Sequence-specific Oligonucleotide Research Articles

Fast and Economic Microarray-Based Detection of Species-, Resistance-, and Virulence-Associated Genes in Clinical Strains of Vancomycin-Resistant Enterococci (VRE)

Today, there is a continuous worldwide battle against antimicrobial resistance (AMR) and that includes vancomycin-resistant enterococci (VRE). Methods that can adequately and quickly detect transmission chains in outbreaks are needed to trace and manage this problem fast and cost-effectively. In this study, DNA-microarray-based technology was developed for this purpose. It commenced with the bioinformatic design of specific oligonucleotide sequences to obtain amplification primers and hybridization probes. Microarrays were manufactured using these synthesized oligonucleotides. A highly parallel and stringent labeling and hybridization protocol was developed and employed using isolated genomic DNA from previously sequenced (referenced) clinical VRE strains for optimal sensitivity and specificity. Microarray results showed the detection of virulence, resistance, and species-specific genes in the VRE strains. Theoretical predictions of the microarray results were also derived from the sequences of the same VRE strain and were compared to array results while optimizing protocols until the microarray result and theoretical predictions were a match. The study concludes that DNA microarray technology can be used to quickly, accurately, and economically detect specifically and massively parallel target genes in enterococci.

Frequencies of Human Leukocyte Antigen Alleles in Turkish Children with Kawasaki Disease

Abstract Objective Kawasaki disease (KD) is an acute systemic vasculitis that is one of the major causes of acquired heart disease especially in young children. The pathogenesis of KD is still unclear. The increased incidence of the disease among Japanese children and siblings of affected patients suggests a genetic component to KD susceptibility. Several reports have studied human leukocyte antigen (HLA) polymorphisms in different populations with KD and found various results. In the present study, we aimed to evaluate the association of HLA-A, -B, -C, -DRB1, and -DQB1 allele frequencies in Turkish children with KD. Methods The study was conducted between January 2016 and February 2018. HLA Class I (A, B, and C) and Class II (DRB1 and DQB1) alleles of patients and healthy controls were studied using the low-resolution DNA-based sequence-specific oligonucleotide method. Results Fifty children with KD and 500 healthy controls were included in this study. In the analysis of HLA-A, HLA-B, HLA-C, and HLA-DRB1 alleles, no statistical difference was found in the frequency of alleles between the patients with KD and the control group. However, a significantly lower frequency of the HLA-DQB1*03 allele was observed in the KD group than in the control group (p < 0.001, odds ratio [OR]: 0.29, 95% confidence interval [CI]: 0.15–0.55). When the patients with KD were divided into two subgroups with or without coronary artery lesions (CALs), the frequency of the HLA-DQB1*03 allele was also found lower in the KD group with CALs than the KD group without CALs (p = 0.008, OR: 0.19, 95% CI: 0.05–0.68). Conclusion The study may guide future studies on HLA-DQB1*03 whether it is a protective allele for KD and CALs in Turkish children.

Associations between end stage renal disease and HLA polymorphisms in the Guangxi Zhuang population

To investigate the genetic relationship between end stage renal disease (ESRD) and human leukocyte antigen (HLA) alleles in the Guangxi Zhuang population. We performed polymerase chain reaction reversed sequence-specific oligonucleotide (PCR-rSSO) in 325 patients with ESRD and genotyped the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. The direct counting method was used to determine the frequencies of HLA alleles, and Arlequin software (version 3.5.2.2) was used for haplotypic frequency analyses to compare the included ESRD patients with 350 healthy donors from the Guangxi Zhuang population. In our study, 120 HLA alleles, 284 HLA-A-B-DRB1 haplotypes, and 332 HLA-A-C-B-DRB1-DQB1 haplotypes were detected. We found that only A*11:01-B*15:02-DRB1*12:02 had a positive association with ESRD (P = 0.001, Pc = 0.020, OR = 3.106, 95% CI = 1.497–6.446) after Bonferroni correction; thus, individuals with this haplotype may be susceptible to ESRD. A*11:01-B*15:02-DRB1*12:02 is a potentially valuable haplotype for evaluating the risk of ESRD in the Guangxi Zhuang population.

Impact of HLA Alleles on COVID-19 Severity in Kidney Transplant Recipients: A Single-Center Study.

