Gilbert Semana

The HLA-G gene is expressed at a low mRNA level in different human cells and tissues

Recently, HLA-G transgenic mice were shown to exhibit transgene transcription in several extraembryonic tissues. To determine whether HLA-G mRNAs are also expressed in other human tissues, we have undertaken Northern blot and RT-PCR assays using HLA-G locus-specific probe and primers. These studies demonstrate that the HLA-G gene is transcribed in a variety of cells and adult tissues obtained from different individuals (peripheral blood leukocytes, placenta, skin, spleen, thymus, prostate, testicle, ovary, small intestine, colon, heart, brain, lung, liver, and kidney), as well as in fetal tissues (heart, lung, liver, and kidney). The HLA-G mRNA level observed in most tissues is orders of magnitude lower than the level of classic class I genes in the same tissues. RT-PCR studies have demonstrated that alternative splicing of the HLA-G primary transcript is different from tissue to tissue and could be regulated in a tissue-specific fashion. Sequencing of keratinocyte transcripts has confirmed previous observations: (a) three different alternative splicing transcripts are produced (a full-length transcript, an mRNA lacking exon 3, and a transcript devoid of exon 3 and 4) and (b) HLA-G polymorphism is limited in the coding regions. In view of this wide HLA-G tissue distribution, a new hypothesis dealing with possible HLA-G function is proposed.

HLA-E is the only class I gene that escapes CpG methylation and is transcriptionally active in the trophoblast-derived human cell line JAR

Polymorphic as well as HLA-F and -G genes are repressed in the human cell line JAR, derived from a tumor of trophoblast origin. By contrast, the HLA-E gene as well as the non-HLA novel coding-sequence, R1, located 5′ to HLA-E, both remain transcriptionally active. We first demonstrated the role of DNA methylation in the repression of class I genes (except HLA-E) in JAR by the use of the 5-Azacytidine demethylating agent. Following treatment, JAR clones reexpressed polymorphic class I transcripts and cell surface α chains. Using methylation-sensitive rare cutter enzymes on JAR genomic DNA, followed by classical or pulse field gel electrophesis and hybridization with HLA locus-specific probes, we found methylated CpG islands in the 5′ region of all class I genes, except for HLA-E. These results, establishing an inverse relationship between states of methylation and transcriptional activity within the MHC class I chromosomal region in JAR, and the observations that the HLA-E and R1 genes were ubiquitously expressed, suggest that the HLA-E chromosomal domain might have functional importance including the presence of housekeeping genes.

TAP2 GENE POLYMORPHISM CONTRIBUTES TO GENETIC SUSCEPTIBILITY TO MULTIPLE SCLEROSIS

MS is an autoimmune demyelinating disease that has been known to be associated with the HLA-DRB1*1501-DQA1*0102-DQB1*0602 haplotype. TAP1 and TAP2, two genes encoded within the MHC class II region between HLA-DP and -DQ loci, display genetic variability and are involved in the transport of antigenic peptides from the cytoplasm to the endoplasmic reticulum. Comparison of 116 MS patients with Caucasoid controls did not reveal any significant correlation between the previously described alleles of the TAP1 and TAP2 genes and MS. We report here an additional TAP2 dimorphism at codon 386, called I and J, corresponding to a silent mutation. An increased frequency of the J variant was observed in the patient population. The J mutation was not found in linkage disequilibrium with the HLA-DRB1*1501 allele and can be considered an additional genetic susceptibility marker of the disease.

Evidence for a polymorphism of HLA-G gene

HLA-G gene polymorphism was analyzed by RFLP using seven restriction enzymes and an HLA-G locus-specific probe. Hybridization of 55 DNAs digested with three enzymes (Taq I, Pst I, and Bgl II) revealed two polymorphic bands in each case. RFLP patterns obtained with Taq I and Pst I corresponded to the same allelic polymorphism and differed from the Bgl II polymorphism. Combining both polymorphisms enabled determination of four alleles. Allelic frequencies were calculated: 40% of the subjects tested had allele 1, 36% had allele 2, 22% had allele 3, and 2% had allele 4. Analyzing the complete HLA class I phenotype revealed strong linkage disequilibrium with the HLA-A locus. The polymorphism described is located in the 3 flanking region of the gene. Moreover, extended HLA-A haplotypes were constructed by combining the HLA-G polymorphism with other class-I-sequence polymorphisms.

