Sequence-specific Oligonucleotide Research Articles

P8.039: Allele, Genotype and Haplotype Frequencies of the HLA System in the Province of Neuquén, Argentina

Knowledge of the bioanthropological and genetic characteristics of the population in Neuquén is limited. Determination of the allele and haplotype frequencies of the HLA system provides data on genetic variability, susceptibility or resistance to diverse disease, immunological phenomena and contributes to the selection of donors and recipients in organ transplantation. 583 samples from patients attending the histocompatibility laboratory between January 2015 and March 2022 have been analyzed retrospectively. HLA typing of locus A, B and DR has been performed on all patients by using medium resolution ssop methodology (sequence specific oligonucleotide probes). It was observed that, in locus A, the predominant allele was A2, with a frequency of 15.5 (42.8%). It was followed by A68, with a frequency of 12.22 (33.2%) and A24, with a frequency of 6.83 (20.8%). In locus B, the majority alleles were B35 and B39, with a frequency of 2.68 (33.3%). They were followed by B7, with a frequency of 2.89 (13.8%) and B40, with a frequency of 2.68 (12.8%). In locus DR, the most frequent allele was DR4, with a frequency of 20.85 (46,5%). The second most frequent allele was DR11, with 9.23 (20.6%), and finally, DR8, with 9.0 (20%). The most frequent genotype in locus A was A2-A68 (9.77%). In locus B, it was B35-B39 (5.83%) and, in locus DR, it was DR4-DR4 (7.55%). The most frequent haplotypes were A2-B39 (15%) and A68-B39 (13,4%). Regarding loci A-B-DR, the most observed haplotype was A68-B39-DR4 (7.5%). Due to the existing demographic growth in the province, according to the last census, Neuquén received population from the whole country (Argentina) and from South American countries. Even though the most frequent alleles were similar to alleles from other studies conducted, substantial discrepancies were also identified. Thus, considering the great number of native peoples present in Neuquén, using these methodologies for the study of closed populations would be convenient in the future. Baradello Stefano.

Genetics: Is LADA just late onset type 1 diabetes?

BackgroundThere is a controversy regarding Latent Autoimmune Diabetes in Adults (LADA) classification and whether it should be considered a slowly progressing form of type 1 (T1) diabetes (DM) or a distinct type of DM altogether.MethodsThis cross-sectional study assessed major genes associated with T1DM (class II HLA, PTPN22 [rs2476601] and INS [rs689]) in patients with LADA, as compared with participants with T1DM (stratified according to age of diagnosis before or after 30) and T2DM. HLA genotyping of the DRB1, DQA1 and DQB1 loci was performed by reverse PCR sequence-specific oligonucleotides. HLA haplotypes were assigned according to those most frequently described in the European population. INS and PTPN22 SNPs were genotyped by real-time PCR.ResultsA total of 578 participants were included: 248 with T1DM (70 diagnosed after the age of 30), 256 with T2DM and 74 with LADA. High risk HLA alleles were significantly more frequent in LADA than in T2DM, whereas the opposite was true for protective alleles. We found a lower frequency of the high-risk DRB1*04-DQB1*03:02-DQA1*03:01 haplotype in LADA (21.1%) than in the overall T1DM (34.7%) (p<0.05), whereas no differences were found between these groups for DRB1*03-DQB1*02:01-DQA1*05:01 or for protective alleles. Only 12% the overall T1DM group had no risk alleles vs 30% of LADA (p<0.0005). However, HLA allele distribution was similar in LADA and T1DM diagnosed after the age of 30. A total of 506 individuals (195 with T1DM [21 diagnosed after age 30] 253 with T2DM and 58 with LADA) were genotyped for the PTPN22 and INS SNPs. The G/A genotype of the PTPN22 rs2476601 was more frequent and the T/T genotype of the INS SNP rs689 was less frequent in T1DM compared to LADA. We did not find any significant differences in the frequency of the mentioned SNPs between LADA and T2DM, or between LADA and T1DM diagnosed after the age of 30.ConclusionIn this relatively small cross-sectional study, the genetic profile of subjects with LADA showed a similar T1DM-related risk allele distribution as in participants with T1DM diagnosed after the age of 30, but fewer risk alleles than those diagnosed before 30. Differences were present for HLA, as well as PTPN22 and INS genes.

Frequency and distribution of HLA-DQB1 alleles from 2076 cord blood samples of the Vietnamese cohort.

Human leucocyte antigen (HLA) alleles are very diverse and characterized by ethnicity. To date, information about the frequencies and distributions of HLA alleles among the Vietnamese population is still limited. In this study, HLA-DQB1 alleles of 2076 cord blood units from individuals belonging to Vietnam's Kinh ethnic people were genotyped using Luminex-based polymerase chain reaction sequence-specific oligonucleotide. The results of the study demonstrated that there were 23 alleles on the locus HLA-DQB1. Among those, there were six alleles with high frequencies of over 5%, including DQB1* 03:01 (35.9%), DQB1* 05:01 (12.8%), DQB1* 03:03 (12.2%); DQB1* 06:01 (7.20%), DQB1* 05:02 (6.62%) and DQB1* 02:01 (5.30%) and five rare alleles with low frequencies of below 0.1%. More importantly, this study for the first time reported the presence of two new rare alleles including DQB1* 01:01 and DQB1* 01:02. Conclusively, this study provided significant information about HLA-DQB1 alleles for further investigations and clinical applications.

