Cross talk between the liver microbiome and epigenome in patients with metabolic dysfunction-associated steatotic liver disease.

BACKGROUND: The pathogenesis of MASLD (metabolic dysfunction-associated steatotic liver disease), including its severe clinical forms, involves complex processes at all levels of biological organization. This study examined the potential link between the liver microbiome profile and epigenetic factors. METHODS: Liver microbial DNA composition was analysed using high throughput 16S rRNA gene sequencing in 116 individuals, with 55% being female, across the spectrum of liver disease severity. Total activity of histone deacetylases (HDACs) and acetyltransferases (HATs) was assayed in nuclear extracts from fresh liver samples. In addition, we measured the global 5-hydroxymethylcytosine (5-hmC) levels of liver DNA. FINDINGS: Patients with MASLD showed a 2.07-fold increase (p = 0.013) in liver total HAT activity. Moreover, a correlation was observed between liver total HAT activity and the score for histological steatosis (Spearman's R = 0.60, p = 1.0E-3) and disease severity (R = 0.40, p = 2.0E-2). Liver HAT and HDAC activities also showed associations with the abundance of several liver bacterial DNAs. Additionally, liver global levels of 5-hmC showed negative correlation with the read number of Bacteroidetes (R = -0.62, p = 9.3E-4) and Gammaproteobacteria (R = -0.43, p = 3.2E-2), while it was positively correlated with the abundance of Acidobacteria (R = 0.42, p = 4.1E-2) and Actinobacteria (R = 0.47, p = 1.8E-2). INTERPRETATION: The host liver epigenome, including the activity of enzymes involved in maintaining the balance between protein acetylation and deacetylation and the global DNA hydroxy-methylation status, may be the target of microbial signals. FUNDING: Agencia Nacional de Promoción Científica y Tecnológica, FonCyT.

The influence of host genetics on liver microbiome composition in patients with NAFLD.

BACKGROUND: Human body microbiotas are influenced by several factors, including the interaction of the host with the environment and dietary preferences. The role of host genetics in modulating the liver microbiota in the context of NAFLD remains unknown. To address this gap, we examined the interplay between the liver metataxonomic profile and host genetics. METHODS: We obtained 16S rRNA gene sequences from liver biopsies and genotypes by Taqman-assays in 116 individuals. We compared taxon abundance at the genus level across host genotypes using dominant models of inheritance. We focused the analysis on variants influencing the risk/ protection against NAFLD-histological severity (PNPLA3-rs738409, TM6SF2-rs58542926, MBOAT7-rs641738, and HSD17B13-rs72613567) and a variant influencing macronutrient intake (FGF21-rs838133). We also explored the variants' combined effect via a polygenic risk score (PRS). FINDINGS: We identified at least 18 bacterial taxa associated with variants in the selected loci. Members of the Gammaproteobacteria class were significantly enriched in carriers of the rs738409 and rs58542926 risk-alleles, including Enterobacter (fold change [FC]=6.2) and Pseudoalteromonas (FC=2) genera, respectively. Lawsonella (1.6-FC), Prevotella_9 (FC=1.5), and Staphylococcus (FC=1.3) genera were enriched in rs838133-minor allele carriers, which is linked to sugar consumption and carbohydrate intake. Tyzzerella abundance (FC=2.64) exhibited the strongest association (p = 0.0019) with high PRS values (>4 risk alleles). The percentage of genus-level taxa variation explained by the PRS was ∼7.4%, independently of liver steatosis score and obesity. INTERPRETATION: We provided evidence that genetic variation may influence the liver microbial DNA composition. These observations may represent potentially actionable mechanisms of disease. FUNDING: This study was partially supported by grants PICT 2018-889, PICT 2019-0528, PICT2016-0135 and PICT 2018-0620 (Agencia Nacional de Promoción Científica y Tecnológica, FONCyT), CONICET Proyectos Unidades Ejecutoras 2017, PUE 0055.

Intrahepatic bacterial metataxonomic signature in non-alcoholic fatty liver disease.

