Tissue Antigens ISSN 0001-2815
Influence of the HLA characteristics of Italian patients on donor search outcome in unrelated hematopoietic stem cell transplantation M. Testi1 , M. Andreani1 , F. Locatelli2 , W. Arcese3 , M. Troiano1 , M. Battarra1 , J. Gaziev3 & G. Lucarelli4 1 Laboratory of Immunogenetics and Transplant Biology, IME Foundation at Polyclinic of Tor Vergata, Rome, Italy 2 Department of Pediatric Hematology-Oncology, IRCCS Bambino Gesù Children’s Hospital, University of Pavia, Rome, Italy 3 Rome Transplant Network, Department of Hematology, Stem Cell Transplant Unit, Tor Vergata University, Rome, Italy 4 International Center for Transplantation in Thalassemia and Sickle Cell Anemia, IME Foundation at Polyclinic of Tor Vergata, Rome, Italy
Key words hematopoietic stem cell transplantation; human leukocyte antigen; matched unrelated donor search Correspondence Manuela Testi Laboratory of Immunogenetics and Transplant Biology IME Foundation Polyclinic of Tor Vergata Viale Oxford, 81 Rome 00133 Italy Tel: +39 06 20900679 Fax: +39 06 20661313 e-mail: [email protected] Received 3 December 2013; revised 11 March 2014; accepted 19 March 2014 doi: 10.1111/tan.12355
Abstract The information regarding the probability of finding a matched unrelated donor (MUD) within a relatively short time is crucial for the success of hematopoietic stem cell transplantation (HSCT), particularly in patients with malignancies. In this study, we retrospectively analyzed 315 Italian patients who started a search for a MUD, in order to assess the distribution of human leukocyte antigen (HLA) alleles and haplotypes in this population of patients and to evaluate the probability of finding a donor. Comparing two groups of patients based on whether or not a 10/10 HLA-matched donor was available, we found that patients who had a fully-matched MUD possessed at least one frequent haplotype more often than the others (45.6% vs 14.3%; P = 0.000003). In addition, analysis of data pertaining to the HLA class I alleles distribution showed that, in the first group of patients, less common alleles were under-represented (20.2% vs 40.0%; P = 0.006). Therefore, the presence of less frequent alleles represents a negative factor for the search for a potential compatible donor being successful, whereas the presence of one frequent haplotype represents a positive predictive factor. Antigenic differences between patient and donor observed at C and DQB1 loci, were mostly represented by particular B/C or DRB1/DQB1 allelic associations. Thus, having a particular B or DRB1 allele, linked to multiple C or DQB1 alleles, respectively, might be considered to be associated with a lower probability of a successful search. Taken together, these data may help determine in advance the probability of finding a suitable unrelated donor for an Italian patient.
Major histocompatibility complex (MHC) is the most polymorphic system in the human genome (1). In particular, the human leukocyte antigen (HLA) region, located on the short arm of human chromosome 6 (6p21.3), contains more than 220 genes (2). Linkage disequilibrium (LD) between alleles at different HLA loci determines which blocks of alleles are inherited together, otherwise known as haplotypes (3), which vary in different populations or ethnic groups. It is well-known that HLA alleles segregation at various loci is not random; in fact, the number of haplotypes observed in the different populations is much smaller than theoretically expected (3). Hematopoietic stem cell transplantation (HSCT) from a matched marrow unrelated donor (MUD) is a suitable option of treatment when patients lack an HLA-matched sibling or a phenotypically HLA-matched family donor (4, 5). Generally, MUD selection relies on HLA-A, -B, -C, -DRB1 and -DQB1 198
high-resolution typing. It has been shown that compatibility in the donor/recipient pair for these five loci reduces the risk of graft-vs-host disease (GVHD) and mortality, this translating into a better probability of disease-free survival (6, 7). Finding an allelic HLA-matched donor is still challenging for many patients, because of the great diversity of HLA alleles and haplotypes (8, 9), although the number of volunteer donors enrolled in the international registries is constantly increasing. Different authors reported that patients possessing common HLA alleles on conserved haplotypes have a higher probability of finding a fully-matched MUD than those carrying rare alleles or low-frequency haplotypes (10–12). In fact, the availability of a matched MUD at the genomic level depends on the inventory of patient genotypes in the International Registries. A useful tool in order to define a potential compatible donor could be the use of computer search algorithms © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2014, 84, 198–205
Testi et al.
Immunogenetic characteristics of Italian patients and MUD search
based on the probability matching approach such as the software developed by different donor registries, i.e. Optimatch or HapLogic. Nevertheless, the knowledge of the HLA characteristics of each population may help to predict the probability of finding a suitable unrelated donor, by the set-up of a customized search algorithm (13). For this reason, it is important to have reliable information on the allele and haplotype frequencies in the patient population. The aim of this study was to retrospectively analyze a group of 315 Italian patients in order to assess the distribution of HLA alleles and haplotypes. Moreover, to better understand which histocompatibility factors might positively or negatively influence a successful MUD search, we compared two patient groups, defined according to the availability of a HLA-A, -B, -C, -DRB1 and -DQB1 (10/10) compatible donor.