Characterization of KRAS Mutational Regression in Oligometastatic Patients.

BackgroundWe previously reported rare regressive genetic trajectories of KRAS pathogenic mutations as a specific hallmark of the genuine oligometastatic status in colorectal cancer (CRC).MethodsSurvival and prognostic impact of disease extent in 140 metastatic CRC patients were evaluated through the Kaplan–Meyer curves and the Log-Rank test. KRAS mutations were assessed through the Illumina NovaSeq 6000 platform and TruSight™ Oncology 500 kit. HLA typing was carried out by PCR with sequence-specific oligonucleotides. Lymphocyte densities in tumors were expressed as cells per square millimeter. NKs isolated and CD8+ from NK-depleted PBMCs were characterized through flow cytometry. CD107a externalization was evaluated as NKs/CD8 cytotoxicity toward human colon cancer cells HT29, SW620, HCT116, and LS174T carrying different KRAS mutations.ResultsThe oligometastatic status was a strong and independent variable for survival (HR: 0.08 vs. polymetastatic disease; 95% CI: 0.02–0.26; p < 0.001). Eighteen oligometastatic patients were selected. Twelve were alive at the last follow-up, and 9 were characterized. Genetic regression of KRAS was observed in 3 patients: patient (PAT)2, PAT5, and PAT8. PAT2 and PAT5 presented the highest levels of GrzB+ lymphocytes in the tumor cores of the metastases (120 ± 11.2 and 132 ± 12.2 cells/mm2, respectively). Six out of 9 patients (67%), including PAT2 and PAT5, expressed HLA-C7. Twopatients (PAT2 and PAT5) presented high CD3+/CD8+-dependent cytotoxicity against HLA-C7+ SW620 cells (p.G12V-mutated cells), which was consistent with their observed mutational regression (p.G12V/p.G13D in primary→p.G13D in metastatic tumor).ConclusionsWe provide evidence that CD3+/CD8+ lymphocytes from oligometastatic CRC patients display differential cytotoxicity against human colon cancer cells carrying KRAS mutations. This could provide an interesting basis for monitoring oligometastatic disease and developing future adoptive immunotherapies.

KIR- Ligand Interactions in Hypertensive Disorders in Pregnancy.

HypothesisThe activity of natural killer (NK) cells is considered an important factor for the tolerance of the fetus during pregnancy. The complications of pregnancy, such as hypertensive disorders (HDP), may be therefore associated with this immune compartment.MethodsThe current study included 41 pregnant women diagnosed with HDPs (Gestational Hypertension; GH or Preeclampsia; PE) and 21 healthy women. All the patients were under continuous obstetric care during the pregnancy and labour. The number of mother-child mismatches within killer immunoglobulin-like receptors (KIRs), their ligands [MM], and missing KIR ligands [MSLs] was assessed. KIRs and their ligands were assessed with Next Generation Sequencing (NGS) and Polymerase Chain Reaction Sequence-Specific Oligonucleotide (PCR-SSO) typing. The subsets of NK cells were assessed with multicolor flow cytometry and correlated to the number of MSLs.ResultsThe number of MSLs was significantly higher in HDP patients when compared to healthy non-complicated pregnancy patients. Some MSLs, such as those with 2DS2 activating KIR, were present only in HDP patients. The percentage of CD56+CD16-CD94+ NK cells and CD56+CD16-CD279+ NK cells correlated with the number of MSLs with inhibiting KIRs only in healthy patients. In HDP patients, there was a correlation between the percentage of CD56-CD16+CD69+ NK cells and the number of MSLs with inhibiting and activating KIRs. As compared to the healthy group, the percentage of CD56+CD16-CD279+ NK cells and CD56-CD16+CD279+ NK cells were lower in HDP patients. HDP patients were also characterized by a higher percentage of CD56+CD16+perforin+ NK cells than their healthy counterparts.ConclusionsPatients with HDP were characterized by a higher number of MSLs within the KIRs receptors. It seemed that the number of MSLs in the healthy group was balanced by various receptors, such as CD94 or inhibitory CD279, expressed on NK cells. Conversely, in HDP patients the number of MSLs was associated with the activation detected as the increased level of CD69+ NK cells.

