Association study of 3-UTR haplotype of HLA-G gene with Lupus

doi:10.21203/rs.3.rs-3010737/v1

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Association study of 3-UTR haplotype of HLA-G gene with Lupus

https://doi.org/10.21203/rs.3.rs-3010737/v1

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Background

Human leukocyte antigen-G (HLA-G) is a protein that plays a critical role in immune regulation and tolerance. Systemic lupus erythematosus (SLE) is a complex autoimmune disease that results from a combination of genetic and environmental factors. Studies have shown that HLA-G polymorphisms and mutations may contribute to the development of SLE.

Objective

The aim of this study was to investigate the association between polymorphisms in the 3'-UTR region of the HLA-G gene and SLE.

Methods

DNA was extracted from 100 SLE patients and 100 control samples, and PCR was used to amplify the target sequence. The allele and genotype frequencies were calculated, and haplotypes were evaluated using Haploview v.4.2 software, with linkage disequilibrium calculated.

Results

The results showed that the + 2960 Ins allele was significantly associated with SLE as a risk factor, while the Del allele was protective. The + 3010 C allele and + 3187 A allele were also significantly associated with SLE at both the allele and genotype level. The + 3142 GG homozygote was significantly associated with SLE at the genotype level. Haplotype block analysis found that the UTR-2 haplotypes were significantly associated with SLE as a risk factor, while the UTR-1 haplotype was protective. These findings provide valuable insights into the genetic factors contributing to the risk of developing SLE.

Conclusion

The study highlights the significance of polymorphisms in the 3'-UTR region of the HLA-G gene in SLE susceptibility and suggests that these variants may have potential as diagnostic or therapeutic targets.

HLA-G

3-UTR

Haplotype

Polymorphism

SLE

HLA-G is a human leukocyte antigen (HLA) gene that encodes a protein involved in immune regulation. HLA-G is primarily expressed by fetal trophoblasts during pregnancy, where it plays a role in protecting the fetus from the maternal immune system. However, HLA-G expression has also been observed in various types of cancer cells and is thought to contribute to immune evasion by these cells. [1, 2].

Autoimmune diseases, on the other hand, occur when the immune system mistakenly attacks healthy cells and tissues in the body. While HLA-G has been implicated in various immune-related processes, there is currently no evidence to suggest that mutations or dysregulation of HLA-G specifically contribute to autoimmune diseases. Rather, autoimmune diseases are thought to result from a complex interplay of genetic, environmental, and immunological factors. [3–5].

Systemic lupus erythematosus (SLE) is an autoimmune disease that disproportionately affects women compared to men, with a prevalence ratio of approximately 13:1. The development of SLE is influenced by multiple genetic and non-genetic factors. Unfortunately, the 20-year survival rate for SLE patients is only around 53 to 61%. The most common symptoms of SLE include fever, malaise, joint pain, muscle pain, and fatigue, while more severe cases may involve seizures, kidney failure, and skin problems [6–8]. There is evidence to suggest that multiple polymorphisms and mutations of HLA-G are associated with SLE. HLA-G is involved in immune regulation, and alterations in its expression or function could potentially contribute to the dysregulation of the immune system that is observed in SLE. Several studies have investigated the correlation between HLA-G polymorphisms and SLE, and while the results are not entirely consistent across all studies, some have reported significant associations [9, 10].

However, the relationship between HLA-G and SLE is complex and not fully understood. The precise mechanisms by which HLA-G polymorphisms contribute to the development or progression of SLE require further investigation. Additionally, while HLA-G may be a promising target for therapeutic interventions, more research is needed to determine the feasibility and efficacy of such approaches in the context of SLE [11].

This study aims to evaluate the association between HLA-G 3'UTR polymorphism and haplotype with SLE in the northwestern population of Iran.

