Integrative common and rare variant analyses provide insights into the genetic architecture of liver cirrhosis.

We report a multi-ancestry genome-wide association study on liver cirrhosis and its associated endophenotypes, alanine aminotransferase (ALT) and γ-glutamyl transferase. Using data from 12 cohorts, including 18,265 cases with cirrhosis, 1,782,047 controls, up to 1 million individuals with liver function tests and a validation cohort of 21,689 cases and 617,729 controls, we identify and validate 14 risk associations for cirrhosis. Many variants are located near genes involved in hepatic lipid metabolism. One of these, PNPLA3 p.Ile148Met, interacts with alcohol intake, obesity and diabetes on the risk of cirrhosis and hepatocellular carcinoma (HCC). We develop a polygenic risk score that associates with the progression from cirrhosis to HCC. By focusing on prioritized genes from common variant analyses, we find that rare coding variants in GPAM associate with lower ALT, supporting GPAM as a potential target for therapeutic inhibition. In conclusion, this study provides insights into the genetic underpinnings of cirrhosis.

Genetics of liver disease in adults.

Chronic liver disease stands as a significant global health problem with an estimated 2 million annual deaths across the globe. Combining the use of next-generation sequencing technologies with evolving knowledge in the interpretation of genetic variation across the human genome is propelling our understanding, diagnosis, and management of both rare and common liver diseases. Here, we review the contribution of risk and protective alleles to common forms of liver disease, the rising number of monogenic diseases affecting the liver, and the role of somatic genetic variants in the onset and progression of oncological and non-oncological liver diseases. The incorporation of genomic information in the diagnosis and management of patients with liver disease is driving the beginning of a new era of genomics-informed clinical hepatology practice, facilitating personalized medicine, and improving patient care.

Advancing diagnosis and management of liver disease in adults through exome sequencing.

BACKGROUND: Whole-exome sequencing (WES) is an effective tool for diagnosis in patients who remain undiagnosed despite a comprehensive clinical work-up. While WES is being used increasingly in pediatrics and oncology, it remains underutilized in non-oncological adult medicine, including in patients with liver disease, in part based on the faulty premise that adults are unlikely to harbor rare genetic variants with large effect size. Here, we aim to assess the burden of rare genetic variants underlying liver disease in adults at two major tertiary referral academic medical centers. METHODS: WES analysis paired with comprehensive clinical evaluation was performed in fifty-two adult patients with liver disease of unknown etiology evaluated at two US tertiary academic health care centers. FINDINGS: Exome analysis uncovered a definitive or presumed diagnosis in 33% of patients (17/52) providing insight into their disease pathogenesis, with most of these patients (12/17) not having a known family history of liver disease. Our data shows that over two-thirds of undiagnosed liver disease patients attaining a genetic diagnosis were being evaluated for cholestasis or hepatic steatosis of unknown etiology. INTERPRETATION: This study reveals an underappreciated incidence and spectrum of genetic diseases presenting in adulthood and underscores the clinical value of incorporating exome sequencing in the evaluation and management of adults with liver disease of unknown etiology. FUNDING: S.V. is supported by the NIH/NIDDK (K08 DK113109 and R01 DK131033-01A1) and the Doris Duke Charitable Foundation Grant #2019081. This work was supported in part by NIH-funded Yale Liver Center, P30 DK34989.

Protocol for enrichment, purification, and cytocentrifugation of mouse liver endothelial cells.

Liver endothelial cells (LECs) are critical in maintaining liver homeostasis. To understand the mechanistic processes occurring in these cells, high-quality isolation protocols must be in place. Here, we present a protocol for LEC enrichment, subsequent LEC purification using fluorescence-assisted cell sorting, and cytocentrifugation of sorted LECs for imaging. We describe steps for isolation of LEC-enriched population from mouse livers, immunolabeling and sorting, and cytospin and immunostaining. We then mention procedures for downstream analysis. For complete details on the use and execution of this protocol, please refer to Drzewiecki et al. (2021).1.