P-367 Centromeric motif AA of the KIR: the best subrogate marker that defines Alloimmune Reproductive Failure

Abstract Study question Should the centromeric and telomeric motifs been studied separately and taked them in to account in women with recurrent reproductive failure? Summary answer The results show that when the classic KIR study is performed, there is a 12,5% of AA centromeric KIR patients with reproductive failure unidentified. What is known already During human pregnancy, the decidua is mainly populated by NK lymphocytes that express immunoglobulin-type receptors (KIR) recognizing their HLA-C ligands from trophoblast. The objective of this study was to analyze the association of KIR-HLAC phenotypes in couples with sterility, recurrent pregnancy loss (RPL), recurrent implantation failure (RIF), and fetal death (FD) in comparison with reference population and fertile control. Study design, size, duration It is a retrospective real-life observational study from 2015 to 2019 Participants/materials, setting, methods The frequency of the KIR and HLA-C genes were analyzed in 397 women and their partners with reproductive failure. We compare them with 33 fertile controls and reference Spanish population. KIR and HLA-C genotypes of the patients and controls were determined by Lifecodes Sequence-Specific Oligonucleotides (SSO) Typing KIT (Diagnostica Longwood®) performed at Centro de Transfusiones de la Comunidad de Madrid and those of controls at the Hospital Universitario Clínico San Carlos. Main results and the role of chance Centromeric (Cen) AA was the most frequent KIR genotype in women with unexplained RPL and RIG with respect to reference Spanish population (p = 0.001 and 0.02, respectively). Moreover, cenAA was associated with 1.51-fold risk of RPL and 1.2-fold risk of RIF. On the other hand, BB KIR showed lower risk of any reproductive failure tan non-BB KIR (0.12; p &amp;lt; 0.001). HLA-C1C1/C1C1 women and partner were significantly lower in RPL-Group (p &amp;lt; 0.001) and RIF-Group (p = 0.04). Indeed, the combination cenAA/C1C1 women with C1C1 partners were significantly higher in the control group compared with RPL (p = 0.009) and RIF (0.04) with 5-fold of successful pregnancy outcome. In our cohort, cenAA KIR was a more accurate biomarker tan classic AA KIR haplotype for RPL- and RIF-associated risk. Limitations, reasons for caution The fact of having stratified a lot by groups means that a small number of patients is obtained in each subgroup, which makes it more difficult to conclude the results. Wider implications of the findings Our study demonstrates the importance of the complete KIR genotype study, which would give us more comprehensive information on the behavior of NK cells, moreover we propose that centromeric KIR classification is superior to the KIR classical haplotypes, better identifying those patients with potential KIR-HLA-C mismatch. Trial registration number Not applicable

Allele and Haplotype Frequencies of HLA-A, -B, -C, and -DRB1 Genes in 3,750 Cord Blood Units From a Kinh Vietnamese Population.

The frequencies and diversities of human leukocyte antigen (HLA) alleles and haplotypes are representative of ethnicities. Matching HLA alleles is essential for many clinical applications, including blood transfusion, stem cell transplantation, and tissue/organ transplantation. To date, the information about the frequencies and distributions of HLA alleles and haplotypes among the Kinh Vietnamese population is limited because of the small sample size. In this study, more than 3,750 cord blood units from individuals belonging to the Kinh Vietnamese population were genotyped using PCR sequence-specific oligonucleotide (PCR-SSO) for HLA testing. The results of the study demonstrated that the most frequently occurring HLA-A, -B, -C, and -DRB1 alleles were A*11:01 (25%), A*24:02 (12.3%), A*02:01 (11.2); A*03:03 (8.95%), A*02:03 (7.81%), A*29:01 (7.03%); B*15:02 (15.1%), B*46:01 (10.7%), B*58:01 (7.65%), B*38:02 (7.29%); C*08:01 (17.2), C*07:02 (16.2%), C*01:02 (15.2), C*03:02 (8.3%), C*15:05 (6.13); DRB1*12:02 (31.0%), DRB1*09:01 (10.47%), DRB1*15:02 (7.54%); DRB1*07:01 (6.68%), DRB1*10:01 (6.63%), respectively, with the highest allele diversity level observed in locus B (93 alleles). The most frequent haplotypes of two-locus combinations of HLA-A–B, HLA-A–C, HLA-A–DRB1, HLA-B–C, HLA-B–DRB1, and HLA-C–DRB1 haplotypes were A*11:01–B*15:02 (7.63%), A*11:01–C*08:01 (7.98%), A*11:01–DRB1*12:02 (10.56%), B*15:02–C*08:01 (14.0%), B*15:02–DRB1*12:02 (10.47%), and C*08:01–DRB1*12:02 (11.38%), respectively. In addition, the most frequent haplotypes of three- and four-locus sets of HLA-A–B–C, HLA-A–B–DRB1, HLA-A–C–DRB1, HLA-B–C–DRB1, and HLA-A–B–C–DRB1 were A*11:01–B*15:02–C*08:01 (7.57%), A*11:01–B*15:02–DRB1*12:02 (5.39%), A*11:01–C*08:01–DRB1*12:02 (5.54%), B*15:02–C*08:01–DRB1*12:02 (10.21%), and A*11:01–B*15:02–C*08:01–DRB1*12:02 (5.45%), respectively. This study provides critical information on the frequencies and distributions of HLA alleles and haplotypes in the Kinh Vietnamese population, accounting for more than 85% of Vietnamese citizens. It paves the way to establish an umbilical cord blood bank for cord blood transplantation programs in Vietnam.