HLA matching
Materials and methods
Results
Patients and donor
HLA allele and haplotype frequencies
Data on 315 Italian patients with an indication to an allograft collected from March 2009 to December 2012 were analyzed. For each patient, all available members of the family were typed by low-resolution molecular methods, in order to establish haplotype inheritance, allowing HLA gene segregation study for both classes I and II alleles. A total of 630 five-loci allelic haplotypes (HLA-A, -B, -C, -DRB1 and -DQB1) were defined. At least one potentially matched MUD was identified as available for confirmatory high-resolution typing for 184 patients, for a total of 308 MUD samples to be further analyzed. Among the 131 patients who did not have an available MUD 30% had a negative search, 42% interrupted the MUD search process for clinical reasons and for 28% of them the identified potential MUD was not available.
In this study, we determined the frequency of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles at high resolution level, HLA-B/C, HLA-DRB1/DQB1 two-allele associations and the most common HLA-A-B-C-DRB1-DQB1 five-loci haplotypes in 315 Italian patients, candidate to receive HSCT. For each patient, all available members of the family were typed by low-resolution molecular methods. We therefore were able to establish haplotype inheritance by analysing the HLA gene segregation for both classes I and II alleles in 630 haplotypes, in one unicentric cohort of Italian unrelated individuals. The frequencies of HLA-A, -B, -C, -DRB1 and -DQB1 alleles, including the relative frequencies inside each allelic group, are listed in Tables 1 and 2. Our data are comparable to those reported for healthy Italian donors recruited in the Italian Bone Marrow Donor Registry (IBMDR) (14). For class I, a total of 37 HLA-A, 52 HLA-B and 31 HLA-C alleles were identified, while in class II we identified 38 DRB1 and 17 DQB1 different alleles. In this survey, we considered frequent the alleles exhibiting a frequency higher than 5% and infrequent those with a frequency lower than 1%. On the basis of this criterion, six HLA-A, five -B, five -C, seven -DRB1 and seven -DQB1 alleles were defined as predominant, representing 68%, 38%, 62%, 65% and 78%, respectively, of all known alleles in our population. By contrast, 22 HLA-A (9%), 28 -B (11%), 12 -C (4%), 18 -DRB1 (6%) and 3 -DQB1 (0.6%) were defined as infrequent alleles. Concerning the DPB1 locus, the most frequent alleles found in our population were DPB1*04:01P (35.8%) and DPB1*04:02P (14.4%), see also Table 2. All HLA-B/C allelic combinations were defined at a high-resolution level (data not shown). As reported in Table 3, some B alleles resulted to be associated with many different C alleles. For example, among the most frequent B alleles, B*51:01 was linked to 12 different C alleles; B*18:01 and B*44:03 with nine and six different C alleles, respectively.
HLA typing
The patients and all available members of the family were initially HLA-A, -B, -C, -DRB1 and -DQB1 typed at low resolution by polymerase chain reaction sequence-specific oligonucleotide probes (PCR-SSOP) using a commercial kit (LAB®Type, One Lambda Inc., Canoga Park, CA). When a suitable MUD was identified, patient and donor were typed for HLA-A, -B, -C, -DRB1 and -DQB1 genes by SBT (Invitrogen, Brown Deer, WI; Atria, Abbott Park, IL) and HD PCR-SSOP method using Luminex technology (One Lambda Inc.). Moreover, DPB1 typing was performed using PCR-SSOP (LAB®Type, One Lambda Inc) or PCR-sequence-specific-primers (SSP) (OlerupSSP, Saltsjöbaden, Sweden). HLA mismatches were taken into account when non-synonymous nucleotide differences were placed at exons 2 and 3 of HLA class I genes and exon 2 of DRB1 and DQB1 loci. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2014, 84, 198–205
HLA matching in the donor/recipient pair was evaluated for allele- and antigen-level mismatches involving HLA-A, -B, -C, -DRB1 and -DQB1 loci. The cohort data were categorized into two HLA-matching levels, defining the patient/donor matching as ‘well-matched’ (zero mismatches) n = 79 (43%) and ‘mismatched’ (1 or more mismatches) n = 105 (57%). Statistical analysis
Allele and haplotype frequencies were obtained by direct counting. Statistical analysis was performed by using VassarStats Website. Analysis was conducted using Fisher two-tailed exact test. For the purpose of this analysis, a stringent P-value less than 0.01 was considered to be statistically significant.
