Impact of HLA-G 14 bp polymorphism and soluble HLA-G level on kidney graft outcome

Vladimira Durmanova; Helena Bandzuchova; Zuzana Zilinska; Jana Tirpakova; Daniel Kuba; Milan Buc; Katarina Polakova

doi:10.1515/biol-2016-0049

Article Open Access

Impact of HLA-G 14 bp polymorphism and soluble HLA-G level on kidney graft outcome

Vladimira Durmanova , Helena Bandzuchova , Zuzana Zilinska , Jana Tirpakova , Daniel Kuba , Milan Buc and Katarina Polakova

Published/Copyright: December 2, 2016

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From the journal Open Life Sciences Volume 11 Issue 1

Abstract

Human leukocyte antigen G (HLA-G) is a non-classical HLA class I protein with various immunosuppressive functions. Besides its profound effect to induce fetal tolerance, HLA-G has been also found to enhance graft acceptance. The aim of the study was to analyse the association between HLA-G 14 bp insertion/deletion polymorphism, soluble HLA-G level and kidney graft outcome in the Slovak population. We investigated 69 kidney transplant recipients aged 27–65 years. Out of this group, 37 recipients developed acute rejection, confirmed by biopsy, and 32 patients had stable allograft function. Plasma was obtained from recipients at 1 day before transplantation and analyzed by ELISA. Genotyping of HLA-G polymorphism was performed by PCR. Significantly higher pre-transplantation levels of sHLA-G were found in the group with stable allograft function in comparison to group with acute rejection (P = 0.0409). In the homozygous −14/−14 recipients with stable allograft function, significantly higher values of sHLA-G were determined in comparison to the recipients with acute rejection (P = 0.0052). The study revealed an association between 14 bp deletion polymorphism and soluble HLA-G level that is proportional to kidney graft acceptance. It is suggested that pre-transplantation levels of soluble HLA-G should be monitored as additional marker to predict kidney graft outcome.

Keywords: soluble HLA-G; genetic polymorphism; kidney transplantation; acute rejection; genotyping

1 Introduction

Human leukocyte antigen G (HLA-G) belongs to the non-classical HLA-class I molecules and is characterized by its immunosuppressive properties. HLA-G is mainly expressed in extravillous cytotrophoblasts, which help to protect the fetus from maternal immune surveillance [1]. HLA-G molecules have also been found in other tissues, such as thymic medulla [2], cornea [3], pancreas [4] and in human mononuclear phagocytes [5]. Expression of HLA-G antigens can be induced under pathological conditions such as transplantation, tumors, viral infections and autoimmune diseases [6-10]. HLA-G mediated immunosuppression seems to have a beneficial effect on transplantation and autoimmune diseases, whereas it is detrimental at tumor diseases or viral infections.

Immunosuppressive activity of the HLA-G molecule is mediated through its interaction with inhibitory receptors of immune cells: ILT-2 receptor present on B cells, APC and some T, and NK cells, ILT-4 receptor present on APC and KIR2DL4 expressed by NK and some T cells [11,12]. Furthermore, the immunosuppressive effect of HLA-G can also be achieved through the destruction of cytotoxic CD8⁺ T cells and NK cells: engagement of the HLA-G molecule to receptor CD8 results in Fas ligand (FasL) production, followed by Fas/FasL - mediated apoptosis in CD8 bearing T lymphocytes and NK cells [13]. To summarize, HLA-G mediates inhibition of cytotoxic activity of uterine and peripheral blood NK cells and CD8⁺ T cells, inhibition of the alloproliferative response of CD4⁺ T cells, inhibition of dendritic cell maturation and activation of regulatory T cells [12].

The HLA-G gene is 4170 bp long and consists of 8 exons. By an alternative splicing of the primary transcript, 7 HLA-G isoforms can be generated. Four isoforms: HLA-G1, -G2, -G3 and -G4 are membrane bound, whereas three isoforms: HLA-G5, -G6 and -G7 are soluble. Of these, only the HLA-G1 molecule has a structure resembling that of other membrane-bound HLA-I molecules [14].

