Polymerase Chain Reaction-sequence Specific Oligonucleotide Probes Research Articles

Risk prediction and function evaluation by T-cell epitope model and expression model of HLA-DPB1 mismatching in unrelated-donor hematopoietic stem cell transplantations

Objective: To evaluate the risk prediction and assessment function of HLA-DPB1 T-cell epitope (TCE) model and expression model in human leukocyte antigen (HLA)-matched unrelated hematopoietic stem cell transplantation (MUD-HSCT) with HLA-DPB1 mismatching. Methods: A total of 364 (182 pairs) potential MUD-HSCT donors and recipients confirmed by HLA high-resolution typing in Shaanxi Blood Center from 2016 to 2019 were analyzed retrospectively. Of the 182 recipients, there were 121 males and 61 females with an average age of (26.3±14.2) years. Of the 182 donors, there were 148 males and 34 females with an average age of (33.7±7.5) years. Polymerase chain reaction-sequence-based typing (PCR-SBT), next-generation sequencing (NGS) and polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSO) based on LABScan®3D platform were used for high-resolution typing of HLA-A, B, C, DRB1, DQB1, DPB1 gene, and PCR-SBT was used for single nucleotide polymorphism (SNP) typing. TCE model and expression model were used to predict and evaluate the HLA-DPB1 mismatch pattern and acute graft-versus-host-disease (aGVHD) risk. Results: A total of 26 HLA-DPB1 alleles and their 3'-UTR rs9277534 SNP genotypes were detected in this study population, and two new alleles HLA-DPB1*1052∶01 and HLA-DPB1*1119∶01 were found and officially named. The overall mismatch rate of HLA-DPB1 in MUD-HSCT donors and recipients was 90.66% (165/182). In TCE model, the HLA-DPB1 mismatch rates of permissible mismatch (PM) and non-permissible mismatch (non-PM) were 47.80% (87/182) and 42.86% (78/182), respectively. The non-PM in GvH direction was 13.73% (25/182), and which in HvG direction was 29.12% (53/182). A total of 73 pairs of donors and recipients in TCE model met the evaluation criteria of expression model. Among of TCE PM group, recipient DP5 mismatches accounted for 34.25% (25/73) were predicted as aGVHD high risk according to expression model. For the TCE non-PM group, both the recipient DP2 mismatches of 6.85% (5/73) and recipient DP5 mismatches of 10.86% (8/73) were predicted to be at high risk for aGVHD. Risk prediction by TCE model and expression model was 27.27% concordant and 16.97% unconcordant. Conclusions: TCE model and expression model are effective tools to predict aGVHD risk of MUD-HSCT. Comprehensive application of the two models is helpful to the hierarchical assessment of HSCT risk.

HLA molecular study of patients in a public kidney transplant program in Guatemala

Guatemala is a country located in Central America, and while it is one of the most populated countries in the region, the genetic diversity of the population has been poorly analyzed. Currently, there are no analyses of the distribution of human leukocyte antigen (HLA) system alleles in mixed ancestry (i.e., ladino) populations in Guatemala. The HLA system exhibits the most extensive polymorphism in the human genome and has been extensively analyzed in a large number of studies related to disease association, transplantation, and population genetics (with particular importance in the understanding of diversity in the human population). Here, we present HLA typing data from 127 samples of unrelated individuals from the kidney transplant program of the San Juan de Dios General Hospital (Guatemala City) using a PCR-SSOP-based (PCR-sequence specific oligonucleotide probes) typing method. We found 16 haplotypes that accounted for 39.76 % of the total haplotype diversity, of which thirteen have been reported previously in Native American populations and three have been reported in European populations. The analyses showed no deviations from Hardy-Weinberg equilibrium, and admixture estimates calculated with k = 3 ancestral components showed that Native American was the most represented component, followed by the European component. The African component was less prominent in the Guatemala mixed ancestry sample in comparison to samples from other countries in Central America. The HLA-based admixture results for Central America showed a continuum in the distribution of Native American, European and African ancestries throughout the region, which is consistent with the complex demographic history of the region.

