Allele and Haplotype Frequencies of HLA-A, -B, -C, and -DRB1 Genes in 3,750 Cord Blood Units From a Kinh Vietnamese Population

The frequencies and diversities of human leukocyte antigen (HLA) alleles and haplotypes are representative of ethnicities. Matching HLA alleles is essential for many clinical applications, including blood transfusion, stem cell transplantation, and tissue/organ transplantation. To date, the information about the frequencies and distributions of HLA alleles and haplotypes among the Kinh Vietnamese population is limited because of the small sample size. In this study, more than 3,750 cord blood units from individuals belonging to the Kinh Vietnamese population were genotyped using PCR sequence-specific oligonucleotide (PCR-SSO) for HLA testing. The results of the study demonstrated that the most frequently occurring HLA-A, -B, -C, and -DRB1 alleles were A*11:01 (25%), A*24:02 (12.3%), A*02:01 (11.2); A*03:03 (8.95%), A*02:03 (7.81%), A*29:01 (7.03%); B*15:02 (15.1%), B*46:01 (10.7%), B*58:01 (7.65%), B*38:02 (7.29%); C*08:01 (17.2), C*07:02 (16.2%), C*01:02 (15.2), C*03:02 (8.3%), C*15:05 (6.13); DRB1*12:02 (31.0%), DRB1*09:01 (10.47%), DRB1*15:02 (7.54%); DRB1*07:01 (6.68%), DRB1*10:01 (6.63%), respectively, with the highest allele diversity level observed in locus B (93 alleles). The most frequent haplotypes of two-locus combinations of HLA-A–B, HLA-A–C, HLA-A–DRB1, HLA-B–C, HLA-B–DRB1, and HLA-C–DRB1 haplotypes were A*11:01–B*15:02 (7.63%), A*11:01–C*08:01 (7.98%), A*11:01–DRB1*12:02 (10.56%), B*15:02–C*08:01 (14.0%), B*15:02–DRB1*12:02 (10.47%), and C*08:01–DRB1*12:02 (11.38%), respectively. In addition, the most frequent haplotypes of three- and four-locus sets of HLA-A–B–C, HLA-A–B–DRB1, HLA-A–C–DRB1, HLA-B–C–DRB1, and HLA-A–B–C–DRB1 were A*11:01–B*15:02–C*08:01 (7.57%), A*11:01–B*15:02–DRB1*12:02 (5.39%), A*11:01–C*08:01–DRB1*12:02 (5.54%), B*15:02–C*08:01–DRB1*12:02 (10.21%), and A*11:01–B*15:02–C*08:01–DRB1*12:02 (5.45%), respectively. This study provides critical information on the frequencies and distributions of HLA alleles and haplotypes in the Kinh Vietnamese population, accounting for more than 85% of Vietnamese citizens. It paves the way to establish an umbilical cord blood bank for cord blood transplantation programs in Vietnam.

The HLA system includes two classes: HLA-A, -B, and -C, which correspond to major histocompatibility complex (MHC) class I, and HLA-DRB1, -DQA1, -DQB1, and -DPB1, which correspond to MHC class II. MHC class I is present on the surface of all cell types and is recognised byT-CD8 + cells (9,10). Incompatibility in genotypes of both HLA classes I and II between the donor and recipient causes organ rejection and graft failure in haematopoietic stem cell transplantation (9). The primary requirement for cord blood grafting is HLA compatibility of at least four of the six loci from MHC classes I and II (10,11).
Cord blood was previously regarded as clinical waste; however, it has recently been recognised as a promising and effective source for treating various diseases, particularly haematopoietic disorder-related diseases (5)(6)(7)(8). When applying stem cells for transplantation, finding compatible donors among the relations is problematic. Therefore, cord blood samples stored in blood banks are an excellent alternative. Graft rejection is a serious complication that may occur after transplantation. Therefore, storing cord blood is beneficial for children and their relatives in the future. Because of its abundant sources, using cord blood in clinics will help reduce the treatment cost and time (3)(4)(5)(6). Cord blood stem cells can differentiate into various cells, including red blood cells, white blood cells, and platelets. Thus, cord blood has become very promising for clinical applications in treating multiple aggressive pathologies, such as anaemia, lymphoma, and leukaemia (1,(5)(6)(7)(8)12). Therefore, we investigated and reported information on the frequency, diversity, and distribution of alleles and haplotypes in four HLA loci, including HLA-A, HLA-B, HLA-C, and HLA-DRB1, in 3,750 cord blood units of the Kinh Vietnamese population, to establish an umbilical cord blood bank at the National Institute of Haematology and Blood Transfusion of Vietnam.

