Genetic variation in transmembrane 6 superfamily member 2 and the risk of nonalcoholic fatty liver disease and histological disease severity.

UNLABELLED: We explored the role of transmembrane 6 superfamily member 2 (TM6SF2) rs58542926 C/T nonsynonymous (p.Glu167Lys) variant in genetic susceptibility to nonalcoholic fatty liver disease (NAFLD) and disease severity. A total of 361 individuals (135 control subjects and 226 patients with histologically proven NAFLD) were included in a sample with 97% power for the additive genetic model. A discrete trait analysis of NAFLD showed that rs58542926 was associated with a modest risk of fatty liver (P = 0.038; odds ratio [OR]: 1.37; 95% confidence interval [CI]: 1.02-1.84); nevertheless, conditioning on patatin-like phospholipase domain-containing 3 (PNPLA3)-rs738409 abolished this effect. We did not observe an interaction between rs738409 and rs58542926 variants on the risk of NAFLD. We observed a significant association of rs58542926 and disease severity (P = 0.027), but not lobular inflammation or fibrosis; rs58542926 was not associated with levels of liver enzymes. An allelic test showed that the T (Lys167) allele was significantly associated with disease progression (P = 0.021; OR, 1.66; 95% CI: 1.08-2.55). A significant association was found with the histological degree of liver steatosis (ß, 0.15; standard error: 0.06; P = 0.0299) that was independent of rs738409. Homozygous carriers of the C (Glu167) allele showed increased risk for cardiovascular disease. TM6SF2 protein expression was decreased markedly in liver of NAFLD patients, compared to controls. In addition, TM6SF2 immunoreactivity was reduced in subjects carrying at least one copy of the T allele, consistent with a difference in liver allele-specific transcript abundance. CONCLUSION: rs58542926 is a low-frequency variant with a modest effect on NAFLD, suggesting that carriers of the T allele are slightly more likely to accumulate fat in the liver and develop nonalcoholic steatohepatitis than those without. TM6SF2 appears to play a significant role in disease biology.

Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis.

OBJECTIVES: We used a screening strategy of global serum microRNA (miRNA) profiling, followed by a second stage of independent replication and exploration of liver expression of selected miRNAs to study: (1) the circulating miRNA signature associated with non-alcoholic fatty liver disease (NAFLD) progression and predictive power, (2) the role of miRNAs in disease biology and (3) the association between circulating miRNAs and features of the metabolic syndrome. METHODS: The study used a case-control design and included patients with NAFLD proven through biopsy and healthy controls. RESULTS: Among 84 circulating miRNAs analysed, miR-122, miR-192, miR-19a and miR-19b, miR-125b, and miR-375 were upregulated >2-fold (p<0.05) either in simple steatosis (SS) or non-alcoholic steatohepatitis (NASH). The most dramatic and significant fold changes were observed in the serum levels of miR-122 (7.2-fold change in NASH vs controls and 3.1-fold change in NASH vs SS) and miR-192 (4.4-fold change in NASH vs controls); these results were replicated in the validation set. The majority of serum miR-122 circulate in argonaute2-free forms. Circulating miR-19a/b and miR-125b were correlated with biomarkers of atherosclerosis. Liver miR-122 expression was 10-fold (p<0.03) downregulated in NASH compared with SS and was preferentially expressed at the edge of lipid-laden hepatocytes. In vitro exploration showed that overexpression of miR-122 enhances alanine aminotransferase activity. CONCLUSIONS: miR-122 plays a role of physiological significance in the biology of NAFLD; circulating miRNAs mirror the histological and molecular events occurring in the liver. NAFLD has a distinguishing circulating miRNA profile associated with a global dysmetabolic disease state and cardiovascular risk.

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.

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.

Importancia pronóstica de las micrometástasis ganglionares en el cáncer de colon y recto / Pronostic significance of lymph node micrometastases in colon and rectal cancer

Antecedentes: Los pacientes con invasión transmural de cáncer colorrectal y ganglios linfáticos negativos (Dukes B), al momento de la resección, pueden presentar micrometástasis ganglionares. Objetivo: Identificar células tumorales en ganglios provenientes de piezas colorrectales, considerados libre de enfermedad por el clásico método de hematoxilina-eosina. Lugar de aplicación: Hospital general. Diseño: Retrospectivo. Población: Se seleccionaron 160 resecciones colónicas y rectales, 54 (34 por ciento) de las cuales correspondieron al estadio B de Dukes. Método: Se efectuaron cortes de los ganglios linfáticos de 3 micrones de espesor. Luego fueron colocados en solución de citrato de buffer (pH 6). La peroxidasa endógena fue inhibida con H2O2 en metanol. Se incubaron con anticuerpo AE1 y AE3. El sistema de detección usado fue el Vestactin Universal Elite ABC. El revelado se efectuó con diaminobencidina al 0,5 por ciento. Resultados: De los 54 casos estudiados, 5 resultaron citoqueratina positiva (9,3 por ciento). De los 5 casos Dukes B con micrometástasis ganglionares, mueren 3 (60 por ciento), de los 49 Dukes B sin micrometástasis, fallecen 4 (8,2 por ciento). Conclusiones: La técnica de inmunomarcación es capaz de identificar micrometástasis en ganglios linfáticos no halladas por técnicas convencionales. La presencia de células citoqueratina positiva, empobrece significativamente el pronóstico de los pacientes Dukes B

Importancia pronóstica de las micrometástasis ganglionares en el cáncer de colon y recto / Pronostic significance of lymph node micrometastases in colon and rectal cancer

Antecedentes: Los pacientes con invasión transmural de cáncer colorrectal y ganglios linfáticos negativos (Dukes B), al momento de la resección, pueden presentar micrometástasis ganglionares. Objetivo: Identificar células tumorales en ganglios provenientes de piezas colorrectales, considerados libre de enfermedad por el clásico método de hematoxilina-eosina. Lugar de aplicación: Hospital general. Diseño: Retrospectivo. Población: Se seleccionaron 160 resecciones colónicas y rectales, 54 (34 por ciento) de las cuales correspondieron al estadio B de Dukes. Método: Se efectuaron cortes de los ganglios linfáticos de 3 micrones de espesor. Luego fueron colocados en solución de citrato de buffer (pH 6). La peroxidasa endógena fue inhibida con H2O2 en metanol. Se incubaron con anticuerpo AE1 y AE3. El sistema de detección usado fue el Vestactin Universal Elite ABC. El revelado se efectuó con diaminobencidina al 0,5 por ciento. Resultados: De los 54 casos estudiados, 5 resultaron citoqueratina positiva (9,3 por ciento). De los 5 casos Dukes B con micrometástasis ganglionares, mueren 3 (60 por ciento), de los 49 Dukes B sin micrometástasis, fallecen 4 (8,2 por ciento). Conclusiones: La técnica de inmunomarcación es capaz de identificar micrometástasis en ganglios linfáticos no halladas por técnicas convencionales. La presencia de células citoqueratina positiva, empobrece significativamente el pronóstico de los pacientes Dukes B (AU)