Detection of novel HLA alleles by Next-Generation Sequencing in the Croatian population.

The introduction of Next-Generation Sequencing (NGS) methodology in the histocompatibility testing for both allo-HSCT and solid organ transplantation enables the sequencing of all HLA genes, which in turn leads to the discovery of many new HLA alleles. Over the last 3 years, we have identified 28 novel alleles (HLA-A*02:1079, A*03:01:01:112, A*11:01:01:83, A*11:01:01:87, A*24:595, A*68:01:01:15, B*07:02:01:107, B*08:01:01:67, B*08:01:01:69, B*13:02:01:25, B*15:01:82, B*15:18:08, B*18:01:01:76, B*27:02:06, B*27:05:02:34, B*40:06:01:17, B*40:517, C*04:01:01:173, C*04:477, C*05:276, C*07:01:01:130, C*12:03:80, C*12:03:01:62, DQA1*05:01:01:10, DPB1*13:01:07, DPB1*1146:01, DPB1*1456:01 and DPB1*1514:01) using the NGS method. The presented data emphasises the benefits gained by the utilisation of the NGS-based techniques in HLA genotyping but also provides new insight on the HLA polymorphism in the Croatian population.

An intermediate-sized donor registry experience: HLA barriers in matching procedures.

The data enabling the estimation of the possibility of finding a matched unrelated donor (MUD) within a relatively short time is important for the success of hematopoietic stem cell transplantation (HSCT). In the present study, 738 unrelated Croatian patients in the program of unrelated HSCT were retrospectively analyzed for gender matching, donor origin (national or international), the distribution of HLA alleles and haplotypes, as well as for the probability of finding a 9-10/10 MUD. Almost 70% of the patients in our study group had a 10/10 MUD, while among the patients with a 9/10 MUD, a 1st field resolution level mismatched donor was selected for 55.0% of patients. The majority of pairs were HLA-A mismatched (33.8%). A comparison of HLA allele frequencies between two subgroups of patients revealed significant differences for 13 alleles. However, after p value correction, the difference in frequency remained significant only for four alleles; three HLA alleles (B*08:01, C*07:01, and DRB1*03:01) demonstrated a significantly higher frequency among patients with a 10/10 MUD (Pcorr < 0.0001, Pcorr = 0.0096, and Pcorr < 0.0001, respectively), while the B*35:08 allele was significantly more present among patients with a 9/10 MUD (Pcorr = 0.0328). The comparison of the distribution of HLA haplotypes between patients with a 10/10 MUD and patients with a 9/10 MUD showed significant differences for a number of two-locus and three-locus haplotypes, as well as for one five-locus haplotype (HLA-A*01:01~B*08:01~C*07:01~DRB1*03:01~DQB1*02:01), which was significantly more present in the group of patients with a 10/10 MUD. At least one HLA haplotype from the group of non-frequent HLA haplotypes (positions >1000) was carried by patients with a 9/10 MUD. The data obtained by the present study will contribute to a better estimation of the probability of finding a suitable 9-10/10 MUD for Croatian patients in need of HSCT.

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

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

Various approaches for accessing the influence of human leukocyte antigens disparity in haploidentical stem cell transplantation.

INTRODUCTION: We investigated the association of HLA on clinical outcomes in our cohort of patients in the haplo-HSCT program using the HLAMatchmaker (EM) and PIRCHE score (PS) algorithms. METHODS: The group comprised 64 patients (male = 35-54.7%, female 29-45.3%; median age 43 years) and their related haplo-HSCT donors (male = 30-46.9%, female 34-53.1%). HLA-A/B/C/DRB1/DQB1/DPB1 loci were analyzed. RESULTS: Multivariate analysis of the association between different HLA or patient/donor-related parameters and clinical outcome revealed the following associations with statistical significance: GvHD and HLA class I PS in the GvH direction (p = .0420) and relapse with diagnosis (p = .0163). For OS, the only variable showing a tendency of association was the source of HSCT (p = .0965). CONCLUSION: Combined results of univariate and multivariate analysis suggest that the patients awaiting the selection of the best haplo-HSCT donor could benefit the most from the combination of all three approaches, in cases when a suitable donor can be chosen from a number of potential donors.