OBJECTIVE: We aimed to characterise the liver tissue bacterial metataxonomic signature in two independent cohorts of patients with biopsy-proven non-alcoholic fatty liver disease (NAFLD) diagnosis, as differences in the host phenotypic features-from moderate to severe obesity-may be associated with significant changes in the microbial DNA profile. DESIGN AND METHODS: Liver tissue samples from 116 individuals, comprising of 47 NAFLD overweight or moderately obese patients, 50 NAFLD morbidly obese patients elected for bariatric surgery and 19 controls, were analysed using high-throughput 16S rRNA gene sequencing. RESULTS: Liver bacterial DNA profile significantly differs between morbidly obese and non-morbidly obese patients with NAFLD. Bacteroidetes (p=1.8e-18) and Firmicutes (p=0.0044) were over-represented in morbidly obese patients and Proteobacteria (p=5.2e-10)-specifically Gammaproteobacteria and Alphaproteobacteria, and Deinococcus-Thermus (p=0.00012)-were over-represented in the non-morbidly obese cohort. Cohort-specific analysis of liver microbial DNA signatures shows patterns linked to obesity. The imbalance in Proteobacteria (Alpha or Gamma) among non-morbidly obese patients, and Peptostreptococcaceae, Verrucomicrobia, Actinobacteria and Gamma Proteobacteria DNA among morbidly obese patients was associated with histological severity. Decreased amounts of bacterial DNA from the Lachnospiraceae family were associated with more severe histological features. Proteobacteria DNA was consistently associated with lobular and portal inflammation scores. Microbial DNA composition corresponded to predicted functional differences. CONCLUSION: This is the first comprehensive study showing that the liver tissue of NAFLD patients contains a diverse repertoire of bacterial DNA (up to 2.5×104 read counts). The liver metataxonomic signature may explain differences in the NAFLD pathogenic mechanisms as well as physiological functions of the host.

Splice variant rs72613567 prevents worst histologic outcomes in patients with nonalcoholic fatty liver disease.

Hydroxysteroid 17-ß dehydrogenase 13 (HSD17B13) is a lipid droplet-associated protein; its gene-encoding variants affect the chronic liver diseases, including nonalcoholic fatty liver disease (NAFLD). To estimate the effect of rs72613567, a splice variant with an adenine insertion (A-INS), on NAFLD susceptibility and severity, we performed a case-control study with 609 individuals. We investigated the effect of carrying the A-INS allele in 356 patients with biopsy-proven disease and explored the relationship between rs72613567 genotypes and the hepatic transcriptome. The A-INS allele protected against NAFLD [odds ratio (OR) per adenine allele = 0.667; 95% CI, 0.486-0.916; P = 0.012]; this effect was nonsignificant when logistic regression analysis included BMI. The A-INS allele protected against nonalcoholic steatohepatitis (NASH) (OR = 0.612; 95% CI, 0.388-0.964; P = 0.033), ballooning degeneration (OR = 0.474; 95% CI, 0.267-0.842; P = 0.01), lobular inflammation (OR = 0.475; 95% CI, 0.275-0.821; P = 0.007), and fibrosis (OR = 0.590; 95% CI, 0.361-0.965; P = 0.035). In patients carrying A-INS, HSD17B13 levels decreased proportionally to allele dosage. Whole-transcriptome genotype profiling showed overrepresented immune response-related pathways. Thus, the rs72613567 A-INS allele reduces the risk of NASH and progressive liver damage and may become a therapeutic target.

A Rare Nonsense Mutation in the Glucokinase Regulator Gene Is Associated With a Rapidly Progressive Clinical Form of Nonalcoholic Steatohepatitis.

We report on the presence of a rare nonsense mutation (rs149847328, p.Arg227Ter) in the glucokinase regulator (GCKR) gene in an adult patient with nonalcoholic fatty liver disease (NAFLD), morbid obesity, and type 2 diabetes; this patient developed a progressive histological form of the disease. Analysis of paired (5 years apart) liver biopsies (at baseline and follow-up) showed progression of simple steatosis to severe nonalcoholic steatohepatitis and cirrhosis. Study design involved an initial exploration that consisted of deep sequencing of 14 chromosomal regions in 96 individuals (64 of whom were patients with NAFLD who were diagnosed by liver biopsy that showed the full spectrum of histological severity). We further performed a replication study to explore the presence of rs149847328 that included a sample of 517 unrelated individuals in a case-control study (n = 390), including patients who were morbidly obese (n = 127). Exploration of sequence variation by next-generation sequencing of exons, exon-intron boundaries, and 5' and 3' untranslated regions of 14 genomic loci that encode metabolic enzymes of the tricarboxylic acid cycle revealed the presence of heterozygosity for the p.Arg227Ter mutation, the frequency of which is 0.0003963 (4:10,000; Exome Aggregation Consortium database). GCKR protein expression was markedly decreased in the liver of the affected patient compared with patients with NAFLD who carry the wild-type allele. Sequencing of the same 14 genomic loci in 95 individuals failed to reveal the rare mutation. The rarity of p.Arg227Ter was confirmed in a more extensive screening. Conclusion: While rare variants/mutations are difficult to detect in even reasonably large samples (frequency of the mutant allele of p.Arg227Ter was ~1:1,000 in our data set), the presence of this mutation should be suspected as potentially associated with NAFLD, particularly in young adults at the extreme of histological phenotypes. Hepatology Communications 2018;0:0-0).

