Genome-Wide Association Study of Late-Onset Myasthenia Gravis: Confirmation of TNFRSF11A and Identification of ZBTB10 and Three Distinct HLA Associations.

To investigate the genetics of late-onset myasthenia gravis (LOMG), we conducted a genome-wide association study imputation of>6 million single nucleotide polymorphisms (SNPs) in 532 LOMG cases (anti-acetylcholine receptor [AChR] antibody positive; onset age≥50 years) and 2,128 controls matched for sex and population substructure. The data confirm reported TNFRSF11A associations (rs4574025, P = 3.9 × 10-7, odds ratio [OR] 1.42) and identify a novel candidate gene, ZBTB10, achieving genome-wide significance (rs6998967, P = 8.9 × 10-10, OR 0.53). Several other SNPs showed suggestive significance including rs2476601 (P = 6.5 × 10-6, OR 1.62) encoding the PTPN22 R620W variant noted in early-onset myasthenia gravis (EOMG) and other autoimmune diseases. In contrast, EOMG-associated SNPs in TNIP1 showed no association in LOMG, nor did other loci suggested for EOMG. Many SNPs within the major histocompatibility complex (MHC) region showed strong associations in LOMG, but with smaller effect sizes than in EOMG (highest OR ~2 versus ~6 in EOMG). Moreover, the strongest associations were in opposite directions from EOMG, including an OR of 0.54 for DQA1*05:01 in LOMG (P = 5.9 × 10-12) versus 2.82 in EOMG (P = 3.86 × 10-45). Association and conditioning studies for the MHC region showed three distinct and largely independent association peaks for LOMG corresponding to (a) MHC class II (highest attenuation when conditioning on DQA1), (b) HLA-A and (c) MHC class III SNPs. Conditioning studies of human leukocyte antigen (HLA) amino acid residues also suggest potential functional correlates. Together, these findings emphasize the value of subgrouping myasthenia gravis patients for clinical and basic investigations and imply distinct predisposing mechanisms in LOMG.

VAV1 and BAFF, via NFκB pathway, are genetic risk factors for myasthenia gravis.

OBJECTIVE: To identify novel genetic loci that predispose to early-onset myasthenia gravis (EOMG) applying a two-stage association study, exploration, and replication strategy. METHODS: Thirty-four loci and one confirmation loci, human leukocyte antigen (HLA)-DRA, were selected as candidate genes by team members of groups involved in different research aspects of MG. In the exploration step, these candidate genes were genotyped in 384 EOMG and 384 matched controls and significant difference in allele frequency were found in eight genes. In the replication step, eight candidate genes and one confirmation loci were genotyped in 1177 EOMG patients and 814 controls, from nine European centres. RESULTS: ALLELE FREQUENCY DIFFERENCES WERE FOUND IN FOUR NOVEL LOCI: CD86, AKAP12, VAV1, B-cell activating factor (BAFF), and tumor necrosis factor-alpha (TNF-α), and these differences were consistent in all nine cohorts. Haplotype trend test supported the differences in allele frequencies between cases and controls. In addition, allele frequency difference in female versus male patients at HLA-DRA and TNF-α loci were observed. INTERPRETATION: The genetic associations to EOMG outside the HLA complex are novel and of interest as VAV1 is a key signal transducer essential for T- and B-cell activation, and BAFF is a cytokine that plays important roles in the proliferation and differentiation of B-cells. Moreover, we noted striking epistasis between the predisposing VAV1 and BAFF haplotypes; they conferred a greater risk in combination than alone. These, and CD86, share the same signaling pathway, namely nuclear factor-kappaB (NFκB), thus implicating dysregulation of proinflammatory signaling in predisposition to EOMG.

Risk for myasthenia gravis maps to a (151) Pro→Ala change in TNIP1 and to human leukocyte antigen-B*08.

