DQB1 Loci Research Articles

High-resolution allele frequencies for NGS based HLA-A, B, C, DQB1 and DRB1 typing of 23,595 bone marrow donors recruited for the Polish central potential unrelated bone marrow donor registry

Next-generation sequencing (NGS)-based typings of HLA-A, B, C, DQB1 and DRB1 loci were performed from 2018 to 2019 in 23 595 newly recruited or re-typed adult potential bone marrow donors registered in Poltransplant Registry to characterize allele and haplotype frequencies of HLA system for loci important for hematopoietic stem cell transplantation. The donors were recruited for registry and not for any other purpose including controls in a disease association study. The population sample was collected in various regions of Poland including all voivodships. The data regarding the degree of relatedness among individuals in the sample were not collected. Typings were supported by public funds as a part of the Polish National Program for Transplant Medicine Development. HLA frequency data are available in the Allele Frequencies Net Database.

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.

Epitopes as characterized by antibody-verified eplet mismatches determine risk of kidney transplant loss

To optimize strategies that mitigate the risk of graft loss associated with HLA incompatibility, we evaluated whether sequence defined HLA targets (eplets) that result in donor-specific antibodies are associated with transplant outcomes. To define this, we fit multivariable Cox proportional hazard models in a cohort of 118 382 United States first kidney transplant recipients to assess risk of death-censored graft failure by increments of ten antibody-verified eplet mismatches. To verify robustness of our findings, we conducted sensitivity analysis in this United States cohort and assessed the role of antibody-verified eplet mismatches as autonomous predictors of transplant glomerulopathy in an independent Canadian cohort. Antibody-verified eplet mismatches were found to be independent predictors of death-censored graft failure with hazard ratios of 1.231 [95% confidence interval 1.195, 1. 268], 1.268 [1.231, 1.305] and 1.411 [1.331, 1.495] for Class I (HLA-A, B, and C), -DRB1 and -DQB1 loci, respectively. To address linkage disequilibrium between HLA-DRB1 and -DQB1, we fit models in a subcohort without HLA-DQB1 eplet mismatches and found hazard ratios for death-censored graft failure of 1.384 [1.293, 1.480] for each additional antibody-verified HLA-DRB1 eplet mismatch. In a subcohort without HLA-DRB1 mismatches, the hazard ratio was 1.384 [1.072, 1.791] for each additional HLA-DQB1 mismatch. In the Canadian cohort, antibody-verified eplet mismatches were independent predictors of transplant glomerulopathy with hazard ratios of 5.511 [1.442, 21.080] for HLA-DRB1 and 3.640 [1.574, 8.416] for -DRB1/3/4/5. Thus, donor-recipient matching for specific HLA eplets appears to be a feasible and clinically justifiable strategy to mitigate risk of graft loss.

Features of distribution HLAalleles and haplotypes in Kalmyks.

Conducted high-resolution HLA-typing loci HLA-A, -B, -C, -DRB1 and -DQB1 by massively parallel sequencing of 150 potential donors of hematopoietic stem cells from the Republic of Kalmykia. In the studied population, four new alleles identified that not previously registered by the International Committee on the Nomenclature of Factors of the HLA-system of WHO. During the HLA-typing identified: 29 alleles at the HLA-A locus, 44 - at the HLA-B locus, 26 - at the HLA-C locus, 15 - at the DQB1 locus, 37 - at the HLA-DRB1 locus. The following alleles have a frequency of more than 10%: HLA-A*02:01 (11,7%), HLA-A*01:01 (11%), HLA-B*51:01 (10,3%), HLA-B*58:01 (10,3%), HLA-C*06:02 (17,7%), HLA-C*03:04 (10,3%), HLA-C*03:02 (10%), HLA-DQB1*03:01 (26,7%), HLA-DQB1*02:02 (10%), HLA-DRB1*07:01 (11,7%). The most common HLA-A-B-C-DQB1-DRB1 haplotype is A*02:05-B*50:01-C*06:02-DQB1*02:02-DRB1*07:01 (3,7%). Deviations from the Hardy - Weinberg equilibrium not identified.

