Dissecting causal relationships between inflammatory factors, plasma metabolites, and nonalcoholic fatty liver disease: a mediating Mendelian randomization study.

ABSTRACTBackground and AimsObservational studies suggest a link between cholecystectomy and nonalcoholic fatty liver disease (NAFLD), but whether cholecystectomy is an independent causal risk factor and the underlying mechanisms remain unclear. Inflammation, a key driver of NAFLD, is a potential mediator of this association. The aims of this study are to determine the independent causal effect of cholecystectomy on NAFLD and to quantify the mediating effect of inflammatory factors in the causal pathway of cholecystectomy and NAFLD.MethodsWe employed a comprehensive Mendelian randomization (MR) framework. Multivariable MR (MVMR) assessed the independent causal effect of cholecystectomy on NAFLD after adjusting for eight common NAFLD risk factors (five lipid levels, body mass index, type 2 diabetes, and hypertension). Second, mediation MR evaluated the potential mediating role of 91 circulating inflammatory proteins in the cholecystectomy–NAFLD pathway. Genetic instruments were derived from European‐ancestry genome‐wide association studies (GWAS). Sensitivity analyses assessed pleiotropy, heterogeneity, and robustness.ResultsCholecystectomy demonstrated a significant independent causal effect on increased NAFLD risk after adjustment for confounders (OR = 12.988, 95% CI = 1.998–84.420, p = 0.007). Among 91 inflammatory proteins, tumor necrosis factor receptor superfamily member 9 (TNFRSF9) mediated 10.76% of the increased risk of NAFLD associated with cholecystectomy (Mediator effect β = 0.522, 95% CI = 0.124–0.919, p = 0.01).ConclusionCholecystectomy is an independent risk factor for NAFLD. Circulating TNFRSF9 as a key inflammatory mediator explains ~10.76% of the increased NAFLD risk following cholecystectomy. These findings highlight TNFRSF9 as a potential biomarker and therapeutic target for preventing postcholecystectomy NAFLD, although further validation is required.

ABSTRACTIncreasingly, emerging research evidence has demonstrated that nonalcoholic fatty liver disease (NAFLD) is a disease closely associated with systemic inflammation. However, the specific upstream inflammatory factors engaged in the pathogenesis of NAFLD remain unclear. Our study aimed to identify the inflammatory regulators causally associated with NAFLD pathogenesis through Mendelian randomisation. A two‐sample Mendelian randomisation method was applied to analyse the causal association between 91 circulating inflammatory proteins and NAFLD. Data on circulating inflammatory proteins were derived from samples of European ancestry (14,824 samples) and NAFLD data were obtained from the FinnGen consortium (2025 cases and 284,826 controls). Instrumental variables were selected from the genetic variance and F‐statistics were calculated to avoid bias. We adopted the random‐effects inverse variance weighting (IVW) method as our primary analytical approach. Supplementary analyses were also implemented, including weighted median, MR‐Egger and weighted mode. Moreover, we conducted pleiotropy and heterogeneity analyses to validate the accuracy of the findings. The application of Mendelian randomisation analysis identified four inflammatory factors that might be causally associated with NAFLD at the genetic level. Elevated levels of eotaxin (or = 1.27, 95% CI: 1.05–1.53, p = 0.014), osteoprotegerin (OPG) (or = 1.29, 1.03–1.60, p = 0.023) and TNFRSF9 (or = 1.32, 95% CI: 1.06–1.64, p = 0.014) may be causally related to an increasing risk of NAFLD. Conversely, heightened leukaemia inhibitory factor (LIF) levels (or = 0.63, 0.44–0.92, p = 0.016) were linked to a lower risk of NAFLD onset. There was no causal relationship between levels of other circulating inflammatory proteins and NAFLD. Our analysis uncovered four upstream inflammatory factors genetically associated with the pathogenesis of NAFLD. These results highlight the potential involvement of inflammation in NAFLD, which provides partial insights for further research in this field in the future.

