HLA Allele Frequencies Research Articles

Immunogenetics and human papillomavirus (HPV) in male genital lichen sclerosus (MGLSc).

BackgroundThe pathogenesis of male genital lichen sclerosus (MGLSc) is controversial. Incriminated factors include infection with human papillomavirus (HPV) and autoimmunity (e.g. Human Leukocyte Antigen [HLA]). To address the roles of HLA and HPV in MGLSc we studied adult Caucasian males with a clinical and histological diagnosis of MGLSc. The men in the study attended two specialised Male Genital Dermatoses Clinics between July 2011 and September 2012 and were selected and phenotyped from the clinical records. DNA was extracted from blood and paraffin-embedded biopsy sections, for HLA and HPV typing, respectively. HLA allele frequencies were compared with those derived from the UK-based Caucasian population. Eighty-eight cases of MGLSc were identified. HPV DNA was detected in 33/88 (37.5%) cases of MGLSc. HPV16 was the most prevalent type found: 11/88 (12.5%) MGLSc. No statistically significant HLA associations were established but HLA-B*35, -B*51, -C*15, -DRB1*04, -DRB1*10 (predisposition) and -DQA1*01 (protection) were revealed as alleles of interest. HPV16-associated MGLSc cases showed no statistically significant association with HLA genotype. The relationship between HPV and MGLSc suggests a passenger effect rather than a pathogenic role. HLA is not associated with MGLSc nor co-existent HPV16.

HLA allele repertoire in Russian chronic lymphocytic leukemia patients with an unfavorable prognosis

Introduction. An unfavorable prognosis in chronic lymphocytic leukemia (CLL) is associated with unmutated status of rearranged IGHV genes. CLL is also characterized by a narrowing of the repertoire of IGHV genes and the formation of quasiidentical (stereotyped) receptors, which is probably associated with antigenic selection of the tumor B-cell clone in the pathogenesis of the disease. The HLA phenotype plays an important role in antigenic selection of B cells. On the other hand, the association of specifi c HLA alleles with various diseases has been described. Aim. To assess the frequencies of HLA alleles in CLL patients with unmutated IGHV genes and the most common stereotyped receptors (SARs). Materials and methods. The study included 100 CLL patients with unmutated IGHV genes - 50 with the most common stereotyped antigen receptors (SARs) and 50 with non-stereotyped antigenic receptors. Control group of healthy donors was also included. Results. Signifi cant differences in HLA-allele repertoire between this two groups of patients and groups of donors were found. B*18 allele group was found much more common in patients with SARs than in donors and in patients without SARs. HLA-B*39 was more frequent for patients with SARs compared to donors; in patients without SARs these alleles were not found. For all patients, the frequency of HLA-B*52 alleles was higher than for donors. HLA-C*12 allelic group was found more frequent in CLL patients than in donors. HLA-DRB1*15 in CLL patients with SARs was found twice as often as in healthy donors or patients without SARs, while HLA-DRB1*13, oppositely, was found twice as rare. HLA-DRB1*16 was signifi cantly more frequent in patients without SARs, compared with donors and the patients with SARs. No signifi cant differences were found in the HLA-A and HLA-DQB1 loci. Conclusion. The association of two HLA alleles with “unmutated” CLL and two others with CLL bearing prognostically unfavorable SARs was found. HLA typing of expanded samples of CLL patients with different prognosis and course of the disease will provide more information on the mechanisms of antigen selection in the pathogenesis of CLL and improve diagnostic and therapeutic approaches.

Identification of HLA-A/B/DRB1 alleles in Iranian patients with Fanconi anemia.

Fanconi anemia includes a number of clinically and genetically diverse disorders all of them being associated with genomic instability. Some previous studies reported higher frequencies of certain HLA alleles in patients with Fanconi anemia. In the current study, we genotyped HLA-A/B/DRB1 alleles in 40 Iranian patients with Fanconi anemia. We also genotyped these alleles in the same number of Iranian sex-matched healthy individuals. The frequency of DRB1*11 was significantly higher in patients compared with controls (OR (95% CI) = 2.143 [1.05, 4.46], P value = 0.036). On the other hand, the frequencies of DRB1*13 and B*13 were lower in patients compared with controls (OR (95% CI) = 0.134 [0.02, 0.55], P value = 0.003 and OR (95% CI) = 0.13 [0.01, 0.89], P value = 0.035, respectively). Assessment of genetic divergence using Fstat test showed complete divergence in HLA-A, -B, -DRB1 alleles and haplotypes between patients and controls. The current study provides evidences for different distribution of HLA alleles between patients with Fanconi anemia and healthy subjects.

Analysis of Putative Epitope Candidates of Mycobacterium tuberculosis Against Pakistani Human Leukocyte Antigen Background: An Immunoinformatic Study for the Development of Future Vaccine.

