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

Human Immunology xxx (2016) xxx–xxx

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The effect of HLA allele and haplotype polymorphisms on donor matching in hematopoietic stem cell transplantation – Croatian experience Zorana Grubic a,⇑, Katarina Stingl Jankovic a, Marija Maskalan a, Ranka Serventi-Seiwerth b, Mirta Mikulic c, Damir Nemet b, Marija Burek Kamenaric a, Boris Labar b, Renata Zunec a a

Tissue Typing Centre, Department of Transfusion Medicine and Transplantation Biology, University Hospital Centre Zagreb, Zagreb, Croatia Department of Haematology, Internal Clinic, University Hospital Centre Zagreb, Zagreb, Croatia c Croatian Bone Marrow Donor Registry, University Hospital Centre Zagreb, Zagreb, Croatia b

a r t i c l e

i n f o

Article history: Received 16 June 2016 Revised 10 October 2016 Accepted 14 October 2016 Available online xxxx Keywords: HLA HSCT Donor search

a b s t r a c t 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. Ó 2016 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

1. Introduction The human leukocyte antigen (HLA) gene family is the most polymorphic system in humans. In particular, the HLA region, located on the short arm of human chromosome 6 (6p21.3), contains more than 220 genes [1]. A main characteristic of the HLA system is linkage disequilibrium (LD). Differences in the distribution of HLA alleles and haplotypes and LD patterns of HLA alleles among various populations and ethnic groups are well documented [2,3]. Because of LD, the number of HLA haplotypes observed in different populations is much smaller than theoretically expected [4].

⇑ Corresponding author at: Tissue Typing Centre, Department of Transfusion Medicine and Transplantation Biology, University Hospital Centre Zagreb, HR1000 Zagreb, Croatia. E-mail address: [email protected] (Z. Grubic).

It is also well known that HLA genes play an important role in the immune response and, for that reason, in the hematopoietic stem cell transplantation (HSCT), too [5,6]. Previous studies have demonstrated the impact of HLA matching on patient outcomes such as graft failure, overall survival (OS), disease free survival (DFS), and graft versus host disease (GvHD) [7–9]. HSCT has been established as a method of therapy for hematologic malignancies and other hematologic or immune disorders. HSC donor selection has been almost exclusively based on selecting an HLA identical donor. At the same time, HSCT from a matched unrelated donor (MUD) is the method of choice when the patient lacks a genotypically or a phenotypically HLA-identical family member donor. At this moment, different criteria for the selection of compatible MUDs are used, but most of them are based on high resolution typing for HLA-A, -B, -C, and -DRB1 alleles (matching 8/8) or HLA-A, -B, -C, -DRB1, and -DQB1 alleles (matching 10/10) [10–12]. It is also reported that patients with frequent HLA alleles

http://dx.doi.org/10.1016/j.humimm.2016.10.004 0198-8859/Ó 2016 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

Please cite this article in press as: Z. Grubic et al., The effect of HLA allele and haplotype polymorphisms on donor matching in hematopoietic stem cell transplantation – Croatian experience, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.10.004

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Z. Grubic et al. / Human Immunology xxx (2016) xxx–xxx

as well as with conserved HLA haplotypes have a higher probability of finding a MUD than those carrying rare HLA alleles or HLA haplotypes with a low frequency [13,14]. A search for a MUD is performed among more than 27 million volunteer donors included in 75 stem cell donor registries from 53 countries from Bone Marrow Donors Worldwide (BMDW) [15]. This number of donors ensures a 50% probability of finding a 10/10 MUD for most patients of European ancestry [11]. Numerous recent studies have emphasized the role of HLA-C compatibility in HSCT [11,16–19]. One of the general conclusions drawn from those studies is that despite matching for both HLA-B alleles and the presence of a strong LD between HLA-B
C loci, unrelated individuals are rather frequently a mismatch (MM) for one or both HLA-C alleles. Moreover, even in the context of frequent haplotypes, HLA-C MMs were often present if those haplotypes included B*18, B*44 or B*51 alleles [17,20]. In view of all of the facts stated above, the knowledge of the HLA characteristics of a patient’s population may help to predict the probability of finding a MUD. This kind of data will also be valuable in creating more specific guidelines for deciding whether the patient’s condition allows a search for a fully MUD or if such a search would be too time-consuming and a mismatched donor is a better option. The first aim of this study was to retrospectively analyze a group of 170 Croatian patients who have undergone the search for a MUD followed by HSCT. The comparison of their HLA allele/ haplotype frequencies with those of healthy controls was performed to point out the presence/absence of differences between those two groups. The second goal was to look into the HLA matching in patient/donor pairs. Furthermore, we aimed to investigate the possible particularities of HLA-B
C linkage in our group of patients and finally to clarify which HLA factors positively or negatively influence a successful MUD search.

