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In delayed-release dimethyl fumarate (DMF)-treated patients, absolute lymphocyte count (ALC) often declines in the first year and stabilizes thereafter; early declines have been associated with development of severe prolonged lymphopenia (SPL). Prolonged moderate or severe lymphopenia is a known risk factor for progressive multifocal leukoencephalopathy (PML); DMF-associated PML is very rare. It is unknown whether genetic predictors of SPL secondary to DMF treatment exist. We aimed to identify genetic predictors of reduced white blood cell (WBC) counts in DMF-treated multiple sclerosis (MS) patients. Genotyping (N = 1,258) and blood transcriptional profiling (N = 1,133) were performed on MS patients from DEFINE/CONFIRM. ALCs were categorized as: SPL, < 500 cells/µL for ≥6 months; moderate prolonged lymphopenia (MPL), < 800 cells/µL for ≥6 months, excluding SPL; mildly reduced lymphocytes, < 910 cells/µL at any point, excluding SPL and MPL; no lymphopenia, ≥910 cells/µL. Genome-wide association, HLA, and cross-sectional gene expression studies were performed. No common variants, HLA alleles, or expression profiles clinically useful for predicting SPL or MPL were identified. There was no overlap between genetic peaks and genetic loci known to be associated with WBC. Gene expression profiles were not associated with lymphopenia status. A classification model including gene expression features was not more predictive of lymphopenia status than standard covariates. There were no genetic predictors of SPL (or MPL) secondary to DMF treatment. Our results support ALC monitoring during DMF treatment as the most effective way to identify patients at risk of SPL.

Neuromyelitis optica (NMO) is a rare autoimmune disease that affects the optic nerve and spinal cord. Most NMO patients ( > 70%) are seropositive for circulating autoantibodies against aquaporin 4 (NMO-IgG+). Here, we meta-analyze whole-genome sequences from 86 NMO cases and 460 controls with genome-wide SNP array from 129 NMO cases and 784 controls to test for association with SNPs and copy number variation (total N = 215 NMO cases, 1244 controls). We identify two independent signals in the major histocompatibility complex (MHC) region associated with NMO-IgG+, one of which may be explained by structural variation in the complement component 4 genes. Mendelian Randomization analysis reveals a significant causal effect of known systemic lupus erythematosus (SLE), but not multiple sclerosis (MS), risk variants in NMO-IgG+. Our results suggest that genetic variants in the MHC region contribute to the etiology of NMO-IgG+ and that NMO-IgG+ is genetically more similar to SLE than MS., Neuromyelitis optica (NMO) is a rare autoimmune condition characterized by inflammation and demyelination of the optic nerve and the spinal cord. Here, Estrada et al. identify NMO susceptibility variants in the MHC region and find that autoantibody-positive NMO genetically overlaps with lupus.

Sickle cell disease (SCD) affects more than five million people worldwide, predominantly in sub-Saharan Africa. Hereditary persistence of fetal hemoglobin (HbF) is an uncommon genetic condition in which production of HbF in early life is not suppressed. SCD symptoms are reduced in patients carrying this condition, suggesting that increased HbF levels may be a promising therapeutic strategy to ameliorate the symptoms of SCD. This is exemplified by the HbF-raising compound hydroxyurea, currently the most commonly used US Food and Drug Administration approved drug treatment for SCD. However, hydroxyurea is only effective in ~70% of patients and carries a black box warning for carcinogenicity, hence there is a need for additional SCD treatments. Previous genome-wide association studies (GWAS) have identified four loci robustly associated with HbF levels, including variants in the BCL11A region. These initial genetic discoveries have led to promising ex vivo gene-editing approaches to silence or reduce levels of BCL11A, which are currently being tested in clinical trials. To identify novel potential therapeutic targets to raise HbF levels, we conducted the largest GWAS of HbF levels in ~11,000 healthy blood donors from the INTERVAL study among whom HbF was measured in whole blood using a mass spectrometry approach. We ran linear mixed models accounting for age, sex, blood group, technical effects and 10 principal components of ancestry for 14,910,742 variants either directly genotyped using the Affymetrix UK Biobank array or imputed from a combined 1000 Genomes/UK10K reference panel. In addition to confirming previously reported signals at the BCL11A, HBS1L-MYB and HBB loci, stepwise conditional analysis identified six novel genomic regions at genome-wide significance (p0.5), including a rare (frequency=0.2%) variant near GRIK2. To identify likely causal genes and mechanisms, we integrated our results with relevant transcriptomic and epigenomic datasets. Preliminary results suggest that a common (frequency=26%) 29 base-pair indel upstream of CHAC2 is highly likely (posterior probability=0.77) to be the causal variant at this locus. The variant maps to a site of active chromatin and GATA1 transcription factor binding specifically in erythroblasts, and the insertion allele contains a GATA motif suggesting that this variant regulates HbF levels by the presence or absence of a second GATA1 binding site. Gene expression data across a range of hematopoietic cells revealed a restricted expression pattern of CHAC2 in erythroblasts, but not for other genes in the region, suggesting CHAC2 as the likely causal gene. CHAC2 encodes the glutathione-specific gamma-glutamylcyclotransferase 2, an enzyme that catalyzes the cleavage of glutathione into 5-oxo-L-proline and a Cys-Gly dipeptide. This finding adds support to the idea that altered erythrocyte glutathione levels play a role in SCD pathogenesis, potentially via HbF modulation. Ongoing work includes characterizing the likely causal mechanisms of the six novel loci, and providing target validation of CHAC2 using genome editing and deep phenotyping of erythroid cells. In summary, our expanded GWAS has identified new loci associated with HbF levels, providing novel potential therapeutic targets for SCD. Disclosures Lessard: Sanofi: Employment. Peters:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment.