This case-control study was approved by the Ethics Committee of the Department of Biology at the University of Tabriz (14000907-198-10) and followed ethical guidelines outlined in the 1975 Declaration of Helsinki. Written consent was obtained from all participants prior to their inclusion in the study. DNA was extracted from 100 lupus patients and 100 control samples using the Ziaviz DNA extraction kit, which employs magnetic bead-based technology to isolate DNA and includes reagents and protocols for efficient and high-yield DNA extraction. The kit is based on nanoparticle bead technology and is designed to optimize DNA extraction from various sample types, including blood and tissue. The control samples were likely obtained from individuals without lupus or other autoimmune diseases. The target sequence that includes the polymorphic region was amplified using specific primers (forward primer 5' GTGCTATGAGGTTTCTTTGACTTCAATG-3' and the reverse primer 5'-GTCTTCCATTTATTTTGTCTCT-3'). PCR amplification was performed using PCR Master Mix and a thermal cycler. PCR amplification was performed using 50 µl of PCR Master Mix (Cinaclone-Iran). The amplification protocol consisted of an initial denaturation step at 95°C for 2 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 40 seconds, and a final extension step at 72°C for 2 minutes. The Corbett CGI-96 Palm-Cycler Thermal cycler was used for PCR amplification.

The sequencing results of the PCR products were analyzed using Chromas software. All the SNPs in the 3'-UTR region of the HLA-G gene were genotyped in all study subjects. After SNP genotyping, allele and genotype frequencies were calculated using the Statistical Package for the Social Sciences (SPSS) software. The frequency of these polymorphisms and the combination of these markers as haplotypes were evaluated using Haploview v.4.2 software, with significance determined by p-values less than 0.05. Additionally, linkage disequilibrium between each marker was calculated.

The objective of this study was to examine the potential association between 24 SNPs located in the 3'-UTR region of the HLA-G gene and the risk of developing systemic lupus erythematosus (SLE). The frequencies of alleles and genotypes for eight of these SNPs were presented in Table 1, while the remaining SNPs had low minor allele frequencies and were not found to have any significant association with SLE as either a risk or protective factor.

Table 1

Alleles and genotypes distribution of the 3′-UTR region of HLA-G polymorphisms in SLE patients compared to controls.
HLA-G	Patients n = 100	Controls n = 200	P-value	OR (95%CI)
rs371194629 (+ 2960)
D	99(49.5%)	256(64%)
I	101(51.5%)	144(36%)	0.001**	1.81(1.29–2.56)
DD	29(27%)	84 (42%)	0.029*	0.56(0.34–0.94)
DI	37 (41%)	88 (44%)	0.247	0.75(0.46–1.22)
II	32(30%)	28 (14%)	0.001**	2.89(1.62–5.16)
rs1707 (+ 3003)
T	172(86%)	340(85%)	0.744	1.08(0.67–1.76)
C	28(14%)	60(15%)
TT	77(77%)	144(72%)	0.355	1.30(0.74–2.28)
TC	18(18%)	54(27%)	0.720	0.91(0.53–1.54)
CC	5(5%)	4(2%)	0.734	1.26(0.33–4.85)
rs1710 (+ 3010)
G	66(33%)	174(43.5%)
C	134(67%)	226(56.6%)	0.014*	1.56(1.01–2.23)
GG	12(12%)	42(22%)	0.060	0.51(0.26–1.01)
GC	42(42%)	90(45%)	0.621	0.89(0.54–1.44)
CC	46(46%)	68(34%)	0.044*	1.65(1.01–2.70)
rs17179101 (+ 3027)
C	179(89.5%)	356(89%)	0.853	1.05(0.61–1.83)
A	21(10.5%)	44(11%)
CC	83 (83%)	157(78.5%)	0.094	1.34(0.72–2.49)
CA	13 (13%)	42(21%)	0.253	0.56(0.29–1.10)
AA	4(2%)	1(0.5%)	0.060	8.29(0.91–75.20)
rs17179108 (+ 3035)
C	151(75.5%)	316(79%)
T	49(24.5%)	84(21%)	0.331	1.22(0.82–1.83)
CC	68(68%)	142(71%)	0.593	0.87(0.52–1.46)
CT	15(15%)	32(16%)	0.822	0.93(0.48–1.80)
TT	17(17%)	26(13%)	0.353	1.37(0.71–2.67)
rs1063320 (+ 3142)
G	132(66%)	232(58%)	0.059	1.41(0.99-2.00)
C	68(34%)	168(42%)
GG	49(49%)	73(36.5%)	0.038*	1.67(1.03–2.72)
GC	34(34%)	86(43%)	0.135	0.68(0.41–1.13)
CC	17(17%)	41(20.5%)	0.047	0.79(0.43–1.48)
rs9380142 (+ 3187)
A	169(84.5%)	304(76%)	0.017*	1.72(1.10–2.69)
G	31(15.5%)	96(24%)
AA	72(72%)	120 (60%)	0.042*	1.71(1.02–2.88)
AG	25 (25%)	64(29%)	0.212	0.71(0.41–1.22)
GG	3(3%)	16(8%)	0.107	0.36(0.10–1.26)
rs1610696 (+ 3196)
C	134(67%)	276 (69%)	0.620	1.10(0.76–1.58)
G	66(33%)	124(31%)
CC	53(53%)	104(52%)	0.870	1.04(0.64–1.68)
CG	28(28%)	68(34%)	0.294	0.75(0.45–1.28)
GG	19(7%)	28(14%)	0.263	1.44(0.76–2.73)