bone marrow registries such as the German National Bone Marrow Donor Registry (ZKRD) or the National Marrow Donor Program (NMDP). In Table 1 are listed patients who were excluded from this study from the beginning due to the decision of the transplantation team. Namely, transplantation team requested a 10/10 MUD for those patients, while BMDW searches did not provide even one potential donor that matches that criteria. For 11 out of 170 patients disease progression occurred during donor search but they later proceeded to SCT. The criteria for the suitability (matching grade, source of stem cells, age of donor, etc.) of the donor were determined primarily by the patient’s physician. As the University Hospital Centre Zagreb is the only hospital in Croatia where unrelated HSCT is performed, the patients originated from different areas of Croatia. The standard protocol and preferable matching for our transplant centre is a 10/10 match at allelic level (HLA-A, -B, -C, -DRB1, and -DQB1). In cases when a 10/10 allele MUD cannot be identified, an allelic MM is firstly accepted, and in case if no such donor exists an antigen MM might be accepted (9/10 MUD or even 8/10 MUD) depending on the clinical condition of the patient. The final decision regarding a suitable MUD is made by the patient’s transplant physician. Demographic and clinical data for patients are presented in Table 2. 2.2. Controls A sample of 4000 volunteer unrelated donors registered in the Croatian Bone Marrow Donor Registry (CBMDR) was randomly chosen as controls. These donors represent around 10% of our entire registry; they were chosen from all 10 different regions of Croatia. Individuals living in the same town and carrying the same last name were excluded from the sample, and as such the donors can be considered as a representative sample of the Croatian population.

2. Material and methods 2.3. DNA Isolation

2.1. Patient group The first part of this study included a group of 170 patients who had undergone a HSCT program at the University Hospital Centre Zagreb. Since a matched donor could not be previously found among family members, a search for an unrelated donor in BMDW was performed for all of them. The median time to suitable donor identification was 6.5 weeks. This group of patients does not represent all the patients referred for the unrelated HSCT program in our transplant centre. Namely, patients included in the present study were selected by the transplantation team which includes physicians, a search coordinator and an immunogeneticist, on the basis of a possibility of finding a suitable donor in reasonable time and according to matching probability prediction provided by different

DNA was isolated from peripheral blood using a commercial kit (MagNA Pure LC DNA, Roche Diagnostics GmbH, Mannheim, Germany).

2.4. HLA typing The patients and MUDs selected for them were initially typed for HLA-A, -B, -C, -DRB1, and -DQB1 loci at antigen recognition domain level by means of the Polymerase Chain Reaction – Sequence Specific Oligos (PCR-SSO) method (Immucor Transplant Diagnostics Inc., Stamford, CT, USA) using Luminex technology (Luminex Corporation, Austin, TX, USA) [21].

Table 1 HLA-A
B
C
DRB1
DQB1 haplotypes of patients excluded from this study due to the lack of a MUD who meets the transplantation team requirements for a 10/10 HLA match. Haplotype HLAPatient

Haplotype (rank#)

A⁄

B⁄

C⁄

DRB1⁄

DQB1⁄

1

a (–) b (–) a (–) b (–) a (119,901) b (10,547) a (23,638) b (–) a (–) b (3003)

01:01 80:01 26:01 69:01 26:01 03:02 32:01 03:02 02:06 32:01

18:01 58:01 35:08 51:01 47:01 35:03 50:01 39:01 40:01 40:02

06:02 07:18 12:03 15:02 06:02 04:01 06:02 12:03 04:01 02:02

03:01 03:01 04:04 04:07 14:54 11:04 04:02 04:02 09:01 16:01

02:01 02:01 03:02 03:02 05:03 03:01 03:02 03:02 03:03 05:02

2 3 4 5 #

Ranking in National Marrow Donor Program data files among European Caucasians (http://www.haplostats.org) [2].

Please cite this article in press as: Z. Grubic et al., The effect of HLA allele and haplotype polymorphisms on donor matching in hematopoietic stem cell transplantation – Croatian experience, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.10.004

Z. Grubic et al. / Human Immunology xxx (2016) xxx–xxx

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Age Range Mean value

0.5–68 yrs 38.1 yrs

(Geno-Vision Inc, West Chester, PA, USA). The number of individuals typed at allelic level was dependent on the frequency of the HLA allelic group among Croatians and the number of known HLA alleles so far, for example the number of individuals tested for HLA-A*02 was higher than the number of individuals tested for HLA-A*30.