Tuberculosis (TB), a chronic disease caused by Mycobacterium tuberculosis (Mtb), is a global health issue across the world. Pakistan ranks fifth among the countries, which are facing, a significantly great number of mortalities and morbidities due to TB. Unfortunately, all previously reported treatments are not successful for the eradication of TB. Here in this study, we report an emerging treatment option for this disease. We have applied immunoinformatics to predict highly conserved B and T-cell epitopes from Mtb, showing significant binding affinities to the frequent HLA alleles in the Pakistani population. A total of ten highly referenced and experimentally validated epitopes were selected from the Immune Epitope Database (IEDB), followed by their conservancy analysis using weblogos. The consensus sequences and variants derived from these sequences were examined, for their binding affinities, with prevalent HLA alleles of Pakistan. Moreover, the antigenic and allergenic natures of these peptides were also evaluated via Vaxijen and AllerTOP, respectively. Consequently, all potentially allergenic and non-antigenic, peptide fragments, were excluded from the analysis. Among all putative epitopes, three CD8 + T-cell epitopes were selected, as ideal vaccine candidates and, population coverage analysis revealed that the combination of these three peptides was covering, 67.28% Pakistani Asian and 57.15% mixed Pakistani populations. Likewise, eleven linear and six conformational or discontinuous B-cell epitopes were also marked as potential vaccine candidates based on their prediction score, non-allergenic nature, and antigenic properties. These epitopes, however, need the final validation via wet-lab studies. After their approval, these epitopes would be effective candidates for the future designing of epitope-based vaccines against Mtb infections in Pakistan.Electronic supplementary materialThe online version of this article (10.1007/s10989-020-10111-w) contains supplementary material, which is available to authorized users.

Identification of Novel Candidate Epitopes on SARS-CoV-2 Proteins for South America: A Review of HLA Frequencies by Country.

Coronavirus disease (COVID-19), caused by the virus SARS-CoV-2, is already responsible for more than 4.3 million confirmed cases and 295,000 deaths worldwide as of May 15, 2020. Ongoing efforts to control the pandemic include the development of peptide-based vaccines and diagnostic tests. In these approaches, HLA allelic diversity plays a crucial role. Despite its importance, current knowledge of HLA allele frequencies in South America is very limited. In this study, we have performed a literature review of datasets reporting HLA frequencies of South American populations, available in scientific literature and/or in the Allele Frequency Net Database. This allowed us to enrich the current scenario with more than 12.8 million data points. As a result, we are presenting updated HLA allelic frequencies based on country, including 91 alleles that were previously thought to have frequencies either under 5% or of an unknown value. Using alleles with an updated frequency of at least ≥5% in any South American country, we predicted epitopes in SARS-CoV-2 proteins using NetMHCpan (I and II) and MHC flurry. Then, the best predicted epitopes (class-I and -II) were selected based on their binding to South American alleles (Coverage Score). Class II predicted epitopes were also filtered based on their three-dimensional exposure. We obtained 14 class-I and four class-II candidate epitopes with experimental evidence (reported in the Immune Epitope Database and Analysis Resource), having good coverage scores for South America. Additionally, we are presenting 13 HLA-I and 30 HLA-II novel candidate epitopes without experimental evidence, including 16 class-II candidates in highly exposed conserved areas of the NTD and RBD regions of the Spike protein. These novel candidates have even better coverage scores for South America than those with experimental evidence. Finally, we show that recent similar studies presenting candidate epitopes also predicted some of our candidates but discarded them in the selection process, resulting in candidates with suboptimal coverage for South America. In conclusion, the candidate epitopes presented provide valuable information for the development of epitope-based strategies against SARS-CoV-2, such as peptide vaccines and diagnostic tests. Additionally, the updated HLA allelic frequencies provide a better representation of South America and may impact different immunogenetic studies.

HLA allele frequencies and susceptibility to COVID-19 in a group of 99 Italian patients.

With the aim to individuate alleles that may reflect a higher susceptibility to the disease, in the present study we analyzed the HLA allele frequency distribution in a group of 99 Italian patients affected by a severe or extremely severe form of COVID‐19. After the application of Bonferroni's correction for multiple tests, a significant association was found for HLA‐DRB1*15:01, ‐DQB1*06:02 and ‐B*27:07, after comparing the results to a reference group of 1017 Italian individuals, previously typed in our laboratory. The increased frequencies observed may contribute to identify potential markers of susceptibility to the disease, although controversial results on the role of single HLA alleles in COVID‐19 patients have been recently reported.

