Development and Validation of a Scoring System, Based on Genetic and Clinical Factors, to Determine Risk of Steatohepatitis in Asian Patients with Nonalcoholic Fatty Liver Disease

doi:10.1016/j.cgh.2020.02.011

Clinical Gastroenterology and Hepatology

Volume 18, Issue 11, October 2020, Pages 2592-2599.e10

https://doi.org/10.1016/j.cgh.2020.02.011 Get rights and content

Background & Aims

There are no biomarkers of nonalcoholic steatohepatitis (NASH) that are ready for routine clinical use. We investigated whether an analysis of PNPLA3 and TM6SF2 genotypes (rs738409 and rs58542926) can be used to identify patients with nonalcoholic fatty liver disease (NAFLD), with and without diabetes, who also have NASH.

Methods

We collected data from the Boramae registry in Korea on 453 patients with biopsy-proven NAFLD with sufficient clinical data for calculating scores. Patients enrolled from February 2014 through March 2016 were assigned to cohort 1 (n = 302; discovery cohort) and patients enrolled thereafter were assigned to cohort 2 (n = 151; validation cohort). DNA samples were obtained from all participants and analyzed for the PNPLA3 rs738409 C>G, TM6SF2 rs58542926 C>T, SREBF2 rs133291 C>T, MBOAT7-TMC4 rs641738 C>T, and HSD17B13 rs72613567 adenine insertion (A-INS) polymorphisms. We used multivariable logistic regression analyses with stepwise backward selection to build a model to determine patients’ risk for NASH (NASH PT) using the genotype and clinical data from cohort 1 and tested its accuracy in cohort 2. We used the receiver operating characteristic (ROC) curve to compare the diagnostic performances of the NASH PT and the NASH scoring systems.

Results

We developed a NASH PT scoring system based on PNPLA3 and TM6SF2 genotypes, diabetes status, insulin resistance, and levels of aspartate aminotransferase and high-sensitivity C-reactive protein. NASH PT scores identified patients with NASH with an area under the ROC (AUROC) of 0.859 (95% CI, 0.817–0.901) in cohort 1. In cohort 2, NASH PT scores identified patients with NASH with an AUROC of 0.787 (95% CI, 0.715–0.860), which was significantly higher than the AUROC of the NASH score (AUROC, 0.729; 95% CI, 0.647–0.812; P = .007). The AUROC of the NASH PT score for detecting NASH in patients with NAFLD with diabetes was 0.835 (95% CI, 0.776–0.895) and in patients without diabetes was 0.809 (95% CI, 0.757–0.861). The negative predictive value of the NASH PT score <–0.785 for NASH in patients with NAFLD with diabetes reached 0.905.

Conclusions

We developed a scoring system, based on polymorphisms in PNPLA3 and TM6SF2 and clinical factors that identifies patients with NAFLD, with or without diabetes, who have NASH, with an AUROC value of 0.787. This system might help clinicians better identify NAFLD patients at risk for NASH.

Section snippets

Patients and Assessment of Liver Histology

We constructed a prospective cohort from the ongoing Boramae NAFLD registry.⁷ Among the eligible study participants, only biopsy-proven NAFLD patients with sufficient clinical data for calculating scores were finally included in this study (N = 453) (Supplementary Methods). They were divided into cohort 1 (N = 302) and cohort 2 (N = 151) according to the period of enrollment. Cohort 1 consisted of individuals enrolled from February 2014 to March 2016 to build a model to determine patients’ risk

Characteristics of the Discovery and Replication Cohorts

In cohort 1 (n = 302) and cohort 2 (n = 151), 145 (48.0%) and 67 (44.4%) patients, respectively, were classified as NASH (P = .464) (Table 1). Cohort 2 showed significantly lower high-density lipoprotein cholesterol, higher AST and alanine aminotransferase, lower albumin, and higher HOMA-IR levels than cohort 1 (P = .019, .016, .038, .001, and < .001, respectively) (Table 1).

