The combination of antibodies to GAD-65 and IA-2ic can replace the islet-cell antibody assay to identify subjects at risk of type 1 diabetes mellitus.

First-degree relatives of type 1 diabetic patients are at increased risk of developing diabetes and, until recently, islet cell antibodies (ICA) have represented the major risk marker used for identification of individuals at increased risk for subsequent progression to diabetes. In order to determine the value of antibodies to GAD-65 and IA-2ic to identify individuals at high risk for type 1 diabetes mellitus, we measured both autoantibodies and ICA in 1436 first-degree relatives of patients with type 1 diabetes. In addition, the sera were analyzed for thyroid, adrenal and gastric-parietal cell autoantibodies as markers for possible polyendocrine involvement. GAD-65 Abs were found in 135 out of 1436 (9.4%) first-degree relatives and in 57 of 98 (58.2%) ICA-positive subjects. IA-2ic were detected in 52 of 1436 (3.6%) first-degree relatives and in 44 of 98 (44.8%) ICA-positive relatives. IA-2ic and/or GAD-65 were detected in 73 of 98 (74.5%) ICA-positive relatives. Interestingly, antibodies to GAD-65 and/or IA-2ic were present in 91.2% of individuals with more than 20JDF-units. Anti-IA-2ic and GAD-65 were positively correlated with high levels of ICA. Anti-IA-2ic and GAD-65 were found in 19% and 48.5% of subjects with ICA levels of 5-20JDF-u but in 68.8% and 76.5% of individuals with ICA of 40JDF-u or more, respectively (p < 0.001), compared to subjects with ICA levels less than 5 JDF-u. When autoantibody frequencies among the relatives were analyzed according to relationship to the proband, the offspring and siblings had a higher frequency of ICA and IA-2ic (p<0.05) than the subgroup of parents. A significant association was observed between IA-2ic and thyroid antibodies. In addition, higher levels of IA-2ic were found in relatives with positive TPO antibodies (p < 0.001); this correlation was particularly strong in offspring and siblings (p < 0.01). Determination of GAD-65 and IA-2ic antibodies may be considered as an alternative to primary ICA-screening, enabling the screening of large populations.     

Islet cell and 64K autoantibodies are associated with plasma IgG in newly diagnosed insulin-dependent diabetic children

There is a high prevalence of islet cell antibodies (ICA) and autoantibodies detected against an islet cell protein of Mr 64,000 at the time of clinical diagnosis of insulin-dependent diabetes (IDDM). In view of the biphasic immune response after antigen presentation, the purpose of this study was to determine the presence of ICA and antibodies against the 64,000 islet antigen after separation of IgM from IgG to prevent interference between the two antibody classes. Plasma samples from 10 newly diagnosed IDDM children and 10 healthy controls were precipitated with polyethylene glycol (PEG), and the crude Ig was subjected to Sephacryl S-300 chromatography to separate IgM and IgG. ICA determined by indirect immunofluorescence on frozen sections of human pancreas showed reduced background immunofluorescence intensity in the purified fractions compared with crude plasma. The number of ICA-positive samples among the IDDM patients increased from 7/10 in plasma to 9/10 in the IgG fraction. There was an increase in the ICA titer in 6/9 of the positive samples. All purified IgM samples were ICA negative. Immunoprecipitation experiments by using Nonidet P-40 detergent lysates of [35S]methionine-labeled neonatal rat islets demonstrated that the 64,000 autoantibodies were in the IgG fraction. We found 7/10 IDDM samples to be positive, whereas all controls were negative. The background in the autoradiographic analysis was markedly reduced in the IgG fractions compared with immunoprecipitates with crude or PEG-purified plasma and the IgM fraction. ICA titers did not correlate to the ability of the IgG fraction to precipitate the 64,000 autoantigen. It is concluded that both the ICA and 64,000 autoantibodies are primarily of the IgG class at the time of clinical onset of IDDM, and that purification of IgG from human IDDM plasma facilitates the detection of the rat islet cell 64,000 antigen.     

Acetylcholine receptor peptide recognition in HLA DR3-transgenic mice: in vivo responses correlate with MHC-peptide binding.