HLA-DRB1∗1502 Is Associated With Anti-N-Methyl-D-aspartate Receptor Encephalitis in Thai Children

BackgroundAnti-N-methyl-d-aspartate receptor encephalitis (anti-NMDARE) is one of the most common types of autoimmune encephalitis. Most patients have no apparent immunologic triggers, which suggests a genetic predisposition. This study was conducted to identify human leukocyte antigen (HLA) class II alleles associated with anti-NMDARE in Thai children. MethodsThis case-control study enrolled patients younger than 18 years who were diagnosed with anti-NMDARE between January 2010 and December 2020. A “good outcome” was determined as a patient with a modified Rankin scale score of less than 2 at any follow-up visit. HLA genotypes were determined at four-digit alleles using reverse sequence-specific oligonucleotide probe hybridization. The HLA class II allele frequency in patients was compared with that in a database of 101 healthy control Thai children. ResultsThirty-four patients were enrolled with a mean age of 12.8 ± 5.6 years (females 85.3%). The HLA-DRB1∗1502 allele frequency was significantly higher in patients than in controls (odds ratio, 2.32; 95% confidence interval, 1.11-4.8, P = 0.023). A good outcome was noted in 14 of 14 (100%) HLA-DRB1∗1502-positive patients (median time to a good outcome, 6 months) and 14 of 17 (82.3%) HLA-DRB1∗1502-negative patients (median time to a good outcome, 3 months). Two (11.8%) HLA-DRB1∗1502-positive patients had one relapse each, and six (35.3%) HLA-DRB1∗1502-negative patients had one to three relapses. ConclusionsHLA-DRB1∗1502 was significantly associated with anti-NMDARE in our patients. Most patients had good outcomes. HLA-DRB1∗1502-positive patients tended to require a longer time to achieve a good outcome but had less frequent relapses than HLA-DRB1∗1502-negative patients.

The Accuracy of Sequence-Specific Oligonucleotide and Real-Time Polymerase Chain Reaction HLA Typing in Determining the Presence of Pre-Transplant Donor-Specific Anti-HLA Antibodies and Total Eplet Mismatches for Deceased Donor Kidney Transplantation

High resolution human leukocyte antigen (HLA) typing is important in establishing eplet compatibility and the specificity of donor-specific anti-HLA antibodies (DSA). In deceased donor kidney transplantation, high resolution donor HLA typing may not be immediately available, leading to inaccuracies during the organ allocation process. We aimed to determine the concordance and agreement of HLA-Class I and II eplet mismatches calculated using population frequency based allelic haplotype association (linkage disequilibrium, LD) from sequence-specific oligonucleotide (SSO) and real-time polymerase chain reaction (rtPCR) donor HLA typing (available at time of donor kidney allocation) compared to high-resolution Next Generation Sequencing (NGS) donor typing. NGS high resolution HLA typing were available for all recipients prior to donor kidney allocation. A cohort of 94 deceased donor-recipient pairs from a single Western Australian center were included (77 individual donors typed, 55 local and 22 interstate). The number of class I (HLA-A+B+C) and class II (HLA-DRB1+DRB3/4/5+DQB1+DQA1+DPB1+DPA1) eplet mismatches were calculated using HLAMatchmaker, comparing LD- and NGS-HLA typing. The accuracy in assigning pre-transplant DSA was compared between methods. The concordance correlation coefficient (95%CI) for HLA-class I and II eplet mismatches were 0.994 (0.992 to 0.996) and 0.991 (0.986 to 0.993), respectively. The 95% limits of agreement for class I were -1.3 (-1.6 to -1.1) to 1.4 (1.2 to 1.7) and -4.8 (-5.7 to -3.9) to 5.0 (4.1 to 5.9) for Class II. Disagreement between the two methods were present for 11 and 37 of the Class I and II donor/recipient pairs. Of which, 5 had a difference of ≥5 class II eplet mismatches. There were 34 (36%) recipients with potential pre-transplant DSA, of which 8 (24% of recipients with DSA) had indeterminate and ultimately false positive DSA assigned by donor LD-typing. While the concordance between NGS- and LD-typing was high, the limits of agreement suggest meaningful differences between these two techniques. The inaccurate assignment of DSA from donor LD-typing may result in associated HLA being considered unacceptable mismatches, inappropriately precluding candidates’ access to transplantation. Accurate imputation of two-field HLA alleles based on LD from SSO and rtPCR HLA typing remains a substantial challenge in clinical practice in-lieu of widely available, rapid, high-resolution methods.

HLA Diversity in Saudi Population: High Frequency of Homozygous HLA Alleles and Haplotypes.