Gender Male Female

106 (62.35) 64 (37.65)

2.5. Statistics

Diagnosis AA ALL AML CLL CML MDS HL NHL MM SCID Other disease

2 (1.18%) 27 (15.88%) 62 (36.47%) 4 (2.35%) 20 (11.76%) 18 (10.59%) 7 (4.12%) 10 (5.88%) 2 (1.18%) 2 (1.18%) 16 (9.41%)

Donor registries ZKRD CBMDR PL5 + PL6 NMDP United Kingdom other registries

94 (55.29%) 37 (21.76%) 13 (7.65%) 9 (5.29%) 7 (4.12%) 10 (5.88%)

Matching grade 10/10 9/10 8/10

117 (68.82%) 45 (26.47%) 8 (4.71%)

Table 2 Demographic and clinical characteristics of patients (N = 170). Patient characteristics

Allele frequencies were calculated using the GeneRate program (http://geneva.unige.ch/ahpd/) [23]. Two-locus and five-locus haplotypes for the patients were determined by segregation. Haplotype analysis for donors from CBMDR was calculated as previously described [22]. The significance of differences in allele and haplotype frequencies between the patients’ group and controls, as well as between populations, was evaluated using the chi square test, while Fisher’s exact test was used if any of the values in 2  2 tables were less than 5. The P value was corrected by the number of alleles observed at each locus (Pcorr). To determine the power of the study and the probability that the test will find a true statistically significant difference between the group of patients and the control group post hoc power analysis calculation was performed [24]. 3. Results

AA – aplastic anemia; ALL – acute lymphoblastic leukemia; AML – acute myelogenous leukemia; CLL – chronic lymphocytic leukemia; CML – chronic myelogenous leukemia; MDS – myelodysplastic syndrome; HL – Hodgkin’s lymphoma; NHL – non Hodgkins’ lymphoma; MM – multiple myeloma; SCID – severe combined immunodeficiency; CBMDR – Croatian Bone Marrow Donor Registry; ZKRD – Zentrales Knochenmarkspender-Register Deutschland – The German Bone Marrow Donor Registry, NMDP – National Marrow Donor Program, PL5 – PolandPOLTransplant; PL6 – Fundacja DKMS Polska; other registries: France, Austria, Italy, Denmark, Canada, Portugal.

This method uses kits for HLA class I, which cover exons 2 and 3, while kits for HLA class II cover exon 2. The 50 ends of upstream primers (included in kits) were labelled with biotin and each PCR product was hybridized with 72 probes for HLA-A, 92 probes for HLA-B, 53 probes for HLA-C, 68 probes for HLA-DRB1 and 48 probes for HLA-DQB1, complementary to the polymorphic sequences. After hybridization, amplicons were labelled with streptavidin-R-phycoerythrin which is a specific fluorescent ligand of biotin and quantified on the Luminex LABScanTM 100 flow analyser (Luminex Corporation, Austin, TX, USA). The patients’ confirmatory typing as well as MUD additional typing was performed using Polymerase Chain Reaction – Sequence Specific Primers (PCR-SSP) at allelic level for each group (GenoVision Inc., West Chester, PA, USA) [19]. Donors from CBMDR were also typed for HLA-A, -B, -C, and -DRB1 loci using the PCR-LABTypeÒ SSO method (Immucor Transplant Diagnostics Inc., Stamford, CT, USA). Only a small number of donors from CBMDR was HLA-DQB1 typed and for that reason HLA-DQB1 locus was excluded from the analysis. After obtaining a typing result at antigen level, a group of randomly chosen individuals was subtyped by the standard PCR-SSP highresolution protocol for different HLA allele families (HLA-A*01, A*02, A*03, A*11, A*23, A*24, A*25, A*26, A*29, A*30, A*31, A*32, A*33, A*66, A*68, B*07, B*08, B*13, B*14, B*15, B*18, B*27, B*35, B*38, B*39, B*40, B*41, B*44, B*50, B*51, B*57, B*58, C*01, C*02, C*03, C*04, C*05, C*06, C*07, C*08, C*12, C*14, C*15, C*16, C*17, DRB1*01, DRB1*03, DRB1*04, DRB1*07, DRB1*08, DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*15 and DRB1*16)

The frequencies of HLA-A, -B, -C, -DRB1, and -DQB1 alleles found in our group of patients are listed in Table 3. Our data are comparable with data reported for donors from CBMDR [22]. We identified 22 HLA-A, 35 HLA-B, 22 HLA-C, 31 HLA-DRB1 and 16 HLA-DQB1 alleles. The most frequent allele at each locus was as follows: A*02:01 (30.0%), B*51:01 (12.7%), C*07:01 (21.8%), DRB1*03:01 (12.9%) and DQB1*03:01 (24.1%). The comparison of allele frequencies at tested HLA loci between the patients’ group and donors from CBMDR did not reveal any significant differences. Eight HLA-A, 6 HLA-B, 4 HLA-C, 9 HLA-DRB1 and 3 HLA-DQB1 alleles were observed only once among our group of patients, but the frequencies of these HLA alleles are low among volunteer donors, too. Two alleles, DRB1*11:28 and DRB1*11:58 present in the patients’ group were not observed among analyzed donors from CBMDR. Two hundred and nineteen different five-locus haplotypes were detected in a total of 150 patients. Specifically, for 20 patients we were not able to determine HLA haplotypes because there were no additional family members. Among the observed haplotypes, 16 haplotypes were observed 3 or more times (Table 4), 12 different haplotypes were present two times, while the remaining 191 haplotypes were detected only once (Post-hoc power analysis 100%). The most frequent one was HLA-A*01:01
B*08:01
C*07: 01
DRB1*03:01
DQB1*02:01, followed by HLA-A*02:01
B*18:0 1
C*07:01
DRB1*11:04
DQB1*03:01 and HLA-A*02:01
B*51:0 1
C*14:02
DRB1*01:01
DQB1*05:01. The search for donors from BMDW was then performed for each patient and, as a result, at least one donor was chosen to proceed with the confirmatory HLA typing and after that for transplantation. The confirmatory HLA typing resulted in at least one suitable, fully MUD for HLA-A, -B, -C, -DRB1, and -DQB1 alleles (10/10) for 117 (68.8%) patients (Table 2). For 45 (26.5%) patients, a 9/10 MUD was identified, while for 8 (4.7%) patients an 8/10 MUD was obtained. The total number of mismatches among patientdonor pairs was 61. The highest number of mismatches was present at the HLA-DRB1 locus (19; 31.1%), followed by mismatches at the HLA-C and HLA-A locus (16 and 15, respectively), while 7 mismatches were found for HLA-B alleles. Only 4 mismatches were detected at the HLA-DQB1 locus (Table 5). However, when the

