Report from the extended HLA-DPA1 ~ promoter ~ HLA-DPB1 haplotype of the 18th international HLA and immunogenetics workshop.

The primary goal of the HLA-DPA1 ~ promoter ~ HLA-DPB1 haplotype component of the 18th IHIWS was to characterise the extended haplotypes within the HLA-DP region and survey the extent of genetic diversity in this region across human populations. In this report, we analysed single-nucleotide polymorphisms (SNPs) in 255 subjects from 6 different cohorts. The results from the HLA-DP haplotype component have validated findings from the initial pilot study. SNPs in this region were inherited in strong linkage, particularly HLA-DPA1, SNP-linked promoter haplotypes and motifs in exon 2 of HLA-DPB1. We reported 17 SNP-linked haplotypes in the promoter region. Together with HLA-DPA1 and HLA-DPB1 alleles, they formed 74 distinct extended HLA-DP haplotypes in 438 sequences. We also observed the presence of region-specific alleles and promoter haplotypes. Our approach involved phasing extended SNPs including promoter SNPs, HLA-DPA1 and HLA-DPB1 alleles, in a 22 kb region, GRCh38/hg38 (chr6:33,064,111-33,086,679), followed by clustering of these SNPs as one extended haplotype. This hierarchical clustering revealed four major clades, suggesting that haplotypes within each clade may have diverged from a common ancestral haplotype and undergone similar evolutionary processes. The correlation between HLA-DPA1 and the promoter region raises questions about the role of HLA-DPA1 antigen in the heterodimer. This finding requires validation on a larger sample size specifically designed for anthropological analysis. Nevertheless, the results from this study highlight the clinical potential of selecting better-matched donors for patients awaiting haematopoietic stem cell transplants from genetically overlapping groups that share common ancestral haplotypes.

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

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

Correlation of Luminex-Based Single Antigen Based Results With Complement-Dependent Cytotoxicity Crossmatch and Flow Cytometry Crossmatch Results: A Single-Center Experience From Istanbul.

BACKGROUND: This study aimed to retrospectively investigate the correlation of mean Class I donor-specific antibody (DSA) intensity values detected in Luminex-based techniques with the results of complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometry crossmatch (FC-XM) results. METHODS: A total of 335 patients with kidney failure and their living donors whose CDC-XM, FC-XM, and single antigen based (SAB) tests were studied between 2018 and 2020 for transplant preparation from living donor candidates were included in the study. Patients were divided into 4 groups according to their mean fluorescence intensity (MFI) values of SAB assay. RESULTS: Anti-HLA antibodies (class I and/or class II) were detected using SAB in 91.6% patients included in the study (MFI >1000). Class I DSA was positive in 34.8% of patients with anti-HLA antibodies. When CDC-XM and FC-XM results were evaluated in the 4 groups separated according to MFI values, 3 patients with DSA MFI <1000 had negative CDC-XM and T-B-FC-XM results. Of 32 patients with DSA-MFI between 1000 and 3000, 93.75% (n = 30) had T-B-FC-XM or CDC-XM-negative results, and 6.25% (n = 2) had B-FC-XM-positive results. The CDC-XM, T, and B-FC-XM were negative in all 17 patients with DSA-MFI between 3000 and 5000. Our results showed that MFI >5834 DSA values were significantly correlated with positive T-FC-XM (P < .001), and MFI >6016 values were significantly correlated with positive CDC-XM (P = .002). In addition, MFI values >5000 were associated with both CDC-XM and FC-XM in our study. CONCLUSIONS: The MFI values >5000 correlated with both CDC-XM and FC-XM.

Blood and bone marrow donor registry of Istanbul medical faculty activity and experience in past 3 years : A cross-sectional documentation of TRIS activity: the first blood and marrow registry in Turkey.