SARS-CoV-2 genomic variations associated with mortality rate of COVID-19

The coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, has rapidly expanded to a global pandemic. However, numbers of infected cases, deaths, and mortality rates related to COVID-19 vary from country to country. Although many studies were conducted, the reasons of these differences have not been clarified. In this study, we comprehensively investigated 12,343 SARS-CoV-2 genome sequences isolated from patients/individuals in six geographic areas and identified a total of 1234 mutations by comparing with the reference SARS-CoV-2 sequence. Through a hierarchical clustering based on the mutant frequencies, we classified the 28 countries into three clusters showing different fatality rates of COVID-19. In correlation analyses, we identified that ORF1ab 4715L and S protein 614G variants, which are in a strong linkage disequilibrium, showed significant positive correlations with fatality rates (r = 0.41, P = 0.029 and r = 0.43, P = 0.022, respectively). We found that BCG-vaccination status significantly associated with the fatality rates as well as number of infected cases. In BCG-vaccinated countries, the frequency of the S 614G variant had a trend of association with the higher fatality rate. We also found that the frequency of several HLA alleles, including HLA-A*11:01, were significantly associated with the fatality rates, although these factors were associated with number of infected cases and not an independent factor to affect fatality rate in each country. Our findings suggest that SARS-CoV-2 mutations as well as BCG-vaccination status and a host genetic factor, HLA genotypes might affect the susceptibility to SARS-CoV-2 infection or severity of COVID-19.

HIV-1 p24Gag adaptation to modern and archaic HLA-allele frequency differences in ethnic groups contributes to viral subtype diversification.

Pathogen-driven selection and past interbreeding with archaic human lineages have resulted in differences in human leukocyte antigen (HLA)-allele frequencies between modern human populations. Whether or not this variation affects pathogen subtype diversification is unknown. Here we show a strong positive correlation between ethnic diversity in African countries and both human immunodeficiency virus (HIV)-1 p24gag and subtype diversity. We demonstrate that ethnic HLA-allele differences between populations have influenced HIV-1 subtype diversification as the virus adapted to escape common antiviral immune responses. The evolution of HIV Subtype B (HIV-B), which does not appear to be indigenous to Africa, is strongly affected by immune responses associated with Eurasian HLA variants acquired through adaptive introgression from Neanderthals and Denisovans. Furthermore, we show that the increasing and disproportionate number of HIV-infections among African Americans in the USA drive HIV-B evolution towards an Africa-centric HIV-1 state. Similar adaptation of other pathogens to HLA variants common in affected populations is likely.

Binding affinities of 438 HLA proteins to complete proteomes of seven pandemic viruses and distributions of strongest and weakest HLA peptide binders in populations worldwide.

We report detailed peptide‐binding affinities between 438 HLA Class I and Class II proteins and complete proteomes of seven pandemic human viruses, including coronaviruses, influenza viruses and HIV‐1. We contrast these affinities with HLA allele frequencies across hundreds of human populations worldwide. Statistical modelling shows that peptide‐binding affinities classified into four distinct categories depend on the HLA locus but that the type of virus is only a weak predictor, except in the case of HIV‐1. Among the strong HLA binders (IC50 ≤ 50), we uncovered 16 alleles (the top ones being A*02:02, B*15:03 and DRB1*01:02) binding more than 1% of peptides derived from all viruses, 9 (top ones including HLA‐A*68:01, B*15:25, C*03:02 and DRB1*07:01) binding all viruses except HIV‐1, and 15 (top ones A*02:01 and C*14:02) only binding coronaviruses. The frequencies of strongest and weakest HLA peptide binders differ significantly among populations from different geographic regions. In particular, Indigenous peoples of America show both higher frequencies of strongest and lower frequencies of weakest HLA binders. As many HLA proteins are found to be strong binders of peptides derived from distinct viral families, and are hence promiscuous (or generalist), we discuss this result in relation to possible signatures of natural selection on HLA promiscuous alleles due to past pathogenic infections. Our findings are highly relevant for both evolutionary genetics and the development of vaccine therapies. However they should not lead to forget that individual resistance and vulnerability to diseases go beyond the sole HLA allelic affinity and depend on multiple, complex and often unknown biological, environmental and other variables.

1677-P: Higher Frequency of HLA DRB1*08:03 and DRB1*04:01 Observed in A-beta+ Ketosis-Prone Type 2 Diabetes in Japanese Subjects

