Polymerase Chain Reaction-sequence Specific Primer Research Articles

Alternative donor strategy in unrelated hematopoietic stem cell transplantation – outcome with mismatched donors

PurposeThis study retrospectively investigated the association between the level of human leukocyte antigen (HLA) mismatches (MMs), direction of disparities and differences at particular HLA locus on clinical outcomes of hematopoietic stem cell transplantation (HSCT). Investigated outcomes were overall survival (OS) and disease-free survival (DFS), graft-versus-host disease (GvHD), relapse and non-relapse mortality (NRM). Patients and methodsStudy cohort included 108 adult patients transplanted between 2011 and 2021 and their 9/10 mismatched unrelated donors (MMUD). All individuals were typed for HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 loci using Polymerase Chain Reaction-Sequence Specific Primers, PCR-Sequence Based Typing and Next-Generation Sequencing. All statistical analyses were done in the MedCalc software, version 19.2.6. ResultsPatients with MMs at HLA-B locus demonstrated worse OS (P ​= ​0.0440, HR ​= ​2.00, n ​= ​20). Absence of HLA-DRB5 was associated with a higher incidence of GvHD (P ​= ​0.0112, HR ​= ​1.93, n ​= ​67). A lower incidence of GvHD was observed in patients with HLA class II MMs compared to patients with HLA class I MMs (P ​= ​0.0166, HR ​= ​1.94, n ​= ​29). Finally, analysis of PIRCHE score (PS) impact revealed that patients with HLA class II PS ​> ​10 in GvH direction showed higher incidence of GvHD compared to patients with HLA class II PS ​< ​10 (P ​= ​0.0073, HR ​= ​2.01, n ​= ​55). ConclusionObtained results undisputedly indicate the necessity to further investigate this matter on a larger patient group, with focus on specific HLA alleles to define precisely priority criteria for selecting the best donor for all patients, thus improving the outcome of HSCT with an MMUD.

Association of MICA Gene Polymorphism in Opisthorchis viverrini-Induced Periductal Fibrosis in Northeastern Thais.

Chronic Opisthorchis viverrini (OV) infection is the cause of advanced periductal fibrosis (APF), subsequently leading to cholangiocarcinoma (CCA). Natural killer (NK) cells can kill hepatic stellate cells (HSCs), the initiating cells for fibrosis formation, by using the interaction between the natural killer group 2 member D (NKG2D) receptor and its ligand on the HSCs. This can inhibit the fibrosis formation. Major histocompatibility complex class I chain-related A (MICA) is the ligand of the NKG2D receptor and has highly polymorphic characteristics that are involved in NKG2D binding and NK cell activation. This study aimed to investigate the polymorphism of MICA in OV-induced fibrosis. MICA typing was performed by polymerase chain reaction- sequence specific primer (PCR-SSP) and sequencing in two groups: OV infection without fibrosis (N = 99) and with fibrosis (N = 290). Six alleles were identified and the MICA*010 allele had the highest frequency in both groups. The MICA*00201-02 allele was a protective factor for fibrosis (OR= 0.508, 95%CI= 0.34-0.76, Pc <0.05), while the MICA*019 allele was suggested to be a risk allele for fibrosis (OR=1.95, 95%CI=1.25-3.03, Pc<0.005). In addition, two motifs, glycine (G) at position 14 and glutamine (Q) at position 251, were negatively associated with fibrosis (G14: OR=0.508, 95%CI=0.34-0.76, Pc <0.05 and Q251: OR=0.586, 95%CI=0.41-0.84, Pc <0.05). Moreover, the distribution of the MICA-129 genotype also showed the protective genotype (Pc<0.05, OR=0.319, 95%CI= 0.12-0.54) for fibrosis. The MICA*00201-02 allele encoded all these motifs, and this suggested that it might lead to strong NK cell activation to kill HSCs, subsequently preventing fibrosis formation. This study described initial evidence suggesting that the polymorphism of the MICA gene might be a marker for OV-derived periductal fibrosis.

HLA-B*57:01 typing in a Malaysian cohort: implications of abacavir hypersensitivity in people living with HIV.

