alpha, and DQ beta. In a study of 34 patients and 34 controls who were not matched for DR type, we found that the DQ beta allele-specific RFLP or allogenotype, termed DQ beta lb, which corresponds at the molecular level to the DQwl serotype, is preferentially associated with MS. A significant disease association with DR2 was demonstrated by serology but this was not confirmed using DR2/Dw2-specific RFLPs. We suggest that DQ beta lb is largely responsible for HLA-associated susceptibility to MS and that the apparent MS-DR2 serological association is due to the strong linkage disequilibrium between DR2 and DQ beta lb. Homozygosity of one of the two allelic bands of the DX alpha gene, usually termed the DX alpha lower (DX alpha L) band (which cross-hybridizes with the DQ alpha probe), correlated with reduced susceptibility to MS. Similarly a 5.3 kb band identified by the DQ alpha probe in Mspl digests showed a negative correlation with MS. In an analysis of 27 DR2+ controls and 26 DR2+ patients it was found that these individuals all had DR2/Dw2-specific RFLPs and all had identical DR2/Dw2-associated DQ beta (DQ beta lb) and DQ alpha (DQ alpha lb) allogenotypes. We detected no polymorphisms of DR beta, DQ alpha, or DQ beta genes among the DR2+ MS patients which distinguished them from normals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence variation in the transforming growth factor-beta1 (TGFB1) gene and multiple sclerosis susceptibility

Genome screenings in multiple sclerosis (MS) have identified multiple susceptibility regions supporting a polygenic model for this disease. Evidence for linkage was consistently observed at ch.19q13 suggesting the presence of an MS gene(s) in this region. Several interesting candidate genes are encoded within this region, including transforming growth factor-beta 1 (TGFB1) and interleukin-11 (IL11). Both are multifunctional cytokines with significant and well-characterized immunomodulatory properties. We performed a comprehensive evaluation of common polymorphisms within the TGFB1 and IL11 loci and three closely flanking microsatellite markers (D19S421, CEA, D19S908) in 161 stringently ascertained and clinically characterized MS multiplex families using tests of both linkage (lod score, sib-pair analysis) and association (pedigree disequilibrium test or PDT). Patients and families were stratified by HLA-DR2 status to search for two-locus interactions. Suggestive evidence for linkage and association to CEA (lod score = 1.25, theta = 0.20, p = 0.015, respectively), located 0.4 cM from TGFB1, was observed in DR2 positive families only. Distinct clinical phenotypes were also examined and an association between a TGFB1 haplotype and a mild disease course was present (p = 0.008), raising the possibility that TGFB1 or a nearby locus may influence disease expression.     

HLA typing in the United Kingdom multiple sclerosis genome screen

The United Kingdom multiple sclerosis genome screen demonstrated a peak maximum lod score of 2.8 in the HLA region, together with statistically significant excess transmission of the 121-base pair (bp) allele of the tumour necrosis factor-a marker. In order to determine whether this association is independent of the established HLA association, or simply a consequence of the 121-bp allele being part of the same haplotype, we HLA-DR and -DQ typed the 227 sibling-pair families used in the original screen. The expected associations of multiple sclerosis with the DR15 (p=8.7E-18), DQ6 (p=2.0E-09) and DR51 (p=2.8E-16) phenotypes were confirmed, and excess transmission of the DRB1*1501 and DQB1*0602 alleles was demonstrated. Combining HLA typing with the original microsatellite data demonstrated extensive linkage disequilibrium between the 121-bp allele and the 1501-0602 haplotype. Outside this extended haplotype (121-1501-0602), none of the alleles demonstrated significant transmission distortion. Having established the importance of this extended haplotype, we reanalysed the entire genome screen data after excluding those sibling pairs sharing the extended haplotype (n=27). Conditioning the full genome screen data on the basis of identity by state sharing showed that some potential linkage regions identified in the original screen clustered in families, in which the extended haplotype was shared (1p, 2p and 17q), whereas others grouped with those in which it was not (5cen, 7p and Xq). This suggests complexity in the genetics of multiple sclerosis. Received: May 22, 1998 / Accepted: July 1, 1998 / Published online: December 9, 1998     

