Edited by: Leonardo M. R. Ferreira, Medical University of South Carolina, United States
Reviewed by: Sina Naserian, Hôpital Paul Brousse (INSERM), France; Vivian Weiwen Xue, Shenzhen University, China
*Correspondence: Ching-Ping Tseng,
This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
In addition to the classical human leukocyte antigen (HLA) genes, the outcomes of post-hematopoietic stem cell transplantation (HSCT) are associated with human leukocyte antigen (HLA)-related genes and non-HLA genes involved in immune regulation. HLA-G gene plays an important role in immune tolerance, assisting immune escape of tumor cells, and decrease of transplant rejection. In this study, we explored the association of genetic variants at the 3’-untranslated region (3’-UTR) and 5’-upstream regulatory region (5’-URR) of HLA-G gene with the adverse outcomes of patients with leukemia receiving HSCT. The genomic DNAs of 164 patients who had acute leukemia and received HSCT were collected for analysis. Nine single nucleotide polymorphisms (SNPs) and six haplotypes in the 3’-UTR and 27 SNPs and 6 haplotypes in the 5’-URR were selected to investigate their relationship with the development of adverse outcomes for patients receiving HSCT, including mortality, relapse, and graft-versus-host disease. Our results revealed that two SNPs (rs371194629 and rs9380142) and one haplotype (UTR-3) located in the 3’-UTR and two SNPs (rs3823321 and rs1736934) and one haplotype (G0104a) located in the 5’-URR of HLA-G were associated with the occurrence of chronic GVHD or development of any forms of GVHD. No SNP was found to associate with the occurrence of mortality and relapse for patients receiving HSCT. These SNPs and haplotypes may play important roles in regulating immune tolerance of allografts post-HSCT that can be used to predict the risk of poor outcomes after receiving HSCT and giving preventive treatment to patients on time.
Hematopoietic stem cell transplantation (HSCT) is a treatment of patients with blood disorders or cancer (
HLA-A, HLA-B, and HLA-C are the human major histocompatibility complex (MHC) class I proteins which are well documented to associate with the outcomes of HSCT (
The 3’-untranslated region (3’-UTR) and the 5’-upstream regulatory region (5’-URR) of HLA-G gene are highly polymorphic. Genetic variations in these regions are known to regulate the levels of HLA-G gene expression and post-transcriptional mRNA stability (
The aim of this study was to investigate the effects of genotype and haplotypes in the 3’-UTR and 5’-URR of HLA-G gene on the mortality, the risk of disease relapse, and the development of GVHD for patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) after receiving HSCT. The significance of the findings in this study was discussed.
The study has been approved by the Institutional Review Board (IRB) of Chang Gung Memorial Hospital (CGMH) and was performed according to the ethical regulations and requirements. The approved ID was 201304949B0 and 202100302B0. Because HLA-G was mainly expressed in the saliva, peritoneal fluid, plasma, thymus, semen, cerebrospinal fluid but not in the immune cells (
Clinical characteristics of patients who enrolled in the study.
Clinical parameters | Number of patients | percentage |
---|---|---|
Patients | 164 | 100% |
Median age (years, range) | 32 (0.8-66) | |
Diagnosis | ||
ALL | 58 | 35% |
AML | 106 | 65% |
Mortality | 71 | 43% |
Relapse | 67 | 41% |
GVHD | ||
acute GVHD I-II | 46 | 28% |
acute GVHD III-IV | 9 | 5% |
chronic GVHD | 85 | 52% |
ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia.
The specimens for analysis were the remnant DNA for short tandem repeats (STR) analysis obtained from the Department of Laboratory Medicine. Briefly, peripheral blood (3 ml) was collected from patients in the blood collection tube with EDTA as the anticoagulant. The buffy coat was collected for genomic DNA extraction by using QIAamp DNA Blood Mini Kit according to the instruction of the manufacturer (Qiagen, Valencia, California, USA). The OD260/280 of the template genomic DNA had to be in the range of 1.8-2.0 with a minimal concentration of 100 ng/μl for genotyping and haplotyping by PCR.
Based on previous studies for the association of HLA-G genetic polymorphisms with the clinical outcomes of various diseases and organ transplantation (
The genetic variants of HLA-G gene for analysis in this study.