OBJECTIVE: The objective of this study is to comprehensively define the genetic basis of early onset myasthenia gravis (EOMG). METHODS: We have carried out a 2-stage genome-wide association study on a total of 649 North European EOMG patients. Cases were matched 1:4 with controls of European ancestry. We performed imputation and conditional analyses across the major histocompatibility complex, as well as in the top regions of association outside the human leukocyte antigen (HLA) region. RESULTS: We observed the strongest association in the HLA class I region at rs7750641 (p = 1.2 × 10(-92) ; odds ratio [OR], 6.25). By imputation and conditional analyses, HLA-B*08 proves to be the major associated allele (p = 2.87 × 10(-113) ; OR, 6.41). In addition to the expected association with PTPN22 (rs2476601; OR, 1.71; p = 8.2 × 10(-10) ), an imputed coding variant (rs2233290) at position 151 (Pro→Ala) in the TNFAIP3-interacting protein 1, TNIP1, confers even stronger risk than PTPN22 (OR, 1.91; p = 3.2 × 10(-10) ). INTERPRETATION: The association at TNIP1 in EOMG implies disease mechanisms involving ubiquitin-dependent dysregulation of NF-κB signaling. The localization of the major HLA signal to the HLA-B*08 allele suggests that CD8(+) T cells may play a key role in disease initiation or pathogenesis.

Preferential expression of AChR epsilon-subunit in thymomas from patients with myasthenia gravis.

The role of antigen expression by thymomas in myasthenia gravis (MG) is not clear. Previous reports of acetylcholine receptor (AChR) mRNA expression by the highly sensitive reverse transcription-polymerase chain reactions (RT-PCR) produced varying results. To try to clarify this issue, we first used RT-PCR but then turned to the more accurate and quantitative RNase protection assays (RPA) to assess AChR subunit mRNA expression in thymomas from 25 patients (22 with MG). By RT-PCR, all five AChR subunits could be detected in many thymomas. However, by RPA, the mRNA for the adult-specific AChR epsilon-subunit was found in 13/25 (52%) thymomas, but not mRNA for the other subunits. AChR epsilon-subunit was more frequently detected in thymomas of A or AB histology (WHO classification) than those with B1-B3 histology. Overall, 6/6 with thymomas of A or AB histology were positive compared with only 8/19 with B histology (p=0.02). Autoantibodies in the two patients with the highest levels of epsilon-subunit mRNA bound better to adult (alpha(2)betadeltaepsilon) AChR than to fetal (alpha(2)betadeltagamma) AChR, whereas the other sera bound better to fetal AChR. The greater abundance of mRNA for AChR epsilon-subunit than for other subunits suggests that the AChR epsilon-subunit may play a distinctive role in autosensitization in MG-associated thymomas, particularly those of type A or AB.

Clonal expansions of CD4+ B helper T cells in autoimmune myasthenia gravis.

The weakness in myasthenia gravis (MG) is mediated by T helper cell (Th)-dependent autoantibodies against neuromuscular epitopes. So far, analyzing Th phenotypes or antigen specificities has yielded very few clues to pathogenesis. Here we adopt an alternative antigen-independent approach, analyzing T cell receptor (TCR) Vbeta usage/expansions in blood from 118 MG patients. We found major expansions (>or= five standard deviations above the mean of 118 healthy, individually age- and sex-matched controls) in diverse Vbeta in 21 patients (17.6%, p<0.001) among CD4+ T cells, and in 45 patients (38.1%, p<0.001) among CD8+ T cells. In informative probands, the expanded CD4+ cells consistently showed a Th cell phenotype (CD57+CXCR5+) and expressed Th1 cytokines. Furthermore, their expression of markers for activation, lymphocyte trafficking and B cell-activating ability persisted for >or=3 years. Surprisingly, we noted a selective decline in the expansions/their CD57 positivity while the probands' MG was improving. CDR3 spectratyping suggested mono- or oligoclonal origins, which were confirmed by the prevalent TCR Vbeta CDR3 sequences of Th cells cloned from repeat bleeds. Thus, our data provide evidence for persistent clonally expanded CD4+ B helper T cell populations in the blood of MG patients. These unexpected CD4+ expansions might hold valuable clues to MG immunopathogenesis.