Evaluating novel effective primers to amplify heterozygous alleles of second, third and fourth exons of HLA-A; -B; -C; -DRB1 and -DQB1 loci using sequencing-based typing

Human leukocyte antigen (HLA) typing is one of the most crucial steps that determines the success of an organ transplant. However, HLA typing is a challenging task due to the diversity of HLA alleles, which is caused by high polymorphism of the region and high number of guanine and cytosine bases that limits the degree of amplification. Low resolution serology typing currently employed in Sri Lanka may fail to identify subtle differences in certain alleles, which may affect the long-term survival of the organ recipient. Therefore, a low cost, high-resolution DNA-based typing method for the HLA loci of Sri Lankans was developed based on polymerase chain reaction (PCR) amplification followed by Sanger sequencing, which is considered to be the gold standard for HLA typing. With minimised PCR bias and equal chances of amplifying all the alleles curated so far, a novel set of primers were designed to amplify the second and third exons of alleles in group specific PCR. To increase the resolution of alleles further, the fourth exon was also amplified using novel primers designed in this study and primers reported in the literature. Touchdown PCR and hot-start PCR were used to optimise PCR conditions so that non-specific amplifications are minimal. SBTengine® (version 3.12.0.2724) software was used in assigning the sequence chromatogram to the allele sequence. Seventeen new primers were designed in this study to ensure the amplification and identification of both alleles in heterozygous individuals that were previously unable to be identified using primers reported in the literature.

HLA-A, -B, -C, -DRB1 and -DQB1 alleles and haplotypes in 194 Southeast Asia Chinese from Peninsular Malaysia

A total of 194 Southeast Asia Chinese from Peninsular Malaysia were genotyped for HLA-A, -B, -C -DRB1, and -DQB1 loci using polymerase chain reaction sequence-specific oligonucleotide probe hybridization methods. In this report, the HLA-B, HLA-DRB1 and HLA-DQB1 were in Hardy-Weinberg proportions (HWEP) (p > 0.05). We observed significant deviation from HWEP in HLA-A (p < 0.05) and HLA-C (p < 0.01) loci. This genotype data was available in Allele Frequencies Network Database (AFND) Dos Santos et al. (2016).

A single center study of protective and susceptible HLA alleles and haplotypes with end-stage renal disease in China

Chronic kidney disease (CKD) is becoming a global public health problem and usually cause End-Stage Renal Disease (ESRD) in the end of progression. To analyze the associations of HLA-A, -B, -C, -DRB1 and -DQB1 alleles at high resolution with ESRD in Jiangsu province of China, a total of 499 unrelated patients with ESRD from the First Affiliated Hospital with Nanjing Medical University and 1584 healthy controls from Jiangsu Branch of Chinese Marrow Donor Program (CMDP) were genotyped at HLA-A, -B, -C, -DRB1 and -DQB1 loci. Statistical analysis was applied to compare the differences of HLA allele frequencies between patients with ESRD and healthy controls. As results, no protective allele at A locus was found and the susceptible alleles were A*11:01 and A*31:01. At B locus, B*15:01, B*55:02 and B*39:05 emerged as susceptible alleles, whereas no protective allele was found. At C locus, C*06:02 and C*07:01 emerged as protective alleles and no susceptible allele was found. At DRB1 locus, six alleles including DRB1*03:01, DRB1*04:03, DRB1*04:04, DRB1*04:05, DRB1*11:01 and DRB1*12:02 emerged as susceptible alleles, while DRB1*15:01 emerged as a protective allele. At DQB1 locus, DQB1*02:01, DQB1*03:01, DQB1*03:02 and DQB1*04:01 emerged as susceptible alleles, while DQB1*06:02 and DQB1*06:09 emerged as protective alleles. Haplotype A*11:01-C*03:03-B*15:01-DRB1*11:01-DQB1*03:01 containing four susceptible alleles was regarded as the most susceptible haplotype. The susceptible alleles and haplotypes might be used as some important risk classification markers. Besides, in the consanguineous renal transplantation, it would be very beneficial for the long-term survival of renal transplant patients to avoid the susceptible alleles and haplotypes in selecting optimal donors.

