Marta Zajacova1, Anna Kotrbova-Kozak2, Pavel Cepek3, Marie Cerna4. 1. Department of General Biology and Genetics, Third Faculty of Medicine, Charles University in Prague, Ruska 87, 100 00 Prague, Czech Republic. Electronic address: marta.zajac17@gmail.com. 2. Department of General Biology and Genetics, Third Faculty of Medicine, Charles University in Prague, Ruska 87, 100 00 Prague, Czech Republic. Electronic address: anna.kotrbova@lf3.cuni.cz. 3. Department of General Biology and Genetics, Third Faculty of Medicine, Charles University in Prague, Ruska 87, 100 00 Prague, Czech Republic. Electronic address: pa.cepek9@gmail.com. 4. Department of General Biology and Genetics, Third Faculty of Medicine, Charles University in Prague, Ruska 87, 100 00 Prague, Czech Republic. Electronic address: marie.cerna@lf3.cuni.cz.
Abstract
OBJECTIVES: Extensive polymorphism of HLA class II genes is not restricted to the coding region of the gene. It extends also to the linked promoter region, where it forms the basis for different levels of individual allele's expression. Differential expression of HLA class II alleles can shape an immune response and influence the risk of developing autoimmune disease. In addition to genetic variability, variation in epigenetic modifications, including DNA methylation, can be another cause of the uneven expression of individual alleles. We aimed to analyze the DNA methylation of promoter sequences and the levels of expression of individual DQA1 gene alleles, interallelic variation of these two characteristics and the relationship between them. METHODS: The 60 healthy donors included into study were HLA-DRB1, HLA-DQB1 and HLA-DQA1 genotyped using PCR-SSP. Genomic DNA was treated by sodium bisulfite and the target segment in the HLA-DQA1 gene promoter was PCR amplified. PCR product was cloned into Escherichia coli and individual clones were sequenced. Transcripts of individual DQA1 alleles in peripheral blood leukocytes were quantified by Real-Time PCR. RESULTS: In this study, we have described detailed DNA methylation profile of promoter area of DQA1 gene alleles. The overall promoter methylation is increased for DQA1*02:01 and DQA1*04:01 alleles, on the other side, DQA1*05:01 allele shows decreased methylation level. Our results suggest that there are only minor interindividual differences in DRA-normalized expression level of specific allele. Furthermore, expression levels of individual alleles followed DQA1*03>*01:03 (in DRB1*13-DQA1*01:03-DQB1*06:03 haplotype)>*01:01,*05:05, and DQA1*03>*02:01>*05:05 hierarchy. The statistically significantly most expressed allele, DQA1*03, comprises part of DQ8 molecule, which is commonly linked to autoimmune diseases. A clear relationship between promoter DNA methylation and mRNA expression level of the DQA1 gene could not be identified.
OBJECTIVES: Extensive polymorphism of HLA class II genes is not restricted to the coding region of the gene. It extends also to the linked promoter region, where it forms the basis for different levels of individual allele's expression. Differential expression of HLA class II alleles can shape an immune response and influence the risk of developing autoimmune disease. In addition to genetic variability, variation in epigenetic modifications, including DNA methylation, can be another cause of the uneven expression of individual alleles. We aimed to analyze the DNA methylation of promoter sequences and the levels of expression of individual DQA1 gene alleles, interallelic variation of these two characteristics and the relationship between them. METHODS: The 60 healthy donors included into study were HLA-DRB1, HLA-DQB1 and HLA-DQA1 genotyped using PCR-SSP. Genomic DNA was treated by sodium bisulfite and the target segment in the HLA-DQA1 gene promoter was PCR amplified. PCR product was cloned into Escherichia coli and individual clones were sequenced. Transcripts of individual DQA1 alleles in peripheral blood leukocytes were quantified by Real-Time PCR. RESULTS: In this study, we have described detailed DNA methylation profile of promoter area of DQA1 gene alleles. The overall promoter methylation is increased for DQA1*02:01 and DQA1*04:01 alleles, on the other side, DQA1*05:01 allele shows decreased methylation level. Our results suggest that there are only minor interindividual differences in DRA-normalized expression level of specific allele. Furthermore, expression levels of individual alleles followed DQA1*03>*01:03 (in DRB1*13-DQA1*01:03-DQB1*06:03 haplotype)>*01:01,*05:05, and DQA1*03>*02:01>*05:05 hierarchy. The statistically significantly most expressed allele, DQA1*03, comprises part of DQ8 molecule, which is commonly linked to autoimmune diseases. A clear relationship between promoter DNA methylation and mRNA expression level of the DQA1 gene could not be identified.
Authors: Quinn Orb; Abigail Pulsipher; Kristine A Smith; Shaelene Ashby; Jeremiah A Alt Journal: Int Forum Allergy Rhinol Date: 2019-01-18 Impact factor: 3.858