Statistical Analysis
Population statistical analyses and diversity of HLA genotypes were calculated using Arlequin 3.5 software. In particular, the individual allele frequencies of each locus and the common haplotype frequencies of two, three, and four loci were determined using the expectation-maximisation (EM) algorithm. An exact test using a Markov chain algorithm was performed to test for deviation from Hardy-Weinberg equilibrium (13,14).

Population and Sample Collection
A cross-sectional study was conducted on a cohort of 3,776 Kinh Vietnamese healthy pregnant women for cord blood samples. The samples were collected between May 2014 and December 2019. Cord blood donors mostly came from every province in northern and north-central Vietnam. Cord blood samples of ≥80 mL in volume, without blood cluster or bizarre colours, collected within 24 h of birth, mean corpuscular volume (MCV) of ≥95 fl, no abnormal haemoglobin confirmed by high-performance liquid chromatography (HPLC) analysis, number of nucleated cells ≥10 9 cells/mL, negative for infectious pathogens including hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), cytomegalovirus (CMV), and bacteria were collected and stored at −196°C in liquid nitrogen tanks at the Umbilical Cord Blood Bank, National Institute of Haematology and Blood Transfusion, Vietnam.

HLA Genotyping
All cord blood samples were used for DNA extraction using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), from which 40-120 ng of genomic DNA was required for each locus typing. Medium-to-high resolution (second field) HLA typing (MHR-2F) of four loci, including A, B, C, and DRB1, was conducted by PCR sequence-specific oligonucleotide (PCR-SSO) using the LIFECODES HLA SSO kit (Luminex, Austin, TX, USA) on a Luminex 200 system, and analysed with Xponent 3.1 software connected to the IMGT (International ImMunoGeneTics) database version 3.43 library to provide us with the well-documented allele (WDA) (15)(16)(17). Blind external controls supplied by UK External Quality Assessment Services (UK EQAS) were used to confirm the accuracy of the four HLA loci identified. The procedure has been standardized under ISO15189:2012 criteria and is routinely used at Stem Cell Bank, Vietnam National Institute of Haematology and Blood Transfusion. Heterogeneous or homogeneous forms of the HLA gene were found. HLA typing was performed from June 2015 to December 2020. A few samples with ambiguous results in the 4-digit assignment were excluded from further investigation in this study. HLA typing was performed from June 2015 to December 2020.

Cohort Haematological Characteristics
In this study, 3,776 cord blood samples were collected. The proportion of ambiguous results in the 4-digit assignment ("medium-to-high" resolution) was 0.68% (26/3,776) among the total 3,776 cord blood units. These 26 samples with ambiguous data were removed, and the remaining 3,750 samples were included in this research for further investigation. The characteristics of the study cohort are shown in Table 1. Among 3,750 donors, there were 1,976 men (52.7%) and 1,774 women (47.3%). The distribution of blood types, including A, B, AB, and O, was almost the same in men and women. The percentage of donors with O-type was the highest at 45.5%, the AB-type was the lowest at 4.8%, and the A-type and Btype contributed to 20.9% and 28.8%, respectively. In particular, the number of donors with the O-type was 2.2-, 1.6-, and 9.5-fold higher than the A-, B-, and AB-types, respectively ( Table 1). This report agrees with previous reports on Kinh Vietnamese population (15,18).

