DR3 Haplotype Research Articles

Genetic Discovery and Risk Prediction for Type 1 Diabetes in Individuals Without High-Risk HLA-DR3/DR4 Haplotypes.

More than 10% of patients with type 1 diabetes (T1D) do not have high-risk HLA-DR3 or -DR4 haplotypes with distinct clinical features, such as later onset and reduced insulin dependence. We aimed to identify genetic drivers of T1D in the absence of DR3/DR4 and improve prediction of T1D risk in these individuals. We performed T1D association and fine-mapping analyses in 12,316 non-DR3/DR4 samples. Next, we performed heterogeneity tests to examine differences in T1D risk variants in individuals without versus those with DR3/DR4 haplotypes. We further assessed genome-wide differences in gene regulatory element and biological pathway enrichments between the non-DR3/DR4 and DR3/DR4 cohorts. Finally, we developed a genetic risk score (GRS) to predict T1D in individuals without DR3/DR4 and compared with an existing T1D GRS. A total of 18 T1D risk variants in non-DR3/DR4 samples were identified. Risk variants at the MHC and multiple other loci genome wide had heterogeneity in effects on T1D dependent on DR3/DR4 status, and non-DR3/DR4 T1D had evidence for a greater polygenic burden. T1D-assocated variants in non-DR3/DR4 were more enriched for regulatory elements and pathways involved in antigen presentation, innate immunity, and β-cells and depleted in T cells compared with DR3/DR4. A non-DR3/DR4 GRS outperformed an existing risk score GRS2 in discriminating non-DR3/DR4 T1D from no diabetes (area under the curve 0.867; P = 7.48 × 10-32) and type 2 diabetes (0.907; P = 4.94 × 10-44). In total, we identified heterogeneity in T1D genetic risk dependent on high-risk HLA-DR3/DR4 haplotype, which uncovers disease mechanisms and enables more accurate prediction of T1D across the HLA background.

HLA Class II (DR, DQ, DP) Genes Were Separately Associated With the Progression From Seroconversion to Onset of Type 1 Diabetes Among Participants in Two Diabetes Prevention Trials (DPT-1 and TN07).

To explore associations of HLA class II genes (HLAII) with the progression of islet autoimmunity from asymptomatic to symptomatic type 1 diabetes (T1D). Next-generation targeted sequencing was used to genotype eight HLAII genes (DQA1, DQB1, DRB1, DRB3, DRB4, DRB5, DPA1, DPB1) in 1,216 participants from the Diabetes Prevention Trial-1 and Randomized Diabetes Prevention Trial with Oral Insulin sponsored by TrialNet. By the linkage disequilibrium, DQA1 and DQB1 are haplotyped to form DQ haplotypes; DP and DR haplotypes are similarly constructed. Together with available clinical covariables, we applied the Cox regression model to assess HLAII immunogenic associations with the disease progression. First, the current investigation updated the previously reported genetic associations of DQA1*03:01-DQB1*03:02 (hazard ratio [HR] = 1.25, P = 3.50*10-3) and DQA1*03:03-DQB1*03:01 (HR = 0.56, P = 1.16*10-3), and also uncovered a risk association with DQA1*05:01-DQB1*02:01 (HR = 1.19, P = 0.041). Second, after adjusting for DQ, DPA1*02:01-DPB1*11:01 and DPA1*01:03-DPB1*03:01 were found to have opposite associations with progression (HR = 1.98 and 0.70, P = 0.021 and 6.16*10-3, respectively). Third, DRB1*03:01-DRB3*01:01 and DRB1*03:01-DRB3*02:02, sharing the DRB1*03:01, had opposite associations (HR = 0.73 and 1.44, P = 0.04 and 0.019, respectively), indicating a role of DRB3. Meanwhile, DRB1*12:01-DRB3*02:02 and DRB1*01:03 alone were found to associate with progression (HR = 2.6 and 2.32, P = 0.018 and 0.039, respectively). Fourth, through enumerating all heterodimers, it was found that both DQ and DP could exhibit associations with disease progression. These results suggest that HLAII polymorphisms influence progression from islet autoimmunity to T1D among at-risk subjects with islet autoantibodies.

HLA-DRB1*15:01 and the MERTK Gene Interact to Selectively Influence the Profile of MERTK-Expressing Monocytes in Both Health and MS.