Please cite this article in press as: Z. Grubic et al., The effect of HLA allele and haplotype polymorphisms on donor matching in hematopoietic stem cell transplantation – Croatian experience, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.10.004

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Z. Grubic et al. / Human Immunology xxx (2016) xxx–xxx

Table 3 HLA-A, -B, -C, -DRB1 and -DQB1 allele frequencies in the sample of 170 patients. HLAA⁄

AF

B⁄

AF

C⁄

AF

DRB1⁄

AF

DQB1⁄

AF

01:01 02:01 02:05 03:01 03:02 11:01 23:01 24:02 24:03 24:07 25:01 26:01 29:01 29:02 30:01 30:04 31:01 32:01 33:01 66:01 68:01 69:01

0.1235 0.3000 0.0029 0.1353 0.0029 0.0500 0.0265 0.1323 0.0029 0.0029 0.0382 0.0471 0.0029 0.0088 0.0029 0.0029 0.0176 0.0382 0.0029 0.0147 0.0382 0.0029

07:02 07:04 07:05 08:01 13:02 14:01 14:02 15:01 15:17 18:01 27:02 27:05 35:01 35:02 35:03 35:08 38:01 39:01 40:01 40:02 41:01 41:02 44:02 44:03 44:05 44:27 49:01 50:01 51:01 52:01 53:01 55:01 56:01 57:01 58:01

0.0735 0.0029 0.0029 0.0971 0.0206 0.0059 0.0176 0.0235 0.0029 0.1147 0.0294 0.0382 0.0735 0.0088 0.0500 0.0118 0.0294 0.0265 0.0118 0.0176 0.0029 0.0118 0.0471 0.0176 0.0088 0.0118 0.0265 0.0029 0.1265 0.0235 0.0029 0.0088 0.0118 0.0294 0.0088

01:02 02:02 03:02 03:03 03:04 04:01 05:01 06:02 07:01 07:02 07:04 07:18 08:02 12:02 12:03 14:02 15:02 16:01 16:02 16:04 17:01 17:03

0.0441 0.1000 0.0059 0.0765 0.0147 0.1559 0.0382 0.0559 0.2177 0.0765 0.0118 0.0029 0.0235 0.0235 0.1059 0.0412 0.0441 0.0059 0.0029 0.0029 0.0029 0.0118

01:01 01:02 03:01 04:01 04:02 04:03 04:04 04:07 04:08 04:15 07:01 08:01 08:04 10:01 11:01 11:02 11:03 11:04 11:11 11:15 11:28 11:58 12:01 13:01 13:02 13:03 14:01 14:54 15:01 15:02 16:01

0.1118 0.0059 0.1294 0.0206 0.0088 0.0147 0.0176 0.0029 0.0059 0.0029 0.0912 0.0147 0.0029 0.0088 0.0824 0.0029 0.0029 0.1118 0.0029 0.0029 0.0029 0.0029 0.0147 0.05 0.0471 0.0118 0.0118 0.0176 0.0941 0.0206 0.0824

02:01 02:02 03:01 03:02 03:03 03:04 03:05 03:19 04:02 05:01 05:02 05:03 06:01 06:02 06:03 06:04 06:09

0.1294 0.0588 0.2412 0.0618 0.0324 0.0029 0.0029 0.0029 0.0147 0.1294 0.0853 0.0294 0.0206 0.0912 0.0559 0.0353 0.0059

AF – allele frequency; in bold – five most frequent HLA alleles for each locus.