Mapping the Human Leukocyte Antigen Diversity among Croatian Regions: Implication in Transplantation.

In the present study, HLA allele and haplotype frequencies were studied using the HLA data of 9277 Croatian unrelated individuals, typed using high-resolution methods for the HLA-A, -B, -C, and -DRB1 loci. The total numbers of observed alleles were 47 for HLA-A, 88 for HLA-B, 34 for HLA-C, and 53 for HLA-DRB1. HLA-A∗02:01 (29.5%), B∗51:01 (10.5%), C∗04:01 (15.8%), and DRB1∗16:01 (10.4%) were the most frequent alleles in the Croatian general population. The three most frequent haplotypes were HLA-A∗01:01~C∗07:01~B∗08:01~DRB1∗03:01 (4.7%), HLA-A∗03:01~C∗07:02~B∗07:02~DRB1∗15:01 (1.7%), and HLA-A∗02:01~C∗07:01~B∗18:01~DRB1∗11:04 (1.5%). Allele and haplotype frequencies were compared between national and regional data, and differences were observed, particularly in the North Croatia region. The data has potential use in refining donor recruitment strategies for national registries of volunteer hematopoietic stem cell donors, solid organ allocation schemes, and the design of future disease and anthropological studies.

The role of HLA in Balkan endemic nephropathy.

Balkan endemic nephropathy (BEN), a progressive chronic tubulointerstitial disease, occurs in the endemic focus of Croatia in a population of about 10,000 inhabitants. One of its most peculiar characteristics is a strong association with upper tract urothelial carcinoma (UTUC). Despite a high number of studies, currently there are insufficient data about the association of BEN and HLA genes. The aim of this study was to investigate the polymorphism of HLA-A, -B, and -DRB1 alleles and haplotypes among BEN patients and to determine whether an association between HLA and BEN exists. In this study, we investigated HLA-A, -B, and -DRB1 alleles and haplotypes in a population of patients with BEN (N = 111) and matched healthy controls (N = 190). All individuals were tested by PCR-SSO and PCR-SSP methods to assess the possible contribution of HLA alleles and haplotypes to the development of/protection from BEN. Our results showed a positive association between the presence of HLA-B*35:02 and DRB1*04:02 alleles and BEN (P = 0.0179 and P = 0.0151, respectively) in contrast to the protective effect of HLA-A*01:01, B*27:05 and B*57:01 alleles (P = 0.0111, P = 0.0330 and P = 0.0318, respectively). Moreover, when BEN patients' HLA haplotypes were compared to controls, two haplotypes were associated with BEN susceptibility among Croatians (HLA-A*02:01~B*08:01~DRB1*03:01 and HLA-A*02:01~B*27:02~DRB1*16:01, P = 0.0064 and P = 0.0023, respectively), while haplotypes HLA-A*02:01~B*27:05~DRB1*01:01 and HLA-A*02:01~B*38:01~DRB1*13:01 each showed a possible protective effect (P = 0.0495). Our results point toward genetic susceptibility to BEN and observed differences in both susceptible/protective HLA profiles indicate the necessity of further studies in order to elucidate the pathogenesis of this disease.

Impact of HLA polymorphisms among cadaveric donors on kidney graft allocation.

This study provides data on HLA-A, -B, and -DRB1 frequencies among 861 end-stage renal disease (ESRD) patients from Croatia and estimates the benefit of the kidney exchange program by comparing HLA distribution and assessing HLA mismatches (MMs) within a group of ESRD patients who received kidney grafts from 707 cadaveric donors (422 from Croatia and 285 from Eurotransplant). Patients positive for HLA-B*07, -B*08, or -B*44 genes more often received a kidney from ET donors, while HLA-DRB1*11 and -DRB1*16 positive patients more frequently received a kidney from CRO donors. ABDR MM 000 was more frequently present in the case of transplantation from ET donors, while MM 222 was significantly more frequent when the donor was from Croatia. Sensitized patients received kidney more frequently from ET donors (P < .0001). A large pool of organ donors with different HLA gene distributions allows for a higher probability of transplantation from HLA highly matched donor.