Control of autoimmunity by "epitope theft".

We all possess T cells with autoaggressive potential. Knowledge of their regulation is crucial for elucidating pathogenetic pathways and designing effective treatments for autoimmune diseases. A novel mechanism of T-cell silencing--in an autoimmune model--has recently been identified and is termed "epitope theft". The "thieves" are naive CD8+ T cells, which apparently "steal" MHC-class-I-antigen complexes from antigen-presenting cells (APCs). The deprived APCs can no longer activate other potentially pathogenic naive CD8+ T cells that are specific for the same epitope. This phenomenon is a previously unrecognized antigen-specific mode of protection against autoimmunity.

Spontaneous production of anti-IFN-alpha and anti-IL-12 autoantibodies by thymoma cells from myasthenia gravis patients suggests autoimmunization in the tumor.

Myasthenia gravis (MG) is mediated by autoantibodies to the acetylcholine receptor (AChR), expressed in muscle and rare thymic myoid cells. Most early-onset cases show thymic lymph node-type infiltrates, including pre-activated plasma cells spontaneously producing anti-AChR antibodies. Since these are not evident in the associated thymomas found in another 10% of MG patients, AChR-specific B cells must be autosensitized elsewhere. Unexpectedly, at diagnosis, >70% of MG/thymoma patients also have high-titer neutralizing autoantibodies to IFN-alpha, and >50% to IL-12; moreover, titers increase strikingly if the thymomas recur, indicating a closer tumor relationship than for anti-AChR. To investigate this, we have measured autoantibody production by cells cultured from thymomas, any available thymic remnants and blood, with or without the B cell stimulant pokeweed mitogen (PWM). To check autoantibody specificity and clonal origins, we isolated Fabs from two combinatorial libraries from producer thymus/thymoma cells. Surprisingly, thymoma cells spontaneously produced antibodies to IFN-alpha and/or IL-12 in >40% of seropositive cases, showing typical plasma cell behavior, whereas they produced anti-AChR only after PWM stimulation. We isolated 15 combinatorial Fabs to IFN-alpha (versus only one to AChR). Their strong binding in radio-immunoprecipitation and Western blots implies high affinities. The four Fabs tested neutralized anti-viral actions of IFN-alpha. The diverse V genes clearly showed ongoing antigen-driven selection. These results imply pre-activation in situ by native IFN-alpha/IL-12 expressed within a 'dangerous' tumor microenvironment. With these molecules, it should be easier to identify provoking cell type(s) that may give novel additional clues to autoimmunization against T-cell epitopes from the more complex AChR.

Major differences in antigen-processing correlate with a single Arg71<-->Lys substitution in HLA-DR molecules predisposing to rheumatoid arthritis and with their selective interactions with 70-kDa heat shock protein chaperones.

Several HLA-DR alleles are genetically associated with rheumatoid arthritis. DRB1*0401 predominates in Northern Europe and has a characteristic (70)QKRAA motif. This sequence contacts bound peptides and the TCR. Further interactions have been suggested with additional proteins during Ag loading. We explored the much stronger processing/presentation of full-length recombinant human acetylcholine receptor alpha subunit to a specific T cell clone by APC from DRB1*0401+ than *0408+ donors. Using DR*04 transfectants, we show that this difference results largely from the single Lys71<-->Arg interchange (0401<-->0408), which scarcely affects epitope binding, rather than from any other associated polymorphism. Furthermore, we proved our recombinant polypeptides to contain the Escherichia coli 70-kDa heat shock protein molecule DnaK and its requirement for efficient processing and presentation of the epitope by DRB1*0401+ cells. According to a recent report, 70-kDa heat shock protein chaperones preferentially bind to the QKRAA, rather than the QRRAA, motif. Variations between the shared epitope motifs QKRAA and QRRAA are emphasized by underlining. We propose that such interactions enhance the intracellular epitope loading of *0401 molecules. They may thus broaden immune responses to pathogens and at least partially explain the distinct contributions of DRB1*0401 and other alleles to disease predisposition.