HLA DR3 is an MHC molecule that reportedly predisposes humans to myasthenia gravis (MG). Though MG is an Ab-mediated autoimmune disease, CD4+ T cells are essential for the generation of high-affinity Abs; hence the specificities of autoreactive CD4+ T cells are important. In this study we report the HLA DR3-restricted T cell determinants on the extracellular region sequence of human acetylcholine receptor alpha subunit. We find two promiscuous determinants on this region 141-160 and 171-190 as defined by their immunogenicity in HLA DR3-, HLA DQ8-, and HLA DQ6-transgenic mice in the absence of endogenous mouse class II molecules. We also studied the minimal determinants of these two regions by truncation analysis, and the MHC binding affinity of a set of overlapping peptides spanning the complete sequence region of human acetylcholine receptor alpha subunit. One of the peptide sequences strongly immunogenic in HLA DR3-transgenic mice also had the highest binding affinity to HLA DR3. Identification of T cell determinants restricted to an MHC molecule known to predispose to MG may be an important step toward the development of peptide-based immunomodulation strategies for this autoimmune disease.     

Functional classification of class II human leukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes

Previous studies have attempted to define human leukocyte antigen (HLA) class II supertypes, analogous to the case for class I, on the basis of shared peptide-binding motifs or structure. In the present study, we determined the binding capacity of a large panel of non-redundant peptides for a set of 27 common HLA DR, DQ, and DP molecules. The measured binding data were then used to define class II supertypes on the basis of shared binding repertoires. Seven different supertypes (main DR, DR4, DRB3, main DQ, DQ7, main DP, and DP2) were defined. The molecules associated with the respective supertypes fell largely along lines defined by MHC locus and reflect, in broad terms, commonalities in reported peptide-binding motifs. Repertoire overlaps between molecules within the same class II supertype were found to be similar in magnitude to what has been observed for HLA class I supertypes. Surprisingly, however, the degree to which repertoires between molecules in the different class II supertypes also overlapped was found to be five to tenfold higher than repertoire overlaps noted between molecules in different class I supertypes. These results highlight a high degree of repertoire overlap amongst all HLA class II molecules, perhaps reflecting binding in multiple registers, and more pronounced dependence on backbone interactions rather than peptide anchor residues. This fundamental difference between HLA class I and class II would not have been predicted on the basis of analysis of either binding motifs or the sequence/predicted structures of the HLA molecules.     

Type 1 diabetes-predisposing MHC alleles influence the selection of glutamic acid decarboxylase (GAD) 65-specific T cells in a transgenic model

The genetic factors that contribute to the etiology of type 1 diabetes are still largely uncharacterized. However, the genes of the MHC (HLA in humans) have been consistently associated with susceptibility to disease. We have used several transgenic mice generated in our laboratory, bearing susceptible or resistant HLA alleles, in the absence of endogenous MHC class II (Abetao), to study immune responses to the autoantigen glutamic acid decarboxylase (GAD) 65 and its relevance in determining the association between autoreactivity and disease pathogenesis. Mice bearing diabetes-susceptible haplotypes, HLA DR3 (DRB1*0301) or DQ8 (DQB1*0302), singly or in combination showed spontaneous T cell reactivity to rat GAD 65, which is highly homologous to the self Ag, mouse GAD 65. The presence of diabetes-resistant or neutral alleles, such as HLA DQ6 (DQB1*0602) and DR2 (DRB1*1502) prevented the generation of any self-reactive responses to rat GAD. In addition, unmanipulated Abetao/DR3, Abetao/DQ8, and Abetao/DR3/DQ8 mice recognized specific peptides, mainly from the N-terminal region of the GAD 65 molecule. Most of these regions are conserved between human, mouse, and rat GAD 65. Further analysis revealed that the reactivity was mediated primarily by CD4(+) T cells. Stimulation of these T cells by rat GAD 65 resulted in the generation of a mixed Th1/Th2 cytokine profile in the Abetao/DR3/DQ8, Abetao/DR3, and Abetao/DQ8 mice. Thus, the presence of diabetes-associated genes determines whether immune tolerance is maintained to islet autoantigens, but autoreactivity in itself is not sufficient to induce diabetes.     