Background: Abacavir (ABC)in combination with other antiretroviral drugs, is used to treat people living with HIV (PLWH). However, it is linked to a fatal hypersensitivity reaction in susceptible individuals, and is strongly associated with the HLA-B*57:01 allele. Materials & methods: A total of 152 patients, 50 PLWH and 102 HIV-1negative patients, were assessed for the HLA-B*57:01 allele through a sequence-specific primer PCR. Results: All PLWH tested negative for the HLA-B*57:01 allele, but two HIV-negative patients were found to have HLA-B*57, with one of them expressing the HLA-B*57:01 allele. Conclusion: Given the low prevalence of this risk allele in the population, testing for the presence of HLA-B*57:01 in PLWH may not provide significant benefit for the reported population.

When the available blood supply mismatches the needs of the patient.

Substantial regional differences in the genetic patterns related to blood group have been observed across different continents. This diversity means that the blood supply, as an essential part of patient care, is increasingly impacted by global migration. Consequently, the Austrian blood donor population does not match the immigrant patient population. This mismatch is likely to result in the formation of alloantibodies to red cell antigens in the chronically transfused. Subsequently, major difficulties in providing compatible blood emerge. The study included patients of African origin (n=290) and Caucasians who represent the Austrian donor population (n=1,017). Genetic typing was performed for up to 69 blood group polymorphisms with a multiplex sequence specific primer-PCR including high frequency antigens and antigens for which antisera are not commercially available. By assessing differences in antigen frequencies between the two populations, and using these data for prophylactic matching, we aim to develop tools to increase the quality of patient care. Results indicate various and significant differences (p<0.0001) in antigen frequencies between African patients and the European donor population, especially in the MNS, Duffy, Knops and Rhesus systems. Our data highlight the importance of matching the donor population to the demographics of the patient population. In addition, it underlines the need to recruit donors of African origin and to focus on the upcoming challenges, such as malaria semi-immunity and a significantly higher rate of infectious disease in this population. It is also recommended to apply extended genetic typing to detect rare blood types, and (cryo)storage of rare blood in national and international rare blood banks. Co-operation with regional blood banks should also be encouraged.

The Association of Human Leucocyte Antigen (HLA) Class I and II Genes with Cutaneous and Visceral Leishmaniasis in Iranian Patients: A Preliminary Case-Control Study.

Leishmaniasis is currently considered a re-emerging or emerging infection based on the geographic region. The outcome of leishmaniasis vastly depends on Leishmania-host interaction. This preliminary study aimed to show the association of human leukocyte antigen (HLA) class I and II genes with healed and non-healed cutaneous leishmaniasis (CL), and symptomatic and asymptomatic visceral leishmaniasis (VL) compared with control groups in Iran. Ninety-five people, including 31 patients versus 64 individuals in the control group, were enrolled. Among them, 20 patients had confirmed CL based on amastigote observation, 10 had improved CL and 10 non-healed CL. Eleven patients were suffering from confirmed VL based on direct agglutination test (Five asymptomatic and six symptomatic VL cases). Besides, they were residents in an endemic area of VL in the northwest of Iran. To select a control group, it was ensured that they had no history of leishmaniasis. Peripheral blood samples were collected from each patient. After DNA extraction, HLA typing was conducted using polymerase chain reaction - sequence-specific priming (PCR-SSP). Subsequently, data were statistically analyzed by SPSS. There was a statistical relationship between the presence of HLA-A26 and CL, healed CL and the existence of the B38 allele, C1 allele and symptomatic VL, as well as B1.4 allele and asymptomatic VL (P<0.05). This primary finding indicates that several HLA genes have a potential role in the susceptibility of Iranian people to CL and VL.

Establishment of a PCR-SSP method for the simultaneous amplification and identification of the presence of KIR genes

To develop a polymerase chain reaction-sequence specific primer (PCR-SSP) method for simultaneous amplification and identification of the KIR genes among Chinese population. Peripheral blood samples from 132 healthy donors who had given blood at Shenzhen Blood Center from January 2015 to November 2015 were selected as the study subjects. Based on the polymorphism and single nucleotide polymorphism (SNP) information of high-resolution KIR alleles in the Chinese population and the IPD-KIR database, specific primers were designed to amplify all the 16 KIR genes and the 2DS4-Normal and 2DS4-Deleted subtypes. The specificity of each pair of PCR primers was verified by using samples with known KIR genotypes. During PCR amplification of the KIR gene, co-amplification the fragment of human growth hormone (HGH) gene by multiplex PCR was used as the internal control to prevent false negative results. A total of 132 samples with known KIR genotypes were randomly selected for blind inspection to verify the reliability of the developed method. The designed primers can specifically amplify the corresponding KIR genes, with clear and bright bands for the internal control and KIR genes. The results of detection are fully consistent with the known results. The KIR PCR-SSP method established in this study can yield accurate results for the identification of the presence of KIR genes.