DISCUSSION
Transplantation rejection is a common issue. Six HLA loci correspond to MHC class I and MHC class II, with three loci of HLA-A, -B, and -C relating to class I and HLA-DRB1, -DQA1, -DQB1, and -DPB1 related to class II. The level of HLA compatibility required varies depending on the transplantation technique used. In organ or stem cell transplantation, the compatibility of five or even six loci between the donor and the recipient is necessary; however, compatibility of at least four of the six loci is acceptable in the case of cord blood transplantation. Approximately 500 cord blood banks have been established in 97 countries worldwide (3)(4)(5).
The Kinh people were the most predominant ethnic community in Vietnam and accounted for more than 85% of the Vietnamese population (15,18). However, information on the frequency and distribution of HLA alleles in the Kinh Vietnamese cord blood is yet to be thoroughly investigated. Several studies have examined HLA alleles and haplotypes in the Kinh Vietnamese, but the sample size was quite limited, with only around 100 donors (14,15). The National Institute of Haematology and Blood Transfusion in Vietnam is one of the largest and most important storage facilities for umbilical cord blood in Vietnam. For future transplantation use, HLA genotypes of every cord blood sample should be tested.
In this study, cord blood units from the Kinh Vietnamese cohort were collected and stored in the cord blood bank at the National Institute of Haematology and Blood Transfusion, and PCR-SSO with Luminex Technology, a routine method in the institute, was used for HLA genotyping of these samples. Actually, there are several studies recorded that Luminex Technology for HLA-typing may cause a few percentage of ambiguous results as the Reviewer 2 have mentioned. However, many laboratories have used this method to identify HLA genotypes at 4-digit assignment (described as "medium-to-high" resolution) with good results (21,24). Typically, Park et al. conducted genotyping of HLA-A, HLA-B and HLA-DRB1 of more than ten thousand (10,918) Koreans by using Luminex Technology and they confirmed that this platform was possible for genotyping most of the samples at the level of 4-digit assignment, there were only few cases with ambiguous results that must be reconfirmed by Sequence-Based Typing (SBT) from Abbot Molecular, USA (21). In this study, because the procedure has been standardized under ISO15189:2012 criteria and is routinely used at Stem Cell Bank, Vietnam National Institute of Haematology and Blood Transfusion, the proportion of ambiguous results in the 4-digit assignment ("medium-tohigh" resolution) was very low, only 0.68% (26/3,776) among the total 3,776 cord blood units. As we concentrated on big data analysiswith a large sample size, therefore, for convenience, we did not include these 26 samples in this report, and the remaining 3,750 samples were analysed.
The findings in this study regarding frequencies and distributions of alleles and haplotypes in the Kinh Vietnamese cohort were similar to those in previous reports (15,18), but there were a few differences. Hoa et al. (15) and Do et al. (18) found that the three most frequent alleles for HLA-A were A * 11:01, A * 24:02, and A * 33:03, with frequencies of 22.77%, 12.87%, 10.89%, and 22.9%, and 13.8% and 11.5%, respectively; however, in this study, the three most frequent alleles in HLA-A were A*11:01, A*24:02, and A*02:01 with frequencies of 25%,          frequent alleles in USA NMDP Han Chinese; three of 10 HLA-A alleles, two of 10 HLA-B alleles, four of 10 HLA-C alleles, and two of 10 HLA-DRB1 alleles in the 10 most frequent alleles in USA NMDP Africa American; and four of 10 HLA-A alleles, two of 10 HLA-B alleles, five of 10 HLA-C alleles, and three of 10 HLA-DRB1 alleles in 10 highest frequent alleles in USA NMDP European Caucasian are the same as those in Kinh Vietnamese (Tables 5-8). These data demonstrated that, except for the USA NMDP Vietnamese, among these populations, the similarity of high-frequency HLA alleles in Kinh Vietnamese and Han Chinese was the highest, followed by South Korean, Japanese, European Caucasian, and African American. These data also indicated that the distributions and the most frequent HLA alleles in the Kinh Vietnamese population differed from those in European American and African American populations. These results suggest that Asian populations have different HLA allele frequencies than the American and European populations. Moreover, the diversity in the frequencies of individual alleles of HLA loci creates large differences in frequencies and distributions of two-, three-, or four-loci haplotypes among various populations (19)(20)(21)(22)(23)(24)(25)(26)(27). This suggests that transplantations between different populations are quite challenging, and therefore, establishing a bank to store cord blood samples for transplantation programs is important.

INSTITUTIONAL REVIEW BOARD STATEMENT
All participants provided informed consent for inclusion before participating in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Hanoi Medical

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further enquiries can be directed to the corresponding author.