An analysis of variation in the long-range genomic organization of the human major histocompatibility complex class II region by pulsed-field gel electrophoresis

The class II region of the human major histocompatibility complex in seven common HLA haplotypes has been analyzed using pulsed-field gel electrophoresis, restriction enzymes that cut genomic DNA infrequently, and Southern blotting. This analysis has revealed that there are differences in the amount of DNA present in the DQ and DR subregions dependent on the haplotype. The class II region of the DR3 haplotype spans approximately 750 kb and has the same amount of DNA as the class II region of the DR5 and DR6 haplotypes. However, the DR2 haplotype has approximately 30 kb more DNA within the DR subregion. The DR4 haplotype has an additional approximately 110 kb of DNA within the DQ or DR subregions compared to the DR3, DR5, and DR6 haplotypes. These haplotype-specific differences could have some bearing both on the analysis of disease susceptibility and on the ability of chromosomes possessing different HLA haplotypes to recombine within the DQ/DR subregions.     

Heterogeneity of non-insulin-dependent diabetes mellitus in HLA types and clinical features: comparison with insulin-dependent diabetes mellitus

We determined HLA types in 110 Japanese patients with non-insulin-dependent diabetes mellitus (NIDDM) and studied the relationship between the HLA phenotypes and clinical features. Sixty-nine patients with insulin-dependent diabetes mellitus (IDDM) and 100 healthy blood donors served as controls. Concerning HLA DR and DQ loci, frequencies of DR4, DRw9 and DQw3.2 were higher, and those of DR2, DRw8, DRw11, DRw12 and DQw1 were lower in patients with IDDM compared than in healthy controls. There were no differences between NIDDM and normal controls in the frequency of a particular HLA DR antigen except for a decreased frequency in DRw11 in the former. The frequency of DQw3.2 antigen in NIDDM was intermediate between IDDM and normal controls. There were some differences between DQw3.2-positive and -negative NIDDM patients in clinical features. Those who showed low C-peptide responses during oral glucose tolerance test were more frequently found among DQw3.2-positive NIDDM patients. These results suggest that Type 1 diabetes mellitus may have a mild clinical course and is found among the Japanese NIDDM population.     

HLA class II influences the immune response and antibody diversification to Ro60/Sjögren's syndrome-A: heightened antibody responses and epitope spreading in mice expressing HLA-DR molecules

Abs to Ro/SSA Ags in the sera of patients with systemic lupus erythematosus and Sj?gren's syndrome are influenced by the HLA class II genes. To investigate the role of individual HLA class II genes in immune responses to human Ro60 (hRo60), mice lacking murine class II molecules and carrying either HLA genes DR2(DRB1*1502), DR3(DRB1*0301), DQ6(DQA1*0103/DQB1*0601), or DQ8(DQA1*0301/DQB1*0302), were immunized with rhRo60. The results show that hRo60 induces strong T and B cell responses in DR2, DR3, and DQ8 mice in comparison to weaker responses in DQ6 mice. In all mice, the majority of the dominant T cell epitopes were located in the amino portion (aa 61-185) and the carboxy portion (aa 381-525) of the hRo60 molecules. In contrast, the early dominant B cell epitopes were located in the middle and carboxy portion of the hRo60 molecule (aa 281-315 and 401-538). In DR2, DR3, and DQ8 mice, the B cell epitopes subsequently spread to the amino and carboxy portion of the hRo60 molecule but were limited to the middle and carboxy portion in DQ6 mice. The DR2 and DR3 mice produced the highest titers of immunoprecipitating Abs against hRo60 and native mouse Ro60. In addition, only DR2 mice exclusively produced immunoprecipitating Abs to native mouse Ro52 and Abs to mouse La by slot blot analysis, whereas in other strains of mice Abs to mouse La were cross-reactive with the immunogen. The results of the present study demonstrate the importance of HLA class II in controlling the immune responses to the Ro-ribonucleoprotein.     

Association of LMP2 and LMP7 genes within the major histocompatibility complex with insulin-dependent diabetes mellitus: population and family studies.

LMP2 and LMP7, two subunits of the proteasomes encoded in the major histocompatibility complex, are speculated to play a role in the generation of endogenous peptides for presentation by class I molecules to cytotoxic T cells. Their possible role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) has not been documented. In this study of Caucasian subjects, we have analyzed the polymorphisms of four genes within the HLA class II region (LMP2, LMP7, and HLA-DRB1 and -DQB1) in 198 unrelated IDDM patients and 192 normal controls ascertained from the southeastern United States. A genomic polymorphism of LMP7 was found strongly associated with IDDM, and the Arg/His-60 polymorphism in LMP2 was found associated with IDDM only in subjects containing an HLA DR4-DQB1*0302 haplotype. To determine whether the apparent associations between LMP genes and IDDM resulted from the strong linkage disequilibria observed between LMP and HLA-DR/DQ genes, we compared LMP gene frequencies in extended LMP-HLA haplotypes derived from control and diabetic families. Our results suggest that LMP genes have independent effects on IDDM susceptibility.     

