Machine learning reveals the contribution of lipoproteins to liver triglyceride content and inflammation.

CONTEXT: Metabolic dysfunction-associated steatotic liver disease (MASLD) is currently the most common chronic liver disease worldwide and is strongly associated with metabolic comorbidities, including dyslipidemia. OBJECTIVE: Herein, we aim to estimate the prevalence of MASLD and metabolic dysfunction-associated steatohepatitis (MASH) in Europeans with isolated hypercholesterolemia and isolated hypertriglyceridemia in the UK Biobank and to estimate the independent contribution of lipoproteins to liver triglyceride content. METHODS: We selected 218,732 Europeans from the UK Biobank without chronic viral hepatitis and other causes of liver disease, of whom 14,937 with liver magnetic resonance imaging (MRI) data available. Next, to examine the relationships between traits in predicting liver triglyceride content, we compared the predictive performance of several machine learning methods and selected the best performing algorithms based on the minimum cross-validated mean squared error (MSE). RESULTS: There was an approximately 3-fold and 4-fold enrichment of MASLD and MASH in individuals with isolated hypertriglyceridemia (p=1.23E-41 and p=1.29E-10, respectively), whereas individuals with isolated hypercholesterolemia had a marginal higher rate of MASLD and no difference in MASH rate compared to control group (p=0.019 and p=0.97, respectively). Among machine learning methods, feed-forward neural network had the best cross-validation MSE on the validation set. Circulating triglycerides, after body mass index (BMI), were the second strongest independent predictor of liver proton density fat fraction (PDFF) with the largest absolute mean Shapley additive explanation (SHAP) value. CONCLUSION: Isolated hypertriglyceridemia is the second strongest, after obesity, independent predictor of MASLD/MASH. Individuals with hypertriglyceridemia, but not with hypercholesterolemia, should be screened for liver disease.

Poor accuracy and sustainability of the first-step FIB4 EASL pathway for stratifying steatotic liver disease risk in the general population.

BACKGROUND AND AIMS: The European Association for the Study of the Liver introduced a clinical pathway (EASL CP) for screening significant/advanced fibrosis in people at risk of steatotic liver disease (SLD). We assessed the performance of the first-step FIB4 EASL CP in the general population across different SLD risk groups (MASLD, Met-ALD and ALD) and various age classes. METHODS: We analysed a total of 3372 individuals at risk of SLD from the 2017-2018 National Health and Nutrition Examination Survey (NHANES17-18), projected to 152.3 million U.S. adults, 300,329 from the UK Biobank (UKBB) and 57,644 from the Biobank Japan (BBJ). We assessed liver stiffness measurement (LSM) ≥8 kPa and liver-related events occurring within 3 and 10 years (3/10 year-LREs) as outcomes. We defined MASLD, MetALD, and ALD according to recent international recommendations. RESULTS: FIB4 sensitivity for LSM ≥ 8 kPa was low (27.7%), but it ranged approximately 80%-90% for 3-year LREs. Using FIB4, 22%-57% of subjects across the three cohorts were identified as candidates for vibration-controlled transient elastography (VCTE), which was mostly avoidable (positive predictive value of FIB4 ≥ 1.3 for LSM ≥ 8 kPa ranging 9.5%-13% across different SLD categories). Sensitivity for LSM ≥ 8 kPa and LREs increased with increasing alcohol intake (ALD>MetALD>MASLD) and age classes. For individuals aged ≥65 years, using the recommended age-adjusted FIB4 cut-off (≥2) substantially reduced sensitivity for LSM ≥ 8 kPa and LREs. CONCLUSIONS: The first-step FIB4 EASL CP is poorly accurate and feasible for individuals at risk of SLD in the general population. It is crucial to enhance the screening strategy with a first-step approach able to reduce unnecessary VCTEs and optimise their yield.

Non-invasive assessment of hepatic steatosis by ultrasound-derived fat fraction in individuals at high-risk for metabolic dysfunction-associated steatotic liver disease.

