HLA-A Haplotypes Research Articles

The humoral and cellular response to mRNA SARS-CoV-2 vaccine is influenced by HLA polymorphisms.

Host genetic variability contributes to susceptibility to SARS-CoV-2 infection and COVID-19 evolution and the role of HLA system has not clearly emerged, suggesting the involvement of other factors. Studying response to vaccination with Spyke protein mRNA represents an ideal model to highlight whether the humoral or cellular responses are influenced by HLA. Four hundred and sixteen workers, vaccinated with Comirnaty beginning 2021, were selected within the Azienda Ospedaliera Universitaria "Città della Salute e della Scienza di Torino." The humoral response was determined with the LIAISON® kit, while the analysis of the cellular response was performed with the Quantiferon SARS-CoV-2 assay, for the S1 (receptor-binding domain; Ag1) and S1 and S2 (Ag2) subunits of the Spyke protein. Six HLA loci were typed by next-generation sequencing. Associations between HLA and vaccine response were performed with univariate and multivariate analyses. An association was found between A*03:01, B*40:02 and DPB1*06:01 and high antibody concentration and between A*24:02, B*08:01 and C*07:01 and low humoral responses. The haplotype HLA-A*01:01 ~ B1*08:01 ~ C*07:01 ~ DRB1*03:01 ~ DQB1*02:01 conferred an increased risk of low humoral response. Considering cellular responses, 50% of the vaccinated subjects responded against Ag1 and 59% against Ag2. Carriers of DRB1*15:01 displayed a higher cellular response both to Ag1 and Ag2 compared to the rest of the cohort. Similarly, DRB1*13:02 predisposed to a robust cellular response to Ag1 and Ag2, while DRB1*11:04 showed an opposite trend. Cellular and humoral responses to Comirnaty are influenced by HLA. Humoral response is mainly associated to class I alleles, with A*03:01, previously associated to protection against severe COVID-19, and response to vaccination, standing out. Cellular response predominantly involves class II alleles, with DRB1*15:01 and DPB1*13:01 prevailing. Affinity analysis for Spyke peptides is generally in line with the association results.

Genetic Markers that Predict the Formation of Certain Body Types and the Development of Motor Qualities

Purpose of the study-development of criteria and techniques for the prognostic selection of athletes based not only on the signs of the phenotype but also on the indicators of genetic markers that predict the development of certain physical qualities and the formation of certain body types. Material and Methods: The assessment of the physical development of athletes was carried out based on anthropometric methods according to the recommendations set forth by E.G. Martirosov, 1982 [1]. The type of constitution was determined by the Heath-Carter method, 1989 with the identification of ecto, endo, and mesomorphic components, calculated using special formulas. The HLA- genetic system was identified by typing in the microcyto-lymphotoxin test according to the method of Yu.M. Zaretskaya, and V.M. Abramov [3]. Research results: Athletes-rowers with the highest sports qualifications - masters of sports of international class and masters of sports, represent a fairly homogeneous sample: 75% of athletes have an endo-mesomorphic type, 16.7% have an ecto-endomorphic type, 8.3% have an ecto-mesomorphic type of. For a more reliable clarification of the role of the HLA system genes in the program of individual development, the associations of sub loco A, B, and C of the HLA complex with the somatotype were studied. So, for endo-mesomorphic somatotypes, significant specific associations with antigens HLA-A3, HLA-A9, HLA-A11, sublocus HLA-B35, as well as HLA-СW4 and HLA-СW6 were revealed. Ecto-mesomorphic somatotypes had significant associations with antigens of the sublocus HLA-CW-5, A-10. It has been established that for athletes specializing in speed-strength sports (rowing, boxing), the haplotype HLA-A1 and B-35 are specific at X2-5.76. Intergroup comparison of frequencies of registration of the antigenic composition revealed that the content of HLA-B7 in athletes - power workers (wrestling) are significantly higher and significantly at x = 6.645, that is, HLA-B7 can be considered as a genetic marker of strength qualities. It has been established that for athletes specializing in speed-strength sports (rowing, boxing), the haplotype HLA-A1 and B-35 are specific at X2-5.76. Conclusions: Specific genetic markers associated not only with the formation of certain types of physique but also predicting the spectrum of development of several physical qualities that are of selective importance in the practice of sports have been established.

In silico and in vitro arboviral MHC class I-restricted-epitope signatures reveal immunodominance and poor overlapping patterns.