Association of Human Leukocyte Antigen (HLA) class II (DRB1 and DQB1) alleles and haplotypes with Rheumatoid Arthritis in Sudanese patients.

The aim of this study was to determine the Human Leukocyte Antigen (HLA) class II (DRB1 and DQB1) alleles and haplotype frequency in Rheumatoid Arthritis (RA) in the Sudanese population. The frequency of HLA-DRB1 and -DQB1 alleles and DRB1-DQB1 haplotypes were determined in 122 RA patients and 100 controls. HLA alleles were genotyped by the polymerase chain reaction-sequence specific primers (PCR-SSP) method. In RA patients, HLA-DRB1*04 and *10 alleles were high in frequency (9.6% vs 14.2%, P = 0.038 and P = 0.042, respectively), and dependently on anti-citrullinated protein antibodies (ACPAs) seropositivity (P = 0.044 and P = 0.027, respectively). In contrast, the frequency of the HLA-DRB1*07 allele was significantly low in patients than in controls (11.7% vs 5.0%, P = 0.010). Moreover, the HLA-DQB1*03 allele was strongly associated with RA risk (42.2%, P = 2.2x10-8), whereas, HLA-DQB1*02 and *06 showed protective effects against RA (23.1% and 42.2%, P = 0.024 and P = 2.2x10-6, respectively). Five different HLA haplotypes, DRB1*03-DQB1*03 (P = 0.00003), DRB1*04-DQB1*03 (P = 0.00014), DRB1*08-DQB1*03 (P = 0.027), DRB1*13-DQB1*02 (P = 0.004), and DRB1*13-DQB1*03 (P = 3.79x10-8) were significantly associated with RA risk, while 3 protective haplotypes, DRB1*03-DQB1*02 (Pc = 0.008), DRB1*07-DQB1*02 (Pc = 0.004), and DRB1*13-DQB1*06 (Pc = 0.02) were identified. This is the first study determining the association between HLA class II alleles and haplotypes and RA risk in our population.

Rapid Identification of Seven Common ABO Alleles by Two-Dimensional Polymerase Chain Reaction Technology

Introduction: The molecular biology detection technology of the human ABO blood group system makes up for the limitations in many aspects compared with conventional serological typing technology. This study aimed to establish a new method to identify seven common ABO alleles (ABO*A1.01, ABO*A1.02, ABO*A2.01, ABO*B.01, ABO*O.01.01, ABO*O.01.02, and ABO*O.02.01) by two-dimensional polymerase chain reaction (2D PCR). 2D PCR can identify multiple target genes in a closed test tube by labeling specific primers with tags homologous to the sequence of fluorescently labeled probes, and melting curve analysis is performed after the fluorescent probes are hybridized with tag complementary sequences in PCR-specific products. In this study, 2D PCR and PCR sequence-specific primer (PCR-SSP) were combined to discriminate different alleles in a single reaction, which has the characteristics of high throughput, and compared with other typing techniques; the typing results can be obtained without additional operations. Methods: The ABO*A1.01 allele genetic sequence was used as the reference sequence. The specific sense and antisense primers for seven common ABO alleles were designed on exons 6 and 7 according to the principle of 2D PCR and PCR-SSP. Single nucleotide polymorphism sites for identifying seven alleles were detected in FAM and HEX channels, respectively. Two hundred sixty DNA samples were enrolled for rapid ABO genotyping by 2D PCR, and 95 of them were selected for Sanger sequencing. The Kappa test was used to analyze the agreement of the methodologies. Results: These 7 alleles each had four characteristic melting valleys at different single nucleotide polymorphism loci. A total of 15 genotypes were detected, including ABO*A1.01/A1.02, ABO*A1.01/O.01.01, ABO*A1.01/O.01.02, ABO*A1.02/A1.02, ABO*A1.02/O.01.01, ABO*A1.02/O.01.02, ABO*B.01/B.01, ABO*B.01/O.01.01, ABO*B.01/O.01.02, ABO*O.01.01/O.01.01, ABO*O.01.01/O.01.02, ABO*O.01.02/O.01.02, ABO*A1.01/B.01, ABO*A1.02/B.01, and ABO*B.01/O.01. v (containing a rare ABO*O allele, based on the sequencing results). The Kappa test showed completely consistent results for 2D PCR and Sanger sequencing (Kappa = 1). Conclusion: The 2D PCR technique could be used for molecular typing of the ABO blood group, which was efficient, rapid, accurate, and economical.

