Oligonucleotide Probe Method Research Articles

Chemical contamination in the extracted DNA affects the results of HLA typing by the PCR-SSOP method.

The purity of the extracted DNA is critical for successful molecular testing. This study aimed to compare the effect of various DNA extraction methods, extraction processes, and sources of consumables, such as microtubes, on PCR results. DNA extraction from whole blood was performed using four different approaches utilizing four types of microtubes: chloroform-based, sodium perchlorate-based, heat-assisted salting out, and solid-phase extraction. Extracted DNA was evaluated by nanodrop spectrophotometry and used in PCR-based methods for human leukocyte antigens (HLA) typing. The lowest and highest concentrations of extracted DNA were observed in the column-based and heat-assisted methods, respectively. The mean ratios of A260/A230, represents chemical contamination, were significantly lower in all extraction methods using all types of microtubes versus "A" microtube. We observed the highest mean ratio of A260/A230 (> 1.9) in the sodium perchlorate method by using the "A" microtube compared to other types of microtubes and extraction methods. Extracted DNA samples using microtube "A" showed acceptable results for HLA typing compared to other microtubes that represented uninterpretable results in the PCR using the sequence-specific oligonucleotide probe (PCR-SSOP) method. Our findings indicate that the chemical contaminations derived mainly from microtubes can decrease the quality of DNA and consequently interfere with the amplification or hybridization reactions in the PCR-SSOP method for HLA typing. Although, technical issues and extraction processes may also influence the quality of DNA.

The role of Killer immunoglobulin-like receptors (KIRs) in the genetic susceptibility to non-celiac wheat sensitivity (NCWS).

Non-celiac wheat sensitivity (NCWS) is an emerging clinical condition characterized by gastrointestinal and extraintestinal symptoms following the ingestion of gluten-containing foods in patients without celiac disease (CD) or wheat allergy. Despite the great interest for NCWS, the genetic risk factors still need to be fully clarified. In this study, we first assessed the possible contribution of KIR genes and KIR haplotypes on the genetic predisposition to NCWS. Fifty patients with NCWS, 50 patients with CD, and 50 healthy controls (HC) were included in this study. KIR genes and KIR genotyping were investigated in all subjects by polymerase chain reaction with the sequence oligonucleotide probe (PCR-SSOP) method using Luminex technology. We found a statistically different distribution of some KIR genes among NCWS, CD, and HC. Specifically, NCWS showed a decreased frequency of KIR2DL1, -2DL3, -2DL5, -2DS2, -2DS3, -2DS4, -2DS5, and -3DS1 genes, and an increased frequency of -3DL1 gene respect to both CD and HC. No difference was detected in the KIR haplotype expression. At the multivariate analysis, KIR2DL5, -2DS4, and -2DS5 were independent predictors of NCWS. Our findings suggest a role of KIR genes in NCWS susceptibility, with KIR2DL5, -2DS4, and -2DS5 having a protective effect. Further large-scale multicentric studies are required to validate these preliminary findings.

Family-based association of HLA-DRB1 and DQB1 alleles and haplotypes in a group of Iranian Type 1 diabetes children.