Metastasis-associated lung adenocarcinoma transcript 1 as a common molecular driver in the pathogenesis of nonalcoholic steatohepatitis and chronic immune-mediated liver damage.

Long noncoding RNAs (lncRNAs) are functional molecules that orchestrate gene expression. To identify lncRNAs involved in nonalcoholic fatty liver disease (NAFLD) severity, we performed a multiscale study that included: (a) systems biology modeling that indicated metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) as a candidate lncRNA for exploring disease-related associations, (b) translational exploration in the clinical setting, and (c) mechanistic modeling. MALAT1 liver profiling was performed in three consecutive phases, including an exploratory stage (liver samples from patients with NAFLD who were morbidly obese [n = 47] and from 13 individuals with normal liver histology); a replication stage (patients with NAFLD and metabolic syndrome [n =49]); and a hypothesis-driven stage (patients with chronic hepatitis C and autoimmune liver diseases, [n = 65]). Liver abundance of MALAT1 was associated with NAFLD severity (P = 1 × 10-6); MALAT1 expression levels were up-regulated 1.75-fold (P = 0.029) and 3.6-fold (P = 0.012) in patients with nonalcoholic steatohepatitis compared to those diagnosed with simple steatosis (discovery and replication set, respectively; analysis of covariance adjusted by age, homeostasis model assessment, and body mass index). Quantification of liver vascular endothelial growth factor A messenger RNA, a target of MALAT1, revealed a significant correlation between the two RNAs (R, 0.58; P = 5 × 10-8). Increased levels of MALAT1 were also associated with autoimmune liver diseases. Interactome assessment uncovered significant biological pathways, including Janus kinase-signal transducers and activators of transcription and response to interferon-γ. Conclusion: Deregulated expression of MALAT1 stratifies patients into the histologic phenotypes associated with NAFLD severity. MALAT1 up-regulation seems to be a common molecular mechanism in immune-mediated chronic inflammatory liver damage. This suggests that convergent pathophenotypes (inflammation and fibrosis) share similar molecular mediators. (Hepatology Communications 2018;2:654-665).

Lack of evidence supporting a role of TMC4-rs641738 missense variant-MBOAT7- intergenic downstream variant-in the Susceptibility to Nonalcoholic Fatty Liver Disease.

Current knowledge on the genetic basis of nonalcoholic fatty liver disease (NAFLD) suggests that variants contributing not only to the disease predisposition but histological severity as well are located in genes that regulate lipid metabolism. We explored the role of rs641738 C/T located in TMC4 (transmembrane channel-like 4) exon 1 (p.Gly17Glu) and 500 bases- downstream of MBOAT7 gene (TMC4/MBOAT7), in the genetic risk for developing NAFLD in a case-control study. Our sample included 634 individuals (372 patients with NAFLD diagnosed by liver biopsy and 262 control subjects); genotyping was performed by a Taqman assay. Genotype frequencies in controls (CC: 84, CT: 137, TT: 41) and patients (CC: 134, CT: 178, TT: 60) were in Hardy-Weinberg equilibrium; minor allele frequency 40.8%. Our sample had 84-99% power if an additive genetic model is assumed for estimated odds ratios of 1.3-1.5, respectively. We found no evidence of association between rs641738 and either NAFLD (Cochran-Armitage test for trend, p = 0.529) or the disease severity (p = 0.61). Low levels of MBOAT7 protein expression were found in the liver of patients with NAFLD, which were unrelated to the rs641738 genotypes. In conclusion, the role of rs641738 in the pathogenesis of NAFLD is inconclusive.