Table 4 HLA-A
B
C
DRB1
DQB1 haplotype frequencies in the sample of 150 patients observed 3 or more times. Haplotype HLARank

A⁄

B⁄

C⁄

DRB1⁄

DQB1⁄

HF

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

01:01 02:01 02:01 01:01 01:01 03:01 03:01 02:01 25:01 02:01 02:01 02:01 02:01 02:01 03:01 11:01

08:01 18:01 51:01 57:01 52:01 07:02 18:01 44:27 18:01 07:02 08:01 27:02 51:01 51:01 07:02 35:01

07:01 07:01 14:02 06:02 12:02 07:02 07:01 07:04 12:03 07:02 07:01 02:02 14:02 15:02 07:02 04:01

03:01 11:04 01:01 07:01 15:02 15:01 11:04 16:01 15:01 15:01 03:01 16:01 16:01 01:01 13:02 01:01

02:01 03:01 05:01 03:03 06:01 06:02 03:01 05:02 06:02 06:02 02:01 05:02 05:02 05:01 06:04 05:01

0.0733 0.0400 0.0233 0.0167 0.0133 0.0133 0.0133 0.0133 0.0133 0.0100 0.0100 0.0100 0.0100 0.0100 0.0100 0.0100

HF-haplotype frequency.

analysis of the MMs was performed with respect to the resolution level the situation was different. To be precise, the highest number of MMs at high resolution level was again observed for the HLADRB1 locus (18; 29.5%), but the MMs at antigen level were found in most cases for the HLA-C locus (14; 22.9%). The presence of DRB1*11 and DRB1*04 allelic groups among patients caused allele-level MMs at the HLA-DRB1 locus in most cases (Table 5). Specifically, 9 patients mismatched for the DRB1*11 allelic group and 5 for the DRB1*04 allelic group.

Analysis of the HLA-B locus MMs revealed that in 5 out of 7 high resolution level MMs at the HLA-B locus the allelic group in question was B*35. On the other hand, the presence of an infrequent HLA-B
C haplotype resulted in an HLA-C MM at antigen level in the majority of cases. In Table 6 are listed all HLA-B allelic groups seen in HLA-C mismatched patient/donor pairs. The selection was based on the presence/absence of the combination among the patients’ group for the given HLA-B allelic group, which caused a mismatch at HLA-C locus. The HLA-B
C haplotype frequencies

Please cite this article in press as: Z. Grubic et al., The effect of HLA allele and haplotype polymorphisms on donor matching in hematopoietic stem cell transplantation – Croatian experience, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.10.004

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Z. Grubic et al. / Human Immunology xxx (2016) xxx–xxx Table 5 The distribution of HLA mismatches among patient/donor pairs. HLA locus

N

a) Patient/donor pairs with 9/10 matching grade A 13 B 3 C 14 DRB1 12 DQB1 3 R 45 b) Patient/donor pairs with 8/10 matching grade A+B 1 A + DRB1 1 B+C 1 B + DRB1 2 C + DRB1 1 Both DRB1 1 DRB1 + DQB1 1 R 8

LR

HR

9 – 12 1 1 23

4 3 2 11 2 22

C C

2

1 1 B 2 DRB1 1 1 14

LR – low resolution (antigen level); HR – high resolution (allelic level).

were compared with those from CBMDR and no significant differences were observed [25].

4. Discussion The present study is an extension of a previous study of HLA polymorphism among patients in the program of unrelated HSCT [26]. Our intention was to determine which HLA factors could play a role in the problem for MUD searching, to predict the difficulties during the searching period and to predict in advance which patient’s HLA alleles/haplotypes will probably result in a successful or an unsuccessful search for 10/10 MUD. For that reason, we retrospectively analyzed 170 Croatian patients for whom the search for a MUD was performed in order to evaluate the distribution of HLA alleles and haplotypes. The design of our study led us to present only the realised donor for the given patient, the one for whom a CT was performed and was finally chosen to proceed with the stem cell harvesting and transplantation. However, it should be mentioned that based only on the HLA matching, a number of patients did indeed have more than one potential MUD and as a result different HLA alleles/linkages could be responsible for a MM for the same patient, depending on which MUD was actually selected. We also analyzed each patient/donor pair, defining the HLA factors that influence the match in our cohort. The comparison of HLA allele distribution between patients and controls did not reveal any significant difference. The 16 most frequent HLA-A
B
C
DRB1
DQB1 haplotypes are also in the group of the most frequent haplotypes in CBMDR. We observed that patients with at least one frequent haplotype are in most cases in the group of patients with a 10/10 MUD [22]. A comparison between haplotype frequencies and haplotype ranking reported for the German Bone Marrow Donor Centre (DKMS) as well as for ‘‘European Caucasians” in the NMDP donor file showed that some of the most frequent haplotypes in our cohort (Table 4; ranks 1, 2, 4, 6, 9, 10, 11, 16) are also very frequent in those two donor inventories [27,28]. The haplotype A*02:01
B*27:02
C*02:02
DRB1*16:01
DQB1*05:02 was frequent among our patients (Table 4; rank 12), but its rank among German donors was low (49). HLA-B*51:01 positive haplotypes (Table 4; ranks 3, 13, 14) were not listed among frequent haplotypes in the DKMS data set, which could be explained by the more frequent occurrence of the B*51:01 allele among Croatians [26]. The high frequency of A*03:01
B*18:01
C*07:01
DRB1*11:04
DQB1*03:01 observed among patients (Table 4; rank 7) corresponds well to its frequency in CBMDR and represent the most marked difference