Allogeneic stem cell transplantation (SCT) offers a potential cure for some hematological malignancies. For those patients without a family donor, unrelated donor (MUD) registries serve for identifying the best donor. In the present study, we aimed to give a cross-sectional report of our registry's activity and experience as the first established national MUD registry in the country. The study is retrospective and covers the period of 2016 to 2019. A total of 1855 donor searches were performed, and 642 were included in the study. All data were electronically obtained from the institutional database system. All SCTs were either 10/10 or 9/10 HLA matched and originated from an international registry. The most preferred stem cell source was peripheral blood (70.2%). A quarter of transplants were performed using bone marrow, and cord blood was used with a rate of 1.4%. The pandemic-related problems were similar for the other two national registries. During the pandemic, 71 of 432 patients who were searched for donors underwent stem cell transplant(SCT). The low number was related mostly with postponing of SCTs and/also difficulties in continuing of volunteering and in achievement of stem cells from international registry. During the Covid19 pandemic, the SCT activity of centers decreased according to the national, and international guidelines. The study revealed an organized, and multidirectional capacity of the registry and also the adaptation to unpredicted conditions such as pandemic. On the other hand, there is a need for more effective strategies for donor recruitment and retention programme.

Recombination frequencies of human leukocyte antigen loci in hematological malignancies among Turkish population.

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (HSCT) is a treatment option with growing performance for leukaemia, aplastic anaemia and genetic disorders. The frequency of MHC (Major Histocompatibility Complex) gene locus recombination is increased at loci close to the telomeres and in the female gender. The aim of the present study is to document the recombination events by pedigree diagrams with the primary goal to determine the frequency of recombination in a different ethnic population from mostly reported studies. METHODS: Altogether 9545 allogeneic HSCT recipients and their family-based potential donors (n:36231) were included in this retrospective study. RESULTS: Recombinations were determined in 118 (F/M:50/68) out of 9545 families enrolled on the study. These were present in 40 of the patients and 78 of healthy donors. The frequency of recombinations was 0.42% and 0.22%, in patients and donors, respectively. Of the 118 recombinations, 60 were detected in A locus (13 inpatients), 14 in B locus (3 inpatients) and 42 in DR locus (22 inpatients). In our study, due to recombinations in HLA (Human Leukocyte Antigen)-A,-B,-DR loci, we found that some patient-donor pairs became 6/5 matched instead of 6/6 (n:45), eliminating the allogeneic HSCT possibility for the patients from the full-matched siblings. CONCLUSION: To our knowledge, this is the first study reporting the recombination frequencies in HLA loci among Turkish population and thus, providing informative data to the clinicians regarding the cross-over possibilities in Turkish patients with haematological malignancies.

A shared motif of hla-dpb1 affecting the susceptibility to pr3-anca positive granulomatosis with polyangiitis: comparative analysis of a Turkish cohort with matched healthy controls.

We aimed to analyse the distribution of HLA Class 2 genotypes which were reported among the genetic risk factors for ANCA-associated vasculitis (AAV) among Turkish patients in comparison with healthy subjects and previously reported data of AAV cohorts. Ninety-eight patients (F/M: 47/51 and mean age: 49 ± 1.14) were enrolled in the study and records of gender and birthplace-matched 196 healthy kidney donors were used as the control group. Patients were classified according to the clinical subgroups and ANCA serotypes (MPO-AAV, PR3-AAV). DNA was isolated from venous blood from all patients, and high-resolution HLA Class 2 genotyping was carried out by using NGS-Omixon Holotype HLA Kit. The frequencies of HLA-DQB1*03:03, - *06:04, and -DPB1*13:01, -*16:01 and -*66:01:00 alleles were significantly higher, and the frequencies of HLA-DQB1*02:02, -DPB1*02:01 and -*04:01 alleles were lower in the PR3-AAV subgroup (n = 53) compared to the controls. Comparison of amino acid sequences of the associated HLA-DPB1 alleles revealed the sequence of D-E-A-V in risk alleles replaced with the G-G-P-M sequence in protective alleles between 84 and 87th positions. Structural analysis of the HLA-DPB1*02:01 showed that this shared position is in the contact area between HLA-DP α and ß chains and within pocket 1 of the antigen-binding groove. First HLA genotyping analysis in Turkish AAV patients revealed a negative correlation between PR3-ANCA positivity and certain HLA-DPB1 alleles contradictory to the results reported from European cohorts. Known functional effects of D-E-A-V sequence on HLA-DPB1 support the importance of our finding, but further studies are needed to reveal its pathogenic mechanisms.