The HLA-G gene is characterized by low polymorphism, namely 53 HLA-G alleles, 18 HLA-G proteins and 2 null alleles have been identified to date (IMGT/HLA database, July 2016). The most polymorphic sites that influence HLA-G expression were identified in the 5´ and 3´ non-coding regions. In the 3´UTR (untranslated region) a 14 bp insertion polymorphism (5´-ATTTGTTCATGCCT-3´) has been described, which affects stability of mRNA causing decreased production of most membrane and soluble isoforms [15]. Other polymorphisms in the 3´UTR that can influence mRNA stability and soluble HLA-G level includes SNP at the +3142 position (C/G), at the +3187 (A/G) and +3196 (C/G) [16].

Several studies have described a protective effect of HLA-G in human allotransplantation such as kidney, liver, heart and lung [6, 17–19]. In kidney transplant patients, increased soluble HLA-G level was significantly associated with better graft acceptance [8, 18, 20–23]. As the expression of sHLA-G may be influenced by the 14 bp insertion polymorphism [15], the possible association between this variant and graft acceptance has been investigated, but the results are controversial. Out of 5 studies, only 2 studies described association between the HLA-G 14-bp insertion (ins) /deletion (del) polymorphism and risk of kidney transplant rejection [24]. Misra et al. [22] evaluated the relationship between the 14 bp ins/del polymorphism of the HLA-G gene and the level of soluble HLA-G in kidney graft acceptance. The level of soluble HLA-G was found to be increased among the non-acute rejection group for 14 bp ins/ins (P < 0.0001), ins/del (P = 0.0171) and del/del (P = 0.0263) genotypes in comparison to the acute rejection group [22]. The present study further investigates the association between HLA-G 14 bp polymorphism, soluble HLA-G level and kidney graft acceptance in the Slovak Caucasian population.

2 Material and methods

2.1 Study subjects

The investigated group included 69 kidney transplant recipients (45 males, 24 females) aged 27–65 years in a period between the years 2010 and 2013. Out of this group, 37 recipients developed acute rejection. All rejection episodes were confirmed by biopsy and classified according to histological Banff 07 criteria [25]. Blood samples were obtained from the recipients at 1 day before transplantation and collected at the National Transplantation Organization in Bratislava, Slovakia. The clinical characteristics of the recipients are shown in Table 1. Immunosuppressive drugs were administered for the first 1–2 days after transplantation as described by Zilinska et al. [26]. Written informed consent for enrolling in the study and for personal data management was obtained from all recipients. The study was approved by the Ethics committee of the University Hospital Bratislava. All the investigations were carried out in accordance with the International Ethical Guidelines and the Declaration of Helsinki.

Tab. 1

Clinical characteristics of kidney transplant recipients

Parameter (Mean ± SD)	Acute rejection	Stable allograft	P
	N = 37	N = 32
Age at Tx (years)	46.89 ± 12.06	45.21 ± 11.37	0.56
Gender
Male	29	16
Female	8	16
Cold ischemic time (1 = 24 h)	0.71 ± 0.21	0.71 ± 0.23	0.98
Duration on dialysis (days)	1411 ± 1305	1256 ± 1006	0.59
Number of Tx
First	35	30
Second	2	2	-
Type of donor
Living	2	2
Deceased	35	30	-
Pre-transplantation PRA
0%	24	19
1-10%	13	11	-
11-50%	0	3
HLA mismatch (MM)
1 MM	1	4
2 MM	5	11
3 MM	16	11	-
> 3 MM	15	6
Type of rejection
AMR	20	0
TCMR	11	0	-
AMR+TCMR	6	0
Immunosupressive treatment
Basil/Dacl, TAC, MMF (MPA), CS	13	14
Basil/Dacl, CyA, MMF (MPA), CS	2	0	-
Thymo, TAC, MMF (MPA), CS	1	1
TAC, MMF (MPA), CS	13	11
CyA, MMF (MPA), CS	8	6

PRA - panel reaction antibodies, Tx - transplantation, AMR - antibody-mediated rejection, TCMR – T cell-mediated rejection, N – number, Basil – basiliximab, Dacl – daclizumab, TAC – tacrolimus, MMF – mycofenolate mofetil, MPA – mycofenolate acid, CS – corticosteroid, CyA – cyclosporine A, Thymo– rabbit anti-thymocyte globulin.