Genomic region | Genetic variants under analysis | ||||
---|---|---|---|---|---|
3’-UTR | rs371194629 | rs1707 | rs1710 | rs17179101 | rs17179108 |
rs1063320 | rs9380142 | rs1610696 | rs1233331 | ||
5’-URR | rs1736936 | rs1736935 | rs3823321 | rs1736934 | rs17875389 |
rs3115630 | rs1632947 | rs1632946 | rs1233334 | rs2249863 | |
rs2735022 | rs35674592 | rs17875391 | rs1632944 | rs201221694 | |
rs112940953 | rs17875393 | rs1736933 | rs149890776 | rs1736932 | |
rs17875394 | rs17875395 | rs17875396 | rs1632943 | rs191630481 | |
rs1233333 | rs17875397 |
The haplotypes in the 3’-UTR of HLA-G under analysis in this study.
Haplotypes | Position, rs number, and sequences of the genetic variants | Frequency (n = 164) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
+2960 | +2989 | +2996 | +3013 | +3021 | +3128 | +3173 | +3182 | +3213 | ||
rs371194629 | rs1707 | rs1710 | rs17179101 | rs17179108 | rs1063320 | rs9380142 | rs1610696 | rs1233331 | ||
UTR-1 | Del | T | G | C | C | C | G | C | G | 0.303 |
UTR-2 | Ins | T | C | C | C | G | A | G | G | 0.082 |
UTR-3 | Del | T | C | C | C | G | A | C | G | 0.252 |
UTR-4 | Del | C | G | C | C | C | A | C | G | 0.006 |
UTR-5 | Ins | T | C | C | T | G | A | C | G | 0.006 |
UTR-7 | Ins | T | C | A | T | G | A | C | G | 0.197 |
The A nucleotide of the ATG start codon was designated as position +1.
The haplotypes in the 5’-URR of HLA-G under analysis in this study.
Haplotypes | Position, rs number and sequences of the genetic variants | Frequency |
||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
-1305 | -1179 | -1155 | -1140 | -1138 | -1121 | -964 | -762 | -725 | -716 | -689 | -666 | -646 | -633 | -545 | -539 | -509 | -486 | -483 | -477 | -443 | -400 | -391 | -369 | -355 | -201 | -56 | ||
rs1736936 | rs1736935 | rs3823321 | rs1736934 | rs17875389 | rs3115630 | rs1632947 | rs1632946 | rs1233334 | rs2249863 | rs2735022 | rs35674592 | rs17875391 | rs1632944 | rs201221694 | rs112940953 | rs17875393 | rs1736933 | rs149890776 | rs1736932 | rs17875394 | rs17875395 | rs17875396 | rs1632943 | rs191630481 | rs1233333 | rs17875397 | ||
G010101a | G | A | G | A | A | C | G | C | C | T | A | G | A | G | 5g | A | C | A | A | C | G | G | G | C | G | G | C | 0.304 |
G010101b | G | A | G | A | A | C | G | C | G | T | A | G | A | G | 5g | A | C | A | A | C | G | G | G | C | G | G | C | 0.006 |
G010102a | A | G | G | T | A | C | A | T | C | G | G | T | A | A | 5g | A | C | C | A | G | G | G | G | A | G | A | C | 0.252 |
G0103a | G | G | G | A | G | C | G | C | T | T | A | G | A | G | 6g | A | G | A | A | G | G | A | A | A | G | G | T | 0.003 |
G0104a | A | G | A | A | A | C | A | T | C | G | G | T | A | A | 5g | A | C | C | A | G | G | G | G | A | G | A | C | 0.211 |
G0104b | A | G | A | A | A | C | A | T | C | G | G | T | A | A | 5g | A | C | C | A | G | A | G | G | A | G | A | C | 0.096 |
The first nucleotide at the 5’ of the ATG start codon was designated as position -1.
Primers (
The primer pairs for amplifying the 3’-UTR and 5’-URR of HLA-G.