EFI region 8 Balkan external proficiency testing for HLA typing: Results of 15 years.

Istanbul Medical Faculty, Department of Medical Biology, as the first EFI-accredited HLA laboratory in Turkey since 1999 has been organizing both national and international quality control tests for HLA typing under the banner of the "Balkan external proficiency testing (BEPT)" encompassing countries from EFI regions 8. The first round of BEPT in 2004 was organized for low-resolution HLA-A,-B typing by molecular methods and 12 centres participated in the exercise. In 2005, low-resolution HLA-DR typing was added, and in 2007, low-resolution HLA-C and DQ typing were added to the exercise and 28 centres participated. In 2015, high-resolution (four digits or higher typing) HLA-A,-B,-C,-DR and -DQ typing added to the exercise and 40 centres participated. In the last 2 years, 2017 and 2018, the number of participating centres increased to 48 and 52, respectively. When the distribution of low-resolution typing methods applied in the exercises were investigated, it was found that 82% of the centres in the first round of BEPT in 2004 used PCR-SSP, whereas in the last round in 2018, 26% of the centres preferred SSP, while the rest used SSO (50%) or SSP and SSO (24%) methods. Methods for high-resolution typing were SBT (41%), NGS (18%), SSO (18%), SSP (6%), SBT + NGS (6%) and SBT + SSP (11%). In 15th trial for BEPT, nomenclature mistake rate was 12%, and erroneous result rate was 6% for HLA samples. The most common mistakes in the exercises were nomenclature mistakes. The EFI Standards Version 7.0 is stated "HLA type can be reported using a hyphen if homozygosity is not proven by family studies." In order to provide standard results among HLA laboratories and since accurate HLA typing leads to significant increase of graft and patient survival, quality control exercises should be performed in all HLA-based tests periodically.

Short Tandem Repeat-Polymerase Chain Reaction (STR-PCR) with Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) Method Using for Chimerism Analysis.

BACKGROUND: Recently molecular chimerism analysis after allogeneic hematopoietic stem cell transplantation (AHSCT) has become more important. The use of quantitative chimerism methods aims to assess the kinetics of engraftment to determine graft rejection and failure or relapse of the underlying disease after AHSCT. An accurate and sensitive determination of chimerism status is mandatory after AHSCT. This study aimed to compare two chimerism methods: Multiplex Short Tandem Repeat-Polymerase Chain Reaction (STR-PCR) and quantitative Real Time-PCR (qRT-PCR). METHODS: Thirty-nine blood samples at +28 day were used to extract DNA. Most patients had been diagnosed with acute leukemia (74.3 %) and other hematological diseases. For Multiplex STR-PCR method, PCR products were separated on an ABI 3130 Genetic Analyzer (Applied Biosystem, USA) and for qRT-PCR, an ABI 7500 (Applied Biosystem, USA) Plate System Real Time Analyzer was used to determine the quantification of chimerism per-centage. RESULTS: Of the 39 analyzed samples, 82% concordant chimerism results were detected for both STR-PCR and qRT-PCR methods. Ten mixed chimerisms (MC) were found by qRT-PCR whereas of only 3 MC cases were detected by STR-PCR. In the discordant group of 7 by qRT-PCR, we observed Acute Graft versus Host Disease (aGVHD). Three MC cases that were detected by both STR- and qRT-PCR methods died because of relapse. CONCLUSIONS: The quantitative chimerism method along with multiplex STR-PCR method is important for early detection of MC. qRT-PCR methods can be valuable options in the prevention of graft failure and assisting with fast and early treatment strategies for patients undergoing AHSCT.