Using next‐generation sequencing for characterising HLA‐DRB1 and DQB1 loci in a cohort of Colombian women

The Colombian population is characterised by a high genetic diversity, secondary to the ethnic mixture arising from colonisation. Unfortunately, few reports are available regarding HLA-DRB1 and DQB1 diversity in Colombia to date. HLA-DRB1 and DQB1 diversity was identified in this study using next-generating sequencing (NGS) on a cohort of Colombian women. Cervical samples taken from 276 women were used for typing DRB1 and DQB1 loci by Illumina MiSeq. Allele and haplotype frequencies were calculated using an expectation-maximisation algorithm. Hardy-Weinberg Equilibrium and linkage disequilibrium (LD) between loci were evaluated. Forty-seven DRB1 alleles and 14 DQB1 alleles were identified. DRB1*04:07:01G and DQB1*03:02:01G alleles occurred most frequently in the target population. Significant LD was found in 44 out of the 144 identified haplotypes, within which DRB1*04:07:01G-DQB1*03:02:01G occurred most frequently (6.56%). The alleles and haplotypes found with NGS agreed with that found in previous reports involving lower resolution for the Colombian population, and greater genetic variability was found, especially concerning DRB1. Comparing allele and haplotype frequency distribution in the target population to that of other populations denoted HLA system intra- and inter-population diversity.

Assessment by Extended-Coverage Next-Generation Sequencing Typing of DPA1 and DPB1 Mismatches in Siblings Matching at HLA-A, -B, -C, -DRB1, and -DQ Loci.

Allogeneic hematopoietic stem cell transplant from an HLA matched sibling donor is usually the preferable choice. The use of next-generation sequencing (NGS) for HLA typing in clinical practice provides broader coverage and higher resolution of HLA genes. We evaluated the frequency of DPB1 crossing-over events among patients and potential related donors typed with NGS. From July 2016 to January 2018, 593 patients and 2385 siblings were typed. We evaluated sibling matching status in 546 patients, and 44.8% of these patients had siblings that matched at HLA-A, -B, -C, -DRB1, and -DQB1 loci. In 306 patient-HLA matched sibling pairs, we found 6 pairs (1.96%) with 1 DPB1 mismatch, and 5 of these pairs included an additional mismatch in DPA1. No additional mismatches were observed at the low expression loci. Using the T cell epitope algorithm, 4 of these DP mismatches were classified as permissive, 1 as nonpermissive in the host-versus-graft direction, and 1 as nonpermissive in the graft-versus-host direction. The frequency of DPB1 and DPA1 mismatches is low, and their impact in related donor transplants is not well established. Although DP typing in related transplants goes beyond guidelines, it is especially relevant for sensitized patients. NGS-based HLA typing provides full gene coverage, and its use in clinical practice can enable better donor selection.

A Pilot Study on Markers of Genetic Predisposition in Tuberculous Pneumonia Patients In Latgale

Abstract Tuberculosis (TB) is still one of the top ten leading causes of death in the world. Compared to other Baltic and Eastern European countries, TB incidence (24.8 new cases per 100 000 people in 2017) in Latvia is relatively high. One of the regions with the highest TB incidence is Latgale (31.1 cases per 100 000 people). The aim of this pilot study was to identify markers of genetic predisposition to TB in Latgale. The study included 26 patients (16 males and 10 females) aged between 18 and 85 with bilateral TB pneumonia and without HIV infection. HLA typing was performed in HLA-DRB1, -DQA1, and -DQB1 loci by a polymerase chain reaction with low resolution sequence-specific primers. HLA-DRB1*07 and HLA-DRB1*11 alleles were identified as risk alleles for TB. HLA-DRB1*15 allele was a protective allele. Due to the limitations of this exploratory study, a broader study needs to be conducted to revealing specific risk and protective HLA Class II alleles for TB in the subpopulation of Latgale.

