Tomas Fernández Gianotti

A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity.

We explored the role of the adiponutrin (PNPLA3) nonsynonymous-rs738409 single nucleotide polymorphism (SNP) in genetic susceptibility to nonalcoholic fatty liver disease (NAFLD) and whether this SNP contributes to the severity of histological disease. Two hundred sixty-six individuals were evaluated in a case-control association study, which included 172 patients with features of NAFLD and 94 control subjects. The rs738409 G allele was significantly associated with NAFLD (P < 0.001; OR 2.8 95%, CI 1.5–5.2), independent of age, sex, body mass index (BMI), and Homeostasis Model Assessment (HOMA) index. When we tested the hypothesis of a relation between the SNP and the histological spectrum of NAFLD, a significant association was observed [chi2 19.9, degree of freedom (df): 2, P < 5 × 10−5, adjusted for HOMA and BMI]. The degree of liver steatosis, as evaluated by liver biopsy, was significantly associated with the rs738409 G allele. Patients with CC genotype showed a lower steatosis score (14.9% ± 3.9) in comparison with the CG genotype (26.3% ± 3.5) and GG genotype (33.3% ± 4.0) (P < 0.005). The proportion of the total variation attributed to rs738409 genotypes was 5.3% (&bgr; 0.23 ± 0.07; P < 0.002). Our data suggest that the rs738409 G allele is associated not only with fat accumulation in the liver but also with liver injury, possibly triggered by lipotoxicity.

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: Impact of liver methylation of the peroxisome proliferator–activated receptor γ coactivator 1α promoter †‡

Insulin resistance (IR) and mitochondrial dysfunction play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). We hypothesized that genetic factors and epigenetic modifications occurring in the liver contribute to the IR phenotype. We specifically examined whether fatty liver and IR are modified by hepatic DNA methylation of the peroxisome proliferator–activated receptor γ coactivator 1α (PPARGC1A) and mitochondrial transcription factor A (TFAM) promoters, and also evaluated whether liver mitochondrial DNA (mtDNA) content is associated with NAFLD and IR. We studied liver biopsies obtained from NAFLD patients in a case–control design. After bisulfite treatment of DNA, we used methylation‐specific polymerase chain reaction (PCR) to assess the putative methylation of three CpG in the PPARGC1A and TFAM promoters. Liver mtDNA quantification using nuclear DNA (nDNA) as a reference was evaluated by way of real‐time PCR. Liver PPARGC1A methylated DNA/unmethylated DNA ratio correlated with plasma fasting insulin levels and homeostasis model assessment of insulin resistance (HOMA‐IR); TFAM methylated DNA/unmethylated DNA ratio was inversely correlated with insulin levels. PPARGC1A promoter methylation was inversely correlated with the abundance of liver PPARGC1A messenger RNA. The liver mtDNA/nDNA ratio was significantly higher in control livers compared with NAFLD livers. mtDNA/nDNA ratio was inversely correlated with HOMA‐IR, fasting glucose, and insulin and was inversely correlated with PPARGC1A promoter methylation. Conclusion: Our data suggest that the IR phenotype and the liver transcriptional activity of PPARGC1A show a tight interaction, probably through epigenetic modifications. Decreased liver mtDNA content concomitantly contributes to peripheral IR. (HEPATOLOGY 2010)

Epigenetic modification of liver mitochondrial DNA is associated with histological severity of nonalcoholic fatty liver disease