The present work sought to identify MHC-I-restricted peptide signatures for arbovirus using in silico and in vitro peptide microarray tools. First, an in-silico analysis of immunogenic epitopes restricted to four of the most prevalent human MHC class-I was performed by identification of MHC affinity score. For that, more than 10,000 peptide sequences from 5 Arbovirus and 8 different viral serotypes, namely Zika (ZIKV), Dengue (DENV serotypes 1-4), Chikungunya (CHIKV), Mayaro (MAYV) and Oropouche (OROV) viruses, in addition to YFV were analyzed. Haplotype HLA-A*02.01 was the dominant human MHC for all arboviruses. Over one thousand HLA-A2 immunogenic peptides were employed to build a comprehensive identity matrix. Intending to assess HLAA*02:01 reactivity of peptides in vitro, a peptide microarray was designed and generated using a dimeric protein containing HLA-A*02:01. The comprehensive identity matrix allowed the identification of only three overlapping peptides between two or more flavivirus sequences, suggesting poor overlapping of virus-specific immunogenic peptides amongst arborviruses. Global analysis of the fluorescence intensity for peptide-HLA-A*02:01 binding indicated a dose-dependent effect in the array. Considering all assessed arboviruses, the number of DENV-derived peptides with HLA-A*02:01 reactivity was the highest. Furthermore, a lower number of YFV-17DD overlapping peptides presented reactivity when compared to non-overlapping peptides. In addition, the assessment of HLA-A*02:01-reactive peptides across virus polyproteins highlighted non-structural proteins as "hot-spots". Data analysis supported these findings showing the presence of major hydrophobic sites in the final segment of non-structural protein 1 throughout 2a (Ns2a) and in nonstructural proteins 2b (Ns2b), 4a (Ns4a) and 4b (Ns4b). To our knowledge, these results provide the most comprehensive and detailed snapshot of the immunodominant peptide signature for arbovirus with MHC-class I restriction, which may bring insight into the design of future virus-specific vaccines to arboviruses and for vaccination protocols in highly endemic areas.

The Association of HLA Alleles and Haplotypes with Age of Disease Onset in Pakistani Psoriatic Patients

Introduction: The human leukocyte antigen (HLA) region on chromosome 6p21 is well known to carry the most important genetic factors in susceptibility to psoriasis. Different HLA alleles and haplotypes have been reported to be associated with psoriasis in different populations. Psoriasis has a variable age of onset and, based on this, it can be classified into two types; type I with age of onset before 40 years of age and type II with age of onset after 40 years of age. The objective of this study was to determine the association of HLA class I and class II alleles and haplotypes with disease and stratification using age of onset in Pakistani psoriatic patients. Methods: A group of 603 individuals (326 cases and 277 controls) were analyzed for HLA class I and II alleles and haplotype association by sequence specific PCR. The association was further analyzed according to the age of onset of the patients. Results: We found that HLA alleles B*57 and Cw*06:02, DQB1*03:03:02 are strongly associated with early onset psoriasis, while alleles B*15, DRB1*13:02 and DQB1*03:03:02 are associated with late-onset psoriasis. Cw*06:02 allele was not associated with late-onset psoriasis patients. Allele DQB1*03:03:02 had the highest odds ratio in all patients. We found a novel association specifically with late-onset psoriasis samples with the haplotype HLA-A*11; B*15; Cw*04; DRB1*15; DQB1*05 (Pc = 3.60 × 10⁻⁷). We also found strong association with previously reported extended haplotype EH-57.1: HLA-B*57; Cw*06:02; DRB1*07:01; DQB1*03:03:02 in all our patients (Pc = 8.34 × 10⁻⁰⁷). Conclusion: Our results show that different HLA class I and II alleles and haplotypes are associated with psoriasis at different age of onset. In this study, we have reported novel alleles and haplotype association with late-onset psoriasis. Our data confirm the previous strong associations with HLA alleles and haplotypes and also reports novel alleles and haplotype association in Pakistani psoriasis patients.

Association of HLA Alleles and HLA Haplotypes with Psoriasis, Psoriatic Arthritis and Disease Severity in a Miscegenated Population.