Selective immunoglobulin A deficiency (IgAD) is the most common primary immunodeficiency in Europeans. Our genome-wide association study (GWAS) meta-analysis of 1,635 patients with IgAD and 4,852 controls identified four new significant (P < 5 × 10-8) loci and association with a rare IFIH1 variant (p.Ile923Val). Peak new variants (PVT1, P = 4.3 × 10-11; ATG13-AMBRA1, P = 6.7 × 10-10; AHI1, P = 8.4 × 10-10; CLEC16A, P = 1.4 × 10-9) overlapped with autoimmune markers (3/4) and correlated with 21 putative regulatory variants, including expression quantitative trait loci (eQTLs) for AHI1 and DEXI and DNase hypersensitivity sites in FOXP3+ regulatory T cells. Pathway analysis of the meta-analysis results showed striking association with the KEGG pathway for IgA production (pathway P < 0.0001), with 22 of the 30 annotated pathway genes containing at least one variant with P ≤ 0.05 in the IgAD meta-analysis. These data suggest that a complex network of genetic effects, including genes known to influence the biology of IgA production, contributes to IgAD.

The discovery that type I interferon (IFN)-inducible genes were strongly upregulated in peripheral blood in SLE over a decade ago sparked interest in understanding the relationship between type I IFN and SLE. Genome-wide association studies provide strong genetic evidence that type I IFNs are important for SLE risk. Of 47 genetic variants associated with SLE, over half (27/47, 57%) can be linked to type I IFN production or signaling. The recent identification of single gene mutations for disorders that share features with SLE – Aicardi–Goutieres syndrome, chilblain lupus, and spondyloenchondrodysplasia – provide additional support for the hypothesis that type I IFNs are central drivers of SLE pathogenesis. These insights provide significant focus for efforts to tackle SLE therapeutically.

DNA methylation is an epigenetic mark that is influenced by environmental factors and is associated with changes to gene expression and phenotypes. It may link environmental exposures to disease etiology or indicate important gene pathways involved in disease pathogenesis. We identified genomic regions that are differentially methylated in T cells of patients with relapsing remitting multiple sclerosis (MS) compared to healthy controls. DNA methylation was assessed at 450,000 genomic sites in CD4+ and CD8+ T cells purified from peripheral blood of 94 women with MS and 94 healthy women, and differentially methylated regions were identified using bumphunter. Differential DNA methylation was observed near four loci: MOG/ZFP57, HLA-DRB1, NINJ2/LOC100049716, and SLFN12. Increased methylation of the first exon of the SLFN12 gene was observed in both T cell subtypes and remained present after restricting analyses to samples from patients who had never been on treatment or had been off treatment for more than 2.5 years. Genes near the regions of differential methylation in T cells were assessed for differential expression in whole blood samples from a separate population of 1,329 women with MS and 97 healthy women. Gene expression of HLA-DRB1, NINJ2, and SLFN12 was observed to be decreased in whole blood in MS patients compared to controls. We conclude that T cells from MS patients display regions of differential DNA methylation compared to controls, and corresponding gene expression differences are observed in whole blood. Two of the genes that showed both methylation and expression differences, NINJ2 and SLFN12, have not previously been implicated in MS. SLFN12 is a particularly compelling target of further research, as this gene is known to be down-regulated during T cell activation and up-regulated by type I interferons (IFNs), which are used to treat MS.

