Sequence-specific Oligonucleotide Research Articles

Association of HLA genotypes with Beta-lactam antibiotic hypersensitivity in children.

Beta-lactam (BL) antibiotics hypersensitivity is common in children. Clinical manifestation of BL hypersensitivity varies from mild to severe cutaneous adverse drug reactions (SCARs). To determine the association of HLA genotype and BL hypersensitivity and the prevalence of true drug allergy in patients with history of BL hypersensitivity. A case-control study was performed in 117 children with aged 1-18 years. Children with history of non-SCARs BL hypersensitivity were evaluated for true drug hypersensitivity including skin test and drug provocation test. Tolerant control patients were children who could tolerate BL for at least 7 days without hypersensitivity reaction. HLA genotype (HLA-A, HLA-B, HLA-C and HLA-DRB1) were performed in 24 cases and 93 tolerant controls using PCR-SSO (polymerase chain reaction - sequence specific oligonucleotide probes). There were association of HLA-C*04:06 (OR = 13.14, 95%CI: 1.3-137.71; p = 0.027), and HLA-C*08:01 (OR = 4.83, 95%CI: 1.93-16.70; p = 0.016) with BL hypersensitivity. HLA-B*48:01 was strongly associated with immediate reaction from BL hypersensitivity (OR = 37.4, 95%CI: 1.69-824.59; p = 0.016) while HLA-C*04:06, HLA-C*08:01 and HLA-DRB1*04:06 were associated with delayed reaction (p < 0.05). Among 71 cases who were newly evaluated for BL hypersensitivity, only 7 cases (9.8%) had true BL hypersensitivity. Less than 10% of children with suspected of BL hypersensitivity have true hypersensitivity. There might be a role of HLA-B, HLA-C and HLA-DRB1 genotype in predicting BL hypersensitivity in Thai children.

An Analysis of Association of Human Leukocyte Antigen and Liver Disease Patients in a Tertiary Care Hospital, India

Background and Objectives: In addition, the role of human leukocyte antigen (HLA) in transplantation, importance of HLA in disease susceptibility, and protection has been widely studied. The aim of the present study was to find out the HLA allelic frequency (HLA-A,-B,-DRB1) and its association with liver diseases. Methods: The present study was done for the duration of 4 years from 2013 to 2017 in a tertiary care center which primarily caters to north Indian patient population. A total of 429 patients were studied. The HLA genotyping was performed by molecular method using commercial available low resolution ABDR sequence-specific oligonucleotide probe-based method (Luminex platform). Results: There was an increased association of HLA-A*01 (n = 9, 18.6%), HLA-B*15, B*35, B*40, B*44 (n = 5, 11.6%), and HLA-DRB1*15 (n = 9, 20.9%) in 43 patients with hepatitis B virus (HBV)-related disease. There was an increased association of HLA-A*24 (n = 9, 20.7%), HLA-B*35, B*40 (n = 14, 15.2%), and HLA-DRB1*15 (n = 17, 18.5%) in 92 patients with alcohol-related disease. An increased association of HLA-A*11 (n = 30, 19.6%), HLA-B*35 (n = 24, 16.8%) and HLA-DRB1*15 (n = 28, 19.6%) in 143 patients with hepatitis C virus (HCV) related disease. Conclusion: This is possibly the first study in the north Indian patients having liver diseases related to HBV, HCV, and alcohol related with respect to A, B, and DR loci previous studies from India were conducted to see disease association of HLA class I and class II with respect to HCV only.