Introduction The coronavirus disease 2019 (COVID-19) pandemic has significantly impacted global health, particularly affecting vulnerable populations, such as organ transplant recipients. Human leukocyte antigens (HLAs) play a critical role in immune response regulation, and understanding their association with COVID-19 can provide insights into disease susceptibility and severity. This study aims to explore the association between HLA allele variability and COVID-19 susceptibility and severity among kidney transplant recipients. Methods In 2023, we conducted a study on 73 kidney transplant recipients who tested positive for COVID-19 via polymerase chain reaction. This study included assessments of clinical status, immunosuppressive drug levels, HLA allele frequencies, and donor-recipient tissue compatibility. Molecular analyses were performed using sequence-specific oligonucleotide typing, and statistical analysis was conducted using IBM SPSS Statistics, version 20.0 (IBM Corp., Armonk, NY). Results Among the participants, 31 were hospitalized and 42 were treated as outpatients. Significant differences were observed in HLA allele distributions, particularly the HLA-A*11 allele, which was more prevalent in outpatient-treated patients, suggesting a potential protective effect. No significant age differences were observed between hospitalized and outpatient groups. Serum tacrolimus levels were notably higher in outpatients. Statistical analyses revealed significant associations between certain HLA groups and the severity of COVID-19 infection. Conclusions This study highlights the importance of HLA allele compatibility in influencing the clinical outcomes of COVID-19 in kidney transplant recipients. The findings suggest that specific HLA alleles may reduce susceptibility or moderate the severity of COVID-19, indicating a need for broader genetic studies across diverse populations to validate these observations and improve management strategies for transplant recipients during pandemics.

Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation.

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting for drug resistance mutations and circulating tumor cell mutations during therapy. Traditional Sanger sequencing has 5%-20% wild-type mutation detection, so its limit of detection cannot meet the corresponding requirements. The wild-type blocking technologies that have been reported to overcome this include blocker displacement amplification (BDA), non-extendable locked nucleic acid (LNA), hot-spot-specific probes (HSSP), etc. These technologies use specific oligonucleotide sequences to block wild-type or recognize wild-type and then combine this with other methods to prevent wild-type amplification and amplify mutant amplification, leading to characteristics like high sensitivity, flexibility, and convenience. This protocol uses BDA, a wild-type blocking PCR combined with Sanger sequencing, to optimize the detection of RHOA G17V low-frequency somatic mutations, and the detection sensitivity can reach 0.5%, which can provide a basis for MRD monitoring of angioimmunoblastic T-cell lymphoma.

Oligonucleotide Therapies for Facioscapulohumeral Muscular Dystrophy: Current Preclinical Landscape.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy, characterized by progressive and asymmetric muscle atrophy, primarily affecting muscles of the face, shoulder girdle, and upper arms before affecting muscles of the lower extremities with age and greater disease severity. FSHD is a disabling condition, and patients may also present with various extramuscular symptoms. FSHD is caused by the aberrant expression of double homeobox 4 (DUX4) in skeletal muscle, arising from compromised epigenetic repression of the D4Z4 array. DUX4 encodes the DUX4 protein, a transcription factor that activates myotoxic gene programs to produce the FSHD pathology. Therefore, sequence-specific oligonucleotides aimed at reducing DUX4 levels in patients is a compelling therapeutic approach, and one that has received considerable research interest over the last decade. This review aims to describe the current preclinical landscape of oligonucleotide therapies for FSHD. This includes outlining the mechanism of action of each therapy and summarizing the preclinical results obtained regarding their efficacy in cellular and/or murine disease models. The scope of this review is limited to oligonucleotide-based therapies that inhibit the DUX4 gene, mRNA, or protein in a way that does not involve gene editing.

A prospective epidemiological investigation of human leukocyte antigen-B*57:01 in HIV-1-infected Moroccan subjects

BackgroundThe occurrence of hypersensitivity reactions to abacavir, a significant adverse effect, affects approximately 5–8% of Caucasians. Multiple studies conducted in diverse populations have underscored the association between Human Leukocyte Antigen-B*57:01 and abacavir-hypersensitivity reactions. In late 2021, the Moroccan National AIDS Program issued new anti-retroviral therapy protocols for HIV management, including abacavir as an option for the first-line ART regimen. However, data regarding the HLA-B*57:01 prevalence of HIV in Morocco is scarce. This study aims to assess the prevalence of HLA-B*57:01 in both HIV-1-infected children and adults in Morocco. MethodsFrom April to December 2022, we screened 292 HIV-1 infected patients, 70 children and 222 adults, for the HLA-B*57:01 allele using sequence-specific oligonucleotide probes reverse hybridization method. The flow cytometry was used to assess the TCD4 lymphocyte count. The virological status was evaluated using quantitative real-time PCR. Sanger sequencing is under process to confirm the results obtained. ResultsOf 292 HIV-1-infected patients, 12 (4.1%) had the HLA-B*57:01 allele (95% CI, 2.1–7.1). 5 of 70 (7.2%) and 7 of 222 (3.2%) of children and adults were respectively, HLA-B*57:01 positive. There was no statistical association between clinical characteristics (TCD4 cell count and VL) and HLA-B*57:01 allele carriage. ConclusionThe prevalence of HLA-B*57:01 in Moroccan patients was 4.1%, comparable with that of other Middle Eastern populations, higher than that in South African populations but lower than that in Caucasians and Southwest Asians. Our findings indicate an urgent need for HLA-B*57:01 screening before abacavir to reduce the risk of hypersensitivity reactions.

