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Department of General, Visceral-, Thoracic-, Transplantation- and Pediatric Surgery, University Medical Center Schleswig-Holstein (UKSH), Campus Kiel, and Christian-Albrecht University (CAU)
Corresponding author. ; Department of Internal Medicine I, University of Bonn; Venusberg Campus 1; D – 53127 Bonn, Germany. ;. Phone: +49 228 287 51417;
The C allele of the LPL rs13702 variant was associated with reduced risk for ALD-HCC
•
This association could be replicated on the population level in the UK Biobank cohort
•
LPL is overexpressed in ALD cirrhosis, in contrast to HCV cirrhosis or non-cirrhotic liver
•
LPL expression was not found in hepatocytes, but in HSCs and LSECs
Abstract
Background and Aims (273/275)
Progression of alcohol-associated liver disease (ALD) is driven by genetic predisposition. The rs13702 variant in the lipoproteinlipase (LPL) gene is linked to non-alcoholic fatty liver disease. We aimed at clarifying its role in ALD.
Methods
Patients with alcohol-associated cirrhosis, with (n=385) and without hepatocellular carcinoma (HCC) (n=656), with HCC due to viral hepatitis C (n=280), controls with alcohol abuse without liver damage (n=366) and healthy controls (n=277) were genotyped regarding the LPL rs13702 polymorphism. Furthermore, the UK Biobank cohort was analysed. LPL expression was investigated in human liver specimen and in liver cell lines.
Results
Frequency of the LPL rs13702 C C genotype was lower in ALD with in comparison to ALD without HCC both in the initial (3.9% vs 9.3%) and the validation cohort (4.7% vs. 9.5%; p<0.05 each) and compared to patients with viral HCC (11.4%), alcohol misuse without cirrhosis (8.7%) or healthy controls (9.0%). This protective effect (OR = 0.5) was confirmed in multivariate analysis including age (OR=1.1/year), male sex (OR=3.0), diabetes (OR=1.8), and carriage of the PNPLA3 I148M risk variant (OR=2.0). In the UK Biobank cohort, the LPL rs13702 C allele was replicated as risk factor for HCC. Liver expression of LPL mRNA was dependent on LPL rs13702 genotype and significantly higher in patients with ALD cirrhosis compared to controls and alcohol-associated HCC. While hepatocyte cell lines showed negligible LPL protein expression, hepatic stellate cells (HSC) and liver sinusoidal endothelial cells (LSEC) expressed LPL.
Conclusion
LPL is upregulated in the liver of patients with alcohol-associated cirrhosis. TheLPLrs13702 high producer variant confers protection against HCC in ALD, which might help to stratify patients for HCC risk.
Lay Summary
Hepatocellular carcinoma is a severe complications of liver cirrhosis influenced by genetic predisposition. We found that a genetic variant in the gene encoding lipoprotein lipase (LPL) reduces the risk for hepatocellular carcinoma in alcohol-associated cirrhosis. This genetic variation may directly affect the liver, because, unlike in healthy adult liver, LPL is produced from liver cells in alcohol associated cirrhosis.
patatin-like phospholipase domain-containing protein 3
PPARγ –
peroxisome proliferator-activated receptor γ
RPL19 –
ribosomal protein L19
T2DM –
type 2 diabetes mellitus
Conflict of interest statement
None of the authors has any conflict of interest in relation to the contents of this manuscript.
Financial support statement
This study was funded by the Deutsche Krebshilfe (70114349) to HDN. FSt was funded by grant SNF 310030_169196 from the Swiss National Fund. JN received funding by the German Research Foundation (DFG SFB/TRR 57 and SPP1937) and the Hector foundation (M88). The funding organizations had no role in the design of the study, and collection, analysis, and interpretation of the data, or writing of the manuscript.
Authors contribution
PL, AK, JF, SB, WS, HI, CB, FSt, MS, MMS collected specimen and data; FSc, CM, HDN, BK and BL performed the experiments, PL, HDN, FSc, HI, SB analyzed the data; PL, HDN, FSc wrote the manuscript; JN, FSt, CPS critically revised the manuscript.
Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Introduction
Heavy alcohol use, a common risk factor for liver disease, is reported by 6.6% of the adult population in the United States.