This family-based study was conducted in a group of Iranians with Type 1 diabetes (T1D) to investigate the transmission from parents of risk and non-risk HLA alleles and haplotypes, and to estimate the genetic risk score for this disease within this population. A total of 240 T1D subjects including 111 parent-child trios (111 children with T1D, 133 siblings, and 222 parents) and 330 ethnically matched healthy individuals were recruited. High-resolution HLA typing for DRB1/DQB1 loci was performed for all study subjects (n = 925) using polymerase chain reaction-sequence-specific oligonucleotide probe method. The highest predisposing effect on developing T1D was conferred by the following haplotypes both in all subjects and in probands compared to controls: DRB1*04:05-DQB1*03:02 (Pc = 2.97e-06 and Pc = 6.04e-10, respectively), DRB1*04:02-DQB1*03:02 (Pc = 5.94e-17 and Pc = 3.86e-09, respectively), and DRB1*03:01-DQB1*02:01 (Pc = 8.26e-29 and Pc = 6.56e-16, respectively). Conversely, the major protective haplotypes included DRB1*13:01-DQB1*06:03 (Pc = 6.99e-08), DRB1*15:01-DQB1*06:02 (Pc = 2.97e-06) in the cases versus controls. Also, DRB1*03:01-DQB1*02:01/DRB1*04:02|05-DQB1*03:02 and DRB1*03:01-DQB1*02:01/DRB1*03:01-DQB1*02:01 diplotypes conferred the highest predisposing effect in the cases (Pc = 8.65e-17 and Pc = 6.26e-08, respectively) and in probands (Pc = 5.4e-15 and Pc = 0.001, respectively) compared to controls. Transmission disequilibrium test showed that the highest risk was conferred by DRB1*04:02-DQB1*03:02 (Pc = 3.26e-05) and DRB1*03:01-DQB1*02:01 (Pc = 1.78e-12) haplotypes and the highest protection by DRB1*14:01-DQB1*05:03 (Pc = 8.66e-05), DRB1*15:01-DQB1*06:02 (Pc = 0.002), and DRB1*11:01-DQB1*03:01 (Pc = 0.0003) haplotypes. Based on logistic regression analysis, carriage of risk haplotypes increased the risk of T1D development 24.5 times in the Iranian population (p = 5.61e-13). Also, receiver operating characteristic curve analysis revealed a high predictive power of those risk haplotypes in discrimination of susceptible from healthy individuals (area under curve: 0.88, p = 5.5e-32). Our study highlights the potential utility of genetic risk assessment based on HLA diplotypes for predicting T1D risk in individuals, particularly among family members of affected children in our population.

Donor-Specific Antibody Detection by Single-Antigen Bead Assay for Renal Transplantation: A 2-Year Experience from South India.

Recipient sensitization against donor human leukocyte antigens (HLA) plays a key role in transplant rejection, and this risk is best minimized by efficient pre transplant antibody detection. Determination of antibody specificity with the highest sensitivity and degree of resolution to the allelic antigen level is achieved by using single-antigen bead (SAB) assay. This study evaluated the correlation of Luminex cross match (LXM) with SAB assay for detection of donor-specific antibodies (DSA). A total of 2075 renal transplant patients were screened for the presence of DSA by LXM, complement-dependent cytotoxicity (CDC) cross match, and 125 patients for SAB from January 2018 to December 2019. There was a male preponderance among recipients (P < 0.0001), and the most affected age group was 21-40 years. HLA typing was done in 550/2075 by DNA PCR-reverse sequence-specific oligonucleotide probes (SSOP) method. HLA DSA by LXM was detected in 16.3% of recipients (338/2075). Majority 180/338 (53.2%) of the patients were class II DSA positive, (P < 0.0001). Among the class II DSA positive patients, 20/180 (11.1%) samples gave false-positive results by LXM. SAB for class I and class II HLA IgG antibodies was done in 125/338 renal transplant recipients, which included 20 recipients with false-positive class II Luminex DSA, to check whether the DSA detected were really donor specific or not. The results showed that although 20/125 patients had some antibodies detected in their serum, they were not against the donor HLA antigens, as per the HLA typing reports of the donors. When compared to SAB assay, LXM showed more discrepant results, particularly to class II DSA. In conclusion, LXM, if used in combination with SAB assay and HLA typing of donors if necessary for virtual cross match, will help in avoiding unnecessary exclusion of donors for renal transplant recipients and also for post transplant monitoring of recipients, especially in cadaveric donor transplants.

New cost-effective human leukocyte antigen testing algorithm for screening of human leukocyte antigen-matched related donor in thalassemia major patient pretransplant workup