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Table 1 Frequency of human leukocyte antigen HLA-A, -B, and -C alleles found in the studied population HLA-A
f (%)
f r (%)
HLA-B
f (%)
f r (%)
HLA-C
f (%)
f r (%)
*01:01 *01:02 *01:03 *02:01 *02:02 *02:05 *02:06 *02:09 *02:17 *02:20 *02:140 *03:01 *03:02 *03:143 *11:01 *23:01 *24:02 *24:03 *25:01 *26:01 *29:01 *29:02 *30:01 *30:02 *30:04 *31:01 *31:48 *32:01 *32:11Q *33:01 *33:03 *66:01 *68:01 *68:02 *68:24 *69:01 *80:01
12.22 0.48 0.16 2.63 0.16 2.22 0.32 0.16 0.16 0.16 0.32 10.48 0.48 0.16 6.19 2.7 13.49 0.48 0.63 3.02 0.95 1.9 3.65 2.54 1.43 1.43 0.32 5.24 0.16 0.95 0.48 0.48 4.29 0.63 0.16 0.63 0.16
95.1 3.7 1.2 85.4 0.7 9.2 1.3 0.7 0.7 0.7 1.3 94.3 4.3 1.4 100 100 96.6 3.4 100 100 33.3 66.7 47.9 33.3 18.8 81.8 18.2 97.1 2.9 66.7 33.3 100 84.4 12.5 3.1 100 100
*07:02 *07:05 *07:06 *07:07 *08:01 *13:02 *14:01 *14:02 *15:01 *15:03 *15:08 *15:17 *15:18 *15:71 *18:01 *18:03 *27:02 *27:05 *27:09 *35:01 *35:02 *35:03 *35:08 *37:01 *38:01 *39:01 *39:06 *39:10 *40:01 *40:02 *40:06 *40:19 *41:01 *41:02 *44:02 *44:03 *44:05 *45:01 *47:01 *48:01 *49:01 *50:01 *51:01 *51:07 *51:08 *52:01 *53:01 *55:01 *56:01 *57:01 *57:03 *58:01
5.08 0.48 0.48 0.16 3.97 4.76 0.63 2.38 2.86 0.32 0.16 2.22 0.79 0.16 9.68 0.48 0.32 0.95 0.16 8.25 4.6 3.17 1.59 1.59 3.49 0.79 0.79 0.32 1.11 0.95 0.16 0.16 1.59 0.16 3.65 3.49 0.16 0.16 0.16 0.16 5.08 1.9 10.0 0.16 0.63 2.7 0.48 1.11 0.32 3.02 0.16 1.9
82.0 7.7 7.7 2.6 100 100 21.0 79.0 43.9 4.9 2.4 34.2 12.2 2.4 95.3 4.7 22.2 66.7 11.1 46.8 26.1 18.0 9.0 100 100 41.7 41.7 16.6 46.8 40.0 6.6 6.6 90.9 9.1 50.0 47.8 2.2 100 100 100 100 100 92.6 1.5 5.9 100 100 100 100 95.0 5.0 100
*01:02 *02:02 *02:10 *03:02 *03:03 *03:04 *04:01 *05:01 *06:02 *06:58 *07:01 *07:02 *07:04 *07:06 *07:18 *08:02 *08:03 *12:02 *12:03 *12:05 *14:02 *14:03 *15:02 *15:05 *15:06 *15:13 *15:24 *16:01 *16:02 *16:04 *17:01
1.75 3.65 0.32 0.63 3.49 1.11 18.41 4.76 11.27 0.16 16.35 6.51 1.43 0.48 1.11 2.86 0.16 2.86 9.68 0.16 2.22 0.16 3.81 0.63 0.32 0.32 0.16 1.43 1.43 0.95 1.43
100 92.0 8.0 12.1 66.7 21.2 100 100 98.6 1.4 63.2 25.2 5.5 1.8 4.3 94.7 5.3 22.5 76.3 1.3 93.3 6.7 72.7 12.1 6.1 6.1 3.0 37.5 37.5 25.0 100
HLA, human leukocyte antigen. f, frequency of HLA-A, -B and -C alleles distribution; f r , frequency inside each allelic group
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© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2014, 84, 198–205
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Table 2 Frequency of human leukocyte antigen HLA-DRB1, -DQB1 and -DPB1 alleles found in the studied population HLA-DRB1
f (%)
f r (%)
HLA-DQB1
f (%)
f r (%)
HLA-DPB1
f (%)
*01:01 *01:02 *01:03 *03:01 *04:01 *04:02 *04:03 *04:04 *04:05 *04:06 *04:07 *07:01 *08:01 *08:03 *08:04 *08:10 *09:01 *10:01 *11:01 *11:02 *11:03 *11:04 *11:36 *12:01 *12:02 *13:01 *13:02 *13:03 *13:05 *14:01 *14:04 *14:19 *14:54 *15:01 *15:02 *15:03 *16:01 *16:02
4.44 1.9 0.16 7.94 1.27 0.95 2.06 1.59 0.79 0.16 0.63 15.08 1.27 0.16 0.32 0.16 0.16 1.59 10.48 0.16 1.75 14.76 0.16 2.38 0.16 2.86 5.71 0.32 0.63 1.27 0.32 0.16 5.24 5.4 1.9 0.16 4.76 0.79
68.3 29.3 2.4 100 17.0 12.8 27.7 21.3 10.6 2.1 8.5 100 66.7 8.3 16.7 8.3 100 100 38.4 0.6 6.4 54.1 0.6 93.8 6.3 30.0 60.0 3.3 6.7 18.2 4.5 2.3 75.0 72.3 25.5 2.1 85.7 14.3