AIMS: Given the increasing number of individuals developing metabolic dysfunction-associated steatotic liver disease (MASLD) and the low rate of those with progressive liver disease, there is a pressing need to conceive affordable biomarkers to assess MASLD in general population settings. Herein, we aimed to investigate the performance of the ultrasound-derived fat fraction (UDFF) for hepatic steatosis in high-risk individuals. METHODS: A total of 302 Europeans with obesity, type 2 diabetes, or a clinical history of hepatic steatosis were included in the analyses. Clinical, laboratory, and imaging data were collected using standardized procedures during a single screening visit in Rome, Italy. Hepatic steatosis was defined by controlled attenuation parameter (CAP) or ultrasound-based Hamaguchi's score. UDFF performance for hepatic steatosis was estimated by the area under the receiver operating characteristic curve (AUC). RESULTS: Overall, median (IQR) UDFF was 12% (7-20). UDFF was positively correlated with CAP (ρ = 0.73, p < 0.0001) and Hamaguchi's score (ρ = 0.79, p < 0.0001). Independent predictors of UDFF were circulating triglycerides, alanine aminotransferase (ALT), and ultrasound-measured visceral adipose tissue (VAT). UDFF AUC was 0.89 (0.85-0.93) and 0.92 (0.88-0.95) for CAP- and ultrasound-diagnosed hepatic steatosis, respectively. UDFF AUC for hepatic steatosis was higher than those of fatty liver index (FLI), hepatic steatosis index (HSI), CAP-score (CAPS), and ALT (p < 0.0001). Lower age, ALT, and VAT were associated with discordance between UDFF and ultrasound. CONCLUSIONS: UDFF may be a simple and accurate imaging biomarker to assess hepatic steatosis and monitor changes in hepatic fat content over time or in response to therapeutic interventions beyond clinical trials.

Partitioned polygenic risk scores identify distinct types of metabolic dysfunction-associated steatotic liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses an excess of triglycerides in the liver, which can lead to cirrhosis and liver cancer. While there is solid epidemiological evidence of MASLD coexisting with cardiometabolic disease, several leading genetic risk factors for MASLD do not increase the risk of cardiovascular disease, suggesting no causal relationship between MASLD and cardiometabolic derangement. In this work, we leveraged measurements of visceral adiposity and identified 27 novel genetic loci associated with MASLD. Among these loci, we replicated 6 in several independent cohorts. Next, we generated two partitioned polygenic risk scores (PRS) based on the mechanism of genetic association with MASLD encompassing intra-hepatic lipoprotein retention. The two PRS suggest the presence of at least two distinct types of MASLD, one confined to the liver resulting in a more aggressive liver disease and one that is systemic and results in a higher risk of cardiometabolic disease.

IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids.

Steatotic liver disease (SLD) prevails as the most common chronic liver disease yet lack approved treatments due to incomplete understanding of pathogenesis. Recently, elevated hepatic and circulating interleukin 32 (IL-32) levels were found in individuals with severe SLD. However, the mechanistic link between IL-32 and intracellular triglyceride metabolism remains to be elucidated. We demonstrate in vitro that incubation with IL-32ß protein leads to an increase in intracellular triglyceride synthesis, while downregulation of IL32 by small interfering RNA leads to lower triglyceride synthesis and secretion in organoids from human primary hepatocytes. This reduction requires the upregulation of Phospholipase A2 group IIA (PLA2G2A). Furthermore, downregulation of IL32 results in lower intracellular type I collagen levels in di-lineage human primary hepatic organoids. Finally, we identify a genetic variant of IL32 (rs76580947) associated with lower circulating IL-32 and protection against SLD measured by non-invasive tests. These data suggest that IL32 downregulation may be beneficial against SLD.

Performance of non-invasive tests for liver fibrosis resolution after bariatric surgery.