Analysis of loss of heterozygosity at HLA loci in a patient with leukemia

To detect loss of heterozygosity (LOH) at human leukocyte antigen (HLA) loci in a Chinese patient with leukemia after haploidentical hematopoietic stem cell transplantation. HLA genotyping was carried out on peripheral blood, hair follicle and buccal swab samples derived from the patient after the transplantation as well as peripheral blood samples from his parents by using PCR-sequence specific oligonucleotide probe method and PCR-sequence based typing method. Short tandem repeat (STR) loci were detected by using a 23 site STR assay kit and a self-developed 6 STR loci assay for the HLA regions. After the transplantation, the HLA genotype of the peripheral blood sample of the patient was identical to his father. The patient was HLA-A*02:01,24:02, C*03:03,03:04, B*13:01,15:01, DRB1*08:03,12:02, DQB1*03:01,06:01 for his hair follicle specimen. However, homozygosity of the HLA loci was found in his buccal swab sample. Only the HLA-A*24:02-C*03:03-B*15:01-DRB1*08:03-DQB1*06:01 haplotype from his father's was present, while the HLA-A*02:01-C*03:04-B*13:01-DRB1*12:02-DQB1*03:01 haplotype from his mother was lost. After the transplantation, the alleles of the 23 STR sites in the patient's peripheral blood sample were consistent to his father, with no allelic loss detected in his buccal swab sample. However, at least 4 STR loci in the HLA region were lost in his buccal swab sample. LOH at the HLA loci has been detected in the buccal swab sample of a patient with leukemia who received haploidentical hematopoietic stem cell transplantation.

MICA and KIR: Immunogenetic Factors Influencing Left Ventricular Systolic Dysfunction and Digestive Clinical Form of Chronic Chagas Disease.

Tissue damage observed in the clinical forms of chronic symptomatic Chagas disease seems to have a close relationship with the intensity of the inflammatory process. The objective of this study was to investigate whether the MICA (MHC class I-related chain A) and KIR (killer cell immunoglobulin-like receptors) polymorphisms are associated with the cardiac and digestive clinical forms of chronic Chagas disease. Possible influence of these genes polymorphisms on the left ventricular systolic dysfunction (LVSD) in patients with chronic Chagas heart disease was also evaluated. This study enrolled 185 patients with positive serology for Trypanosoma cruzi classified according to the clinical form of the disease: cardiac (n=107) and digestive (n=78). Subsequently, patients with the cardiac form of the disease were sub-classified as with LVSD (n=52) and without LVSD (n=55). A control group was formed of 110 healthy individuals. Genotyping was performed by polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP). Statistical analyzes were carried out using the Chi-square test and odds ratio with 95% confidence interval was also calculated to evaluate the risk association. MICA-129 allele with high affinity for the NKG2D receptor was associated to the LVSD in patients with CCHD. The haplotype MICA*008~HLA-C*06 and the KIR2DS2-/KIR2DL2-/KIR2DL3+/C1+ combination were associated to the digestive clinical form of the disease. Our data showed that the MICA and KIR polymorphisms may exert a role in the LVSD of cardiac patients, and in digestive form of Chagas disease.

Spectrum of cutaneous adverse reactions to aromatic antiepileptic drugs and human leukocyte antigen genotypes in Thai patients and meta-analysis

