R. Zunec

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.

Distribution of KIR genes in the Croatian population

The KIR locus with genes involved in immune processes is among the most polymorphic and structurally diverse human loci. KIR genes encode activating and inhibitory receptors that differ in specificity for HLA class I ligands and signaling potential. These receptors are expressed principally by natural killer (NK) cells and subpopulations of T cells. This study represents the first report of the distribution of KIR genes, KIR genotypes and KIR/HLA pairs in 121 unrelated healthy Croatian individuals. Twenty-three different genotypes were observed in the Croatian population and all 16 KIR genes known to date were found. The most frequent KIR genotype was the AA genotype. All individuals had at least one inhibitory KIR/HLA pair with the majority of individuals with three inhibitory KIR/HLA pairs. The most frequent KIR/HLA pair was the KIR2DL3/C1 group. Our results demonstrated the similarity of the Croatian populations KIR repertoire with other Caucasian populations reported so far.

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.

Repetitive DNA polymorphisms in following chimerism after allogeneic bone marrow transplantation

Abstract:  Information about the chimeric status of patients is of great importance in comparison of different conditioning and prophylactic regimens as well as for the post‐bone marrow transplantation (BMT) therapies. In some cases, mixed chimerism (MC) can also be predictive of relapse. Analysis of the short tandem repeats (STR) loci by polymerase chain reaction (PCR) is a choice method for this purpose. In this study, we monitored 15 patients after BMT. Twelve of them underwent classical‐conditioning regimen while the remaining three patients were subjected to non‐myeloablative conditioning (minitransplantation). Evaluation of chimerism was performed using five STR and one variable number of tandem repeats (VNTR) locus. Four additional loci were PCR‐amplified in cases of minitransplantation. Samples were analyzed by electrophoresis in an ALFexpress sequencer. MC was detected in seven cases of which it was predictive of relapse for two patients, who suffered from acute lymphocytic leukemia (ALL). The PCR‐STR method proved to be a fast and relatively simple method, while the tested STR loci showed a high level of informativeness.

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.

Diversity of HLA-B*35 Alleles and Haplotypes among Croatians

Objectives: Investigation of HLA-B*35 subtypes and haplotypes among Croatians. Methods: 4000 randomly selected unrelated donors from Croatian Bone Marrow Donor Registry were typed for HLA-A, -B and -DRB1 by PCR-LabType®SSO method. Results: Four different B*35 alleles (*35:01, *35:02, *35:03, *35:08) were found. Three of them were with similar frequencies as in other European Caucasians, while HLA-B*35:03 was two times more frequent in our population. Predicted haplotypes were also in accordance with present data from other populations. Conclusions: This study should be of benefit in the unrelated haematopoietic stem cell transplantation (HSCT) program. It should facilitate the recipient-donor matching and the selection of a suitable population for further searches.

A case of maternal–foetal chimerism identified during routine histocompatibility testing for hematopoietic stem cell transplantation

This report describes a case of maternal‐foetal chimerism identified in a boy diagnosed with SCID, who underwent HLA testing in preparation for HSCT. The first analysis was carried out on DNA from peripheral blood and included HLA‐A, HLA‐B, HLA‐DRB1 typing using PCR‐SSO. The patients HLA‐B typing results were noninterpretable. All samples were re‐typed for HLA‐B using PCR‐SSP, again resulting in noninterpretable typing of patients HLA‐B. In both cases, several weak positive probes/reactions interfered with the interpretation when using commercial software. Next round of HLA typing, using PCR‐SSP and PCR‐SSO methods, included the patients bone marrow sample and HLA‐C locus, but interpretation was again not possible. The PCR‐STR analysis performed on both peripheral blood and bone marrow samples revealed seven STRs for which two maternal and one paternal allele were detected. Retrospective manual interpretation of HLA‐B and HLA‐C typing revealed that weak positive reactions were indeed owed to paternal HLA‐B and HLA‐C alleles and that the patient had both maternal and one paternal allele. Retyping of HLA‐B and HLA‐C loci and STR analysis on the patients buccal cells sample revealed the expected one maternal/one paternal allele pattern. In summary, the combination of several different typing methods and manual interpretation were necessary to obtain the patients HLA typing results.

The possible role of the tumour necrosis factor polymorphisms and human leucocyte antigens in the development of prostate cancer

The cause of prostate cancer (PC), one of the most common cancers found among ageing men, remains unclear, but genetic predisposition is believed to play a major role in its aetiology. The aim of the study was to examine HLA genes polymorphism and TNF polymorphisms in PC development. Patients diagnosed with PC (N = 113) and 150 healthy individuals were tested for HLA‐A, HLA‐B and HLA‐DRB1 genes and for TNFa, TNFb and TNFd microsatellites. The comparison of patients and controls revealed a positive association of HLA‐DRB1*12, TNFa2 and TNFb5, and a negative association of HLA‐DRB1*13 and TNFb4 with PC. A division of patients into groups according to age, pre‐operative PSA level, Gleason score (GS) and involvement of prostatic capsule, seminal vesicles or bladder neck and perineural invasion of PC demonstrated the following: a positive correlation of HLA‐DRB1*12 and a negative correlation of HLA‐DRB1*13 with younger patients (<65 years), GS > 7 and the positive association of prostatic capsule, seminal vesicles, bladder neck and perineural invasion of PC; TNFb4 alleles negative association with older patients displaying higher PSA levels, higher GS and positive surrounding tissue involvement; positive association of TNFb5 allele for both older and younger patients. Investigation of HLA genes and TNF microsatellites demonstrated a possible role of HLA‐DRB1 and TNF regions in PC aetiology.

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.

Heterogeneity of HLA‐DRB1*04 alleles and haplotypes in the Croatian population

Analysis of allele distribution at the HLA-DRB1*04 gene, as one of the frequent ones among Croatians, and their HLA-A-B-DRB1 haplotypes in the Croatian population was performed in this study. Using LABType® SSO and PCR-SSP method, 11 DRB1*04 subtypes were observed, of which DRB1*04:01 was the most frequent (28.0%) followed by DRB1*04:02 (26.3%), DRB1*04:03 (22.3%), and DRB1*04:04 (14.2%). The significant haplotypes (with highest P value) for given DRB1*04 allele were the following combinations: HLA-B*15:01-DRB1*04:01, HLA-B*38:01-DRB1*04:02, HLA-B*35:03-DRB1*04:03, HLA-B*35:03-DRB1*04:08, HLA-B*14:01-DRB1*04:04, and HLA-B*49-DRB1*04:05. Marked differences in the distribution of our most frequent haplotypes of HLA-B-DRB1*04 (HLA-B*38:01-DRB1*04:02 and HLA-B*15:01-DRB1*04:01) were found in comparison to other European populations investigated so far. Additionally, comparison of HLA-A-B-DRB1*04 haplotypes showed that although there are similarities in the haplotype structure between our and other populations, there are also noteworthy differences. In summary, the identification of conserved and unusual DRB1*04 haplotypes in the present study of Croats should have important clinical implications for donor-recipient matching in the hematopoietic stem cell transplantation program, help in the understanding of HLA polymorphisms in different European populations, and also prove to be very useful in the determination of possible susceptibility genes involved in HLA-DRB1*04-associated diseases.