Volume 24, Issue 5 (Dec - Jan 2021)                   2021, 24(5): 398-409 | Back to browse issues page


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Najafi Z, Khalaj-Kondori M, Hosseinpour Feizi M A, Abbasaliizadeh S. Haplotype Effect of Two Human Leukocyte Antigen-G Polymorphisms of rs1736933 and rs2735022 on the Recurrent Pregnancy Loss. Journal of Inflammatory Diseases. 2021; 24 (5) :398-409
URL: http://journal.qums.ac.ir/article-1-3004-en.html
1- Department of Animal Sciences, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran.
2- Department of Animal Sciences, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran. , khalaj@tabrizu.ac.ir
3- Department of Gynecology &Obstetrics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
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1. Introduction
Recurrent Pregnancy Loss (RPL) is the most common complication of pregnancy and is defined as the occurrence of two or more miscarriages before the 20th weeks of gestation or before the fetus weighing 500 grams [2, 3, 4, 5], which affects about 1%-3% of couples who plan to have children [3]. From an immunological point of view, pregnancy requires the production of inhibitory factors that prevent the rejection of foreign fetal antigens by the maternal immune system [7]. Human Leukocyte Antigen-G (HLA-G), a non-classical HLA class I molecule, is expressed in the cytotrophoblast stem cells at the fetal-maternal interface [8]. This molecule inhibits immune responses and induces the production of inhibitory and regulatory cells [9]. Changes in HLA-G expression have been reported in the tissues of RPL patient [13]. Nucleotide changes in the promoter may affect HLA-G expression level by altering binding affinity that target transcription factors [16]. The rs2735022 (-486 A>C) and rs1736933 (-689 A>G) polymorphisms, located in the HLA-G promoter, may affect its expression [16]. Given that the allele abundance of polymorphisms depends on the genetic background of each population [18, 24, 25], this study aims to evaluate the allele and genotype frequencies of rs2735022 and rs1736933 polymorphisms and analyze their association with RPL in Iranian women.

2. Materials and Methods
This case-control study was conducted during 2018-2019 in northwestern Iran. Peripheral blood samples were taken from control (n=80) and RPL (n=100) groups. DNA was extracted by using Miller’s salting-out method [26] and DNA quantity and quality was determined using a spectrophotometer and an electrophoresis system. Amplification of DNA fragments containing rs2735022 and rs1736933 polymorphisms found in the upstream of HLA-G gene was performed by forward primers (5-GACTCACACGGAAACTTAGG-3) and reverse primers (5-ACACAGGTTAGGAGAAGGAG-3). The Polymerase Chain Reaction (PCR) was initiated at first denaturation for 5 min at 95°C and then amplified in 40 cycles, each containing denaturation at 95°C for 30 seconds, annealing at 58 °C for 30 seconds, and extension at 72°C for 30 seconds. The final extension was performed for 5 minutes at 72°C. The PCR products for sequencing were sent to Microsynth Company in Zurich, Switzerland and the sequences were edited in Chromas 2.6.6 software. Frequencies in both groups were compared by using t-test in SPSS v. 22 software, considering a significance level of P≤ 0.05. The haplotypes were determined using PHASE 2.1 and Haploview 4.2 applications

3. Results
Genotype and allele frequencies of rs2735022 polymorphism 
Comparison of allele and genotype frequencies of rs2735022 between RPL and control groups showed that C allele (OR=1.897) and CC genotype (OR=1.932) were significantly different in the two groups (P<0.05), and they had a positive relationship with the RPL (Table 1).

Genotype and allele frequencies of rs1736933 polymorphism 
Comparison of allele and different genotype frequencies of rs1736933 polymorphism in RPL and control groups showed that G allele (OR=1.948) and GG genotype (OR=1.988) were significantly different in the two groups (P<0.05), and they had a positive correlation with the RPL (Table 2).

Haplotypes of rs2735022 and rs1736933 polymorphisms
Comparing the frequencies of two polymorphisms between the two study groups showed that GC haplotype was associated with RPL. The Linkage Disequilibrium (LD) analysis results showed that the linkage of the studied haplotypes was high (LD =94) for rs2735022 and rs1736933 polymorphisms (Figure 1).
4. Discussion and Conclusion
 The HLA-G promoter shows several peculiarities and the amount of protein produced by HLA-G is affected by several polymorphisms in the HLA-G gene, including polymorphisms rs2735022 and rs1736933 [27]. In our study, a comparison of genotype frequencies between the two study groups in terms of rs2735022 polymorphism showed that the homozygous CC genotype had a positive significant relationship with the RPL (P=0.015). It was also found that the C allele had a positive significant relationship with the RPL (OR=1.897, P=0.019). For rs1736933 polymorphism, results showed that the homozygous GG genotype had a positive significant relationship with the RPL (P=0.012). The G allele also had a positive association with the RPL (OR=1.948, P =0.013). Consistent with our results, Ober et al. observed that not only rs2735022 and rs1736933 polymorphisms are associated with RPL, but also the serum HLA-G level decreases in women with RPL [19]. The main haplotype reported in our study was GC which may be an important factor in RPL in northwestern Iran 

Ethical Considerations
Compliance with ethical guidelines

This study obtained its ethical approval from the Research Ethics Committee of Tabriz University of Medical Sciences (Code: IR-TBZMED.REC.1398.208).

Funding
This study was extracted from the MA. thesis of first author at Department of Genetics, Faculty of Natural Sciences, University of Tabriz, Tabriz.

Authors' contributions
Conceptualization, methodology, resources, initial draft preparation: Zahra Najafi; data analysis, editing & review, final approval, and supervision: Mohammad Khalaj-Kondori; project administration and giving consultation: Mohammad Ali Hosseinpour Feizi and Shamsi Abbasaliizadeh.

Conflict of interest
The authors declare no conflict of interest.

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Type of Study: Research | Subject: Genetics

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