BackgroundThe study of HLA classes I and II in Brazilian psoriasis patients may contribute to a better understanding of their association with the disease.ObjectiveTo describe HLA classes I and II of Brazilian patients with psoriasis, with or without arthritis, compare them to controls and correlate HLA markers with epidemiological and evolutional aspects of psoriasis.MethodsA total of 55 patients with more than 5 years of psoriasis, with or without arthritis, answered a questionnaire on ethnic background and disease severity. A total of 134 bone marrow donors were controls. HLA class I and II genotyping was determined by PCR-SSP.ResultsMean age was 42.4 years; 23 women and 32 men. HLA-B*57 was present in 23.6% patients and in 7.5% controls (p=0.00200, OR= 3.8381), and HLA-C*06 in 29.1% patients and in 16.4% controls (p= 0.04832, OR=2.0886). HLA-B*57 and HLA-C*18 were significantly present in patients with arthritis (p=0.00104, OR=6.6769 and p=0.00269, OR=16.50, respectively). HLA-B*57 was significantly present in patients with history of erythroderma (p=0.00548, OR= 5.1059), as was HLA-C*06 (p=0.02158, OR=3.0545). HLA-B*57 was also frequent in patients with history of hospital internment due to psoriasis (p= 0.00094, OR=7.8909) and in the ones with history of systemic treatment for psoriasis (p= 0.00011, OR= 5.3733). Haplotype HLA-A*02 B*57 C*06 DRB1*07DQB1*03 was the most common among the patients (p= 0.00069, OR= 3.528).ConclusionHLA-B*57 and HLA-C*06 were significantly increased in the patients indicating risk for psoriasis. HLA-B*57 remained high in patients with history of erythroderma, hospital internment, systemic treatment, and psoriatic arthritis, showing association with disease severity. HLA-C*18 was significantly high only in patients with psoriatic arthritis. HLA-B*57 and HLA-C*06 and haplotype HLA-A*02B*57Cw*06DRB1*07 DQB1*03 seen in this study were already described before, associated with psoriasis. HLA-Cw*18 was not described in other populations in association with psoriasis.

Immunogenetic characteristics of potential donors of hemapoietic stem cells recruited in the North Caucasus

Aim of the study was to investigate the immunogenetic characteristics of the potential donors of hematopoietic stem cells recruited in the North Caucasus and the distribution features of HLA alleles and multilocus haplotypes. Material and methods . Next Generation Sequencing technology was used to identify HLA-A, -B, -C, -DRB1 and -DQB1 alleles from 2663 unrelated bone marrow volunteers living in the Chechen Republic, Stavropol region, Republic of Dagestan. Mass parallel sequencing was performed using the MiSeq™ System («Illumina Inc.», USA). HLA allele and haplotype frequencies were estimated via maximum-likelihood analysis from genotypic data through an expectationmaximization (EM) algorithm for unknown gametic phase using Arlequin v. 3.5.2.2. Results and discussion . In studied population, 47 HLA-A, 77 HLA-B, 39 HLA-C, 54 HLA-DRB1, 22 HLA-DQB1 alleles were selected. Thirteenth alleles, HLA-A*02:01, HLA-A*01:01, HLA-B*13:02, HLA-B*51:01, HLA-C*06:02, HLA-C*04:01, HLA-C*07:02, HLADRB1*07:01, HLA-DRB1*13:01, HLA-DQB1*03:01, HLA-DQB1*02:02, HLA-DQB1*06:03, DQB1*03:02 exhibit frequencies over 10 %. The highest frequency extended haplotype HLA-A*02:01-B*13:02-C*06:02-DRB1*07:01DQB1*02:02, was observed frequencies of 4,5 %. Routine HLA typing allowed us to define 13 new HLA alleles.

Human Leukocyte Antigen Complex and Other Immunogenetic and Clinical Factors Influence Susceptibility or Protection to SARS-CoV-2 Infection and Severity of the Disease Course. The Sardinian Experience.

AimSARS-CoV-2 infection is a world-wide public health problem. Several aspects of its pathogenesis and the related clinical consequences still need elucidation. In Italy, Sardinia has had very low numbers of infections. Taking advantage of the low genetic polymorphism in the Sardinian population, we analyzed clinical, genetic and immunogenetic factors, with particular attention to HLA class I and II molecules, to evaluate their influence on susceptibility to SARS-CoV-2 infection and the clinical outcome.Method and MaterialsWe recruited 619 healthy Sardinian controls and 182 SARS-CoV-2 patients. Thirty-nine patients required hospital care and 143 were without symptoms, pauci-symptomatic or with mild disease. For all participants, we collected demographic and clinical data and analyzed the HLA allele and haplotype frequencies.ResultsMale sex and older age were more frequent in hospitalized patients, none of whom had been vaccinated during the previous seasonal flu vaccination campaignes. Compared to the group of asymptomatic or pauci-symptomatic patients, hospitalized patients also had a higher frequency of autoimmune diseases and glucose-6-phosphate-dehydrogenase (G6PDH) deficiency. None of these patients carried the beta-thalassemia trait, a relatively common finding in the Sardinian population. The extended haplotype HLA-A*02:05, B*58:01, C*07:01, DRB1*03:01 [OR 0.1 (95% CI 0–0.6), Pc = 0.015] was absent in all 182 patients, while the HLA-C*04:01 allele and the three-loci haplotype HLA-A*30:02, B*14:02, C*08:02 [OR 3.8 (95% CI 1.8–8.1), Pc = 0.025] were more frequently represented in patients than controls. In a comparison between in-patients and home care patients, the HLA-DRB1*08:01 allele was exclusively present in the hospitalized patients [OR > 2.5 (95% CI 2.7–220.6), Pc = 0.024].ConclusionThe data emerging from our study suggest that the extended haplotype HLA-A*02:05, B*58:01, C*07:01, DRB1*03:01 has a protective effect against SARS-CoV-2 infection in the Sardinian population. Genetic factors that resulted to have a negative influence on the disease course were presence of the HLA-DRB1*08:01 allele and G6PDH deficiency, but not the beta-thalassemic trait. Absence of influenza vaccination could be a predisposing factor for more severe disease.