Objective & design Nonalcoholic fatty liver disease (NAFLD) is a clinical condition that refers to progressive histological changes ranging from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH). We evaluated the status of cytosine methylation (5mC) of liver mitochondrial DNA (mtDNA) in selected regions of the mtDNA genome, such as D-loop control region, and mitochondrially encoded NADH dehydrogenase 6 (MT-ND6) and cytochrome C oxidase I (MT-CO1), to contrast the hypothesis that epigenetic modifications play a role in the phenotypic switching from SS to NASH. Methods We studied liver biopsies obtained from patients with NAFLD in a case-control design; 45 patients and 18 near-normal liver-histology subjects. Results MT-ND6 methylation was higher in the liver of NASH than SS patients (p<0.04) and MT-ND6 methylated DNA/unmethylated DNA ratio was significantly associated with NAFLD activity score (p<0.02). Liver MT-ND6 mRNA expression was significantly decreased in NASH patients (0.26±0.30) versus SS (0.74±0.48), p<0.003, and the protein level was also diminished. The status of liver MT-ND6 methylation in NASH group was inversely correlated with the level of regular physical activity (R=-0.54, p<0.02). Hepatic methylation levels of D-Loop and MT-CO1 were not associated with the disease severity. DNA (cytosine-5) methyltransferase 1 was significantly upregulated in NASH patients (p<0.002). Ultrastructural evaluation showed that NASH is associated with mitochondrial defects and peroxisome proliferation. Conclusion Hepatic methylation and transcriptional activity of the MT-ND6 are associated with the histological severity of NAFLD. Epigenetic changes of mtDNA are potentially reversible by interventional programs, as physical activity could modulate the methylation status of MT-ND6.

Association of the C-344T aldosterone synthase gene variant with essential hypertension: a meta-analysis.

Background The CYP11B2 gene (CYP11B2) encoding aldosterone synthase has been associated with essential hypertension and some, but not all, studies have reported that the C−344T variant may influence the risk of the disease. Objective We performed a systematic review of the literature by means of a meta-analysis to evaluate the influence of the C−344T CYP11B2 polymorphism on arterial hypertension and intermediate phenotypes. Methods From 485 reports, we included 42 observational studies, case–control and cohort at baseline. Fixed and random effect models were used to pool data from individual studies. Results From 19 heterogeneous studies including 5343 essential hypertensive and 5882 control subjects, we found a significant association between hypertension and the C−344T variant in fixed but not in random effect models [for homozygous CC: odds ratio (OR), 0.834; 95% confidence interval (CI), 0.760–0.914; P < 0.0001, n = 11 225]. Besides, homozygous CC subjects had lower plasma renin activity (D, −0.161; 95% CI, −0.279 to −0.043; P < 0.01, n = 1428) but no difference in plasma aldosterone levels (D, −0.006; 95% CI, −0.081 to 0.07; P = 0.88, n = 2872). Limiting the quantitative analysis of blood pressure to 13 studies including only untreated individuals, no significant association was found for systolic arterial blood pressure (D, 0.042; 95% CI, −0.057 to 0.141; P = 0.41, n = 1775) and diastolic arterial blood pressure (D, 0.026; 95% CI, −0.073 to 0.125; P = 0.61, n = 1775). Conclusion Homozygous individuals for the −344C CYP11B2 allele are at 17% lower risk of hypertension with respect to homozygous TT subjects.

Circulating levels and hepatic expression of molecular mediators of atherosclerosis in nonalcoholic fatty liver disease

OBJECTIVES We evaluated circulating levels of biomarkers of atherosclerosis (soluble intercellular adhesion molecule: sICAM-1, plasminogen activator inhibitor: PAI-1 and soluble CD40 ligand: sCD40L) in patients with NAFLD proven through biopsy and control subjects, and correlated them with the histological disease severity. We further explored liver protein expression of ICAM-1, CD40 and PAI-1 in patients with different histological forms of NAFLD and control liver biopsies. PATIENTS AND METHODS We included 215 individuals: 113 patients with NAFLD (simple steatosis n=45 and NASH n=68) and 102 control subjects. Circulating levels of the biomarkers were measured by ELISA. Liver expression of ICAM-1, CD40 and PAI-1 was assessed by immunohistochemistry using monoclonal antihuman antibodies. RESULTS Patients with NAFLD, in comparison with control subjects, showed significantly higher circulating levels of sICAM-1 (605.3+/-34.6ng/ml vs. 356.5+/-24.6ng/ml, p=5.9 x 10(-6)), PAI-1 (22.8+/-1.7ng/ml vs. 19.0+/-2.1ng/ml, p=0.0149) and sCD40L (1347.5+/-513.7pg/ml vs. 804.5+/-396.1pg/ml, p=0.0229), results expressed as mean+/-SE. sICAM-1 was a strong predictor of histological severity of NAFLD, after adjusting for potential confounders. In addition, patients with NAFLD showed significantly higher liver staining scores for ICAM-1 and PAI-1 than control liver biopsies. ICAM-1 immunoreactivity in lobular inflammatory infiltrate showed high scores in NASH patients; a significant correlation was found between both the degree of liver steatosis and the severity of necroinflammatory activity and liver ICAM-1 expression. CONCLUSIONS Our study shows that NAFLD is associated with elevated circulating levels and abnormal liver expression of molecular mediators of atherosclerosis. Additionally, ICAM-1 may be involved in liver damage and inflammation.