*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
7.78 11.9 32.22 5.24 3.33 0.16 0.32 0.16 2.22 8.41 6.19 6.67 1.9 3.81 4.29 3.97 1.43
39.5 60.5 77.8 12.6 8.0 0.4 0.8 0.4 100 39.6 29.1 31.3 12.4 24.7 27.8 25.8 9.3
*01:01 *02:01P *02:02 *03:01P *04:01P *04:02P *05:01P *06:01 *09:01 *10:01 *11:01 *13:01P *14:01 *15:01 *16:01 *17:01P *19:01 *20:01 *23:01P *30:01 *35:01 *41:01 *81:01
1.77 18.58 0.22 8.63 35.84 14.40 1.0 1.11 0.88 2.88 1.33 3.98 2.88 0.44 0.22 4.2 0.11 0.22 0.77 0.11 0.11 0.22 0.11
f , frequency of HLA-DRB1, -DQB1 and DPB1 alleles distribution; f r, frequency inside each allelic group
Similarly, we identified for HLA class II specific DRB1 alleles associated with multiple DQB1 antigens, as reported in Table 4. After HLA haplotype segregation analysis, we identified 514 unique haplotypes in our patient population. The overall results revealed the presence of eight different 5-loci haplotypes out of 630, showing an haplotype frequency (Hf) higher than 0.5% (range 0.63%–2%) and therefore considered to be common in our survey (Table 5). Patient/MUD comparison
After the initial search, 184 of 315 patients identified at least one MUD for confirmatory high-resolution typing. A total of 308 donor samples from either the IBMDR or other International Registries were received (average: 1.67 per patient; 78 pts = 1 MUD; 88 pts = 2 MUD; 18 pts = 3 MUD). All the selected © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2014, 84, 198–205
donors were matched for HLA-A, -B at low resolution and for -DRB1 at high resolution. After confirmatory HLA allele assignment, one suitable donor fully matched for HLA-A, -B, -C, -DRB1 and -DQB1 loci (10/10) was identified for 79 patients (43%) (Table 6). Among them, 16% were also DPB1 matched (12/12). For what concern the remaining 105 patients, 65% showed antigenic disparities at the C locus, while 21% of them were different at the DQB1 locus. We moreover detected incompatibilities at the allelic level in 32% of patients for locus A and in 51% of them for locus B. On the other hand, changing the required level of HLA-matching to a 9/10 compatibility criterion, the proportion of patients with a suitable unrelated donor would rise, in our survey to 75% (138/184). Of 59 patients characterized by a 9/10 compatibility level, 40.7% and 15.2% were mismatched at antigenic level for HLA-C and for 201
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Table 4 Relevant HLA-DRB1/DQB1 associations
Table 3 Relevant HLA-B/C associations HLA-B
HLA-C
n
%
DRB1
DQB1
n
%
*18:01
*07:01 *12:03 *05:01 *01:02 *03:03 *07:04P *12:02 *12:05 *15:02 *04:01P *16:01 *07:06 *02:02 *14:03 *16:04 *15:02 *14:02 *16:02 *02:02 *07:01 *12:03 *01:02 *07:02 *15:06 *15:13 *06:02 *15:24
26 19 10 1 1 1 1 1 1 8 8 3 1 1 1 20 13 6 5 4 4 3 2 2 2 1 1
42.62 31.15 16.39 1.64 1.64 1.64 1.64 1.64 1.64 36.36 36.36 13.64 4.55 4.55 4.55 31.75 20.63 9.52 7.94 6.35 6.35 4.76 3.17 3.17 3.17 1.59 1.59
*04:03
*03:02 *03:04 *03:05 *03:02 *04:02 *02:02 *03:01 *03:03 *05:01 *06:04 *06:09 *05:02 *06:02 *06:03P
10 1 2 7 3 75 1 20 2 25 9 3 24 7
76.9 7.7 15.4 70 30 78.1 1.1 20.8 5.6 69.4 25.0 8.8 70.6 20.6
*44:03
*51:01
HLA, human leukocyte antigen. n, number of B–C haplotypes; %, proportion of C alleles associated with B specific alleles.