HLA alleles, haplotypes frequencies, and their association with hematological disorders: a report from 1550 families whose patients underwent allogeneic bone marrow transplantation in Egypt.

HLA alleles are representative of ethnicities and may play important roles in predisposition to hematological disorders. We analyzed DNA samples for HLA-A, -B, -C, -DRB1, and -DQB1 loci, from 1550 patients and 4450 potential related donors by PCR-SSO (Polymerase chain reaction sequence-specific oligonucleotides) and estimated allele frequencies in donors and patients from 1550 families who underwent bone marrow transplantation (BMT) in Egypt. We also studied the association between HLA allele frequencies and incidence of acute myeloid leukemia, acute lymphoblastic leukemia, and severe aplastic anemia. The most frequently observed HLA class I alleles were HLA- A*01:01 (16.9%), A*02:01 (16.1%), B*41:01 (8.7%), B*49:01 (7.3%), C*06:02 (25.1%), and C*07:01 (25.1%), and the most frequently observed class II alleles were HLA-DRB1*11:01 (11.8%), DRB1*03:01 (11.6%), DQB1*03:01 (27.5%), and DQB1*05:01 (18.9%). The most frequently observed haplotypes were A*33:01~B*14:02 ~ DRB1*01:02 (2.35%) and A*01:01~B*52:01~DRB1*15:01 (2.11%). HLA-DRB1*07:01 was associated with higher AML odds (OR, 1.26; 95% CI, 1.02-1.55; p = 0.030). Only HLA-B38 antigen showed a trend towards increased odds of ALL (OR, 1.52; 95% CI, 1.00-2.30; p = 0.049) HLA-A*02:01, -B*14:02, and -DRB1*15:01 were associated with higher odds of SAA (A*02:01: OR, 1.35; 95% CI, 1.07-1.70; p = 0.010; B*14:02: OR, 1.43; 95% CI, 1.06-1.93; p = 0.020; DRB1*15:01: OR, 1.32; 95% CI, 1.07-1.64; p = 0.011). This study provides estimates of HLA allele and haplotype frequencies and their association with hematological disorders in an Egyptian population.

Genetic study of a rare Chinese pedigree with a recombination occurring between the HLA-A/C loci in both parents

To analyze a Chinese pedigree with a recombination occurring between the HLA-A/C loci in both parents. A patient who was planning to undergo hematopoietic stem cell transplantation due to "aplastic anemia" in February 2022 was selected as the study subject. Peripheral blood samples were collected from the patient, his parents and brother. HLA-A/C/B/DRB1/DQB1 high-resolution typing was carried out by using sequence-based typing and sequence-specific oligonucleotides. The recombination was identified by pedigree analysis. The HLA haplotype of each individual was identified by genealogical analysis. The parentage possibility was determined by short tandem repeat analysis. HLA-A/C/B/DRB1/DRB345/DQA1/DQB1/DPA1/DPB1 were determined with next-generation high-throughput sequence-based typing. The recombination sites were analyzed by family study. The high parentage possibilities of the family was confirmed by short tandem repeat analysis. Recombination was found between the HLA-A*24:02 A*33:03/C*14:03 in the paternally transmitted haplotype, whilst HLA-A*01:01 A*03:01/C*08:02 was found in the maternally transmitted haplotype, which had resulted in two novel HLA haplotypes in the proband. A rare case with simultaneous recombination of the paternal and maternal HLA-A/C loci has been discovered, which may facilitate further study of the mechanisms of the HLA recombination.

Unraveling the Complexity of DNA Radiation Damage Using DNA Nanotechnology.