Ketosis-prone type 2 diabetes, especially islet-associated autoantibody negative and beta-cell function reserved (A-beta+; KPD) group, is recognized as atypical diabetes. Because we detected insulin peptide reactive T cells in these KPD subjects, we realized that KPD possesses characteristics of type 1 diabetes (ADA meeting, 2017). Therefore, in this study, we examined HLA types. After institutional review board permission was obtained, written informed consent and blood samples were obtained from the enrolled Japanese subjects (KPD: 22 males and 0 female, mean onset age 36.4 years of age; acute onset type 1 diabetes (AT1D): 21 males and 22 females, mean onset age 36.3 years of age; slowly progressive type 1 diabetes (SP): 10 males and 10 females, mean onset age 49.0 years of age). Then the HLA allele frequency was compared to normal controls (http://hla.or.jp/med/frequency_search/ja/allele/search/R/). The frequencies of HLA DRB1*04:05 and DRB1*09:01, which are known susceptible alleles in Japanese type 1 diabetes, were not higher in KPD (13.6%, 27.3%, respectively) subjects as compared to controls (13.5%, 14.3%, respectively), whereas were significantly higher in AT1D (41.9%, 53.5%, respectively; both p<0.001) and SP subjects (45.0%, 40.0%, respectively; both p<0.002). However, interestingly, the frequency of HLA DRB1*08:03 was significantly higher in KPD (34.8%, p<0.001) subjects as compared to controls (8.3%), whereas it was not the case both in AT1D (4.7%) and SP (10.0%) subjects. Moreover, the frequency of HLA DRB1*04:01 which is known susceptible alleles in Caucasian type 1 diabetes was significantly higher in KPD (18.2%, p<0.001) subjects as compared to controls (0.9%), whereas it was not the case both in AT1D (2.3%) and SP (5.0%) subjects. In conclusion, although KPD subjects possess similar insulin peptide T cell responses to type 1 diabetes, different genetic background seems to exist probably leading to different clinical phenotype. Disclosure A. Shimada: Advisory Panel; Self; Astellas Pharma Inc. Speaker’s Bureau; Self; Eli Lilly Japan K.K., Novo Nordisk Inc., Sanofi-Aventis. A. Satomura: None. S. Suzuki: None. A. Haisa: None. Y. Oikawa: None. Funding Saitama Medical University

Increasing accuracy of HLA imputation by a population-specific reference panel in a FinnGen biobank cohort.

The HLA genes, the most polymorphic genes in the human genome, constitute the strongest single genetic susceptibility factor for autoimmune diseases, transplantation alloimmunity and infections. HLA imputation via statistical inference of alleles based on single-nucleotide polymorphisms (SNPs) in linkage disequilibrium (LD) with alleles is a powerful first-step screening tool. Due to different LD structures between populations, the accuracy of HLA imputation may benefit from matching the imputation reference with the study population. To evaluate the potential advantage of using population-specific reference in HLA imputation, we constructed an HLA reference panel consisting of 1150 Finns with 5365 major histocompatibility complex region SNPs consistent between genome builds. We evaluated the accuracy of the panel against a European panel in an independent test set of 213 Finnish subjects. We show that the Finnish panel yields a lower imputation error rate (1.24% versus 1.79%). More than 30% of imputation errors occurred in haplotypes enriched in Finland. The frequencies of imputed HLA alleles were highly correlated with clinical-grade HLA allele frequencies and allowed accurate replication of established HLA–disease associations in ∼102 000 biobank participants. The results show that a population-specific reference increases imputation accuracy in a relatively isolated population within Europe and can be successfully applied to biobank-scale genome data collections.

Frequency of HLA Class I and Class II Alleles in Patients with CVID from Turkey

ABSTRACT Background: Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency. Certain gene loci are pointed out in several studies in CVID patients. Until now, monogenic defects have been identified in only 2-10% of CVID patients; therefore, association of the disease with HLA alleles may be important for elucidating immunological and genetic mechanisms behind CVID. The aim of this study is to investigate the relationship between CVID and HLA alleles. Methods: HLA class I/II alleles were analyzed in 65 patients with CVID and alleles that may be related to disease susceptibility were determined by comparing with 300 healthy controls. We also evaluated HLA allele frequencies in CVID patients with gastrointestial system (GIS) involvement and autoimmune manifestations. Results: When compared with controls, frequencies of B*27, B*35, C*04, and DRB1*04 alleles were significantly different in patients with CVID (p < .05). Frequencies of C*12, DRB1*13, and DRB1*15 alleles were more frequent in controls, indicating protective alleles (p < .05). There was a statistically significant difference for DQ2 and DQ8 haplotypes between patients with GIS involvement and controls. Conclusion: In comparison with literature, distinctive HLA alleles found in our study may originate from the diversity in gene pool between the populations. These data may provide clues for disease susceptibility.

Protective and causative killer Ig-like receptor (KIR) and metalloproteinase genetic patterns associated with Herpes simplex virus 1 (HSV-1) encephalitis occurrence