HLA-DQ primarily confers protection and HLA-DR susceptibility in type I (insulin-dependent) diabetes studied in population-based affected families and controls.

The association between HLA-DR and -DQ and insulin-dependent diabetes mellitus (IDDM) in a defined high-incidence area was analyzed in a total of 58 population-based patients, representing 77% of IDDM patients with age at onset below 16 years, and in 92 unrelated parents in control families without IDDM. HLA haplotypes were confirmed by analyzing first-degree relatives in both groups. Seven different methods were used to analyze risk: (1) odds ratio, (2) absolute risk, (3) haplotype relative risk, (4) transcomplementation relative risk, (5) relative predisposing effects, (6) stratification analysis, and (7) test of predisposing allele on haplotype. DQB1*0302 indicated somewhat higher risk than did DR4, while DR3 had a higher risk than DQB1*0201; however, the 95% confidence intervals of the risk estimates overlapped. The positive association between IDDM and the DQB1*0201-DQA1*0501-DR3 haplotype seems to be due to DR3 or to an unknown linked gene. More important, DQA1*0301 was present among 93% of the patients, and this allele in various transcomplementation combinations with DQB1 alleles showed closer association to IDDM than did any other alleles. The strong negative association of the DQB1*0602 allele also in the presence of either DR4 or DQB1*0302 or both suggests that, in a high-risk population for IDDM, HLA-DQ primarily confers protection, perhaps by induction of tolerance. Consistent with known functions, HLA-DR may primarily confer susceptibility, perhaps by induction of autoreactive T lymphocytes.     

A novel HLA class II molecule

Molecular evidence has been obtained for a novel monomorphic HLA class II molecule distinct from HLA-DP/DQ/DR using a panel of lymphoblastoid cells which include HLA-loss mutants. The expression of this molecule was investigated using monomorphic affinity-purified mouse monoclonal antibodies (mAbs), including one of the IgG_2a subclass designated EDUA. This antibody reacts strongly in a cell-binding radioimmunoassay with HLA-DR and -DQ loss mutants derived from a lymphoblastoid parental cell. The EDU-1 mAb also reacted with a local panel of homozygous Epstein-Barr virus-transformed cell lines. The reactive molecules were further detected on allostimulated T-cell clones and various leukemic cells including those of myeloid origin which lack surface expression of HLA-DQ molecules. Thus the class II molecule described in this study corresponds to a monomorphic HLA class II determinant expressed on a variety of cells of different origin and HLA phenotypes. Moreover, this antigen structure is distinct from that of HLA-DP/DQ/DR as shown by direct immunoprecipitation, serial immunodepletion experiments, and two-dimensional gel electrophoresis. The molecule could be specified by new class II genes between DP and DQ. An alternative explanation for the genetic basis of the novel molecule is the existence of isotypic associations between alpha and beta chains of various class II molecules (DP, DX, DZ, and DO) but not DR and DQ as the mutant cells tested lack the latter genes.     

The study of HLA class II and autoimmune diabetes

Many autoimmune diseases have genetic associations with the Major Histocompatibility Complex (MHC) class II loci. Susceptibility to Type 1 diabetes mellitus (TIDM) is particularly associated with Human Leucocyte Antigen (HLA) DR3, 4 and associated DQ2, 8 alleles and this is well documented in genetic association studies. These molecules play an important role in presentation of peptide antigens after intracellular processing to CD4 T lymphocytes. During the last decade, a number of approaches have been used to elucidate the molecular basis for the association of particular alleles with susceptibility to or protection from TIDM. These studies have focused on investigating the structure of the antigen presenting molecules, together with their peptides. Through binding studies, peptide elution, molecular modelling and crystallization of the peptide MHC complex, it has been possible to define the peptide binding regions and examine the stability of binding of peptides from putative autoantigens. This knowledge has also facilitated the development of reagents such as multimeric MHC-peptide complexes that will help to track the low frequency, potentially pathogenic antigen specific cells. Recently, HLA transgenic mice have been generated and used to study T cell epitopes. In addition, although it is clear that the presence of HLA molecules alone does not by itself cause disease, these transgenic mice will develop diabetes when there is an islet "insult", even if the islet "insult" is, itself, not sufficient to precipitate disease in the absence of the HLA class II transgene. These mice will allow further study of the role of these HLA molecules in vivo. We now have a much greater general understanding of the possible reasons why particular molecules may encode susceptibility to or protection from disease. All these studies will provide information to ultimately define a rational basis for the development of targeted immunotherapy.     