HLA-DRB1*15:01 (DR15) and MERTK are 2 risk genes for multiple sclerosis (MS). The variant rs7422195 is an expression quantitative trait locus for MERTK in CD14+ monocytes; cells with phagocytic and immunomodulatory potential. We aimed to understand how drivers of disease risk and pathogenesis vary with HLA and MERTK genotype and disease activity. We investigated how proportions of monocytes vary with HLA and MERTK genotype and disease activity in MS. CD14+ monocytes were isolated from patients with MS at relapse (n = 40) and 3 months later (n = 23). Healthy controls (HCs) underwent 2 blood collections 3 months apart. Immunophenotypic profiling of monocytes was performed by flow cytometry. Methylation of 35 CpG sites within and near the MERTK gene was assessed in whole blood samples of individuals experiencing their first episode of clinical CNS demyelination (n = 204) and matched HCs (n = 345) using an Illumina EPIC array. DR15-positive patients had lower proportions of CD14+ MERTK+ monocytes than DR15-negative patients, independent of genotype at the MERTK SNP rs7422195. Proportions of CD14+ MERTK+ monocytes were further reduced during relapse in DR15-positive but not DR15-negative patients. Patients homozygous for the major G allele at rs7422195 exhibited higher proportions of CD14+ MERTK+ monocytes at both relapse and remission compared with controls. We observed that increased methylation of the MERTK gene was significantly associated with the presence of DR15. DR15 and MERTK genotype independently influence proportions of CD14+ MERTK+ monocytes in MS. We confirmed previous observations that the MERTK risk SNP rs7422195 is associated with altered MERTK expression in monocytes. We identified that expression of MERTK is stratified by disease in people homozygous for the major G allele of rs7422195. The finding that the proportion of CD14+ MERTK+ monocytes is reduced in DR15-positive individuals supports prior data identifying genetic links between these 2 loci in influencing MS risk. DR15 genotype-dependent alterations in methylation of the MERTK gene provides a molecular link between these loci and identifies a potential mechanism by which MERTK expression is influenced by DR15. This links DR15 haplotype to MS susceptibility beyond direct influence on antigen presentation and suggests the need for HLA-based stratification of approaches to MERTK as a therapeutic target.

Smith-specific regulatory T cells halt the progression of lupus nephritis

Antigen-specific regulatory T cells (Tregs) suppress pathogenic autoreactivity and are potential therapeutic candidates for autoimmune diseases such as systemic lupus erythematosus (SLE). Lupus nephritis is associated with autoreactivity to the Smith (Sm) autoantigen and the human leucocyte antigen (HLA)-DR15 haplotype; hence, we investigated the potential of Sm-specific Tregs (Sm-Tregs) to suppress disease. Here we identify a HLA-DR15 restricted immunodominant Sm T cell epitope using biophysical affinity binding assays, then identify high-affinity Sm-specific T cell receptors (TCRs) using high-throughput single-cell sequencing. Using lentiviral vectors, we transduce our lead Sm-specific TCR into Tregs derived from patients with SLE who are anti-Sm and HLA-DR15 positive. Compared with polyclonal mock-transduced Tregs, Sm-Tregs potently suppress Sm-specific pro-inflammatory responses in vitro and suppress disease progression in a humanized mouse model of lupus nephritis. These results show that Sm-Tregs are a promising therapy for SLE.

Next-generation sequencing reveals additional HLA class I and class II alleles associated with type 1 diabetes and age at onset.

Type 1 diabetes is an autoimmune disease with an significant genetic component, played mainly by the HLA class II genes. Although evidence on the role of HLA class I genes in developing type 1 diabetes and its onset have emerged, current HLA screening is limited to determining DR3 and DR4 haplotypes. This study aimed to investigate the role of HLA genes on type 1 diabetes risk and age of onset by extensive typing. This study included 115 children and young adults with type 1 diabetes for whom typing of HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 genes was conducted using Next Generation Sequencing. We observed that 13% of type 1 diabetes subjects had non-classical HLA haplotypes that predispose to diabetes. We also found that compared to type 1 diabetes subjects with classical HLA haplotypes, non-classical HLA subjects had a significantly higher frequency of HLA-B*39:06:02 (p-value=0.01) and HLA-C*07:02:01 (p-value=0.03) alleles, known to be involved in activating the immune response. Non-classical HLA subjects also presented peculiar clinical features compared to classical HLA subjects, such as multiple diabetic antibodies and the absence of other autoimmune diseases (i.e., coeliac disease and thyroiditis). We also observed that subjects with early onset had a higher frequency of DQ2/DQ8 genotype than late-onset individuals. Moreover, subjects with late-onset had a higher frequency of alleles HLA-B*27 (p-value=0.003), HLA-C*01:02:01 (p-value=0.027) and C*02:02:02 (p-value=0.01), known to be associated with increased protection against viral infections. This study reveals a broader involvement of the HLA locus in the development and onset of type 1 diabetes, providing insights into new possible disease prevention and management strategies.