. Development, progression and complications of alcohol-associated and non-alcoholic liver disease are, next to lifestyle factors, strongly influenced by genetic predisposition.
. Several common genetic variants that increase the risk for liver disease development have been identified. The most important is the I148M variant in the PNPLA3 gene, which is a risk factor both for cirrhosis and hepatocellular carcinoma, but whose molecular background is still poorly understood.
, while others, such as the rs708113 polymorphism in the WNT3A-WNT9A locus are only associated with the risk for HCC(5). Still, also protective genetic variants, e.g. the rs429358 polymorphism in apolipoprotein E, the rs72613567 HSD17B13 locus and rs2242652 in TERT have been unravelled.
Stickel F, Lutz P, Buch S, Nischalke HD, Silva I, Rausch V, et al. Genetic variation in HSD17B13 reduces the risk of developing cirrhosis and hepatocellular carcinoma in alcohol misusers. Hepatol. Baltim. Md. 2019;
. Knowledge of these factors may both lead to a better understanding of the molecular pathways underlying liver disease development and the identification of potential drug targets, but also facilitate risk stratification of patients, e.g. for HCC screening programs.
In a genome-wide association study, the rs13702 locus in the lipoprotein lipase (LPL) gene has recently been linked to the risk of NAFLD(9). In presence of the C allele at this locus, downregulation of LPL transcription by miR-410 is impaired,
. LPL is a triglyceride lipase which is expressed in different metabolically active tissues, in particular on vascular endothelium, but only in fetal, not in adult healthy liver. It acts as an important modulator of lipid metabolism by hydrolyzing triglycerides, which are transported in the blood in very low density lipoprotein (VLDL) and chylomicrons, releasing free fatty acids, which can be taken up by parenchymal cells.
Genes involved in fatty acid partitioning and binding, lipolysis, monocyte/macrophage recruitment, and inflammation are overexpressed in the human fatty liver of insulin-resistant subjects.
. Presence of a minor C allele ofLPLrs13702 has been associated with lower triglyceride and higher high-density lipoprotein (HDL) levels in the general population
MicroRNA-410 regulated lipoprotein lipase variant rs13702 is associated with stroke incidence and modulated by diet in the randomized controlled PREDIMED trial.
Common Variants in Lipid Metabolism-Related Genes Associate with Fat Mass Changes in Response to Dietary Monounsaturated Fatty Acids in Adults with Abdominal Obesity.
Associations of Lipoprotein Lipase Gene Polymorphisms With Longitudinal Plasma Lipid Trends in Young Adults: The Coronary Artery Risk Development in Young Adults (CARDIA) Study.
We aimed at analysing if genetically modulated expression of LPL may be associated with development of cirrhosis or HCC in alcohol-associated liver disease.
Materials/patients and methods
Patients
As initial cohort, blood and clinical data were collected from 485 patients with alcohol-associated liver cirrhosis, 152 of them with HCC, from the University Hospital Bonn and the Berlin Department of Hepatology and Gastroenterology. The validation cohort, collected at the Division of Hepatology of the Leipzig University Medical Centre, comprised 323 patients with ALD and 233 patients with HCC due to ALD. In addition, 277 healthy subjects, 366 alcohol misusers without cirrhosis and 280 patients with chronic hepatitis C and HCC were included in the study. Patients were considered as alcohol misusers without cirrhosis when a minimal amount of 60 g alcohol/day for women and 80 g alcohol/day for men was consumed for at least 10 years without resulting liver damage. Healthy controls included subjects from blood donation and cancer screening programs who did not show clinical or laboratory signs of liver disease. Diagnosis of cirrhosis was based on liver biopsy or on a consistent clinical presentation supported by laboratory and imaging findings. We recorded demographic standard laboratory and clinical data such as sex, age and etiology of cirrhosis. Patients were allocated to alcohol-associated cirrhosis if their history indicated average alcohol consumption to exceed 300 g ethanol per week after exclusion of other causes of cirrhosis such as HBV or HCV infection or hemochromatosis. Hepatocellular carcinoma was diagnosed according to international guidelines.
European Association For The Study Of The Liver, European Organisation For Research And Treatment Of Cancer EASL-EORTC clinical practice guidelines: management of hepatocellular carcinoma.