BACKGROUND & AIMS: The value of non-invasive tests for monitoring the resolution of significant liver fibrosis after treatment is poorly investigated. We compared the performances of six non-invasive tests to predict the resolution of significant fibrosis after bariatric surgery. METHODS: Participants were individuals with obesity submitted to needle liver biopsy at the time of bariatric surgery, and 12 and/or 60 months after surgery. We calculated the fibrosis-4 index (FIB-4), NAFLD fibrosis score (NFS), AST to platelet ratio index (APRI), Hepatic fibrosis score (HFS), Fibrotic NASH index (FNI), and Liver risk score (LRS) at each time point, and compared their performances for predicting significant fibrosis (F ≥ 2) and its resolution following surgery. RESULTS: At baseline, 2436 patients had liver biopsy, including 261 (10.7 %) with significant fibrosis. Overall, 672 patients had pre- and post-operative biopsies (564 at M12 and 328 at M60). The fibrosis stage decreased at M12 and M60 (p < 0.001 vs M0). Resolution of significant fibrosis occurred in 58/121 (47.9 %) at M12 and 32/50 (64 %) at M60. The mean value of all tests decreased after surgery, except for FIB-4. Performances for predicting fibrosis resolution was higher at M60 than at M12 for all tests, and maximal at M60 for FNI and LRS: area under the curve 0.843 (95%CI 0.71-0.95) and 0.92 (95%CI 0.84-1.00); positive likelihood ratio 3.75 (95 % CI 1.33-10.59) and 4.58 (95 % CI 1.65-12.70), respectively. CONCLUSIONS: Results showed the value and limits of non-invasive tests for monitoring the evolution of liver fibrosis after an intervention. Following bariatric surgery, the best performances to predict the resolution of significant fibrosis were observed at M60 with tests combining liver and metabolic traits, namely FNI and LRS.

Predictors of controlled attenuation parameter in metabolic dysfunction.

BACKGROUND & AIMS: Hepatic fat content can be non-invasively estimated by controlled attenuation parameter (CAP) during transient elastography. The aim of this study was to examine the determinants and predictors of CAP values in individuals with metabolic dysfunction. METHODS: We enrolled 1230 consecutive apparently healthy individuals (Liver-Bible-2022 cohort) with ≥3 metabolic dysfunction features. CAP was measured by Fibroscan. CAP determinants and predictors were identified using backward stepwise analysis and introduced in generalized linear models. RESULTS: Participants were predominantly males (82.9%), mean age was 53.8 ± 6.4 years, 600 (48.8%) had steatosis (CAP ≥ 275 dB/m), and 27 had liver stiffness measurement (LSM) ≥ 8 kPa. CAP values correlated with LSM (p < 10-22). In multivariable analysis, fasting insulin and abdominal circumference (AC) were the main determinants of CAP (p < 10-6), together with body mass index (BMI; p < 10-4), age, diabetes, triglycerides, ferritin, and lower HDL and thyroid stimulating hormone (TSH; p < 0.05 for all). In a subset of 592 participants with thyroid hormone measurement, we found an association between higher free triiodothyronine levels, correlating with lower TSH, and CAP values, independent of TSH and of levothyroxine treatment (p = 0.0025). A clinical CAP score based on age, BMI, AC, HbA1c, ALT, and HDL predicted CAP ≥ 275 dB/m with moderate accuracy (AUROC = 0.73), which was better than that of the Fatty Liver Index and of ALT (AUROC = 0.70/0.61, respectively) and validated it in multiple cohorts. CONCLUSION: Abdominal adiposity and insulin resistance severity were the main determinants of CAP in individuals with metabolic dysfunction and may improve steatotic liver disease risk stratification. CAP values were modulated by the hypophysis-thyroid axis.

Endocrine aspects of metabolic dysfunction-associated steatotic liver disease (MASLD): Beyond insulin resistance.

While the association of metabolic dysfunction-associated steatotic liver disease (MASLD) with obesity and insulin resistance is widely appreciated, there are a host of complex interactions between the liver and other endocrine axes. While it can be difficult to definitively distinguish direct causal relationships and those attributable to increased adipocyte mass, there is substantial evidence of the direct and indirect effects of endocrine dysregulation on the severity of MASLD, with strong evidence that low levels of growth hormone, sex hormones, and thyroid hormone promote the development and progression of disease. The impact of steroid hormones, e.g. cortisol and dehydroepiandrosterone, and adipokines is much more divergent. Thoughtful assessment, based on individual risk factors and findings, and management of non-insulin endocrine axes is essential in the evaluation and management of MASLD. Multiple therapeutic options have emerged that leverage various endocrine axes to reduce the fibroinflammatory cascade in MASH.