FRI608 Secondary Prevention Of Diabetes Type 1 With Oral Calcitriol And Analogs, The Precal Study

Abstract Disclosure: D.T. Papadimitriou: None. E. Dermitzaki: None. P. Christopoulos: None. M. Papagianni: None. K. Kleanthous: None. C. Marakaki: None. A. Papadimitriou: None. G. Mastorakos: None. Type 1 diabetes (T1D) hits about 1:300 with rising incidence affecting increasingly younger children. Population screening at ages 2 and 6 yrs with T1D associated autoantibodies (T1Ab) has been recently proven sensitive. While potential treatments to prevent or delay T1D are currently in development, a population based cost-effective preventive strategy is still lacking. Hence, 2000 IU cholecalciferol daily in a large birth cohort study published in 2001 reduced by 80% the risk of T1D in 1 yr. A pilot clinical trial 10 yrs ago, demonstrated negativation of T1Ab within 0.6 (0.4-2.1) yrs under oral calcitriol in 12 children. To further pursue this hypothesis, we initiated a prospective interventional non-randomized clinical trial, the PRECAL study (ISRCTN17354692). Between 2010-2022, 50 children (26 boys, 24 girls) aged 0.65-16,37 yrs identified as at high risk for T1D were included: 44 were positive for T1Ab (Insulin auto-antibodies, IAA; anti-tyrosinase abs, IA2, anti-glutamic decarboxylase abs, GAD; islet auto-abs, ICA; anti-Zn8 if available) and 6 with negative T1Abs had predisposing HLA A DQ DR haplotypes and genotypes. Nine were diagnosed with varied impaired glucose tolerance (IGT) ( all T1Ab+), 4 with pre-T1D (3 T1Ab+ and 1 HLA+), and 9 had new-onset T1D (all T1Ab+). Serum T1Ab levels and fasting plasma glucose, HbA1c, c-peptide, Ca (tolerated ≤11.5 mg/dl), P, ALP, Ca/Cr 2-hr urine morning sample, 25(OH)D, 1-25(OH)2D with renal ultrasound in cases with hypercalcemia/hypercalciuria (tolerated ≤50%, or at the upper normal for age) were determined before and q3-6 months after initiation of calcitriol (0.05 mcg/Kg/day) 0.25-0.5 x 1-3/day or its synthetic analogue, paricalcitol (thrice the calcitriol dose) 1-4 mcg x 1-3/day p.o. under cholecalciferol repletion. Measurements are given as median and range. Available data on 42 (7 dropouts, 1 follow-up < 3 m). All 26 without pre-T1D/T1D followed 3.06 (0.5-10) yrs negativized T1Ab (15 IAA, 3 IA2, 4 ICA, 2 GAD, 1 IAA/GAD, 1 ICA/GAD) within 0.57 (0.32-1.3) yrs or did not progress to T1D [5 +HLA, follow-up 3 (1-4) yrs]. From 4 pre-T1D, 1 negativized T1Ab (follow-up 1 yr), 1 with +HLA did not progress to T1D (follow-up 3.3 yrs) and 2 with +T1Ab developed T1D in 6m/3yrs. Three out of 8 children with T1D at presentation progressed immediately to overt disease, 5 showed complete remission for 1 yr (1m-2yrs). Five with +T1Ab relapsed and negativized again after resuming therapy. Four (aged < 3 yrs) negativized anti-TPO/TG, and 2 anti-transglutaminase-IgA. Eight presented mild hypercalciuria/hypercalcemia, resolving with dose titration/discontinuation. Calcitriol, and it’s less-calcemic analogue paricalcitol were 100% effective and reasonably safe in negativizing T1Ab in healthy children, possibly preventing the otherwise probably inevitable evolution towards clinical development of T1D, at least if started soon enough after seroconversion. Presentation: Friday, June 16, 2023

138-OR: Characterization and Prediction of Genetic Risk of T1D in Individuals without HLA-DR3 or HLA-DR4

The Major Histocompatibility (MHC) class II haplotypes HLA-DR3 and HLA-DR4 confer substantial genetic risk for type 1 diabetes (T1D), where 90% of individuals with T1D carry at least one copy of DR3 and/or DR4. We sought to understand whether the remaining 10% of T1D individuals without DR3 or DR4 haplotypes (non-DR3/DR4) have differences in genetic risk and whether current risk scores are effective for these individuals. We tested for T1D association using 12,716 non-DR3/DR4 samples from five European ancestry cohorts using genotypes imputed into the Michigan HLA and TOPMed v2 reference panels and identified independent signals using stepwise conditional analyses. There were 21 signals with significant effects on T1D, including 13 at the MHC locus and 8 genome-wide, of which 5 were novel. Across all T1D signals, 14 lead variants exhibited significant heterogeneity in effects on T1D in the non-DR3/DR4 subset compared to the full T1D population, including HLA-B*39 and the PTPN22 locus. Nearly 50% of non-DR3/DR4 T1D would be misclassified by a current T1D genetic risk score (T1D GRS1), which is weighted heavily by DR3/DR4 tag variants. We developed a new GRS using the 21 independent signals and determined whether this GRS could better discriminate T1D in individuals with non-DR3/DR4 haplotypes. The non-DR3/DR4 T1D GRS effectively differentiated T1D case and control samples (AUC=0.87) including in samples not in the initial discovery set (AUC=0.86), and improved prediction compared to GRS1 (AUC=0.52 using all controls, AUC=0.79 using only non-DR3/DR4 controls). These findings reveal a subset of T1D cases with heterogeneity in genetic risk and which are not as well represented by current risk scores, and argue that risk scores developed within heterogeneous subsets may enable even more accurate prediction of T1D. Disclosure C.Mcgrail: None. K.J.Gaulton: Consultant; Genentech, Inc., Stock/Shareholder; Neurocrine Biosciences, Inc., Vertex Pharmaceuticals Incorporated. Funding National Institutes of Health (DK120429, DK122607)

Complete sequences of six major histocompatibility complex haplotypes, including all the major MHC class II structures.