Human leukocyte antigens (HLA) diversity has a tremendous impact on shaping the transplantation practices, transfusion-associated graft versus host disease prevention strategies, and host–pathogen interactions. Here, we conducted a retrospective study of HLA class I and class II homozygosity at allelic and haplotype levels in unrelated individuals genotyped from 2012 to 2016 in a tertiary hospital in the capital of Saudi Arabia. Among 5,000 individuals, 2,773 individuals meet inclusion criteria and were retrospectively analyzed for HLA-A, -B, -C–DRB1, and -DQB1 homozygosity at allelic and haplotype levels. HLA molecular typing was performed using a commercial reverse sequence-specific oligonucleotide (rSSO) kit. We were able to identify 15 HLA-A, 20 HLA-B, 11 HLA-C, 13 HLA-DRB1, and five HLA-DQB1 homozygous alleles demonstrating a very low genetic diversity in the Saudi population. The highest homozygosity in HLA class I was found in locus C followed by A and B (20.3% > 16.1% > 15.5%; p < 0.001) where the most homozygote alleles were A*02 (9.2%), B*51 and B*50 (5.7% and 3.7%), and C*07, C*06, and C*15 (7.2%, 5.48%, and 3.3%) and in HLA class II, the highest homozygosity was found in locus DQB1 compared to DRB1 (31.71% > 19.2%; p < 0.001), with the most common homozygote alleles being DRB1*07 and DRB1*04 (5.33% and 4.2%) and DQB1*02, DQB1*06, and DQB1*03 (13.55%, 7.92%, and 7.64%). The frequency of finding an individual with one homozygote allele was (24.6%), two homozygote alleles (13.5%), three homozygote alleles (4.7%), four homozygote alleles (3.4%), and five alleles were (4.8%). The most frequent homozygote haplotypes are A*23∼C*06∼B*50∼DRB1*07∼DQB1*02 and A*02∼C*06∼B*50∼DRB1*07∼DQB1*02. This study shows low diversity of both class I and II alleles and haplotypes in the Saudi population, which would have a significant impact on shaping the transplantation practices, transfusion-associated graft versus host disease prevention strategies, and host–pathogen interactions.

Natalizumab therapy in patients with pediatric-onset multiple sclerosis in Greece: clinical and immunological insights of time-long administration and future directions-a single-center retrospective observational study.

Pediatric-onset multiple sclerosis (MS, POMS) accounts for 3-5% of all MS cases and is characterized by a highly inflammatory profile, often warranting treatment with high-efficacy agents. Our aim is to present real-world data of a series of 18 Hellenic POMS patients treated with natalizumab (NTZ) either as adolescents or as adults, after high disease activity has efficiently subsided. Clinical and imaging/laboratory data from 18 POMS patients who have received at least one NTZ infusion were selected in this single-center retrospective observational study. Human leukocyte antigen (HLA) genotyping was performed with standard low-resolution sequence-specific oligonucleotide techniques. Eighteen patients with a mean age of disease onset of 15.3 ± 2.4years were treated with NTZ with a mean of 51.7 ± 46.4 infusions, 6 as adolescents and 12 as adults. 22.2% were treatment naïve. At the end of the observational period, patients of both groups remained relapse-free, with no radiological activity and significantly reduced disability accumulation. No evidence of disease activity (NEDA)-3 status was achieved in 66.7% of all patients, 58.3% in the adult-treated, and 83.3% in the adolescent-treated POMS patients. NTZ was generally well tolerated. Only 5 adverse events were observed, in 3 patients who were carriers of the HLA-DRB1*15 (HLA-DRB1*15/HLA-DRB1*11 and HLA-DRB1*15/HLA-DRB1*13 genotypes), 1 homozygous for the HLA-DRB1*03 allele and 1 heterozygous for HLA-DRB1*04 and HLA-DRB1*16 alleles. NTZ is highly efficacious and mostly safe for POMS patients with high disease activity in all age groups. The role of immunogenetics in personalized patient evaluation and treatment needs to be further investigated.

OA35 Efficacy of secukinumab and HLA-B27 subtypes: results from a Phase 3b randomised controlled trial in axial SpA