Radiation cancer therapies use different ionizing radiation qualities that damage DNA molecules in tumor cells by a yet not completely understood plethora of mechanisms and processes. While the direct action of the radiation is significant, the byproducts of the water radiolysis, mainly secondary low-energy electrons (LEEs, <20 eV) and reactive oxygen species (ROS), can also efficiently cause DNA damage, in terms of DNA strand breakage or DNA interstrand cross-linking. As a result, these types of DNA damage evolve into mutations hindering DNA replication, leading to cancer cell death. Concomitant chemo-radiotherapy explores the addition of radiosensitizing therapeutics commonly targeting DNA, such as platinum derivatives and halogenated nucleosides, to enhance the harmful effects of ionizing radiation on the DNA molecule. Further complicating the landscape of DNA damage are secondary structures such as G-quadruplexes occurring in telomeric DNA. These structures protect DNA from radiation damage, rendering them as promising targets for new and more selective cancer radiation treatments, rather than targeting linear DNA. However, despite extensive research, there is no single paradigm approach to understanding the mysterious way in which ionizing radiation causes DNA damage. This is due to the multidisciplinary nature of the field of research, which deals with multiple levels of biological organization, from the molecular building blocks of life toward cells and organisms, as well as with complex multiscale radiation-induced effects. Also, intrinsic DNA features, such as DNA topology and specific oligonucleotide sequences, strongly influence its response to damage from ionizing radiation. In this Account, we present our studies focused on the absolute quantification of photon- and low-energy electron-induced DNA damage in strategically selected target DNA sequences. Our methodology involves using DNA origami nanostructures, specifically the Rothemund triangle, as a platform to expose DNA sequences to either low-energy electrons or vacuum-ultraviolet (VUV, <15 eV) photons and subsequent atomic force microscopy (AFM) analysis. Through this approach, the effects of the DNA sequence, incorporation of halogenated radiosensitizers, DNA topology, and the radiation quality on radiation-induced DNA strand breakage have been systematically assessed and correlated with fundamental photon- and electron-driven mechanisms underlying DNA radiation damage. At lower energies, these mechanisms include dissociative electron attachment (DEA), where electrons attach to DNA molecules causing strand breaks, and dissociative photoexcitation of DNA. Additionally, further dissociative processes such as photoionization and electron impact contribute to the complex cascade of DNA damage events induced by ionizing radiation. We expect that emerging DNA origami-based approaches will lead to a paradigm shift in research fields associated with DNA damage and suggest future directions, which can foster the development of technological applications in nanomedicine, e.g., optimized cancer treatments or the molecular design of optimized radiosensitizing therapeutics.

A lateral flow assay for miRNA-21 based on CRISPR/Cas13a and MnO2 nanosheets-mediated recognition and signal amplification.

The point-of-care testing (POCT) of miRNA has significant application in medical diagnosis, yet presents challenges due to their characteristics of high homology, low abundance, and short length, which hinders the achievement of quick detection with high specificity and sensitivity. In this study, a lateral flow assay based on the CRISPR/Cas13a system and MnO2 nanozyme was developed for highlysensitive detection of microRNA-21 (miR-21). The CRISPR/Cas13a cleavage system exhibits the ability to recognize the specific oligonucleotide sequence, where two-base mismatches significantly impact the cleavage activity of the Cas13a. Upon binding of the target to crRNA, the cleavage activity of Cas13a is activated, resulting in the unlocking of the sequence and initiating strand displacement, thereby enabling signal amplification to produce a new sequence P1. When applying the reaction solution to the lateral flow test strip, P1 mediates the capture of MnO2 nanosheets (MnO2 NSs) on the T zone, which catalyzes the oxidation of the pre-immobilized colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) on the T zone and generates the blue-green product (ox-TMB). The change in gray value is directly proportional to the concentration of miR-21, allowing for qualitative detection through visual inspection and quantitative measurement using ImageJ software. This method achieves the detection of miR-21 within a rapid 10-min timeframe, and the limit of detection (LOD) is 0.33pM. With the advantages of high specificity, simplicity, and sensitivity, the lateral flow test strip and the design strategy hold great potential for the early diagnosis of related diseases.

HLA variations in patients with diffuse large B-cell lymphoma and association with disease risk and prognosis: a case-control study.