HLA-DQB1, respectively, while at allelic level the differences were found in 18.7%, 23.7% and 1.7% for HLA-A, -B and -C, respectively. Table 6 shows the rate of transplantation according to the HLA matching of 91 of 184 patients who after the search process were effectively transplanted, with a sharp decrease in the ratio of transplanted patients with a decrease in the matching grade. On the basis of the allele and haplotype distribution found in our Italian cohort of patients, the relevant immunogenetic information was used in order to compare the two groups of patients defined by the availability or not of a 10/10 matched donor. When allele frequencies were analyzed, we observed that 40% (42/105) of patients without a fully matched donor were characterized by the presence of at least one HLA-A, -B or -C infrequent allele (
Influence of the HLA characteristics of Italian patients on donor search outcome in unrelated hematopoietic stem cell transplantation M. Testi1 , M. Andreani1 , F. Locatelli2 , W. Arcese3 , M. Troiano1 , M. Battarra1 , J. Gaziev3 & G. Lucarelli4 1 Laboratory of Immunogenetics and Transplant Biology, IME Foundation at Polyclinic of Tor Vergata, Rome, Italy 2 Department of Pediatric Hematology-Oncology, IRCCS Bambino Gesù Children’s Hospital, University of Pavia, Rome, Italy 3 Rome Transplant Network, Department of Hematology, Stem Cell Transplant Unit, Tor Vergata University, Rome, Italy 4 International Center for Transplantation in Thalassemia and Sickle Cell Anemia, IME Foundation at Polyclinic of Tor Vergata, Rome, Italy
Key words hematopoietic stem cell transplantation; human leukocyte antigen; matched unrelated donor search Correspondence Manuela Testi Laboratory of Immunogenetics and Transplant Biology IME Foundation Polyclinic of Tor Vergata Viale Oxford, 81 Rome 00133 Italy Tel: +39 06 20900679 Fax: +39 06 20661313 e-mail: [email protected] Received 3 December 2013; revised 11 March 2014; accepted 19 March 2014 doi: 10.1111/tan.12355
Abstract The information regarding the probability of finding a matched unrelated donor (MUD) within a relatively short time is crucial for the success of hematopoietic stem cell transplantation (HSCT), particularly in patients with malignancies. In this study, we retrospectively analyzed 315 Italian patients who started a search for a MUD, in order to assess the distribution of human leukocyte antigen (HLA) alleles and haplotypes in this population of patients and to evaluate the probability of finding a donor. Comparing two groups of patients based on whether or not a 10/10 HLA-matched donor was available, we found that patients who had a fully-matched MUD possessed at least one frequent haplotype more often than the others (45.6% vs 14.3%; P = 0.000003). In addition, analysis of data pertaining to the HLA class I alleles distribution showed that, in the first group of patients, less common alleles were under-represented (20.2% vs 40.0%; P = 0.006). Therefore, the presence of less frequent alleles represents a negative factor for the search for a potential compatible donor being successful, whereas the presence of one frequent haplotype represents a positive predictive factor. Antigenic differences between patient and donor observed at C and DQB1 loci, were mostly represented by particular B/C or DRB1/DQB1 allelic associations. Thus, having a particular B or DRB1 allele, linked to multiple C or DQB1 alleles, respectively, might be considered to be associated with a lower probability of a successful search. Taken together, these data may help determine in advance the probability of finding a suitable unrelated donor for an Italian patient.
Major histocompatibility complex (MHC) is the most polymorphic system in the human genome (1). In particular, the human leukocyte antigen (HLA) region, located on the short arm of human chromosome 6 (6p21.3), contains more than 220 genes (2). Linkage disequilibrium (LD) between alleles at different HLA loci determines which blocks of alleles are inherited together, otherwise known as haplotypes (3), which vary in different populations or ethnic groups. It is well-known that HLA alleles segregation at various loci is not random; in fact, the number of haplotypes observed in the different populations is much smaller than theoretically expected (3). Hematopoietic stem cell transplantation (HSCT) from a matched marrow unrelated donor (MUD) is a suitable option of treatment when patients lack an HLA-matched sibling or a phenotypically HLA-matched family donor (4, 5). Generally, MUD selection relies on HLA-A, -B, -C, -DRB1 and -DQB1 198
high-resolution typing. It has been shown that compatibility in the donor/recipient pair for these five loci reduces the risk of graft-vs-host disease (GVHD) and mortality, this translating into a better probability of disease-free survival (6, 7). Finding an allelic HLA-matched donor is still challenging for many patients, because of the great diversity of HLA alleles and haplotypes (8, 9), although the number of volunteer donors enrolled in the international registries is constantly increasing. Different authors reported that patients possessing common HLA alleles on conserved haplotypes have a higher probability of finding a fully-matched MUD than those carrying rare alleles or low-frequency haplotypes (10–12). In fact, the availability of a matched MUD at the genomic level depends on the inventory of patient genotypes in the International Registries. A useful tool in order to define a potential compatible donor could be the use of computer search algorithms © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2014, 84, 198–205
Testi et al.
Immunogenetic characteristics of Italian patients and MUD search
based on the probability matching approach such as the software developed by different donor registries, i.e. Optimatch or HapLogic. Nevertheless, the knowledge of the HLA characteristics of each population may help to predict the probability of finding a suitable unrelated donor, by the set-up of a customized search algorithm (13). For this reason, it is important to have reliable information on the allele and haplotype frequencies in the patient population. The aim of this study was to retrospectively analyze a group of 315 Italian patients in order to assess the distribution of HLA alleles and haplotypes. Moreover, to better understand which histocompatibility factors might positively or negatively influence a successful MUD search, we compared two patient groups, defined according to the availability of a HLA-A, -B, -C, -DRB1 and -DQB1 (10/10) compatible donor.