Nonalcoholic fatty liver disease (NAFLD), an increasingly prevalent paediatric disorder, is diagnosed and managed not only by both pediatric gastroenterologists/hepatologists but also frequently by the general pediatrician. This article updates recent advances in diagnostic and therapeutic approach, which may be applied to everyday practice. Diagnosis of NAFLD takes into account the risk factor profile and is a diagnosis of exclusion. Techniques such as transient elastography and specific biomarkers aimed at improving diagnosis and monitoring of NAFLD need further validation in the pediatric population. Defining the risk to develop cirrhosis seems to be of primary importance already in childhood and a combination of genetic, clinical, and environmental factors can help in monitoring and making decisions on therapy. Weight reduction therapy should be the aim of treatment approach, but the compliance is poor and pharmacological treatment would be helpful; docosahexaenoic acid, some probiotics, and vitamin E are to be considered, but evidence is not sufficient to recommend widespread use.

Aim: The cytokine receptor tumor necrosis factor receptor superfamily member 9 (TNFRSF9) is mainly considered to be a co-stimulatory activation marker in hematopoietic cells. Several preclinical models have shown a dramatic beneficial effect of treatment approaches targeting TNFRSF9 with agonistic antibodies. However, preliminary clinical phase I/II studies were stopped after the occurrence of several severe deleterious side effects. In a previous study, it was demonstrated that TNFRSF9 was strongly expressed by reactive astrocytes in primary central nervous system (CNS) tumors, but was largely absent from tumor or inflammatory cells. The aim of the present study was to address the cellular source of TNFRSF9 expression in the setting of human melanoma brain metastasis, a highly immunogenic tumor with a prominent tropism to the CNS. Methods: Melanoma brain metastasis was analyzed in a cohort of 78 patients by immunohistochemistry for TNFRSF9 and its expression was correlated with clinicopathological parameters including sex, age, survival, tumor size, number of tumor spots, and BRAF V600E expression status. Results: Tumor necrosis factor receptor superfamily member 9 was frequently expressed independently on both melanoma and endothelial cells. In addition, TNFRSF9 was also present on smooth muscle cells of larger vessels and on a subset of lymphomonocytic tumor infiltrates. No association between TNFRSF9 expression and patient survival or other clinicopathological parameters was seen. Of note, several cases showed a gradual increase in TNFRSF9 expression on tumor cells with increasing distance from blood vessels, an observation that might be linked to hypoxia-driven TNFRSF9 expression in tumor cells. Conclusion: The findings indicate that the cellular source of TNFRSF9 in melanoma brain metastasis largely exceeds the lymphomonocytic pool, and therefore further careful (re-) assessment of potential TNFRSF9 functions in cell types other than hematopoietic cells is needed. Furthermore, the hypothesis of hypoxia-driven TNFRSF9 expression in brain metastasis melanoma cells requires further functional testing.

See article in J. Gastroenterol. Hepatol. 2010; 25: 1687–1691.

Biomarkers in Fatty Liver Disease—Here is the Skinny

This study explored the causal connection among the plasma lipidome, nonalcoholic fatty liver disease (NAFLD), and potential metabolome mediators through Mendelian randomization (MR). We obtained summary statistics for 179 plasma lipidome traits (N = 7,174), 1,400 plasma metabolome traits (N = 8,299), and one NAFLD trait from publicly available genome-wide association studies. A two-sample MR analysis was conducted to infer causality. Additionally, multiple sensitivity analyses were conducted to assess the heterogeneity, horizontal pleiotropy, and robustness of the MR findings. MetaboAnalyst 6.0 was used for the pathway analysis of the identified lipids and metabolites. Furthermore, we used mediation analysis to assess whether the effect of plasma lipidome on NAFLD was mediated by plasma metabolome. The MR analysis predicted a genetically determined causal relationship between plasma lipidomes and NAFLD. No compelling proof was found that genetically predicted NAFLD influenced the risk of the five plasma lipidomes mentioned earlier. Based on established causal relationships between lipids and metabolites, we identified that eight metabolic pathways are closely associated with NAFLD. Our mediation analysis revealed six mediation relationships, indicating the causal pathway from plasma lipids to NAFLD mediated by five specific metabolites. No potential pleiotropy was found in the sensitivity analysis. In summary, our study identified causal relationships between plasma lipidomes, plasma metabolomes, and NAFLD. Certainly, the impact of plasma lipidomes on NAFLD is not limited to plasma metabolomes, indicating a need to further investigate into other possible mediators. These identified factors may become new biomarkers of the NAFLD contributing to its prevention, diagnosis, and treatment.