Aromatic antiepileptic drugs (AEDs)-induced cutaneous adverse drug reactions (cADRs) add up to the limited use of the AEDs in the treatment and prevention of seizures. Human leukocyte antigen-B (HLA-B) alleles have been linked to AEDs-induced cADRs. We investigated the association between cADRs (including Stevens–Johnson syndrome; SJS/toxic epidermal necrolysis; TEN, drug reaction with eosinophilia and systemic symptoms; DRESS, and Maculopapular eruption; MPE) caused by AEDs (phenytoin, carbamazepine, lamotrigine, phenobarbital and oxcarbazepine) and HLA-B alleles in Thai population. Through the case-control study, 166 patients with AEDs-induced cADRs, 426 AEDs-tolerant patients (AEDs-tolerant controls), and 470 healthy subjects (Thai population) were collected. The HLA genotypes were detected using the polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) method. We also performed a meta-analysis with these data and other populations. The carrier rate of HLA-B*15:02 was significantly different between AEDs-induced cADRs group and AEDs-tolerant group (Odds ratio; OR 4.28, 95% Confidence interval; CI 2.64–6.95, p < 0.001), AEDs-induced cADRs group and Thai population (OR 2.15, 95%CI 1.41–3.29, p < 0.001). In meta-analysis showed the strong association HLA-B*15:02 with AEDs-induced cADRs (OR 4.77, 95%CI 1.79–12.73, p < 0.001). Furthermore, HLA-B*15:02 was associated with SJS/TEN induced by AEDs (OR 10.28, 95%CI 6.50–16.28, p < 0.001) Phenytoin (OR 4.12, 95%CI 1.77–9.59, p = 0.001) and carbamazepine (OR 137.69, 95%CI 50.97–371.98, p < 0.001). This study demonstrated that genetic association for AEDs-induced cADRs was phenotype-specific. A strong association between HLA-B*15:02 and AEDs-induced SJS/TEN was demonstrated with an OR of 10.79 (95%CI 5.50–21.16, p < 0.001) when compared with AEDs-tolerant group. On the other hand, the carrier rates of HLA-B*08:01, HLA-B*13:01, and HLA-B*56:02 were significantly higher in the DRESS group compared with the AEDs-tolerant group (p = 0.029, 0.007, and 0.017, respectively). The HLA-B*15:02 allele may represent a risk factor for AEDs-induced cADRs.

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.

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 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.

Optimization of HLA-B*27 ALLELE Genotyping by PCR-SSP

OBJECTIVES:HLA-B27 is strongly associated with ankylosing spondylitis (AS) and its presence helps to confirm AS diagnosis. Due to the high HLA polymorphism and the differentiated contribution of alleles and molecules encoded by them, HLA-B*27 allele identification is relevant in the clinical follow-up, diagnosis, and treatment of this spondyloarthropathy. Inexpensive genotyping techniques with high specificity and sensitivity are of great interest in histocompatibility laboratories. This work aimed to optimize HLA-B*27 genotyping by Polymerase Chain Reaction Sequence-specific Primer (PCR-SSP), which is an accessible and inexpensive technique.METHODS:The PCR-SSP was standardized using 26 HLA-B*27 positive and 3 HLA-B*27 negative samples previously defined by Polymerase Chain Reaction Sequence-specific Oligonucleotide Probes (PCR-SSOP) (medium resolution, One Lambda®) and primers described by Duangchanchot et al. (2009). For validating the technique, 397 samples were genotyped using PCR-SSP as well as PCR-SSOP.RESULTS:The PCR-SSP technique was standardized for identifying the alleles HLA-B*27:02, HLA-B*27:CAFRW (05/13/16/17/28/37/38/39/42), HLA-B*27:CAFRZ (08/26/40), HLA-B*27:09 and HLA-B*27:12, which were found in 90 positive samples (22.67%). There was 100% agreement between the two techniques for heterozygous samples; however, two homozygous samples could not be detected by PCR-SSP.CONCLUSION:The HLA-B*27 genotyping using PCR-SSP, an easy-to-use, specific, and affordable technique, was optimized for heterozygous samples. This technique may contribute to AS diagnosis.

Human leukocyte antigen polymorphism of HIV infected persons without disease progress for long-term in Henan province, 2011-2016

Objective: To understand the disease progression and human leukocyte antigen (HLA) gene polymorphism of HIV-infected persons without disease progress for long term, also known as long-term non-progressors (LTNPs), in Henan province. Methods: A retrospective study was conducted in 48 LTNPs with complete detection and follow-up information during 2011-2016 in Henan. Changes of CD(4)(+)T cells counts (CD(4)) and viral load (VL) during follow-up period were discussed. Polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) was used for the analyses of HLA-A, HLA-B and HLA-DRB1 alleles between LTNPs and healthy controls. Results: From 2011 to 2016, forty-eight LTNPs showed a decrease of the quartile (P(25)-P(75)) of CD(4) from 601.00 (488.50-708.72)/μl to 494.00 (367.00-672.00)/μl, and the difference was significant (P<0.05). The increase of the quartile (P(25)-P(75)) of log(10)VL from 3.40 (2.87-3.97) to 3.48 (2.60-4.37), but the difference was not significant (P>0.05). HLA polymorphism analysis revealed that HLA-B*13:02 and HLA-B*40:06 were more common in LTNPs (P<0.05), while HLA-B*46:01 and HLA-DRB1*09:01 were more common in healthy controls (P<0.05). Conclusions: The CD(4) of LTNPs in Henan showed a downward trend year by year. HLA-B*13:02 and B*40:06 might be associated with delayed disease progression for HIV infected persons in Henan.