HLA matching
Materials and methods
Results
Patients and donor
HLA allele and haplotype frequencies
Data on 315 Italian patients with an indication to an allograft collected from March 2009 to December 2012 were analyzed. For each patient, all available members of the family were typed by low-resolution molecular methods, in order to establish haplotype inheritance, allowing HLA gene segregation study for both classes I and II alleles. A total of 630 five-loci allelic haplotypes (HLA-A, -B, -C, -DRB1 and -DQB1) were defined. At least one potentially matched MUD was identified as available for confirmatory high-resolution typing for 184 patients, for a total of 308 MUD samples to be further analyzed. Among the 131 patients who did not have an available MUD 30% had a negative search, 42% interrupted the MUD search process for clinical reasons and for 28% of them the identified potential MUD was not available.
In this study, we determined the frequency of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles at high resolution level, HLA-B/C, HLA-DRB1/DQB1 two-allele associations and the most common HLA-A-B-C-DRB1-DQB1 five-loci haplotypes in 315 Italian patients, candidate to receive HSCT. For each patient, all available members of the family were typed by low-resolution molecular methods. We therefore were able to establish haplotype inheritance by analysing the HLA gene segregation for both classes I and II alleles in 630 haplotypes, in one unicentric cohort of Italian unrelated individuals. The frequencies of HLA-A, -B, -C, -DRB1 and -DQB1 alleles, including the relative frequencies inside each allelic group, are listed in Tables 1 and 2. Our data are comparable to those reported for healthy Italian donors recruited in the Italian Bone Marrow Donor Registry (IBMDR) (14). For class I, a total of 37 HLA-A, 52 HLA-B and 31 HLA-C alleles were identified, while in class II we identified 38 DRB1 and 17 DQB1 different alleles. In this survey, we considered frequent the alleles exhibiting a frequency higher than 5% and infrequent those with a frequency lower than 1%. On the basis of this criterion, six HLA-A, five -B, five -C, seven -DRB1 and seven -DQB1 alleles were defined as predominant, representing 68%, 38%, 62%, 65% and 78%, respectively, of all known alleles in our population. By contrast, 22 HLA-A (9%), 28 -B (11%), 12 -C (4%), 18 -DRB1 (6%) and 3 -DQB1 (0.6%) were defined as infrequent alleles. Concerning the DPB1 locus, the most frequent alleles found in our population were DPB1*04:01P (35.8%) and DPB1*04:02P (14.4%), see also Table 2. All HLA-B/C allelic combinations were defined at a high-resolution level (data not shown). As reported in Table 3, some B alleles resulted to be associated with many different C alleles. For example, among the most frequent B alleles, B*51:01 was linked to 12 different C alleles; B*18:01 and B*44:03 with nine and six different C alleles, respectively.
HLA typing
The patients and all available members of the family were initially HLA-A, -B, -C, -DRB1 and -DQB1 typed at low resolution by polymerase chain reaction sequence-specific oligonucleotide probes (PCR-SSOP) using a commercial kit (LAB®Type, One Lambda Inc., Canoga Park, CA). When a suitable MUD was identified, patient and donor were typed for HLA-A, -B, -C, -DRB1 and -DQB1 genes by SBT (Invitrogen, Brown Deer, WI; Atria, Abbott Park, IL) and HD PCR-SSOP method using Luminex technology (One Lambda Inc.). Moreover, DPB1 typing was performed using PCR-SSOP (LAB®Type, One Lambda Inc) or PCR-sequence-specific-primers (SSP) (OlerupSSP, Saltsjöbaden, Sweden). HLA mismatches were taken into account when non-synonymous nucleotide differences were placed at exons 2 and 3 of HLA class I genes and exon 2 of DRB1 and DQB1 loci. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2014, 84, 198–205
HLA matching in the donor/recipient pair was evaluated for allele- and antigen-level mismatches involving HLA-A, -B, -C, -DRB1 and -DQB1 loci. The cohort data were categorized into two HLA-matching levels, defining the patient/donor matching as ‘well-matched’ (zero mismatches) n = 79 (43%) and ‘mismatched’ (1 or more mismatches) n = 105 (57%). Statistical analysis
Allele and haplotype frequencies were obtained by direct counting. Statistical analysis was performed by using VassarStats Website. Analysis was conducted using Fisher two-tailed exact test. For the purpose of this analysis, a stringent P-value less than 0.01 was considered to be statistically significant.
