Vitamin D Responsive Elements within the HLA-DRB1 Promoter Region in Sardinian Multiple Sclerosis Associated Alleles

Abstract

Vitamin D response elements (VDREs) have been found in the promoter region of the MS-associated allele HLA-DRB1*15∶01, suggesting that with low vitamin D availability VDREs are incapable of inducing *15∶01 expression allowing in early life autoreactive T-cells to escape central thymic deletion. The Italian island of Sardinia exhibits a very high frequency of MS and high solar radiation exposure. We test the contribution of VDREs analysing the promoter region of the MS-associated DRB1 *04∶05, *03∶01, *13∶01 and *15∶01 and non-MS-associated *16∶01, *01, *11, *07∶01 alleles in a cohort of Sardinians (44 MS patients and 112 healthy subjects). Sequencing of the DRB1 promoter region revealed a homozygous canonical VDRE in all *15∶01, *16∶01, *11 and in 45/73 *03∶01 and in heterozygous state in 28/73 *03∶01 and all *01 alleles. A new mutated homozygous VDRE was found in all *13∶03, *04∶05 and *07∶01 alleles. Functionality of mutated and canonical VDREs was assessed for its potential to modulate levels of DRB1 gene expression using an in vitro transactivation assay after stimulation with active vitamin D metabolite. Vitamin D failed to increase promoter activity of the *04∶05 and *03∶01 alleles carrying the new mutated VDRE, while the *16∶01 and *03∶01 alleles carrying the canonical VDRE sequence showed significantly increased transcriptional activity. The ability of VDR to bind the mutant VDRE in the DRB1 promoter was evaluated by EMSA. Efficient binding of VDR to the VDRE sequence found in the *16∶01 and in the *15∶01 allele reduced electrophoretic mobility when either an anti-VDR or an anti-RXR monoclonal antibody was added. Conversely, the Sardinian mutated VDRE sample showed very low affinity for the RXR/VDR heterodimer. These data seem to exclude a role of VDREs in the promoter region of the DRB1 gene in susceptibility to MS carried by DRB1* alleles in Sardinian patients.

Phase-defined complete sequencing of the HLA genes by next-generation sequencing

“…The complete cDNA sequencing can automatically define the HLA gene haplotype structures of the exons without inference [18,19], however, preparation of cDNA is not practical, especially considering the clinical application. The exon-centric analyses are efficient in data-analyses, however, two fundamental shortcomings need to be reminded: First, it has been reported that expression level of HLA genes is associated with disease phenotype [20,21], thus genetic variants in regulatory element such as promoter or even introns need to be extensively analyzed. In fact, a variant of intron 2 of HLA-A was reported as causality of low expression of the HLA gene, furthermore, variants in intron 2 of HLA-A , introns 2 and 4 of HLA-B cause null expression by cryptic splicing activation [22-24].…”

“…Also, cDNA amplicons of HLA genes were extensively sequenced [18,19] and these exon-centric analyses are successful in determining genotypes after consulting with the IMGT/HLA database to detect the closest HLA gene sequence. However, non-coding regions that may have impact on gene regulation [20,21], or mRNA splicing [22-24] are ignored. Most recently, 8-digit sequencing of HLA-genes is partially achieved using a combination of long-range PCR and Roche GS Junior sequencer and/or IonPGM sequencer [25].…”

Immunologic Effects of Vitamin D on Human Health and Disease

“…Individuals carrying certain human leukocyte antigen (HLA) alleles such as HLA-DRB1*1501 have a significantly higher risk of developing MS [ 111 ]. Interestingly, vitamin D response elements have been identified in the promoter region of the HLA-DRB1 gene, and its expression can be altered by activation of VDR by 1,25(OH) 2 D, strengthening the link between vitamin D and MS [ 112 , 113 ].…”

“…Genetic polymorphisms in the vitamin D-binding protein (DBP) ( GC ) gene was shown to influence the ratio between circulating total and free 25(OH)D [ 197 ]. Finally, it has been demonstrated that the variations of vitamin D responsive elements (VDRE) located in the promotor regions of VDR target genes could significantly affect the binding affinity to VDR–RXR complex, resulting in varying expression of the target gene in response to VDR signaling [ 112 , 113 , 198 ]. Thus, it is possible that individuals carrying different polymorphisms of VDR or GC genes among others or VDRE in the target genes may have varying genomic responses to the comparable levels of total serum 25(OH)D and therefore might benefit from vitamin D differently.…”

In Phase HLA Genotyping by Next Generation Sequencing — A Comparison Between Two Massively Parallel Sequencing Bench-Top Systems, the Roche GS Junior and Ion Torrent PGM

“…Although the IMGT/HLA database has sequences mostly of exons 2 and 3 [74,75], the promoter/enhancer, intron, and 3'UTR variants should not be ignored for more comprehensive HLA typing now and into the future. The genetic variants in a regulatory element such as promoter or even introns need to be extensively analyzed because autoimmune [76] and infectious diseases [77] have been associated with the differential expression levels of the HLA genes in different haplotypes [78]. In addition, null alleles resulting from intronic polymorphisms warrant investigation and resolution to better understand their functional effects [79][80][81][82].…”