Genomic region | Primer sequences | PCR product (bp) |
---|---|---|
3’-UTR | 5’-TCACCCCTCACTGTGACTGATA-3’ | 366 |
5’-CTGTGGAAAGTTCTCATGTCTTCC-3’ | ||
5’-URR | 5’-GCCTGACATTCTAGAAGCTTCACAAGAAT-3’ | 1402 |
5’-ATCCTTGGCGTCTGGGGAGAAT-3’ |
The definition of outcomes post-HSCT has been described previously (
Patients were grouped as case or control for statistical analyses of the association between the genotype and haplotype of patients and the effectiveness of HSCT. For mortality analysis, patients who died and lived at the end of this study were grouped as case and control, respectively. For relapse analysis, patients who relapsed and did not relapse at the end of this study were grouped as the case and control, respectively. For GVHD analysis, patients who developed stages I-II aGVHD, stages III-IV of aGVHD, cGVHD, and any forms of GVHD were considered as case and patients without development of GVHD were considered as control.
The PLINK software was used for genotyping analysis (
The haplotypes with frequency less than 1% were excluded from the analysis. The dosage of a specific haplotype was first defined as 2, 1, and 0 when the indicated haplotype was homozygous, heterozygous, and not present in the recipient genomic DNA, respectively. Analysis was performed by either heterozygous or homozygous haplotype models. For heterozygous haplotype model, the occurrence of adverse outcomes for patients with two copies of the same haplotypes were compared with patients with one copy of the haplotype. For homozygous haplotype model, the occurrence of adverse outcomes for patients with two copies of the same haplotypes were compared with patients without the specific haplotype.
The SPSS 17.0 software was used for statistical analysis. A p < 0.05 was considered as statistical significance for all tests. Odds ratio (OR) and 95% confidence interval (CI) were also provided.
A total of 9 genetic polymorphisms in the 3’-UTR of HLA-G gene were analyzed to determine the association of these genotypes with the risks of adverse outcomes post-HSCT, including mortality, disease relapse, and GVHD. The complete genotype and allele frequency datasets for all recipients are summarized in the
Association of genetic variants in 3’-UTR of HLA-G with the adverse outcomes post-HSCT.
SNP | No. of patients (%) | Additive p | Model | Logistic regression p | OR (95% CI) | ||
---|---|---|---|---|---|---|---|
Chronic GVHD | |||||||
rs371194629 | del/del | del/ins | ins/ins | 0.05 | Heterozygous | 0.03* | 2.98 (1.10-8.07) |
Cases |
33 | 43 | 9 | Homozygous | 0.25 | 4.36 (0.51-37.48) | |
Controls |
16 | 7 | 1 | Dominant | 0.02* | 3.15 (1.21-8.18) | |
Recessive | 0.45 | 2.72 (0.33-22.65) | |||||
rs9380142 | A/A | A/G | G/G | 0.08 | Heterozygous | 0.03* | 3.01 (1.08-8.42) |
Cases | 29 | 47 | 9 | Homozygous | 1.00 | 1.01 (0.26-3.88) | |
Controls | 13 | 7 | 4 | Dominant | 0.08 | 2.28 (0.91-5.73) | |
Recessive | 0.48 | 0.59 (0.17-2.12) | |||||
Any form of GVHD | |||||||
rs371194629 | del/del | del/ins | ins/ins | 0.12 | Heterozygous | 0.07 | 2.40 (0.92-6.22) |
Cases | 62 | 65 | 13 | Homozygous | 0.45 | 3.36 (0.41-27.59) | |
Controls | 16 | 7 | 1 | Dominant | 0.04* | 2.52 (1.01-6.26) | |
Recessive | 0.70 | 2.35 (0.29-18.88) | |||||
rs9380142 | A/A | A/G | G/G | 0.13 | Heterozygous | 0.05* | 2.62 (0.98-7.03) |
Cases | 51 | 72 | 17 | Homozygous | 1.00 | 1.08 (0.31-3.77) | |
Controls | 13 | 7 | 4 | Dominant | 0.10 | 2.06 (0.86-4.94) | |
Recessive | 0.52 | 0.69 (0.21-2.27) |
Cases: patients with the event; Controls: patients without GVHD.*, p < 0.05.
The association of 27 genetic polymorphisms in 5’-URR of HLA-G gene with post-HSCT adverse outcomes of patients were also investigated. Because of the insufficient amount of genomic DNA for PCR, the samples of three patients including one patient with ALL (the patient had mortality and chronic GVHD, but without relapse) and two patients with AML (both patients had mortality and chronic GHVD, and one had relapse) failed to be tested at 5’-URR. No association of these polymorphisms with mortality and relapse was found (
Association of genetic variants in 5’-URR of HLA-G with the adverse outcomes post-HSCT.