Apart from blood samples, liver specimens from cirrhotic liver explants from patients with alcohol-associated and viral disease with and without HCC and tumor-free liver tissue gained by surgical resection of liver metastases were used for investigation of mRNA expression.
All controls and patients were of Caucasian ethnicity. Details of the study cohorts are shown in table 1.
Table 1Demographic, clinical and laboratory details on the study cohorts.
Alcohol-associated cirrhosis Bonn/Berlin
Alcohol-associated cirrhosis Leipzig
Viral HCC
Alcohol misuse without cirrhosis
Healthy controls
without HCC
with HCC
without HCC
with HCC
Total number
333
152
323
233
280
366
277
Age, mean (range)
58.1 (27-92)
63.9 (36-87)
54.8 (28-87)
63.1 (47-79)
59.6 (24-83)
44.0 (28-81)
65.0 (28-94)
Sex (% male/female)
64.2/35.8
81.5/18.5
77.0/23.0
93.2/6.8
71.8/28.2
75.4/24.6
44.4/55.6
Bilirubin [mg/dl], (Mean ± SD)
3.91±6.34
3.84±5.56
4.17±7.08
2.30±3.25
2.74±4.38
0.74±0.63
—
ALT [IU/l], (Mean ± SD)
49.1±151.5
52.4±59.9
37.0±37.0
48.0±55.6
97.6±148.3
45.2±45.8
—
AST [IU/l], (Mean ± SD)
82.8±237.7
90.5±100.4
63.7±48.8
88.0±114.2
131.4±172.8
55.6±64.4
—
GGT [IU/l], (Mean ± SD)
199.9±213.9
221.4±185.5
194.0±240.4
294.1±379.3
138.2±128.8
212.6±396.1
—
Platelet count [*103/μl], (Mean ± SD)
154.9±143.9
140.2±75.8
148.6±82.3
153.2±95.1
124.6±91.9
228.8±80.5
—
MELD (Mean ± SD)
16.3±7.0
17.0±8.1
17.9±7.4
12.5±5.9
15.8±6.2
—
—
Legend: ALT: alanine aminotransferase; AST: aspartate aminotransferase; GGT: Gamma glutamyl transferase; HCC hepatocellular carcinoma; MELD: model for end-stage liver disease.
The study protocol followed the ethical guidelines of the Helsinki Declaration and had been approved by the local ethics committees (Bonn: number 536/20; Leipzig: number 357/19-ek). Written informed consent was obtained from the patients prior to inclusion in this study.
Determination of LPL and PNPLA3 genotypes
Genomic DNA was isolated from 200μl EDTA-blood using the QIAamp Blood Mini Kit (Qiagen, Hilden, Germany) as suggested by the manufacturer’s protocol. Determination of the polymorphisms was performed by melting curve analysis using LightSNiP (SimpleProbe) assays from TIB-MolBiol (Berlin, Germany). 1μl of sample DNA was mixed with 0.5μl of the LightSNiP reagent mix, 5μl of Blue Probe qPCR 2x Mix (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) and 3.5μl PCR grade water (Invitrogen, Paisley, UK). The real time PCR was performed by the LightCycler® 96 (Roche, Germany) according to the manufacturer’s protocol.
Cell culture
PLC/PRF/5, and LX-2 cells were cultured in RPMI 1640 medium containing L-glutamine (Thermo Fisher, Schwerte, Germany), 1% penicillin/streptomycin (PAN-Biotech GmbH, Aidenbach, Germany) and 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany). Primary Hepatocytes, liver sinusoidal endothelial cells and primary hepatic stellate cells were purchased from ScienCell™ (Carlsbad, CA, USA) and cultivated in the appropriate media according to the manufacturer's instructions. All cell lines were tested negative for mycoplasma contamination.
Analysis of mRNA expression
RNA was isolated from liver explants with the GeneJET RNA Purification Kit (Thermo Scientific, Lithuania) according to manufacturer’s protocol. RNA was stored at -80 °C until cDNA synthesis was performed. Reverse transcription of the RNA was carried out with the QuantiTect® Reverse Transcription Kit (Qiagen, Germany) resulting in cDNA without contamination of genomic DNA. 1μl of cDNA was used as a template for the following real time PCR on the LightCycler® 96 as recommended by the manufacturer. 5μl of Blue S’Green qPCR 2x Mix (Biozym Scientific GmbH, Hessisch Oldendorf, Germany), 1μl of designated primers (5μM) and 3μl of PCR grade water were added for the amplification of the desired gene products. LPL (forward: 5’-CTG CTG GCA TTG CAG GAA GTC T-3’, reverse: 5’-CAT CAG GAG AAA GAC GAC TCG G-3’) and the housekeeping primers RPL19 (forward: 5‘-TGG GCA TAG GTA AGC GGA-3’, reverse: 5’-GCC TTG TCT GCC TTC AGC-3’) were purchased from IDT (Leuven, Belgium).