BackgroundThe cerebral innate immune system has a critical role in control processes of viral replication in the brain after primary infactivo and immunologic disregulation and inflammation has been reported as a primary determinant of pathogenesis and prognosis of subsequent HSV-1 related encephalitis (HSE). Interaction linking LTR3-activated DCs is also represented by the killer Ig-like receptor (KIR) + pathways on NK cells. Only a few studies analyzed the role of of MMP-9 activity regulating genetic polymorphism on clinical outcome of viral infections. Susceptibility to symptomatic encephalitis depends on SNC viral invasion and BBB disruption. We hypothesize that certain KIR genes and MMP allele may help to characterize a risk profile of developing an acute encephalitis due to HSV 1. Aim of the studyAnalyze the frequency of KIR genes and the C(−1562)T MMP-9 allels in subjects with HSV-1 encephalitis and to analyze their interaction with regard of the risk of occurrence of a symptomatic encephalitis. Materials and methodsBetween November 2014 and January 2019, all consecutive patients with symptomatic acute encephalitis were recruited from three wards (Internal Medicine, Neurology, and Infectious Diseases) of “P. Giaccone” University Hospital, Palermo. ResultsPatients with acute viral encephalitis in comparison to controls showed a higher frequency AA KIR haplotype, HLA-C2 and of HLA-A-Bw4 alleles. With regard of HLA allele frequency patients with acute viral encephalitis In comparison to controls also showed a higher frequency of HLA-C2 and of HLA-A-Bw4 alleles. With regard of MMP-9 alleles, subjects with acute viral encephalitis were more likely to have the TT genotype. The multiple logistic regression analysis considering variables predictive of the occurrence of acute viral encephalitis showed the detrimental effect of AA KIR, HLAC1, HLA-A-BW4 and HLA-B-BW4T and of TT aplotype of MMP-9 genotype. ConclusionsOur study shows that in immunocompetent adult subjects there is an association between some KIR genes, MMP-9 alleles and HLA-ligand alleles and susceptibility to develop a symptomatic acute viral encephalitis. Definition of the genetic and immunological background of acute viral encephalitis can play a key role to determine personalized medicine.

Proteogenomics Uncovers a Vast Repertoire of Shared Tumor-Specific Antigens in Ovarian Cancer.

High-grade serous ovarian cancer (HGSC), the principal cause of death from gynecologic malignancies in the world, has not significantly benefited from advances in cancer immunotherapy. Although HGSC infiltration by lymphocytes correlates with superior survival, the nature of antigens that can elicit anti-HGSC immune responses is unknown. The goal of this study was to establish the global landscape of HGSC tumor-specific antigens (TSA) using a mass spectrometry pipeline that interrogated all reading frames of all genomic regions. In 23 HGSC tumors, we identified 103 TSAs. Classic TSA discovery approaches focusing only on mutated exonic sequences would have uncovered only three of these TSAs. Other mutated TSAs resulted from out-of-frame exonic translation (n = 2) or from noncoding sequences (n = 7). One group of TSAs (n = 91) derived from aberrantly expressed unmutated genomic sequences, which were not expressed in normal tissues. These aberrantly expressed TSAs (aeTSA) originated primarily from nonexonic sequences, in particular intronic (29%) and intergenic (22%) sequences. Their expression was regulated at the transcriptional level by variations in gene copy number and DNA methylation. Although mutated TSAs were unique to individual tumors, aeTSAs were shared by a large proportion of HGSCs. Taking into account the frequency of aeTSA expression and HLA allele frequencies, we calculated that, in Caucasians, the median number of aeTSAs per tumor would be five. We conclude that, in view of their number and the fact that they are shared by many tumors, aeTSAs may be the most attractive targets for HGSC immunotherapy.

Computational MHC-I epitope predictor identifies 95% of experimentally mapped HIV-1 clade A and D epitopes in a Ugandan cohort

BackgroundIdentifying immunogens that induce HIV-1-specific immune responses is a lengthy process that can benefit from computational methods, which predict T-cell epitopes for various HLA types.MethodsWe tested the performance of the NetMHCpan4.0 computational neural network in re-identifying 93 T-cell epitopes that had been previously independently mapped using the whole proteome IFN-γ ELISPOT assays in 6 HLA class I typed Ugandan individuals infected with HIV-1 subtypes A1 and D. To provide a benchmark we compared the predictions for NetMHCpan4.0 to MHCflurry1.2.0 and NetCTL1.2.ResultsNetMHCpan4.0 performed best correctly predicting 88 of the 93 experimentally mapped epitopes for a set length of 9-mer and matched HLA class I alleles. Receiver Operator Characteristic (ROC) analysis gave an area under the curve (AUC) of 0.928. Setting NetMHCpan4.0 to predict 11-14mer length did not improve the prediction (37–79 of 93 peptides) with an inverse correlation between the number of predictions and length set. Late time point peptides were significantly stronger binders than early peptides (Wilcoxon signed rank test: p = 0.0000005). MHCflurry1.2.0 similarly predicted all but 2 of the peptides that NetMHCpan4.0 predicted and NetCTL1.2 predicted only 14 of the 93 experimental peptides.ConclusionNetMHCpan4.0 class I epitope predictions covered 95% of the epitope responses identified in six HIV-1 infected individuals, and would have reduced the number of experimental confirmatory tests by > 80%. Algorithmic epitope prediction in conjunction with HLA allele frequency information can cost-effectively assist immunogen design through minimizing the experimental effort.