Table 6 The list of all HLA-B allelic groups seen in HLA-C mismatched patient/donor pairs and their distribution among donors from CBMDR. HLA-B
C

Patients (N = 7)%

CBMDR (N = 200)%

15:01
01:02 02:02# 03:03# 03:04 04:01# 05:01 07:01 12:03

0 14.29 42.86 28.57 14.29 0 0 0

2.50 2.50 39.00 26.50 15.50 0.50 13.00 0.50

18:01
02:02 04:01 05:01# 06:02 07:01 12:03

Patients (N = 39)% 0 0 2.56 0 66.67 30.77

CBMDR (N = 600)% 1.33 0.67 2.67 0.33 57.33 37.67

35:01
01:02 04:01 14:02

Patients (N = 27)% 0 96.30 3.70

CBMDR (N = 92)% 1.09 97.82 1.09

35:02
04:01

Patients (N = 5)% 100

CBMDR (N = 21)% 100

35:03
01:02 04:01 07:01# 12:03

Patients (N = 15)% 0 73.33 6.67 20.00

CBMDR (N = 85)% 1.18 63.52 1.18 34.12

35:08
04:01 12:03

Patients (N = 4)% 75.00 25.00

CBMDR (N = 16)% 93.75 6.25

44:02
01:02 03:03 04:01# 05:01# 06:02 07:01 07:04# 16:04

Patients (N = 16)% 0 0 6.25 81.25 0 0 6.25 6.25

CBMDR (N = 132)% 2.27 1.51 0 81.82 0.76 0.76 0.76 12.12

44:03
04:01 14:02 16:01 16:02#

Patients (N = 6)% 66.67 0 16.67 16.67

CBMDR (N = 82)% 71.95 1.22 19.51 7.32

44:05
02:02 05:01

Patients (N = 4)% 100.00 0

CBMDR (N = 45)% 97.80 2.20

44:27
07:04

Patients (N = 4)% 100.00

CBMDR (N = 31)% 100.00

51:01
01:02# 02:02# 03:03 03:04 04:01 05:01 06:02 07:01 07:02 12:03 14:02 15:02# 15:04 16:01 16:02

Patients (N = 42)% 26.19 7.14 0 0 0 2.38 2.38 0 0 7.14 26.19 26.19 0 2.38 0

CBMDR (N = 600)% 20.83 7.83 1.83 0.50 2.50 1.67 1.17 1.83 0.50 3.68 21.00 31.83 0.17 2.83 1.83

CBMDR – Croatian Bone Marrow Donor Registry. # HLA-B
C haplotype of HLA-C mismatched patient(s).

between our study cohort and donors from Germany [22,27]. It is interesting to note that the A*01:01
B*52:01
C*12:02
DR

Please cite this article in press as: Z. Grubic et al., The effect of HLA allele and haplotype polymorphisms on donor matching in hematopoietic stem cell transplantation – Croatian experience, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.10.004

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B1*15:02
DQB1*06:01 haplotype (Table 4; rank 5) is not mentioned among frequent donor haplotypes from DKMS, although it has been reported for Italian and Turkish minorities in the DKMS data set. In NMDP it was found at position 58 among European Caucasians [3,22,27–28]. On the other hand, A*03:01
B*07:02
B*07:02
DRB1*13:02
DQB1*06:04 was not reported for donors from Germany but this haplotype is present among NMDP European Caucasians [27,28]. Finally, for the A*02:01
B*44:27
C*07: 04
DRB1*16:01
DQB1*05:02 haplotype no data is available so far, neither for the German donors nor for the NMDP European Caucasians [27,28]. Moreover, this haplotype is not currently reported for any of the populations listed at allelefrequencies.net [3]. The reason for the missing population data is that no systematically typed populations for HLA-B variations outside of exon 2 and 3 exist. Our findings regarding this haplotype fit well into data reported by Vidan Jeras about B*44:02:01G allelic variability among 12 European populations [29]. As a result of the comparison of the percentage of Croatian patients with a 10/10 MUD with data reported from other studies, we found that the frequency of our patients with a 10/10 MUD was higher than in other investigations, while the percentage of patients with a 9/10 MUD was similar [13,30–32]. A few possible explanations for this finding exist. The first reason for such a high percentage of 10/10 MUDs in comparison to other similar studies is that the patients with low probability (for example: prediction probability < 60% for 8/10 MUD) after preliminary search results, were excluded from this study and switched to other protocols such as haplo-identical transplantation. Also, patients who did not have a single potentially MUD upon direct interrogation of the BMDW database and for whom a previous decision of their physician was that only a 10/10 MUD is acceptable were also not included in the present study; the number of excluded patients was 5. And the last reason lies in the homogeneity of the Croatian population in comparison to the populations included in the above mentioned studies. Namely, in the period of time when the patients were selected we did not have any patient of nonEuropean origin. In this study we did not observe any difference in the search duration between the patients with frequent or rare HLA haplotypes. The analysis of HLA-A allele frequencies among patients showed that the presence of non-frequent HLA-A alleles such as A*02:05, A*24:07, A*30:04, A*66:01 or A*69:01 resulted in a 9/10 MUD. The presence of non-frequent HLA alleles does not automatically mean that the search for the 10/10 MUD is abandoned, but a discussion with the transplantation team members is always performed in such cases and the decision with respect to time constraints and patient condition is made, and a period in which a search for a 10/10 MUD can be performed is identified. The search for a 10/10 donor is stopped after that period and a 9/10 donor will be considered. On the other hand, the presence of non-frequent alleles at the HLA-B locus in our sample did not cause a mismatch except when the B*35 allelic group was in question. This finding fits well with the data from other studies which also reported that the presence of HLA-B*35:02/*35:03/*35:08 confers a higher risk of a B*35 allele MM [19], or that as much as 61.7% of HLA-B allelic incompatibilities were due to the HLA-B*35 allelic group [30]. Additionally, mismatch at the HLA-C locus could also be present when patients are B*35:03 positive because they have a higher possibility of carrying the HLA-C*12:03 allele instead of the HLA-C*04 allelic group. Namely, the well-known strong linkage disequilibrium between B*35 and C*04, which is reported for many populations worldwide, weakens for the HLA-B*35:03 allele [18]. The HLA-B*35
C haplotype analysis in our population demonstrated that 7.84% of B*35 haplotypes are C*12:03 positive. More in depth analysis revealed that 20.0% of B*35:03 positive

