The rs599839 A>G Variant Disentangles Cardiovascular Risk and Hepatocellular Carcinoma in NAFLD Patients

Abstract

Background and aims: Dyslipidemia and cardiovascular diseases (CVD) are comorbidities of nonalcoholic fatty liver disease (NAFLD), which ranges from steatosis to hepatocellular carcinoma (HCC). The rs599839 A>G variant, in the CELSR2-PSRC1-SORT1 gene cluster, has been associated CVD, but its impact on metabolic traits and on the severity liver damage in NAFLD has not been investigated yet.

Methods: We evaluated the effect of the rs599839 variant in 1426 NAFLD patients (Overall cohort) of whom 131 had HCC (NAFLD-HCC), in 500,000 individuals from the UK Biobank Cohort (UKBBC), and in 366 HCC samples from The Cancer Genome Atlas (TCGA). Hepatic PSRC1, SORT1 and CELSR2 expressions were evaluated by RNAseq (n = 125).

Results: The rs599839 variant was associated with reduced circulating LDL, carotid intima-media thickness, carotid plaques and hypertension (p < 0.05) in NAFLD patients and with protection against dyslipidemia in UKBBC. The minor G allele was associated with higher risk of HCC, independently of fibrosis severity (odds ratio (OR): 5.62; 95% c.i. 1.77-17.84, p = 0.003), poor prognosis and advanced tumor stage (p < 0.05) in the overall cohort. Hepatic PSRC1, SORT1 and CELSR2 expressions were increased in NAFLD patients carrying the rs599839 variant (p < 0.0001). SORT1 mRNA levels negatively correlated with circulating lipids and with those of genes involved in lipoprotein turnover (p < 0.0001). Conversely, PSRC1 expression was positively related to that of genes implicated in cell proliferation (p < 0.0001). In TCGA, PSRC1 over-expression promoted more aggressive HCC development (p < 0.05).

Conclusions: In sum, the rs599839 A>G variant is associated with protection against dyslipidemia and CVD in NAFLD patients, but as one it might promote HCC development by modulating SORT1 and PSRC1 expressions which impact on lipid metabolism and cell proliferation, respectively.

Keywords: HCC; NAFLD; PSRC1; genetic variants; lipid metabolism.

Figure 1

The rs599839 variant affects circulating lipids, carotid IMT, plaque formation and incidence of hypertension in patients with NAFLD. Circulating total cholesterol (TC) (mmol/L) (A), LDL (mmol/L) (B), HDL (mmol/L) (C) were evaluated in NAFLD patients from the Overall cohort (n = 1426) and stratified by the presence of the rs599839 G allele. Boxes span from 25° to 75° percentile, while whiskers indicate the 10° and 90° percentile. p < 0.0001 at one-way ANOVA. Association of the rs599839 variant with IMT (D), plaque presence (E) and hypertension (F) in NAFLD patients from the Overall cohort (n = 1426). Multivariable generalized linear model (for IMT) or nominal logistic regression analysis (for Plaques and Hypertension) adjusted for age, sex, BMI, T2D, presence of TM6SF2 E167K, statin use and active smoking at † recessive model. p < 0.01 at one-way ANOVA.

Figure 2

The rs599839 variant influences the risk of HCC in NAFLD patients. Frequencies distribution of the rs599839 variant across the 404 individuals from 1000 genomes European non-Finnish cohort, Hepatology service cohort (n = 1295) and NAFLD-HCC cohort (n = 131) (A). Association of the rs599839 variant with HCC (n = 131 cases from the NAFLD-HCC cohort and n = 1295 controls with NAFLD from the Hepatology service cohort). Multivariable nominal logistic regression analysis adjusted for age, sex, BMI, T2D, presence of PNPLA3 I148M, TM6SF2 E167K and MBOAT7 T alleles at ° additive or † recessive model (B). Kaplan-Meier survival analysis of patients carrying the rs599839 in homozygosity (n = 16; GG rs599839—red line) vs. the others (n = 115; AA*AG rs599839—blue line). X axis indicates the months of survival. Log-rank Test p-Value: 0.04 (C).

Figure 3

PSRC1, SORT1 and CELSR2 expressions increase in NAFLD patients carrying the rs599839 variant. PSRC1 (A), SORT1 (B) and CELSR2 (C) mRNA levels were evaluated by transcriptome analysis on liver biopsies (n = 125) and stratified by the presence of the rs599839 G allele. mRNA levels were represented as log transformed. Boxes span from 25° to 75° percentile, while whiskers indicate the 10° and 90° percentile. p < 0.001 at one-way ANOVA. Correlation analyses between PSRC1 and SORT1 (D), CELSR2 (E) and between SORT1 and CELSR2 (F).

Figure 4

SORT1 mRNA levels correlate with the concentration of circulating lipids and with the expression of genes involved in lipoprotein release and lipid synthesis. Correlation analyses between hepatic SORT1 gene expression evaluated by transcriptome analysis on liver biopsies (n = 125) and circulating total cholesterol (TC) (mmol/L) (A), LDL (mmol/L) (B), HDL (mmol/L) (C), and triglycerides (mmol/L) (D). Correlation analyses between hepatic SORT1 gene expression and APOA1 (E), APOE (F), APOB (G), MTTP (H), TM6SF2 (I), LPL (L), SREBP1 (M), DGAT2 (N) mRNA levels evaluated by transcriptome analysis on liver biopsies (n = 125).

Figure 5

PSRC1 mRNA levels correlate with that of genes involved in cell proliferation and its overexpression reduced survival in HCC patients. Correlation analyses between hepatic PSRC1 gene expression and PCNA (A) and TP53 (B) mRNA levels evaluated by transcriptome analysis on liver biopsies (n = 125). PSRC1 mRNA levels were evaluated by transcriptome analysis on HCC samples from TCGA dataset (n = 366). Among the 366 HCC samples, 25 patients (7%) overexpressed hepatic PSRC1 (altered group) compared to the others (n = 341, unaltered group). mRNA levels were represented as log transformed. Boxes span from 25° to 75° percentile, while whiskers indicate the 10° and 90° percentile. p < 0.0001 at two-tailed Student t-test (C). Kaplan-Meier survival analysis of patients with PSRC1 overexpressed (n = 25; altered group—red line) vs. the others (n = 341; unaltered—blue line). X axis indicates the months of survival. Log-rank Test p value: 0.0128 (D). Reactome pathways enriched for 6353 genes co-regulated with PSRC1 expression in 366 samples from TCGA dataset. The statistical significance level (p-Value) was negative 10-based log trans-formed (E). Volcano plot illustrates the differential frequency distribution of mutations, observed in patients belonging to the altered group (n = 25) compared to the unaltered one (n = 341), represented as log fold changes (log ratio, on x axis) and the distribution of transformed p values (−log10 of p, y axis) (F). The frequency of mutations in the 10 genes most enriched in mutations in both groups are stratified according to the presence of PSRC1 overexpression (n = 25, altered group in red) or not (n = 341, unaltered group in blue) (G).