Transplantation of Dystrophin Expressing Chimeric Human Cells of Myoblast/Mesenchymal Stem Cell Origin Improves Function in Duchenne Muscular Dystrophy Model.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in dystrophin gene. Currently, there is no cure for DMD. Cell therapies are challenged by limited engraftment and rejection. Thus, more effective and safer therapeutic approaches are needed for DMD. We previously reported increased dystrophin expression correlating with improved function after transplantation of dystrophin expressing chimeric (DEC) cells of myoblast origin in the mdx mouse models of DMD. This study established new DEC cell line of myoblasts and mesenchymal stem cells (MSC) origin and tested its efficacy and therapeutic potential in mdx/scid mouse model of DMD. Fifteen ex vivo cell fusions of allogenic human myoblast [normal myoblasts (MBN)] and normal human bone marrow-derived MSC (MSCN) from normal donors were performed using polyethylene glycol. Flow cytometry, confocal microscopy, polymerase chain reaction (PCR)-short tandem repeats, polymerase chain reaction-reverse sequence-specific oligonucleotide probe assessed chimeric state of fused MBN/MSCN DEC cells, whereas Comet assay assessed fusion procedure safety testing genotoxicity. Immunofluorescence and real-time PCR assessed dystrophin expression and myogenic differentiation. Mixed lymphocyte reaction (MLR) evaluated DEC's immunogenicity. To test MBN/MSCN DEC efficacy in vivo, gastrocnemius muscle of mdx/scid mice were injected with vehicle (n = 12), nonfused MBN and MSCN (n = 9, 0.25 × 106/each) or MBN/MSCN DEC (n = 9, 0.5 × 106). Animals were evaluated for 90 days using ex vivo and in vivo muscle strength tests. Histology and immunofluorescence staining assessed dystrophin expression, centrally nucleated fibers and scar tissue formation. Post-fusion, MBN/MSCN DEC chimeric state, myogenic differentiation, and dystrophin expression were confirmed. MLR reveled reduced DEC's immune response compared with controls (P < 0.05). At 90 days post-DEC transplant, increase in dystrophin expression (20.26% ± 2.5%, P < 0.05) correlated with improved muscle strength and function in mdx/scid mice. The created human MBN/MSCN DEC cell line introduces novel therapeutic approach combining myogenic and immunomodulatory properties of MB and MSC, and as such may open a universal approach for muscle regeneration in DMD.

Differential Frequencies of HLA-DRB1, DQA1, and DQB1 Alleles and Haplotypes Are Observed in the Arbovirus-Related Neurological Syndromes.

We took advantage of the 2015-2016 Brazilian arbovirus outbreak (Zika [ZIKV]/dengue/chikungunya viruses) associated with neurological complications to type HLA-DRB1/DQA1/DQB1 variants in patients exhibiting neurological complications and in bone marrow donors from the same endemic geographical region. DRB1/DQA1/DQB1 loci were typed using sequence-specific oligonucleotides. In silico studies were performed using X-ray resolved dimer constructions. The DQA1*01, DQA1*05, DQB1*02, or DQB1*06 genotypes/haplotypes and DQA1/DQB1 haplotypes that encode the putative DQA1/DQB1 dimers were overrepresented in the whole group of patients and in patients exhibiting peripheral neurological spectrum disorders (PSD) or encephalitis spectrum disorders (ESD). The DRB1*04, DRB1*13, and DQA1*03 allele groups protected against arbovirus neurological manifestation, being underrepresented in whole group of patients and ESD and PSD groups. Genetic and in silico studies revealed that DQA1/DQB1 dimers (1) were primarily associated with susceptibility to arbovirus infections; (2) can bind to a broad range of ZIKV peptides (235 of 1878 peptides, primarily prM and NS2A); and (3) exhibited hydrophilic and highly positively charged grooves when compared to the DRA1/DRB1 cleft. The protective dimer (DRA1/DRB1*04) bound a limited number of ZIKV peptides (40 of 1878 peptides, primarily prM). Protective haplotypes may recognize arbovirus peptides more specifically than susceptible haplotypes.

Frequency analysis of HLA-B allele in leukemia patients from a North Indian population: A case-control study