Abstract Background/Aims Human Leukocyte Antigen (HLA)-B is strongly associated with axial spondyloarthritis (axSpA); over 100 subtypes of HLA-B27 are currently recognized and designated as HLA-B∗2701 to HLA-B∗27106. The association of these subtypes with clinical features of axSpA patients or their response to therapy has not been determined. This post hoc analysis explored the potential association of the HLA-B27 subtypes with the effect of secukinumab in axSpA patients from the SKIPPAIN trial (NCT03136861). Methods SKIPPAIN, a 24-week, randomised, double-blind, placebo-controlled, multicentre trial, enrolled adult axSpA patients with active disease fulfilling ASAS classification criteria (Bath Ankylosing Spondylitis Disease Activity Index [BASDAI] score ≥4 and average spinal pain numerical rating scale [NRS] score &amp;gt;4 at baseline) and inadequate response to ≥ 2 NSAIDs for ≥4 weeks. Patients were randomised (3:1) to receive subcutaneous secukinumab 150 mg or placebo weekly followed by every 4 weeks (q4w) starting at Week 4. At Week 8, placebo patients were re-randomised to secukinumab 150 or 300 mg q4w up to Week 24. HLA-B27 subtypes were tested by PCR-reverse sequence-specific oligonucleotide probe on a baseline blood sample. Average spinal pain scores were analysed using a repeated measures analysis of covariance model. Results Overall, 380 patients with axSpA (269 [70.8%] AS; 111 [29.2%] nr-axSpA) were randomised to secukinumab 150 mg (n = 285) or placebo (n = 95). Most patients were HLA-B27 positive in both treatment groups (233 [81.8%] SEC vs 76 [80.0%] placebo). Distribution of HLA subtypes was consistent with what is expected in a typical European population, with HLA-B*27:05:02G as the most common allele (secukinumab 150 mg, n = 172 [60.4%]; placebo, n = 48 [50.5%]). In the HLA-B*27 homozygous group, a higher proportion of patients had uveitis, peripheral arthritis, enthesitis and a family history of spondyloarthritis versus the heterozygous group; disease severity or burden of disease was similar between the two groups. Male predominance was more evident in the HLA-B*27 homozygous group, who were younger by 1 year and reported longer duration of symptoms by an average of 8 months. Disease severity was comparable while disease burden was higher in the B*27:05:02G versus the B27:02:01G heterozygous group. There was a significant least square (LS) mean difference (standard error [SE]) in back pain response between secukinumab and placebo in HLA-B27 positive patients (-0.9 [±0.27], 95% confidence interval [CI] -1.47 to -0.42, P = 0.0004) and HLA-B27 heterozygous (B*27:05:02G) patients (-0.9 [±0.37], 95% CI − 1.62 to -0.17, P = 0.0152), differences in other HLA-B27 subtypes were non-significant. Conclusion Although secukinumab has shown efficacy in both HLA-B27 positive and negative patients, presence of HLA-B27 and homozygosity had a distinct positive effect on response. Further studies or pooled analyses are warranted to investigate associations of HLA-B27 subtypes with the clinical features of axSpA or of less prevalent HLA-B27 subtypes with treatment response. Disclosure H. Marzo-Ortega: Consultancies; AbbVie, Amgen, BMS, Biogen, Celgene, Gilead, Janssen, Lilly, Novartis, Pfizer, Samsung Bioepis, Sanofi-Aventis, UCB. Grants/research support; Amgen, Lilly, Janssen, Pfizer, Sandoz, Sanofi, Galapagos. D. Poddubnyy: Consultancies; AbbVie, Biocad, BMS, Eli Lilly, MSD, Novartis, Pfizer, Samsung Bioepis, UCB, Gilead. Member of speakers’ bureau; AbbVie, BMS, Lilly, MSD, Novartis, Pfizer, UCB. Grants/research support; AbbVie, MSD, Eli Lilly, Novartis, Pfizer. E. Pournara: Shareholder/stock ownership; Novartis. Other; Employee of Novartis. B. Schulz: Other; Employee of Novartis. A. Deodhar: Consultancies; AbbVie, Amgen, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Eli Lilly, Gilead, GSK, Janssen, Novartis, Pfizer, UCB. Member of speakers’ bureau; AbbVie, Amgen, Boehringer Ingelheim, Bristol Myer Squibb, Eli Lilly, GSK, Janssen, Novartis, Pfizer, UCB. Grants/research support; AbbVie, Eli Lilly, GSK, Novartis, Pfizer, UCB. X. Baraliakos: Consultancies; AbbVie, BMS, Celgene, Chugai, Galapagos, Gilead, MSD, Novartis, Pfizer, UCB. Member of speakers’ bureau; AbbVie, BMS, Celgene, Chugai, MSD, Novartis, Pfizer, UCB. Grants/research support; AbbVie, Novartis.

Characterization of a novel HLA-A*11:335 allele resulting from a rare interlocus recombination involving HLA-A*11:01:01:01/126 and HLA-H*02:07/14/18 alleles with nanopore sequencing, in a volunteer from the China Marrow Donor Program

BackgroundThe major histocompatibility complex (MHC) in humans includes three classical class I loci (A, B, and C), which are important biomarkers for the transplantation of organs and hematopoietic stem cells. In the MHC, polymorphism is known to be extremely high while interlocus recombination is rare. We report a rare interlocus recombination between HLA-A and HLA-H, which was analyzed using next generation sequencing and nanopore sequencing.MethodsIn the sample, the genotypes of HLA-A, B, C, DRB1, and DQB1 were firstly determined using the methods of sequence-specific primer, sequence-specific oligonucleotide, Sanger’s sequencing, and NGS; however, HLA-A could not be phased. Nanopore sequencing was finally utilized to distinguish the sequence of the novel allele.ResultsFinally, the novel HLA-A*11:335 allele was identified as an interlocus recombination involving HLA-A*11:01:01:01/126 and HLA-H*02:07/14/18 alleles; this was mainly achieved by nanopore sequencing.ConclusionsThe identification of the interlocus recombination indicated that nanopore sequencing can be helpful in the characterization of novel alleles with complex rearrangements. Interlocus recombination has been identified as one of the mechanisms involved in the generation of novel HLA alleles.

