Marija Maskalan

HLA-A, HLA-B and HLA-DRB1 allele and haplotype diversity among volunteer bone marrow donors from Croatia

The determination of human leucocyte antigen (HLA)‐A, HLA‐B and HLA‐DRB1 alleles in the routine procedure of a volunteer hematopoietic stem cell (HSC) donors registration in the Croatian Bone Marrow Donor Registry (CBMDR) is performed to enhance the odds of finding a suitable HLA compatible donor for patients in need of a HSC transplantation worldwide. However, besides its original purpose, it also provides valuable information about the HLA polymorphism among Croats. The aim of the present study was to analyse the HLA allele and haplotype frequencies in a sample of 4000 donors from CBMDR. The distribution of HLA‐A, HLA‐B and HLA‐DRB1 alleles did not demonstrate significant differences from the data reported for other European populations. The higher frequency of B*40:02 allele in comparison with B*40:01 and DRB1*11:04 in comparison with DRB1*11:01 is interesting because it represents a difference in comparison with the Western and Northern European populations which are a main source of donors for Croatian patients. The haplotype frequencies show a greater variation and difference in comparison with data from other registries and populations; however, due to a lack of high‐resolution haplotype data, comparison was possible only with a very limited number of other populations.

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.

Identification of the novel HLA-B*18:37:02 allele in a Croatian individual

The new allele HLA‐B*18:37:02 differs from HLA‐B*18:37:01 by one nucleotide substitutions in exon 2.

Identification of the novel HLA-A*01:200 allele by sequence-based typing in a Croatian individual.

The new allele HLA‐A*01:200 differs from A*01:12 by four nucleotide substitutions in exon 3.

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 patients 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.

The distribution of the DRB4*01:03:01:02N null allele in HLA‐DRB1~DQB1 haplotypes in the Croatian population

The aim of the present study was to investigate frequency and haplotype distribution of DRB4 alleles in the Croatian population. The investigated sample consisted of 288 cadaveric donor samples positive for one of the DR53 alleles. HLA‐A, ‐B, ‐C, ‐DRB1, and ‐DQB1 typing was performed using the polymerase chain reaction‐sequence specific primers (PCR‐SSP) low resolution method, while HLA‐DRB4 and selected HLA class II specificities typing was performed using PCR‐SSP at the allelic level. Three different DRB4 alleles were observed among DRB1*04 samples; DRB4*01:02 (2.38%), DRB4*01:03 (91.27%), and DRB4*01:03:01:02N (6.35%). The DRB4*01:03:01:02N allele was predominantly observed among DRB1*04:02‐positive samples, while DRB4*01:02 and DRB4*01:03 alleles did not associate preferably with any of the DRB1*04 subtypes. Among DRB1*04~DRB4~DQB1 haplotypes, the predominant DQB1 allele was DQB1*03:02 (69.94%). Seven different DRB4 alleles were found among DRB1*07:01‐positive samples. The analysis of DRB1*07~DRB4~DQB1 haplotypes showed that DRB4*01:03 was found in the majority of HLA‐DRB1*07:01~DQB1*02:02 (49.09%) haplotypes while DRB1*07:01~DQB1*03:03 haplotypes carried the DRB4*01:03:01:02N allele almost exclusively (98.21%). Among six DRB1*09:01‐positive samples, HLA‐DRB1*09:01~DRB4*01:03~DQB1*03:03 was the only detected haplotype. The extended haplotype analysis showed a high frequency of HLA‐B*15(B62)~C*03(Cw9)~DRB1*04:02~DRB4*01:03:01:02N~DQB1*03:02 and HLA‐B*57~C*06~DRB1*07:01~DRB4*01:03:01:02N~DQB1*03:03 haplotypes. In conclusion, the data presented in this study should prompt other population studies focused on DRB3/4/5 genes and be used as a basis for future investigations of the clinical relevance of these genes in transplantation setting.

The distribution of HLA-DRB3 alleles among HLA-DRB1*03:01-positive haplotypes

HLA‐DRB3 allelic polymorphism on HLA‐DRB1*03:01‐positive haplotypes was investigated among 104 cadaveric donors typed for HLA‐A, ‐B, ‐DRB1, and ‐DRB3. Only HLA‐DRB3*01:01:02 and ‐DRB3*02:02:01:01 alleles were detected among HLA‐DRB1*03:01‐positive individuals and their distribution depended on HLA‐B*08 presence: nearly all HLA‐B*08‐positive samples carried DRB3*01:01:02, while HLA‐DRB3*02:02:01:01 was more frequent among HLA‐B*08‐negative subjects.

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.

Association of HLA alleles and haplotypes with CYP21A2 gene p. V282L mutation in the Croatian population

The CYP21A2 mutations that are in linkage disequilibrium with particular HLA‐A, ‐B, ‐DRB1 alleles/haplotypes, cause deficiency of the 21‐hydroxylase enzyme (21‐OHD) and account for the majority of congenital adrenal hyperplasia (CAH) cases. The aim of this study was to investigate those associations with the p.V282L mutation linked to the non‐classical (NC) form of CAH among Croatians. The study included parents of patients with the NC form of CAH, positive for the p.V282L mutation (N = 55) and cadaveric donor samples (N = 231). All subjects were HLA‐A, ‐B, and ‐DRB1 typed and tested for the presence of the p.V282L mutation. Among parents of patients, 92.73% of subjects were positive for the B*14:02 allele and almost half of them carried the HLA‐A*33:01‐B*14:02‐DRB1*01:02 haplotype. Among cadaveric samples 77 out of 96 subjects positive for the B*14:02 allele had the p.V282L mutation. Among them, 37 were positive for the HLA‐A*33:01‐B*14:02‐DRB1*01:02 haplotype, 23 had the HLA‐A*33:01‐B*14:02‐DRB1*03:01 haplotype, 8 had the B*14:02‐DRB1*01:02 combination and 5 were carrying the HLA‐A*68:02‐B*14:02‐DRB1*13:03 haplotype. Only 4 of these subjects were positive for the B*14:02 allele. HLA‐B*14:02 was the only single allele with association that reached statistically significant P value (RR = 12.00; P = 0.0024). Haplotypes B*14:02‐DRB1*01:02 (P < 0.001) and HLA‐A*68:02‐B*14:02‐DRB1*13:03 (P < 0.001) as well as HLA‐A*33:01‐B*14:02‐DRB1*01:02 and HLA‐A*33:01‐B*14:02‐DRB1*03:01 showed high relative risks (RR = 45.00, RR = 41.63 and RR = 36.96, respectively). Our data support the previously documented association of the HLA‐A*33:01‐B*14:02‐DRB1*01:02 haplotype with the p.V282L mutation, but also point out a high frequency of the p.V282L mutation among Croatians with HLA‐A*33:01‐B*14:02‐DRB1*03:01 and HLA‐A*68:02‐B*14:02‐DRB1*13:03 haplotypes.