HLA and prognosis in multiple sclerosis

The patients of a multiple sclerosis (MS) incidence cohort with 25 years of longitudinal follow-up were typed for HLA-DR and DQ. This type of cohort provides reliable data for gene frequencies and prognostic studies. The influence of sampling bias, mainly due to mortality during the long follow-up, was accounted for. A positive association between MS and DR15,DQ6 was confirmed, but this haplotype did not influence prognosis. There was no difference in haplotype frequency between relapsing-remitting and primary chronic progressive MS. DR17,DQ2 was significantly over-represented in the quartile with the most malignant course. The haplotype DR1,DQ5, which was found rather less frequently in MS patients, also tended to be associated with a poorer prognosis.     

Chromosome 19q13 and multiple sclerosis susceptibility in Finland: a linkage and two-stage association study

Several studies have previously provided some albeit weak evidence for linkage or association between chromosome 19q13 and multiple sclerosis (MS) susceptibility. We performed a two-stage association analysis with 19 markers spanning 7 Mb/5.5 cM of 19q13. In stage 1 analysis (135 MS families) allelic and haplotypic associations were found with markers within or close to the ApoE-ApoC subregion. These observations were taken as a hypothesis, which was tested in stage 2 in 125 families. However, none of the initial associations were replicated suggesting that they were most likely due to chance. Linkage analysis was performed in 27 Finnish multiplex families using 10 microsatellites spanning 23 Mb/24 cM of 19q13. DNA was available from 72 MS patients and 150 unaffected relatives. Parametric and non-parametric linkage analyses did not provide evidence for linkage when all families were tested. After stratifying the families according to HLA-DR15 there was weak evidence for linkage to the 19q13.1 subregion in DR15 negative families (LOD(max)=1.8). Taken together these results do not support a major role of chromosome 19q13.2-q13.3 in MS susceptibility among Finnish MS patients, whereas conclusions on the 19q13.1 subregion are less clear and this region requires further study.     

Evidence for the genetic role of human leukocyte antigens in low frequency DRB1*1501 multiple sclerosis patients in Israel

A strong association exists between multiple sclerosis (MS) and the DRB1*1501 haplotype, in most populations. Linkage of Multiple Sclerosis (MS) with the MHC or HLA region on chromosome 6p21 has previously been observed in DRB1*1501 positive MS families. A group of 13 Israeli multiplex MS families with a very low frequency of DRB1*1501 haplotype were examined in this study. Association and a linkage test were performed in order to identify a non-DRB1*1501 effect of HLA on susceptibility for MS. MS multiplex families and healthy controls were molecularly typed for six highly polymorphic markers located within the MHC region: DRB1, DQA1 and DQB1, BAT-2, MIB and D6S248. Data analyses included: (a) an association study comparing the patient group with both healthy relative, and healthy control groups (b) a transmission test for linkage disequilibrium (TDT) of the MS-associated alleles in the multiplex families, and (c) multipoint non-parametric linkage (NPL) and parametric LOD score analyses using the GENEHUNTER program. The DRB1*1303 allele was significantly more frequent among the MS patients. There was a trend towards transmission disequilibrium of DRB1*1303, but was not statistically significant. Allele sharing and LOD score analyses revealed no evidence for linkage. The high frequency of DRB1*1303 observed in our family patients provides evidence to support the association with this allele that previously described in sporadic non-Ashkenazi MS patients. Thus, DRB1*1303 may serve as genetic risk factor for MS. Our study exemplifies the genetic heterogeneity in MS as there is a genetic effect of HLA on MS susceptibility in our low frequency DRB1*1501 patients.     