Genetic affinity of Muslim population in South India based on HLA-DQB1 and relationship with other Indian Populations

The present study made an attempt to observe genetic affinity of the Muslim population in South India with other neighbor populations. In this regard, DQB1 loci of HLA class II gene as a common genetic marker in phylogenetic assessment has been examined in 45 unrelated healthy individuals using sequence-based typing. The result of this study indicates a close genetic similarity among Indian sub-populations, in spite of segregation with other Muslim populations in North India. Although results of present study indicates genetic relationship of selected populations, all HLA loci or at least all loci of each classes to be assessed in order to attain highly probability of estimates.Keywords: South Indian Muslims, Anthropology, HLA

HLA DRB1 and HLA DQB1 Alleles in Bulgarian Patients with Primary and Secondary Antiphospholipid Syndrome

Abstract Antiphospholipid syndrome (APS) is an autoimmune disease with multifactorial and polygenic pathogenesis. Recently, the genetic predisposition in APS has been subjected to wide discussion. The aim of this study is to determine the prevalence of DRB1 and DQB1 loci in Bulgarian population of healthy persons and patients with primary (PAPS) and secondary (SAPS) APS. Patients are divided in 5 groups: I-29 patents with systemic lupus erythematosus (SLE) with SAPS, II-35 patients with PAPS, III-32 women with spontaneous abortions without aPL, IV-15 patients with different thrombosis (deep venous thromboses, pulmonary embolism, mesenterial thrombosis, myocardial infarction, stroke) without laboratory data for APS, and V-16 SLE patients without clinical and laboratory data for APS. SAPS patients have more frequently DRB1*03 and DQB1*02 and more rarely DRB1*11 and DQB1*03 in comparison with healthy subjects and patients with PAPS. Patents with PAPS, those with spontaneous abortions and patients with thrombotic events but without antiphospholipid antibodies (aPL) have DRB1*03, DRB1*11, DQB1*02 and DQB1*03 alleles similar to the general population. There are no differences between group I (SLE+APS) and group V (SLE) in DRB1* and DQB1*alleles.

The Impact of HLA-DPB1 Mismatch in T-Cell Replete Unrelated Donor Allogeneic Stem Cell Transplantation

High-resolution matching of donor-recipient HLA improves outcome in allogeneic stem cell transplants. Matching for HLA-A, -B, -C, –DRB1 and –DQ is mandatory in our transplant centre, to identify 10/10 or 9/10 matched unrelated donors. High resolution matching for DPB1 has been added over the last 10-15 years. However, the role of DPB1 matching is not yet clearly defined. In this study, we retrospectively analyzed the impact of HLA-DPB1 matching on the outcome of T cell replete allogeneic hematopoietic stem cell transplants with Cya/Mtx- and without ATG as GVHD prophylaxis in patients with hematological malignancies at Oslo University Hospital between 2005 and 2017. 301 patients with an unrelated donor fully matched (10/10) at HLA-A, -B, -C, -DRB1, and –DQB1 loci were included. Further, 87 patient-recipient pairs were also fully matched on DPB1 (12/12); 118 had permissive and 96 had non-permissive mismatches of one or two DPB1 alleles. The three groups were comparable with respect to diagnosis, gender, age, cytomegalovirus serostatus and conditioning regimen (Table 1). Cumulative incidence of relapse at 5 years were significantly higher in the DPB1 matched pairs compared with the permissive and non-permissive mismatched ones, at 40% vs 22% and 13% (p Figure 1-2 ). No difference in frequency of acute GVHD grade II-IV between the three groups were found; DP match 43%, permissive mismatch 40% and non-permissive mismatched 48% (p=0.49). Neither was there a difference seen in GVHD grade III-IV; 12% versus 17% versus 11%, respectively. Finally, there were similar outcomes between the three groups regarding chronic GVHD and TRM (Figure 3). In corrected multivariate analyses, only DP matching had significant influence on mortality and survival. Our results show a favorable relapse free and overall survival following a MUD allotransplant with a DBP1 permissive or non-permissive mismatched donor compared to a fully DPB1 matched. This is likely due to an increased GvL-effect in the DPB1 mismatched groups without the counterbalance of increased acute GVHD and TRM.