Introduction: India has a huge disease burden of thalassemia major with an estimated 40 million carriers and over a million thalassemia major patients. Very few patients are optimally treated, and the standard of care “hematopoietic stem cell transplant” (HSCT) is out of reach for most patients and their families. The cost of HSCT is significant, and a substantial proportion of it goes to human leukocyte antigen (HLA) testing of family members (HLA screening) in hope of getting a matched related donor (MRD) for HSCT. The aim of this study was to establish that a new proposed testing algorithm of HLA typing would be more cost-effective as compared to the conventional HLA screening within MRD families for possible HSCT. Material and Methods: Buccal swab samples of 177 thalassemia patients and their prospective family donors (232) were collected. Using a new HLA testing algorithm, samples were tested for HLA typing in a sequential manner (first HLA-B, then HLA-A, and finally HLA-DR) using the sequence-specific oligonucleotide probe method on the Luminex platform. Results: The new sequential HLA-A, HLA-B, and HLA-DRB1 testing algorithm showed a 49.1% reduction in cost compared to the conventional HLA testing algorithm. Furthermore, 40 patients (22.59%) were found to have HLA-MRD within the family among other samples that were tested. Conclusion: The new HLA testing algorithm proposed in the present study for identifying MRD for HSCT resulted in a substantial reduction in the cost of HSCT workup.

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.

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.

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.

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

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

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.

Association of HLA genotypes with phenytoin induced severe cutaneous adverse drug reactions in Thai children

PurposePhenytoin (PHT) is a common causative drug for severe cutaneous adverse drug reactions (SCARs) in children. SCARs, including drug reaction with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), are associated with a variation in HLA genotypes. Blood screening for specific HLA allele before PHT prescription would help in the reduction of the incidence of PHT induced SCARs. This study was to investigate the association between variations of HLA genotypes and PHT induced SCARs in Thai children. MethodsCases were Thai children aged between 0–18 years diagnosed with SCARs from PHT. Control groups were Thai children of corresponding age who had taken PHT for a least 12 weeks without any hypersensitivity reaction and healthy population controls. Blood samples from both groups were collected for HLA genotyping using a reverse-sequence specific oligonucleotide (SSO) probes method. Carrier rates of HLA alleles were compared between 22 cases (17 DRESS and 5 SJS-TEN), 60 tolerant controls and 649 population controls. ResultsTwo HLA alleles includingHLA-B*51:01 and HLA-C*14:02 were significantly associated with PHT induced DRESS (OR 5.83; 95 % CI 1.36–25.00, p = 0.022 and OR 5.85; 95 % CI 1.16–29.35, p = 0.039). HLA-B*38:02 was significantly associated with PHT induced SJS-TEN (OR12.67; 95 % CI 1.50–106.89, p = 0.044). Haplotype analysis demonstrated the association of HLA haplotype A*11:01-B*51:01-C*14:02 and PHT induced DRESS compared to tolerant controls and the healthy population control group (OR 8.92; 95 % CI 1.47–54.02, p = 0.019, and OR 10.2; 95 % CI 3.04–34.21, p = 0.002). HLA haplotype B*38:02-C*07:01 in PHT induced SJS-TEN was significantly higher than those in tolerant controls and the healthy population control group (40 % vs 3.3 % vs 0.3 %; OR 19.33; 95 % CI 1.98–188.59, p = 0.027 and OR 215.67; 95 % CI 22.40–2076.04, p = 0.0003. HLA-B*15:02 was not associated with PHT induced SCARs. SignificanceAn association betweenHLA-B*51:01 and HLA-C*14:02 and PHT induced DRESS and HLA-B*38:02 and PHT induced SJS-TEN has been demonstrated in Thai children.