Detection of novel and confirmation of very rare and rare HLA alleles by next generation sequencing in Croatia.

Routine HLA typing in clinical practice encompassing solid organ and hematopoietic stem cells transplantation programs, disease association typing, volunteer marrow donor typing and population studies, provided a large dataset for studying HLA allele polymorphism in the Croatian population which led to the identification of new, very rare and rare HLA alleles. Over the last 4 years we have identified six new HLA alleles (HLA-A*01:200, A*02:836, A*11:01:01:44, B*08:251, B*18:169 and C*05:46:01:02) and a number of very rare (HLA-B*08:78, DRB1*12:39, DRB1*13:23:02 and DQB1*06:09:04) or rare (HLA-A*24:41, B*39:40:01N, B*51:78:01, DRB1*01:31 and DRB1*14:111) alleles using sequence-based typing methods. The reported data enhance the knowledge about HLA polymorphisms in the Croatian population and provide a foundation for further studies in population genetics.

The MHC gamma block matching: Impact on unrelated hematopoietic stem cell transplantation outcome.

Matching for HLA-A, -B, -C, -DRB1, -DQB1 alleles is the gold standard in hematopoietic stem cell transplantation (HSCT). However, this is often not enough to prevent postransplantational complications. The HLA mismatches (MM) have been associated with higher risk of acute graft versus host disease (GvHD). Gamma block (GB) is located in central HLA region, between HLA-B/C and HLA-DRB/DQB blocks and contains many inflammatory and immune regulatory genes. Single nucleotide polymorphisms (SNPs) within that block can be considered as markers for MHC haplotype matching. Aim of the research was to test whether MM in GB impact the outcome of HSCT in 51 patients transplanted with HLA 10/10 matched unrelated donor. The recipient-donor pairs were typed using PCR SSP kit that detects 25 SNPs within GB. Fifteen out of 51 (29.41%) pairs were GT matched (GT-M) while 36 out of 51 pairs (70.59%) were mismatched (GT-MM). In a univariate analysis, GT-MM was a significant risk factor associated with aGvHD (P = 0.041), although this association was not seen in multivariate analysis. No significant difference in overall survival and relapse occurrence was seen. These results were obtained on a small sample, and it is necessary to further test the possible role of GT matching in unrelated HSCT.

Quantitative polymerase chain reaction technology in chimerism monitoring after hematopoietic stem cell transplantation: One center experience.

Chimerism status evaluation is a routine test performed in post-hematopoietic stem cell transplantation (HSCT) period. The aim of the study was to evaluate a quantitative polymerase chain reaction (qPCR) method (GenDx, Utrecht, the Netherlands) applicability for this purpose. The study included 74 recipient/donor pairs tested for informative markers: median of four and six informative markers was found for patients (related and unrelated donor, respectively). Higher sensitivity of qPCR method was confirmed by analysis of recipient post-HSCT samples (N = 800) among which microchimerism (0.1%-1% recipient DNA) was detected in 21.8% of cases. The ability to detect less than 1% of minor population, as opposed to the short tandem repeat (STR) method for which 1% is the limit, translated into earlier identification of a disease relapse for four patients in our study sample.

Human Leukocyte Antigen class II polymorphisms among Croatian patients with type 1 diabetes and autoimmune polyglandular syndrome type 3 variant.

This study included 161 patients: 92 patients had type 1 diabetes (T1D) while 69 patients had a combination of T1D and autoimmune thyroiditis, the so-called autoimmune polyglandular syndrome type 3 variant (APS3v). Those patients, as well as 93 controls, were typed for HLA-DRB1 and -DQB1 genes to assess their possible contribution to the development/protection of T1D with/without autoimmune thyroiditis. Both HLA-DRB1*04 and -DRB1*03 frequencies were significantly higher among T1D and APS3v patients than in controls. The frequencies of HLA-DRB1*11 and -DRB1*15 were lower among T1D patients, while HLA-DRB1*07 and -DRB1*11 occurred significantly less frequently among APS3v patients in comparison to controls. HLA-DQB1*03:01 and -DQB1*03:02 were associated with a higher risk of developing T1D and APS3v; HLA-DQB1*02 was significantly more present among APS3v patients while HLA-DQB1*03:03 was observed with a significantly lower frequency only among T1D patients. HLA-DRB1*03~DQB1*02 and HLA-DRB1*04~DQB1*03:02 were associated with both diseases. The higher frequency of HLA-DRB1*03/DRB1*03 among APS3v patients was the only significant difference in genotype frequency when compared to T1D patients, while high risk (HLA-DRB1*03/DRB1*04) and medium risk genotypes for T1D (HLA-DRB1*04/DRB1*04) occurred with similar frequencies in both patient groups. Although some of the results point toward shared genetic susceptibility of T1D and APS3v, observed differences in both susceptible/protective HLA profiles indicate the necessity of further studies in order to elucidate the pathogenesis of these diseases.