CLEC16A, a putative immunoreceptor, was recently established as a susceptibility locus for type I diabetes and multiple sclerosis. Subsequently, associations between CLEC16A and rheumatoid arthritis (RA), Addison’s disease and Crohn’s disease have been reported. A large comprehensive and independent investigation of CLEC16A variation in RA was pursued. This study tested 251 CLEC16A single-nucleotide polymorphisms in 2542 RA cases (85% anti-cyclic citrullinated peptide (anti-CCP) positive) and 2210 controls (N = 4752). All individuals were of European ancestry, as determined by ancestry informative genetic markers. No evidence for significant association between CLEC16A variation and RA was observed. This is the first study to fully characterize common genetic variation in CLEC16A including assessment of haplotypes and gender-specific effects. The previously reported association between RA and rs6498169 was not replicated. Results show that CLEC16A does not have a prominent function in susceptibility to anti-CCP-positive RA.

Background: An association between major histocompatibility complex (MHC) genes, particularly those within the class II HLA region, and rheumatoid arthritis (RA) is well established, and accounts for an estimated 30% of the genetic component in RA. The MHC class II transactivator gene ( MHC2TA ) on chromosome 16p13 has recently emerged as the most important transcription factor regulating genes required for class II MHC-restricted antigen presentation. Previous studies of a promoter region polymorphism (−168A/G, rs3087456) in the MHC2TA gene and RA have yielded conflicting results. Objective: To assess the association of the MHC2TA −168A/G polymorphism (rs3087456) and risk for RA by meta-analysis. Methods: Meta-analysis was performed for 6861 patients with RA and 9270 controls from 10 case–control studies. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for each study. Summary ORs and 95% CIs were calculated for random effects models. Results: No effect was observed for the G risk allele (OR 1.02, 95% CI 0.93 to 1.12, p = 0.70) or the GG risk genotype (OR 1.14, 95% CI 0.95 to 1.36, p = 0.16). Conclusions: Our results indicate that the MHC2TA −168A/G polymorphism (rs3087456) is not associated with RA yet underscore the importance of including shared epitope allele carrier status, secondary phenotypes and more complete characterisation of MHC2TA variation in future studies.

The major histocompatibility complex (MHC) class II transactivator gene (CIITA) encodes an important transcription factor regulating genes required for human leukocyte antigen (HLA) class II MHC-restricted antigen presentation. Major histocompatibility complex (MHC) genes, particularly HLA class II, are strongly associated with risk of developing rheumatoid arthritis (RA). Given the strong biological relationship between CIITA and HLA class II genes, a comprehensive investigation of CIITA variation in RA was conducted. This study tested 31 CIITA SNPs in 2542 RA cases and 3690 controls (N = 6232). All individuals were of European ancestry, as determined by ancestry informative genetic markers. No evidence for association between CIITA variation and RA was observed after a correction for multiple testing was applied. This is the largest study to fully characterize common genetic variation in CIITA, including an assessment of haplotypes. Results exclude even a modest role for common CIITA polymorphisms in susceptibility to RA.