DISTRIBUTION OF HLA-G ALTERNATIVE mRNAs INCLUDING SOLUBLE FORMS IN NORMAL LYMPHOCYTES AND IN LYMPHOID CELL-DERIVED LEUKEMIA

The non‐classical HLA‐G gene is the only class I antigen expressed in trophoblasts at the maternofetal interface. In placenta, the HLA‐G gene produces several alternatively spliced isoforms encoding bound‐membrane proteins (G1, G2, G3 and G4) lacking, respectively, exon 7; exons 7 and 3; exons 7, 3 and 4, and exons 7 and 4. In addition, two isoforms (G1s and G2s) containing an intron 4 sequence are able to encode soluble antigens. We have recently reported that the HLA‐G gene is transcriptionally active in lymphocytes and is not transcribed in CD34+ cells, polynuclear cells or monocytes. To investigate the functional significance of the different isoforms in lymphocytes, we studied their distribution in normal T and B lymphocytes and in malignant lymphoid cells by using the RT‐PCR technique followed by hybridization with exon‐specific probes and sequencing assays. In transcriptionally active lymphocytes, the HLA‐G primary transcript is the major form and is differentially spliced in B and T lymphocytes: (i) G1s is found in several samples of T and B cells whereas G2s is only transcribed in T lymphocytes, (ii) the G4 isoform is never detected in B lymphocytes. In addition, we have shown that HLA‐G is inactive in some samples of lymphocytes. Our data suggest that HLA‐G transcription is regulated at the initiation level and at the subsequent splicing. These two levels of regulation may be dysregulated in some cases of T‐ALL and CLL. The potential functions of the HLA‐G alternative forms in lymphocytes, such as peptide binding and modulation of the immune response, are discussed.

Performance Characteristics of Updated INNO-LiPA Assays for Molecular Typing of Human Leukocyte Antigen A (HLA-A), HLA-B, and HLA-DQB1 Alleles

ABSTRACT We carried out a multicenter performance evaluation of three new DNA-based human leukocyte antigen (HLA) typing assays: INNO-LiPA HLA-A Update, INNO-LiPA HLA-B Update, and INNO-LiPA HLA-DQB1 Update. After optimization, the accuracy rates were all 100%, and the final observed resolutions were 99.4, 92.4, and 85.6%, respectively. These rapid and easy-to-perform assays yielded results fully concordant with other DNA-based tissue typing tests.

Implication of the HLA-DRB3 gene in graves' disease: Predominance of Allele Dw24

Using RFLP, the present study sets off to determine the MHC class II gene polymorphism in Graves disease, in order to define the HLA-related genetic susceptibility. Considering the preferential link between Graves disease and the HLA-DR3 antigen, 42 HLA-DR3 Graves disease patients were studied and compared with 42 HLA-DR-matched controls. Hybridization with a DQ alpha probe of DNAs digested by Taq I revealed a polymorphism of the DR3 haplotype with an overrepresentation of a 2.1 kb(U) fragment in patients, but this was merely a sign of the linkage disequilibrium between U and B8DR3. Hybridization with the DR beta probe of DNAs digested by Taq I yielded more facts. It revealed the overrepresentation of the Dw24 specificity (Taq I:9.8 kb) in DR3 Graves disease patients. This study thus enabled us to determine precisely the susceptibility linked to the DR3 haplotype, implicating the DRB3 gene and its Dw24 allele, which appear to be the most reliable markers of the disease, providing a higher relative risk than B8DR3.

Genetic and environmental factors in Systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a multifaceted autoimmune disorder (Shoenfeld and Isenberg 1989). Its diagnosis relies on a set of criteria established by the American College of Rheumatology (Tan et al 1982). There is compelling evidence that the genetic background plays an important role, not only in the susceptibility to the disease, but also in its clinical and serological expression (Arnett and Reveille 1992). The first association of SLE with a genetic trait was reported in 1971 with the HLA class I allele B8 (Grumet et al 1971). Subsequently a large number of family and population studies have confirmed this finding. However it is also known that environmental factors play an important role.