HLA-DPB1 matching in unrelated hematopoietic stem cell transplantation program contributes to a higher incidence of disease relapse.

The impact of patient/donor matching for HLA-A, -B, -C, -DRB1 and -DQB1 genes in hematopoietic stem cell transplantation (HSCT) is well-recognized, but typing for additional genes, such as HLA-DPB1, is still controversial. Based on defined T-cell epitope (TCE) groups, all HLA-DPB1 mismatches can be classified as permissive or non-permissive. In this retrospective study we analysed 82 patient-matched unrelated donor (MUD) pairs who underwent HSCT, and explored the impact of HLA-DPB1 matches, permissive and non-permissive mismatches on transplantation outcomes. Patient-MUD pairs matched for HLA-DPB1 alleles in univariate analysis were associated with a significantly higher incidence of disease relapse compared to pairs who were permissive/non-permissive HLA-DPB1 mismatched according to the TCE3 and TCE4 algorithms (P=0.025 and P=0.026, respectively), although the significance was lost in multivariate analysis. The analysis did not reveal any significant influence of HLA-DPB1 alleles on overall survival (OS), non-relapse mortality (NRM) or graft-versus-host disease (GvHD) incidence. In conclusion, our study presents evidence that HLA-DPB1 matching influenced the relapse rate in patients after HSCT so the HLA-DPB1 alleles should be implemented in the MUD search algorithm as a transplantation determinant.

The impact of KIR2DS4 gene on clinical outcome after hematopoietic stem cell transplantation.

Killer cell immunoglobulin-like receptors (KIR) are a family of inhibitory/activating receptors expressed on NK cells. Interactions of KIR receptors with KIR ligands have been shown to modify hematopoietic stem cell transplantation (HSCT) outcome. The aim of this research was to determine the KIR2DS4 allele variants distribution among 111 patients with different hematological malignancy who underwent HSCT and their donors, and to evaluate KIR2DS4 alleles' impact on HSCT outcome. The KIR gene frequency analysis showed a significantly higher incidence of full-length KIR2DS4 alleles among patients. The impact of KIR2DS4 alleles on transplantation outcomes revealed that donors' full-length KIR2DS4 alleles is associated with lower overall survival rates, higher risk of GVHD and higher relapse incidence. The expression of full-length KIR2DS4 allele variants may contribute to a worse clinical outcome after HSCT. KIR typing for KIR2DS4 could be used as an additional criterion for selecting suitable donors in cases when more than one HLA identical donor is identified for a specific patient.

The effect of HLA allele and haplotype polymorphisms on donor matching in hematopoietic stem cell transplantation - Croatian experience.

The knowledge of HLA characteristics of a patient's population helps to predict the probability of finding a MUD. The study included 170 transplanted patients for whom a search for a MUD in BMDW was performed and a sample of 4000 volunteer unrelated donors from the Croatian Bone Marrow Donor Registry (CBMDR). Patients and their MUDs were typed for HLA-A, -B, -C, -DRB1, and -DQB1 loci using PCR-SSO and PCR-SSP methods while donors were typed for HLA-A, -B, -C, and -DRB1 loci using the PCR-SSO method. A comparison of allele frequencies at tested HLA loci between patients and donors from CBMDR did not reveal significant differences. The majority of patients (117, 68.8%) had a 10/10 MUD, 45 (26.5%) patients had a 9/10 MUD and eight (4.7%) patients had an 8/10 MUD. The highest number of mismatches (MM) was present at HLA-DRB1 (19; 31.1%). The presence of DRB1*11 and DRB1*04 allelic groups among patients caused allelic MMs at HLA-DRB1 in most cases. The presence of an infrequent HLA-B∼C haplotype resulted in the HLA-C MM at antigen level in the majority of cases. The present study clarified HLA factors that cause difficulties in searching for a 10/10 MUD for Croatian patients.