CD45RA, CD8β, and IFNγ Are Potential Immune Biomarkers of Human Cognitive Function.

There is increasing evidence that in humans the adaptive immunological system can influence cognitive functions of the brain. We have undertaken a comprehensive immunological analysis of lymphocyte and monocyte populations as well as of HLA molecules expression in a cohort of elderly volunteers (age range, 64–101) differing in their cognitive status. Hereby, we report on the identification of a novel signature in cognitively impaired elderly characterized by: (1) elevated percentages of CD8+ T effector-memory cells expressing high levels of the CD45RA phosphate receptor (Temra hi); (2) high percentages of CD8+ T cells expressing high levels of the CD8β chain (CD8βhi); (3) augmented production of IFNγ by in vitro activated CD4+ T cells. Noteworthy, CD3+CD8+ Temra hi and CD3+CD8βhi cells were associated with impaired cognition. Cytomegalovirus seroprevalence showed that all volunteers studied but one were CMV positive. Finally, we show that some of these phenotypic and functional features are associated with an increased frequency of the HLA-B8 serotype, which belongs to the ancestral haplotype HLA-A1, Cw7, B8, DR3, DQ2, among cognitively impaired volunteers. To our knowledge, this is the first proof in humans linking the amount of cell surface CD45RA and CD8β chain expressed by CD8+ Temra cells, and the amount of IFNγ produced by in vitro activated CD4+ T cells, with impaired cognitive function in the elderly.

The role of HLA in Balkan endemic nephropathy

Balkan endemic nephropathy (BEN), a progressive chronic tubulointerstitial disease, occurs in the endemic focus of Croatia in a population of about 10,000 inhabitants. One of its most peculiar characteristics is a strong association with upper tract urothelial carcinoma (UTUC). Despite a high number of studies, currently there are insufficient data about the association of BEN and HLA genes. The aim of this study was to investigate the polymorphism of HLA-A, -B, and -DRB1 alleles and haplotypes among BEN patients and to determine whether an association between HLA and BEN exists. In this study, we investigated HLA-A, -B, and -DRB1 alleles and haplotypes in a population of patients with BEN (N=111) and matched healthy controls (N=190). All individuals were tested by PCR-SSO and PCR-SSP methods to assess the possible contribution of HLA alleles and haplotypes to the development of/protection from BEN. Our results showed a positive association between the presence of HLA-B*35:02 and DRB1*04:02 alleles and BEN (P=0.0179 and P=0.0151, respectively) in contrast to the protective effect of HLA-A*01:01, B*27:05 and B*57:01 alleles (P=0.0111, P=0.0330 and P=0.0318, respectively). Moreover, when BEN patients' HLA haplotypes were compared to controls, two haplotypes were associated with BEN susceptibility among Croatians (HLA-A*02:01~B*08:01~DRB1*03:01 and HLA-A*02:01~B*27:02~DRB1*16:01, P=0.0064 and P=0.0023, respectively), while haplotypes HLA-A*02:01~B*27:05~DRB1*01:01 and HLA-A*02:01~B*38:01~DRB1*13:01 each showed a possible protective effect (P=0.0495). Our results point toward genetic susceptibility to BEN and observed differences in both susceptible/protective HLA profiles indicate the necessity of further studies in order to elucidate the pathogenesis of this disease.