Accurate and comprehensive immunogenetic reference panels are key to the successful implementation of population-scale immunogenomics. The 5Mbp Major Histocompatibility Complex (MHC) is the most polymorphic region of the human genome and associated with multiple immune-mediated diseases, transplant matching and therapy responses. Analysis of MHC genetic variation is severely complicated by complex patterns of sequence variation, linkage disequilibrium and a lack of fully resolved MHC reference haplotypes, increasing the risk of spurious findings on analyzing this medically important region. Integrating Illumina, ultra-long Nanopore, and PacBio HiFi sequencing as well as bespoke bioinformatics, we completed five of the alternative MHC reference haplotypes of the current (GRCh38/hg38) build of the human reference genome and added one other. The six assembled MHC haplotypes encompass the DR1 and DR4 haplotype structures in addition to the previously completed DR2 and DR3, as well as six distinct classes of the structurally variable C4 region. Analysis of the assembled haplotypes showed that MHC class II sequence structures, including repeat element positions, are generally conserved within the DR haplotype supergroups, and that sequence diversity peaks in three regions around HLA-A, HLA-B+C, and the HLA class II genes. Demonstrating the potential for improved short-read analysis, the number of proper read pairs recruited to the MHC was found to be increased by 0.06%-0.49% in a 1000 Genomes Project read remapping experiment with seven diverse samples. Furthermore, the assembled haplotypes can serve as references for the community and provide the basis of a structurally accurate genotyping graph of the complete MHC region.

The phenotype of type 1 diabetes in sub-Saharan Africa.

The phenotype of type 1 diabetes in Africa, especially sub-Saharan Africa, is poorly understood. Most previously conducted studies have suggested that type 1 diabetes may have a different phenotype from the classical form of the disease described in western literature. Making an accurate diagnosis of type 1 diabetes in Africa is challenging, given the predominance of atypical diabetes forms and limited resources. The peak age of onset of type 1 diabetes in sub-Saharan Africa seems to occur after 18-20 years. Multiple studies have reported lower rates of islet autoantibodies ranging from 20 to 60% amongst people with type 1 diabetes in African populations, lower than that reported in other populations. Some studies have reported much higher levels of retained endogenous insulin secretion than in type 1 diabetes elsewhere, with lower rates of type 1 diabetes genetic susceptibility and HLA haplotypes. The HLA DR3 appears to be the most predominant HLA haplotype amongst people with type 1 diabetes in sub-Saharan Africa than the HLA DR4 haplotype. Some type 1 diabetes studies in sub-Saharan Africa have been limited by small sample sizes and diverse methods employed. Robust studies close to diabetes onset are sparse. Large prospective studies with well-standardized methodologies in people at or close to diabetes diagnosis in different population groups will be paramount to provide further insight into the phenotype of type 1 diabetes in sub-Saharan Africa.

Haplotypes of Dirofilaria repens from Poland and selected countries of Central, North-Eastern Europe and the Middle East: An evaluation on the relation between the genetic diversity and the geographic distribution of the fast-spreading parasite

Subcutaneous dirofilariosis is a fast-spreading infection of dogs, and occasionally of other carnivores and humans. Several factors contribute to its spread, including climate change, which facilitates development and survival of Dirofilaria repens in the mosquito vector. Movement/relocation of infected definitive hosts (dogs) from endemic regions to non-endemic regions is another possible cause of local emergence and the presence of a wide variety of wild reservoirs of the parasite may also contribute to its spread. The main aim of this study was to evaluate the genetic diversity of D. repens from different regions of Europe and to evaluate the spread of identified haplotypes and their geographic origin. A total of 95 D. repens isolates were obtained from Central and Eastern Europe (Poland, Belarus, Ukraine, Austria, Romania), NE Europe (Lithuania, Latvia, Estonia), Italy and Israel. All but two positive samples were obtained from the blood of dogs while one positive sample was obtained from an adult worm from a human case from the Lublin area in SE Poland and one sample was obtained from Anopheles plumbeus mosquito from Austria. Genetic diversity in D. repens isolates was evaluated by PCR amplification and sequencing of three genetic markers, including two mitochondrial genes (mtDNA): the cytochrome c oxidase subunit I (COI) and dehydrogenase subunit I (NADH). Additionally, the genomic marker, internal transcribed spacer 1 (ITS-1) was amplified and sequenced. Haplotypes were differentiated based on sequence alignments by identifying Single Nucleotide Polymorphism (SNPs) using DnaSP and Mega X. PopArt was used to construct a haplotype network including all identified haplotypes. Both mtDNA sequences (COI and NADH) were combined together for phylogenetic and network analyses. Altogether 18 haplotypes (DR1-DR18) were identified in combined mtDNA markers among 95 analysed samples. Haplotype DR1 was the most common encompassing 66 isolates: 42 isolates from Poland (41 from dogs and one from a human), 13 from Lithuania, 4 from Latvia, 2 from Ukraine and 5 from Romania. All other haplotypes grouped around haplotype DR1 separated by 1–5 SNPs, forming a star-like shape. Haplotype DR2 was the second most common haplotype, formed by six isolates from Romania. Interestingly, haplotype DR3 was represented only by four isolates from Israel. The remaining 15 haplotypes were represented by 1–4 isolates of different origins. Our study showed that only minor genetic diversity was found in D. repens since all isolates appear to have clustered in or branched out from haplotype DR1 with 1–5 SNP differences. The genetic diversity appears to be governed by geographic origin since isolates from neighbouring populations (countries) appear to share unique haplotypes while other populations that are geographically distant from individual haplotypes.