Validation of a novel single-drop rapid human leukocyte antigen-DQ2/-DQ8 typing method to identify subjects susceptible to celiac disease.

Background and AimHuman leukocyte antigen (HLA)‐DQ2 and/or ‐DQ8 is an essential risk factor for celiac disease (CD). About 90–95% of patients with CD carry HLA‐DQ2/‐DQ8 alleles, and HLA‐DQ typing is considered an additional diagnostic test. Conventional polymerase chain reaction (PCR)‐based HLA‐DQ typing methods are expensive, complex, and a time‐consuming process. We assessed the efficacy of a novel HLA‐DQ typing method, “Celiac Gene Screen,” for the detection of CD‐associated HLA haplotypes.MethodsTo assess the diagnostic performance of the Celiac Gene Screen test, 100 ethylenediaminetetraacetic acid (EDTA) blood samples, already characterized by the conventional HLA‐DQ typing method, that is, PCR sequence‐specific oligonucleotide probes (PCR‐SSOP), a concordance between both the methods were explored. For validity, a further 300 EDTA blood samples with unknown HLA‐DQ status were genotyped using the Celiac Gene Screen test, including 141 samples from CD, 56 first‐degree relatives (FDRs) of CD and 103 samples from controls.ResultsOf the 100 samples with known status of HLA‐DQ alleles, 79 samples were HLA‐DQ2 and/or ‐DQ8 positive, and 21 samples were HLA‐DQ2 and/or ‐DQ8 negative by conventional PCR. These 100 samples were re‐typed using the Celiac Gene screen kit; all 79 positives were typed positive, and 21 negatives were typed negative for HLA‐DQ alleles. Among 300 samples with unknown HLA‐DQ status, 118 of 141 (84%) patients with CD, 48 of 56 (86%) FDRs of CD, and 52 of 103 (50%) controls typed positive for HLA‐DQ alleles.ConclusionsThe Celiac Gene Screen HLA‐DQ typing method showed excellent concordance with the conventional HLA‐DQ typing method and could be a cost‐reducing and effective method for CD‐associated HLA screening.

Association of HLA alleles (A, B, DRB1) and HIV-1 infection in the Han population of Hubei, China.

The HIV susceptibility and resistance alleles in the HLA genes were determined by investigating the distribution characteristics of the HLA alleles (A, B, and DRB1) in HIV-infected individuals of the Han population in Hubei, and by comparing these alleles with HIV-negative individuals from the same area. A cohort of 424 HIV-1 infected individuals were chosen as study subjects, and 836 HIV-negative healthy subjects from the same area served as the control population. HLA-A, B, and DRB1 allele typing was performed using polymerase chain reaction-sequence-specific oligonucleotide probes (PCR-SSOP) and polymerase chain reaction-sequencing based typing (PCR-SBT) techniques. Arlequin ver3.0 was used to analyze the allele and haplotype frequencies of HLA-A, B, and DRB 1, whereas Epi Info 7 and SPSS18.0 was used to analyze the differences in the HLA alleles between the HIV-1 positive and HIV-1 negative groups. A*02:03, DRB1*01:01, and DRB1*15:01 alleles and their haplotypes as well as the HLA_Bw4-Bw6 hybrid showed a protective effect on HIV-1 infection. After adjusting for confounding factors such as age and sex, multivariate logistic regression analysis revealed that B*15:02G, DRB1*01:01, and DRB1*15:01 subtypes were the resistance genes of HIV-1 infection, while B*13:01 might increase susceptibility to HIV-1 infection. The correlation between A*02:06 and B*15:01G subtypes and HIV-1 susceptibility was independent of the age and sex of the host. This study demonstrated the influence of genetic factors in humans such as HLA polymorphism on individuals to resist HIV-1 infection. Association studies of HLA polymorphism, susceptibility/resistance to HIV-1 infection, and hosts' genetic background are of significant importance for research on HIV-1 pathogenesis and vaccine design.