Multiple CD59 Polymorphisms in Chinese Patients with Mycobacterium tuberculosis Infection.

A case-control study was conducted to investigate the SNPs at CD59 rs1047581, rs7046, rs2231460, rs184251026, rs41275164, rs831633, rs704700, rs41275166, and rs10768024 by sequence-specific primer-polymerase chain reaction (SSP-PCR) in 900 tuberculosis patients and 1,534 controls. The minor allele frequencies at rs2231460, rs184251026, rs41275164, and rs41275166 were extremely low both in the Cases (0.00%-0.61%) and in the Controls (0.07%-0.43%), comparatively at rs1047581, rs7046, rs831633, rs704700, and rs10768024 were notably higher both in the Cases (8.23%-48.39%) and in the Controls (8.57%-47.16%). Among the nine SNPs, only homozygous CC genotype at rs10768024 showed a significant protective effect against TB than homozygous TT genotype (OR(95% CI) = 0.59(0.38, 0.91), χ 2 = 5.779, P = 0.016), and homozygous TT and heterozygous CT genotypes showed a significant risk of TB infection in the recessive model (OR(95% CI) = 1.68(1.10, 2.56), χ 2 = 5.769, P = 0.016). Further analysis verified that rs10768024 CC genotype independently related to TB susceptibility (OR(95% CI) = 0.60(0.39, 0.91), Wald χ 2 = 5.664, P = 0.017) in multivariate logistic regression analysis, and its genetic mutation was independent of the other SNPs (r 2 = 0.00-0.20) in haplotype analysis. The first investigation of the CD59 gene and susceptibility to TB suggests a significant risk with homozygous TT and heterozygous CT genotypes at rs10768024 loci. The homozygous CC mutation at rs10768024 loci showed a significant protection against TB susceptibility.

The Influence of TNF-α, IL-6, TGF-β1, IFN-γ, IL-10 Polymorphisms on Predisposition to Diabetes Mellitus among Jordanian Patients.

Diabetes mellitus is a long-term disorder with high prevalence globally. It can be classified into two types: Type 1 diabetes and Type 2 diabetes mellitus. Diabetes mellitus is considered a multifactorial disorder in which genetic factors such as cytokines play a major role. Cytokines play a role in immune modulation and are associated with the development of diabetes mellitus. Single nucleotide polymorphisms in cytokines were studied extensively in different populations to determine their association with a predisposition to diabetes mellitus. The aim of this study was to estimate the frequency of single nucleotide polymorphisms in the cytokine genes TNF-α, TGF-β, IL-6, IL-10, and IFN-γ in 102 Jordanian diabetes mellitus patients in comparison to 50 controls and their association to diabetes mellitus susceptibility. Analysis was performed using the highly specific polymerase chain reaction-sequence specific primers methodology. Our findings showed that the IL-10-1082 G/G genotype (P = 0.02) and the TGF-β1 codon 25*G allele (P < 0.01) may be considered risk factors for type 2 diabetes mellitus. In addition, the IFN-γ-874*A allele (P = 0.04) seems to increase the predisposition to type 1 diabetes. Our study showed that the IL-10-1082 G/G genotype and TGF-β1 codon 25*G allele are associated with type 2 diabetes mellitus while the IFN- γ -874*A allele is associated with type 1 diabetes. Our findings may help in the early detection of diabetes mellitus, which would in turn help in undergoing the needed preventative measures to delay the onset of diabetes mellitus.