The nuclear receptor PXR gene variants are associated with liver injury in nonalcoholic fatty liver disease.

Objective To explore the contribution of gene variants and derived haplotypes of the pregnane X receptor (NR1I2) to the severity of nonalcoholic fatty liver disease (NAFLD). Methods A total of 290 individuals were evaluated in a case-control association study, including 188 NAFLD patients with different stages of disease severity and 102 healthy individuals. Four tag single nucleotide polymorphisms (SNPs; rs12488820 C/T, rs2472671 C/T, rs2461823 A/G, and rs1054191 A/G) encompassing 36 kb in chromosome 3 and representing 33 polymorphic sites (r2>0.8) were genotyped. Four additional SNPs (rs3814055, rs3814057, rs6785049, and rs7643645) were also included because they showed earlier evidence of functionality. Results Genotypic tests for single SNPs showed that rs7643645 and rs2461823 were significantly associated with disease severity by ordinal multinomial analysis (P<0.0015 and 0.039, respectively). A significant association was also observed under the additive model for both variants (P<0.00038 and 0.012, respectively). Consistent with the analysis of individual markers, we observed that the multimarker composed of rs2461823/A-rs7643645/G was significantly associated with disease severity (P<6.9×10−5, &bgr;: 0.45). In addition, the rs7643645/G variant was significantly associated with ALT level (P<0.026), a surrogate marker of severe liver injury. Finally, in univariate analysis rs7643645/G was significantly associated with fatty liver disease (P<0.04), with an odds ratio of 1.457 (95% confidence interval: 1.018–2.086). Conclusion Our study suggests that pregnane X receptor polymorphisms and related haplotypes may contribute to disease severity in NAFLD by influencing the individual susceptibility to progress to more severe stages of the disease.

Short Allele of Serotonin Transporter Gene Promoter Is a Risk Factor for Obesity in Adolescents

Obesity and hypertension are increasing medical problems in adolescents. Serotonin transporter (5‐HTT) is involved in mood and eating disturbances. Encoded by the gene SLC6A4, the promoter shows functional insertion/deletion alleles: long (L) and short (S). Because individuals who are carriers for the short version are known to be at risk for higher levels of anxiety, we hypothesized that this variant may be associated with overweight. Data and blood samples were collected from 172 adolescents out of a cross‐sectional, population‐based study of 934 high school students. To replicate the findings, we also included 119 outpatients from the Nutrition and Diabetes Section of the Childrens County Hospital. We found that the S allele was associated with overweight (BMI > 85th percentile), being a risk factor for overweight independently of sex, age, and hypertension [odds ratio (OR): 1.85; 95% confidence interval (CI): 1.13, 3.05; p < 0.02]. Additionally, in the outpatient study, compared with the homozygous LL subjects, S allele carriers showed a higher BMI z‐score (1.47 ± 1.09 vs. 0.51 ± 1.4; p < 0.002) and were more frequent in overweight children. In conclusion, the S allele of the SLC6A4 promoter variant is associated with overweight being an independent genetic risk factor for obesity.