BackgroundHLA-B gene is reported to have the highest number of allelic variants among the other HLA class I genes. It is suggested that the HLA-B gene has a particular ability to affect the activity of some immune responses mainly those related to lymphoproliferative neoplasia triggered by intracellular pathogens. Although, several studies have been conducted to study HLA polymorphism and distribution of HLA alleles in different populations and ethnic groups, the scarcity of comparable findings in India encouraged us to examine the possible HLA-B leukemia association in the North Indian population. ObjectiveTo determine the allelic frequencies of HLA-B and their association with leukemia in a North Indian population. Material and methodsA total of 583 patients (case) diagnosed with leukemia as well as 1188 healthy individuals (control) were studied for a period of 4 years (February 2015–March 2019). All these samples (cases and controls) were typed for HLA-B allele by polymerase chain reaction- sequence-specific oligonucleotide probe (PCR-SSOP) method based on Luminex platform. ResultsWe found an increased frequency of HLA-B*07:02 (5.75% versus 4.42%) in cases as compared to the control group. Similarly, frequency of HLA-B*15:01 (2.3% versus 1.38%), B*15:25 (0.43% versus 0.08%), B*18:01 (3.60% versus 2.81%) and B*44:03 (8.75% versus 6.01%) were found to be increased in cases as compared to the controls. These results suggest that HLA-B*15:25 (p = 0.048, OR = 4.49, 95% CI), 38:02 (p = 0.002, OR = 3.97, 95% CI), 44:03 (p = 0.003, OR = 1.48, 95% CI) have a positive association whereas HLA-B*08:01 (p ≤0.001, OR = 0.48, 95% CI) has a negative association with leukemia. ConclusionsOur results provide the first evidence of the HLA-B allele association with leukemia in the North Indian population. Further, it is suggested that HLA-B*15:25, B*38:02, and HLA-B*44:03 alleles may play a presumptive predisposing factor while the HLA-B*08: 01 allele could be a protective genetic factor against leukemia in the North Indian population.

Analysis of HLA-B allele polymorphism in North Indian population: Experience at tertiary care centre

BackgroundThe diverse polymorphism among human leukocyte antigen (HLA) and its association with genetic population structure, transplantation immunology and with different disease pathology has been the objective of several studies. Since, HLA-B shows various polymorphic forms in comparison to others among the HLA class I molecules, therefore, studies correlating to the diversity, occurrence and compositions of HLA allele in a particular population may be a useful tool for transplantation and anthropological studies as well. ObjectiveTo determine and analyze the distribution of HLA-B allele polymorphism in North Indian population and understand its uniqueness and diversity in comparison to other parts of the globe. Material and methodsIn this study, sequence-specific oligonucleotide probe (SSOP) typing of the HLA-B locus along with a comprehensive analysis of its allele polymorphism and distribution of all collected 1188 normal individuals were performed. ResultsIn the studied samples, a total of 60 HLA-B allele variants were identified. HLA-B*15 was having a maximum number of allelic variants (13), followed by HLA-B*35 (5), B*40 (5) and B*27 (4). HLA-B*08:01 (10.39%), B*40:06 (9.46%), B*51:01 (9.38%), 35:03 (7.99%) and B*52:01(6.52%) were found to be the most frequent HLA-B allele. A total of 376 HLA-B allelic combinations (genotypes) were identified. The most frequent genotypic combination identified were HLA*B-08:01–40:06 (1.94%) and B*40:06–51:01(1.94%) followed by HLA-B*08:01–51:01(1.77%) and 08:01–08:01 (1.68%). ConclusionPresent population-based study highlights the diversity of HLA-B allele distribution, polymorphism and its uniqueness when compared to other parts of the globe. The HLA-B diversity among North Indian population exhibits its own characteristic and uniqueness from rest of the world. Our study also demonstrates the importance of studying such types of profiling of HLA genes in detail which will not only help in identification but also in selection of most compatible donor for matched unrelated stem cell transplant in addition to solid organ transplant.

Association of HLA polymorphisms and acetaminophen-related Steven-Johnson syndrome with severe ocular complications in Thai population

Background/aimsTo investigate the association of genetic polymorphisms of human leucocyte antigens (HLA) class I and II genes with acetaminophen-related Steven-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) who developed severe ocular complications...