Normalization was performed with the Lightcycler® 96 S W 1.1 analysis software expressing the relative mRNA expression of LPL as 2(-ΔCt).
Western blot
Liver tissue and cell lines were homogenized in RIPA buffer (SERVA Electrophoresis GmbH, Heidelberg, Germany) supplemented with Halt™ Protease-Inhibitor-Cocktail 100x (Life Technologies GmbH, Darmstadt, Germany) followed by a centrifugation step for 5 minutes at 300 × g. The protein concentration of the supernatant was determined via the DC Protein Assay Kit from Bio-Rad Laboratories GmbH (Feldkirchen, Germany) as per manufacturer’s instructions. 30μg of protein for cell lines and 20μg of protein for liver tissue, respectively, were mixed with ROTI®Load 1 4x concentrate (Carl Roth GmbH, Karlsruhe, Germany) and heated for 5 minutes at 95 °C. Equal samples volumes were size-fractionated by 10 % SDS Page and transferred onto a 0.2μm Whatman™ Protran nitrocellulose membrane (Whatman GmbH, Dassel, Germany). Membranes were blocked for 30 minutes in 3 % BSA (PAN-Biotech GmbH, Aidenbach, Germany) in phosphate-buffered saline (Fisher Scientific GmbH, Schwerte, Germany) with 0.1% Tween20 (Carl Roth, Karlsruhe, Germany) and incubated overnight with the appropriate antibody. Membranes were probed with a LPL mouse monoclonal antibody (clone ID: OTI2C12, OriGene Technologies, Rockville, US), a human/mouse LPL polyclonal antibody (AF7197, R&D Systems, Minneapolis, USA) and a GAPDH antibody (sc-32233, Santa Cruz, Heidelberg, Germany) 1:500 in 3% BSA. Then, membranes were incubated with Anti-mouse IgG (A9044, Merck, Darmstadt, Germany) (diluted 1:10.000 in 3% BSA for GAPDH and LPL monoclonal antibody) and with Anti-goat IgG-HRP (sc-2020, Santa Cruz Biotechnology, Heidelberg, Germany) (diluted 1:5000 in 3% BSA for LPL polyclonal antibody). The protein quantification was carried out with the Healthcare Amersham™ ECL Prime Western-Blot detection reagent (Fisher Scientific GmbH, Schwerte, Germany). For the detection of chemiluminescence and the following densiometric analysis the ChemDocTM MP Imaging System and the corresponding ImageLab Software (BioRad, Feldkirchen, Germany) was used.
UK biobank cohort (population-based cohort)
In addition, we leveraged data from the UK Biobank (UKB) cohort - a prospective community cohort comprised of approximately half a million middle-aged individuals from the UK. Participants were interviewed between May 2006 to July 2010, where they completed a comprehensive health questionnaire, a physical examination and donated biological specimens
. Information on health outcome events occurring after enrolment are supplied through linkage to UK mortality, hospital admission and cancer registries.
Statistical analysis
The data from clinical cohorts were analyzed with IBM SPSS Statistics software version 24 (IBM, New York, USA). Significant deviations from the Hardy-Weinberg-equilibrium were ruled out using an exact test (https://ihg.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl).