Hepatology Genome Rounds: An interdisciplinary approach to integrate genomic data into clinical practice.

In the last decade, the utility of whole-exome sequencing in uncovering genetic aetiologies of a variety of liver diseases has been demonstrated. These new diagnoses have guided the management, treatment, and prognostication of previously undiagnosed patients, largely thanks to improved insight into the underlying pathogenesis of their conditions. Despite its clear benefits, the uptake of genetic testing by hepatologists has been limited, in part due to limited prior genetic training and/or opportunities for continuing education. Herein, we show that Hepatology Genome Rounds, an interdisciplinary forum highlighting hepatology cases of clinical interest and educational value, are an important venue for integrating genotypic and phenotypic information to enable accurate diagnosis and appropriate management, dissemination of genomic knowledge within the field of hepatology, and ongoing education to providers and trainees in genomic medicine. We describe our single-centre experience and discuss practical considerations for clinicians interested in launching such a series. We foresee that this format will be adopted at other institutions and by additional specialties, with the aim of further incorporating genomic information into clinical medicine.

Genomic analysis of lean individuals with NAFLD identifies monogenic disorders in a prospective cohort study.

Background & Aims: Lean patients with non-alcoholic fatty liver disease (NAFLD) represent 10-20% of the affected population and may have heterogeneous drivers of disease. We have recently proposed the evaluation of patients with lean NAFLD without visceral adiposity for rare monogenic drivers of disease. Here, we aimed to validate this framework in a well-characterised cohort of patients with biopsy-proven NAFLD by performing whole exome sequencing. Methods: This prospective study included 124 patients with biopsy-proven NAFLD and paired liver biopsies who underwent standardised research visits including advanced magnetic resonance imaging (MRI) assessment of liver fat and stiffness. Results: Six patients with lean NAFLD were identified and underwent whole exome sequencing. Two lean patients (33%) were identified to have monogenic disorders. The lean patients with monogenic disorders had similar age, and anthropometric and MRI characteristics to lean patients without a monogenic disorder. Patient 1 harbours a rare homozygous pathogenic mutation in ALDOB (aldolase B) and was diagnosed with hereditary fructose intolerance. Patient 2 harbours a rare heterozygous mutation in apolipoprotein B (APOB). The pathogenicity of this APOB variant (p.Val1856CysfsTer2) was further validated in the UK Biobank and associated with lower circulating APOB levels (beta = -0.51 g/L, 95% CI -0.65 to -0.36 g/L, p = 1.4 × 10-11) and higher liver fat on MRI (beta = +10.4%, 95% CI 4.3-16.5%, p = 8.8 × 10-4). Hence, patient 2 was diagnosed with heterozygous familial hypobetalipoproteinaemia. Conclusions: In this cohort of well-characterised patients with lean NAFLD without visceral adiposity, 33% (2/6) had rare monogenic drivers of disease, highlighting the importance of genomic analysis in this NAFLD subtype. Impact and Implications: Although most people with non-alcoholic fatty liver disease (NAFLD) are overweight or obese, a subset are lean and may have unique genetic mutations that cause their fatty liver disease. We show that 33% of study participants with NAFLD who were lean harboured unique mutations that cause their fatty liver, and that these mutations had effects beyond the liver. This study demonstrates the value of genetic assessment of NAFLD in lean individuals to identify distinct subtypes of disease.

Fulminant Viral Hepatitis in Two Siblings with Inherited IL-10RB Deficiency.