Human leukocyte antigen (HLA) polymorphisms have been associated with the development of various autoimmune diseases, as well as malignant neoplasms. Non-Hodgkin lymphomas (NHLs) are a heterogenous group of lymphoid malignancies in which a genetic substrate has been established and is deemed to play a crucial role in disease pathogenesis. This study aimed to identify whether variations in the HLA gene region were associated with diffuse large B-cell lymphoma (DLBCL) risk and prognosis. We defined HLA class I (HLA-A, HLA-B, HLA-C) and class II (HLA-DRB1, HLA-DQB1) alleles in 60 patients with DLBCL and compared the results to those found by 236 healthy adult donors from the bone marrow bank of Northern Greece. HLA typing was performed by two molecular methods, Sequence - Specific Oligonucleotide HLA typing (SSO) and Sequence - Specific Primer HLA typing (SSP), from white blood cells recovered from peripheral blood. The phenotypic frequencies of HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 between patients and controls were compared with the 2-sided Fisher's exact test. Results with p-value <0.05 were considered statistically significant. Odds Ratios with 95% Confidence Intervals were calculated to further strengthen the results. The 2-sided Fisher's exact test was also applied to alleles found only in one of the two groups, while the odds ratios together with the confidence intervals were corrected with Haldane-Anscombe method. Among the studied HLA polymorphisms, the frequency HLA-C*12 allele was significantly lower in patients with DLBCL compared with control subjects (6.7% vs. 34.7%, OR = 0.16, 95% CI: 0.04-0.44). Frequency of HLA-B*39 was significantly lower in patients with DLBCL compared with controls, but due to the low frequency of this polymorphism in the studied population and small sample size, determinations regarding the significance of this findings were limited. Survival analysis revealed that the presence of HLA-C*12 was not associated with improved or worsened overall and progression-free survival. No statistically significant associations were observed in the phenotypic frequencies of HLA-A, HLA-DQB1, HLA-DRB1 and the rest of HLA-B alleles between the control and DLBCL groups. Collectively, our results provide valuable insight regarding the role of HLA variations on DLBCL risk. Further studies are required to consolidate our findings and ascertain the clinical implications of these genetic variations on DLBCL management and prognosis.

Analysis of transfusion effect of different platelet matching schemes in patients with platelet transfusion refractoriness

Objective: To analyze the transfusion effect of different platelet matching schemes in patients with platelet transfusion refractoriness (PTR). Methods: A total of 94 patients with PTR received by Taiyuan Blood Center from January to December 2021 were retrospectively analyzed, including 26 males and 68 females, aged 53(34,66) years. Platelet antibody screening was performed by enzyme-linked immunosorbent assay (ELISA). For patients with positive human leukocyte antigen (HLA) class Ⅰ antibodies, Luminex platform liquid chip assay was used to identify the specificity of antibodies, and platelets with missing allelic expression antigen corresponding to their specific antibodies were found in the platelet donor gene database established in our laboratory. For patients with negative class HLA-Ⅰ antibody screening, medium and high-resolution HLA-A and B alleles were genotyped by polymerase chain reaction restriction sequence specific oligonucleotide (PCR-SSO), and the compatible platelets were searched from the platelet donor gene database by HLA cross-reactive group genotype matching scheme or directly selected by serological cross-matching. The PCI compliance rate and total transfusion effective rate of different mismatch site groups and different matching scheme groups were statistically analyzed. Results: Platelet antibody was detected in 39 of 94 PTR patients with a positive rate of 41.5%, and all of them were HLA-Ⅰ antibodies, and 1 case was accompanied by human platelet antigen (HPA) antibody. A total of 134 times of compatible platelets were supplied to 39 patients with HLA-Ⅰ antibody positive by using antibody avoidance matching method. And the total effective rate of transfusion was 97.8% (131/134); The PCI compliance rates of HLA-A antigen mismatch, HLA-B antigen mismatch and HLA-A and B antigen mismatch groups were 81.6% (31/38), 86.5% (32/37) and 78.6% (22/28), respectively. The total effective rate of transfusion was 97.4% (37/38), 94.6% (35/37) and 100% (28/28), respectively, with no statistical significance (all P>0.05). A total of 118 times of compatible platelets were provided by HLA antigen cross-reaction group genotype matching and serological cross-matching, 90 transfusion effects were collected during follow-up, and the total effective rate was 76.7% (69/90). Conclusion: The combination of different platelet matching schemes can improve the PCI compliance rate and the total effective rate of transfusion in PTR patients.