The genotypic distributions of PNPLA3 rs738409, TM6SF2 rs58542926, SREBF2 rs133291, MBOAT7-TMC4 rs641738, and HSD17B13

Conclusions

In the current study, we successfully developed a NASH-identifying polygenic risk scoring model incorporating 2 genetic risk variants, such as PNPLA3 rs738409 and TM6SF2 rs58542926, from our biopsy-proven NAFLD cohort and validated it using an independent replication dataset. Its AUROC for differentiating NASH from NAFL was 0.859 and 0.787 in the discovery and replication cohorts, respectively. In the replication cohort, the AUROC of the newly developed model (NASH PT score) was significantly

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It is important to note that the rs626283 SNP is in strong linkage disequilibrium with rs641738 across numerous studies (23). Although the majority of large studies find a clear association between MBOAT7 loss of function and NAFLD progression, it is important to point out that not all studies have found statistically significant associations (24–35). Although consistent trends in association exist, several independent studies looking at NAFLD-related indices in adults (24–31) and children (32–35) have failed to conclude that the rs641738 SNP is associated with NAFLD progression.
Advanced liver diseases account for approximately 2 million deaths annually worldwide. Roughly, half of liver disease-associated deaths arise from complications of cirrhosis and the other half driven by viral hepatitis and hepatocellular carcinoma. Unfortunately, the development of therapeutic strategies to treat subjects with advanced liver disease has been hampered by a lack of mechanistic understanding of liver disease progression and a lack of human-relevant animal models. An important advance has been made within the past several years, as several genome-wide association studies have discovered that an SNP near the gene encoding membrane-bound O-acyltransferase 7 (MBOAT7) is associated with severe liver diseases. This common MBOAT7 variant (rs641738, C>T), which reduces MBOAT7 expression, confers increased susceptibility to nonalcoholic fatty liver disease, alcohol-associated liver disease, and liver fibrosis in patients chronically infected with viral hepatitis. Recent studies in mice also show that Mboat7 loss of function can promote hepatic steatosis, inflammation, and fibrosis, causally linking this phosphatidylinositol remodeling enzyme to liver health in both rodents and humans. Herein, we review recent insights into the mechanisms by which MBOAT7-driven phosphatidylinositol remodeling influences liver disease progression and discuss how rapid progress in this area could inform drug discovery moving forward.
Genetic and epigenetic factors determining NAFLD risk
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The scoring system (NASH PNPLA3-TM6SF2 score, NASH-PT) was based on risk alleles of PNPLA3 rs738409 (C > G) and TM6SF2 rs58542926 (G > A), diabetes status, insulin resistance, and levels of AST and C-reactive protein. NASH-PT scores identified patients with NASH with a ROC-AUC between 0.787 and 0.859 [92]. In a cohort of 514 obese children and adolescents in which almost 70% of the participants were diagnosed with NAFLD by ultrasonography, Zusi et al. tested 11 genes and detected highly significant associations with risk alleles of PNPLA3 rs738409 (C > G), TM6SF2 rs58542926 (G > A), and GCKR rs1260326 (C > T) and a weaker association with a polymorphism of ELOVL2 rs2236212 (G > C) with a higher risk of NAFLD [93].
Hepatic steatosis is a common chronic liver disease that can progress into more severe stages of NAFLD or promote the development of life-threatening secondary diseases for some of those affected. These include the liver itself (nonalcoholic steatohepatitis or NASH; fibrosis and cirrhosis, and hepatocellular carcinoma) or other organs such as the vessels and the heart (cardiovascular disease) or the islets of Langerhans (type 2 diabetes). In addition to elevated caloric intake and a sedentary lifestyle, genetic and epigenetic predisposition contribute to the development of NAFLD and the secondary diseases.
We present data from genome-wide association studies (GWAS) and functional studies in rodents which describe polymorphisms identified in genes relevant for the disease as well as changes caused by altered DNA methylation and gene regulation via specific miRNAs. The review also provides information on the current status of the use of genetic and epigenetic factors as risk markers.
With our overview we provide an insight into the genetic and epigenetic landscape of NAFLD and argue about the applicability of currently defined risk scores for risk stratification and conclude that further efforts are needed to make the scores more usable and meaningful.
The HSD17B13 rs72613567 variant is associated with lower levels of albuminuria in patients with biopsy-proven nonalcoholic fatty liver disease
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These investigators also showed that the A/A genotype of HSD17B13 was significantly associated with lower rates of liver-related mortality, and with lower serum alanine aminotransferase levels, especially in people with obesity, in heavy drinkers and in individuals carrying three or four steatogenic alleles in the PNPLA3 and TM6SF2 genes [15]. In our study, we did not find any significant association between the HSD17B13 rs72613567 variant and the presence of NASH or the individual histological components of NASH, which is also consistent with the results recently reported by Koo et al. in a cohort of 453 South Korean individuals with biopsy-proven NAFLD [29]. Currently, a significant association between the HSD17B13 rs72612567 variant and NAFLD has been reported only in Caucasian individuals [13,30,31].
Several susceptibility gene variants predisposing to nonalcoholic fatty liver disease (NAFLD) have been identified in chronic kidney disease (CKD). Evidence supports that 17-beta hydroxysteroid dehydrogenase 13 (HSD17B13) rs72613567 plays a role in NAFLD development by affecting lipid homeostasis. Since lipid droplets may accumulate in the kidneys and contribute to renal injury, we investigated the association between the HSD17B13 rs72613567 variant and markers of renal function/injury in NAFLD.
We measured estimated glomerular filtration rate (eGFR), urinary/serum neutrophil gelatinase-associated lipocalin (NGAL), and urinary albumin-to-creatinine ratio (u-ACR) in individuals with biopsy-proven NAFLD. Multivariable regression analyses were undertaken to examine the associations between the HSD17B13 rs72613567 variant and markers of renal function/injury. Individuals were stratified by HSD17B13 rs72613567 genotypes into −/−, A/- and A/A groups. HSD17B13 rs72613567 genotypes were not significantly associated with eGFR and urinary/serum NGAL levels. Conversely, the prevalence of abnormal albuminuria in the A/- + A/A group was lower than in the −/− group (4.92% vs. 19.35%, p = 0.001). Additionally, the mean u-ACR levels were lower among carriers of the A/- or A/A genotypes with coexisting hypertension or diabetes, than among those with the −/− genotype. The risk of abnormal albuminuria (adjusted-odds ratio 0.16, p = 0.001) remained significantly lower in the A/- + A/A group after adjustment for established renal risk factors and histologic severity of NAFLD.
HSD17B13 rs72613567: A allele is associated with a lower risk of having abnormal albuminuria, but not with lower eGFR or urinary/serum NGAL levels, in patients with biopsy-proven NAFLD.
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: CRediT Authorship Contributions Bo Kyung Koo (Data curation: Lead; Formal analysis: Lead; Investigation: Lead; Validation: Lead; Writing – original draft: Lead; Writing – review and editing: Lead) Sae Kyung Joo (Data curation: Lead; Formal analysis: Equal; Investigation: Lead; Resources: Lead) Donghee Kim (Conceptualization: Supporting; Supervision: Equal; Writing – original draft: Supporting; Writing – review and editing: Equal) Seonhwa Lee (Investigation: Supporting; Methodology: Supporting; Resources: Supporting) Jeong Mo Bae (Formal analysis: Supporting; Investigation: Lead; Resources: Lead) Jeong Hwan Park (Formal analysis: Supporting; Investigation: Lead; Resources: Equal) Jung Ho Kim (Formal analysis: Equal; Investigation: Lead; Resources: Equal) Mee Soo Chang (Formal analysis: Equal; Investigation: Equal; Resources: Supporting; Supervision: Equal) Won Kim, MD, PhD (Data curation: Lead; Formal analysis: Lead; Funding acquisition: Lead; Investigation: Lead; Methodology: Lead; Project administration: Lead; Resources: Lead; Supervision: Lead; Validation: Lead; Writing – original draft: Lead; Writing – review and editing: Lead)

