Allele-specific DNA methylation reinforces PEAR1 enhancer activity

Abstract

Key Points• Rs12041331 is the first functional CpG-SNP related to platelet function whose regulatory mechanism depends on DNA methylation. • Rs12041331 marks allelespecific methylation at the CpG island encompassing the first untranslated exon during megakaryopoiesis.Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 59-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation. (Blood. 2016;128(7):1003-1012

Jedi-1 deficiency increases sensory neuron excitability through a non-cell autonomous mechanism

“…Previous reports indicate that Jedi-1 is a negative regulator of proliferation in endothelial cells lines 14 , bone marrow mononuclear cells 21 , and megakaryocyte precursors 22 . However, we did not find any significant difference between genotypes in the proliferation rate of SGCs or perineurial cells based on Ki67 staining (Fig.…”

“…Altered excitability of peripheral DRG neurons is associated with changes in nociception, raising the possibility that Jedi-1 KO mice and humans with loss-of-function (LOF) Jedi-1 SNPs such as rs12041331 22 may have altered pain responses. Consistent with this possibility, our patch clamp electrophysiology experiments show that small diameter DRG neurons isolated from Jedi-1 KO animals exhibit enhanced excitability as demonstrated by the smaller rheobase and increase in the proportion of cells exhibiting a tonic rather than phasic firing pattern.…”

Variants of PEAR1 Are Associated With Outcome in Patients With ACS and Stable CAD Undergoing PCI

“…In another study, they found a strong association between rs12566888 in PEAR1 and ADP ( p = 1.51 × 10 −7 ) and thrombin-induced ( p = 1.91 × 10 −6 ) platelet reactivity in platelet rich plasma (Eicher et al, 2016a ). Several studies on genetic variants of PEAR1 provided evidence that the intronic SNP rs12041331 alters PEAR1 protein expression and platelet function (Faraday et al, 2011 ; Kauskot et al, 2012 ; Kunicki et al, 2012 ; Qayyum et al, 2015 ) and Western blotting and ELISA confirmed dose-response relation between the number of major G alleles at rs12041331 and expression of PEAR1 protein (Faraday et al, 2011 ; Izzi et al, 2016 ). For ADP-induced aggregation, the PEAR1 minor allele was associated with a decreased response (Kunicki et al, 2012 ).…”

Patient blood management programs: how to spread the word?

“…Patient blood management (PBM) programs have been developed worldwide in order to optimize the utilization of blood components and, as a result, an up to 40% reduction in RBC units transfused per patient has been achieved [ 9 – 17 ]. Lack of such hospital PBM programs results in an extensive liberal RBC transfusion practice, as was shown in a large Danish study [ 18 ] and in a study we performed in three hospitals in Jerusalem, Israel [ 19 ]. In order to succeed with reducing the utilization of RBC units, the PBM program needs to include several important elements: Clear hospital transfusion guidelines including single unit transfusion policy, laboratory “gatekeeping” and the use of an electronic ordering system for blood products (identifying the clinician who ordered blood products is important for feedback and audit).…”