Programmed cell death 1 genetic variant and liver damage in nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Programmed cell death 1/programmed cell death-ligand 1 (PD-1/PDL-1) axis has been reported to modulate liver inflammation and progression to hepatocellular carcinoma (HCC) in patients with nonalcoholic fatty liver disease (NAFLD). Here, we examined whether the PDCD1 variation is associated with NAFLD severity in individuals with liver biopsy. METHODS: We examined the impact of PDCD1 gene variants on HCC, as robust severe liver disease phenotype in UK Biobank participants. The strongest genetic association with the rs13023138 G>C variation was subsequently tested for association with liver damage in 2889 individuals who underwent liver biopsy for suspected nonalcoholic steatohepatitis (NASH). Hepatic transcriptome was examined by RNA-Seq in a subset of NAFLD individuals (n = 121). Transcriptomic and deconvolution analyses were performed to identify biological pathways modulated by the risk allele. RESULTS: The rs13023138 C>G showed the most robust association with HCC in UK Biobank (p = 5.28E-4, OR = 1.32, 95% CI [1.1, 1.5]). In the liver biopsy cohort, rs13023138 G allele was independently associated with severe steatosis (OR 1.17, 95% CI 1.02-1.34; p = .01), NASH (OR 1.22, 95% CI 1.09-1.37; p < .001) and advanced fibrosis (OR 1.26, 95% CI 1.06-1.50; p = .007). At deconvolution analysis, rs13023138 G>C allele was linked to higher hepatic representation of M1 macrophages, paralleled by upregulation of pathways related to inflammation and higher expression of CXCR6. CONCLUSIONS: The PDCD1 rs13023138 G allele was associated with HCC development in the general population and with liver disease severity in patients at high risk of NASH.

Fatty liver disease, heart rate and cardiac remodelling: Evidence from the UK Biobank.

BACKGROUND AND AIMS: Growing evidence supports an association between fatty liver disease (FLD) and cardiac dysfunction and remodelling, leading to cardiovascular disease and heart failure. Herein, we investigated the independent contribution of FLD to cardiac dysfunction and remodelling in participants from the UK Biobank with cardiac magnetic resonance (CMR) data available. METHODS: A total of 18 848 Europeans without chronic viral hepatitis and valvular heart diseases, with liver magnetic resonance imaging and CMR data were included in the analyses. Clinical, laboratory and imaging data were collected using standardized procedures. Multivariable regression models were used to test the association between FLD and CMR endpoints, after adjusting for several cardiometabolic risk factors. Linear regression models with regularization (Least Absolute Shrinkage and Selection Operator [LASSO], Ridge and Elastic Net) were used to generate predictive models for heart-related endpoints. RESULTS: FLD was independently associated with higher average heart rate, higher cardiac remodelling (higher eccentricity ratio and lower remodelling index), lower left and right ventricular volumes (end-systolic, end-diastolic and stroke volumes) as well as with lower left and right atrial maximal volumes (p < 0.001). FLD was the strongest positive predictor for average heart rate, followed by age, hypertension and type 2 diabetes. Male sex was the strongest positive predictor for eccentricity ratio followed by FLD, age, hypertension and BMI. For LV volumes, FLD was the strongest negative predictor along with age. CONCLUSIONS: FLD is an independent predictor of higher heart rate and early cardiac remodelling associated with reduced ventricular volumes.