: Conflicts of interest The authors disclose no conflicts.

: Funding This work was supported by a National Research Foundation of Korea grant funded by the Korea government (MEST) (2016R1D1A1B04934590), and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute funded by the Ministry of Health & Welfare, Republic of Korea (H I17C0912). The funding did not affect the collection, analysis, or presentation of the data.

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Original ArticlePancreas, Biliary Tract, and LiverDevelopment and Validation of a Scoring System, Based on Genetic and Clinical Factors, to Determine Risk of Steatohepatitis in Asian Patients with Nonalcoholic Fatty Liver Disease

Background & Aims

Methods

Results

Conclusions

Section snippets

Patients and Assessment of Liver Histology

Characteristics of the Discovery and Replication Cohorts

Conclusions

Gastroenterology

Lancet

J Hepatol

J Hepatol

J Hepatol

J Lipid Res

J Hepatol

Gastroenterology

J Lipid Res

Gastroenterology

J Biol Chem

Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis

N Engl J Med

Elafibranor, an agonist of the peroxisome proliferator-activated receptor-alpha and -delta, induces resolution of nonalcoholic steatohepatitis without fibrosis worsening

Gastroenterology

EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease

J Hepatol

The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases

Hepatology

Additive effects of PNPLA3 and TM6SF2 on the histological severity of non-alcoholic fatty liver disease

J Gastroenterol Hepatol

Original Article
Pancreas, Biliary Tract, and Liver
Development and Validation of a Scoring System, Based on Genetic and Clinical Factors, to Determine Risk of Steatohepatitis in Asian Patients with Nonalcoholic Fatty Liver Disease