haplotypes carried the C*12:03 allele. The highest number of MMs at the HLA-C locus among our patients was observed in cases of haplotypes with B*51:01 (four times), followed by haplotypes with B*15:01 (three times) and this observation fits very well with the conclusions from Tiercy who marked these alleles as factors that contribute to an unsuccessful search for a 10/10 MUD [19]. Haplotypes with HLA-B*51 were also the cause of HLA-C mismatches in the highest number of patients in the Italian study [30]. It is interesting to note that one patient carried a nonfrequent combination B*44:02
C*07:04, while his donor carried a common combination B*44:02
C*05:01. Namely, in our population within the B*44:02:01G group, B*44:02 is observed with a frequency of 78% which is similar to the data reported for Slovenians and Bulgarians [29] and forms a haplotype with C*05:01 allele in more than 80% of cases. At the same time, the B*44:27 allele is found almost exclusively in combination with the C*07:04 allele. Since, based on the results of in vitro functional tests, it has been suggested that B*44:02 vs. B*44:27 mismatch might be permissive [33], a donor positive for B*44:27
C*07:04 was selected in order to avoid an HLA-C mismatch. It should be noted that the numbers of positive patients for some of the HLA-B
C combinations mentioned before was small (<5), which is in concordance with their presence among donors from CBMDR. Still, all detected associations among patients demonstrate similar frequencies as observed in the larger group of donors from CBMDR. In our sample, a donor with a mismatch at the HLA-C locus was selected in 9.4% cases, while the total number of HLA-C MMs in the total number of MMs was 26.2%. These frequencies are quite similar to data observed in multi-centre studies [9,18,34], but they differ from the data by the Italian study in which the authors have discovered 65% of disparities at the HLA-C locus [30]. 87.5% of HLA-C mismatches found among our patient/donor pairs are on the antigen level which is in concordance with the data reported in previously mentioned studies [9]. Regarding the MMs at the high resolution level for HLA-DRB1 alleles (n = 18), the most prominent challenge for matching proved to be patients with the DRB1*11 and DRB1*04 allelic groups. This study again confirmed our previous data on a smaller sample of patients that the DRB1*11:04 allele, which is one of the common alleles in our population, belongs in the same category as B*35 alleles in our group of patients. Namely, patients positive for the DRB1*11:04 allele were more difficult to match than patients carrying the DRB1*11:01 allele. Among those DRB1*11:04 allele positive patients four (22.2%) were transplanted with a DRB1*11:01 allele positive donor. The explanation for this lies in the fact that these two alleles are present among Croats with an almost equal frequency, which is in contrast with the German registry (the source of more than 50% of donors for our patients) where the DRB1*11:01 allele is predominant, as well as in the populations of the Central, North and West Europe [3]. Among 25 patients positive for the DRB1*04 allelic group, 20% received a DRB1*04 mismatched transplant. The difficulty of matching those patients might be a result of the fact that this allelic group contains a number of alleles with similar frequencies. Namely, 11 different DRB1*04 alleles have been observed in our population so far and the distribution of DRB1*04:01, 04:02, 04:03, 04:04 among Croats is as follows: 28.0%, 26.3%, 22.3%, and 14.2%. This is similar to other European populations [3]. It is interesting that in our cohort of patients positive for one of the two allelic groups (DRB1*13 and DRB1*14) from the same broad serological specificity HLA-DR6, the less frequent (2.9%) DRB1*14 caused four MMs. More precisely, among patients positive for the DRB1*13 allelic group (n = 41; 12.1%) no MMs at HLA-DRB1 were found, while at the same time the DRB1*14 allelic group caused mismatches in 4 out of 10 DRB1*14:01/54 positive patient/donor pairs. Our findings are similar to the results reported