Genetic segregation of multiple sclerosis and histocompatibility (HLA) haplotypes

Summary The possibility that a gene determining susceptibility to multiple sclerosis (MSS) may be closely linked to the major histocompatibility locus (HLA) is suggested by observation of a loose association between multiple sclerosis (MS) and certain HLA determinants. In the present study, the possible association was analyzed by studying the segregation of MS and the HLA haplotypes in families with more than one case of MS. Analysis of 48 published families revealed that the haplotype shared by those with MS within the family was also shared by those without clinical signs of MS at close to the 50% frequency expected by chance. Thus, we were unable to demonstrate that MS is associated with one HLA defined parental haplotype. We discussed reasons for this apparent failure to demonstrate existence of an MSS gene using available multiplex MS families.Supported in part by a grant from the Kroc Foundation     

Linkage and association analysis of susceptibility regions on chromosomes 5 and 6 in 106 Scandinavian sibling pair families with multiple sclerosis.

In the genetically homogeneous Scandinavian population, we have investigated chromosome 5 and the HLA (human leukocyte antigen) region on chromosome 6p21 by applying linkage and association analyses on 106 white sibling pair families with multiple sclerosis. The importance of genes within the HLA region for the susceptibility of multiple sclerosis has previously been reported. More recently, findings have suggested importance of regions on chromosome 5. Half of chromosome 5 was analyzed by using 14 microsatellite markers and a susceptibility region with a maximum LOD score of 1.1 was identified. Chromosome 6 was analyzed by HLA-DR typing and using the TNF alpha microsatellite marker. A peak maximum LOD score of 2.0 was found at the HLA-DR marker. Association studies were made for all the markers, comparing 106 probands from the sibling pairs with 100 unrelated controls. None of the markers on chromosome 5 showed significant association with multiple sclerosis, whereas strong association between multiple sclerosis and DR2 was found, with an odds ratio of 3.7 (p < 10(-5)). It is surprising that association was not seen for any of the TNF alpha alleles including the 121-bp allele, although this allele was in positive linkage disequilibrium with DR2 in both patients and controls. Our results support the existence of multiple sclerosis susceptibility loci on chromosomes 5p and 6p21.     

Fine mapping of the multiple sclerosis susceptibility locus on 5p14-p12

Linkage analyses have identified four major MS susceptibility loci in Finns. Here we have fine mapped the region on chromosome 5p in 28 Finnish MS families. Marker D5S416 provided the highest pairwise LOD score, and multipoint and haplotype analyses restrict the critical region to about 5.3 Mb on 5p15 between markers D5S1987 and D5S416. Ascertaining for HLA type and geographical origin indicated that families with and without the HLA DR15 risk haplotype, as well as families within and outside an internal high-risk region, contributed to the linkage to 5p, implying the general significance for this locus in Finnish MS families.     

Multiple sclerosis and HLA class II susceptibility and resistance genes

Probes to the HLA class II genes DRβ, DQβ, and DQα were used to study DNA from unrelated Caucasian multiple sclerosis (MS) patients by sequential restriction fragment length polymorphism (RFLP) analysis in Taq1 restriction enzyme digests. Comparison of 104 patients and 108 controls, who were not matched for DR type, has identified for the first time a linked series of allele-specific RFLPs or allogenotypes which form an extended haplotype that is preferentially associated with MS. These allogenotypes include DRw15 or DR2(15);DQβlb, which corresponds at the DNA level to the DQwl (DQw6) serotype; a DQA1 allogenotype termed DQα1b; and a 2.2kb DX (DQA2) allogenotype termed DXαU (DQA2U). The role of HLA class II genes in susceptibility to MS was found to be complex. First, 23 of 104 MS patients showed DR-DQ linkages which were not observed in our control population. We suggest these anomalous associations may be important in the pathogenesis of MS. Second, homozygosity of a 2.0 KB DX (DQA2) gene, termed DXαL (DQA2L), Showed a strong negative association with MS. DXαL (DQA2L) is in strong linkage disequilobrium with DR1, 5(w11)7, and a subset of DR4, all of which also showed a negative association with MS, Since DXαL (DQA2L) does not code for any known product, DR1, 5(11), 4, and 7 become candidates for disease resistance genes. Third, in EcoR1 and EcoRV digests of DNA from both controls and patients homozygous for DQβ1b a number of different RFLP patterns were identified and these RFLPs patterns were identified and these RFLPs were associated with either relapsing-remitting or progressive MS. This suggests there may be HLA sequence differences between individuals bearing a particular class II allele and these may correlate with the clinical course of MS.     