ODP019 A Case Study of the Association of Autoimmune Polyglandular Syndrome and Pulmonary Artery Hypertension

Abstract We report a patient with combined pulmonary arterial hypertension (PAH) and autoimmune polyglandular syndrome (APS) type 2. A 41 y/o woman with hypothyroidism presented for right heart (RHC) in the work up of PAH. Pt had a medical history of PAH diagnosed by transthoracic echocardiogram (TTE) and hypothyroidism. She reported symptoms of fatigue, shortness of breath with increased pigmentation of her skin progressive for several years. Preadmission lab found hyponatremia of 126 and potassium of 6.6. She was admitted and treated with IV insulin with D50, kayexalate, IV fluids. A random 11 AM cortisol was 0.57 mcg/dl N: AM: 6. 0–30. 0 mcg/dl; PM: 3. 0–16. 0 mcg/dl. Acosyntropin test was ordered. Pt failed the stimulation test (baseline 0.39, 30 min 0.49, 60 min 0.50, with an ACTH at baseline of 1423 pg/ml N: 7.2-63.3). Pt was started on hydrocortisone 20 mg AM/10 mg PM with fludrocortisone 0.1mg. On follow up at 2 weeks, her energy, mood, appetite and breathing tolerance had improved. Follow up RHC in 4 weeks found normalization of right heart pressures. APS is a collection of autoimmune diseases. Type I APS, caused by a defect in the autoimmune regulator (AIRE) gene, is diagnosed with two of three conditions, chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal cortical insufficiency. Type II APS (formally "Schmidt's Syndrome"), associated with HLA haplotypes DR3 (DQB*0201) and DR4 (DQB1*0302), is diagnosed with chronic autoimmune adrenal insufficiency with either Type 1 DM or chronic autoimmune thyroid disease (commonly Hashimotos but can be Graves). It typically presents later in life compared to APS I, age 30-40s. The association between PAH is rare but has been noted in several case studies. It has been associated with APS I in Korniszewski et al. and Bhansali et al. For APS II, the first association was reported by Barrou et al. of a patient with hypothyroidism, hypogonadism, and adrenal insufficiency who later developed PAH. Two cases reported by García-Hernández et al found PAH with positive adrenal antibodies without adrenal insufficiency. In 2009 Walid R. et al. reported a case of severe adrenal insufficiency (hypotension, hypokalemia) with PAH. She improved with steroids, but her PAH was present on TTE four years after treatment started. Pt in that case declined RHC. The patient in this case study proceeded with a RHC which did show normalization of right heart pressures, the first reported case to document normalization of PAH with replacement steroid treatment. Presentation: No date and time listed

GAD65 Antibody Epitopes and Genetic Background in Latent Autoimmune Diabetes in Youth (LADY).

Epitope-specific GAD65Abs and HLA-DR-DQ gene assays help improve the value of risk stratification in autoimmune diabetes mellitus and protect islet function. Identification and early intervention are important for latent autoimmune diabetes in youth (LADY). The aims of this study were to investigate 1) the frequencies of the epitope-specific GAD65Abs and HLA-DR-DQ genes in LADY and 2) the association between HLA-DR-DQ genes and epitope-specific GAD65Abs. Higher frequencies of GAD65-CAb and multiepitope GAD65Abs were observed in young type 1 diabetes, LADY, and old type 1 diabetes subjects than those in latent autoimmune diabetes in adult (LADA) patients. The frequencies of the specific susceptible HLA haplotype DR3, total susceptible HLA haplotypes, and high-risk genotypes were higher in type 1 diabetes and LADY patients than those in LADA patients. In contrast, type 1 diabetes and LADY patients had lower frequencies of low/no genetic risk genotypes (DRX/X) than those of LADA patients. Logistic regression analysis suggested that the susceptible HLA haplotypes were risk factors for glutamic acid decarboxylase antibody (GADA) multiepitope positivity in autoimmune diabetes mellitus. LADY may be more severe than LADA, and LADY seemed to be a transitional type of type 1 diabetes and LADA. GADA epitope and HLA-DR-DQ gene assays are important for risk stratification in autoimmune diabetes mellitus and protection of islet function.