Fulminant viral hepatitis (FVH) caused by hepatitis A virus (HAV) is a life-threatening disease that typically strikes otherwise healthy individuals. The only known genetic etiology of FVH is inherited IL-18BP deficiency, which unleashes IL-18-dependent lymphocyte cytotoxicity and IFN-γ production. We studied two siblings who died from a combination of early-onset inflammatory bowel disease (EOIBD) and FVH due to HAV. The sibling tested was homozygous for the W100G variant of IL10RB previously described in an unrelated patient with EOIBD. We show here that the out-of-frame IL10RB variants seen in other EOIBD patients disrupt cellular responses to IL-10, IL-22, IL-26, and IFN-λs in overexpression conditions and in homozygous cells. By contrast, the impact of in-frame disease-causing variants varies between cases. When overexpressed, the W100G variant impairs cellular responses to IL-10, but not to IL-22, IL-26, or IFN-λ1, whereas cells homozygous for W100G do not respond to IL-10, IL-22, IL-26, or IFN-λ1. As IL-10 is a potent antagonist of IFN-γ in phagocytes, these findings suggest that the molecular basis of FVH in patients with IL-18BP or IL-10RB deficiency may involve excessive IFN-γ activity during HAV infections of the liver. Inherited IL-10RB deficiency, and possibly inherited IL-10 and IL-10RA deficiencies, confer a predisposition to FVH, and patients with these deficiencies should be vaccinated against HAV and other liver-tropic viruses.

A multiancestry genome-wide association study of unexplained chronic ALT elevation as a proxy for nonalcoholic fatty liver disease with histological and radiological validation.

Nonalcoholic fatty liver disease (NAFLD) is a growing cause of chronic liver disease. Using a proxy NAFLD definition of chronic elevation of alanine aminotransferase (cALT) levels without other liver diseases, we performed a multiancestry genome-wide association study (GWAS) in the Million Veteran Program (MVP) including 90,408 cALT cases and 128,187 controls. Seventy-seven loci exceeded genome-wide significance, including 25 without prior NAFLD or alanine aminotransferase associations, with one additional locus identified in European American-only and two in African American-only analyses (P < 5 × 10-8). External replication in histology-defined NAFLD cohorts (7,397 cases and 56,785 controls) or radiologic imaging cohorts (n = 44,289) replicated 17 single-nucleotide polymorphisms (SNPs) (P < 6.5 × 10-4), of which 9 were new (TRIB1, PPARG, MTTP, SERPINA1, FTO, IL1RN, COBLL1, APOH and IFI30). Pleiotropy analysis showed that 61 of 77 multiancestry and all 17 replicated SNPs were jointly associated with metabolic and/or inflammatory traits, revealing a complex model of genetic architecture. Our approach integrating cALT, histology and imaging reveals new insights into genetic liability to NAFLD.

Heterozygosity of the Alpha 1-Antitrypsin Pi*Z Allele and Risk of Liver Disease.

The serpin family A member 1 (SERPINA1) Z allele is present in approximately one in 25 individuals of European ancestry. Z allele homozygosity (Pi*ZZ) is the most common cause of alpha 1-antitrypsin deficiency and is a proven risk factor for cirrhosis. We examined whether heterozygous Z allele (Pi*Z) carriers in United Kingdom (UK) Biobank, a population-based cohort, are at increased risk of liver disease. We replicated findings in Massachusetts General Brigham Biobank, a hospital-based cohort. We also examined variants associated with liver disease and assessed for gene-gene and gene-environment interactions. In UK Biobank, we identified 1,493 cases of cirrhosis, 12,603 Z allele heterozygotes, and 129 Z allele homozygotes among 312,671 unrelated white British participants. Heterozygous carriage of the Z allele was associated with cirrhosis compared to noncarriage (odds ratio [OR], 1.53; P = 1.1×10-04); homozygosity of the Z allele also increased the risk of cirrhosis (OR, 11.8; P = 1.8 × 10-09). The OR for cirrhosis of the Z allele was comparable to that of well-established genetic variants, including patatin-like phospholipase domain containing 3 (PNPLA3) I148M (OR, 1.48; P = 1.1 × 10-22) and transmembrane 6 superfamily member 2 (TM6SF2) E167K (OR, 1.34; P = 2.6 × 10-06). In heterozygotes compared to noncarriers, the Z allele was associated with higher alanine aminotransferase (ALT; P = = 4.6 × 10-46), aspartate aminotransferase (AST; P = 2.2 × 10-27), alkaline phosphatase (P = 3.3 × 10-43), gamma-glutamyltransferase (P = 1.2 × 10-05), and total bilirubin (P = 6.4 × 10-06); Z allele homozygotes had even greater elevations in liver biochemistries. Body mass index (BMI) amplified the association of the Z allele for ALT (P interaction = 0.021) and AST (P interaction = 0.0040), suggesting a gene-environment interaction. Finally, we demonstrated genetic interactions between variants in PNPLA3, TM6SF2, and hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13); there was no evidence of epistasis between the Z allele and these variants. Conclusion: SERPINA1 Z allele heterozygosity is an important risk factor for liver disease; this risk is amplified by increasing BMI.