Clinical significance of HLA-A, -B, -C, -DRB1, -DQB1 haplotype gene frequencies

目的研究单倍型移植中HLA-A、-B、-C、-DRB1、-DQB1单倍型频率，并探讨其临床意义。方法回顾性分析2012–2017年在苏州大学附属第一医院拟行亲缘HLA单倍型移植的3 568个家系，同时采用基因测序（PCR-SBT）和寡核苷酸探针杂交（PCR-SSOP）进行HLA基因分型检测，并进行家系单倍型分析。结果全部3 568个家系可分为3种情况：组1为1 422个完整家系，组2为1 310个仅有患者与一个亲代或子代的家系，组3为836个仅有患者与一个HLA≥5/10基因型相同同胞的不完整家系。组1和组2合并统计后建立单倍型总频率表。总频率≥0.1%的单倍型中：A*11∶01-B*40∶01-C*03∶04-DRB1*11∶01-DQB1*03∶01、A*02∶07-B*51∶01-C*14∶02-DRB1*09∶01-DQB1*03∶03的单倍型频率在组1和组2间差异有统计学意义（P值分别为0.029、0.033）；A*11∶01-B*46∶01-C*01∶02∶01G-DRB1*09∶01-DQB1*03∶03的频率在组1和组3间差异有统计学意义（P=0.035）；A*02∶01-B*40∶01-C*07∶02-DRB1*09∶01-DQB1*03∶03的频率在组1和组2、组1和组3间差异均有统计学意义（P值分别为0.034、0.034）；A*24∶02-B*13∶01-C*03∶04-DRB1*12∶02-DQB1*03∶01的频率在组2和组3间差异有统计学意义（P=0.046）。结论本研究建立了HLA-A、-B、-C、-DRB1、-DQB1单倍型频率总表，同时列举完整家系单倍型研究中分析基因型相同但单倍型完全不合，却被拟作为潜在供者移植的临床病例。家系不完整的供受者可通过本表进行单倍型分析，结合家系中其他成员HLA分型检测结果，为选择最佳供者及检索非血缘供者提供参考。

Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR

BackgroundPatients with metastatic colorectal cancer can benefit from anti-EGFR therapy, such as cetuximab and panitumumab. However, colorectal cancers harboring constitutive activating mutations in KRAS, NRAS and BRAF genes are not responsive to anti-EGFR therapy. To select patients for appropriate treatment, genetic testing of these three genes is routinely performed.MethodsWe applied bridged nucleic acid-clamp real-time PCR (BNA-clamp PCR) to detect somatic hotspot mutations in KRAS, NRAS and BRAF. PCR products from BNA-clamp PCR were subsequently analyzed Sanger sequencing. We then compared results with those from the PCR–reverse sequence-specific oligonucleotide probe (PCR-rSSO) method, which has been used as in vitro diagnostic test in Japan. To validate the mutation status, we also performed next generation sequencing using all samples.ResultsIn 50 formalin-fixed paraffin-embedded tissues, KRAS mutations were detected at frequencies of 50% (25/50) and 52% (26/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively, and NRAS mutations were detected at 12% (6/50) and 12% (6/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively. The concordance rate for detection of KRAS and NRAS mutations between the two was 94% (47/50). However, there were three discordant results. We validated these three discordant and 47 concordant results by next generation sequencing. All mutations identified by BNA-clamp PCR with Sanger sequencing were also identified by next generation sequencing. BNA-clamp PCR detected BRAF mutations in 6% (3/50) of tumor samples.ConclusionsOur results indicate that BNA-clamp PCR with Sanger sequencing detects somatic mutations in KRAS, NRAS and BRAF with high accuracy.

A Comparative Study: Flow Cytometry, Complement-dependent Cytotoxicity, and Luminex Methods

AimThe cell-based flow cytometric and bead-based Luminex crossmatch methods have been used alongside the standard complement-dependent cytotoxic crossmatch (CDCXM) test to detect donor specific anti-HLA antibodies. In this study, it was aimed to compare flow cytometric crossmatch (FCXM), CDCXM, and Luminex donor-specific crossmatch (LM-XM) tests for pre-transplant assessment of patients who applied to Tepecik Education and Research Hospital for kidney transplantation from related or deceased donors. MethodHLA tissue typing of 1120 patients were tested using the sequence specific oligonucleotide probe method with low resolution. FCXM and LM-XM were performed according to the manufacturer's instructions. The CDCXM test was performed according to the standard procedure. The results were analyzed using SPSS version 21.0 software (IBM, Armonk, NY, United States). P < .05 was accepted as statistically significant. ResultsFCXM, CDCXM, and LM-XM tests were performed on 58.2% (n = 652), 91% (n = 1019), and 55.4% (n = 620) of 1120 patients. There were statistically significant differences between FCXM/CDCXM, LM-XM/CDCXM, and FCXM/LM-XM (P < .0001), although there was also a moderate correlation between them (for class I, r = .599, r = .693, and r = .507; for class II, r = .546, r = .471, and r = .495, respectively). The results obtained according to donor type were compatible with the total study group. ConclusionThe utilization of FCXM and/or LM-XM tests together with the CDCXM method before kidney transplantations from related and/or deceased donor may facilitate the determination of target cells of donor-specific antibodies or their antibody class, which may increase the success of transplantation.