No evidence to support CTLA-4 as a susceptibility gene in MS families: the Canadian Collaborative Study

Two polymorphisms of the CTLA-4 gene were genotyped in 232 sibling pairs affected with multiple sclerosis (MS) from 185 families. The CTLA-4 polymorphisms genotyped were a 3' untranslated (AT)(n) microsatellite and an alanine/threonine RFLP of exon 1. There was no evidence observed for linkage by either identity-by-descent (ibd) or identity-by-state (ibs) methods. A transmission disequilibrium test (TDT) was performed and no preferential transmission of alleles was observed. Upon stratification of patients, there was no preferential transmission observed based upon gender, by presence or absence of HLA*DRB1*15, by ethnicity or by clinical course of the disease. CTLA-4 does not appear to be a major MS susceptibility locus in Canadian multiplex families.     

Genetic control of multiple sclerosis: Increased production of lymphotoxin and tumor necrosis factor-α by HLA-DR2+ T cells

Lymphotoxin (LT) and tumor necrosis factor-α (TNF-α) play an important role in the pathogenesis of multiple sclerosis (MS). MS is associated with the HLA-DR2, Dw2, DQ6 HLA class II haplotype. Because both LT and TNF-α are encoded in the HLA region, the HLA association of MS may be related to the production of these cytokines. To test this hypothesis, we investigated the production of LT, TNF-α, and interferon-γ (IFN-γ) by CD4⁺ T-cell lines (TCLs) specific for myelin basic protein (MBP) or tetanus toxoid (TT) isolated from MS patients and normal controls. After stimulation with specific antigen but not mitogen, TCLs from HLA-DR2⁺ donors produced significantly more LT and TNF-α, than TCLs from DR2^? donors. In contrast, HLA-DR2⁺ and DR2^? TCLs did not differ in the production of IFN-γ, a cytokine also produced by T cells but not encoded in the HLA region. Increased secretion of LT and TNF-α was unrelated to the specificity (MBP vs TT), MHC restriction (HLA-DR2 vs other DR molecules), or source (MS vs normal) of the TCLs. There was no significant association of the cytokine production with individual LT or TNF-α alleles, indicating that the increased production of these cytokines may be linked to other polymorphic genes in this region. The results suggest that the association of MS with HLA-DR2 implies a genetically determined propensity of T cells to produce increased amounts of LT and TNF-α.     

Sardinian multiple sclerosis is associated with HLA-DR4: a serologic and molecular analysis

HLA haplotypes in 45 unrelated Sardinian multiple sclerosis patients and in six multiplex families were defined, using both serologic and restriction fragment length polymorphism (RFLP) analysis. In unrelated MS patients, we found an association with HLA-DR4 (p less than 0.01, relative risk = 2.5) and DQw3 (p less than 0.04, relative risk = 2.2). Using a beta-DR cDNA probe, we observed no variation of the DR4 RFLP profile in sporadic or related MS patients compared with DR4-specific pattern in controls. Using a beta-DQ cDNA probe, we identified two DQw3 patterns (DQw3.1 and DQw3.2) with similar frequency in patients and in controls. No specific RFLPs were observed in association with different disease courses. The frequency of haplotype sharing in affected members of multiplex families was not different from that expected by chance. This study shows that Sardinian MS patients carry predominantly the HLA-DR4 allele, in contrast to the DR2 prevalence reported in Caucasian populations. The lack of association with HLA haplotypes in affected members of multiplex families may indicate that genetic factors outside the HLA system play a substantial role in families with MS.     