Frequency of classes I and II HLA alleles in deceased donors of solid organs in Espírito Santo, Brazil

ABSTRACT Introduction: The Brazilian population has a great racial mix, originating mainly from Europeans, Africans and Indians. Human leukocyte antigens (HLA) specificities vary among different population and ethnic groups, since they demonstrate greater similarity in populations of common origin. Objective: To determine the genetic structure of HLA classes I and II alleles in deceased donors of solid organs in the state of Espirito Santo, Brazil, from 2011 to 2015. Methods: The present study covered 208 brain-dead donors of solid organs, from 2011 to 2015, in the state of Espirito Santo, Brazil. HLA typing was performed by polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSP) using kits for generic classes I and II SSP DNA typing test, A, B and DR loci. Results: The profile of deceased donors found in Espirito Santo, Brazil, in the period from 2011 to 2015, was brown men, aged over 40 years. The most frequent HLA alleles were A2 (24.28%), B44 (10.34%) and DR15 (14.18%); and the least frequent, A43 (0.24%), B46, B47, B70, B75 and B78 (0.24%) and DR9 (1.68%), DR12 and DR18 (2.16%). Conclusion: The present study contributed with significant information about the frequency of HLA antigens in deceased donors of solid organs in the Brazilian population that will contribute to optimize the transplantation process in the country.

Association between HLA-B alleles and nevirapine-induced allergies among Indonesian HIV patients

This study aimed to investigate the association between human leukocyte antigen-B (HLA-B) alleles and nevirapine allergy in HIV patients in Indonesia. A case control study was conducted involving 147 HIV patients comprising of 50 patients with and 97 patients without nevirapine allergy as control. The HLA-B allele typing was conducted by using polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP), followed by sequencing. Bivariate analysis using Chi-square (X2) test and multivariate logistic regression with significance level at p<0.05 were applied to analysis the data. Among 147 subjects, 34 with HLA-B alleles were identified. Bivariable analysis showed that HLA-B*58 allele was the most significant risk factor for the nevirapine allergy (OR = 3.67; 95% CI: 1.79 to 7.54), while HLA-B*35 allele was a protective factor (OR = 0.18; 95% CI: 0.08 to 0.42). Multivariate logistic regression analysis showed that young men and HLA-B*58 allele were the significant risk factors of nevirapine allergy (OR: 4.63; 95% CI: 2.02 to 10.61), while older women with the HLA-B*35 was able to reduce the risk of nevirapine allergy approximately 81% (OR: 0.19; 95% CI: 0.08 to 0.49). In conclusion, young male with the HLA-B*58 allele are the high risk factor for nevirapine allergy in Indonesian HIV patients.

Killer immunoglobulin-like receptor repertoire analysis in a Caucasian Spanish cohort with inflammatory bowel disease.

Immunological molecules are implicated in inflammatory disorders, including inflammatory bowel disease (IBD; Crohn disease [CD] and ulcerative colitis [UC]). Killer cell immunoglobulin-like receptors (KIRs) are also genetically variable proteins involved in immune function. They are expressed by NK cells and certain T lymphocytes, regulate specificity and function by interaction with HLA Class I molecules, may be either inhibitory or activating and are polymorphic both in terms of alleles and haplotype gene content. Genetic associations between activating KIRs and certain autoimmune and inflammatory diseases have been reported; however, a possible association between KIR and IBD remains unclear. The aim of this study was to determine the relationship between KIR repertoire and IBD pathologies in a Spanish cohort. KIR variability was analyzed using PCR-sequence specific oligonucleotide probes (SSOP). Inhibitory KIR2DL5 was found more frequently in UC and IBD patient groups than in healthy controls (P = 0.028 and P = 0.01, respectively), as was activating KIR2DS1 (P = 0.02, Pc > 0.05, UC vs. Controls; P = 0.001, Pc = 0.01, IBD vs Controls; P = 0.01, Pc > 0.05, Controls vs CR), KIR2DS5 (P = 0.0028, Pc = 0.04, Controls vs UC; P = 0.0001, Pc = 0.0017, Controls vs IBD; P = 0.01, Pc > 0.05, Controls vs CD) and KIR3DS1 (P = 0.012, Pc > 0.05, Controls vs IBD). Our data suggest that imbalance between activating and inhibitory KIR may partially explain the different pathogeneses of these IBDs and that there is a hypothetical role for the telomeric B region (which contains both KIR2DS5 and KIR2DS1) in these diseases.