Association of High-Affinity Autoantibodies With Type 1 Diabetes High-Risk HLA Haplotypes.

Electrochemiluminescence (ECL) assays are high-affinity autoantibody (Ab) tests that are more specific than Abs detected by traditional radiobinding assays (RBA) for risk screening and prediction of progression to type 1 diabetes. We sought to characterize the association of high-risk human leukocyte antigen (HLA) haplotypes and genotypes with ECL positivity and levels in relatives of individuals with type 1 diabetes. We analyzed 602 participants from the TrialNet Pathway to Prevention Study who were positive for at least 1 RBA diabetes-related Ab [glutamic acid decarboxylase autoantibodies (GADA) or insulin autoantibodies (IAA)] and for whom ECL and HLA data were available. ECL and RBA Ab levels were converted to SD units away from mean (z-scores) for analyses. Mean age at initial visit was 19.4 ± 13.7 years; 344 (57.1%) were female and 104 (17.3%) carried the high-risk HLA-DR3/4*0302 genotype. At initial visit 424/602 (70.4%) participants were positive for either ECL-GADA or ECL-IAA, and 178/602 (29.6%) were ECL negative. ECL and RBA-GADA positivity were associated with both HLA-DR3 and DR4 haplotypes (all Ps < 0.05), while ECL and RBA-GADA z-score titers were higher in participants with HLA-DR3 haplotypes only (both Ps < 0.001). ECL-IAA (but not RBA-IAA) positivity was associated with the HLA-DR4 haplotype (P < 0.05). ECL-GADA positivity is associated with the HLA-DR3 and HLA-DR4 haplotypes and levels are associated with the HLA-DR3 haplotype. ECL-IAA positivity is associated with HLA-DR4 haplotype. These studies further contribute to the understanding of genetic risk and islet autoimmunity endotypes in type 1 diabetes.

ПРОГНОСТИЧЕСКИЕ МАРКЕРЫ У ПАЦИЕНТОВ С ТИМУСНЕЗАВИСИМОЙ И ТИМУСЗАВИСИМОЙ МИАСТЕНИЕЙ

Objective. To assess the presence of specific markers in patients with thymus-independent and thymus-dependent myasthenia gravis for choosing treatment tactics. Methods. The presence of specific markers was assessed in 138 patients with thymus-independent (M - myasthenia gravis without thymus changes) and thymus-dependent (MH - myasthenia gravis with thymus hyperplasia, MT - myasthenia gravis with thymoma). The method ELISA (the content of antibodies to subunits 1 and 7 nAChR in blood serum, to 7 nAChR subunit in thymocyte mitochondria, a detectablelevel of antinuclear antibody(ANA), immunofluorescence (ANA glow) and flow cytometry (expression of CD14+CD11c+and CD14 + HLA-DR +) has been used. Results. The relationship between the clinical phenotypes of myasthenia gravis and the variants of HLA diplotypes was revealed: in young patients with thymus-independent myasthenia gravis (M), a high heterogeneity of the genotypic markers HLA-DR (DR1, DR2, DR3, DR5, DR7) was detected. Patients with thymus-dependent myasthenia (MT) had only the HLA DR2 and HLA DR7 diplo- and haplotypes. The presence of HLA DR2 and HLA DR7 haplotypes in some young patients with progressive thymus-independent myasthenia gravis (M) led to the development of myasthenia gravis with thymoma (MT) in the elderly people. The pathogenic role also belongs to infection (СMV, EBV, HBV, HCV, HSV-1, HSV-2, HHV-6, mycoplasma) and food intolerance (IgE and IgG4) in the development and progression of myasthenia gravis. A four-fold prevalence of α7 subunit nicotinic acetylcholine receptors on the thymocyte mitochondria as an additional targets of autoimmune aggression in myasthenia gravis was determined. Specific antinuclear antibodies to centromere chromosome proteins were visualized in the elderly people with thymoma. Conclusion. The prognosis of the myasthenia gravis progression and the development of remission can be made using genomic (the presence of certain HLA-DR haplotypes) and molecular (ANA antibodies to centromere chromosome proteins, expression of CD20+, CD14+CD11c+, CD14+HLA-DR+) biomarkers, that can be used for the choice of treatment tactics. What this paper adds The change of complex biomarkers has been firstly studied for prognosis and choice of complex treatment tactics for young patients with progressive thymus-independent myasthenia gravis and for the elderly patients with thymus-dependent myasthenia gravis. In the presence of certain HLA-DR haplotypes, antinuclear antibodies to centromere chromosome proteins, an increasing expression of CD20+, CD14+CD11c+ and CD14+HLADR+ in some young patients with thymus-independent myasthenia gravis, analogically to biomarkers in thymus-dependent myasthenia gravis with thymoma, to perform a thymectomy is of high dangerous. potentialrisksof the procedure.

