Genome-wide association study for circulating FGF21 in patients with alcohol use disorder: Molecular links between the SNHG16 locus and catecholamine metabolism

Abstract

Objective: Alcohol consumption can increase circulating levels of fibroblast growth factor 21 (FGF21). The effects of FGF21 in the central nervous system are associated with the regulation of catecholamines, neurotransmitters that play a crucial role in reward pathways. This study aims to identify genetic variants associated with FGF21 levels and evaluate their functional role in alcohol use disorder (AUD).

Methods: We performed a genome-wide association study (GWAS) using DNA samples from 442 AUD subjects recruited from the Mayo Clinic Center for the Individualized Treatment of Alcoholism Study. Plasma FGF21 levels were measured using Olink proximity extension immunoassays. Alcohol consumption at time of entry into the study was measured using the self-reported timeline followback method. Functional genomic studies were performed using HepG2 cells and induced pluripotent stem cell (iPSC)-derived brain organoids.

Results: Plasma FGF21 levels were positively correlated with recent alcohol consumption and gamma-glutamyl transferase levels, a commonly used marker for heavy alcohol use. One variant, rs9914222, located 5' of SNHG16 on chromosome 17 was associated with plasma FGF21 levels (p = 4.60E-09). This variant was also associated with AUD risk (β: -3.23; p:0.0004). The rs9914222 SNP is an eQTL for SNHG16 in several brain regions, i.e., the variant genotype was associated with decreased expression of SNHG16. The variant genotype for the rs9914222 SNP was also associated with higher plasma FGF21 levels. Knockdown of SNHG16 in HepG2 cells resulted in increased FGF21 concentrations and decreased expression and enzyme activity for COMT, an enzyme that plays a key role in catecholamine metabolism. Finally, we demonstrated that ethanol significantly induced FGF21, dopamine, norepinephrine, and epinephrine concentrations in iPSC-derived brain organoids.

Conclusions: GWAS for FGF21 revealed a SNHG16 genetic variant associated with FGF21 levels which are associated with recent alcohol consumption. Our data suggest that SNHG16 can regulate FGF21 concentrations and decrease COMT expression and enzyme activity which, in turn, have implications for the regulation of catecholamines. (The ClinicalTrials.gov Identifier: NCT00662571).

Keywords: Alcohol use disorder; Catecholamine metabolism; FGF21; GWAS.

Conflict of interest statement

Conflict of interest Dr. Weinshilboum is a co-founder and stockholder in OneOme LLC, a pharmacogenomics decision-support company. Dr. Choi is a scientific advisory board member for Peptron Inc. Dr. Frye reports Grant Support from Assurex Health, Mayo Foundation, and Medibio. All other authors have no conflicts to declare.

Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.

Figures

Figure 1

Plasma FGF21 concentrations were associated with recent alcohol use phenotypes. Plasma FGF21 levels were positively correlated with recent alcohol use as determined by TLFB 90 days prior to blood collection i.e. (A) the number of drinking days, (B) the number of heavy drinking days, and (C) total drinks. (D) Plasma FGF21 levels were also positively correlated with blood levels of gamma-glutamyl transferase (GGT) which is commonly used as a marker for heavy alcohol use.

Figure 2

(A) Schematic outline of our proteomics-informed genomics research strategy. (B–C) Q–Q plot and Manhattan plot for GWAS of plasma FGF21 levels. (D) The locus zoom plot displays that the top SNPs on chromosome 17 map within a gene cluster: CYGB, PRCD and SNHG16. The SNP most highly associated with plasma FGF21 levels in patients with AUD was rs9914222 (p: 4.6E-09).

Figure 3

Biological functions of the rs9914222 SNP. (A) SNP-dependent plasma FGF21 levels in patients with AUD demonstrating that rs9914222 is an eQTL. (B) rs9914222 is associated with SNHG16 mRNA expression in several brain regions. https://gtexportal.org/home/snp/rs9914222.

Figure 4

Biological functions of the SNHG16 gene. (A) FGF21 concentration was measured after the knockdown of SNHG16 using siRNA in HepG2 cells. Relative mRNA expression of SNHG16 and COMT was determined after the knockdown of SNHG16. COMT enzyme activity was also measured after the knockdown of SNHG16 in HepG2 cells. At least three independent experiments were performed. ∗p < 0.05. (B) FGF21 concentration was determined before and after ethanol treatment of HepG2 cells. Relative mRNA expression of SNHG16 and COMT was determined in response to ethanol treatment. COMT enzyme activity was then measured using HepG2 cells treated with ethanol. ∗A p value ≤ 0.05 was considered statistically significant (two tailed paired t test). Three independent experiments were performed. All values are mean ± S.E.M.

Figure 5

Ethanol induced FGF21 in iPSC-derived brain organoids and activated the release of catecholamines. (A) A schematic outline of procedures used during the differentiation of iPSC-derived brain organoids. The panel below the schematic displays representative examples of staining for tyrosin hydroxylase (TH), and Neuron-specific class III beta-tubulin (TUJ1). (B) the effect of ethanol on FGF21 concentration was measure using ELISA. Dopamine, norepinephrine, and epinephrine concentrations were measured using UPLC–Tandem Mass Spectrometry. (C) A schematic outline of the catecholamine biosynthesis pathway. (D) mRNA expression of SNHG16, MAOA, MAOB, COMT, TH, COMT, DDC, DBH, and PNMT in response to ethanol treatment (25 mM) for 7 days. Realtime PCR experiments were performed in iPSC-derived brain organoids (n = 3). The expression of these genes was determined after exposure to drug for 7 days. ∗A p value ≤ 0.05 was considered statistically significant (two tailed paired t test). All values shown are mean ± S.E.M.

Figure 6

Gene expression of iPSC-derived brain organoids. (A) Schematic diagram illustrating the effects of ethanol on FGF21 which have implications for alcohol use. Specifically, FGF21 can be induced by ethanol. Our GWAS for plasma FGF21 showed that a SNP located 5′ of SNHG16 is associated with AUD. SNHG16 could regulate COMT expression and activity, which play a role in catecholamine metabolism. Finally, we determined that ethanol induced both FGF21 and catecholamines, including dopamine, norepinephrine and epinephrine using iPSC-derived brain organoids. (B–G) mRNA expression of SNHG16, TH, COMT, DDC, DBH, PNMT in response to drug treatment. Realtime PCR experiments were performed in iPSC-derived brain organoids (n = 3). The expression of those genes was determined after exposure to drugs for 7 days. ∗A p value ≤ 0.05 was considered statistically significant (two tailed paired t test). All values are mean ± S.E.M.