Circulating adipokine concentrations and risk of non-alcoholic fatty liver disease: A Mendelian randomization study

We aimed to elucidate the frequency of polymorphic genotypes and alleles of patatin-like phospholipase domain containing 3 rs738409 polymorphism and its possible associations with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis in a cohort from Turkey.We enrolled 200 patients diagnosed with NAFLD and genotyped for rs738409 I148M polymorphism by real-time polymerase chain reaction, particularly by melting curve analysis. SPSS analysis software was used for statistical significance. Continuous variable values were expressed as mean ± standard deviation. Significant statistical level was chosen as p = 0.05.Our results demonstrate in a cohort from Turkey that rs738409 C > G polymorphism (I148M) of patatin-like phospholipase domain containing 3 gene is significantly able to affect individuals to have NAFLD in unadjusted regression model.Consistent with the previous studies in other populations, our study group showed a significantly higher risk of having NAFLD in unadjusted regression model but not in the adjusted model indicating that non-genetic factors such as age and sex may be responsible for the association. However, independent studies need to validate our findings with a larger group of NAFLD patients, as well as in different ethnic cohorts.

Answer questions and earn CME Content available: Author Interview and Audio Recording India is the seventh largest and second most populous country in the world. It has a rapidly developing economy with an estimated gross domestic product of US $2.87 trillion. Easy access to calorie-dense food and sedentary lifestyle together with the modern epidemics of diabetes mellitus (DM) and obesity have catapulted nonalcoholic fatty liver disease (NAFLD) into a substantial public health problem in India as in other parts of the world. NAFLD has emerged as one of the leading causes of cirrhosis, hepatocellular carcinoma (HCC), and liver transplant in India.1 Given its enormous population, the burden of NAFLD in India is likely to be substantial, which may significantly impact the limited health care resources in the country. The prevalence of NAFLD among the general population in India ranges from 9% to 53%.1, 2 One of the caveats in interpreting epidemiological data on NAFLD from India is that many of the studies have been conducted in the hospital setting and are therefore liable to referral bias. Although differences in diagnostic techniques for NAFLD may partly account for the wide variation in reported prevalence, a possible rural-urban divide and geographical variation are evident from the available data (Fig. 1). Most studies from urban centers have reported a higher prevalence as compared with those that cater to a largely rural population. One of the earlier population-based studies from India that showed a prevalence rate of 8.7% in predominantly nonobese populations was from rural West Bengal (Table 1).1 More recently, a population-based study from coastal south India reported an overall NAFLD prevalence rate of 49.8%; urban domicile was found to be associated with a higher risk for NAFLD after adjusting for sex, body mass index (BMI), DM, and metabolic syndrome (adjusted odds ratio [OR], 1.21; P = 0.048).3 As a part of the ongoing community-based Prospective Urban Rural Epidemiology (PURE) cohort study in north India, prevalence of NAFLD was found to be higher in urban communities (53.7%) in comparison with rural communities (30.2%) (P < 0.001).4 Among the high-risk groups, prevalence has been reported to be higher among those with type 2 DM, prediabetes, obesity, and metabolic syndrome.1 One of the multicenter studies across 101 Indian cities estimated the prevalence rate of NAFLD as 56.5% (n = 522) among 924 patients with type 2 DM.5 Further worrisome are the recent data showing a high prevalence of NAFLD in obese Indian children. More important than the mere presence of fatty liver is the prevalence of progressive nonalcoholic steatohepatitis (NASH) with or without hepatic fibrosis that adds to the significant liver disease and extrahepatic disease burden. Even though earlier data had suggested a mild liver histology in Indian patients with NAFLD,1 