Association of Human Leukocyte Antigen Haplotypes With End-Stage Renal Disease in Vietnamese Patients Prior to First Transplantation

IntroductionThe prevalence of chronic kidney failure is significantly increasing in Vietnam, causing a burden for health care. This study assessed the relationship of HLA-A, -B, and –DRB1 alleles with end-stage renal disease (ESRD). MethodA retrospective, cross-sectional study and a comparative study using secondary data analysis were conducted on 196 ESRD patients and 187 controls from 2009 to 2017. The patient and donor profiles were collected from medical records, including age, sex, etiology of renal failure, and HLA phenotypes. HLA-A*, -B*, and -DRB1* typing were done by polymerase chain reaction-sequence specific primers. ResultThe most frequent HLA alleles in Vietnamese patients with ESRD were HLA-A*02, -A*11, -B*15, -B*46, -DRB1*04, -DRB1*09, and -DRB1*12. The haplotypes HLA-A*0233 (odds ratio [OR] = 0.40, 95% CI: 0.15–0.98) had a negative association for ESRD. The haplotypes HLA-B*1515 (OR = 4.14, 95% CI: 1.52–11.26) and HLA-DRB1*1212 (OR = 2.01, 95% CI: 1.06–3.81) had a positive association for ESRD. The haplotypes HLA-B*1515 (OR = 4.69, 95% CI: 1.69–13.03) and -DRB1*1212 (OR = 2.15, 95% CI: 1.10–4.21) had a positive association for ESRD related to glomerulonephritis. The HLA-B*1557 (OR = 17.34, 95% CI: 2.70–11.49) had a positive association for ESRD related to hypertension. ConclusionThe haplotypes of HLA class I and II had significant relationships with ESRD. The results of our study should be confirmed in further investigations.

Human leucocyte antigen but not KIR alleles and haplotypes associated with chronic HCV infection in a Chinese Han population.

The host immune system plays a key role in the elimination of infected cells which depend on killer-cell immunoglobulin-like receptors (KIR), human leucocyte antigen (HLA) class I molecules and their combinations. To evaluate the roles of HLAclass I, KIR genes and their combination in Chronic hepatitis C virus (HCV) infection (CHC), a total of 301 CHCs and 239 controls in a Chinese Han population were included for HLA and KIR genotyping using next-generation sequencing and multiplex PCR sequence-specific priming, respectively. The allele frequency of HLA-C*08:01 was significantly higher in the CHCs than that of the controls (0.088 vs. 0.040, OR=2.332, 95%CI: 1.361-3.996, p=0.022), while the frequencies of B*13:01 (0.032 vs. 0.084, OR=0.357, 95%CI: 0.204-0.625, p=0.009) and C*08:04 (0.008 vs. 0.038, OR=0.214, 95%CI: 0.079-0.581, p=0.022) were significantly lower in the CHCs. The frequencies of haplotype A*11:01-C*08:01 were higher in the CHCs (0.058 vs. 0.019, OR=3.096, 95%CI: 1.486-6.452, p=0.026), while haplotype B*13:01-C*03:04 were lower in the CHCs compared to the controls (0.028 vs. 0.071, OR=0.377, 95%CI: 0.207-0.685, p=0.012). No association of CHC with KIR genes, genotypes, or haplotypes, as well as HLA/KIR combinations was observed. Our results indicated that HLA-C*08:01 was a risk factor for CHC, while HLA-C*08:04 and HLA-B*13:01 were protective factors against CHC. Haplotypes HLA-A*11:01-C*08:01 could increase susceptibility to CHC, while HLA-B*13:01-C*03:04 could be protective against CHC in the Chinese Han population.

HLA typing using next-generation sequencing for Chinese juvenile- and adult-onset myasthenia gravis patients

To compare HLA typing between juvenile- and adult-onset myasthenia gravis (MG), we enrolled 101 children (age ≤12 years) and 168 adults (age ≥20 years) with MG. We excluded patients with histories of thymoma, thyroid disease, or other autoimmune disease. We selected 41 seronegative juvenile-onset patients with ocular symptoms only, and 41 seropositive adult-onset patients with generalized symptoms. We used next-generation sequencing for typing and analysis of HLA genes (Loci: A, B, C, DPA1, DPB1, DQA1, DQB1 and DRB1). Haplotypes HLA-A∗02:07:01-B∗46:01:01-C∗01:02:01-DQA1∗01:01:01-DQB1∗03:03:02-DRB1∗09:01:02, HLA-A∗11:01:01, HLA-A∗24:02:01, and HLA-DPA1∗02:02:02 were found to be related to juvenile-onset MG and HLA-A∗01:01:01, HLA-A∗02:03:01, HLA-C∗03:04:01, and HLA-DQB1∗06:02:01 to adult-onset MG. Therefore, our findings suggested that HLA typing might determine the heterogeneity between AChR-Ab negative juvenile-onset and AChR-Ab positive adult-onset Chinese MG patients.

