Studies On Gene Mutation Screening Of WNT6 And Gene Polymorphism Of KIR2Dl4 In Unexplained Recurrent Miscarriage

Background Recurrent miscarriage (RM) is referred to women who have three or more consecutive pregnancy losses with the same partner, no matter if they have a live birth before. The incidence of RM is about 1% to 3%. The pathogenesis is very complex. There still have 50% of the patients with unknown reasons. Some known reasons have been proved, such as genetic disorders, uterine pathologies, endocrine abnormalities, autoimmune dysfunction, acquired and inherited thrombophilia as well as infectious diseases. Chromosomal abnormalities were used to be thought of the most important reason for recurrent miscarriage, but by now, only abnormal structure and numerical aberration of chromosomes can be diagnosed in RM in clinic. However, the molecular level of chromosomal abnormality is still in the research stage. The level of molecular genetics research of unexplained RM (URM) were mainly concentrated in cytokines, angiogenic mediators, and the polymorphism of hormones gene. And most of these conclusions were not consistent.Recent researches focused on endometrial decidualization in URM. The endometrial decidualization is crucial in the process of establishing and maintaining of normal pregnancy, which involved in stromal cell proliferation, decidualization and apoptosis. Some researches have shown that Wnt proteins involved in the process of endometrial decidualization and played an important role in it. Wnt (wingless-type MMTV integration site family) gene families, with highly conserved gene sequnences in some species, were studied in the regulation of organ development in embryogenesis and controls of mulllerian duct development in women. At present, some studies showed that Wnt family secreted Wnt proteins through Wnt-β-catenin signaling pathway, and participated in the process of endometrial decidualization. If the inactivation of Wnt-(3-catenin signaling pathway can limit the blastocyst implantation and development. In the latest research, Wnt6 was proved that it played an essential role in the process of endometrial decidualization and proliferation in mice. It also found that endometrial stromal cell significantly slowed down the growth cycle in Wnt6- mice. The result explained that WNT6 was very important in stromal decidualization, also gave a hint that WNT6 may be associated with recurrent miscarriage. The relationship between the URM and WNT6 gene in humans has not been reported yet. In the present study, the purpose is aimed to screen mutations of WNT6 gene in patients with unexplained RM in Han population.On the other hand, reproductive immune balance of maternal-fetal interface is a hotspot in the study of RM. Many researches have shown that the tolerance of mother to fetus is disappeared when mother has imbalance of immune system, and it could damage the functions of NK cells and leaded to miscarriage. KIR2DL4 is belonged to KIRs family, which is one of the NK receptors. Its structure is complex that has dual characteristics which include activated function and inhibited function. Recent studies have indicated that KIR2DL4 expressed on the surface of all NK cells, especially was rich in endometrial decidual cells in the early pregnancy. These fetures and functional studies of KIR2DL4 pointed out that KIR2DL4 could play a role in the pathogenesis of spontaneous miscarriage. It is also reported that KIR2DL4 gene polymorphisms were closely related to the function of NK cells activities. Studies on the genotype frequency in transmembrane regions of KIR2DL4 (intron6 and exon 7) in RM showed that is significantly different between control group and case group. And studies have also shown that 10 A in exon 7 (10 consecutive adenine in transmembrane area) encodes the full length of KIR2DL4, but when it changed into 9A, it will produce a termination code ahead of the completion of translation, at the same time, the study found that KIR2DL4 expression can be detected in the surface of a small amount of CD56Bright NK cells in at least one 10 A allele existed in the subjects. In vitro, studies confirmed that at least one 10 A allele can activate NK cells, and a 9 A homozygous type can not activate NK cells. So far, the association between KIR2DL4 gene polymorphism and unexplained RM has not been reported yet in Han populaton, especially the 10A/9A polymorphic site. It is worth further studying in the present study.The past genetic researches were more inclined to focus on the female patients with unexplained RM, ignored the male partner. And on the other hand, due to the lower incidence of RM, case collections are very difficult. So the number of the cases is usually small. In this study, we chose the qualified clinical samples strictly from the sample bank of Reproductive hospital affiliated to Shandong university, which can enlarged the numbers of cases. And at the meanwhile, both partners screened gene mutations and gene polymorphism to evaluate the association between KIR2DL4 and WNT6 genes and unexplained RM.Part ⅠGene mutations screening in WNT6 coding sequneces in couples with unexplained RMObjective To explore the connections between WNT6 mutations and unexplained RM in Chinese Han population.Methods A total number of 100 Chinese couples with unexplained RM were screened by using PCR amplification and Sanger sequencing technology, and 100 Chinese couples with normal fertilities were as controls. Four coding exons and related gene sequences of WNT6 were detected. And compared with a normal sequence alignment that Ensembl provided, sequences were analysed, which to find out whether there were abnormal changes in the bases, such as variation, insertion or deletion. If novel mutations are found, and subsequently, the control numbers will be expanded to verify if the mutations existed only in the case group furtherly. we predicted gene mutations function by using different online softwares.Results Four novel mutatios in WNT6 were found in different female samples respectively:one was located in the border of intron1 and exon 1, which is nonsense; two in exon 3, which one is a missense mutation and the other is a synonymous mutations; one in 3’UTR region, which the meaning of this mutation was unknown. Males in RM group and the couples in control group cannot find the same mutations. The missense mutation lies in c.443 T>G. According to protein sequence alignment, the sequences of mutation are highly conserved in mammals, and it also lies in the function domain area of WNT6. To further validate the findings, we continued to screen this missense mutation in another 100 normal fertile female blood samples. There was still no positive result. There were no findings in any of the paternal or control group samples.Conclusions There is a possible connection between the missense mutation in WNT6 and unexplained RM, but further studies are warranted to investigate the role of WNT6 gene in uRM.Part ⅡThe correlation research on KIR2DL4 gene polymorphism in unexplained RMObjective The purpose of this study is to detect the KIR2DL4 gene polymorphism in 96 Chinese couples with unexplained RM to explore the connection between KIR2DL4 gene polymorphism and unexplained RM.Methods Exon 3, exon 5, exon 7, exon 8 and exon 9 of KIR2DL4 gene were considered as two fragments to carry out the PCR amplification by using two pairs of primers. And four pairs of primers were used to sequence the five exons. The frequency of gene polymorphism in the total population were calculated, and allele frequencies of 9 A or 10 A were analysed between the case group and control group.Results The sequencing results showed that genotype distributions of KIR2DL4 were consisted with the genotype distribution reproted in Chinese Han population. About 10 A/9 A polymorphic loci, its genotype frequency in the study population was no statistical difference compared with controls. The allele frequency of 9A or 10A in the study population is no difference with allele frequency been reported in Chinese han population; the allele genotype frequency in couples or in males has no significant statistical difference. The 9 A allele frequency in females in unexplained RM (19.3%) is higher than in control group (14.9%), but there is no statistic significance.Conclusions KIR2DL4 gene polymorphism has no correlation with unexplained RM. Although the 9A allele frequency tends to be at a higher level in unexplained RM, it still need to expand the sample numbers to validate its meanings furtherly.