Epstein Barr virus infection and immune defense related to HLA‐DR15: consequences for multiple sclerosis

MS is a multifactorial disease in which a series of genetic and non‐genetic, environmental factors plays a role in its etiology. In particular, HLA class II alleles, mainly HLADRB1*15:01 (HLA‐DR15), increase the risk for this disease. Out of several environmental factors, and with regard to infections, EBV remains to be a strong candidate, and may synergize with HLA‐DR15 thus increasing the risk for MS. In this issue of the European Journal of Immunology, Zdimerova et al. present highly interesting experimental data using EBV infection in immune‐deficient mice engrafted with human immune cells, either HLA‐DR15+ or HLA‐DRB1*04:01 (HLA DR4), here after denoted as HLA‐DR15−. As a result of EBV infection, the viral load and CD8+ cell expansion were conspicuously higher in mice engrafted with HLA‐DR15+ compared to HLA‐DR15− mice; and myelin basic protein specific T cells emerged in mice engrafted with HLA‐DR15 bearing cells. This study sheds light on how EBV and the class II DR15 haplotype may jointly predispose and synergize in the etiology of MS.

The association of HLA-DP loci with autoimmune diabetes in Chinese

AimsTo determine if HLA-DP loci independently contribute to classic type 1 diabetes (T1D) of all ages, childhood-onset T1D and latent autoimmune diabetes in adults (LADA) among Chinese Han population. MethodsA total of 518 patients with classic T1D (Among them 180 participants manifested T1D between 1 and 14 years), 519 patients with LADA and 527 normal controls were genotyped for both HLA-DPA1 and -DPB1 loci. The frequencies of DP alleles and haplotypes in patients were directly compared to those in controls, followed by adjustment for linkage disequilibrium (LD) with DR-DQ haplotypes. ResultsIn the direct comparison, DPA1*01:03, DPB1*04:01 and DPA1*01:03-DPB1*04:01 showed disease-predisposing effects in both the overall T1D group and the childhood-onset T1D group mainly due to their conjunction with the known susceptible DR3 haplotype. Conditioning on DR-DQ haplotypes, only DPA1*02:02-DPB1*02:02 significantly increased T1D risk among those diagnosed during childhood (OR = 2.02, 95% CI = 1.35–3.01). Whether or not adjusted for LD, no statistically significant HLA-DP association could be observed for LADA. ConclusionHLA-DP is implicated in the pathogenesis of childhood-onset T1D in Chinese, independent of the predominant DR-DQ loci and might serve as additional markers in genetic models for the recognition of those genetically at-risk individuals.

1617-P: Association of High-Affinity Autoantibodies with High-Risk HLA Haplotypes

Electrochemiluminescence (ECL) assays are high-affinity autoantibodies (Abs) that are more specific than radiobinding assay (RBA) Abs for the prediction of type 1 diabetes (T1D) in natural history studies screening for risk and progression to T1D. We analyzed 603 subjects from the TrialNet Pathway to Prevention Study who were positive for at least one RBA diabetes related Ab and for whom ECL and HLA data were available. We sought to characterize ECL-GADA and ECL-IAA positivity and levels (z-scores) by HLA genotype and haplotypes. Mean age at initial visit was 19.4 ± 13.7 years; 345 (57.2%) were female and 104 (17.2%) carried the high-risk HLA- DR3/4*0302 genotype. At initial visit 425 (70%) of subjects were positive for either ECL-GADA or ECL-IAA, with 207 (34.3%) ECL-IAA positive subjects and 367 (60.9%) ECL-GADA positive subjects. For subjects with high-risk HLA-DR3/4 genotype (n=104), 80 (76.9%) were positive for ECL-GADA, while 42 (40.4%) were positive for ECL-IAA. ECL and RBA-GADA z-scores were higher in subjects with HLA-DR3 haplotypes, while ECL-IAA (but not RBA-IAA) z-scores were associated with HLA-DR4 haplotypes. Associations of ECL and RIA Abs with high-risk HLA haplotypes are shown in the Table. In conclusion, ECL and RBA-GADA positivity are associated with both HLA-DR3 and DR4 haplotypes, whereas only ECL-IAA (but not RBA-IAA) positivity and levels are associated with HLA-DR4 haplotype. Disclosure T.M. Triolo: None. L. Pyle: None. L. Yu: None. P. Gottlieb: Advisory Panel; Self; Janssen Scientific Affairs, LLC., Tolerion, Inc., Viacyte, Inc. Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Janssen Pharmaceuticals, Inc., JDRF, Leona M. and Harry B. Helmsley Charitable Trust, Mercia/Nova Laboratories, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc. Stock/Shareholder; Self; IM Therapeutics. A. Steck: None. Funding American Diabetes Association (1-14-CD-17 to A.S.); National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of Allergy and Infectious Diseases; Eunice Kennedy Shriver National Institute of Child Health and Human Development; JDRF