Comparison of genotypes was performed using the Pearson’s goodness-of-fit chi2 test. Fisher’s exact test, student’s test, Wilcoxon-Mann-Whitney-U test and Spearman’s correlation were applied as appropriate and indicated. Univariate analysis followed by a multivariate forward binary regression was used to confirm the independency of the HCC risk factors with p<0.05 for inclusion and p>0.1 for exclusion of parameters. In the UK Biobank cohort, HCC cases were identified by the presence of the ICD10:C22.0 code recorded in an in-patient hospital admission, cause of death, or a cancer registration record, either before or after enrolment into UKB. Individuals without HCC were defined as controls. The association between rs13702 genotype and HCC case/control status was calculated via logistic regression, under an additive genetic model. Statistical adjustment was included for age, sex and the first five principal components of genetic ancestry. We further extended this UKB analysis to capture: a) ten additional phenotypes of interest beyond HCC; and 2) two additional genetic variants: rs328 (LPL) and rs738409 (PNPLA3). The ten additional phenotypes were: cirrhosis status; type 2 diabetes (T2DM), body mass index (BMI), alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol, fibrosis 4 index (FIB4), glycated hemoglobin (HBA1C), liver fat content and triglycerides. The fields/methods used to derive these phenotypes are outlined in the supplements (Supplementary Tables S1 and S2). For binary phenotypes (e.g. HCC and diabetes), effect sizes are presented on the log odds ratio scale, which are symmetrical around the line of null effect. This was to ensure that our forest plots give equal weight (visually) to associations implying protection vs associations implying harm. Genotype associations with continuous phenotypes (e.g. ALT and BMI) were determined using linear regression. Continuous phenotypes were first log-transformed to achieve approximate normality and then standardized into a z-score (i.e. with mean of zero and a standard deviation of one). This means the effect size represents the change in the phenotype (in terms of standard deviations) for each additional copy of the effect allele carried. In this way, the effect sizes for diverse continuous phenotypes can be compared “like for like”. All UKB associations were adjusted for age, sex and the first 5 principal components of genetic ancestry. Analyses of UKB data were performed using Stata version 17.
Results
Study population
Details of the study cohorts, comprising the initial and the validation cohort of patients with alcohol-associated cirrhosis, with and without HCC, alcohol misusers without cirrhosis and healthy controls as well as patients with HCC due to viral hepatitis C, are shown in table 1.
Genotype distribution
The distribution of LPL rs13702 genotypes was similar between healthy controls with a minor allele frequency (MAF) of 0.30, alcohol misuse without cirrhosis (MAF=0.32) and the genotype distribution reported for European population (MAF=0.32).
. Similarly, irrespective of HCC, MAF was 0.29 in the initial, 0.28 in in the validation cohort and 0.30 among patients with viral HCC. Genotype distribution of LPL rs13702 and rs238 are given in detail in table 2.
Table 2Genotype distribution of theLPLandPNPLA3polymorphisms.
Genotype
Alcohol-associated cirrhosis Bonn/Berlin (n=485)
Alcohol-associated cirrhosis Leipzig (n=556)
Viral HCC (n=280)
Alcohol misuse without cirrhosis (n=366)
Healthy controls (n=277)
LPL rs13702
without HCC n=333
with HCC n=152
without HCC n=323
with HCC n=233
TT
160 (48.0%)
80 (52.6%)
171 (52.9%)
119 (51.1%)
142 (50.7%)
164 (44.8%)
138 (49.8%)
TC
142 (42.6%)
66 (43.4%)
121 (37.5%)
103 (44.2%)*
106 (37.9%)
170 (46.4%)
114 (41.2%)
CC
31 (9.3%)
6 (3.9%)*&
31 (9.6%)
11 (4.7%)*&
32 (11.4%)
32 (8.7%)
25 (9.0%)
LPL rs328
CC
281 (84.4%)
127 (83.5%)
275 (85.1%)
190 (81.5%)
224 (80.0%)
293 (80.1%)
230 (83.0%)
CG
51 (15.3%)
25 (16.5%)
46 (14.3%)
42 (18.1%)
51 (18.2%)
70 (19.1%)
44 (15.9%)
GG
1 (0.3%)
0 (0.0%)
2 (0.6%)
1 (0.4%)
5 (1.8%)
3 (0.8%)
3 (1.1%)
PNPLA3 rs738409
CC
130 (39.0%)
38 (25.0%)
140 (43.3%)
59 (25.3%)
152 (54.3%)
212 (58.0%)
165 (59.6%)
GC
155 (46.5%)### §§§ &
72 (47.4%)### §§§ &
144 (44.6%)## §§ &
126 (54.1%)*** ### §§§ &
100 (35.7%)
129 (35.2%)
97 (35.0%)
GG
48 (14.4%)### §§§
42 (27.6%)*** ### §§§ &
39 (12.1%)### §§§
48 (20.6%)** ### §§§ &
28 (10.0%)
25 (6.8%)
15 (5.4%)
Legend: HCC hepatocellular carcinoma; LPL lipoprotein lipase; PNPLA3: Patatin-like phospholipase domain-containing protein 3 Statistical analysis with Pearson’s goodness-of-fit chi2 test.