Interleukin -37 in rheumatoid arthritis: Correlation with clinical severity and genetic polymorphisms in Mosul city, Iraq

The contribution of anti-inflammatory cytokines to rheumatoid arthritis (RA) is not fully comprehended. In the current research we assessed the serum concentration of interleukin (IL)-37 anti-inflammatory cytokine in RA, studied its association to disease activity score 28 (DAS28) and investigated single nucleotide polymorphism (rs2723176) of IL-37 gene as a threat for RA development. The case-control study included 60 RA patients and 30 normal control individuals. Serum IL-37 was assessed by ELISA and genotyped by "sequence-specific primer-polymerase chain reaction (SSP-PCR)". The mean IL-37 was elevated in RA patients (69.42 ng /l ± 62.99) compared to control individuals (14.66 ng /l ± 23.58, p < 0.001). IL-37 tended to increase with age where highest levels were noted in patients more than 60 years (p = 0.037). No Gender influence was found on IL-37 level (p>0.05). At best cut-off value of 31.5 ng/ l, IL-37 had a sensitivity of 73.3% and specificity of 83.3%. No correlation of IL-37 with DAS 28 score was observed (r=0.1497, p=0.2535). For IL-37 (rs2723176) gene polymorphism, C/C genotype was prevailing in both RA (90%) and normal controls (93.3%) compared to A/C or A/A. Also, no variation was found between patents and controls in regard to C/C genotype (OR = 0.643, 95% CI (0.122-3.39, p=0.603). The mean IL-37 concentration in RA patients with C/C genotype (59.70± 67.92) was not different from AC genotype (80.54± 94.18, p=0.4748). We concluded that serum IL-37 had the implication as a diagnostic marker in RA. However, it did not correlate with clinical severity of the disease. Meanwhile, IL-37 (rs2723176) gene polymorphism did not seem to be as a risk factor for RA, nor contributed to the increase of IL-37 level among patients.

The association of HLA-DRB1 alleles and MBL2 gene variant in pediatric acute lymphoblastic leukemia patients

IntroductionEpidemiologic studies on pediatric acute lymphoblastic leukemias (ALL) have been conducted to evaluate the possible risk factors including genetic, infectious and environmental factors with the objective of idenfying the etiology. Mannose-binding lectin 2 (MBL2) plays an important role in first-line immune defense. HLA DRB1 alleles play a role in presentation of peptides to T cells and in activation of the adaptive immune response. ObjectiveIn our study, we aimed to investigate both the MBL2 gene variant and HLA-DRB1 alleles in pediatric ALL patients. MaterialsIn this study, 86 high-risk ALL patients and 100 controls were included. Polymerase Chain Reaction (PCR)-Restriction Fragment Length Polymorphism (PCR-RFLP) and PCR-sequence specific primer (SSP) methods were used for detection of polymorphism of the MBL2 and HLA-DRB1 alleles, respectively. ResultsThe frequency of the MBL2 AB genotype was lower in female ALL patients, compared to male ALL patients (p = 0.034). An association was found between the MBL2 BB genotype and DRB1*07 and among patients with the MBL2 BB genotype; those who also carried the DRB1*07 and *04 alleles were significantly higher than those without the DRB1*07 and *04 alleles. (p = 0.048, p = 0.022, respectively). ConclusionThis is the first study suggesting that the MBL2 BB genotype in association with the DRB1*07 or co-inheritance of the HLA-DRB1*04 and HLA DRB1*07 may have an impact on the etiopathogenesis of the disease.

Genotyping for Scianna blood group system polymorphism in Pakistan.

Objective: To establish the occurrence of Scianna blood group alleles via genotyping in Pakistani blood donors. Study Design: Cross-sectional Analytical study. Setting: Department of Hematology, Army Medical College (AMC) in association with the Armed Forces Institute of Transfusion (AFIT) Rawalpindi. Period: January 2019 and January 2020. Material &amp; Methods: In this study 300 blood donors were incorporated after taking informed consent; DNA was extracted from their EDTA anticoagulated blood samples and tested for SC*01 and SC*02 alleles by Sequence-Specific Primer Polymerase Chain Reaction (PCR-SSP), which was ensued by gel electrophoresis. The allele frequencies were established and Hardy Weinberg equilibrium was determined. Chi-square test was applied to compare the observed and expected genotype frequencies as well as for comparison with published allele frequencies of other populations. We used SPSS version 22 for the analysis of study data. Results: All donors were homozygous for SC*01, demonstrating allele frequency for SC*01 nearly 1.00. Genotype frequencies were congruent with the Hardy Weinberg equilibrium. The difference between the allele frequencies of SC*01 and SC*02 alleles of population of Pakistan was not statistically significant when compared to those of other populations. Conclusion: This maiden research illustrates Scianna blood group distribution in Pakistani community by PCR-SSP. This method is cheap and proficient and can be applied in developing nations. The findings of this study can aid in the production of local red cell panels, detection of uncommon blood types, and setting up of a domestic rare blood donor platform.