the prevalence of the + 2960 Ins allele was found to be significantly higher (p < 0.001) in SLE patients compared to controls. The Ins/Ins genotype was also found to be significantly more common (p < 0.001), while the Del/Del genotype was less prevalent (p = 0.029) in patients, indicating that the Ins allele may act as a risk factor and the Del allele may offer protection against SLE within this population.

Our results indicate that not only were the + 3010 C allele (p = 0.014) and + 3187 A allele (p = 0.017) significantly associated with the SLE patient group, but these associations were also significant at the genotype level, specifically in individuals with the + 3010CC (p = 0.044) and + 3187AA (p = 0.042) genotypes.

Regarding the + 3142 polymorphism, the results of this study show that although there is a higher frequency of the G allele, this difference was not statistically significant. Nevertheless, at the genotype level, the + 3142 GG homozygote was found to be significantly associated with SLE (p = 0.038).

The remaining SNPs had low minor allele frequencies and showed no significant association with SLE as either a risk or protective factor. A haplotype block consisting of the 24 SNPs was constructed using Haploview v.4.2 software, and a linkage disequilibrium plot was generated for all 24 SNPs (Fig. 1) and for a subset of nine selected SNPs (Fig. 2).

The findings presented in Table 2 indicate that the UTR-2 haplotypes were significantly associated with SLE, as evidenced by their p-values (p = 0.021) and OR (95%CI) [1.68(1.08–2.61)]. Conversely, the UTR-1 haplotype was found to be a protective factor for SLE, with a significant association (p = 0.016) and an OR (95%CI) of [0.57(0.37–0.90)].

Table 2

The HLA-G 3′-UTR haplotypes frequency in breast cancer and healthy control samples
dbSNP	rs371194629	rs1707	rs1710	rs17179101	rs17179108	rs1063320	rs9380142	rs1610696	SLE 2n = 200	Healthy sample 2n = 400	p-value	OR (95%CI)
HLA-G position	2960	3003	3010	3027	3035	3142	3187	3196
UTR-1	Del	T	G	C	C	C	G	C	0.150	0.235	0.016*	0.57(0.37–0.90)
UTR-2	Ins	T	C	C	C	G	A	G	0.215	0.140	0.021*	1.68(1.08–2.61)
UTR-3	Del	T	C	C	C	G	A	C	0.135	0.160	0.422	0.82(0.50–1.33)
UTR-4	Del	C	G	C	C	C	A	C	0.115	0.125	0.724	0.91(0.54–1.54)
UTR-5	Ins	T	C	C	T	G	A	C	0.055	0.040	0.405	1.40(0.64–3.07)
UTR-6	Del	T	G	C	C	C	A	C	0.035	0.030	0.742	1.17(0.45–3.03)
UTR-7	Ins	T	C	A	T	G	A	C	0.095	0.070	0.284	1.39(0.76–2.56)
UTR-10	Del	T	C	C	C	G	A	G	0.040	0.040	1	1(0.42–2.38)
UTR-15	Ins	T	C	C	C	G	A	C	0.020	0.010	0.324	2.02(0.50–8.16)
UTR-18	Del	T	G	C	C	C	A	C	0.015	0.015	1	1(0.25–4.04)
Others									0.125	0.135

This study is the first to investigate the potential association between 24 SNPs and haplotypes in the 3' untranslated region (UTR) of the HLA-G gene and the risk of developing systemic lupus erythematosus (SLE) in the population of northwestern Iran. By identifying genetic markers associated with SLE, the study aims to contribute to a better understanding of the disease and potentially develop new diagnostic or therapeutic approaches.