Frequencies of HLA-B alleles in Indonesian Malay Ethnic

BackgroundThe HLA-B alleles have been used as a marker to predict drug-induced adverse reactions and as a major contributor to hypersensitivity reactions. We examined the feasibility of HLA-B alleles as pharmacogenomic markers of drug-induced hypersensitivity in an Indonesian Malay Ethnic. MethodsFifty-eight Indonesian individuals of Malay ethnicity were enrolled in this study. HLA-B alleles were determined using reverse sequence-specific oligonucleotide probe coupled with xMAP technology. ResultsHLA-B*15:02 (15.52%), HLA-B*35:05 (9.48%), and HLA-B*07:05 (7.76%) were frequent alleles in the Indonesian Malay ethnic populations. We discovered at least eight pharmacogenomics markers of drug-induced hypersensitivity: HLA-B*15:02, HLA-B*15:21, HLA-B*13:01, HLA-B*35:05, HLA-B*38:02, HLA-B*51:01, HLA-B*57:01, and HLA-B*58:01. HLA-B*15:02 was in the same serotype group with HLA-B*15:21, which is a B-75 serotype associated with genetic predisposition for carbamazepine-induced Stevens–Johnson syndrome and toxic epidermal necrolysis. The Indonesian population, represented by Malay, Javanese, and Sundanese ethnicities, was similar to South East Asian, Han Chinese, and Taiwanese populations based on HLA-B*15:02 frequency as the most common allele found in Malay ethnics. ConclusionWe provided valuable information on the frequency of drug hypersensitivity-associated HLA-B alleles in Indonesian Malay ethnic population, which can improve treatment safety.

Distribution of the Prevalence of Human Leukocyte Antigen (HLA)-B*57:01 Positivity in HIV-1 Infected Individuals and Its Effects on Treatment: Türkiye Map-Buhasder Working Group

Human immunodeficiency virus (HIV)/acquired immundeficiency syndrome (AIDS) is a critical global public health problem that significantly affects both life expectancy and the overall quality of life of individuals in all age groups. The landscape of HIV infection has changed significantly in recent years due to the introduction of effective combination antiretroviral therapies (ART). A key component of first-line ART regimens for HIV treatment is abacavir, a nucleoside HIV reverse transcriptase inhibitor. Although abacavir is effective in suppressing viral replication and managing disease, its clinical utility is overshadowed by the potential for life-threatening hypersensitivity reactions in HLA-B*57:01-positive patients. In our country, local data obtained from various centers regarding the prevalence of HLA-B*57:01 in HIV-1-infected patients are available. In this study, it was aimed to determine the prevalence of the HLA-B*57:01 genotype in HIV-infected patients who were followed up and treated in many regions of our country. This retrospective study consists of the data of the patients aged 18 years and over diagnosed with HIV-1 infection between 01.01.2019 and 31.07.2022. Age, gender, place of birth, mode of transmission of the disease, death status, CD4+ T cell count and HIV RNA levels at the first clinical presentation, HLA-B*57:01 positivity, and the method used, clinical stage of the disease, virological response time with the treatment they received were recorded from the patient files. Data were collected from 16 centers and each center used different methods to detect HLA-B*57:01. These methods were sequence-specific oligonucleotide probe hybridization (SSOP), DNA sequence-based typing (SBT), single-specific primer-polymerase chain reaction (SSP-PCR), allele-specific PCR (AS-PCR) and quantitative PCR (Q-PCR). A total of 608 HIV-infected individuals, 523 males (86%) and 85 females (14%), were included in the study. The mean age of the patients was 36.9 ± 11.9 (18-73) years. The prevalence of HLA-B*57:01 allele was found to be 3.6% (22 patients). The number of CD4+ T lymphocytes in HLA-B*57:01 allele-positive patients was > 500/ mm3 in 10 patients (45.5%), while the number of CD4+ T lymphocytes in HLA-B*57:01 negative patients was > 500/mm3 in 216 patients (36.9%) (p> 0.05). Viral load at the time of diagnosis was found to be lower in patients with positive HLA-B*57:01 allele but it was not statistically significant (p> 0.05). Although different treatment algorithms were used in the centers following the patients, it was observed that the duration of virological response was shorter in HLA-B*57:01 positive patients (p= 0.006). Although the presence of the HLA-B*57:01 allele has a negative impact due to its association with hypersensitivity, it is likely to continue to attract interest due to its association with slower progression of HIV infection and reduced risk of developing AIDS. In addition, although the answer to the question of whether it is cost-effective to screen patients for HLA-B*57:01 before starting an abacavir-containing ART regimen for the treatment of HIV infection is being sought, it seems that HIV treatment guidelines will continue to recommend screening to identify patients at risk in this regard.

HLA-B*51 Frequency in Transplant Patients and Donors.