P073The common uncommon extended HLA haplotype: Do two weak associations make it strong?

Aim Genetic recombination occurs at specific hotspots within the MHC, leading to widely dispersed block-like structures; the alpha block containing HLA-A, beta block (HLA-C and HLA-B) and delta block (HLA-DR and HLA-DQ). Linkage disequilibrium (LD) is much stronger within than across these blocks. The HLA-A locus (most telomeric) has weaker LD with extended HLA haplotypes. Additionally, a recombination hotspot exists between HLA-DP genes and other class II loci, which leads to an HLA-DPB1 match rate of only 10–15% in 10/10 HLA-matched donor/recipient pairs. This report describes an extended 9 locus HLA haplotype found in 2 HCT and 1 kidney transplant recipients and their potential donors (8, 2 and 1 donors, respectively). Methods 1. Case review of 2 unrelated HCT recipients, both European Caucasian (CAU) and their related and unrelated potential donors. 2. Family study of HCT recipient and a sibling to identify the haplotype by segregation. 3. Determine frequency of observed haplotype in different populations through Haplostats.org (6 locus at 2 field resolution could yield higher estimate than the 9 locus 3 field resolution haplotype described here). 4. Searching lab database for incidence of this haplotype. Results All identified cases shared the following haplotype HLA-A*31:01:02∼C*07:02:01∼B*07:02:01∼DRB5*01:01:01∼DRB1*15:01:01∼DQA1*01:02:01∼DQB1*06:02:01∼DPA1*01:03:01∼DPB1*04:01:01. Although most alleles in this haplotype are fairly common, with HLA-A*31:01 least common (∼5%), Haplostats indicated that the haplotype HLA-A*31:01∼C*07:02∼B*07:02∼DRB5*01:01∼DRB1*15:01∼DQB1*06:02 ranks 61898th & 170477th in CAU & African Americans (AFA), respectively and is not seen in other populations. Searching our laboratory database for subjects A31 positive, 382 subjects out of 8340 typed between 2015 & 2017 were identified. Of those, 40 were positive for the haplotype HLA-A*31:01∼C*07:02∼B*07:02∼DRB5*01:01∼DRB1*15:01∼DQB1*06:02 and of these 17 were typed for HLA-DPB1. All were positive for the entire extended 9 locus haplotype and were either CAU (n = 6) or of unknown ethnicity (n = 11). Conclusions Although the 6 locus haplotype portion of this extended haplotype seems fairly uncommon, the extended 9 locus subset of that haplotype seems to be conserved especially in Caucasians.

HLA and killer cell immunoglobulin-like receptor (KIRs) genotyping in patients with acute viral encephalitis.

IntroductionThe HLA genes, as well as the innate immune KIR genes, are considered relevant determinants of viral outcomes but no study, to our knowledge, has evaluated their role in the clinical setting of acute viral encephalitis.ResultsSubjects with acute viral encephalitis in comparison to subjects without acute viral encephalitis showed a significantly higher frequency of 2DL1 KIR gene and AA KIR haplotypes and of HLA-C2 and HLA-A-Bw4 alleles. Subjects without acute viral encephalitis showed a higher frequency of interaction between KIR2DL2 and HLAC1. Multiple logistic regression analysis showed the detrimental effect of HLA-A haplotype and HLA-C1, HLA-A-BW4 HLA-B-BW4T alleles, whereas multiple logistic regression showed a protective effect of AB+BB KIR haplotype and a detrimental effect of interaction between KIR3DL1 and HLA-A-Bw4.DiscussionOur findings of a lower frequency of activating receptors in patients with acute encephalitis compared to controls could result in a less efficient response of NK cells. This finding could represent a possible pathogenetic explanation of susceptibility to acute symptomatic encephalitis in patients with viral infection from potentially responsible viruses such as Herpes virus.Materials and Methods30 Consecutive patients with symptomatic acute viral encephalitis and as controls, 36 consecutive subjects without acute encephalitis were analyzed. The following KIR genes were analyzed, KIR2DL1, 2DL2, 2DL3, 2DL5, 3DL1, 3DL2, 3DL3, 2DL4, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DS1, 2 pseudogenes (2DP1 and 3DP1) and the common variants of KIR2DL5 (KIR2DL5A, KIR2DL5B).

HLA Class I-Mediated HIV-1 Control in Vietnamese Infected with HIV-1 Subtype A/E.