The MHC class II transactivator gene (CIITA) is an important transcription factor regulating gene required for HLA class II MHC-restricted antigen presentation. Association with HLA class II variation, particularly HLA-DRB1*1501, has been well-established for multiple sclerosis (MS). In addition, the -168A/G CIITA promoter variant (rs3087456) has been reported to be associated with MS. Thus, a multi-stage investigation of variation within CIITA, DRB1*1501 and MS was undertaken in 6108 individuals. In stage 1, 24 SNPs within CIITA were genotyped in 1320 cases and 1363 controls (n = 2683). Rs4774 (missense +1614G/C; G500A) was associated with MS (P = 4.9 x 10(-3)), particularly in DRB1*1501 +individuals (P = 1 x 10(-4)). No association was observed for the -168A/G promoter variant. In stage 2, rs4774 was genotyped in 973 extended families; rs4774*C was also associated with increased risk for MS in DRB1*1501+ families (P = 2.3 x 10(-2)). In a third analysis, rs4774 was tested in cases and controls (stage 1) combined with one case per family (stage 2) for increased power. Rs4774*C was associated with MS (P = 1 x 10(-3)), particularly in DRB1*1501+ cases and controls (P = 1 x 10(-4)). Results obtained from logistic regression analysis showed evidence for interaction between rs4774*C and DRB1*1501 associated with risk for MS (ratio of ORs = 1.72, 95% CI 1.28-2.32, P = 3 x 10(-4)). Furthermore, rs4774*C was associated with DRB1*1501+ MS when conditioned on the presence (OR = 1.67, 95% CI = 1.19-2.37, P = 1.9 x 10(-3)) and absence (OR = 1.49, 95% CI = 1.15-1.95, P = 2.3 x 10(-3)) of CLEC16A rs6498169*G, a putative MS risk allele adjacent to CIITA. Our results provide strong evidence supporting a role for CIITA variation in MS risk, which appears to depend on the presence of DRB1*1501.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by autoantibodies to nuclear and cell surface antigens. Although the etiology of SLE remains unknown, evidence for genetic susceptibility is well established. The HLA class II alleles DRB1*1501, *0301, and *0801 and the HLA class I alleles A*01 and B*08 in the major histocompatibility complex (MHC) region are consistently associated with SLE. HLA loci may also influence SLE through additional inherited or noninherited mechanisms. Differences in maternal and paternal transmission rates, or parent-of-origin effects, have not been previously examined in SLE. One potential mechanism influencing disease susceptibility is “genomic imprinting” due to epigenetic modification of the genome. This modification results in unequal transcription of parental alleles and subsequent allele expression, depending on whether alleles were transmitted from the mother or from the father. Increased HLA compatibility between a mother and her offspring is hypothesized to contribute to the risk of autoimmune disease. Maternal–offspring effects can present as excess HLA compatibility between the mother and affected offspring (Figure 1). In mice, HLA similarity between mother and fetus has been shown to promote the persistence of maternal cells in the offspring (maternal microchimerism) following pregnancy (1). Figure 1 Maternal–offspring HLA compatibility relationships. The developing immune system of the fetus is also directly exposed to noninherited maternal antigens (NIMAs) in utero (2). Exposure to NIMAs can have a lifelong influence on the immune system and has been theorized to tolerize or predispose to autoimmune reactions. A tolerogenic effect may explain the longer survival of renal transplants from sibling donors expressing NIMAs versus noninherited paternal HLA. Decreased B cell responses to HLA class I NIMAs in humans have been reported (3). Recent data suggest that fetuses may also develop T cell tolerance to NIMAs in utero through tolerogenic fetal regulatory T cells that are maintained throughout life (4). The aim of this study was to test the hypothesis that the DRB1 locus influences SLE through these novel biologic mechanisms in addition to genetic transmission of particular HLA risk alleles.

Aim: Type 1 diabetes (T1D) is a complex trait for which variation in the classical human leucocyte antigen (HLA) loci within the Major Histocompatibility Complex (MHC) significantly influences disease risk. To date, HLA class II DR-DQ genes confer the strongest known genetic effect in T1D. HLA loci may also influence T1D through additional inherited or non-inherited effects. Evidence for the role of increased maternal–offspring HLA compatibility, and both parent-of-origin (POO) and non-inherited maternal HLA (NIMA) effects in autoimmune disease has been previously established. The current study tested hypotheses that classical HLA loci influence T1D through these mechanisms, in addition to genetic transmission of particular risk alleles. Methods: The Type 1 Diabetes Genetics Consortium (T1DGC) cohort was of European descent and consisted of 2271 affected sib-pair families (total n = 11 023 individuals). Class I genes HLA-A, Cw and B, and class II genes HLA-DRB1, DQA1, DQB1, DPA1 and DPB1 were studied. The pedigree disequilibrium test was used to examine transmission of HLA alleles to individuals with T1D. Conditional logistic regression was used to model compatibility relationships between mother–offspring and father–offspring for all HLA loci. POO and NIMA effects were investigated by comparing frequencies of maternal and paternal transmitted and non-transmitted HLA alleles for each locus. Analyses were also stratified by gender of T1D-affected offspring. Results: Strong associations were observed for all classical HLA loci except for DPA1, as expected. Compatibility differences between mother–offspring and father–offspring were not observed for any HLA loci. Furthermore, POO and NIMA HLA effects influencing T1D were not present. Conclusions: Maternal–offspring HLA compatibility, POO and NIMA effects for eight classical HLA loci were investigated. Results suggest that these HLA-related effects are unlikely to play a major role in the development of T1D.