SNP | No. of patients (%) | Additive p | Model | Logistic regression p | OR (95% CI) | |||
---|---|---|---|---|---|---|---|---|
Chronic GVHD | ||||||||
rs3823321 | G/G | A/G | A/A | <0.01* | Heterozygous | 0.75 | 0.84 (0.28-2.49) | |
Cases | 46 | 30 | 6 | Homozygous | <0.01* | 0.15 (0.04-0.53) | ||
Controls | 9 | 7 | 8 | Dominant | 0.11 | 0.47 (0.18-1.20) | ||
Recessive | <0.01* | 0.16 (0.05-0.52) | ||||||
rs1736934 | A/A | A/T | T/T | 0.07 | Heterozygous | 0.04* | 2.77 (1.02-7.54) | |
Cases | 33 | 40 | 9 | Homozygous | 0.25 | 4.36 (0.51-37.48) | ||
Controls | 16 | 7 | 1 | Dominant | 0.02* | 2.97 (1.14-7.73) | ||
Recessive | 0.45 | 2.84 (0.34-23.59) | ||||||
Any form of GVHD | ||||||||
rs3823321 | G/G | A/G | A/A | 0.01* | Heterozygous | 0.99 | 1.01 (0.35-2.88) | |
Cases | 69 | 54 | 14 | Homozygous | 0.01* | 0.23 (0.08-0.69) | ||
Controls | 9 | 7 | 8 | Dominant | 0.25 | 0.59 (0.24-1.44) | ||
Recessive | 0.01* | 0.23 (0.08-0.63) |
Cases: patients with the event; Controls: patients without GVHD.*, p < 0.05.
The association of haplotypes in 3’-UTR with the adverse outcomes of patients receiving HSCT was further analyzed. Six different haplotypes including UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, and UTR-7 were classified according to the sequence combination of 8 SNPs (rs371194629, rs1707, rs1710, rs17179101, rs17179108, rs1063320, rs9380142, rs1610696, and rs1233331) located in 3’-UTR. Among patients under analysis, the frequency of UTR-1 (30.3%) was the highest followed by UTR-3 (25.2%) and UTR-7 (19.7%) (
During haplotype analysis (
Association of haplotypes in 3’-UTR of HLA-G with adverse outcomes post-HSCT.
Haplotype | No. of patients | Additive |
Model | Logistic regression |
OR (95% CI) | ||
---|---|---|---|---|---|---|---|
2 |
1 |
0 |
|||||
Chronic GVHD | |||||||
UTR-3 | |||||||
Cases | 6 | 30 | 49 | <0.01* | Heterozygous (2 vs 1) | 0.01* | 5.71 (1.50-21.84) |
Controls | 8 | 7 | 9 | Homozygous (2 vs 0) | <0.01* | 7.26 (2.03-25.98) | |
Any form of GVHD | |||||||
UTR-3 | |||||||
Cases | 13 | 55 | 72 | 0.01* | Heterozygous (2 vs 1) | 0.02* | 4.84 (1.49-15.75) |
Controls | 8 | 7 | 9 | Homozygous (2 vs 0) | 0.01* | 4.92 (1.61-15.10) |
Dose of haplotype: 2 means that the haplotype of UTR-3 is present in both strands of DNA. 1 means that the haplotype of UTR-3 is present in one strand of DNA, and the other strand of DNA carries the other type of haplotype. 0 means that the haplotype of UTR-3 is absence in both strands of DNA.
Cases: patients with the event; Controls: patients without GVHD.*, p < 0.05.
The association of haplotypes in 5’-URR with the adverse outcomes of patients receiving HSCT was also analyzed (
Association of haplotypes in 5’-URR of HLA-G with adverse outcomes post-HSCT.
Haplotype | No. of patients | Additive |
Model | Logistic regression |
OR (95% CI) | ||
---|---|---|---|---|---|---|---|
2 |
1 |
0 |
|||||
Chronic GVHD | |||||||
0104a | |||||||
Cases | 2 | 22 | 58 | <0.01* | Heterozygous (2 vs 1) | 0.01* | 9.43 (1.54-57.74) |
Controls | 6 | 7 | 11 | Homozygous (2 vs 0) | <0.01* | 15.82 (2.82-88.80) | |
Any form of GVHD | |||||||
0104a | |||||||
Cases | 6 | 39 | 92 | <0.01* | Heterozygous (2 vs 1) | 0.02* | 5.57 (1.39-22.33) |
Controls | 6 | 7 | 11 | Homozygous (2 vs 0) | <0.01* | 8.36 (2.30-30.47) |
Dose of haplotype: 2 means that the haplotype of 0140a is present in both strands of DNA. 1 means that the haplotype of 0104a is present in one strand of DNA, and the other strand of DNA carries the other type of haplotype. 0 means that the haplotype of 0104a is absence in both strands of DNA.