HIV-1-specific cytotoxic T cells (CTLs) play an important role in the control of HIV-1 subtype B or C infection. However, the role of CTLs in HIV-1 subtype A/E infection still remains unclear. Here we investigated the association of HLA class I alleles with clinical outcomes in treatment-naive Vietnamese infected with subtype A/E virus. We found that HLA-C*12:02 was significantly associated with lower plasma viral loads (pVL) and higher CD4 counts and that the HLA-A*29:01-B*07:05-C*15:05 haplotype was significantly associated with higher pVL and lower CD4 counts than those for individuals without these respective genotypes. Nine Pol and three Nef mutations were associated with at least one HLA allele in the HLA-A*29:01-B*07:05-C*15:05 haplotype, with a strong negative correlation between the number of HLA-associated Pol mutations and CD4 count as well as a positive correlation with pVL for individuals with these HLA alleles. The results suggest that the accumulation of mutations selected by CTLs restricted by these HLA alleles affects HIV control.IMPORTANCE Most previous studies on HLA association with disease progression after HIV-1 infection have been performed on cohorts infected with HIV-1 subtypes B and C, whereas few such population-based studies have been reported for cohorts infected with the Asian subtype A/E virus. In this study, we analyzed the association of HLA class I alleles with clinical outcomes for 536 HIV-1 subtype A/E-infected Vietnamese individuals. We found that HLA-C*12:02 is protective, while the HLA haplotype HLA-A*29:01-B*07:05-C*15:05 is deleterious. The individuals with HIV-1 mutations associated with at least one of the HLA alleles in the deleterious HLA haplotype had higher plasma viral loads and lower CD4 counts than those of individuals without the mutations, suggesting that viral adaptation and escape from HLA-mediated immune control occurred. The present study identifies a protective allele and a deleterious haplotype for HIV-1 subtype A/E infection which are different from those identified for cohorts infected with HIV-1 subtypes B and C.

P134 Hot recombinant point between human leukocyte antigen A and C in the Saudi stem cell registry

Aim Recombinant points are frequent in the human genome, where parts of homologous chromosomes are exchanged resulting in recombinant chromosomes. In the literature, several recombinant points have been reported in the HLA region. One of the previously reported common haplotype is HLA-A∗02, C∗06, B∗50, DRB1∗07, DQB1∗02 1 . Saudi families are known to have very high consanguineous marriages 2 . They also have higher than expected homozygosity in the HLA genes 3 . The aim of this study was to determine the most frequent HLA haplotype among the donors of the Saudi Stem Cell Donor Registry ( n = 36670). Methods We analysed our Saudi registry’s HLA typing by listing all homozygous haplotypes. Results Homozygosity of the haplotype HLA-A∗02, C∗06, B∗50, DRB1∗07, DQB1∗02 occurred in only 0.27% of our donors (99/36770). Furthermore, homozygosity of HLA-C∗06, B∗50, DRB1∗07, DQB1∗02 occurred in 1.1% of our donors (395/36770). This was also true for other frequent haplotypes such as HLA-A∗02, C∗07, B∗08, DRB1∗03, DQB1∗02. However, homozygosity of HLA-C∗07, B∗08, DRB1∗03, DQB1∗02, was much more frequent (0.39% or 144/36770), thus indirectly suggesting the presence of a hot recombinant point between HLA-A and HLA-C in the Saudi population. Conclusions The data presented here suggest that hot point between HLA-A and HLA-C may affect the chances of finding full HLA match in the local registry.

Population variation of HLA genes in rural communities in Brazil, the Quilombos from the Vale do Ribeira, São Paulo – Brazil

In the present study, we characterized the allelic and haplotypic profile of the genes HLA-A, -B, -C and -DRB1 (PCR-SBT) in a population sample of 144 highly admixed individuals, coming from rural communities in Brazil (Quilombos from Vale do Ribeira, in the State of São Paulo). Furthermore, we identified three individuals with a new null allele in the HLA-C gene (HLA-C∗02:105N), associated with the haplotype HLA-A∗80: 01∼B∗18: 01:01G∼DRB1∗ 07:01.