Human leukocyte antigen associations with acute leukemia: An indian perspective

Abstract Objective: Acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) are neoplastic blood disorders in which the cancerous white blood cells accumulate, resulting in a significant morbidity and mortality. Human leukocyte antigen (HLA) association is observed as one of the factors in the development of leukemia. The objective of the present study was to analyze the allele frequency of HLA Class I (HLA-A, HLA-B, and HLA-C) and Class II (HLA-DRB1 and HLA-DQB1) in Indian acute leukemia patients and to compare them with the frequencies in healthy, unrelated Indian individuals. Materials and Methods: We included 500 Indian leukemic patients (AML = 324 and ALL = 176) and 1000 unrelated, healthy, Indian individuals as controls. The HLA typing was performed using polymerase chain reaction with sequence-specific oligonucleotide probes. Results: On univariate analysis, allele frequencies of HLA-AFN*0111 and HLA-DRB1FN*0111 were lower in patients with ALL (P = 0.0181 and P = 0.0025, respectively). Whereas of HLA-AFN*0111, HLA-DRB1FN*0111, and HLA-BFN*0151, these frequencies were relatively lower in patients with acute leukemia (AML + ALL) (P = 0.0382, P = 0.0093 and P = 0.0384, respectively) and HLA-CFNx0101 (P = 0.0304) in AML when compared with control individuals. In contrast, the HLA-BFN*0139 and HLA-CFN*0107 allele frequency was higher in acute leukemia (P = 0.00372 and P = 0.0463, respectively) and in AML (P = 0.0010 and P = 0.0178, respectively) than that in controls. On multivariate analysis, BFNx0139 showed positive associations with acute leukemia (P = 0.006) and AML (P = 0.002). HLA-AFN*0111 and-DRB1FN*0111 showed a negative association with acute leukemia (P = 0.009 and P &lt; 0.0001, respectively) and ALL (P = 0.013 and P &lt; 0.0001, respectively). Conclusions: The HLA-BFN*0139 has a positive association with AML and acute leukemia, whereas HLA-AFN*0111 and HLA-DRB1FN*0111 alleles have negative association with ALL and HLA-BFN*0151 along with these two alleles with acute leukemia. No positive association was observed with ALL. HLA-CFN*0101 frequency was lower in AML patients than that in controls.

HCP5 rs2395029 is a rapid and inexpensive alternative to HLA-B*57:01 genotyping to predict abacavir hypersensitivity reaction in Spain.

Abacavir (ABC) is an HIV nucleotide-analogue reverse transcriptase inhibitor that can produce a severe hypersensitivity reaction (ABC-HSR) in about 5% of the patients. The HLA-B*57:01 allele is associated with the development of ABC-HSR. Therefore, HLA-B*57:01 genotyping is required prior to the prescription of ABC. The technique routinely used in our laboratory is the sequence-specific oligonucleotide probes (SSOP) reverse hybridization method followed by Sanger sequencing. This technique is time-consuming and expensive. The single-nucleotide polymorphism (SNP) HCP5 rs2395029 was described to be in complete linkage disequilibrium with HLA-B*57:01. In this study, we aimed to assess the linkage disequilibrium between HCP5 rs2395029 and HLA-B*57:01 in patients receiving medical assistance at our hospital. We selected 226 HIV-infected patients from our hospital who had been routinely genotyped since 2009 with the SSOP and Sanger sequencing method: 49 HLA-B*57:01 positives and 177 negatives. We genotyped them for HCP5 rs2395019 by real time PCR (qPCR). We exploratory performed two copy number variation assays flanking HCP5 rs2395019 to explore possible deletions that could break the linkage disequilibrium with HLA-B*57:01. The concordance between HLA-B*57:01 and the HCP5 rs2395029 G allele was absolute, with a specificity and sensitivity of 100% (95% confidence interval: 93.0-100.0% and 98.0-100.0%, respectively) and estimated positive and negative predictive values of 84.4% (48.1-93.9%) and 99.9% (99.4-100.0%), respectively. No deletions were found at HCP5 flanking regions. The duration and cost of the SSOP-based method was considerably higher than the SNP-based method. Therefore, the HCP5 rs2395029 genotyping method may be alternatively used in the clinical practice.

Human Leukocyte Antigen Examination Using Luminex®-Based Assays for Donor-Recipient Compatibility Assessment in Kidney Transplantation: Our Preliminary Experience