Association of HLA-DRB1 alleles with rheumatoid arthritis in Split-Dalmatia County in southern Croatia.

The aim of this study was to investigate the distribution of HLA-DRB1 alleles in patients with rheumatoid arthritis (RA) in the Sinj Region (SR) and the rest of the Split-Dalmatia County (SDC) in Croatia and to determine their relationship with disease severity. Atotal of 74RA patients and 80healthy controls from the SR, and 74RA patients and 80healthy controls from the rest of the SDC were genotyped using sequence-specific oligonucleotide primed PCR. High-resolution typing of HLA-DRB1*04 alleles was performed using the single specific primed polymerase chain reaction (PCR-SSP) method. Serum anti-CCP, rheumatoid factor, C‑reactive protein, and erythrocyte sedimentation rate were measured in all RA patients, whereas disease activity was assessed by DAS-28 and functional status by the Health Assessment Questionnaire Disability Index. The HLA-DRB1*04 allele was more frequent in patients with RA from the SR than that in patients from the rest of the SDC (18.2% vs. 9.5%; P = 0.014), whereas the HLA-DRB1*15 allele was more frequent in patients with RA from the rest of the SDC than in patients from the SR (16.2% vs. 7.4%; P = 0.010). Shared epitope (SE) positive patients from the SR had significantly higher serum anti-CCP and RF antibody levels (P = 0.014 and P = 0.004, respectively), higher disease activity (P = 0.043), and worse functional status (P < 0.001), than SE-positive patients from the rest of the SDC. The observed higher incidence of more severe forms of RA in the SR in comparison to the rest of the SDC might be associated with the higher incidence of HLA-DRB1*04 allele in the SR.

HLA class I and II alleles profile in Indian patients with aplastic anemia

Several HLA allele associations with aplastic anemia have been reported around the world. However, due to a lack of such association studies in the Indian population, the current study looked at the frequency of HLA class I (A, B, and C loci) and II (DRB1 and DQB1 loci) with aplastic anemia to see if there was an association between the HLA allele and the disease. A total of 49 aplastic anemia patients (cases) and 100 healthy participants (controls) were included in the study. All of these samples were HLA typed for HLA-A, -B, -C, -DRB1, and DQB1 loci using a polymerase chain reaction sequence-specific oligonucleotide probe approach. A positive association of HLA-DRB1*15:02 (p = 0.00001269, OR = 4.275), was found with AA. Whereas HLA-C*07:01 (p = 0.0017318, OR = 0.179) and HLA-DQB1*02:01 (p = 0.00017033, OR = 0.371) were found to be negatively associated. Our findings are the first to show an association between class I and II HLA alleles and aplastic anemia in the Indian population, and they can be used to further investigate aplastic anemia studies.

Human leucocyte antigens profiling in Malay female patients with systemic lupus erythematosus: are we the same or different?

ObjectiveSLE is a heterogeneous autoimmune disease, in terms of clinical presentation, incidence and severity across diverse ethnic populations. We investigated the human leucocyte antigens (HLA) profile (ie, HLA-A, HLA-B and...

Association of HLA-DRB1 and -DQB1 Alleles with Susceptibility to IgA Nephropathy in Korean Patients.

BackgroundAssociations between IgA nephropathy (IgAN) and HLA-DRB1 and -DQB1 alleles have been reported in several ethnic groups. We investigated the association of HLA-DRB1 and -DQB1 alleles with the predisposition for IgAN and disease progression to end-stage kidney disease (ESKD) in Korean patients.MethodsWe analyzed HLA-DRB1 and -DQB1 genotypes in 399 IgAN patients between January 2000 and January 2019 using a LIFECODES sequence-specific oligonucleotide (SSO) typing kit (Immucor, Stamford, CT, USA) or a LABType SSO Typing Test (One Lambda, Canoga Park, CA, USA). Alleles with a significant difference in two-digit resolution were further analyzed using in-house sequence-based typing and sequence-specific primer PCR. As controls, 613 healthy hematopoietic stem cell donors were included. Kidney survival was analyzed in 281 IgAN patients with available clinical and laboratory data using Cox regression analysis. Where needed, P-values were adjusted using Bonferroni correction.ResultsThe allele frequencies of HLA-DRB1*0405 (corrected P [Pc]<0.001), -DQB1*0401 (Pc=0.048), and -DQB1*0302 (Pc=0.021) were significantly higher in IgAN patients than in controls, whereas those of HLA-DRB1*0701, -DRB1*1501, -DQB1*0202, and -DQB1*0602 (Pc<0.001 for all) were significantly lower in IgAN patients than in controls. The allele frequency of HLA-DQB1*0503 (Pc=0.016) was significantly lower in the ESKD group than in the non-ESKD group; however, there was no significant difference for ESKD progression between these groups.ConclusionsWe report novel associations of HLA-DRB1*1501, DQB1*0202, -DQB1*0302, and -DQB1*0401 with IgAN. Further studies of HLA alleles associated with IgAN progression in a larger cohort and in various ethnic groups are needed.