Gorgan (Iran) population HLA genetics and anthropology

Gorgan (Iran) have been studied for HLA-A, -B, -C, -DRB1 and -DQB1 genes for the first time. They are Turkmen and originated in East Asia around Altai Mts; they originally spoke a Turk language classified within the Turkish-Oguz group. Peripheral blood samples were collected from Gorgan City (Iran) and HLA typed by standard methodology. HLA allele frequencies were compared with 7984 chromosomes of other World populations and it was shown existence of admixture of Siberian and Mediterranean HLA characters in this population, probably due to longlasting contact with Persians. Three new HLA extended haplotypes were found: A*01:01-B*35:01-DRB1*03:01-DQB1*02:01, A*30:01-B*13:01-DRB1*15:01-DQB1*02:01 and A*31:01-B*35:01-DRB1*15:01-DQB1*03:01. Gorgan (Iran) were most close to Chuvashians (Noth Caspian Sea, Russia) and Siberians, like Tuvinians, Mansi and Buryats in Neighbour Joining and Vista analyses. It is established a relationship of this population with Kurgan (Gorgan, Iran) archaeological mounds culture. However, their kinship with Scythians (2nd century BC) and Sarmatians (4th century AD) is obscure although both of them spoke a Persian language.

MGeND: an integrated database for Japanese clinical and genomic information

To promote the implementation of genomic medicine, we developed an integrated database, the Medical Genomics Japan Variant Database (MGeND). In its first release, MGeND provides data regarding genomic variations in Japanese individuals, collected by research groups in five disease fields. These variations consist of curated SNV/INDEL variants and susceptibility variants for diseases established by genome-wide association study analysis. Furthermore, we recorded the frequencies of HLA alleles in infectious disease populations.

Spectrum and Clinical Significance of HLA Class I Alleles and Their Somatic Mutations in Immune Aplastic Anemia

Clonal hematopoiesis associated with loss of HLA class I alleles due to somatic mutations and/or 6p loss of heterozygosity (LOH) is frequent in immune aplastic anemia (AA). HLA-B*40:02 is more likely to be involved in HLA loss in Japanese AA patients, suggesting a role for this allele in immune pathophysiology (Zaimoku Y et al, Blood 2017). Mutations in non-B*40:02 HLA class I alleles have been reported in a limited number of patients from the United States (Babushok D et al, Blood Adv 2017) and Japan (Mizumaki H et al, 60th ASH meeting), but their prevalence and clinical significance are not well characterized. We investigated somatic mutations of HLA class I alleles, HLA allele frequencies, and their correlations with outcomes of therapy in a total of 532 AA patients, aged 2 years or older, treated on various Hematology Branch protocols (clinicaltrials.gov NCTs 00001964, 00061360, 00195624, 00260689, 00944749, 01193283, and 01623167). HLA allele-lacking (HLA-) monocytes from cryopreserved peripheral blood mononuclear cells were screened by flow cytometry after staining with allele-specific monoclonal antibodies for HLA-A and/or HLA-B (HLA-flow) in 172 AA patients. HLA- monocytes accounting for 0.5% to 100% (median 9.5%) of total monocytes were detected in 49 (28%) of the 172 patients and in 59 (15%) of 382 alleles analyzed (Figure 1). Loss of cell surface expression was frequent for HLA-B14 (46%), B27 (33%), B49 (33%), A68 (26%), A2 (23%), B40 (21%), and B8 (21%). One percent to 60% (median, 8.9%) of glycosylphosphatidylinositol-linked protein-negative (GPI-) monocytes were also present in 43% (21 of 49) of the patients with HLA- monocytes, but GPI- clones had normal HLA cell surface expression. Deep sequencing of HLA-A, HLA-B and HLA-C on sorted HLA- and HLA+GPI+ monocytes was performed in 42 of the 48 patients from whom adequate cells were available. Somatic mutations and/or LOH corresponding to the lacking alleles were detected in all 42 cases (Figure 1): 9 had both somatic mutations and LOH, 20 had somatic mutations only, and 13 had LOH only. Among the 13 patients who showed only LOH in the absent allele, 6 had somatic mutations in other alleles of HLA+ monocytes that was not analyzable of HLA expression, and 2 had a breakpoint of LOH between HLA-A and HLA-C, leading to loss of a single HLA-A allele. Somatic mutations or LOH involving only one allele were present in 37 patients among 6 HLA-A alleles (in 02:01 [7 patients], 02:05 [1], 02:06 [3], 02:11 [1], 68:01 [2], 68:02 [2]) and 10 HLA-B alleles (07:02 [1], 08:01 [4], 14:01 [1], 14:02 [7], 27:05 [1], 35:02 [1], 35:05 [1], 40:01 [1], 40:02 [3], 45:01 [1]), but were not found in HLA-C alleles. HLA allele frequencies in AA patients, including 271 white Americans, 120 African-Americans, and 99 Hispanics and Latinos, were compared with ethnicity-matched individuals in bone marrow donor datasets of the National Marrow Donor Program, and underwent random-effects meta-analyses. HLA-B*07, B*14, and B*40 were overrepresented in AA, while A*02, A*68, and B*08 frequencies were similar to those of healthy donors (Figure 2). In 164 severe AA patients who were initially treated with horse antithymocyte globulin (hATG), cyclosporine, and eltrombopag between 2012 and 2018, 36 and 79 were positive and negative for HLA- monocytes, respectively, and 49 were not tested by HLA-flow. There was no significant difference in overall and complete response rates at six months among the three groups (Figure 3). Clonal evolution, defined as acquisition of abnormal bone marrow cytogenetics or morphology, especially high-risk evolution to chromosome 7 abnormalities, complex cytogenetics, or morphological MDS/AML, tended to be more frequent in patients with HLA- monocytes, compared to the other two groups, but the difference did not reach statistical significance. Clinical outcomes were also assessed according to the presence of specific HLA alleles in 400 severe AA patients who were treated with hATG-based initial immunosuppressive therapy from 2000 to 2018: there was no significant differences in probabilities of response and clonal evolution according to the alleles associated with somatic mutations. Our study revealed that somatic mutations in HLA genes in AA are broadly distributed, but some alleles are preferentially affected. Inconsistent with previous studies, we found that outcomes of therapy did not significantly correlate with HLA gene mutations or with distinct HLA alleles. Disclosures No relevant conflicts of interest to declare.