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Immunogenetic characteristics of Italian patients and MUD search
Table 1 Frequency of human leukocyte antigen HLA-A, -B, and -C alleles found in the studied population HLA-A
f (%)
f r (%)
HLA-B
f (%)
f r (%)
HLA-C
f (%)
f r (%)
*01:01 *01:02 *01:03 *02:01 *02:02 *02:05 *02:06 *02:09 *02:17 *02:20 *02:140 *03:01 *03:02 *03:143 *11:01 *23:01 *24:02 *24:03 *25:01 *26:01 *29:01 *29:02 *30:01 *30:02 *30:04 *31:01 *31:48 *32:01 *32:11Q *33:01 *33:03 *66:01 *68:01 *68:02 *68:24 *69:01 *80:01
12.22 0.48 0.16 2.63 0.16 2.22 0.32 0.16 0.16 0.16 0.32 10.48 0.48 0.16 6.19 2.7 13.49 0.48 0.63 3.02 0.95 1.9 3.65 2.54 1.43 1.43 0.32 5.24 0.16 0.95 0.48 0.48 4.29 0.63 0.16 0.63 0.16
95.1 3.7 1.2 85.4 0.7 9.2 1.3 0.7 0.7 0.7 1.3 94.3 4.3 1.4 100 100 96.6 3.4 100 100 33.3 66.7 47.9 33.3 18.8 81.8 18.2 97.1 2.9 66.7 33.3 100 84.4 12.5 3.1 100 100
*07:02 *07:05 *07:06 *07:07 *08:01 *13:02 *14:01 *14:02 *15:01 *15:03 *15:08 *15:17 *15:18 *15:71 *18:01 *18:03 *27:02 *27:05 *27:09 *35:01 *35:02 *35:03 *35:08 *37:01 *38:01 *39:01 *39:06 *39:10 *40:01 *40:02 *40:06 *40:19 *41:01 *41:02 *44:02 *44:03 *44:05 *45:01 *47:01 *48:01 *49:01 *50:01 *51:01 *51:07 *51:08 *52:01 *53:01 *55:01 *56:01 *57:01 *57:03 *58:01
5.08 0.48 0.48 0.16 3.97 4.76 0.63 2.38 2.86 0.32 0.16 2.22 0.79 0.16 9.68 0.48 0.32 0.95 0.16 8.25 4.6 3.17 1.59 1.59 3.49 0.79 0.79 0.32 1.11 0.95 0.16 0.16 1.59 0.16 3.65 3.49 0.16 0.16 0.16 0.16 5.08 1.9 10.0 0.16 0.63 2.7 0.48 1.11 0.32 3.02 0.16 1.9
82.0 7.7 7.7 2.6 100 100 21.0 79.0 43.9 4.9 2.4 34.2 12.2 2.4 95.3 4.7 22.2 66.7 11.1 46.8 26.1 18.0 9.0 100 100 41.7 41.7 16.6 46.8 40.0 6.6 6.6 90.9 9.1 50.0 47.8 2.2 100 100 100 100 100 92.6 1.5 5.9 100 100 100 100 95.0 5.0 100
*01:02 *02:02 *02:10 *03:02 *03:03 *03:04 *04:01 *05:01 *06:02 *06:58 *07:01 *07:02 *07:04 *07:06 *07:18 *08:02 *08:03 *12:02 *12:03 *12:05 *14:02 *14:03 *15:02 *15:05 *15:06 *15:13 *15:24 *16:01 *16:02 *16:04 *17:01
1.75 3.65 0.32 0.63 3.49 1.11 18.41 4.76 11.27 0.16 16.35 6.51 1.43 0.48 1.11 2.86 0.16 2.86 9.68 0.16 2.22 0.16 3.81 0.63 0.32 0.32 0.16 1.43 1.43 0.95 1.43
100 92.0 8.0 12.1 66.7 21.2 100 100 98.6 1.4 63.2 25.2 5.5 1.8 4.3 94.7 5.3 22.5 76.3 1.3 93.3 6.7 72.7 12.1 6.1 6.1 3.0 37.5 37.5 25.0 100
HLA, human leukocyte antigen. f, frequency of HLA-A, -B and -C alleles distribution; f r , frequency inside each allelic group
200
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2014, 84, 198–205
Testi et al.