a recent retrospective review of 1000 liver biopsy-proven patients with NAFLD showed histological NASH in more than 60% of patients and advanced fibrosis (≥F3) in 35% of patients.6 Further, an interim analysis of an ongoing real-life, multicentric observational study (Indian Consortium on NAFLD [ICON-D]) in approximately 3000 patients with NAFLD showed the presence of significant fibrosis in 19%, 21%, and 29% of patients as assessed by Fibrosis-4 (FIB-4), aspartate aminotransferase (AST)-to-platelet ratio index (APRI), and FibroScan, respectively.7 The presence of metabolic risk factors and data on explant pathology also suggest NAFLD to be the predominant cause of cryptogenic cirrhosis and cryptogenic HCC in India (Table 2). Similar to data from the West, a recent Indian study has also shown a trend of NASH as the increasing cause of HCC over the years.8 Data from India corroborate that NAFLD is associated with several extrahepatic conditions, such as cardiovascular disease, chronic kidney disease, polycystic ovarian syndrome, obstructive sleep apnoea, vitamin D deficiency, and hypothyroidism.1 NAFLD also has been shown to affect quality of life, particularly in overweight/obese patients with NAFLD. As in the rest of the world, both environmental and genetic factors have been shown to be involved in the pathogenesis of Indian patients with NAFLD. Globally, multiethnic studies have suggested that Indians are more predisposed to insulin resistance and its consequences, including NAFLD. Most of the data from India suggest the presence of insulin resistance in patients with NAFLD; however, a small study suggested occurrence of NAFLD without insulin resistance.9 Earlier data from India had suggested certain subtle differences between Indian patients with NAFLD and their Western counterparts, with Indian patients having lower BMI and fewer cases of morbid obesity, diabetes, hypertension, or metabolic syndrome.1 However, most patients (85%-90%) with NAFLD in India are still overweight or obese as per the Asia-Pacific cutoffs for BMI, and around 10% to 15% of the patients are "lean" with a normal BMI (<23 kg/m2) (Table 1). The interim results of the ongoing real-life study from India (ICON-D) in approximately 3500 patients (mean BMI, 27.6 ± 5.7 kg/m2) showed the presence of overweight (BMI, 23-24.9 kg/m2) in 16%, obesity (BMI ≥ 25 kg/m2) in 73%, and lean NAFLD (BMI < 23 kg/m2) in 10.6% of patients7 (Table 1). Overall, metabolic syndrome was present in 43%, and at least one metabolic risk factor was present in 93% of patients with NAFLD (the commonest being central obesity in 84%).7 Indian data in lean patients with NAFLD suggest that although their total body fat is comparable with lean individuals without NAFLD, they are metabolically unhealthy, with an expanded visceral adipose tissue mass similar to overweight or obese patients with NAFLD.1 In addition to metabolic risk factors, studies from India have also suggested the role of small intestinal bacterial overgrowth, endotoxemia, and toll-like receptor expression in the pathogenesis of NAFLD.10 Dietary constituents and cooking medium vary greatly in different geographic regions of India. A substantial proportion of Indians consume a purely vegetarian diet. The influence of diet on the risk for NAFLD is an underexplored area. A small study from the rural area of Maharashtra state suggested that the risk for NAFLD did not differ between those consuming vegetarian and mixed diets.11 Among the genetic studies, earlier Indian data had suggested the lack of association of NAFLD with HFE gene mutations.1 PNPLA3 and TM6SF2 gene polymorphisms have been shown to be closely associated with prevalence and severity of NAFLD in India. A recent exome-wide association study showed a novel association of nuclear polymorphism rs4788084 with hepatic fat content, which regulates the expression of IL-27, an immune-regulatory gene.12 A novel variant of phosphatidylethanolamine N-methyltransferase (involved in fatty acid metabolism), identified using whole-exome sequencing, was shown to confer a three times greater risk for NAFLD in lean individuals.13 There are some data that suggest that the genetic predisposition to NAFLD may vary according to ancestral ethnicity. A recent study found