Clinical Impact of KIR2DS3 and KIR2DL3 Genes in Neuroblastoma Patients

Objective: Neuroblastoma is a common fatal tumor of childhood. Natural killer (NK) cells can exert direct cytotoxicity on tumor cells. The killer immunoglobulin-like receptor (KIR) family of NK cell receptors is involved in activation/inhibition of NK cells. In the KIR gene cluster, six of them (3DS1, 2DS1–5) encode receptors triggering activation, while seven of them (3DL1–3, 2DL1–3, 2DL5) encode receptors triggering inhibition. We aimed to assess the distribution of genetic polymorphisms of KIRs on the clinical course of neuroblastoma and provide guidance on potential therapeutic options. Methods: Our study group included 50 neuroblastoma patients and 100 healthy children as controls. Twenty-eight patients were boys, and twenty-two were girls; median age was 36 months. Fourteen patients had stage 1, 2, 3, or 4S disease, and 36 patients had stage 4 disease. Isolated DNA from the peripheral blood was amplified for sequence-specific oligonucleotide probe analysis of 16 KIR genes. The Fisher’s exact test was used to evaluate the variation of KIR gene distribution. Results: All patients had a lower frequency of KIR2DS3 compared to the control group (p = 0.005). Evaluation of individual KIR genes/genotypes in patients with early stages (stage 1, 2, 3, and 4S) versus stage 4 disease revealed that the frequency of KIR2DS3 was increased in early stages (p = 0.023). Inhibitory KIR2DL3 was increased in the patient group compared to controls (p = 0.038). Furthermore, the frequency of KIR2DL3 was higher in stage 4 neuroblastoma patients compared to the patients with early stages (p = 0.023). Conclusion: Our data suggest a role for KIR2DS3 and KIR2DL3 in development of neuroblastoma. Thus, modulation of KIR2SD3 and/or KIR2DL3 expression or function might present a novel therapeutic strategy for neuroblastoma.

Human leukocyte antigen Class II alleles associated with acral lentiginous melanoma in Mexican Mestizo patients: A case-control study.

Background Melanoma is an aggressive cutaneous cancer. Acral lentiginous melanoma is a melanoma subtype arising on palms, soles, and nail-units. The incidence, prevalence and prognosis differ among populations. The link between expression of major histocompatibility complex Class II alleles and melanoma progression is known. However, available studies report variable results regarding the association of melanoma with specific HLA Class II loci. Aims The aim of the study was to determine HLA Class II allele frequencies in acral lentiginous melanoma patients and healthy Mexican Mestizo individuals. Methods Eighteen patients with acral lentiginous melanoma and 99 healthy controls were recruited. HLA Class II typing was performed based on the sequence-specific oligonucleotide method. Results Three alleles were associated with increased susceptibility to develop acral lentiginous melanoma, namely: HLA-DRB1*13:01; pC = 0.02, odds ratio = 6.1, IC95% = 1.4-25.5, HLA-DQA1*01:03; pC = 0.001, odds ratio = 9.3, IC95% = 2.7-31.3 and HLA-DQB1*02:02; pC = 0.01, odds ratio = 3.7, IC95% = 1.4-10.3. Limitations The small sample size was a major limitation, although it included all acral lentiginous melanoma patients seen at the dermatology department of Dr. Manuel Gea González General Hospital during the study period. Conclusion HLA-DRB1*13:01, HLA-DQB1*02:02 and HLA-DQA*01:03 alleles are associated with increased susceptibility to develop acral lentiginous melanoma in Mexican Mestizo patients.

SERS and advanced chemometrics – Utilization of Siamese neural network for picomolar identification of beta-lactam antibiotics resistance gene fragment

The enormous development and expansion of antibiotic-resistant bacterial strains impel the intensive search for new methods for fast and reliable detection of antibiotic susceptibility markers. Here, we combined DNA-targeted surface functionalization, surface-enhanced Raman spectroscopy (SERS) measurements, and subsequent spectra processing by decision system (DS) for detection of a specific oligonucleotide (ODN) sequence identical to a fragment of blaNDM-1 gene, responsible for β-lactam antibiotic resistance. The SERS signal was measured on plasmonic gold grating, functionalized with capture ODN, ensuring the binding of corresponded ODNs. Designed DS consists of a Siamese neural network (SNN) coupled with robust statistics and Bayes decision theory. The proposed approach allows manipulation with complex multicomponent samples and predefine the desired detection level of confidence and errors, automatically determining the number of required spectra and samples. In constant to commonly used classification-type SNN, our method was applied to analyze samples with compositions previously “unknown” to DS. The detection of targeted ODN was performed with ≥99% level of confidence up to 3 × 10−12 M limit on the background of 10−10 M concentration of similar but not targeted ODNs.