Genetic Variation in the Mitochondrial Glycerol-3-Phosphate Acyltransferase Is Associated With Liver Injury.

BACKGROUND AND AIMS: Most of the genetic basis of chronic liver disease remains undiscovered. APPROACH AND RESULTS: To identify genetic loci that modulate the risk of liver injury, we performed genome-wide association studies on circulating levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin across 312,671 White British participants in the UK Biobank. We focused on variants associated with elevations in all four liver biochemistries at genome-wide significance (P < 5 × 10-8 ) and that replicated using Mass General Brigham Biobank in 19,323 European ancestry individuals. We identified a genetic locus in mitochondrial glycerol-3-phosphate acyltransferase (GPAM rs10787429) associated with increased levels of ALT (P = 1.4 × 10-30 ), AST (P = 3.6 × 10-10 ), ALP (P = 9.5 × 10-30 ), and total bilirubin (P = 2.9 × 10-12 ). This common genetic variant was also associated with an allele dose-dependent risk of alcohol-associated liver disease (odd ratio [OR] = 1.34, P = 2.6 × 10-5 ) and fatty liver disease (OR = 1.18, P = 5.8 × 10-4 ) by International Classification of Diseases, 10th Revision codes. We identified significant interactions between GPAM rs10787429 and elevated body mass index in association with ALT and AST (P = 7.1 × 10-9 and 3.95 × 10-8 , respectively), as well as between GPAM rs10787429 and weekly alcohol consumption in association with ALT, AST, and alcohol-associated liver disease (P = 4.0 × 10-2 , 1.6 × 10-2 , and 1.3 × 10-2 , respectively). Unlike previously described genetic variants that are associated with an increased risk of liver injury but confer a protective effect on circulating lipids, GPAM rs10787429 was associated with an increase in total cholesterol (P = 2.0 × 10-17 ), LDL cholesterol (P = 2.0 × 10-10 ), and HDL cholesterol (P = 6.6 × 10-37 ). Single-cell RNA-sequencing data demonstrated hepatocyte-predominant expression of GPAM in cells that co-express genes related to VLDL production (P = 9.4 × 10-103 ). CONCLUSIONS: Genetic variation in GPAM is associated with susceptibility to liver injury. GPAM may represent a therapeutic target in chronic liver disease.

Inherited PD-1 deficiency underlies tuberculosis and autoimmunity in a child.

The pathophysiology of adverse events following programmed cell death protein 1 (PD-1) blockade, including tuberculosis (TB) and autoimmunity, remains poorly characterized. We studied a patient with inherited PD-1 deficiency and TB who died of pulmonary autoimmunity. The patient's leukocytes did not express PD-1 or respond to PD-1-mediated suppression. The patient's lymphocytes produced only small amounts of interferon (IFN)-γ upon mycobacterial stimuli, similarly to patients with inborn errors of IFN-γ production who are vulnerable to TB. This phenotype resulted from a combined depletion of Vδ2+ γδ T, mucosal-associated invariant T and CD56bright natural killer lymphocytes and dysfunction of other T lymphocyte subsets. Moreover, the patient displayed hepatosplenomegaly and an expansion of total, activated and RORγT+ CD4-CD8- double-negative αß T cells, similar to patients with STAT3 gain-of-function mutations who display lymphoproliferative autoimmunity. This phenotype resulted from excessive amounts of STAT3-activating cytokines interleukin (IL)-6 and IL-23 produced by activated T lymphocytes and monocytes, and the STAT3-dependent expression of RORγT by activated T lymphocytes. Our work highlights the indispensable role of human PD-1 in governing both antimycobacterial immunity and self-tolerance, while identifying potentially actionable molecular targets for the diagnostic and therapeutic management of TB and autoimmunity in patients on PD-1 blockade.