Correlation Between HLA-A, B and DRB1 Alleles and Severe Fever with Thrombocytopenia Syndrome

ObjectiveSevere fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever caused by a tick-borne bunyavirus (SFTSV) in East Asian countries. The role of human leukocyte antigen (HLA) in resistance and susceptibility to SFTSV is not known. We investigated the correlation of HLA locus A, B and DRB1 alleles with the occurrence of SFTS.MethodsA total of 84 confirmed SFTS patients (patient group) and 501 unrelated non-SFTS patients (healthy individuals as control group) from Shandong Province were genotyped by PCR-sequence specific oligonucleotide probe (PCR-SSOP) for HLA-A, B and DRB1 loci.Allele frequency was calculated and compared using χ2 test or the Fisher's exact test. A corrected P value was calculated with a bonferronis correction. Odds Ratio (OR) and 95% confidence intervals (CI) were calculated by Woolf’s method.ResultsA total of 11 HLA-A, 23 HLA-B and 12 HLA-DRB1 alleles were identified in the patient group, whereas 15 HLA-A, 30 HLA-B and 13 HLA-DRB1 alleles were detected in the control group. The frequencies of A*30 and B*13 in the SFTS patient group were lower than that in the control group (P = 0.0341 and 0.0085, Pc = 0.5115 and 0.252). The ORs of A*30 and B*13 in the SFTS patient group were 0.54 and 0.49, respectively. The frequency of two-locus haplotype A*30-B*13 was lower in the patient group than in the control group(5.59% versus 12.27%, P = 0.037,OR = 0.41, 95%CI = 0.18–0.96) without significance(Pc>0.05). A*30-B*13-DRB1*07 and A*02-B*15-DRB1*04 had strong associations with SFTS resistance and susceptibility respectively (Pc = 0.0412 and 0.0001,OR = 0.43 and 5.07).ConclusionThe host HLA class I polymorphism might play an important role with the occurrence of SFTS. Negative associations were observed with HLA-A*30, HLA-B*13 and Haplotype A*30-B*13, although the associations were not statistically significant. A*30-B*13-DRB1*07 had negative correlation with the occurrence of SFTS; in contrast, haplotype A*02-B*15-DRB1*04 was positively correlated with SFTS.

HLA-C*18:01: A Rare Allele in the European Caucasian Population Coinciding with Difficult-to-Treat Plaque Psoriasis

Advances in knowledge about the metabolic pathways involved in the pathogenesis of psoriasis and related diseases have led to a search for new therapeutic targets and the development of new biological drugs. Several studies have focused on HLA-C*06 and investigated correlations between the genetic risk factors of psoriasis and clinical parameters such as the severity of the disease and the response to treatment. Our objective is to share experience from our institution in the observation of two patients with severe chronic plaque psoriasis who were unresponsive to any anti-TNF-α treatment and only partially responsive to ustekinumab. The patients are carriers of a rare allele of HLA-C that occurs in Caucasians. The patients with moderate-to-severe chronic plaque psoriasis, and candidates for biological therapy were typed for the HLA-C locus at high resolution via polymerase chain reaction sequence-specific oligonucleotide probes (PCR-SSOP) using a commercial kit (LAB(®)Type, One Lambda Inc., Canoga Park, CA, USA). The socio-demographic variables and clinical data were collected. In our cohort of 134 patients, only two showed the presence of the allele HLA-C*18:01. To our knowledge, a coincidence between HLA-C*18 and severe psoriasis in Caucasian patients has not previously been described. The fact that both of these patients showed the same clinical history (unresponsive to any anti-TNF-α treatment and partially responsive to ustekinumab) cannot be attributed to a random observation because HLA-C*18 is extremely rare in Europe. As a consequence, at this latitude, it probably indicates a severe disease for which the proper therapy has still not been identified.