Background: Detection of anti-HLA antibodies (HLAabs) in organ transplantation recipients is vital to determine whether the recipient has specific HLAabs against the donor’s HLA molecules (donor-specific anti-HLA antibodies [DSA]). Methods: This preliminary study involved seven subjects: five without prior HLA sensitization and two with a history of possible HLA sensitization (kidney transplantation and pregnancy). Two of the subjects were siblings, two of the subjects were mother and son, and two of them were first cousins once removed. All the subjects underwent HLA typing, and four of the subjects underwent HLAabs assays using the sequence-specific oligonucleotide probes (SSOP) method. Results: There were 10/16 HLA matches between subjects who were siblings and between subjects who were mother and son, and 6/16 HLA matches were found between subjects who were first cousins once removed. Subjects with previous kidney transplantation developed 59 HLA-Abs toward class I and class II HLA (the mean fluorescence intensity (MFI) value range: 1,003.36 to 12,404.77). HLAabs were also found among subjects without prior HLA sensitization, albeit with relatively lower MFI values. Conclusions: HLA typing and HLAabs detection for donor-recipient compatibility assessment using the SSOP method offers more detailed, semi-quantitative results. Further research involving larger subjects in cohort settings will be useful for profiling MFI cut-off values for HLAabs.

Association between human leukocyte antigen gene polymorphisms and multiple EPIYA-C repeats in gastrointestinal disorders

BACKGROUNDPolymorphisms of human leukocyte antigen (HLA) genes are suggested to increase the risk of gastric cancer (GC).AIMTo investigate the HLA allele frequencies of patients with GC relative to a control group in terms of CagA+ multiple (≥ 2) EPIYA-C repeats.METHODSThe patient group comprised 94 patients [44 GC and 50 duodenal ulcer (DU) patients], and the control group comprised 86 individuals [(50 non-ulcer dyspepsia patients and 36 people with asymptomatic Helicobacter pylori (H. pylori)]. Polymerase chain reaction was performed for the amplification of the H. pylori cagA gene and typing of EPIYA motifs. HLA sequence-specific oligonucleotide (SSO) typing was performed using Lifecodes SSO typing kits (HLA-A, HLA-B HLA-C, HLA-DRB1, and HLA-DQA1-B1 kits).RESULTSThe comparison of GC cases in terms of CagA+ multiple (≥ 2) EPIYA-C repeats showed that only the HLA-DQB1*06 allele [odds ratio (OR): 0.37, P = 0.036] was significantly lower, but significance was lost after correction (Pc = 0.1845). The HLA-DQA1*01 allele had a high ratio in GC cases with multiple EPIYA-C repeats, but this was not significant in the univariate analysis. We compared allele frequencies in the DU cases alone and in GC and DU cases together using the same criterion, and none of the HLA alleles were significantly associated with GC or DU. Also, none of the alleles were detected as independent risk factors after the multivariate analysis. On the other hand, in a multivariate logistic regression with no discriminative criterion, HLA-DQA1*01 (OR = 1.848), HLA-DQB1*06 (OR = 1.821) and HLA-A*02 (OR = 1.579) alleles were detected as independent risk factors for GC and DU.CONCLUSIONNone of the HLA alleles were detected as independent risk factors in terms of CagA+ multiple EPIYA-C repeats. However, HLA-DQA1*01, HLA-DQB1*0601, and HLA-A*2 were independent risk factors with no criterion in the multivariate analysis. We suggest that the association of these alleles with gastric malignancies is not specifically related to cagA and multiple EPIYA C repeats.

An internal amino-terminal FLAG-tag octapeptide alters oligomerization of expressed surfactant protein-A

Pulmonary surfactant protein-A (SP-A) is expressed by lung alveolar and bronchiolar epithelial cells and plays a critical role in innate immunity of the lung. Exposure of the lung to various environmental insults alters SP-A homeostasis. To investigate the cellular mechanisms involved in these alterations, we added the FLAG octapeptide (DYKDDDDK) to the carboxy-terminus (SP-A/C-FLAG) or near the amino-terminus (SP-A/N-FLAG) of mouse SP-A (WT-SP-A) to tag specific pools of protein. We hypothesized that addition of FLAG would have negligible effects on SP-A expression, oligomerization and secretion. Analysis of Chinese hamster ovary cells expressing these proteins indicated that tagged SP-A mRNA could be distinguished from WT-SP-A by northern analysis and RT-PCR using sequence-specific oligonucleotides. Tagged SP-A protein could be differentiated from WT-SP-A by western analysis using antibodies specific for the FLAG epitope. Subcellular fractionation and immunocytochemistry indicated the majority of each protein was present in punctuate (presumably endocytic) vesicles, and all forms of SP-A protein were secreted. These results suggest that a FLAG epitope added to the carboxy-terminus or inserted into the amino-terminus of the mature SP-A protein has little effect on its expression and cellular processing. However, disruptions of the amino-terminal end of SP-A prevents proper oligomerization, suggesting that this region of mature SP-A is critical in proper oligomeric assembly and is not useful for studies intended to define mechanisms underlying SP-A homeostasis.