Association between HLA gene polymorphism and cutaneous adverse reactions caused by antiepileptic drugs.

The association between cutaneous adverse drug reactions (cADRs) caused by antiepileptic drugs (AEDs) and human leukocyte antigen-A (HLA-A) and HLA-B genes in Chinese Han population in Shanghai was investigated. Through the case-control study, 30 child patients with AED-induced cADRs (cADRs group), 60 AED-tolerant child patients (AED-tolerant group) and 60 normal children not taking AEDs (normal group) were collected. The HLA-B*15:02 and HLA-A*31:01 genotypes were detected using the polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO) probe method, and the correlation of HLA-B*15:02 and HLA-A*31:01 genes with the incidence of cADRs was analyzed. The positive rate of HLA-B*15:02 gene was 83.33% in the cADRs group, which was significantly increased compared with that in the AED-tolerant and normal groups (P<0.01). The positive rate of HLA-A*31:01 gene was 63.33% in the cADRs group, which was obviously increased compared with that in the AED-tolerant and normal groups (P<0.01). There were no significant differences in HLA-B*15:02 and HLA-A*31:01 genotypes between the AED-tolerant and normal groups (P>0.05). The results showed that HLA-B*15:02 and HLA-A*31:01 are significantly associated with cADRs in a Chinese Han population in Shanghai, suggesting that HLA-B*15:02 and HLA-A*31:01 genotypes should be detected in the application of AEDs.

Human leukocyte antigen, allele, and eplet mismatches in liver transplantation; observations from a small, single center cohort

BackgroundThe impact of human leukocyte antigen (HLA) matching on outcomes in liver transplantation is controversial. Varying levels of HLA matching resolutions were examined in a uniform patient population with no pre-transplant DSA from a small, single center cohort. MethodsRetrospective chart review from a single center yielded 131 patients, 67 of which were confirmed to be DSA negative, all of which received induction immunotherapy and post-operative immunosuppression. HLA typing was achieved by sequence specific oligonucleotide probe (SSOP) method using LABType® kits. Eplet mismatch analysis was conducted using HLAMatchMaker software. ResultsThe mean number of HLA-A antigen mismatches was significantly higher in patients experiencing acute rejection (1.8 vs 1.6, p = 0.006). Rejection patients more frequently possessed two HLA-A mismatches compared to their non-rejection counterparts (77% vs 43%, p = 0.071). Patient survival was found to be non-significantly decreased in patients with a higher eplet mismatch load at the HLA-A locus (p = 0.155). No other loci were found to be predictive. ConclusionIn conclusion, HLA mismatches were found to increase acute rejection and be associated with decreased patient survival. The outcomes of this study suggest an involvement of HLA-A locus mismatches in predicting liver transplant rejection and patient survival.

A novel analysis strategy for HLA typing using a sequence-specific oligonucleotide probe method.

The technique of reverse sequence-specific oligonucleotide probes (SSOPs) is commonly used in human leukocyte antigen (HLA) typing. In the conventional method for data analysis (exact pattern matching, EPM), the larger is the number of mismatched probes, the longer the time for final typing assignment. A novel strategy, filtering and scoring (FnS), has been developed to easily assign the best-fit allele pair. In the FnS method, candidate alleles and allele pairs were filtered based on (1) subject's ethnicity, and (2) the measured partial reaction pattern with only definitely negative or positive probes. Then, the complete reaction pattern for all probes (CRPoAPs) were compared between the raw sample and expected residual allele pairs to obtain mismatch scores. To compare the FnS and EPM methods, each analysis time (minutes:seconds) for reverse SSOP HLA typing with intermediate resolution (n = 507) was measured. The analysis time with FnS method was shorter than that of the EPM method [00:21 (00:08-01:47) and 01:04 (00:15-23:45), respectively, P < .05]. In addition, the analysis time of the FnS method was relatively constant, regardless of the number of mismatched probes. The alternative approach of filtering based only on definite probes (neglecting ethnicity) took a long time for analysis. However, this approach did not compromise the accuracy. The FnS method showed improved accuracy and efficiency of HLA typing in a comprehensive and quantitative comparison between measured and expected CRPoAPs of candidate allele pairs. Therefore, this analysis strategy might be useful in a clinical setting.