HLA-B*27 – Frequency of clinical signs in Brazilian patients with spondyloarthritis

Spondyloarthritis presents clinical features, laboratory findings, and similar images, but their clinical manifestations reveal great heterogeneity in patients HLA-B*27 positive and negative. This study compared the frequencies of the clinical manifestations in the presence and absence of HLA-B*27. From the 156 patients with clinical suspicion of spondyloarthritis, 73 had a diagnosis of spondyloarthritis confirmed. The HLA-B*27 gene was identified by polymerase chain-reaction sequence-specific oligonucleotide probe (PCR-SSOP). The Student t test was used to calculate the values of mean and the Fisher's exact test was used to compare proportions. The values of odds ratio (OR) and confidence interval (CI) at 95% were also calculated (p < 0.05). The spondyloarthritis found were: ankylosing spondylitis (n = 47, 64.4%), psoriatic spondyloarthritis (n = 9, 12.3%), undifferentiated spondyloarthritis (n = 9, 12.3%), enteropathic spondyloarthritis (n = 6; 8.2%) and reactive spondyloarthritis (n = 2, 2.7%). Overall, 35 (47.9%) patients were HLA-B*27 positive and 38 (52.1%) were negative. This gene was associated with ankylosing spondylitis (OR: 5.37, 95% CI: 1.813–15.905, p = 0.003) but not with enteropathic spondyloarthritis (OR: 0.07, 95% CI: 0.003–1.301, p = 0.025). The sacroiliitis was associated with HLA-B*27 positive (OR: 10.552, 95% CI: 1.260–88.256, p = 0.014) and intestinal injury with HLA-B*27 negative (OR: 0.195, 95% CI: 0.038–0.978, p = 0.048). The image signals sacroiliitis were associated with the HLA-B*27 gene while intestinal involvement was not associated with this gene.

HLA Genotyping: Methods for the Identification of the HLA-DQ2,-DQ8 Heterodimers Implicated in Celiac Disease (CD) Susceptibility.

In this chapter we will present the principal technical methods to genotype the HLA-DQA1* and -DQB1* alleles associated with celiac disease (CD), corresponding to the serological heterodimers HLA-DQ2 and -DQ8. We will present the methods specific for the genotyping of these heterodimers, which represents a common request from consultant doctors. Because these alleles are also common in healthy subjects, their presence is not diagnostic for CD. Conversely, their absence is more important because it excludes the disease, since CD patients negative for these heterodimers are very rare. Accordingly, HLA typing has been included as a useful test to exclude celiac disease in the ESPGHAN guidelines for diagnosis of celiac disease. The methods for HLA typing described in the present chapter are based on the following techniques: PCR-SSP (Polymerase Chain Reaction-Sequence Specific Primers): PCR with primers specific for HLA alleles encoding the CD risk heterodimers, whose presence is revealed through the electrophoresis of PCR products. Reverse PCR-SSOP (PCR-Sequence Specific Oligonucleotide Probes): PCR with primers specific for a single locus or a large group of alleles followed by hybridization with enzyme-conjugated probes specific for a single allele, immobilized on different supports (i.e., nitrocellulose strips), in which DNA-probes binding is revealed by the production of a colored precipitate derived from the enzymatic modification of a specific substrate. Real-Time PCR (RT-PCR): PCR with locus or allelic specific primers whose amplification is revealed by particular probes (i.e., Taqman probes) hybridizing the DNA template within the two PCR primers and emitting fluorescent while the PCR reaction occurs.

Identification of a novel HLA allele HLA-DRB1*09:01:07

To identify and confirm a novel HLA allele in a Chinese Han individual. HLA typing was performed by polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) for HLA-A, -B and -DRB1 in a registered donor of China Marrow Donor Program(CMDP). Sequencing-based typing (SBT) was carried out to further confirm the novel allele of HLA-DRB1. The SSOP result showed HLA-DRB1*09:03,15GEP, but an unusual pattern that could not be defined indicated potential presence of a novel allele. The SBT result showed the novel sequence has 1nt change from its closest allele DRB1*09:01:02 at nt306 where C to T(codon 73 GCC to GCT) resulting in no amino acid change. 73A was not changed. A novel HLA allele, HLA-DRB1*09:01:07, has been identified and named officially by WHO Nomenclature Committee for Factors of the HLA System.