2.2 Genotyping of HLA-G 14 bp polymorphism at exon 8 (3´ UTR)

Genomic DNA was isolated from EDTA-treated peripheral blood samples (2 ml) by a modified salting-out procedure [27]. Genotyping of HLA-G 14-bp polymorphism (rs16375) was performed as described by Hviid et al. [28]. Briefly, DNA was amplified by forward primer 5′GTGATGGGCTGTTTAAAGTGTCACC-3′ and reverse primer 5′GGAAGGAATGCAGTTCAGCATGA-3′ using a PCR cycler (Biometra). A reaction mixture with a total volume of 25 ml contained 50 ng of template DNA, 0.2 mM of each dNTP (Thermofisher), 1 unit of Taq DNA polymerase (Thermofisher), 1.5 mmol MgCl₂ (Thermofisher) and 10 pmol of each specific primer. PCR conditions were 95°C for 3 minutes, followed by 30 cycles (denaturation at 95°C for 1 min, annealing at 64°C for 1 minute and elongation at 72°C for 1 minute) and final elongation at 72°C for 10 minutes. The PCR products were run in 3% agarose gel for 20 minutes and then visualized under UV-light. Fragment size was confirmed using the 100 bp DNA ladder (SBS). PCR fragments of 224 bp (14 bp insertion) and PCR fragments of 210 bp (14 bp deletion) were identified (Figure 1).

Fig. 1

HLA-G 14 bp ins/del genotyping by PCR. HLA-G 14 bp ins/del polymorphism in kidney transplant recipients was assessed by PCR [28]. PCR fragment of 224 bp (14 bp insertion, +14) and PCR fragment of 210 bp (14 bp deletion, −14) was identified.

2.3 Analysis of soluble HLA-G level

Plasma samples were obtained from transplant recipients (N = 69) 1 day before transplantation. Levels of soluble HLA-G antigens (shed HLA-G1 and secreted HLA-G5) were determined by sandwich enzyme-linked immunosorbent assay (ELISA) using sHLA-G ELISA assay kit (Exbio, Prague, Czech Republic) according to the manufacturer’s instructions. Optical density was measured at 450 nm. The limit of sensitivity was 3 U/ml. 100 U sHLA-G corresponds to 40–50 ng of protein.

2.4 Data analysis

Allele and genotype frequencies were calculated by chisquare test using SNPstats web software available at http.//bioinfo.iconcologia.net/snpstats/start.htm. The odds ratios (OR) and 95% confidence intervals (95% CI) were also calculated. For comparison of the mean values of sHLA-G unpaired Student’s t test with Welch correction was used. This statistical analysis was performed by InStat statistical software (GraphPad Software, Inc., San Diego, USA. P value of < 0.05 was considered as statistically significant.

3 Results

3.1 Characteristics of kidney transplant recipients

The clinical characteristics of the recipients are shown in Table 1. The study group of 69 kidney transplant recipients (45 males, 24 females) was divided into recipients with acute rejection (N = 37) and recipients with stable graft function (N = 32). The comparison of demographic and clinical data didn’t reveal any significant differences between these analyzed groups (Table 1). The mean age of kidney transplant recipients was 46.05 ± 11.72 years. Out of 69 kidney transplant recipients, 65 underwent first transplantation, whereas 4 recipients underwent second transplantation. Four recipients received kidney from living donors, whereas other recipients received kidney from deceased donors. The mean cold ischemic time was 0.71 ± 0.22 (1 = 24 h). Out of 37 recipients with acute rejection, 20 developed antibody-mediated rejection (AMR), 11 developed T cell - mediated rejection (TCMR) and 6 recipients developed both types of rejection. In relation to HLA mismatch between donor and recipient, 7.25% of kidney transplant recipients had 1 HLA mismatch, 23.19% had 2 HLA mismatches, 39.13% had 3 HLA mismatches and 30.43% had more than 3 HLA mismatches. Immunosuppressive treatment of patients consisted of cyclosporine (N = 17) or tacrolimus (N = 53); mycophenolate mofetil/mycophenolic acid (N = 69); and prednisone (N = 69). 29 patients received immunosuppression as monoclonal antibodies against CD25 and 2 patients received anti-thymocyte globuline.