The polymorphisms of HLA class Ⅰ and Ⅱalleles were not associated with severe acute respiratory syndrome among recovered patients in Beijing: a case-control study

Severe acute respiratory syndrome (SARS) is a viral respiratory illness caused by a novel coronavirus (SARS-CoV), which emerged as a pandemic in 2003. The mechanism of the immune reaction initiated by SARS-CoV still remains unclear. Here we aimed to describe the genetic patterns of high-resolution HLA-A, -B, -C, -DRB1, and -DQB1, loci in recovered SARS patients from Beijing and examine the association between HLA genes and susceptibility or resistance to SARS. A total of 70 recovered Chinese Han SARS patients were recruited to donate convalescent plasma in 2003. HLA high-resolution typing was carried out using sequence based typing (SBT). Allele frequencies were calculated by direct counting, and were compared with the frequencies of HLA alleles of donors recruited by the China Marrow Donor Program between 2002 and 2015 using Fisher's exact test. Significance of association was defined according to the Bonferroni method for multiple comparisons. We observed 20, 35, 21, 25, and 17 alleles respectively at HLA-A, -B, -C, -DRB1, and -DQB1 loci among the 70 recovered patients. We identified 12 alleles (HLA-A*02:10, -A*02:93, -A*03:02, -B*08:01, -B*15:152, -B*37:01, -DRB1*10:01, -DRB1*11:03, -DRB1*14:10, -DRB1*14:12, -DRB1*15:02, and -DQB1*05:10) showing a nominal association with SARS (P<0.05), but none remained significant after Bonferroni correction. The study suggests that high-resolution HLA alleles are unlikely to contribute significantly to the susceptibility or resistance to SARS-CoV infection in the northern Chinese population.

Analysis on polymorphism of high-resolution HLA-A, -B, -C, -DRB1 and -DQB1 in hematopoietic stem cells donors of Chinese Han population from Southern China