In silico design of a universal carrier protein: A potential solution to the variable immunogenicity observed in past vaccines against drugs of abuse

Abstract Vaccines against drugs of abuse (VADAs) show promise as a possible therapeutic for the alleviation of addiction due to their ability to modulate intake behavior via the altering of drug pharmacokinetics. In the past, however, these vaccines have failed to show clinical efficacy due to extensive variability in the magnitude of antibody response between individual vaccinees. Correspondingly, the future success of VADAs hinges directly on whether or not researchers can find ways to engineer more broadly effective vaccines. Differences in response must, to some extent, be the result of variable capacities for each individual’s immune system to process and/or present antigens. As such, we speculate that future VADAs will need to target the three key stages of vaccine processing, recognition, processing, and presentation, in order to achieve clinical effectiveness. Here, we describe a project aiming to engineer a broadly effective carrier protein based on this concept. Using haplotype frequency data provided by Be the Match, HLA-DQ and -DR haplotypes covering 99% of the population were selected for analysis. Next, the most common carrier proteins used in conjugate vaccine formulations were analyzed for MHC II epitope content using these haplotypes and NetMHCIIPan 3.2 prediction software. Finally, a modified version of the prediction output was used to excise the most likely MHC II epitopes. The highest-ranked epitopes were then stitched together using linkers designed with chemical conjugation sites and cathepsin cleavage sequences and subsequently tethered to cholera toxin subunit B. These results present the successful design of an in silico derived, broadly effective carrier protein to be used in future VADA formulations.

ADVISABILITY OF THYMECTOMY IN YOUNG PATIENTS WITH THYMUS-INDEPENDENT MYASTHENIA GRAVIS IN THE PRESENCE OF BIOMARKERS SPECIFIC FOR PATIENTS WITH THYMOMA

Summary. The aim is to assess the presence of specific markers in patients with thymus-independent and thymus-dependent myasthenia gravis for choosing treatment tactics.&#x0D; Materials and methods. The presence of specific markers was assessed in 138 patients with thymus-independent (M – myasthenia gravis without thymus changes) and thymus-dependent (MH – myasthenia gravis with thymus hyperplasia, MT – myasthenia gravis with thymoma) for the choice of treatment tactics. We used methods of enzyme immunoassay, spectrophotometry, light and fluorescence microscopy.&#x0D; Results and discussion. The relationship between the clinical phenotypes of myasthenia gravis and the variants of HLA leukocyte antigen diplotypes was revealed: in young patients with thymus-independent myasthenia gravis (M), a high heterogeneity of the genotypic markers HLA-DR (DR1, DR2, DR3, DR5, DR7) was observed. Patients with thymus-dependent myasthenia (MT) had only the HLA DR2 and HLA DR7 diplo- and haplotypes. The presence of HLA DR2 and HLA DR7 haplotypes and certain changes in the complex of biomarkers (anti-nuclear antibodies, innate immunity and humoral sensitization) in some young patients with M with disease progression led to the development of myasthenia gravis with thymoma (MT) at an older age.&#x0D; Conclusions. The prognosis of the progression of myasthenia gravis and the development of remission can be made using genomic (the presence of certain HLA-DR haplotypes) and molecular (anti-nuclear antibodies ANA, different fractions of medium molecular weight peptides, circulating immune complexes) biomarkers, that can be used for the choice of treatment tactics.

Is multiple sclerosis progression associated with the HLA-DR15 haplotype?

BackgroundThe prevalence of multiple sclerosis is associated with the major histocompatibility complex class II DR15 haplotype HLA-DRB1*15:01∼HLA-DRB5*01:01.ObjectiveTo assess whether multiple sclerosis progression is associated with the main susceptibility haplotype HLA-DRB1*15:01∼HLA-DRB5*01:01.MethodsPatients (n = 1230) and healthy controls (n = 2110) were genotyped for HLA-DRB1 and HLA-DRB5. The baseline Expanded Disability Status Scale (EDSS) score was determined and patients were followed for at least 3 years.ResultsAfter follow-up of the consecutive cohort 349 patients were classified as having clinical isolated syndrome and 881 patients as having multiple sclerosis. The susceptibility allele HLA-DRB1*15:01 was more frequent in clinical isolated syndrome (odds ratio 1.56) and multiple sclerosis (odds ratio 3.17) compared to controls. HLA- DRB1*15:01 was the only enriched HLA-DRB1 allele in multiple sclerosis patients. Comparison of clinical characteristics between HLA-DRB1*15:01∼HLA-DRB5*01:01 negative and positive patients with multiple sclerosis showed that baseline EDSS score, disease duration and frequency of the category secondary progressive multiple sclerosis with relapse were increased in the HLA-DRB1*15:01∼HLA-DRB5*01:01 positive group.ConclusionThe study confirmed HLA-DRB1*15:01 and HLA-DRB5*01:01 as the main susceptibility alleles and showed weak indirect evidence for a role in progression of the disease.