A simple PCR-SSP method for detection of HLA-B*15:02, *15:13, and *15:21

Abstract Objectives This study aims to develop a simple polymerase chain reaction (PCR) sequence-specific primer method, which will be used to genotype HLA-B*15:02, HLA-B*15:13, and HLA-B*15:21. Methods DNA was extracted from whole blood using a commercial DNA extraction kit. New specific primers were designed. The PCR was optimized for reproducibility and specificity. Parameters investigated included concentrations of MgCl2, primers concentration, and annealing temperature, to produce specific bands of interest. Known DNA samples were selected at random and tested using the PCR method. Results Simultaneously six duplex PCRs were successfully optimized using 1.0 mM MgCl2 and an annealing temperature of 62 °C. The method was also reproducible and specific. The results from the method showed 100% concordance with known DNA samples. Conclusions This study has successfully developed a simple and specific method to simultaneously genotype HLA-B*15:02, HLA-B*15:13, and HLA-B*15:21 using in-house developed PCR-SSP.

Correlation between gene polymorphisms of killer cell immunoglobulin-like receptors and their ligands and Graves' disease

Objective: To explore the relationship between gene polymorphism of killer cell immunoglobulin-like receptor (KIR) and its ligand-specific human leukocyte antigen C (HLA-C) and Graves' disease (GD). Methods: Case-control study. A total of 118 unrelated GD patients (GD group) admitted to Shandong Provincial Hospital from January 2011 to December 2017 and 108 age-and sex-matched healthy controls (healthy control group) were included. The KIR genotype and its ligand HLA-C allele were detected by polymerase chain reaction sequence-specific primers (PCR-SSP). The distribution of KIR/HLA-C gene combination in GD patients and control population was analyzed to explore its association with the occurrence of GD. Results: In GD group, there were 29 males and 89 females, aged (38±14) years. In the healthy control group, there were 28 males and 80 females, aged (37±13) years. Compared with the healthy control group, the occurrence frequency of HLA-Cw01 was higher in GD group[36.4%(43/118) vs 18.5%(20/108), P=0.003], and the occurrence frequency of HLA-Cw03 and HLA-Cw06 was lower in GD group[11.9%(14/118) vs 39.8%(43/108), P<0.001; 9.3%(11/118) vs 18.5%(20/108), P=0.045]. The frequency of KIR2DL1/HLA-C2 gene combination in GD group was lower than that in control group [17.8%(21/118) vs 34.3%(37/108), P=0.005]. Logistic regression analysis showed that KIR2DL1/HLA-C2 gene combination was a protective factor for GD occurrence (OR=0.308, 95%CI: 0.126-0.752, P=0.010). Conclusions: The polymorphism of KIR/HLA-C gene is related to GD. The low expression of KIR2DL1/HLA-C2 in GD patients may be a protective factor for GD.

Utility of human leukocyte antigen DQ2 and DQ8 genotypes in Celiac disease: Two sides of the coin