Cirugía Metabólica: experiencia inicial en la República Argentina / Metabolic surgery: initial experience in the Argentine Republic

Es bien conocida la relación entre obesidad y Diabetes Mellitus tipo 2 (DM2) se ha reportado desde hace más de cuatro décadas la remisión o mejoría de la DM2 en pacientes diabéticos que fueron sometidos a gastrectomía. En el año 2015 el Consenso Argentino de Cirugía Metabólica sienta las bases para el tratamiento quirúrgico de la DM2. Entre Noviembre de 2011 y Agosto de 2012 intervenimos quirúrgicamente 17 pacientes diabéticos, 12 mujeres y 5 hombres. El Índice de Masa Corporal (IMC) fue de 32.5 Kg/m2, el tiempo de evolución de la DM2 fue entre 2 y 15 años. La Hemoglobina Glicosilada fue 8.7%, la glucemia 170 mg/dl y el perímetro de cintura de 106 cm, todos valores promedio. Se realizó un Bypass gástrico en Y de Roux (BPGYR) de asa corta. A los 4 años 11 de los 17 pacientes operados presentan remisión de la enfermedad (64.7%), los 6 pacientes restantes presentaron mejoría franca de su DM2. Cuatro recurrieron a los 18 meses de la cirugía y dos no remitieron pero dejaron de requerir insulina. Ninguno de los pacientes requiere insulina en la actualidad, recordando que la tasa de uso de insulina era de 65% en el preoperatorio. La Hba1c es menor a 6.5 en todos los casos, la glucemia en ayunas es de 112 mg/dl y el perímetro de cintura se redujo a 92 cm en promedio. La cirugía metabólica brinda una remisión prolongada en un alto porcentaje de pacientes con DM2, con bajas tasas de complicaciones, y una mejoría franca en aquellos pacientes en quienes no se logra la remisión. El BPGYR es la técnica quirúrgica que presenta los resultados más predecibles, la más utilizada y universalmente aceptada.

Consenso Argentino de Cirugía Metabólica / ARGENTINE CONSENSUS ON METABOLIC SURGERY

Introducción: la creciente pandemia de obesidad y diabetes tipo 2 (DM2) demanda opciones terapéuticas más efectivas para lograr un adecuado control metabólico y disminuir la morbimortalidad cardiovascular en este grupo de pacientes. En este contexto, la cirugía metabólica (CM) constituye una herramienta innovadora, segura y eficaz que complementa pero no reemplaza a los cambios necesarios del estilo de vida y tratamiento médico. Objetivos: el objetivo del Consenso es la fundamentación y acuerdo de utilización de una técnica quirúrgica, específicamente el Bypass Gástrico en Y de Roux (BPGYR), en el tratamiento de pacientes con DM2 que presentan un índice de masa corporal (IMC) entre 30-35 kg/m2 e inadecuado control metabólico. Conclusiones: el Consenso Argentino de Cirugía Metabólica elaborado por la Sociedad Argentina de Diabetes (SAD), la Sociedad Argentina de Nutrición (SAN) y la Sociedad Argentina de Cirugía de la Obesidad (SACO) expresa la opinión de expertos sobre la evidencia científica disponible y propone considerar a la CM en el algoritmo terapéutico de pacientes con DM2 e IMC 30-35 kg/m2 que no logran adecuado control metabólico con tratamiento médico convencional. Se describen los criterios a tener en cuenta en la selección de pacientes para CM, y se destaca el rol del equipo multidisciplinario liderado por médicos especialistas en enfermedades endocrino-metabólicas en la selección, evaluación, preparación y seguimiento de estos pacientes.

Efecto de la ligadura del conducto pancreático principal en la pancreatits necrohemórragica experimental / Effect of the ligation of the pancreatic duct in experimental necrohemorrhagic prancreatitis

Se presenta una serie compuesta por 10 perros, a quienes de 20 minutos de provocarles una pancreatitis aguda grave mediante la inyección intracanalicular de 2 a 4 cm3 de cloruro de calcio, se procedió a ligar el conducto pancreático principal. Se quiso comenzar a investigar el nivel del árbol canalicular en donde el bloqueo desempeña su acción. Al morir los 10 animales se demostró que la simple obturación del conducto por la ligadura no reemplaza la acción obtenida prolamina o Tissucol

Efecto de la ligadura del conducto pancreático principal en la pancreatits necrohemórragica experimental / Effect of the ligation of the pancreatic duct in experimental necrohemorrhagic prancreatitis

Se presenta una serie compuesta por 10 perros, a quienes de 20 minutos de provocarles una pancreatitis aguda grave mediante la inyección intracanalicular de 2 a 4 cm3 de cloruro de calcio, se procedió a ligar el conducto pancreático principal. Se quiso comenzar a investigar el nivel del árbol canalicular en donde el bloqueo desempeña su acción. Al morir los 10 animales se demostró que la simple obturación del conducto por la ligadura no reemplaza la acción obtenida prolamina o Tissucol (AU)