* = p<0.05; ** = p<0.01; *** = p<0.001 vs cirrhosis without HCC.
LPL rs13702 C C genotype is protective against HCC in alcohol-associated cirrhosis
When we stratified patients with ALD concerning the presence of HCC, we found a significantly decreased frequency of the minor CC rs13702 LPL genotype compared to patients without HCC. This difference was confirmed in the validation cohort. Patients with viral HCC did not display a decreased frequency of the CC genotype (figure 1). When we compared histologically assessed levels of liver steatosis in ALD patients between the genotypes, carriers of the CC genotype had lower liver fat content (Figure 2A). Concerning liver enzymes, GGT was significantly decreased in carriers of the CC genotype (Figure 2B), whith a numerical similar effect for ALT (Figure 2C). Serum triglyceride levels decreased while HDL levels increased with the number of C alleles without meeting statistical significance (Figure 2D, E). In the clinical cohorts, no association of the LPL rs13702 C C genotype to diabetes (p = 0.78) or obesity (p = 0.45), was found.
Fig. 1Frequency of the homozygousLPLrs13702 C C genotype among the study cohorts. The homozygousLPLrs13702 C C genotype was less frequent among patients with HCC in alcohol-associated cirrhosis compared to patients without HCC and to cirrhotic patients with HCC due to viral hepatitis. Statistical analysis with Chi-square test. HCC: hepatocellular carcinoma; LPL: lipoprotein lipase
Fig. 2Liver enzymes, hepatic steatosis and lipid parameters in alcohol-associated cirrhosis according to theLPLrs13702 genotypes. Carriers of theLPLrs13702 C C genotype display numerically lower levels of liver enzymes and triglyceride levels and decreased liver fat. A) liver steatosis as assessed by histology B) Gamma-Glutamyl transferase (GGT) C) alanine aminotransferase (ALT) C) D) serum triglyceride levels and E) serum high-density lipoprotein levels according to the LPL rs13702 genotypes. Statistical analysis with Wilcoxon-Mann-Whitney-U test
The LPL rs328 polymorphism was not associated to alcoholic HCC in our cohort (table 2)
The protective effect of rs13702 LPL CC genotype is connected to PNPLA3 I148M genotype.
As expected, carriage of at least one minor PNPLA3 rs738409 allele was replicated as a risk factor both for alcohol-associated cirrhosis and alcohol-associated HCC in our study cohorts (table 2). Interestingly, the protective effect of the LPL rs13702 C C genotype was attenuated in patients carrying the high risk homozygous PNPLA3 GG genotype (figure 3). However, we found no statistical proof of interaction betweenLPLrs13702 C C andPNPLA3I148M concerning the risk to develop HCC (p=0.129;Supplementary Table S3).
Fig. 3Frequency of theLPLrs13702 C C genotype in patients with alcohol-associated cirrhosis stratified for carriage ofPNPLA3I148M genotype. The significant lower frequency of theLPLrs13702 C C genotype among patients with HCC was not found in carriers of the high riskPNPLA3148MM genotype. Statistical analysis with Chi-square test. HCC: hepatocellular carcinoma; LPL: lipoprotein lipase; PNPLA3: patatin-like phospholipase domain-containing protein 3
The LPL rs13702 C C genotype is independently associated with HCC
To verify if carriage of the LPL rs13702 C C genotype is independently associated with occurrence of HCC in patients with alcohol-associated cirrhosis, we performed univariate and multivariate analysis of known risk factors for HCC. Univariate analyses in both cohorts of patients with alcohol-associated cirrhosis combined showed that in addition to the CC genotype, age, male sex, diabetes and presence of the PNPLA3 148 M variant were associated with HCC (table 3). Multivariate analysis confirmed age, male sex, diabetes and PNPLA3 148 M as independent risk factors for HCC, while carriage of the LPL rs13702 C C genotype led to a decreased HCC risk (OR=0.5; p=0.03) (table 3).