GIMAP5 maintains liver endothelial cell homeostasis and prevents portal hypertension.

Portal hypertension is a major contributor to decompensation and death from liver disease, a global health problem. Here, we demonstrate homozygous damaging mutations in GIMAP5, a small organellar GTPase, in four families with unexplained portal hypertension. We show that GIMAP5 is expressed in hepatic endothelial cells and that its loss in both humans and mice results in capillarization of liver sinusoidal endothelial cells (LSECs); this effect is also seen when GIMAP5 is selectively deleted in endothelial cells. Single-cell RNA-sequencing analysis in a GIMAP5-deficient mouse model reveals replacement of LSECs with capillarized endothelial cells, a reduction of macrovascular hepatic endothelial cells, and places GIMAP5 upstream of GATA4, a transcription factor required for LSEC specification. Thus, GIMAP5 is a critical regulator of liver endothelial cell homeostasis and, when absent, produces portal hypertension. These findings provide new insight into the pathogenesis of portal hypertension, a major contributor to morbidity and mortality from liver disease.

Undiagnosed liver diseases.

The landscape of chronic liver disease has drastically changed over the past 20 years, largely due to advances in antiviral therapy and the rise of metabolic syndrome and associated non-alcoholic fatty liver disease (NAFLD). Despite advances in the diagnosis and treatment of a variety of liver diseases, the burden of chronic liver disease is increasing worldwide. The first step to addressing any disease is accurate diagnosis. Here, we discuss liver diseases that remain undiagnosed, either because they are difficult to diagnose or due to hepatic manifestations of an unrecognized systemic disease. Additionally, their underlying etiology may remain unknown or they represent previously uncharacterized and therefore novel liver diseases. Our goal is to provide a framework for approaching undiagnosed liver diseases which elude standard hepatic diagnostic work-up and whose patterns of disease are often overlooked.

Deep mining of oxysterols and cholestenoic acids in human plasma and cerebrospinal fluid: Quantification using isotope dilution mass spectrometry.

Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography - tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient.

Oral and cranio-maxillofacial trauma in children and adolescents in an emergency setting at a Brazilian hospital.

BACKGROUND/AIMS: Very few studies have been performed to evaluate cranio-maxillofacial trauma diagnosed in hospitals in children and adolescents. The aim of this study was to perform an analysis of oral and cranio-maxillofacial trauma in the aforementioned population. MATERIALS AND METHODS: A hospital-based retrospective study, which reviewed 1438 patient records, was conducted at the "Teresina Emergency Hospital", Brazil. Data regarding demographics, day of the week on which trauma occurred, type of injury, etiology, anatomic trauma site, time of hospital admission, and associated comorbidities (or injuries) were collected. RESULTS: There were 1092 (75.9%) males and 346 (24.1%) females. The largest group was adolescents aged between 13 and 18 years (956, 66.5%). The majority lived in urban areas (69%). Trauma occurred most frequently during the week. The most prevalent etiology was road traffic accidents involving motorcycles (771, 53.6%) causing facial and skull fractures (598, 41%). The most prevalent soft tissue lesions were facial abrasions (49%), followed by injuries to the cheek (16.7%). Comorbidities associated with craniofacial trauma were present in 82%, with complications from traumatic brain injuries being the most prevalent (65.6%) Dental trauma was recorded in only 81 cases (5.6%). CONCLUSION: Male adolescents living in urban areas were most affected by craniofacial trauma. The most common cause was road traffic accidents involving motorcycles, resulting in facial and skull fractures. Complications from traumatic brain injuries were the most common associated injuries.