HLA Alleles Repertoire in Russian CLL Patients

Introduction. Chronic lymphocytic leukemia (CLL) is one of the most common lymphoproliferative diseases in Europe and the USA. The mutational status of the immunoglobulin heavy chain variable region genes (IGHV) has long been known as an important factor for long-term prognosis in CLL. The variety of the rearranged IGHV structure variants in CLL cells is very limited and differs significantly from that in normal B cells. About 30% of all cases of CLL have immunoglobulin receptors with very similar nucleotide sequences. These quasi-identical receptors are called stereotyped. The most common 20 subtypes of stereotyped antigen receptors (SARs) are presented in 12% of all CLL cases. HLA-complex plays a central role in the formation of the immune response. Associations of specific HLA-alleles with different malignant, autoimmune and infectious diseases have been described. However, possible relationship of HLA phenotype to the development of CLL and the association of HLA alleles with disease are not completely understood. Aim. To study the repertoire of HLA alleles in Russian CLL patients with the most common SARs. Patients and methods. The study included 50 CLL patients with the most common IGHV genes and SARs, followed up at the National Research Center for Hematology from 2008 to 2019. Two groups of healthy donors were selected as control - one from Moscow (1507) and other from Eastern Siberia (296). The mutational status of IGHV and the SARs were determined according to European Research Initiative in CLL (ERIC) criteria. HLA-typing in 5 loci (HLA-A, -B, -C, -DRB1, -DQB1) was performed using Luminex 200 system (Luminex, TX, USA) with Lifecodes HLA SSO typing kits (Immucor, CT, USA). HLA frequencies were compared using chi-square test. ARLEQUIN software package, version 3.5 was used for statistical analysis. Results. All patients enrolled in the study had unmutated IGHV genes. In 43 of them, homology with the germline gene was 100%, and in 7 - it was in the range of 98.6-99.7%. IGHV genes of 46 patients (92%) belonged to the 1st family. In 14 (28%) patients CLL#1 SAR, associated with the most aggressive course of the disease, was expressed. Nine patients (18%) each belonged to the CLL#3 and CLL#6 subtypes. The remaining patients expressed CLL#5, 7H, 12 and 28A SARs. No significant differences in the frequency of HLA-A alleles were found between CLL patients and both donor groups. HLA-B*18, HLA-B*52 and HLA-C*12:02 were found significantly more often in CLL patients than in donors. In the DRB1 locus, differences were observed in the two allelic groups. In CLL patients HLA-DRB1*15 was twice more frequent than in healthy donors, HLA-DRB1*13, on the contrary, was twice less frequent. No significant differences were found for DQB1 locus. Most common HLA haplotype frequencies were also calculated. DRB1*15-DQB1*06 haplotype was significantly 2 times more frequent in CLL patients than in donors. Furthermore, A*02-B*07-C*07-DRB1*15-DQB1*06 and A*25-B*18-C*12-DRB1*15-DQB1* 06 were more frequent, but these data were not statistically significant probably due to the small patient sample (Table 1). No significant differences were found between the SAR subtypes of CLL patients in the studied cohort. Discussion. Despite a small sample, we were able to detect 4 predictive and 1 protective HLA allele for CLL patients. Our data disagree with the data of researchers from the United States and France and partially match those obtained by Iranian scientists. This might be explained by two reasons. First, HLA allele frequencies are differ between various populations. Second, we studied a small selected sample - all patients had unmutated IGHV genes, mainly from the same V gene family, and belonged to a cohort with an aggressive course of the disease. Further studies on extended samples of CLL patients are required to assess possible associations of HLA alleles with CLL subtypes and the course of the disease. Disclosures No relevant conflicts of interest to declare.