Genomic characterization of MICA gene using multiple next generation sequencing platforms: A validation study.

We have developed a protocol regarding the genomic characterization of the MICA gene by next generation sequencing (NGS). The amplicon includes the full length of the gene and is about 13 kb. A total of 156 samples were included in the study. Ninety‐seven of these samples were previously characterized at MICA by legacy methods (Sanger or sequence specific oligonucleotide) and were used to evaluate the accuracy, precision, specificity, and sensitivity of the assay. An additional 59 DNA samples of unknown ethnicity volunteers from the United States were only genotyped by NGS. Samples were chosen to contain a diverse set of alleles. Our NGS approach included a first round of sequencing on the Illumina MiSeq platform and a second round of sequencing on the MinION platform by Oxford Nanopore Technology (ONT), on selected samples for the purpose of either characterizing new alleles or setting phase among multiple polymorphisms to resolve ambiguities or generate complete sequence for alleles that were only partially reported in the IMGT/HLA database. Complete consensus sequences were generated for every allele sequenced with ONT, extending from the 5′ untranslated region (UTR) to the 3′ UTR of the MICA gene. Thirty‐two MICA sequences were submitted to the IMGT/HLA database including either new alleles or filling up the gaps (exonic, intronic and/or UTRs) of already reported alleles. Some of the challenges associated with the characterization of these samples are discussed.

Frequent transfusion and specific HLA-DRB1 alleles correlate independently with Miltenberger blood group alloimmunization in dialysis patients.

Anti-"Mia " is the most frequent irregular RBC antibody in Taiwan due to high prevalence of Miltenberger antigens. Dialysis patients, a special patient group that needs frequent transfusions of RBCs, may have the greatest risk for developing anti-"Mia " antibodies. The aim of this study was to investigate the association between specific HLA-DRB1 alleles and Mi alloimmunization among dialysis patients. A cohort of 267 maintenance dialysis patients who had ever received at least one RBCs transfusion was enrolled. Anti-"Mia " was identified in patients' serum using the manual polybrene technique and HLA-DRB1 genotyping was carried out using polymerase chain reaction and sequence-specific oligonucleotide probe nonradioactive hybridization. Twenty-one (7.9%) of patients had positive anti-"Mia " tests and had received significantly more units of RBC transfusions than those without anti-"Mia " antibodies (11.3 ± 14.5 U vs 4.5 ± 10.1 U, P = .005). DRB1*04, *07, and *09 alleles were also more prevalent in patients with anti-"Mia " compared to those without Mi III alloimmunization. The multivariate logistic regression analysis showed that the number of RBC transfusions and the presence of DRB1*04, *07, and *09 phenotypes correlated independently with Mi III immunization (Odds ratios [OR] 1.05 (P = .001) for each unit of RBCs transfused; ORs 4.80 (P = .006), 12.29 (P = .005), and 5.42 (P = .003) for presence of DRB1*04, *07, and *09, respectively). This study is the first to demonstrate markedly high prevalence of Mi III alloimmunization in dialysis patients of Taiwan. Extending Mi III matching for RBC transfusions in susceptible dialysis patients may help to reduce the risk of Mi III alloimmunization in this patient population.

HLA-DPB1*03 as Risk Allele and HLA-DPB1*04 as Protective Allele for Both Early- and Adult-Onset Multiple Sclerosis in a Hellenic Cohort.