HLA molecules play a crucial role in transplantation. The best treatment modality in patients with end-stage renal disease is renal transplant. HLA mismatches between patients and donors can prolong time for renal transplant therapy, reduce graft survival, and increase mortality. HLA region is the most polymorphic genetic region and is essential for antigen presentation. The main target of the recipient's immune system is HLA molecules on the surface of donor cells. HLA-B*51 is associated with Behcet disease, a rare multisystemic disease characterized by autoimmunity and inflammatory processes. In transplant recipients, inflammation and vasculitis are immunologic mechanisms that are responsible for damage of graft tissue. In this retrospective study, we aimed to investigate the frequency of HLA-B*51 in patients diagnosed with end-stage renal disease and in controls and to investigate correlations with rejection episodes. Patients who applied to Baskent University Adana Dr. Turgut Noyan Research and Medical Center (between 2010 and 2022) with end-stage renal disease (n = 1732) and a control group (n = 5277) received HLA typing for class I (HLA-A, HLA-B). Sequence-specific primers or sequencespecific oligonucleotides were used. Among patients diagnosed with end-stage renal disease, 321 had kidney transplant. Frequency of HLA-B*51 was 25.92% in patients and 25.22% in controls. No significant differences were found between patients and controls in the frequency of HLA-B*51. Among kidney transplant recipients with HLA-B*51 (n = 72), 38.89% had rejection episodes and 61.11% had no rejection. No significant association was found between HLA-B*51 allele positivity and rejection. No significant relationship was shown between patients diagnosed with end-stage renal disease and HLA-B*51 positivity. Previous studies support frequency of the HLA-B*51 allele in the control group. Although Behçet disease is known to cause renal vasculitis, HLA-B*51 positivity alone was not associated with vasculitis or inflammation.

Human leukocyte antigen association in systemic sclerosis patients: our experience at a tertiary care center in North India.

Systemic sclerosis (SSc) is a chronic multisystem autoimmune rheumatic disease of unknown etiology. Several studies have established that SSc is triggered by a dynamic interplay between genetic factors and environmental stimuli. In the present study, we aimed to study the association of human leukocyte antigen (HLA) with familial and non-familial SSc patients [limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc)] from North India. The HLA-A, B, DRB1, and DQB1 genotyping of 150 (70 lcSSc and 80 dcSSc) adult-onset SSc patients and 150 age-gender-matched healthy controls were performed with sequence-specific oligonucleotide (SSO) typing kits using the luminex platform. HLA typing for HLA class I (A, B, and C) and II (DRB1, DQB1, and DPB1) in five North Indian families consisting of parent-child/sibling pairs affected with SSc or overlap syndrome was performed by Next Generation Sequencing (NGS) with Illumina MiniSeq. Among the non-familial SSc patients, HLA- DRB1*11 (P = 0.001, OR: 2.38, P c = 0.01) was identified as a risk allele, and DRB1*12 (P = .0001, OR: 0.00, P c = 0.001) as a protective allele. There was no statistical association found with HLA-DQB1*. Also, no significant association was observed between HLA antigens and different clinical subsets (lcSSc and dcSSc) of SSc. Two cases of familial SSc patients had the DRB1*11 allele. The DRB1*12 allele was absent in all the familial SSc patients. HLA DRB1*11 (risk allele) and DRB1*12 (protective allele) were found to be strongly associated with non-familial SSc patients and partially explain the disease's familial clustering, supporting the susceptible genetic background theory for SSc development. The study also indicates the HLA allele as a common genetic risk factor in distinct autoimmune diseases contributing to overlap syndrome or polyautoimmunity.

HLA Alleles, Genotype and Haplotype Analyzes from Central Anatolia Region of Turkey.