Development and Validation of a Score for Fibrotic Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: Noninvasive assessment of histological features of nonalcoholic fatty liver disease (NAFLD) has been an intensive research area over the last decade. Herein, we aimed to develop a simple noninvasive score using routine laboratory tests to identify, among individuals at high risk for NAFLD, those with fibrotic nonalcoholic steatohepatitis (NASH) defined as NASH, NAFLD activity score ≥4, and fibrosis stage ≥2. METHODS: The derivation cohort included 264 morbidly obese individuals undergoing intraoperative liver biopsy in Rome, Italy. The best predictive model was developed and internally validated using a bootstrapping stepwise logistic regression analysis (2000 bootstrap samples). Performance was estimated by the area under the receiver operating characteristic curve (AUROC). External validation was assessed in 3 independent European cohorts (Finland, n = 370; Italy, n = 947; England, n = 5368) of individuals at high risk for NAFLD. RESULTS: The final predictive model, designated as Fibrotic NASH Index (FNI), combined aspartate aminotransferase, high-density lipoprotein cholesterol, and hemoglobin A1c. The performance of FNI for fibrotic NASH was satisfactory in both derivation and external validation cohorts (AUROC = 0.78 and AUROC = 0.80-0.95, respectively). In the derivation cohort, rule-out and rule-in cutoffs were 0.10 for sensitivity ≥0.89 (negative predictive value, 0.93) and 0.33 for specificity ≥0.90 (positive predictive value, 0.57), respectively. In the external validation cohorts, sensitivity ranged from 0.87 to 1 (negative predictive value, 0.99-1) and specificity from 0.73 to 0.94 (positive predictive value, 0.12-0.49) for rule-out and rule-in cutoff, respectively. CONCLUSION: FNI is an accurate, simple, and affordable noninvasive score which can be used to screen for fibrotic NASH in individuals with dysmetabolism in primary health care.

Effect of common genetic variants on the risk of cirrhosis in non-alcoholic fatty liver disease during 20 years of follow-up.

BACKGROUND AND AIMS: Several genotypes associate with a worse histopathological profile in patients with non-alcoholic fatty liver disease (NAFLD). Whether genotypes impact long-term outcomes is unclear. We investigated the importance of PNPLA3, TM6SF2, MBOAT7 and GCKR genotype for the development of severe outcomes in NAFLD. METHOD: DNA samples were collected from 546 patients with NAFLD. Advanced fibrosis was diagnosed by liver biopsy or elastography. Non-alcoholic steatohepatitis (NASH) was histologically defined. Additionally, 5396 controls matched for age, sex and municipality were identified from population-based registers. Events of severe liver disease and all-cause mortality were collected from national registries. Hazard ratios (HRs) adjusted for age, sex, body mass index and type 2 diabetes were estimated with Cox regression. RESULTS: In NAFLD, the G/G genotype of PNPLA3 was associated with a higher prevalence of NASH at baseline (odds ratio [OR] 3.67, 95% CI = 1.66-8.08), but not with advanced fibrosis (OR 1.81, 95% CI = 0.79-4.14). After up to 40 years of follow-up, the PNPLA3 G/G genotype was associated with a higher rate of severe liver disease (adjusted hazard ratio [aHR] 2.27, 95% CI = 1.15-4.47) compared with the C/C variant. NAFLD patients developed cirrhosis at a higher rate than controls (aHR 9.00, 95% CI = 6.85-11.83). The PNPLA3 G/G genotype accentuated this rate (aHR 23.32, 95% = CI 9.14-59.47). Overall mortality was not affected by any genetic variant. CONCLUSION: The PNPLA3 G/G genotype is associated with an increased rate of cirrhosis in NAFLD. Our results suggest that assessment of the PNPLA3 genotype is of clinical relevance in patients with NAFLD to individualize monitoring and therapeutic strategies.

