Genetic and epigenetic regulation of gene expression in fetal and adult human livers.

BACKGROUND: The liver plays a central role in the maintenance of homeostasis and health in general. However, there is substantial inter-individual variation in hepatic gene expression, and although numerous genetic factors have been identified, less is known about the epigenetic factors. RESULTS: By analyzing the methylomes and transcriptomes of 14 fetal and 181 adult livers, we identified 657 differentially methylated genes with adult-specific expression, these genes were enriched for transcription factor binding sites of HNF1A and HNF4A. We also identified 1,000 genes specific to fetal liver, which were enriched for GATA1, STAT5A, STAT5B and YY1 binding sites. We saw strong liver-specific effects of single nucleotide polymorphisms on both methylation levels (28,447 unique CpG sites (meQTL)) and gene expression levels (526 unique genes (eQTL)), at a false discovery rate (FDR) < 0.05. Of the 526 unique eQTL associated genes, 293 correlated significantly not only with genetic variation but also with methylation levels. The tissue-specificities of these associations were analyzed in muscle, subcutaneous adipose tissue and visceral adipose tissue. We observed that meQTL were more stable between tissues than eQTL and a very strong tissue-specificity for the identified associations between CpG methylation and gene expression. CONCLUSIONS: Our analyses generated a comprehensive resource of factors involved in the regulation of hepatic gene expression, and allowed us to estimate the proportion of variation in gene expression that could be attributed to genetic and epigenetic variation, both crucial to understanding differences in drug response and the etiology of liver diseases.

Unraveling the regulatory mechanisms underlying tissue-dependent genetic variation of gene expression.

It is known that genetic variants can affect gene expression, but it is not yet completely clear through what mechanisms genetic variation mediate this expression. We therefore compared the cis-effect of single nucleotide polymorphisms (SNPs) on gene expression between blood samples from 1,240 human subjects and four primary non-blood tissues (liver, subcutaneous, and visceral adipose tissue and skeletal muscle) from 85 subjects. We characterized four different mechanisms for 2,072 probes that show tissue-dependent genetic regulation between blood and non-blood tissues: on average 33.2% only showed cis-regulation in non-blood tissues; 14.5% of the eQTL probes were regulated by different, independent SNPs depending on the tissue of investigation. 47.9% showed a different effect size although they were regulated by the same SNPs. Surprisingly, we observed that 4.4% were regulated by the same SNP but with opposite allelic direction. We show here that SNPs that are located in transcriptional regulatory elements are enriched for tissue-dependent regulation, including SNPs at 3' and 5' untranslated regions (P = 1.84×10(-5) and 4.7×10(-4), respectively) and SNPs that are synonymous-coding (P = 9.9×10(-4)). SNPs that are associated with complex traits more often exert a tissue-dependent effect on gene expression (P = 2.6×10(-10)). Our study yields new insights into the genetic basis of tissue-dependent expression and suggests that complex trait associated genetic variants have even more complex regulatory effects than previously anticipated.

Endogenous formation of Nε-(carboxymethyl)lysine is increased in fatty livers and induces inflammatory markers in an in vitro model of hepatic steatosis.

BACKGROUND & AIMS: Increased lipid peroxidation and inflammation are major factors in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). A lipoxidation product that could play a role in the induction of hepatic inflammation is N(ε)-(carboxymethyl)lysine (CML). The aim of the present study was to investigate the relationship between steatosis and CML and to study the role of CML in hepatic inflammation. METHODS: We included 74 obese individuals, which were categorized into 3 groups according to the grade of hepatic steatosis. CML accumulation in liver biopsies was assessed by immunohistochemistry and plasma CML levels were measured by mass spectrometry. Plasma CML levels were also determined in the hepatic artery, portal, and hepatic vein of 22 individuals, and CML fluxes across the liver were calculated. Hepatocyte cell lines were used to study CML formation during intracellular lipid accumulation and the effect of CML on pro-inflammatory cytokine expression. Gene expression levels of the inflammatory markers were determined in liver biopsies of the obese individuals. RESULTS: CML accumulation was significantly associated with the grade of hepatic steatosis, the grade of hepatic inflammation, and gene expression levels of inflammatory markers PAI-1, IL-8, and CRP. Analysis of CML fluxes showed no release/uptake of CML by the liver. Lipid accumulation in hepatocytes, induced by incubation with fatty acids, was associated with increased CML formation and expression of the receptor for advanced glycation endproducts (RAGE), PAI-1, IL-8, IL-6, and CRP. Pyridoxamine and aminoguanidine inhibited the endogenous CML formation and the increased RAGE, PAI-1, IL-8, IL-6, and CRP expression. Incubation of hepatocytes with CML-albumin increased the expression of RAGE, PAI-1, and IL-6, which was inhibited by an antibody against RAGE. CONCLUSIONS: Accumulation of CML and a CML-upregulated RAGE-dependent inflammatory response in steatotic livers may play an important role in hepatic steatosis and in the pathogenesis of NAFLD.