3.2 HLA-G 14 bp polymorphism in kidney transplant recipients

HLA-G 14 bp insertion/deletion polymorphism in kidney transplant recipients were determined by genotyping. 2 groups of recipients were analyzed: those with acute rejection (N = 37) and those with stable allograft function (N = 32). Allele and genotype frequencies of the HLA-G 14 bp deletion/insertion polymorphism (-14/+14) are shown in Table 2. Genotype frequencies fit the Hardy-Weinberg equilibrium (P = 0.34). No statistically significant differences were found between the HLA-G 14 bp deletion/insertion allele and genotypes between the rejection group and non- rejection group (P > 0.05). Nevertheless, in the group with non-rejection higher frequencies of homozygous −14/−14 bp genotype and decreased frequencies of heterozygous −14/+14 bp genotype were observed in comparison to recipients with acute rejection (43.75% vs. 27.03% and 31.25% vs. 54.05%). After adjustment for age and gender there were no significant changes in the distributions of the HLA-G 14 bp deletion/ insertion polymorphism (Table 2).

Tab. 2

HLA-G 14 bp allele and genotypes frequencies in kidney transplant recipients.

Allele/ genotype	Acute rejection (N = 37)	Stable allograft (N = 32)	Univariate analysis		Multivariate analysis
			P	OR (95%CI)	P	OR (95%CI)
−14	40 (54.05%)	38 (59.38%)
+14	34 (45.95%)	26 (40.62%)	0.53	0.81 (0.41-1.58)	-	-
−14/−14	10 (27.03%)	14 (43.75%)		1.00		1.00
−14/+14	20 (54.05%)	10 (31.25%)	0.15	2.80 (0.92-8.51)	0.41	2.47 (0.37-16.31)
+14/+14	7 (18.92%)	8 (25.00%)	0.54	1.22 (0.33-4.49)	0.34	0.51 (0.03-9.24)
−14/−14	10 (27.03%)	14 (43.75%)		1.00		1.00
−14/+14 and +14/+14	27 (73. 97%)	18 (56.25%)	0.15	2.10 (0.77-5.75)	0.5	1.85 (0.31-10.97)
−14/−14 and −14/+14	30 (81.08%)	24 (75.00%)		1.00		1.00
+14/+14	7 (18.92%)	8 (25.00%)	0.54	0.70 (0.22-2.21)	0.34	0.28 (0.02-4.13)
−14/−14 and +14/+14	17 (45.95%	22 (68.75%)		1.00		1.00
−14/+14	20 (54.05%)	10 (31.25%)	0.06	2.59 (0.96-6.95)	0.21	2.97 (0.53-16.63)

Allele and genotype frequencies are presented as absolute numbers with percentages in parentheses. OR - odds ratio; CI - confidence interval. Univariate analysis is based on χ² test. Multivariate analysis is adjusted by sex and age. N - number, −14 - HLA-G 14 bp deletion, +14 - HLA-G 14 bp insertion.

3.3 Analysis of soluble HLA-G level in kidney transplant recipients

Levels of soluble HLA-G were analyzed by ELISA in kidney transplant recipients plasma 1 day before transplantation. Plasma from 69 recipients was evaluated. Significantly higher levels of sHLA-G were found in the group with stable allograft function in comparison to group with acute rejection (22.723 ± 17.063 vs. 15.625 ± 9.172 U/ml, P = 0.0409, Figure 2). Analysis of sHLA-G values in the group with acute rejection revealed no association between the values and type of rejection (AMR: 13.439 ± 6.628 U/ml, TCMR: 17.377 ± 12.143 U/ml, AMR+TCMR: 19.705 ± 9.900 U/ ml, P > 0.05, Figure 3).

Fig. 2

Pre-transplantation sHLA-G levels in kidney transplant recipients. sHLA-G values were expressed as mean±SD. P values were calculated using unpaired Student’s t test with Welch correction. * P value of < 0.05 was considered as statistically significant. AR - acute rejection, STA - stable allograft function, N – number

Fig. 3

Pre-transplantation sHLA-G levels in kidney transplant acute rejection recipients. sHLA-G values were expressed as mean ± SD. P values were calculated using Student’s t test with Welch correction. * P value of < 0.05 was considered as statistically significant. AMR – antibody - mediated rejection, TCMR – T cell - mediated rejection, N – number

