HLA-G and HLA-E Gene Polymorphism in Patients with Idiopathic Recurrent Pregnancy Loss in Gaza strip

Abstract

The causes of recurrent spontaneous abortion (RSA) remain unknown in the large proportion of cases. Physiologically, pregnancy is a condition characterized by persistent local tolerance of the maternal immune system to the paternal HLA antigens expressed by the fetus. This induction of maternal local tolerance to the fetus during pregnancy has provoked the reproductive immunologists to find out the causes of this tolerance. For more than a decade, investigators have known that membrane-bound and soluble isoforms of the HLA class Ib molecule, HLA-G and HLA-E, are present at the maternal–fetal interface. Although it is clear that extravillous cytotrophoblast cells are major producers, other cells may also contribute. Recent findings regarding aspects of HLA-G and HLA-E polymorphism, the possible significance of this polymorphism in respect to HLA-G and HLA-E function and certain complications of pregnancy such as pre-eclampsia and recurrent spontaneous abortions are discussed. The results, however, were contradictory. Objective: This study is conducted to determine the association between three HLA-G alleles ( namely, HLA-G*0103, HLA-G*0104 and HLA-G*0105N) and two HLA-E alleles (HLA-E*0101, HLA-E*0103) frequency and genotypes along with linkage disequilibrium between mentioned HLA-G and HLA-E alleles and idiopathic RPL in Palestinian women residing in Gaza strip. Method: This study is an association study with a case- control design. The study population consisted of 40 women who suffered from unexplained RPL, and 40 non pregnant healthy women matched for age and without previous history or RPL. blood sample collection were carried out during the period March 2012 to July 2012.blood sample collected from each subject, DNA was extracted from whole blood sample. The PCR product digested by RFLP method where they digested using specific restriction enzymes then separated electrophically using 3% agarose gel. Exon 3 of HLA-G gene amplified using specific primers then digested by PpuM-I and BseR-I restriction enzymes, in order to detect HLA-G*0105N and HLA-G*0104 alleles respectively. While exon 2 of HLA-G gene amplified using specific primers then digested with Hinf-I restriction enzyme to detect HLA-G*0103 allele. Exon 3 of HLA-E gene amplified with specific primers then digested with HpaII restriction enzyme for detection of HLA-E*0101 and HLA-E*0103 alleles. Genotypes for the mentioned alleles determined and tested for their association with RPL patients. Results: None of the HLA-G alleles showed significant association with increased risk of RPL patients. HLA-G*0105N and HLA-G*0104 was present in almost similar frequencies in RPL patients and fertile controls, without significant difference between them ( ORs: 2.0323, 1.7576 respectively ). HLA-G*0103 allele was absent in our study. Aforementioned alleles genotyped, and two genotypes were encountered in both groups (HLA-G*0105N/0105N and G*0104/0104) whereas the third genotype (HLA-G*0105N/0104) was absent in the control group. HLA-G*0105N/0104 genotype was interesting as it was evident in 4 patients (100%) whereas none of the fertile controls showed this genotype. Other genotypes presented in both RPL patients and fertile controls. HLA-G*0105N/0105N genotype showed similar frequency (50%) with odds ratio (1) in both groups, 52.2% of the patients revealed HLA-G*0104/ 0104 genotype whereas 47.8% of the control group harbored this genotype with odds ratio 1.1298. For HLA-E alleles also none of them showed significant association with increased risk of RPL. Odds ratios was 1.1481 , 0.8709 respectively for HLA-E* 0101 and HLA-E*0103 alleles. Genotyping of these alleles was absolutely unexpected where all participants showed increased homozygosity, but the results did not reach significant difference between RPL patients and fertile controls. Anew attempt for Linkage disequilibrium between examined HLA-G and HLA-E alleles. Results showed two haplotypes (HLA-G*0104:0105N/E*0101:0101, G*0104:0105N/ E*0103:0103) present only in RPL patients (100%) (OR= ∞ ). Conclusion: Even that examined alleles were not associated with increased risk of RPL, did not exclude the hypothesis of HLA-G or HLA-E association with RPL. The study showed possible association of HLA-G*0104:0105N genotype along with two haplotypes (HLA-G*0104:0105N/E*0101:0101, G*0104:0105N/ E*0103:0103), which may determine a new insight to the role of these alleles together in RPL increased risk. Also linkage disequilibrium of these alleles with other alleles or other genes may determine another subset of patients, which need more investigation. Moreover another polymorphisms along the HLA-G gene may have stronger association with increased risk of RPL.