THE IMPACT OF 14-BP INSERTION/DELETION POLYMORPHISM IN EXON 8 OF HLA-G AND ITS EXPRESSION BY REAL TIME PCR AND IMMUNOHISTOCHEMICAL METHODS IN UNEXPLAINED MISCARRIAGE IRAQI WOMEN

Reema M. Abed, Hasan F. Al-Azzawie* and Kareem Al-Kazazz

ABSTRACT

To study the association of the 14-bp insertion/deletion (INDEL) polymorphism with the risk of RSA, PCR amplification was used Insertion or deletion of the 14-bp sequence in HLA-G generated PCR products of length 224 or 210 bp, respectively were observed and the three different genotypes (+14 bp⁄+14 bp, +14 bp⁄−14 bp, and −14 bp/−14 bp) were distinguishable by 3% agarose gel electrophoresis. Our results showed that the frequencies of the homozygous genotypes (+14 bp/+14 bp) were not observed in women with recurrent abortion. However, the frequency of homozygous genotypes (-14 bp/-14 bp) was significantly increased in women with RSA compared with the normal fertile control. There were a significant differences in allele frequencies of polymorphism between controls and URSA women (OR=1.9259, 95%CI=1.1269-2.3309 and P=0.0179). Real time PCR technique was evaluated in this work, using Smart Cycler system, to detect 14bp insertion/deletion polymorphism in exon 8 of 3́untranslated region of the HLA-G gene. The results showed the accumulation of PCR product was monitored by measuring the level of fluorescence. The results revealed there is no progress of PCR with extracted DNA as the template and the PCR product was not observed when detecting 14bp deletion in patients with RSA, indicating that when 14bp was deleted from DNA of HLA-G will cause defect in HLA-G protein and this lead to women’s abortion. Results of Immunohistochemical experiment was observed that HLA-G expression intensity was decreased in the outer layer of trophoblast cells in the placenta tissue in the first trimester of women with recurrent spontaneous abortion comparing with control based on the 4H84 monoclonal antibody (mAb) against HLA-G protein. This was suggesting that RSA is associated with a lack of expression of HLA-G protein by trophoblast cells, so remains possible that the HLA-G protein expressed by trophoblast cells in women with RSA may be functionally defective.

Keywords: To study Results of Immunohistochemical trophoblast defective.