Abstract 4270: NGS based microhaplotype counting for ultrasensitive human DNA detection

Abstract Introduction: Fields of forensics, paternity, and hematopoietic stem cell transplantation (HSCT) require human identity testing. The polymorphic nature of short tandem repeats (STRs) makes them most frequently used but they are relatively insensitive. In 2000, we demonstrated proof-of-principle for the use of SNPs for bone marrow engraftment testing; however, due to the high error rate of NGS platforms, use of a single SNP for this purpose is not possible. Here, we hypothesized that using multiple SNPs on a short amplicon (microhaplotype) could overcome the inherently high error rate of NGS, and use it for ultrasensitive detection of human DNA mixes. Methods: As proof-of-principle, we identified a 119 bp region on HLA-A Exon 3 that contains a cluster of 18 SNPs. Using cell lines with known HLA-A haplotypes, we made serial dilutions from 1% to 0.001%. By sequencing each dilution at least twice we were able to determine accuracy and limit of detection. Samples collected from patients after BMT that tested as all donor were analyzed using this approach. Sample preparation and sequencing was done per manufacturer's protocol using the Ion Torrent Personal Genome Machine and 200 base chemistry. Additionally, we analyzed the 1000 Genomes database, using 300 bp windows, and identified many other loci that contained clusters of at least 9 SNPs. Results: Alignment and analysis of common European HLA-A haplotypes of this region show mean number of differences of ∼6 SNPs. Homozygous samples with known HLA-A genotypes that varied by multiple SNPs were analyzed and demonstrated no cross-talk between them. Analyzing serially diluted samples we were able to easily achieve a limit of detection of 1 in 10,000 molecules (0.01%). In BMT samples that tested as all donor by STR analysis, we were able to detect patient DNA of up to 1.5%. From 1000 Genomes data, we identified 4,349 loci that contained at least 9 SNPs within a 300 bp window. Informativity of identified loci was determined for 3 major ethnic groups using 1000 Genomes phased haplotype data. Conclusions: Ultrasensitive detection of human DNA mixes can be achieved using multiple SNPs within a short amplicon despite the high error rates associated with NGS. This assay may be useful to detect early relapse following bone marrow transplantation and other clinical applications. Citation Format: Marija Debeljak, Donald N. Freed, Jane A. Welch, Lisa Haley, Katie Beierl, Brian S. Iglehart, Aparna Pallavajjala, Christopher D. Gocke, Mary S. Leffell, Ming-Tseh Lin, Laura D. Wood, Jonathan Pevsner, Sarah J. Wheelan, James R. Eshleman. NGS based microhaplotype counting for ultrasensitive human DNA detection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4270. doi:10.1158/1538-7445.AM2015-4270

Low occurrence of the HLA-C*04:09N allele in a large Hungarian cohort.

The presence of null alleles may affect the outcome of stem cell transplantation. HLA-C*04:09N was defined as 'common' with a frequency of 2-5/1000 in Caucasians, and its presence is routinely tested as part of haplotypes HLA-A*02:01/A*23:01-B*44:03-DRB1*07:01-DQB1*02:01. We aimed to investigate HLA-C*04:09N in a representative Hungarian cohort. HLA-typing data of 7345 unrelated persons were analyzed. The presence of HLA-C*04:09N was excluded in 157 chromosomes with either serology typing or with an allele-specific polymerase chain reaction for HLA-C*04:09N. HLA-C*04:09N was identified in a single chromosome with HLA-A*02, B*44, C*04, DRB1*07 resulting in a HLA-C*04:09N allele frequency of 0.0068% (1/14,690). This is approximately a 10- to 40-fold lower frequency compared with the previous data. Our results emphasize the need of precise local population-specific HLA-data, allowing appropriate modifications of local HLA-typing protocols.

Quantifying the lower limit of antibody-dependent cell cytotoxicity using a TCRm against a WT1 peptide/MHC epitope (VAC3P.1058)

Abstract The WT1 protein is an ideal cancer antigen, but therapeutically targeting this transcription factor has proven difficult. We have discovered a therapeutic TCR mimic antibody that specifically binds to a WT1-derived peptide (RMFPNAPYL) in the context of the MHC class I haplotype HLA-A2. This high affinity mAb, called ESK, has demonstrated significant anti-cancer activity in animal models of human cancers and leukemias. Studies in vitro and in vivo have shown the dominant role of ADCC in ESK activity. While commercially available mAbs target tens to hundreds of thousands of sites on a cell, ESK binds as few as ~500 epitopes and still promotes cancer cell clearance in vivo. Therefore, we believe ADCC can be a more sensitive mode of killing than currently estimated. To probe this question, we developed a biotinylated RMF peptide that can be pulsed onto HLA-A2 on T2 cells at various concentrations and detected using labeled streptavidin probes. By using 3D confocal microscopy photon scanning, we can detect single molecules of the streptavidin probe on a cell surface. Therefore, using a CFP-DEVD-YFP FRET reporter expressed in the pulsed T2 cells, we can measure the number of peptides bound by ESK in the immunological synapse between a target T2 cell and a human NK cell required to induce T2 cell death. Identifying this number will impact patient selection for ESK clinical trials and will influence the development of therapeutic mAbs against other low-density epitopes.