Please cite this article in press as: Z. Grubic et al., The effect of HLA allele and haplotype polymorphisms on donor matching in hematopoietic stem cell transplantation – Croatian experience, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.10.004

Z. Grubic et al. / Human Immunology xxx (2016) xxx–xxx

by Pasi et al. who found a 26.0% DRB1*14 mismatch rate among DRB1*14:01:01G positive patient/donor pairs [35]. In our cohort of investigated patients, only 2.4% of recipient/donor pairs were incompatible at the HLA-DQB1 locus which is in concordance with the results from other studies [30,36]. In particular, most differences were a result of the presence of uncommon DRB1
DQB1 combinations in patients (DRB1*15:01
DQB1*05:02 and DRB1*15:01
DQB1*06:03) as well as the presence of the DRB1*07:01 allele which associated with different DQB1 alleles (DQB1*02:02 and DQB1*03:03). Four of our eight patients with 8/10 MUD received combinations of HLA class I/class II MMs. This was done in concordance with recent studies which reported that if more than one MM is present, it is better to choose a donor with a combined HLA class I/class II incompatibility [9,37]. It is also important to underline that the final decision lies on the patient’s transplant physician. Finally, some limitations of this study should be mentioned; the cohort of patients included in the study is not a large one since it encompasses the patients referred to the only hospital which performs unrelated HSCT and covers the entire country. Approximately 40 unrelated HSCT per year in the last two years were performed, and the studied patients’ cohort was collected in the period from 2010 to 2014. The patients for whom the search was performed in the first few years were not in the same situation as those in the later years since the CBMDR has been expanding at a high rate in the recent period (the total number of donors has been increased by 30% in the last three years). For that reason we can speculate that more of those patients could have found a donor in our registry. One important fact of this study is that on the basis of our results approximately 30% of our patients obtained a 10/10 MUD in CBMDR. Considering the percentage of our population which is included in the CBMDR and the comparison with other registries (ZKRD for example) the size of our registry should be four times larger in order to find a donor for approximately 80% of our patients based on the calculation by Schmidt et al. [27]. In conclusion, the present study clarified HLA factors that may cause problems in searching for a 10/10 MUD in our patients. Although randomized trials would be ideal for this kind of investigation, our intention was to present patients who were transplanted. The results should be of value for transplantation centers in our region with similar HLA population profile. This data will also serve for the modification of our current MUD searching protocol, especially in cases of difficult searches, as well as in cases when the clinical status of a patient presents a time-limiting factor in finding a better MUD. References [1] R. Horton, L. Wilming, V. Rand, R.C. Lovering, E.A. Bruford, V.K. Khodiyar, M.J. Lush, S. Povey, C.C. Talbot Jr, M.W. Wright, H.M. Wain, J. Trowsdale, A. Ziegler, S. Beck, Gene map of the extended human MHC, Nat. Rev. Genet. 5 (2004) 889–899. [2] HaploStats. http://www.haplostats.org/ (accessed on 17 November 2015). [3] F.F. Gonzales-Galarza, S. Christmas, D. Middleton, A.R. Jones, Allele frequency net: a database and online repository for immune gene frequencies in worldwide populations, Nucleic Acids Res. 39 (2011) D913. [4] E.J. Yunis, C.E. Larsen, M. Fernandez-Vina, Z.L. Awdeh, T. Romero, J.A. Hansen, C. A. Alper, Inheritable variable sizes of DNA stretches in the human MHC: conserved extended haplotypes and their fragments or blocks, Tissue Antigens 62 (2003) 1–20. [5] S. Morishima, S. Ogawa, A. Matsubara, T. Kawase, Y. Nannya, K. Kashiwase, M. Satake, H. Saji, H. Inoko, S. Kato, Y. Kodera, T. Sasazuki, Y. Morishima, Japan Marrow Donor Program, Impact of highly conserved HLA haplotype on acute graft-versus-host disease, Blood 115 (2010) 4664–4670. [6] B.E. Shaw, R. Arguello, C.A. Garcia-Sepulveda, J.A. Madrigal, The impact of HLA genotyping on survival following unrelated donor haematopoietic stem cell transplantation, Br. J. Haematol. 150 (2010) 251–258. [7] P. Loiseau, M. Busson, M.L. Balere, A. Dormoy, J.D. Bignon, K. Gagne, L. Gebuhrer, V. Dubois, I. Jollet, M. Bois, P. Perrier, D. Masson, A. Moine, L. Absi, D. Reviron, V. Lepage, R. Tamouza, A. Toubert, E. Marry, Z. Chir, J.P. Jouet, D. Blaise, D. Charron, C. Raffoux, HLA Association with hematopoietic stem cell

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