Objective To explore the human leukocyte antigen (HLA)-A, -B, -C, -DRB1 and -DQB1 high-resolution alleles polymorphism in hematopoietic stem cells donors of Chinese Han population from Southern China. Methods A total of 4 262 cases hematopoietic stem cell donors of chinese Han population in Shenzhen China Marrow Donor Program (CMDP) from January 2016 to December 2017 were selected as subjects. HLA-A, -B, -C, -DRB1 and -DQB1 loci for high resolution typing of these 4 262 cases hematopoietic stem cell donors were detected by PCR-sequence-specific oligonucleotide probes (SSO) and next-generation sequencing (NGS) analysis. The frequencies of common, well-documented and rare alleles were studied by direct counting method. The χ2 test was used to determine whether the distribution of HLA-A, -B, -C, -DRB1 and -DQB1 alleles of these 4 262 cases hematopoietic stem cell donors accorded with the Hardy-Weinberg equilibrium. Results ① Compared observed values of HLA-A, -B, -C, -DRB1 and -DQB1 alleles in these 4 262 cases hematopoietic stem cell donors of Chinese Han population from Southern China with the expected values, there were not statistically significance (χ2=295.068, 572.482, 355.205, 362.584, 312.150; P>0.05), which indicated that the distribution of HLA-A, -B, -C, -DRB1 and -DQB1 alleles of all hematopoietic stem cell donors in this study conformed with Hardy-Weinberg equilibrium. ② The alleles of HLA-A, -B, -C, -DRB1 and -DQB1 loci in 4 262 cases hematopoietic stem cells donors of Chinese Han population from Southern China were 49, 97, 37, 48 and 22, respectively. Among them, the common alleles with frequencies higher than 0.05 included HLA-A*11∶01, 24∶02, 02∶07, 02∶01, 33∶03 and 02∶03; HLA-B*40∶01, 46∶01, 58∶01, 13∶01 and 51∶01; HLA-C*01∶02, 07∶02, 03∶04, 08∶01, 03∶02, 06∶02 and 03∶03; HLA-DRB1*09∶01, 15∶01, 12∶02, 07∶01, 08∶03, 03∶01, 11∶01 and 04∶05; and HLA-DQB1*03∶01, 03∶03, 06∶01, 05∶02, 03∶02, 02∶01, 05∶03, 06∶02 and 02∶02. For each of the locus, the cumulative allele frequencies of common alleles were 0.767 1, 0.471 6, 0.782 5, 0.656 4 and 0.877 6, respectively. ③ The well-documented and rare alleles of HLA-A, -B, -C, -DRB1 and -DQB1 loci were detected as 22, 42, 14, 11 and 7. Among them, the rare alleles of HLA-A*29∶03; HLA-B*18∶04, 15∶296 and 54∶03; HLA-C*12∶12; HLA-DRB1*04∶101 and HLA-DQB1*02∶10 were not included in the Common Alleles and Well-Documented Alleles (CWD) v2.3 of CMDP. Conclusions Relatively complete high-resolution allelic distribution data of HLA-A, -B, -C -DRB1 and -DQB1 loci in hematopoietic stem cells donors of Chinese Han population from Southern China were obtained in this study. And HLA well-documented and rare alleles were recorded. The results of this study contribute to the accumulation on population distribution and polymorphism data of HLA gene in hematopoietic stem cell donors, and provide reference for improving CWD of CMDP. Key words: HLA antigens; Alleles; Gene frequency; Polymerase chain reaction; High resolution genotyping; Common alleles; Well-documented alleles; Rare alleles; Polymerase chain reaction-sequence-specific oligonudeotide; Next-generation sequencing

HLA Polymorphism Affects Risk of de novo Mutation of dystrophin Gene and Clinical Severity of Duchenne Muscular Dystrophy in a Southern Chinese Population.

Immune-mediated pathology has been thought to be an important factor contributing to Duchenne muscular dystrophy (DMD). Allele frequencies of certain HLA types are known to differ between patients with dystrophinopathies and healthy controls with low-resolution HLA gene typing data in limit reports. Using Polymerase chain reactionsequence based typing (PCR-SBT) to genotype 64 children with DMD in HLA-A, -B,-C, -DRB1, and -DQB1 locus and 503 healthy controls in HLA-A, -B, -DRB1 locus, this study aimed to investigate associations of specific HLA alleles with, and their possible roles in the development and clinical phenotypic severity of DMD. The χ2 test was used to evaluate the distribution of allele frequencies in HLA-A, -B, -DRB1 locus between the patients and healthy controls. A significantly higher frequency of HLA-B*07:05 was found in children with DMD compared to that in controls (OR = 16.2, 95%CI = 2.9–89.3, P < 0.046). More importantly, significantly higher frequencies of HLA-A*29:01 (OR = 77.308, 95%CI = 6.794–879.731, P < 0.0160) and HLA-B*07:05 (OR = 60.240, 95%CI = 9.637–376.535, P < 2.41*10−3) was found in patients with de novo mutations (n = 14) compared to controls while no difference of HLA alleles frequency ware indicated between patients with inherited mutation and control. The result indicates that HLA alleles is associated with pathogenesis of DMD especially DMD with de novo mutation. We use Vignos scale to estimate the lower limb motor function of patients. The impact of HLA alleles on score of Vignos scale of DMD children was estimated by multiple linear regression. Our study indicates that HLA-A*02:01 may have a dampening effect on the clinical phenotypic severity of DMD, evidenced by the presence of HLA-A*02:01 being associated with lower Vignos score. Our study demonstrates that certain HLA alleles are indeed associated with the pathogenesis and clinical phenotypic severity of DMD.

