Molecular studies of trophoblast HLA‐G: polymorphism, isoforms, imprinting and expression in preimplantation embryo

Abstract

There is considerable interest in human HLA-G arising from the observation that it is expressed selectively on the surface of extravillous trophoblast, the fetal cell population directly in contact with the mother. We investigated several aspects of the molecular biology of this unusual molecule. Limited polymorphism at the nucleotide level, and even more restricted variation at the amino acid level, was found in our Caucasian population. A further unusual aspect of HLA-G is the occurrence of alternatively spliced mRNAs. Spliced messages that could give rise to either membrane-bound or soluble proteins have been reported and six of these alternative forms were detected in all first trimester and term placentae, highly purified villous and extravillous trophoblast and the cell lines, JEG-3 and 221-G. An additional novel splice variant involving loss of part of the 3'-untranslated region was observed with two alleles. Using a sensitive RNase protection assay higher levels of the membrane-bound RNAs as compared to the soluble forms were detected in first trimester and term placentae as well as in JEG-3. Contrary to previous findings our term samples taken from the maternal aspect showed higher levels of both mRNA species when compared to first trimester placenta. The question of imprinting was addressed through the detection of heterozygotes both in placental tissue and, more tellingly, in the purified trophoblast cells. There was no evidence of imprinting. In addition we did not find mRNA for HLA-G in human two to eight-cell embryos or in blastocyst or in sperm samples.

Differential Expression of Alternatively Spliced Transcripts of HLA-G in Human Preimplantation Embryos and Inner Cell Masses

“…Surprisingly, no data on the proportions of embryos expressing HLA-G in each group was given, so comparison with our study is not possible. In another report, Hiby et al (16) found no HLA-G mRNA in 11 preimplantation embryos ranging from the 2 cell to the blastocyst stage using nested primers for full-length HLA-G. Their methodology differed from ours in that they isolated RNA from zona intact embryos rather than removing the zona first and used standard phenol-chloroform extraction instead of the magnetic bead method, both of which may affect the yields of RNA. The primers used in both the studies of Jurisicova and Hiby would have amplified all the different isoforms, giving the total HLA-G mRNA expression, but the hemi-nested RT-PCR system used by Jurisicova et al (4,5) was probably less sensitive than our specific nested RT-PCR.…”

HLA-G gene repression is reversed by demethylation

“…Duplex PCR was carried out for 40 amplification rounds in the presence of TaqMan Universal PCR Master Mix, using the predeveloped TaqMan assay reagent GAPDH as an endogenous control [probe with VIC reporter and 6-carboxytetramethylrhodamine (TAMRA) quencher (Applied Biosystems)], HLA-G-specific probe located in exon 5 [200 nM; Applied Biosystems: 5Ј-CACTGGAGCTG-CGGTCGCTGCT; 6-carboxyfluorescein (FAM) reporter and TAMRA quencher] and HLA-G-specific primers [300 nM (Qbiogene, Illkirch, France): forward 5Ј-CTGGTTGTCCTTG-CAGCTGTAG; reverse 5Ј-CCTTTTCAATCTGAGCTCT-TCTTTCT] we designed, using PRIMER EXPRESS software. The primers were selected to amplify all alternative forms of HLA-G transcripts, including those deleted in the 3ЈUTR because of the presence of a 14-bp polymorphism (57). The specificity of HLA-G cDNA amplifications was assessed on various cell lines that did not express classical and nonclassical HLA class I genes and that did or did not express HLA-class II genes (not shown).…”

HLA-G polymorphisms in women with squamous intraepithelial lesions harboring human papillomavirus

“…19 In addition, a 14 bp insertion/deletion polymorphism has been reported in the 3 0 -untranslated region (UTR) of exon 8. 20 HLA-G alleles exhibiting the 14 bp insertion ( þ 14 bp) undergo an alternative splicing that removes 92 bases from 3 0 UTR, 21 influencing the stability of HLA-G mRNA. 22 Although removal of the 92 bases produces a more stable mRNA, the presence of þ 14 bp has been associated with a lower mRNA production.…”