3.4 Analysis of soluble HLA-G level in association with 14 bp ins/del polymorphism in kidney transplant recipients

Pre-transplantation sHLA-G level was also analyzed, in association with HLA-G 14 bp ins/del polymorphism genotype in kidney transplant recipients. First, the level of soluble HLA-G was determined in all kidney transplant recipients regardless of rejection (Figure 4). Significantly higher levels of sHLA-G were found in the homozygous −14/−14 recipients in comparison to the homozygous +14/+14 recipients (25.115 ± 18.422 vs. 14.455 ± 6.831 U/ml, P = 0.0153) and to the heterozygous −14/+14 individuals (25.115 ± 18.422 vs. 16.190 ± 10.199, P = 0.0408). In the group with stable allograft function, significantly higher values of sHLA-G were found in the homozygous −14/−14 individuals in comparison to the acute rejection recipients (32.939 ± 20.578 vs. 14.162 ± 5.201, P = 0.0052, Figure 5). However, there was no association of sHLA-G values and +14/+14 genotype or −14/+14 genotype in the group with stable allograft function as compared to the group with acute rejection (14.519 ± 6.502 vs. 14.381 ± 7.717, P = 0.9172; 14.985 ± 8.343 vs. 16.793 ± 11.164, P = 0.6235).

Fig. 4

HLA-G 14 bp ins/del polymorphism and pre-transplantation sHLA-G levels in kidney transplant recipients. sHLA-G values were expressed as mean±SD. P values were calculated using unpaired Student’s t test with Welch correction. * P value of <0.05 was considered as statistically significant. +14 – HLA-G 14 bp insertion in exon 8, −14 – HLA-G 14 bp deletion in exon 8, N – number

Fig. 5

Comparison of HLA-G 14 bp ins/del polymorphism and pre-transplantation sHLA-G levels in kidney transplant recipients. sHLA-G values were expressed as mean ± SD. P values were calculated using unpaired Student’s t test with Welch correction. * P value of < 0.05 was considered as statistically significant. +14 - HLA-G 14 bp insertion in exon 8, −14 - HLA-G 14 bp deletion in exon 8, AR - acute rejection, STA - stable allograft function, N - number

4 Discussion

HLA-G is a non-classical HLA-class I molecule that suppress the immune response. The HLA-G molecule inhibits cytotoxic activity of NK cells and cytotoxic T cells, alloproliferative response of CD4+T cells, dendritic cells maturation and activation of regulatory T cells [12]. In relation to these characteristics, a protective effect of HLA-G in human allotransplantation such as kidney, liver, heart and lung has been described [6, 17–19]. As gene polymorphism can influence protein expression level, the association between HLA-G 14 bp ins/del polymorphism in the 3´UTR, soluble HLA-G level and kidney graft acceptance in the Slovak population was analyzed. It was found that the HLA-G 14 bp insertion polymorphism in the 3´UTR is responsible for a decrease in the mRNA level of most membrane and soluble isoforms [15]. The possible impact of the HLA-G 14 bp variant on kidney graft acceptance has been investigated by various authors, but the results are controversial. Findings of HLA-G 14bp genotyping in kidney transplant recipients of various origins are summarized in Table 3. In the present study, 69 kidney graft recipients of Caucasian origin were genotyped. No statistically significant differences were found between the HLA-G 14 bp deletion/insertion allele and genotypes between kidney transplant rejection group and non-rejection group. This observation is in agreement with data reported by others [29, 30]. In the group with non kidney graft rejection higher frequencies of homozygous −14/−14 bp genotype were observed in comparison to recipients with acute rejection, as confirmed by others [21, 22]. A statistically significant increase of the HLA-G 14 bp ins/ins genotype in the acute rejection group, as reported by [21, 22], was not confirmed in this study.

Tab. 3

Comparison of HLA-G 14 bp genotypes frequencies in kidney transplant recipients of various populations