Human Leukocyte Antigen (HLA) DQ2 and DQ8 are the known risk genotypes for Celiac Disease. Diagnosis of celiac disease requires serology testing and intestinal biopsy however, genetic testing could also be implemented because of its high negative predictive value. Our study, initiated prior to the publication of 2020 guidelines, evaluated the utility of HLA DQ2 and DQ8 genotyping in the cohort of 537 children with celiac disease and their 1420 first-degree relatives. We attempted to evaluate its applicability in low-middle-income countries like India. Prevalence of celiac disease was observed at 18.52% and 15.68% based on serological and histopathological diagnosis in first-degree relatives. HLA DQA1*0501 and DQB1*0201 alleles were the most frequently observed alleles in index cases (84.4% versus 86.4%), biopsy proven first-degree relatives (77.9% vs 72.15) and serology negative first-degree relatives (67.7% vs 58.3%) (p&lt;0.001). A strong association of DQA1*0501 and DQA1*0301 alleles was observed with high serology positivity (p&lt;0.05). The majority of the subjects in our cohort had histopathologic scores of 3c (54.80%), 3b (22.13%), 3a (22%) and grade 2 (1.05%) (p&lt;0.001). HLA DQB1*0201 was observed as 100% in cases with Marsh grade 2, 72.5% in grade 3a, 82.7% in grade 3b and 85.6% in grade 3c (p=0.017) mucosal lesions. HLA DQA1*0501 and DQB1*0201 alleles of the DQ2 genotype also predicted the severity of intestinal mucosal damage assessed by Marsh grading when used in conjunction with anti-tTG-IgA. When performed in-house using polymerase chain reaction-sequence specific primers method, the assay was economical in identification of mucosal severity in index cases and the first-degree relatives. Its value as a model for additive predictive value in the diagnostic algorithm of cases with tTG-IgA less than 10 times of the upper limit of normal needs further evaluation.

Utility of human leukocyte antigen DQ2 and DQ8 genotypes in Celiac disease: Two sides of the coin

Human Leukocyte Antigen (HLA) DQ2 and DQ8 are the known risk genotypes for Celiac Disease. Diagnosis of celiac disease requires serology testing and intestinal biopsy however, genetic testing could also be implemented because of its high negative predictive value. Our study, initiated prior to the publication of 2020 guidelines, evaluated the utility of HLA DQ2 and DQ8 genotyping in the cohort of 537 children with celiac disease and their 1420 first-degree relatives. We attempted to evaluate its applicability in low-middle-income countries like India. Prevalence of celiac disease was observed at 18.52% and 15.68% based on serological and histopathological diagnosis in first-degree relatives. HLA DQA1*0501 and DQB1*0201 alleles were the most frequently observed alleles in index cases (84.4% versus 86.4%), biopsy proven first-degree relatives (77.9% vs 72.15) and serology negative first-degree relatives (67.7% vs 58.3%) (p&lt;0.001). A strong association of DQA1*0501 and DQA1*0301 alleles was observed with high serology positivity (p&lt;0.05). The majority of the subjects in our cohort had histopathologic scores of 3c (54.80%), 3b (22.13%), 3a (22%) and grade 2 (1.05%) (p&lt;0.001). HLA DQB1*0201 was observed as 100% in cases with Marsh grade 2, 72.5% in grade 3a, 82.7% in grade 3b and 85.6% in grade 3c (p=0.017) mucosal lesions. HLA DQA1*0501 and DQB1*0201 alleles of the DQ2 genotype also predicted the severity of intestinal mucosal damage assessed by Marsh grading when used in conjunction with anti-tTG-IgA. When performed in-house using polymerase chain reaction-sequence specific primers method, the assay was economical in identification of mucosal severity in index cases and the first-degree relatives. Its value as a model for additive predictive value in the diagnostic algorithm of cases with tTG-IgA less than 10 times of the upper limit of normal needs further evaluation.

Juvenile Idiopathic Arthritis and Its HLA Association: A Study from Bangladesh

Background: HLA alleles may have association with overall JIA including specific sub-types. Determation of HLA DRB1, DPB1, DQA1, DQB1 and B27, may be helpful to diagnose JIA cases where diagnostic dilemma is present. The aim of the study was to find out the association of HLA alleles with JIA and its subtypes. Methods: This cross sectional study was conducted in the department of Pediatrics, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, from April 2020 to March 2021. A total of 50 cases and 25 controls attending pediatric rheumatology clinic and pediatric OPD were enrolled in the study. Data were collected by a pre-designed questionnaire containing socio-demographic, clinical and laboratory parameters. HLA DR-B1, DQ-A1, DQ-B1 and B27 typing were done for the cases and controls from blood samples by polymerase chain reaction sequence specific primer (PCR-SSP) method. Data were analyzed by SPSS version 26. Frequency, percentage, chi square test and Fisher exact tests were done for statistical analysis. Results: HLA DR-B1_10 had significant positive association whereas HLA DR-B1_03 and B1_16 had negative association with overall JIA cases. Poly-articular JIA, oligo-articular JIA and ERA had association with HLA DRB1_01, B1_08 and B1_12 respectively. HLA DQA1-01 and A1_02 had positive association. However, DQ-A1_06 was negatively associated with JIA cases. Oligo JIA was positively associated with HLA DQ-A1_01, whereas poly JIA and ERA had association with A1_02. Though, HLA DQ_B1 had no association with overall JIA cases, analysis showed association of poly JIA with DQ-B1_04. Frequency of HLA DQ-B1_02 and 03 were higher among ERA cases, though not significant, but they had strong association with HLA B27. Conclusion: HLA associations were present with overall JIA and each sub-groups had different patterns of HLA associations including some protective roles.