Rare ATG7 genetic variants predispose patients to severe fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disorders and has a strong heritable component. The aim of this study was to identify new loci that contribute to severe NAFLD by examining rare variants. METHODS: We performed whole-exome sequencing in individuals with NAFLD and advanced fibrosis or hepatocellular carcinoma (n = 301) and examined the enrichment of likely pathogenic rare variants vs. the general population. This was followed by validation at the gene level. RESULTS: In patients with severe NAFLD, we observed an enrichment of the p.P426L variant (rs143545741 C>T; odds ratio [OR] 5.26, 95% CI 2.1-12.6; p = 0.003) of autophagy-related 7 (ATG7), which we characterized as a loss-of-function, vs. the general population, and an enrichment in rare variants affecting the catalytic domain (OR 13.9; 95% CI 1.9-612; p = 0.002). In the UK Biobank cohort, loss-of-function ATG7 variants increased the risk of cirrhosis and hepatocellular carcinoma (OR 3.30; 95% CI 1.1-7.5 and OR 12.30, 95% CI 2.6-36, respectively; p <0.001 for both). The low-frequency loss-of-function p.V471A variant (rs36117895 T>C) was also associated with severe NAFLD in the clinical cohort (OR 1.7; 95% CI 1.2-2.5; p = 0.003), predisposed to hepatocellular ballooning (p = 0.007) evolving to fibrosis in the Liver biopsy cohort (n = 2,268), and was associated with liver injury in the UK Biobank (aspartate aminotransferase levels, p <0.001), with a larger effect in severely obese individuals in whom it was linked to hepatocellular carcinoma (p = 0.009). ATG7 protein localized to periportal hepatocytes, particularly in the presence of ballooning. In the Liver Transcriptomic cohort (n = 125), ATG7 expression correlated with suppression of the TNFα pathway, which was conversely upregulated in p.V471A carriers. CONCLUSIONS: We identified rare and low-frequency ATG7 loss-of-function variants that promote NAFLD progression by impairing autophagy and facilitating ballooning and inflammation. LAY SUMMARY: We found that rare mutations in a gene called autophagy-related 7 (ATG7) increase the risk of developing severe liver disease in individuals with dysmetabolism. These mutations cause an alteration in protein function and impairment of self-renewal of cellular content, leading to liver damage and inflammation.

A Genetic and Metabolic Staging System for Predicting the Outcome of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is an emerging cause of liver-related events (LREs). Here, we have assessed the ability of a composite score based on clinical features, metabolic comorbidities, and genetic variants to predict LREs. A total of 546 consecutive patients with NAFLD were recruited and stratified according to the fibrosis-4 (FIB-4) index. LREs were defined as occurrence of hepatocellular carcinoma or hepatic decompensation. Cox regression multivariate analysis was used to identify baseline variables associated with LREs. The UK Biobank was used as the validation cohort, and severe liver disease (incidence of cirrhosis, decompensated liver disease, hepatocellular carcinoma, and/or liver transplantation) was used as the outcome. LREs were experienced by 58 patients, only one of whom was in the cohort of patients with a FIB-4 score < 1.3. Multivariate Cox regression analysis of 229 patients with a FIB-4 score ≥ 1.3 highlighted clinical variables independently associated with the development of LREs, including older age, low platelet count, low albumin, low high-density lipoprotein cholesterol, certain genetic factors, and interactions between genetic factors and sex or diabetes. The area under the curve (AUC) for the model was 0.87 at 1, 3, and 5 years. Our novel Genetic and Metabolic Staging (GEMS) scoring system was derived from the Cox model linear predictor, ranked from 0 to 10, and categorized into five classes (0-5, 5-6, 6-7, 7-8, and 8-10). The risk of LREs increased from 4% in patients in the best class (GEMS score 0-5) to 91% in the worst (GEMS score 8-10). GEMS score was associated with incident severe liver disease in the study population (hazard ratio, 1.56; 95% confidence interval, 1.48-1.65; P < 0.001) as well as in the UK Biobank cohort where AUCs for prediction of severe liver disease at 1, 3, and 5 years were 0.70, 0.69, and 0.67, respectively. Conclusion: The novel GEMS scoring system has an adequate ability to predict the outcome of patients with NAFLD.

PSD3 downregulation confers protection against fatty liver disease.

Fatty liver disease (FLD) is a growing health issue with burdening unmet clinical needs. FLD has a genetic component but, despite the common variants already identified, there is still a missing heritability component. Using a candidate gene approach, we identify a locus (rs71519934) at the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene resulting in a leucine to threonine substitution at position 186 of the protein (L186T) that reduces susceptibility to the entire spectrum of FLD in individuals at risk. PSD3 downregulation by short interfering RNA reduces intracellular lipid content in primary human hepatocytes cultured in two and three dimensions, and in human and rodent hepatoma cells. Consistent with this, Psd3 downregulation by antisense oligonucleotides in vivo protects against FLD in mice fed a non-alcoholic steatohepatitis-inducing diet. Thus, translating these results to humans, PSD3 downregulation might be a future therapeutic option for treating FLD.