Association of HLA genotypes with phenobarbital hypersensitivity in children.

Phenobarbital hypersensitivity is one of the common drug hypersensitivity syndromes in children. Clinical symptoms of phenobarbital hypersensitivity vary from maculopapular rashes (MPs) to severe cutaneous adverse drug reactions (SCARs) including drug reactions with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN). Drug hypersensitivity has been demonstrated to be associated with variations in the HLA genotypes. This study was to investigate the association between the variations of HLA genotypes and phenobarbital hypersensitivity in Thai children. The cases were Thai children, between 0 and 18 years of age, who were diagnosed with phenobarbital hypersensitivity, which included SCARs and MPs. The control patients were Thai children of a corresponding age who had taken phenobarbital for at least 12 weeks without any hypersensitivity reaction. Blood samples were collected for HLA genotyping by using a reverse-sequence-specific oligonucleotide (SSO) probes method. The carrier rates of HLA alleles were compared between 47 cases (27 SCARs and 20 MPs) and 54 controls. The carrier rates of HLA-A*01:01 and HLA-B*13:01 were significantly higher in the phenobarbital-induced SCARs than in the tolerant controls (18.5% vs. 1.85%, p = 0.01, odds ratio [OR] 11.66, 95% confidence interval [CI] 1.21-578.19; 37.04% vs. 11.11%, p = 0.009, OR 4.60, 95%CI 1.29-17.98). There was a trend of a higher carrier rate of HLA-C*06:02 in the phenobarbital-induced SCARs when compared with those in the tolerant controls (29.63% vs. 11.11%, p = 0.059, OR 3.31, 95% CI 0.88-13.31). In contrast to the phenobarbital-induced SCARs, only the HLA-A*01:01 carrier rate in the phenobarbital-induced MPs was significantly higher than those in the tolerant controls (20% vs. 1.85%, p = 0.017, OR 12.69, 95% CI 1.15-661.62). An association between phenobarbital hypersensitivity and HLA-A*01:01 and HLA-B*13:01 has been demonstrated in Thai children.

HLA-A, -B, -DRB1 allele and haplotype frequencies of 920 cord blood units from Central Chile

We present human leukocyte antigen (HLA) haplotype and allele/antigenic group frequencies derived from a data set of 920 umbilical cord blood units collected in Central Chile. HLA-A and -B genotypes were typed using sequence specific oligonucleotide probe methods while HLA-DRB1 genotypes were obtained from sequencing-based typing. The most frequent haplotype is A*29~B*44~DRB1*07:01 with an estimated frequency of 2.1%.

HLA-B allele dropout in PCR sequence-specific oligonucleotide probe typing due to intronic polymorphism in the novel B*58:01:01:02 allele.

Currently, Luminex technology based on the PCR sequence-specific oligonucleotide (SSO) probe method has been widely used for HLA genotyping in the immunogenetics laboratories. Here, we reported a case with HLA-B allele dropout by Luminex technology. The initial HLA-B result of the Luminex method with a commercial agent kit was inconclusive, and then, the result of PCR-SBT technology indicated the dropout as a HLA-B*58 allele. Subsequently, the full-length sequence of HLA-B allele was determined by TOPO-TA cloning, and a novel allele B*58:01:01:02 was identified in the individual. Compared with HLA-B*58:01:01:01, the novel allele showed some nucleotides difference at 509 C>T, 521 T>G and CCC insertion in position 503 of intron 2. According to the full-length sequence, the new mutations of intron 2 were contributed to HLA-B locus allele dropout in the sample. Our results indicated multiplatform should be used to improve the HLA typing accuracy when a conclusive HLA genotype cannot be determined.