HLA common and well-documented alleles in China.

The American Society for Histocompatibility and Immunogenetics HLA common and well-documented (CWD) catalog, CWD 2.0.0 catalog and European Federation for Immunogenetics (EFI) CWD catalog have been published, which are useful for improving the accuracy of HLA genotyping in laboratories. Here, we studied the Chinese HLA CWD catalog. A total of 812 211 unrelated volunteer donors from the China Marrow Donor Program (CMDP) were analyzed. Six hundred seventy-six alleles at the HLA-A, -B, -C, -DRB1, and -DQB1 loci were defined as CWD alleles in the Chinese population, including 159 common and 517 well-documented alleles. The distribution of HLA alleles in the Chinese CWD catalog is different from that in the EFI CWD catalog. Thirty-two percent (215/676) of CWD alleles in the Chinese CWD catalog are shared with those in the EFI CWD catalog. Fifty-six percent (380/676) of alleles in the Chinese CWD catalog are not found in the EFI CWD catalog, while 655 alleles in the EFI CWD catalog are neither common nor well-documented alleles in the Chinese CWD catalog. The Chinese CWD catalog described in this study may help to improve high-resolution histocompatibility testing for CMDP-accredited laboratories in China. However, to accommodate an increasing number of HLA alleles, this Chinese CWD catalog should be regularly updated.

Analysis of allelic drop-out at autosomal short tandem repeat loci in composite amplification

Objective To investigate the characteristics and causes of allelic drop-out at the autosomal short tandem repeat (STR) loci, and to explore the confirmation strategy of suspected mutations at the STR loci alleles in paternity. Methods From January to June 2017, nine family members from 3 families of three-banded patterns at Shenzhen Blood Center were selected as subjects. Subject inclusion criteria: subjects′ initial test results of the STR loci alleles did not conform to the Mendels laws of genetics, which were suspected allelic drop-out at STR loci. GlobalFiler™ Express kits from ABI, USA were initially used to detect genotype of 21 autosomal STR loci alleles from all blood spot samples. All blood spot samples suspected for having allelic drop-out were verified with the PowerPlex® 21 kits from Promega, USA. Supplemental experiment was used human leukocyte antigen (HLA)-A/-B/-C/-DR/-DQB1 loci genotype test results with PCR-sequence-based typing (SBT) method, and then the haplotype of the subjects was deduced by the family analysis to complete the individual identification verification. This study met the requirements of World Medical Association Declaration of Helsinki revised in 2013. Informed consent was obtained from every subject. Results ① In this study, the allelic drop-out in 6 subjects occurred in 3 STR loci of CSF1PO, D1S165 and TH01, respectively, and the number of mutation steps were different. These allelic drop-out could be either paternal or maternal. ② In family 1, the initial test result of CSF1PO locus was homozygous in both mother and child. The verification test result of CSF1PO locus was heterozygous, and allelic drop 10-locus were confirmed. ③ In family 2, the initial test result of D1S165 locus was homozygous in both child and father. The verification test result of D1S165 locus was heterozygous, and allelic drop-out 16-locus was confirmed. ④ In family 3, the initial test result of TH01 locus was homozygous in both the mother and child. The verification test result of TH01 locus was heterozygous, and allelic drop-out 9-locus was confirmed. ⑤ Twelve HLA-A/-B/-C/-DRB1/-DQB1 loci haplotypes were detected in the DNA samples of 9 subjects, and the HLA locus haplotypes were detected in accordance with Mendels laws of genetics. Conclusions For the suspected STR allelic mutations in STR composite amplification, a single test has the possibility of allelic drop-out risk. Different kits could be used to verify the authenticity of allele mutations, and more reliable results could be obtained. Key words: Loss of heterozygosity; Alleles; Tandem repeat sequences; HLA antigens; Paternity