Trans-eQTLs reveal that independent genetic variants associated with a complex phenotype converge on intermediate genes, with a major role for the HLA.

For many complex traits, genetic variants have been found associated. However, it is still mostly unclear through which downstream mechanism these variants cause these phenotypes. Knowledge of these intermediate steps is crucial to understand pathogenesis, while also providing leads for potential pharmacological intervention. Here we relied upon natural human genetic variation to identify effects of these variants on trans-gene expression (expression quantitative trait locus mapping, eQTL) in whole peripheral blood from 1,469 unrelated individuals. We looked at 1,167 published trait- or disease-associated SNPs and observed trans-eQTL effects on 113 different genes, of which we replicated 46 in monocytes of 1,490 different individuals and 18 in a smaller dataset that comprised subcutaneous adipose, visceral adipose, liver tissue, and muscle tissue. HLA single-nucleotide polymorphisms (SNPs) were 10-fold enriched for trans-eQTLs: 48% of the trans-acting SNPs map within the HLA, including ulcerative colitis susceptibility variants that affect plausible candidate genes AOAH and TRBV18 in trans. We identified 18 pairs of unlinked SNPs associated with the same phenotype and affecting expression of the same trans-gene (21 times more than expected, P<10(-16)). This was particularly pronounced for mean platelet volume (MPV): Two independent SNPs significantly affect the well-known blood coagulation genes GP9 and F13A1 but also C19orf33, SAMD14, VCL, and GNG11. Several of these SNPs have a substantially higher effect on the downstream trans-genes than on the eventual phenotypes, supporting the concept that the effects of these SNPs on expression seems to be much less multifactorial. Therefore, these trans-eQTLs could well represent some of the intermediate genes that connect genetic variants with their eventual complex phenotypic outcomes.

Co-expressed immune and metabolic genes in visceral and subcutaneous adipose tissue from severely obese individuals are associated with plasma HDL and glucose levels: a microarray study.

BACKGROUND: Excessive accumulation of body fat, in particular in the visceral fat depot, is a major risk factor to develop a variety of diseases such as type 2 diabetes. The mechanisms underlying the increased risk of obese individuals to develop co-morbid diseases are largely unclear.We aimed to identify genes expressed in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) that are related to blood parameters involved in obesity co-morbidity, such as plasma lipid and glucose levels, and to compare gene expression between the fat depots. METHODS: Whole-transcriptome SAT and VAT gene expression levels were determined in 75 individuals with a BMI >35 kg/m2. Modules of co-expressed genes likely to be functionally related were identified and correlated with BMI, plasma levels of glucose, insulin, HbA1c, triglycerides, non-esterified fatty acids, ALAT, ASAT, C-reactive protein, and LDL- and HDL cholesterol. RESULTS: Of the approximately 70 modules identified in SAT and VAT, three SAT modules were inversely associated with plasma HDL-cholesterol levels, and a fourth module was inversely associated with both plasma glucose and plasma triglyceride levels (p < 5.33 x 10(-5)). These modules were markedly enriched in immune and metabolic genes. In VAT, one module was associated with both BMI and insulin, and another with plasma glucose (p < 4.64 x 10(-5)). This module was also enriched in inflammatory genes and showed a marked overlap in gene content with the SAT modules related to HDL. Several genes differentially expressed in SAT and VAT were identified. CONCLUSIONS: In obese subjects, groups of co-expressed genes were identified that correlated with lipid and glucose metabolism parameters; they were enriched with immune genes. A number of genes were identified of which the expression in SAT correlated with plasma HDL cholesterol, while their expression in VAT correlated with plasma glucose. This underlines both the singular importance of these genes for lipid and glucose metabolism and the specific roles of these two fat depots in this respect.