Analysis of the molecular specificities of anti-class II monoclonal antibodies by using L cell transfectants expressing HLA class II molecules

Expressible HLA class II alpha- and beta-chain cDNA were used for DNA-mediated gene transfer to produce L cell transfectants expressing single types of human class II molecules. Cloned transfectants expressing nine different class II molecules were isolated: DR alpha: DR1 beta I, DR alpha: DR4 beta I, DR alpha: DR5 beta I, DR alpha: DR5 beta III (DRw52), DR alpha: DR7 beta I, DR alpha: DR4/7 beta IV (DRw53), DQ7 alpha: DQw2 beta, DQ7 alpha: DQw3 beta, and DPw4 alpha: DPw4 beta. These class II-expressing transfectants were used to analyze by flow cytometry the molecular specificities of 20 anti-class II mAb. These analyes indicate that some mAb are more broadly reactive than was previously thought based on immunochemical studies. In contrast, the narrow molecular specificities of other anti-class II mAb were confirmed by this approach. Transfectants expressing human class II molecules should be valuable reagents for studies of B cell and T cell defined epitopes on these molecules.     

Association of MHC and rheumatoid arthritis. Regulatory role of HLA class II molecules in animal models of RA: studies on transgenic/knockout mice

Human leucocyte antigen (HLA) class II molecules have been shown to be associated with predisposition to rheumatoid arthritis (RA). We generated HLA-DR and DQ transgenic mice that lacked endogenous class II molecules to study the interaction between the DR and DQ molecules and define the immunologic mechanisms in rheumatoid arthritis. Using collagen-induced arthritis (CIA) as an experimental model for inflammatory polyarthritis, we show that both DQ and DR are involved in predisposition or resistance to arthritis. Our studies suggest that polymorphism in DQB1 genes may determine predisposition to RA while the DRB1 polymorphism may dictate severity/protection of the disease. These mice provide powerful tools to develop immunotherapeutic protocols.     

Association of MHC and rheumatoid arthritis: Regulatory role of HLA class II molecules in animal models of RA - studies on transgenic/knockout mice

Human leucocyte antigen (HLA) class II molecules have been shown to be associated with predisposition to rheumatoid arthritis (RA). We generated HLA-DR and DQ transgenic mice that lacked endogenous class II molecules to study the interaction between the DR and DQ molecules and define the immunologic mechanisms in rheumatoid arthritis. Using collagen-induced arthritis (CIA) as an experimental model for inflammatory polyarthritis, we show that both DQ and DR are involved in predisposition or resistance to arthritis. Our studies suggest that polymorphism in DQB1 genes may determine predisposition to RA while the DRB1 polymorphism may dictate severity/protection of the disease. These mice provide powerful tools to develop immunotherapeutic protocols.     

Structure and function of human transplantation antigens

Human transplantation antigens encoded in the major histocompatibility complex (MHC) region play a key role in regulating the immune responses. Here, we will describe the summary of our analyses on the structure and function of the human MHC molecules, HLA antigens as follows. 1) The genomic organization of the HLA antigen region was examined by cosmid cloning and pulsed-field gel electrophoresis technique. The HLA antigen region spans over at least 3,000 kb, and constitutes a multigene family. 2) Genetic polymorphisms in the HLA gene region were analyzed by Southern hybridization with restriction endonuclease digested genomic DNA using the class II cDNAs as probes (RFLP) and found to be tightly associated with each allo specificity. 3) The functional expression of the HLA class II gene product were observed after transfer of their cloned genes into the mouse fibroblast and human lymphocytes. 4) Narcolepsy is completely associated with HLA-DR2 Dw2, but no difference in the sequence of the DQ beta 1 domain could be found between narcoleptic and healthy individuals. This fact suggests that narcolepsy is not caused by mutation in the DQ beta gene. Based on results, it was inferred that one or both of the two Asps within the second variable region in the first domain of the DR beta chain is directly correlated with predisposition to narcolepsy.     