that the TM6SF2 variant (rs58542926) was significantly associated with NAFLD susceptibility in individuals from South Indian ethnicity (OR, 1.9; 95% confidence interval [CI]: 1.5-3), while the PNPLA3 variant (rs2281135) conferred a higher risk for NAFLD in those of North East Indian ancestry (OR, 2.7; 95% CI: 1.37-5.3).14 Concomitant variants in both genes were common in patients with NAFLD irrespective of ethnicity, and the authors concluded that the presence of an additional variant compounded the risk for NAFLD.14 The diagnosis and treatment of patients with NAFLD in India has largely been on the same lines as suggested by various international societies and Indian National Association for the Study of the Liver.1 However, because of the limitations in resources, separate guidelines have been suggested for the management and referral of patients from primary health care level to secondary and tertiary care levels.15, 16 Among the various noninvasive scores, APRI has been found to be more accurate than FIB-4 in ruling out significant fibrosis in the community setting. True to the concept of population-based differences, different cutoffs for the Indian population have been suggested for controlled attenuation parameter, FIB-4, and FibroScan-AST scores for the assessment of hepatic steatosis, hepatic fibrosis, and NASH.17, 18 The large real-life data from the country suggest that in clinical practice, liver biopsy is not a well-accepted modality for determining disease severity, and the practice of liver biopsy in NAFLD in India may be restricted to only tertiary care centers.7 Lifestyle interventions are the primary modality for the management of NAFLD and have been shown to improve biochemical and histological outcomes in Indian patients.1 A study with paired liver biopsies in 58 morbidly obese patients showed improvement in all histological parameters of NAFLD, including steatosis, ballooning, lobular inflammation, NAFLD Activity Score, and fibrosis, at 1-year follow-up after bariatric surgery.19 Of various endoscopic bariatric therapies, only a small amount of data for eight patients described the utility of intragastric balloon for inducing weight loss in morbidly obese patients with cirrhosis (4-cryptogenic cirrhosis) on the transplant wait list.20 Pharmacotherapy in patients with NASH was earlier restricted to the use of vitamin E and pioglitazone.1 However, based on the recent data, the drug controller general of India has approved the use of saroglitazar, a dual peroxisome proliferator-activated receptor α/γ agonist, in a dosage of 4 mg/day for use in patients with NASH with F1-3 fibrosis.21 Although not recommended, the data on the use of vitamin D supplementation, high-potency multistrain probiotic, glucagon-like peptide-1 (GLP-1) agonists, and sodium-glucose co-transporter-2 (SGLT-2) inhibitors also have been encouraging in Indian patients with NAFLD. NASH-related decompensated cirrhosis and HCC are leading indications for liver transplant in India; however, there is a paucity of Indian data on transplant outcomes in patients with NASH. Given the high prevalence of NAFLD among the general population in India, donor steatosis in the living donor liver transplantation program is also a vexing problem.22 With NAFLD being a lifestyle disease, efforts for prevention and control are required not only at the individual and family level but also at the government and administrative level. The recent integration of NAFLD into the National Program on Prevention and Control of Cancer, Diabetes, Cardiovascular disease and Stroke by the Ministry of Health and Family Welfare of India is an encouraging step in this direction.16 In fact, India has become the first country to include NAFLD in one of its national programs. NAFLD has emerged as a major public health issue in India that is responsible for significant burden of hepatic and extrahepatic disease. Education of healthy lifestyle to children and adolescents in schools and colleges may be the need of the hour. Efforts are also required to change the perception of both physicians and the public toward this ongoing silent pandemic. Although much progress has been witnessed in the last one or two decades in NAFLD research in India, a lot more needs to be done.