Cases: patients with the event; Controls: patients without GVHD.*, p < 0.05.
As revealed by additive analysis, the dosage of 0104a haplotype was reversely associated with the development of cGVHD (p < 0.01) and any forms of GVHD (p < 0.01) post-HSCT. Patients with heterozygous 0104a and without 0104a had 9.43 times (95% CI = 1.54 - 57.74, p = 0.01) and 15.82 times (95% CI = 2.82 - 88.80, p < 0.01) higher risk for development of cGVHD when compared to patients with homozygous 0104a, respectively. Patients with heterozygous 0104a and without 0140a had 5.57 times (95% CI = 1.39 - 22.33, p = 0.02) and 8.36 times (95% CI = 2.30 - 30.47, p < 0.01) higher risk for development of any forms of GVHD when compared to patients with homozygous 0104a, respectively.
Genetic polymorphisms and haplotypes of HLA-G have been reported to associate with various diseases (
There are soluble and membrane-bound forms of HLA-G. Through alternative splicing, three major soluble forms of HLA-G (HLA-G5, -G6, and -G7) are expressed in the body fluid such as saliva, plasma, and cerebrospinal fluid (
According to the findings of this study, genetic polymorphisms of rs371194629 and rs9380142, and the UTR-3 haplotype located in the 3’-UTR were associated with the risk for the development of cGVHD and any forms of GVHD. These polymorphisms have been shown to involve in the regulation of HLA-G expression (
Similar to our findings, the association of rs371194629 with the development of GVHD and other post-transplant complications have been reported (
We further demonstrated in this study that the genotypes of rs3823321 and rs1736934 and the G0104a haplotype located at the 5’-URR of HLA-G gene are associated with the risk for occurrence of cGVHD and development of any forms of GVHD. Patients with G/G genotype of rs3823321 is associated with higher risk for cGVHD as well as development of any forms of GVHD, while T-allele of rs1736934 is associated with higher risk for cGVHD. Notably, G0104a haplotype had the A-allele at rs3823321 and rs1736934. The roles of 5’-URR in the regulation of HLA-G gene expression and in immune tolerance and disease development are not yet well studies. Previous study showed that rs3823321 G was a binding site for SOX5 and progesterone receptor, and rs1736934 T is a binding site for POU2F1 (
In conclusion, this study showed that six genotypes and haplotypes in 3’-UTR and 5’-URR of HLA-G gene were associated with the risk for development of any forms of GVHD and cGVHD in patients post-HSCT. The findings of this study are applicable to predict the risk of adverse outcomes post-HSCT that allow physicians to prescribe preventive plan or treatment for patients on time. Moreover, effective prevention of GVHD is depended on the induction of peripheral immune tolerance. The role of HLA-G gene polymorphism in the regulation of immune tolerance and the development of GVHD post-HSCT is worthy to investigate further.
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in
The studies involving human participants were reviewed and approved by Institutional Review Board (IRB) of Chang Gung Memorial Hospital. The patients/participants provided their written informed consent to participate in this study.
D-PC conceived and designed the experiments and reviewed the final draft. P-NW contributed the materials. A-LH, D-PC, and C-PT analyzed and interpreted data. D-PC, F-PH, W-TL, and C-PT wrote draft of the manuscript. W-TW performed the experiments. All authors contributed to the article and approved the submitted version.
This study was supported by grants to D-PC from the Chang Gung Memorial Hospital (CMRPG3L0771-2), and to C-PT from the Chang Gung Memorial Hospital (CMRPD1M0071-2, CMRPD1L0281-2, CORPD1K0031, and BMRP466) and the Ministry of Science and Technology (109-2320-B-182-031-MY3). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The excellent consulting assistance and sample resources from Molecular Diagnosis Laboratory of Chang Gung Memorial Hospital are gratefully acknowledged.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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