HLA transgenic mice provide evidence for a direct and dominant role of HLA class II variation in modulating the severity of streptococcal sepsis

Our epidemiologic studies on invasive Group A Streptococci (GAS) infections identified specific HLA class II haplotypes/alleles conferring high-risk or protection from streptococcal toxic shock syndrome with a strong protection conferred by the DRB1*15/DQB1*06 haplotype. We used HLA-transgenic mice to provide an in vitro and in vivo validation for the direct role of HLA class II allelic variation in streptococcal toxic shock syndrome. When splenocytes from mice expressing the protective HLA-DQB1*06 (DQ6) allele were stimulated with a mixture of streptococcal superantigens (SAgs), secreted by the prevalent M1T1 strain, both proliferative and cytokine responses were significantly lower than those of splenocytes from mice expressing the neutral DRB1*0402/DQB1*0302 (DR4/DQ8) alleles (p < 0.001). In crisscross experiments, the presentation of SAgs to pure T cells from either the DQ6 or the DR4/DQ8 mice resulted in significantly different levels of response depending on the HLA type expressed on the APCs. Presentation by HLA-DQ6 APCs elicited significantly lower responses than the presentation by HLA-DR4/DQ8 APCs. Our in vitro data were supported by in vivo findings, as the DQ6 mice showed significantly longer survival post-i.v. infection with live M1T1 GAS (p < 0.001) and lower inflammatory cytokine responses as compared with the DR4/DQ8 mice (p < 0.01). The data presented here provide evidence for a direct role of HLA class II molecules in modulating responses to GAS SAgs and underscore the dominant role of HLA class II allelic variation in potentiating the severity of GAS systemic infections.     

Expression of class II antigens by subsets of activated T cells

Gene products coded for within the HLA complex play an important role in the control of immune responses. Class I antigens, coded for by the HLA-A, B, and C loci, are expressed by virtually all mononuclear blood cells. Class II antigens, coded for by the DR, DQ, and DP loci, have a more limited tissue distribution. They are expressed by B cells, monocytes, and by activated, but not by resting, T cells. The class II molecules of B cells and antigen-presenting cells have long been of interest to immunologists, since they are involved in the presentation of antigen, in communication between T cells and B cells and between T cells and adherent cells, and in susceptibility to certain diseases. The class II antigens expressed by activated T cells, however, remain largely uncharacterized in terms of their specificity, functional significance, and molecular nature. We have studied the expression of DR and DQ antigens by activated T cells and then examined the expression of DR versus DQ antigens by Leu 2a and Leu 3a subsets of mitogen-activated populations. Our results demonstrated that, as for class II-positive macrophages, the intensity of staining with monoclonal antibodies directed against DR antigens was much greater than that obtained with those directed against DQ antigens. Interestingly, the percentages of Leu 2a- and Leu 3a-positive cells which expressed DR antigens were quite similar, as were the percentages of Leu 2a and Leu 3a cells which expressed DQ. Thus, there does not seem to be preferential expression of DR versus DQ antigens by mitogen-activated T-cell subsets. Finally, though both DR-positive-DQ-positive and DR-positive-DQ-negative populations were detected, few or no DR-negative-DQ-positive cells were observed in these populations.     

Association between Protective and Deleterious HLA Alleles with Multiple Sclerosis in Central East Sardinia

The human leukocyte antigen (HLA) complex on chromosome 6p21 has been unambiguously associated with multiple sclerosis (MS). The complex features of the HLA region, especially its high genic content, extreme polymorphism, and extensive linkage disequilibrium, has prevented to resolve the nature of HLA association in MS. We performed a family based association study on the isolated population of the Nuoro province (Sardinia) to clarify the role of HLA genes in MS. The main stage of our study involved an analysis of the ancestral haplotypes A2Cw7B58DR2DQ1 and A30Cw5B18DR3DQ2. On the basis of a multiplicative model, the effect of the first haplotype is protective with an odds ratio (OR) = 0.27 (95% confidence interval CI 0.13–0.57), while that of the second is deleterious, OR 1.78 (95% CI 1.26–2.50). We found both class I (A, Cw, B) and class II (DR, DQ) loci to have an effect on MS susceptibility, but we saw that they act independently from each other. We also performed an exploratory analysis on a set of 796 SNPs in the same HLA region. Our study supports the claim that Class I and Class II loci act independently on MS susceptibility and this has a biological explanation. Also, the analysis of SNPs suggests that there are other HLA genes involved in MS, but replication is needed. This opens up new perspective on the study of MS.