Next-Generation Sequencing of HLA Loci Identifies Predisposing and Protective Factors for Immune-Mediated Thrombotic Thrombocytopenic Purpura in a Japanese Population

Background Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by neutralizing anti-ADAMTS13 autoantibodies. This condition leads to life-threatening microvascular thrombi in systemic organs due to an imbalance between ADAMTS13 and von Willebrand factor. While several European groups reported that HLA-DRB1*11 was one of the strongest genetic risk factors for iTTP, no large-scale studies have analyzed which HLA alleles convey greater risk for iTTP in genetically isolated Japanese. Therefore, we analyzed ten HLA loci using next-generation sequencing. Materials and Methods We performed a prospective cohort study among Japanese patients with iTTP. The diagnosis of iTTP was suspected in cases of thrombocytopenia and hemolytic anemia with unknown cause, and was confirmed by both severe deficiency of ADAMTS13 (below 10% of normal) and the presence of anti-ADAMTS13 inhibitor. Fifty-six patients with iTTP from 19 institutes were enrolled in this study from August 2017 to March 2019, and 52 patients were finally eligible. An EDTA-treated serum sample was obtained from each patient at enrollment, and was analyzed via next-generation sequencing to identify the HLA alleles at following loci: A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, and DPB1. Then, we compared allele frequencies at each locus in iTTP patients with those in a healthy Japanese population as reported by Nakajima et al in 2001. Additionally, we collected data on laboratory tests and clinical response to treatment during the acute phase. The HLA allele frequency of patients with iTTP and controls were compared by Fisher's exact test, and Bonferroni correction was applied to observed P-values. In this study, we multiplied the P-value by the number of alleles in each locus whose frequency was more than 1% in control to calculate the corrected P-value (Pc). This study was approved by the IRB of each referring institute. Results The male/female ratio of enrolled patients was 22/30. The median age was 58 years (range: 1-81). Based on the underlying disease, iTTP patients were classified as having primary TTP (n=38), autoimmune disease (n=12), malignancy (n=1), or acute pancreatitis (n=1). Plasma levels of ADAMTS13 activity were less than 3% in all cases, and the median concentration of ADAMTS13 autoantibodies was 2.5 Bethesda units/mL (range: 0.5-113). All patients showed severe thrombocytopenia (median platelet count: 12×109/L) and hemolytic anemia (median hemoglobin level: 8.0 g/dL, and median LDH level: 995 U/L). During the acute phase, 49 of 52 patients received combination treatment with plasma exchange (PEX) using fresh frozen plasma and corticosteroids. Additional immunosuppressants were administered in several cases, as follows: rituximab in 23 cases, cyclosporin A in 7 cases, cyclophosphamide in 5 cases, vincristine in 3 cases, and mycophenolate mofetil in 1 case. The median number of PEX treatments was 11 (range: 3-42). The allele frequencies in iTTP patients and controls are shown in Table 1. Regarding HLA class II alleles (DRB1, DRB3/4/5, DQA1, and DQB1), we identified the following alleles as risk factors for iTTP in the Japanese population: DRB1*08:03 (odds ratio [OR]: 3.06, Pc=0.005), DRB3/4/5*blank (OR: 2.3, Pc=0.007), DQA1*01:03 (OR: 2.25, Pc=0.006), and DQB1*06:01 (OR: 2.41, Pc=0.003). The estimated haplotype consisting of these 4 alleles was more frequent in the iTTP group than in the control group (30.8% vs. 6.0%, Pc&lt;0.001). By contrast, DRB5*01:01 was associated with a reduced risk of iTTP (OR: 0.23, Pc=0.03). Among the A, B, C, and DPB1 loci, no significant difference was found between the 2 groups. Surprisingly, DRB1*11 was not identified as a risk factor for iTTP in Japanese, unlike in Caucasians. Additional analysis showed that this haplotype did not influence platelet count, hemoglobin level, lactate dehydrogenase level, ADAMTS13 activity, or the concentration of ADAMTS13 autoantibodies. Conclusion We found that a Japanese-specific haplotype, DRB1*08:03-DRB3/4/5(blank)- DQA1*01:03-DQB1*06:01, was a significant risk factor for iTTP in a Japanese population, and that DRB5*01:01 was a protective factor against iTTP development. Disclosures No relevant conflicts of interest to declare.