Background: Human Leucocyte Antigens (HLA) represent the genetic loci most strongly linked to Multiple Sclerosis (MS). Apart from HLA-DR and HLA–DQ, HLA-DP alleles have been previously studied regarding their role in MS pathogenesis, but to a much lesser extent. Our objective was to investigate the risk/resistance influence of HLA-DPB1 alleles in Hellenic patients with early- and adult-onset MS (EOMS/AOMS), and possible associations with the HLA-DRB1*15:01 risk allele. Methods: One hundred MS-patients (28 EOMS, 72 AOMS) fulfilling the McDonald-2010 criteria were enrolled. HLA genotyping was performed with standard low-resolution Sequence-Specific Oligonucleotide techniques. Demographics, clinical and laboratory data were statistically processed using well-defined parametric and nonparametric methods and the SPSSv22.0 software. Results: No significant HLA-DPB1 differences were found between EOMS and AOMS patients for 23 distinct HLA-DPB1 and 12 HLA-DRB1 alleles. The HLA-DPB1*03 allele frequency was found to be significantly increased, and the HLA-DPB1*02 allele frequency significantly decreased, in AOMS patients compared to controls. The HLA-DPB1*04 allele was to be found significantly decreased in AOMS and EOMS patients compared to controls. Conclusions: Our study supports the previously reported risk susceptibility role of the HLA-DPB1*03 allele in AOMS among Caucasians. Additionally, we report for the first time a protective role of the HLA-DPB1*04 allele among Hellenic patients with both EOMS and AOMS.

Association of HLA-DR-DQ alleles, haplotypes, and diplotypes with type 1 diabetes in Saudis.

Type 1 diabetes (T1D) is an autoimmune disease that affects many children worldwide. Genetic factors and environmental triggers play crucial interacting roles in the aetiology. This study aimed to assess the contribution of HLA-DRB1-DQA1-DQB1 alleles, haplotypes, and genotypes to the risk of T1D among Saudis. A total of 222 children with T1D and 342 controls were genotyped for HLA-DRB1, -DQA1, and -DQB1 using reverse sequence-specific oligonucleotide (rSSO) Lab Type high definition (HD) kits. Alleles, haplotypes, and diplotypes were compared between cases and controls using the SAS statistical package. DRB1*03:01-DQA1*05:01-DQB1*02:01 (32.4%; OR = 3.68; Pc < .0001), DRB1*04:05-DQA1*03:02-DQB1*03:02 (6.6%; OR = 6.76; Pc < .0001), DRB1*04:02-DQA1*03:01-DQB1*03:02 (6.0%; OR = 3.10; Pc = .0194), DRB1*04:01-DQA1*03:01-DQB1*03:02 (3.7%; OR = 4.22; Pc = .0335), and DRB1*04:05-DQA1*03:02-DQB1*02:02 (2.7%; OR = 6.31; Pc = .0326) haplotypes were significantly increased in cases compared to controls, whereas DRB1*07:01-DQA1*02:01-DQB1*02:02 (OR = 0.41; Pc = .0001), DRB1*13:01-DQA1*01:03-DQB1*06:03 (OR = 0.05; Pc < .0001), DRB1*15:01-DQA1*01:02-DQB1*06:02 (OR = 0.03; Pc < .0001), and DRB1*11:01-DQA1*05:05-DQB1*03:01 (OR = 0.07; Pc = .0291) were significantly decreased. Homozygous DRB1*03:01-DQA1*05:01-DQB1*02:01 genotypes and combinations of DRB1*03:01-DQA1*05:01-DQB1*02:01 with DRB1*04:05-DQA1*03:02-DQB1*03:02, DRB1*04:02-DQA1*03:01-DQB1*03:02, and DRB1*04:01-DQA1*03:01-DQB1*03:02 were significantly increased in cases than controls. Combinations of DRB1*03:01-DQA1*05:01-DQB1*02:01 with DRB1*07:01-DQA1*02:01-DQB1*02:02 and DRB1*13:02-DQA1*01:02-DQB1*06:04 showed low OR values but did not remain significantly decreased after Bonferroni correction. HLA-DRB1-DQA1-DQB1 alleles, haplotypes, and diplotypes in Saudis with T1D are not markedly different from those observed in Western and Middle-Eastern populations but are quite different than those of East Asians.