Genotyp	Acute rejection	Stable allograft	P	Population	Reference
	N = 37	N = 32
−14/−14	10 (27.0%)	14 (43.8%)	> 0.05
−14/+14	20 (54.1%)	10 (31.2%)	> 0.05	Slovak Caucasian	Our study
+14/+14	7 (18.9%)	8 (25.0%)	> 0.05
	N = 42	N = 102
−14/−14	7 (16.7%)	35 (34.3%)	0.002
−14/+14	18 (42.9%)	49 (48.0%)	> 0.05	Chinese	[21]
+14/+14	17 (40.4%)	18 (17.7%)	0.004
	N = 35	N = 148
−14/−14	9 (25.7%)	56 (37.8%)	> 0.05
−14/+14	12 (34.3%)	69 (46.6%)	> 0.05	Indian	[22]
+14/+14	14 (40.0%)	23 (15.6%)	0.004
	N = 10	N =53
−14/−14	3 (30.0%)	13 (25.0%)	> 0.05
−14/+14	2 (20.0%)	25 (47.0%)	> 0.05	Brasilian	[29]
+14/+14	5 (50.0%)	15 (28.0%)	> 0.05
	N = 72	N = 72
−14/−14	33 (45.8%)	26 (36.1%)	> 0.05
−14/+14	33 (45.8%)	38 (52.8%)	> 0.05	Iranian	[30]
+14/+14	6 (8.3%)	8 (11.1%)	> 0.05

Genotype frequencies are presented as absolute numbers with percentages in parentheses.

N – number, −14 – HLA-G 14 bp deletion, +14 – HLA-G 14 bp insertion. P<0.05 is statistically significant

Many studies observed that higher serum HLA-G level or HLA-G mRNA expression is associated with kidney graft acceptance [8, 18, 20–23, 26, 31]. The authors have suggested that soluble HLA-G could be used as additional marker to monitor the graft acceptance outcome. In the present study also, significantly higher pre-transplantation levels of sHLA-G were found in the group without rejection in comparison to group with acute rejection. Rebmann et al. [18] showed that pre-transplantation level of sHLA-G < 11.5 ng/ml (sensitivity 60%; specificity 80.4%) is significantly related to rejection. In the present study the pre-transplantation level of sHLA-G < 15.625 ± 9.172 U/ml was significantly related to kidney graft rejection.

The association between the HLA-G 14 bp ins/del polymorphism, soluble HLA-G level and graft acceptance has been analyzed only by few studies to date [22, 32, 33]. Torres et al. [32] and Misra et al. [22] found increased pre-transplantation levels of sHLA-G for 14 bp del/del genotypes in the non-acute kidney and heart graft rejection group as compared to acute rejection recipients. However, data by Waterhouse et al. [33] suggest a weak involvement of 14-bp polymorphism on HLA-G gene expression and sHLA-G concentration in allogeneic hematopoietic cell transplantation outcome. In the present study, significantly higher pre-transplantation values of soluble HLA-G in the homozygous −14/−14 individuals were found in comparison to the homozygous +14/+14 individuals followed by heterozygous −14/+14 individuals. In the group with stable allograft function significantly higher pre-transplantation levels of sHLA-G were detected in the association with 14 bp deletion polymorphism genotype. However, in the group with acute rejection no such HLA-G genotype association was found. It can be hypothesized that the level of HLA-G in these recipients is influenced by other biological factors. It was shown that HLA-G expression can be up-regulated by stressors such as hypoxia, cytokines like IFN-g, TNF, IL-10, TGF-b, immunosuppressive agents or progressive doses of progesterone [34, 35]. In relation to HLA-G expression, the time-course of serum HLA-G in patients after kidney transplantation has been examined by only few studies to date [18, 31]. First, the decrease of pre-transplantation sHLA-G in the first 2 weeks after renal transplantation was observed. In the period between 1–12 months after transplantation, an increase of sHLA-G level was found in patients without rejection while no change was observed in recipients with graft rejection. The explanation of such HLA-G expression is mostly unknown and needs to be elucidated more precisely. It was observed that individual immunosuppressive agents can decrease HLA-G expression; however the sHLA-G levels are still higher in patients without rejection [35].

To conclude, this is the first study examining the association between the HLA-G 14 bp insertion/deletion polymorphism, soluble HLA-G level and kidney graft acceptance in the Slovak Caucasian population. The results have shown that increased soluble HLA-G level is associated with 14 bp deletion polymorphism leading to kidney graft acceptance. Further detailed HLA-G analysis is planned and it is suggested that the pre-transplantation level of sHLA-G should be monitored as an independent soluble marker to predict kidney graft acceptance.

Acknowledgment

This work was supported by the VEGA grant No. 2/0080/15 from the Scientific Grant Agency of Ministry of Education of Slovak Republic and Slovak Academy of Sciences.

Declaration of interest: The authors report no conflict of interests.

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