The rs371194629 polymorphism affects HLA-G mRNA expression and has been studied for its association with systemic lupus erythematosus (SLE), an autoimmune disease. However, the results of these studies have been inconsistent, with some finding a significant association and others finding no significant association [12–17].

While the majority of reports, including the current study, suggest an association between the Ins allele and Ins/Ins genotype with SLE, inconsistent results across some studies may be attributed to differences in study populations, sample sizes, and other factors. Further research is needed to fully understand the role of this polymorphism in the development and progression of SLE and to determine whether it could be a potential target for future therapies [13–17]. Despite the inconsistencies, the investigation of this polymorphism remains important in our understanding of the genetic factors contributing to SLE susceptibility. Further studies may provide greater clarity on this association and its potential implications in the development of new diagnostic or therapeutic approaches for SLE.

The rs1710 polymorphism, another SNP located in the 3' UTR of the HLA-G gene, has also been investigated for its association with SLE susceptibility, but the results have been conflicting. These findings suggest that the association between the rs1710 polymorphism and SLE susceptibility may vary by population, highlighting the importance of considering population-specific genetic differences in SLE susceptibility. Further studies may provide greater clarity on the potential implications of this polymorphism in the development of new diagnostic or therapeutic approaches for SLE [16–19].

Recent studies have implicated several microRNAs in the association between the rs1710 polymorphism and SLE susceptibility. rs1710 polymorphism affected the binding of microRNA-148a and microRNA-148b to the 3' UTR of the HLA-G gene, potentially contributing to the development of SLE [20]. However, the specific mechanisms underlying the interaction between microRNAs, the rs1710 polymorphism, and HLA-G expression in SLE remain unclear and require further investigation.

Another SNP investigated for its potential association with SLE susceptibility was rs1063320 (+ 3142), located in the 3' UTR of the HLA-G gene. Although some studies have suggested a potential association between GG genotype and SLE susceptibility [18, 21].

Apart from the single nucleotide polymorphisms, the association between haplotypes of polymorphisms in the UTR-1 and UTR-2 regions of the HLA-G gene and SLE susceptibility also remains controversial [13–16]. While some studies have reported an association between specific haplotypes in these regions and increased SLE susceptibility, others have found no significant association.

Overall, further research is needed to clarify the potential role of genetic variants, including SNPs and haplotypes in the 3' UTR of the HLA-G gene, in SLE susceptibility. The inconsistent results across studies may be attributed to differences in study populations, sample sizes, and other factors, and more rigorous studies with standardized genotyping methods and larger sample sizes are needed to better understand the potential role of these genetic variants in SLE susceptibility. It is also worth noting that the 3' UTR of the HLA-G gene is involved in post-transcriptional regulation of HLA-G expression, and genetic variants in these regions may affect HLA-G expression levels, which could potentially influence SLE susceptibility[15–22].

In conclusion, while some studies suggest a potential association between certain genetic variants in the 3' UTR of the HLA-G gene and SLE susceptibility, the results have been inconsistent across different populations. Further research is needed to better understand the potential role of these genetic variants in the pathogenesis of SLE and to develop novel diagnostic and therapeutic strategies for this complex autoimmune disease.

Ethics approval and consent to participate: This study was approved by the Department of Biology committee at the University of Tabriz (14000907-198-10). All stages of sample collection have been done with the consent of patients and healthy people, and written consent forms have been obtained.

Competing interests: The authors confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome.
Authors' contributions: IA.C. Experimented and wrote the manuscript with support from M.H and MA.HF. M.H. supervised the work and contributed to the design and implementation of the research, the analysis of the results, and the writing of the manuscript. M.KK. and MA.HF. Helped supervise the project and contributed to data analysis. MR.A. Participated in clinical diagnoses and identification and collection of samples. All authors discussed the results and contributed and reviewed the final manuscript.

Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Availability of data and materials: All data analyzed during this study are included in this published article. Further raw data from the current study are available from the corresponding author upon request.

Acknowledgments: We thank all patients and individuals for participating in this project.

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Association study of 3-UTR haplotype of HLA-G gene with Lupus

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Abstract

Background

Objective

Methods

Results

Conclusion

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Introduction

Methods and materials

Results

Discussion

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References

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