The Link between HLA-B Alleles and Causative Drugs in Vietnamese Patients with Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis

BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe cutaneous adverse drug reactions. Human leukocyte antigens (HLA) may play an important role in the pathogenesis of SJS/TEN.&#x0D; AIMS: This study aims to identify HLA-B alleles in Vietnamese patients with SJS/TEN and to investigate the possible link between HLA-B alleles and causative drugs.&#x0D; MATERIALS AND METHODS: Sixty patients including SJS (30 patients) and TEN (30 patients) were enrolled in a cross-sectional descriptive study at two hospitals in Hanoi, Vietnam, from July 2018 to July 2019. Clinical features and laboratory findings were noted, HLA-B alleles were analyzed by the polymerase chain reaction (PCR)-sequence-specific oligonucleotide assay and LuminexTM Multiplex Technology.&#x0D; RESULTS: The most common HLA-B allele was HLA-B*15:02 (41.7%) followed by HLA-B*58:01 (25%) and HLA-B*46:01 (15%). Of the 25 patients possessing HLA-B*15:02 allele, culprit medicines were carbamazepine (13 patients; 52%), traditional medicine (two patients; 8%), and unknown drugs (seven patients; 28%). Of the 15 patients carrying HLA-B*58:01 allele, there were 13 patients whose offending medicine was allopurinol. Of the eight patients whose culprit drug was traditional medicine, there were 6 patients (75%) carrying HLA-B*51:02. Patients who carry HLA-B*15:02 were found to have 4 times higher risk of developing carbamazepine-induced SJS/TEN as compared with the tolerant control group (OR=4.17; 95% CI=2.07–8.37; p &lt; 0.001).&#x0D; CONCLUSION: HLA-B*15:02 was the most common HLA-B allele in Vietnamese patients with SJS/TEN. In traditional medicine-induced SJS/TEN patients, HLA-B*51:02 allele might play an important role. The link between the HLA-B genotypes and causative drugs may suggest physicians to avoid risk medications for certain patients.

Analysis and verification of a HLA-DQB1*03:90N allele with a single base deletion

To verify a HLA-DQB1*03:90N allele and method to improve the accuracy of HLA typing. A total of 2265 hematopoietic stem cell donors from Shenzhen Branch of China Marrow Donor Program in 2018 were initially detected by a PCR sequence-specific oligonucleotide probe (SSOP) method. Among these, a rare HLA-DQB1 allele was identified by sequence-based tying (SBT) and Ion Torrent S5 next generation sequencing (NGS). The SSOP typing result suggested the HLA-DQB1 to be a rare allele, while an insertion and a deletion was suspected in its exon 2 by SBT, which were confirmed by NGS as DQB1*03:90N and DQB1*06:01, respectively. Rare alleles suspected by the SSOP method should be verified by other methods to ensure the accuracy of HLA genotyping. Rare alleles formed by deletions can be detected by NGS with accuracy.

Distinctive germline expression of class I human leukocyte antigen (HLA) alleles and DRB1 heterozygosis predict the outcome of patients with non-small cell lung cancer receiving PD-1/PD-L1 immune checkpoint blockade

BackgroundNivolumab is a human monoclonal antibody against programmed cell death receptor-1 (PD-1) able to rescue quiescent tumor infiltrating cytotoxic T lymphocytes (CTLs) restoring their ability to kill target cells expressing...

Clinical utility of next generation sequencing based HLA typing for disease association and pharmacogenetic testing

HLA associations have been linked to many diseases and are important for risk assessment of drug hypersensitivity reactions. The increasing number of HLA alleles discovered generated a list of ambiguities that cannot be resolved with the current clinical assays, which commonly include sequence-specific oligonucleotide probe (SSOP) genotyping, and real-time PCR with melting curve analysis. HLA typing by next-generation sequencing (NGS) has recently been adopted by clinical laboratories for transplantation testing, as it provides unambiguous and cost-effective HLA typing. The goal of this study was to evaluate the feasibility of using NGS-based HLA-B and DQ genotyping for clinical HLA disease association testing, and provide direct comparison with the currently used clinical tests, including SSOP genotyping, and real-time PCR with melting curve analysis. While the real-time PCR method is easy and inexpensive to perform, ambiguities are rapidly increasing as more and more HLA alleles are discovered. SSOP genotyping identifies the alleles present but limitations include ambiguities and underreporting less common alleles. Our data show that HLA typing by NGS is superior to the existing clinical methods for identifying HLA alleles associated with disease or drug hypersensitivity, and offers a viable approach for high volume clinical diagnostic laboratories.