DNA restriction fragment length polymorphism of HLA-DR2: Correlation with HLA-DR2-associated functions

HLA-DR2 can be divided into at least 3 distinct HLA-D clusters which correlate with structural differences within the HLA-D region. Further, a functional counterpart of this subdivision has been previously identified. The presence of a particular DR beta 2 polypeptide chain correlated precisely with the susceptibility of measles virus-infected HLA-DR2 homozygous typing cell lines to lysis by measles virus-specific, HLA class II-restricted CTL clones. To determine if a genetic basis for these functional differences could be detected, the degree of polymorphism at the DNA level within the serologically defined HLA-DR2 haplotype has been examined. By using DNA probes for DR beta and DQ beta 4 of the 5 HLA-D clusters of HLA-DR2 could be distinguished and a RFLP pattern was identified which correlates with known immunological functions associated with these various D types. In addition, this technique of 'molecular genotyping' was used to investigate a limited panel of patients with multiple sclerosis (MS) who were HLA typed as either HLA-DR2,2 or HLA-DR2,blank. The RFLP profile of the HLA-DR2 Dw2 D type was found in all of these MS patients.     

The HLA locus and multiple sclerosis in Spain. Role in disease susceptibility,clinical course and response to interferon-beta

The HLA-DR2 haplotype (DRB1*1501, DQB1*0602) on chromosome 6p21 has consistently demonstrated both association and linkage with multiple sclerosis (MS) in case-control and family studies, particularly in Caucasians of Northern European descent. However, the role of a gene within this region in determining clinical features or response to immunotherapy remains largely unknown. A new familial MS data set from the Mediterranean Spanish Basin was collected according to rigorous ascertainment criteria. We confirm, primarily in the cohort originating from Continental Spain, that similar to other high-risk groups, there was a significant association with HLA-DR2. No other DR or DQ alleles were found to be associated with disease susceptibility nor were alleles at the class I A and B loci. Overall, the effect of HLA appears to be less substantial than that observed in a reference US population with a higher disease incidence. No effect of the HLA-DR2 haplotype on age of onset, initial clinical symptoms and disease course was observed. Similarly, no difference in the distribution of responders and nonresponders to interferon-beta (IFNB) therapy, as defined by primary and secondary end points, was observed when individuals were stratified according to HLA-DR2 status.     

DNA analysis of HLA-DR4B1 subtypes in multiple sclerosis by specific oligonucleotide probes

Conversely to the well-established association of DR2/Dw2 with multiple sclerosis (MS) susceptibility in Caucasoids, several studies have found an association of DR4 in populations from Mediterranean origin. We have studied the distribution of the different DR4B1 subtypes in Spanish MS patients. Oligonucleotide probes were selected in order to type samples amplified by polymerase chain reaction (PCR) from Spanish DR4+ MS patients (25) and controls (28). No DR4B1 subtypes were found to be increased in MS. MS susceptibility linked to DR4 may be due to the presence of shared functional epitopes common to the different HLA-DR4B1 subtypes.     

HLA and multiple sclerosis in Italy: a review of the literature

Summary HLA antigens of locus A, C, B, DR and DQ were typed in 104 Italian multiple sclerosis patients and in 905 healthy controls; the results have been compared with those published in the Italian literature. The Italian studies have been reviewed regarding the ethnic origin of the typed population and the corresponding prevalence of the disease. The data suggest a lack of association between A3 and B7 antigens and Italian multiple sclerosis and a relevance of other DR locus antigens (mainly DR4 and DR5), in addition to DR2, in the susceptibility to the disease.     

No evidence for association of multiple sclerosis with the complement factors C6 and C7.

Four genome screens in multiple sclerosis have been completed and each has identified evidence for linkage in the pericentromeric region of chromosome 5. This region encodes a number of candidate genes including those for the complement components C6, C7 and C9. We have used a multiplexed oligoligation assay (OLA) to test single nucleotide polymorphisms (SNPs) from the C6 and C7 genes for evidence of association with multiple sclerosis in our sibling pair families. There was no statistically significant difference in the allele frequencies of these polymorphisms in the index cases from our families when compared with locally derived controls. No evidence for transmission distortion was seen with any of the polymorphisms, or with the haplotype built from the three SNPs from the C7 gene. Despite offering themselves as potential candidates these complement genes appear not to confer susceptibility to multiple sclerosis.