A Decreased Mitochondrial DNA Content Is Related to Insulin Resistance in Adolescents

The aim of this study was to investigate whether mitochondrial DNA (mtDNA) content is associated with insulin resistance (IR) in a sample of adolescents with features of metabolic syndrome. We further studied the link between polymorphisms in three genes involved in mitochondrial biogenesis and the presence of deleted mtDNA and mtDNA content. Data and blood samples were collected from 175 adolescents out of a cross‐sectional, population‐based study of 934 high school students. On the basis of the median value of homeostasis model assessment of IR (HOMA‐IR) of the whole sample (2.2), the population was divided into two groups: noninsulin resistance (NIR) and IR. mtDNA quantification using nuclear DNA (nDNA) as a reference was carried out using a real‐time quantitative PCR method. Genotyping for peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) (pro12Ala), PPAR‐ γ coactivator‐1α (PGC‐1α) (Gly482Ser), and Tfam (rs1937 and rs12247015) polymorphisms was performed by PCR‐based restriction fragment length polymorphism. Long‐extension PCR was performed to amplify the whole mitochondrial genome. The mtDNA/nDNA ratio was significantly lower in the IR group (median: 9.08, range: 68.94) in comparison with the NIR group (12.24, 71.92) (P < 0.03). Besides, the mtDNA/nDNA ratio was inversely correlated with HOMA (R: −0.18, P < 0.02), glucose (R: −0.21, P < 0.008), and uric acid (R: −0.18, P < 0.03). Genotypes for the PPAR‐ γ, PGC‐1α, and Tfam variants were not associated with the mtDNA/nDNA ratio. Long‐extension PCR did not show significant levels of mtDNA deletions. In conclusion, our findings indicate that reduced mtDNA content in peripheral leukocytes is associated with IR. This result seems not to be related with the previously mentioned variants in genes involved in the regulation of mitochondrial biogenesis.

Liver transcriptional profile of atherosclerosis-related genes in human nonalcoholic fatty liver disease

OBJECTIVES AND DESIGN Epidemiological studies have suggested a role of nonalcoholic fatty liver disease (NAFLD) in the development of cardiovascular disease. We evaluated liver mRNA expression of 84 genes encoding proteins involved in the atherosclerosis pathway in patients with NAFLD proven through biopsy in a case-control design, and examined the putative role of the histological disease severity in the molecular events associated with the atherogenic profile. RESULTS Nonalcoholic steatohepatitis (NASH), when compared with simple steatosis (SS), significantly increases the expression of TGFB1 (6.8, p<0.005), angiotensin I-converting enzyme (ACE) (2.1, p<0.007), LAMA1 (2.1, p<0.007), SERPINB2 (2.1, p<0.007), CSF2 (2.5, p<0.002), IL1A (2.5, p<0.005), IL3 (2.1, p<0.007), IL4 (2.1, p<0.007), LIF (2.1, p<0.007), and MMP1 (2.1, p<0.007), and decreases the transcript levels of genes involved in the negative regulation of cell-death pathways. A post hoc analysis of liver biopsies of NASH patients who were treated with enalapril monotherapy because of arterial hypertension showed a significant association with lower fibrosis scores in comparison with untreated patients. BIRC3, a severe hypoxia-activated gene, was significantly increased in SS (8.2, p<0.004), when compared with the controls. NASH, but not SS, was also associated with a significant increase in platelet abundance of TGFB1 mRNA. Systems biology analysis revealed highly scored pathways involved in the regulation of programmed cell death, angiogenesis, and immune system, in which TGFB1 was mostly involved. CONCLUSION NASH, but not SS, may increase atherosclerotic and cardiovascular risk by local overexpression of mediators of atherogenesis, endothelial damage, and regulators of blood pressure; this observation may have therapeutic implications, because ACE inhibitors may improve both cardiovascular outcomes and liver fibrosis. Hepatocyte hypoxia seems to have an important role in the molecular events activated by liver steatosis.

Contribution of the Functional 5-HTTLPR Variant of the SLC6A4 Gene to Obesity Risk in Male Adults

Background: A polymorphism in the promoter region of the serotonin transporter (5‐HTTLPR) gene SLC6A4 shows functionally important 44‐bp insertion/deletion alleles: long (L) and short (S). We have previously found that the S allele is a genetic risk factor for obesity in adolescents.