Assessing causal relationship between non-alcoholic fatty liver disease and risk of atrial fibrillation

Patients with Sjögren's syndrome (SS) are at significantly increased risk of developing non-Hodgkin lymphoma (NHL). However, effective biomarkers to identify the subgroup of SS patients who will progress to lymphoma are currently lacking. This study aims to elucidate the causal mechanisms linking SS to NHL and to identify circulating inflammatory protein biomarkers for risk stratification. Employing a Mendelian randomization framework and leveraging summary data from large-scale genome-wide association studies, we systematically evaluated the causal effect of SS on NHL (total effect) and the potential mediating roles of 91 circulating inflammatory proteins. The analysis comprised three core steps: total effect assessment, mediator screening, and validation. Genetic evidence confirms that SS is a causal risk factor for NHL (OR = 1.400, 95% CI 1.012-1.938). Among the 91 candidate proteins, 14 showed a causal association with SS. Further validation, however, identified tumor necrosis factor receptor superfamily member 9 (TNFRSF9) as the sole protein that was both significantly upregulated by SS and independently capable of increasing NHL risk (OR = 1.797, 95% CI 1.087-2.970). Mediation analysis quantified its clinical relevance: TNFRSF9 mediates approximately 18% of the SS-associated lymphoma risk. This study not only establishes a causal relationship between SS and NHL but, more importantly, identifies circulating TNFRSF9 as a key functional biomarker linking autoimmunity to lymphomagenesis. This finding suggests that circulating TNFRSF9 levels emerge as a candidate biomarker worthy of further investigation. Future prospective clinical studies are needed to correlate serum TNFRSF9 levels with lymphoma development. Key Points • First genetic evidence establishing a causal link between Sjögren's syndrome and increased risk of non-Hodgkin lymphoma using Mendelian randomization. • TNFRSF9 is identified as a key inflammatory mediator, upregulated by Sjögren's syndrome and independently driving lymphoma risk. • Mediation analysis quantifies that TNFRSF9 accounts for approximately 18% of the total causal effect of Sjögren's syndrome on lymphoma. • A novel biomarker and potential therapeutic target, circulating TNFRSF9 may enable risk stratification in Sjögren's syndrome patients.

Non-alcoholic fatty liver disease: Not time for an obituary just yet!

Nonalcoholic fatty liver disease (NAFLD) is defined by liver fat accumulation exceeding 5% in individuals who do not consume significant amounts of alcohol. This condition can advance to more severe outcomes, including fibrosis, cirrhosis, and liver cancer. Although numerous factors contribute to the progression of NAFLD, the influence of psychological elements, especially anxiety, remains inadequately explored. This study applied Mendelian randomization (MR) using genome-wide association data from 4761 NAFLD cases and 373 227 controls to investigate the causal relationship between psychological factors and NAFLD. We conducted both multivariable and mediation MR analyses to determine how anxiety influences NAFLD through pathways involving gut microbiota and metabolites. Furthermore, we examined datasets related to anxiety and NAFLD from the Gene Expression Omnibus, identified differentially expressed genes, and conducted enrichment analyses on the genes shared between these two conditions. The MR analysis established a direct causal relationship between genetically predicted anxiety and the development of NAFLD (β=0.229, 95% confidence interval = 1.11-1.41, P = 0.0002). This association was confirmed by multivariable MR, independent of BMI and type 2 diabetes. Mediation MR revealed that specific metabolites and fatty acid-related gut microbiota mediate the relationship between anxiety and NAFLD. Additionally, enrichment analysis confirmed the involvement of fatty acids in genes common to both anxiety and NAFLD. This study suggests that genetically predicted anxiety contributes to the development of NAFLD by influencing specific gut microbiota and metabolites, underscoring the vital role of mental health in mitigating NAFLD risk.

NAFLD vs. MAFLD – It is not the name but the disease that decides the outcome in fatty liver