Determination of HLA-A, -B, and -DRB1 Allele and Haplotype Frequencies in the Croatian Population Based on a Family Study.

In the present study, HLA allele and haplotype frequencies among Croatian families were investigated to evaluate valuable information about HLA genotypes and to compare them with data from the Croatian Bone Marrow Donor Registry (CBMDR). A total of 350 families which have been typed for the purpose of HSCT were included. All individuals were tested using PCR-SSO or PCR-SSP methods for HLA-A, -B, and -DRB1 alleles. The HLA-A-B-DRB1 haplotypes were determined by segregation and directly counted. A total of 30 HLA-A, 54 HLA-B, and 38 HLA-DRB1 alleles and 716 different HLA-A-B-DRB1 haplotypes were identified. Of these, the three most frequent alleles at HLA-A, -B, and -DRB1 loci, respectively, were A*02:01 (30.39%), A*11:01 (13.37%), A*24:02 (10.91%); B*51:01 (12.48%), B*18:01 (8.35%), B*08:01 (8.06%); DRB1*03:01 (11.20%), DRB1*01:01 (9.84%), DRB1*16:01 (9.63%). The following HLA alleles were detected only once: A*02:09, A*24:03, A*24:04, A*24:07; B*07:04, B*15:07, B*15:08, B*39:04, B*39:10, B*39:24, B*40:04; DRB1*08:03, DRB1*11:06, DRB1*13:32, DRB1*14:05. Five most frequent haplotypes were: A*01:01-B*08:01-DRB1*03:01 (5.34%), A*02:01-B*18:01-DRB1*11:04 (1.57%), A*02:01-B*27:02-DRB1*16:01 (1.50%), A*02:01-B*27:05-DRB1*01:01 (1.42%) and A*02:01-B*44:02:01G-DRB1*16:01 (1.28%). The haplotype frequencies based on the family study were compared with the frequencies from CBMDR, and similar results were obtained for all except for the HLA-A*26:01-B*38:01-DRB1*04:02 haplotype. A significantly higher frequency (P = 0.0017) of this haplotype was observed among family individuals. Nine haplotypes were unique and data about their frequencies do not exist in current databases. The data obtained in this study could be useful for anthropology, transplantation and disease association studies.

HLA allele and haplotype polymorphisms among Croatian patients in an unrelated hematopoietic stem cell donor search program.

The aim of the present study was to investigate HLA alleles and haplotypes among Croatian patients in an unrelated HSCT program, and to analyze HLA matching in patient/donor pairs. Analysis was performed on a group of 105 patients and their donors, and 4000 unrelated donors from our registry (CBMDR) served as controls. PCR-SSO and PCR-SSP high-resolution methods for HLA-A, -B, -C, -DRB1, and -DQB1 loci were used for typing patient/donor pairs. Donors from CBMDR were tested for HLA-A, -B, and -DRB1 by PCR-SSO. No difference in frequency at HLA tested loci among patients and donors from CBMDR was observed. A fully matched donor (10/10) was found for 68 (64.8%) patients, and the highest number of mismatches was found for HLA-DRB1 and HLA-C alleles. The presence of HLA-B alleles (B*15:01, B*18:01, and B*51:01) associated with two or more HLA-C alleles as well as the presence of unusual HLA-B/HLA-C (B*35:01-C*07:01 and B*35:01-C*14:02) combinations resulted in mismatches at the HLA-C locus. Additionally, mismatches at the DRB1 locus were in most cases found for DRB1*11 alleles. The results suggest that the DRB1*11:04 allele might be considered as a limiting factor in finding a 10/10 matched donor. These data may help in the improvement of the searching protocol for unrelated donors for Croatian patients.