Pancreatitis aguda: analisis de la evolución de 132 pacientes / Acute pancreatitis: analysis of the development of 132 patients

Se analizan 3 series consecutivas de pacientes portadores de cuadros compatibles con pancreatitis aguda, que totalizan 132 casos. En las 2 primeras se incluyeron sólo aquellos con confirmación operatoria de lesión glandular, y en la 3ª a todos los pacientes que ingresaron con diagnóstico presuntivo de pancreatitis aguda. Se destaca la importancia de determinar al ingreso el posible origen biliar de la enfermedad. La ecografia permitió demostrar litiasis vesicular en el 89,8% de los enfermos y mediante la colangiografía transparietohepática se pudo observar litiasis coledociana en el 45% de los casos. Es aconsejable la aplicación de los índices pronósticos, ya que advierten el posible curso evolutivo de la enfermedad. Se reconoce la importancia de la indicación del tratamiento medicamentoso. Se analizan los hallozgos, las tácticas empleadas y la morbimortalidad, teniendo en cuenta las conductas de cada época. En la última serie descendió la mortalidad global al 10,3% al adoptar una conducta terapéutica más selectiva. Se enumeran las pautas que actualmente rigen en el Servicio, donde se mantiene el temor a los cálculos retenidos en la vía biliar y se promueve su rápida extracción

Pancreatitis aguda: analisis de la evolución de 132 pacientes / Acute pancreatitis: analysis of the development of 132 patients

Se analizan 3 series consecutivas de pacientes portadores de cuadros compatibles con pancreatitis aguda, que totalizan 132 casos. En las 2 primeras se incluyeron sólo aquellos con confirmación operatoria de lesión glandular, y en la 3¬ a todos los pacientes que ingresaron con diagnóstico presuntivo de pancreatitis aguda. Se destaca la importancia de determinar al ingreso el posible origen biliar de la enfermedad. La ecografia permitió demostrar litiasis vesicular en el 89,8% de los enfermos y mediante la colangiografía transparietohepática se pudo observar litiasis coledociana en el 45% de los casos. Es aconsejable la aplicación de los índices pronósticos, ya que advierten el posible curso evolutivo de la enfermedad. Se reconoce la importancia de la indicación del tratamiento medicamentoso. Se analizan los hallozgos, las tácticas empleadas y la morbimortalidad, teniendo en cuenta las conductas de cada época. En la última serie descendió la mortalidad global al 10,3% al adoptar una conducta terapéutica más selectiva. Se enumeran las pautas que actualmente rigen en el Servicio, donde se mantiene el temor a los cálculos retenidos en la vía biliar y se promueve su rápida extracción (AU)

Pancreatitis aguda: comprobación del estado de la vía biliar mediante la colangiografía transparietohepática / Acute pancreatitis: verification of the status of the bile duct transhepatic cholangiography

Se presenta una serie de 61 enfermos con cuadros clínicos compatibles con pancreatitis aguda que fueron estudiados mediante colangiografía transparietohepática con aguja ultrafina, a fin de comprobar la participación de la vía biliar en el proceso. Se logró opacificar el árbol biliar en 53 oportunidades, lo que representó 86,8% de efectividad. El valor del estudio radicó en que permitió identificar tempranamente aquellos enfermos con litiasis coledociana retenida (41,5%). Al mismo tiempo, se pudo individualizar 8 pacientes con vía biliar dilatada pero sin cálculos y en los 24 restantes se logró demonstrar la indemnidad radiológica del hepatocolédoco. No se registró morbilidad atribuible al método

Pancreatitis aguda: comprobación del estado de la vía biliar mediante la colangiografía transparietohepática / Acute pancreatitis: verification of the status of the bile duct transhepatic cholangiography

Se presenta una serie de 61 enfermos con cuadros clínicos compatibles con pancreatitis aguda que fueron estudiados mediante colangiografía transparietohepática con aguja ultrafina, a fin de comprobar la participación de la vía biliar en el proceso. Se logró opacificar el árbol biliar en 53 oportunidades, lo que representó 86,8% de efectividad. El valor del estudio radicó en que permitió identificar tempranamente aquellos enfermos con litiasis coledociana retenida (41,5%). Al mismo tiempo, se pudo individualizar 8 pacientes con vía biliar dilatada pero sin cálculos y en los 24 restantes se logró demonstrar la indemnidad radiológica del hepatocolédoco. No se registró morbilidad atribuible al método (AU)