Immunogenetic characteristics of Italian patients and MUD search
Table 2 Frequency of human leukocyte antigen HLA-DRB1, -DQB1 and -DPB1 alleles found in the studied population HLA-DRB1
f (%)
f r (%)
HLA-DQB1
f (%)
f r (%)
HLA-DPB1
f (%)
*01:01 *01:02 *01:03 *03:01 *04:01 *04:02 *04:03 *04:04 *04:05 *04:06 *04:07 *07:01 *08:01 *08:03 *08:04 *08:10 *09:01 *10:01 *11:01 *11:02 *11:03 *11:04 *11:36 *12:01 *12:02 *13:01 *13:02 *13:03 *13:05 *14:01 *14:04 *14:19 *14:54 *15:01 *15:02 *15:03 *16:01 *16:02
4.44 1.9 0.16 7.94 1.27 0.95 2.06 1.59 0.79 0.16 0.63 15.08 1.27 0.16 0.32 0.16 0.16 1.59 10.48 0.16 1.75 14.76 0.16 2.38 0.16 2.86 5.71 0.32 0.63 1.27 0.32 0.16 5.24 5.4 1.9 0.16 4.76 0.79
68.3 29.3 2.4 100 17.0 12.8 27.7 21.3 10.6 2.1 8.5 100 66.7 8.3 16.7 8.3 100 100 38.4 0.6 6.4 54.1 0.6 93.8 6.3 30.0 60.0 3.3 6.7 18.2 4.5 2.3 75.0 72.3 25.5 2.1 85.7 14.3
*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
7.78 11.9 32.22 5.24 3.33 0.16 0.32 0.16 2.22 8.41 6.19 6.67 1.9 3.81 4.29 3.97 1.43
39.5 60.5 77.8 12.6 8.0 0.4 0.8 0.4 100 39.6 29.1 31.3 12.4 24.7 27.8 25.8 9.3
*01:01 *02:01P *02:02 *03:01P *04:01P *04:02P *05:01P *06:01 *09:01 *10:01 *11:01 *13:01P *14:01 *15:01 *16:01 *17:01P *19:01 *20:01 *23:01P *30:01 *35:01 *41:01 *81:01
1.77 18.58 0.22 8.63 35.84 14.40 1.0 1.11 0.88 2.88 1.33 3.98 2.88 0.44 0.22 4.2 0.11 0.22 0.77 0.11 0.11 0.22 0.11
f , frequency of HLA-DRB1, -DQB1 and DPB1 alleles distribution; f r, frequency inside each allelic group
Similarly, we identified for HLA class II specific DRB1 alleles associated with multiple DQB1 antigens, as reported in Table 4. After HLA haplotype segregation analysis, we identified 514 unique haplotypes in our patient population. The overall results revealed the presence of eight different 5-loci haplotypes out of 630, showing an haplotype frequency (Hf) higher than 0.5% (range 0.63%–2%) and therefore considered to be common in our survey (Table 5). Patient/MUD comparison
After the initial search, 184 of 315 patients identified at least one MUD for confirmatory high-resolution typing. A total of 308 donor samples from either the IBMDR or other International Registries were received (average: 1.67 per patient; 78 pts = 1 MUD; 88 pts = 2 MUD; 18 pts = 3 MUD). All the selected © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2014, 84, 198–205
donors were matched for HLA-A, -B at low resolution and for -DRB1 at high resolution. After confirmatory HLA allele assignment, one suitable donor fully matched for HLA-A, -B, -C, -DRB1 and -DQB1 loci (10/10) was identified for 79 patients (43%) (Table 6). Among them, 16% were also DPB1 matched (12/12). For what concern the remaining 105 patients, 65% showed antigenic disparities at the C locus, while 21% of them were different at the DQB1 locus. We moreover detected incompatibilities at the allelic level in 32% of patients for locus A and in 51% of them for locus B. On the other hand, changing the required level of HLA-matching to a 9/10 compatibility criterion, the proportion of patients with a suitable unrelated donor would rise, in our survey to 75% (138/184). Of 59 patients characterized by a 9/10 compatibility level, 40.7% and 15.2% were mismatched at antigenic level for HLA-C and for 201
Testi et al.
Immunogenetic characteristics of Italian patients and MUD search
Table 4 Relevant HLA-DRB1/DQB1 associations
Table 3 Relevant HLA-B/C associations HLA-B
HLA-C
n
%
DRB1
DQB1
n
%
*18:01
*07:01 *12:03 *05:01 *01:02 *03:03 *07:04P *12:02 *12:05 *15:02 *04:01P *16:01 *07:06 *02:02 *14:03 *16:04 *15:02 *14:02 *16:02 *02:02 *07:01 *12:03 *01:02 *07:02 *15:06 *15:13 *06:02 *15:24
26 19 10 1 1 1 1 1 1 8 8 3 1 1 1 20 13 6 5 4 4 3 2 2 2 1 1
42.62 31.15 16.39 1.64 1.64 1.64 1.64 1.64 1.64 36.36 36.36 13.64 4.55 4.55 4.55 31.75 20.63 9.52 7.94 6.35 6.35 4.76 3.17 3.17 3.17 1.59 1.59
*04:03
*03:02 *03:04 *03:05 *03:02 *04:02 *02:02 *03:01 *03:03 *05:01 *06:04 *06:09 *05:02 *06:02 *06:03P
10 1 2 7 3 75 1 20 2 25 9 3 24 7
76.9 7.7 15.4 70 30 78.1 1.1 20.8 5.6 69.4 25.0 8.8 70.6 20.6
*44:03
*51:01
HLA, human leukocyte antigen. n, number of B–C haplotypes; %, proportion of C alleles associated with B specific alleles.
HLA-DQB1, respectively, while at allelic level the differences were found in 18.7%, 23.7% and 1.7% for HLA-A, -B and -C, respectively. Table 6 shows the rate of transplantation according to the HLA matching of 91 of 184 patients who after the search process were effectively transplanted, with a sharp decrease in the ratio of transplanted patients with a decrease in the matching grade. On the basis of the allele and haplotype distribution found in our Italian cohort of patients, the relevant immunogenetic information was used in order to compare the two groups of patients defined by the availability or not of a 10/10 matched donor. When allele frequencies were analyzed, we observed that 40% (42/105) of patients without a fully matched donor were characterized by the presence of at least one HLA-A, -B or -C infrequent allele (