P001 Validation of ALLType™ next-generation sequencing in Brazilian samples

Aim Validation of HLA typing by next-generation sequencing (NGS) demands evidence of the assay’s accuracy compared to Sanger sequencing. We evaluated the AllType™ NGS (One Lambda) and TypeStreamVisual™NGS analysis software (One Lambda) for accuracy on HLA genotyping. Methods 48 Brazilian DNA samples selected for allelic diversity were sequenced for HLA class I and class II genes following AllType™ NGS protocol using automation of the Ion Chef and S5 sequencing system (One Lambda). Sequencing data was analyzed by TypeStreamVisual™ software 1.0.0 with IMGTHLA database 3.27.0.8. All samples were previously typed for HLA-A, B, C, DRB1, DQB1 and DPB1 by SeCore Sequence-Based Typing kits (One Lambda) using uTYPE HLA sequence analysis software (One Lambda).Evaluation was done comparing NGS results with previously assigned by Sanger’s method. Results AllTypeTM NGS and SeCore results were 100% concordant for HLA- A, B, C, DRB1, DQB1 and DPB1 loci. Following ambiguities were detected for class I and class II:B*15:01:01 B*15:03:01/B*15:39:01 B*15:54; B*07:02:01 B*08:01:01/B*08:156 B*42:01:01; DRB1*12:01:01 DRB1*13:02:01/DRB1*12:10 DRB1*13:02:01; DQB1*06:03:01 DQB1*06:09:01/DQB1*06:41 DQB1*06:88; DPB1*04:01:01 DPB1*04:02:01/DPB1*105:01 DPB1*126:01 Allelic imbalance cases occurred most in DQB1 locus, all involving DQB1*02. All null alleles required identification by the National Marrow Donor Program could be ruled out as potential results. Reproducibility was validated by 3 samples tested 3 times in the same assay run and 4 samples tested in 3 different runs. 20 blind samples sequenced also by NGS were tested and approved up to 3-field result for all loci. Conclusions We concluded that AllType™ NGS is reliable to be introduced in clinical routine for HLA-A, B, C, DRB1, DQB1 and DPB1 loci. Although HLA-B ambiguities needed additional tests, there was enormous reduction in the number of ambiguities detected by AllType™ providing highest cost benefit than Sanger sequencing. Download : Download high-res image (548KB) Download : Download full-size image

P020 Identification of a new C*06 null allele by SBT typing in an arabic family

A 12 y/o female from Saudi Arabia with chronic granulomatous disease was evaluated at Duke for HSCT. Patient and sibling typing were performed by SBT method (Protrans, Germany) which separates alleles by group-specific PCR before sequencing. The patient was identical to her sibling at HLA-A, B, DRB1, DRB3/4/5 and DQB1 loci. However, the analysis software could not assign alleles at HLA-C locus for both the patient and sibling due to the presence of double peaks in exon 3 electropherogram. For both the patient and the sibling, exon 2 and exon 4 sequences showed no polymorphism. Sequence with forward primer of exon 3 was good 1–224 nt in exon 3 but became double peaks after 224. Reverse primer sequence of exon 3, however, had double peaks 1–224 nt then good after 224. Analyzing the results by ignoring the double peak sequences showed a perfect match to C*06:02 in the database. This indicated there were C*06:02 and another variant of C*06:02 with a G deletion at 224 of exon 3. This corresponds to nt 568 in cDNA which changes codon 166 from GAG to.AG and is predicted to be a null allele due to shift of open reading frame. In order to investigate whether the new variant was inherited from the family, we also obtained the samples from the parents. It turned out the C*06 new null allele was carried by the father. The sequence was submitted to GenBank (accession number MH031344). The patient’s sibling was identified to be the 10/10 matched donor and HSCT was performed in 2018 successfully.