Although human leukocyte antigen (HLA) data for the Turkish population has been reported, there are no statistics on the HLA-DPB1 locus, which has recently received significant attention, particularly in hematopoietic stem cell transplantation. In addition, there is no study that has reported the 2-6 loci HLA haplotype distribution, 8-digit HLA allele frequency, and genotype frequency in the Turkish population. To evaluate the low and high resolution (2-4-8 digits) HLA-A, -B, -C, -DRB1, -DQB1, -DPB1 allele data using the data of 6100 healthy individuals from the Central Anatolian region of Turkey. Retrospective cross-sectional study. All tests were performed using molecular HLA techniques: low-resolution DNA-based sequence-specific oligonucleotides, low/high-resolution DNA-based sequence-specific primer, and high-resolution next generation sequencing. A total of 6100 healthy donors with a minimum of 3 loci (HLA-A, -B, -DRB1) were analyzed for their HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 data. Pypop and HLA-net GENE[RATE] were used to analyze the data. Among the HLA class I alleles, the following were the most frequently observed alleles: for HLA-A, A*02, A*24, A*03, and A*01; for HLA-B, B*35, B*51, and B*44; and for HLA-C, C*07, C*04, and C*12. Among the HLA class II alleles, the following alelles were the most frequently observed: for HLA-DRB1, DRB1*11, DRB1*04, and DRB1*13; for HLA-DQB1, DQB1*03, DQB1*05, and DQB1*06; and for HLA- DPB1, DPB1*04, DPB1*02, and DPB1*03. The most common alleles among HLA-DPB1 in the 4-digit evaluation were DPB1*04:01, DPB1*02:01, and DPB1*04:02. Among the HLA classes I and II, the following were the most frequently observed 8-digit alleles in HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 were A*02:01:01:01, B*49:01:01:01, C*04:01:01:06, DRB1*07:01:01:01, DQB1*03:01:01:02, and DPB1*02:01:02:05, respectively. The most common 6 loci haplotype was A*02~B*35~C*04~DRB1*11~DQB1*03~DPB1*04 (2.71%). Our study has included the highest number of healthy individuals from Turkey till date. Furthermore, we have reported the low- and high-resolution HLA-DPB1 allele frequency, 6 loci haplotype frequency, and genotype frequency in the Turkish population for the first time. This new data may be used to map HLA in our country and provide insight for potential future studies.

Development of cost-effective and fast KIR genotyping by multiplex PCR-SSP.

Killer-cell immunoglobulin-like receptors (KIR) control natural killer (NK) cell functions by recognizing HLA molecules and modulating the activity of NK cells. The KIR gene cluster contains polymorphic and highly homologous genes. Diversity of the KIR region is achieved through differences in gene content, allelic polymorphism, and gene copy number, which result in unrelated individuals having different KIR genotypes and individualized immune responses that are relevant to multiple aspects of human health and disease. Therefore, KIR genotyping is increasingly used in epidemiological studies. Here, we developed multiplex polymerase chain reaction with sequence-specific primers (PCR-SSP) to compensate for the shortcomings of the conventional PCR-SSP method, which is most commonly used for KIR analysis. Multiplex PCR-SSP method involves six multiplex reactions that detect 16 KIR genes and distinguish variant types of some KIR genes by adding two reactions. The assay was evaluated in a blind survey using a panel of 40 reference DNA standards from the UCLA KIR Exchange Program. The results are 100% concordant with the genotype determined using Luminex-based reverse sequence-specific oligonucleotide typing systems. Additionally, we investigated the currently known 16 KIR genes and their common variants in 120 unrelated Korean individuals. The results were consistent with the KIR genotype previously reported by Hwang et al. This multiplex PCR-SSP is an efficient method for analyzing KIR genotypes in both small- and large-scale studies with minimal labor, reagents, and DNA. Furthermore, by providing a better definition of KIR polymorphisms it can contribute to developments in immunogenetics.

Association of HLA-DRB1 alleles with anti-D alloimmunization in RhD negative pregnant women in India

BackgroundHuman leukocyte antigen (HLA) restriction plays an important role in the susceptibility to alloimmunization against red blood cell (RBC) antigens. The prevalence of anti-D alloimmunization in RhD negative pregnancy is still quite high in our population. Thus, we planned this study to determine the association of HLA-DRB1 alleles with anti-D alloimmunization in RhD negative pregnant women. Material and methodsRBC antibody screen (ABS) was performed for RhD negative pregnant women attending the antenatal clinic our institute. Those with a negative result were included in the ‘non-alloimmunized’ (NAL) group (‘Control’ group), while those with anti-D alloantibody on performing antibody identification were included in the alloimmunized (AL) group of the study (n = 50 each). ABS and identification were done using column agglutination technique. The HLA-DRB1 typing was done by Luminex based reverse sequence specific oligonucleotide probing (SSOP) using commercial kits. The HLA-DRB1 allele frequency was compared in both the groups. ResultsThere was a significant difference between the two groups in terms of gravida (p < 0.001) and history of anti-D immunoprophylaxis (p < 0.001). The frequency of HLA-DRB1*03 and HLA-DRB1*04 alleles was significantly higher in the AL group than the NAL group: 40 % versus 18 % [Odds Ratio (OR): 3.04, 95 % CI: 1.21–7.6; p = 0.015] for HLA-DRB1*03 alleles and 18 % versus 4 % (OR: 5.27, 95 % CI: 1.08–25.78, p = 0.025) for HLA-DRB1*04 alleles. ConclusionThe frequency of HLADRB1*03 and HLADRB1*04 alleles was significantly higher in RhD negative pregnant women alloimmunized with anti-D alloantibody.