Baseline Data and HLA Alleles and Haplotypes Diversity and Panel Reactive Antibody in Kidney Transplant Candidates in Southwest China.

To investigate the baseline data characteristic, human leukocyte antigen (HLA) polymorphisms, and panel reactive antibody (PRA) in end-stage kidney disease (ESKD) patients awaiting kidney transplantation in Southwest China. HLA genotyping was performed using the real-time PCR sequence-specific primer. PRA was detected by enzyme-linked immunosorbent assay. The patients' medical records were extracted from the hospital information database. A total of 281 kidney transplant candidates with ESKD were analyzed. The average age was 35.7 ± 13.8 years. There were 61.6% patients had hypertension, 40.2% patients had dialysis ≥ 3 times per week, 47.3% patients had moderate or severe anemia, 30.2% patients with albumin < 35 g/L, 49.1% patients had serum ferritin < 200 ng/mL, 40.5% patients had serum calcium in target range (2.23 - 2.80 mmol/L), 43.4% patients had serum phosphate in target range (1.45 - 2.10 mmol/L), and 93.6% patients with parathyroid hormone > 88.00 pg/mL. In total, 15 HLA-A, 28 HLA-B, 15 HLA-DRB1, and 8 HLA-DQB1 allelic groups were identified. The most frequent alleles for each locus were HLA-A*02 (33.63%), HLA-B*46 (14.41%), HLA-DRB1*15 (21.89%), and HLA-DQB1*05 (39.50%). The most frequent haplotypes were HLA-A*33-B*58-DRB1*17-DQB1*02. A total of 9.60% of patients tested positive for PRAs - Class I or Class II. The data from this study provide some new insights into baseline data, the distribution of HLA polymorphisms, and PRA results in the population of Southwest China. This is of great significance in this region, and indeed in the country as a whole, in comparison with other populations and in the process of organ transplant allocation.

KIR genotype and haplotype frequencies in the multi-ethnic population of Malaysia

Killer cell immunoglobulin-like receptors (KIR) genotype and haplotype frequencies have been reported to vary distinctly between populations, which in turn contributes to variation in the alloreactivity of natural killer (NK) cells. Utilizing the diverse KIR genes to identify suitable transplant donors would prove challenging in multi-ethnic countries, even more in resource-limited countries where KIR genotyping has not been established. In this study, we determined the KIR genotypes from 124 unrelated Malaysians consisting of the Malays, Chinese, Indians, and aboriginal people through polymerase chain reaction sequence-specific primer (PCR-SSP) genotyping and employing an expectation–maximization (EM) algorithm to assign haplotypes based on pre-established reference haplotypes. A total of 27 distinct KIR haplotypes were discerned with higher frequencies of haplotype A (55.2%) than haplotype B (44.8%). The most frequent haplotypes were cA01:tA01 (55.2%), cB01:tB01 (18.1%), and cB02:tA01 (13.3%), while the least frequent haplotypes were cB03:tB01 (1.2%), cB04:tB03 (0.4%), and cB03:tA01 (0.4%). Several haplotypes were identified to be unique to a specific ethnic group. The genotype with the highest frequency was genotype AB (71.8%), followed by AA (19.4%), and BB (8.9%). The Indians exhibited the lowest genotype AA but the highest genotype BB, whereas genotype BB was absent in the aboriginal people. Despite the limitations, the genotype and haplotypes in the Malaysian population were successfully highlighted. The identification of ethnic-specific KIR genotypes and haplotypes provides the first step to utilizing KIR in identifying suitable transplant donors to further improve the transplant outcome in the Malaysian population.