Isolated extramedullary cutaneous relapse despite concomitant severe graft-vs.-host disease and tissue chimerism analysis in a patient with acute lymphoblastic leukemia after allogeneic hematopoietic stem cell transplantation: A case report.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment option for patients with acute lymphoblastic leukemia (ALL). The curative potential of allo-HSCT for ALL is, in part, due to the graft-vs.-leukemia (GVL) effect, in addition to the intensive conditioning chemo-radiotherapy. However, relapse remains the major cause of treatment failure following allo-HSCT for ALL. In the allo-HSCT setting, testing for genetic markers of hematopoietic chimerism has become a part of the routine diagnostic program. Routine chimerism analysis is usually performed in peripheral blood or bone marrow; in fact, little is known about the value of tissue chimerism in patients with extramedullary relapse (EMR) after the allo-HSCT setting. The present study reports on, a case of a patient with ALL who experienced isolated cutaneous EMR despite ongoing graft-vs.-host disease (GVHD), and the results of peripheral blood and skin tissue chimerism studies using multiplex polymerase chain reaction (PCR) of short tandem repeats (STR-PCR). The present case demonstrates that, although complete remission and/or chimerism may be achieved in the bone marrow, chimerism achieved at the tissue level, and the subsequent GVL effect, may be limited, despite concomitant severe GVHD following allo-HSCT. Our tissue chimerism analysis results provide a good example of how skin tissue may be a 'sanctuary' site for effector cells of GVL, despite active GVHD and complete hematopoetic chimerism.

Do the aryl hydrocarbon receptor interacting protein variants (Q228K and Q307R) play a role in patients with familial and sporadic hormone-secreting pituitary adenomas?

AIM: The aim of this study was to examine mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene by sequence analysis of exons 1-6 using leukocyte genomic DNA obtained from a cohort of familial isolated pituitary adenoma (FIPA) and apparent sporadic functional pituitary adenoma Turkish patients. METHODS: Fourteen FIPA and 90 sporadic pituitary adenoma (somatotrophinoma, prolactinoma, and corticotrophinoma) patients, 1 sporadic gigantism case, and 70 healthy controls were included in the study. RESULTS: We did not detect AIP mutations in patients with FIPAs or sporadic pituitary adenomas, including the gigantism case. Only two exonic homozygous missense single-nucleotide polymorphisms (rs641081 [Q228K] and rs4930195 [Q307R]) were identified in the AIP locus. Minor allele frequencies of the Q307R and Q228K variants were significantly higher in FIPA patients compared to controls. In addition, the minor allele frequency of the Q228K variant was significantly increased in patients with sporadic somatotrophinomas compared to controls, whereas the minor allele frequency of the Q307R variant was significantly increased in corticotrophinoma patients compared to controls. Conversely, the minor allele frequencies of Q228R and Q307R variants were similar between patients with prolactinomas and controls. No AIP gene mutation or variant was observed in the sporadic gigantism patient. These results suggest that Q228K and Q307R variants in the AIP gene might be involved in the genetic susceptibility to familial and sporadic pituitary adenomas (somatotrophinoma and corticotrophinoma) in the Turkish population.

Prognostic importance of single-nucleotide polymorphisms in IL-6, IL-10, TGF-ß1, IFN-γ, and TNF-α genes in chronic phase chronic myeloid leukemia.

The aim of this study was to explore the association between polymorphisms of five cytokine genes and clinical parameters in patients with Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) treated with imatinib. We analyzed five cytokine genes (interleukin [IL]-6, IL-10, gamma interferon [IFN-γ], transforming growth factor beta-1 [TGF-ß1], and tumor necrosis factor-alpha [TNF-α]) in 60 cases with Ph+ CML and 74 healthy controls. Cytokine genotyping was performed by the polymerase chain reaction-sequence-specific primer. All data were analyzed using the de Finetti program and SPSS version 14.0 for Windows. No significant differences were detected between the CML group and healthy controls with respect to the distributions and numbers of genotypes and alleles in TNF-α, TGF-ß1, IL-10, and IFN-γ. However, the GG genotype associated with high expression in IL-6 was found to be significantly more frequent in CML as compared to controls (p=0.010). The median follow-up time was 49.3 months (range 6.1-168.4) and the median duration of imatinib treatment was 39.5 months (range 5.2-103.4) for these patients. On multivariateanalysis, only IL-10 GCC/GCC highly produced haplotypes were significantly associated with a shorter event-free survival. The relationship between cytokine genotypes/haplotypes and clinical parameters in CML has not been investigated before. Our results suggest that IL-10 may be a useful marker for CML prognosis and theGG genotype of the IL-6 gene may be associated with susceptibility.