Table 3Regression Analysis for possible HCC risk factors.
Univariate Analysis
95% CI
Parameter
P
OR
Lower
Upper
Age
0.000
1.084
1.067
1.101
Sex (male)
0.000
3.047
2.111
4.399
Diabetes
0.000
2.709
2.034
3.608
PNPLA3 148 M
0.000
2.077
1.573
2.742
LPL rs13702 C C
0.003
0.442
0.254
0.769
Multivariate Analysis*
95% CI
Parameter
P
OR
Lower
Upper
Age
0.000
1.075
1.056
1.095
Sex (male)
0.000
3.040
2.000
4.620
Diabetes
0.000
1.832
1.325
2.532
PNPLA3 148 M
0.000
2.002
1.433
2.796
LPL rs13702 C C
0.033
0.493
0.258
0.943
Legend: LPL lipoprotein lipase; PNPLA3: Patatin-like phospholipase domain-containing protein 3 Statistical analysis: Univariate analysis by Pearson’s goodness-of-fit chi2test followed by multivariate forward binary regression analysis.
Carriage of the LPL rs13702 C C genotype is not related to stage or mortality in HCC
We analyzed if presence of the LPL rs13702 C C genotype might be associated with stage or outcome in HCC patients. However, we did not find any association to BCLC stage at diagnosis, AFP-level at diagnosis or survival (data not shown).
Liver LPL expression is increased in patients with alcohol-associated cirrhosis compared to controls
Because LPL is not expressed in healthy liver, we investigated if this held also true for alcohol-associated cirrhosis, given the association between HCC and the rs13702 variant influencing tissue expression.
. LPL mRNA expression was significantly increased in the liver of alcohol-associated cirrhosis compared to viral cirrhotic livers, tumor-free, non-cirrhotic liver tissue gained by surgical resection of liver metastases and ALD livers with HCC (figure 4A). A similar effect was seen on the protein level (Figure 4B); however, only 2 specimen of ALD liver were available for protein analysis. Hepatic LPL mRNA expression in alcohol-associated cirrhosis was stepwise increased with the number of C alleles at the rs13702 locus (figure 4B).
Fig. 4LPL expression in human liver tissue hepatic cells lines and LPL mRNA expression is significantly increased in liver tissue from alcohol-associated cirrhosis compared to non-cirrhotic liver, HCV cirrhosis and cirrhotic liver with HCC; is associated to the number of C alleles at theLPLrs13702 locus and not found in hepatocytes, but HSC and LSEC. A) LPL mRNA expression in human liver tissue. B) LPL protein expression in human tissue C) LPL mRNA expression correlated with the number ofLPLrs13702 C alleles. D) representative Western blot for protein analysis in hepatic cell lines and E) quantified LPL protein expression in hepatic cell lines revealed negligible amounts in hepatocytes. Statistical analysis with student’s t-test (A, C) and Spearman’s correlation (B). ALD: alcohol-associated liver disease; HCC: hepatocellular carcinoma; HCV: hepatitis C virus; HSC: hepatic stellate cells; LPL: lipoprotein lipase; LSEC: liver sinusoidal endothelial cells
. To verify this finding, we investigated LPL protein expression in various human liver cell lines. While we found only minimal LPL expression in the hepatocyte cell line PLC/PRF/5 and in primary hepatocytes, LPL was expressed at much higher levels in LSEC, primary HSC and the hepatic stellate cell line LX-2 on the protein level (figure 4D, E).
Validation at a population level in the UK biobank cohort
In the UKB cohort, rs13702:C was associated with a reduced risk of HCC (aOR: 0.83; 95%CI: 0.72-0.97; P=0.016) (Figure 5). Several metabolic traits and transaminase levels were also associated with rs13702:C, albeit the effect size was generally very small. An exception to this rule was the association between rs13702 and triglycerides where the effect size was much larger (beta: -0.12 decrease per C allele carried).
Fig. 5Association of theLPLrs13702,LPLrs328 andPNPLA3 rs738409 minor alleles with liver and metabolic related phenotypes in the UK Biobank cohort. Panel A and B include binary and continuous phenotypes, respectively. All associations are adjusted for age, sex and the first five principal components of genetic ancestry. Details of the statistical analysis are provided in the methods section. T2DM: type 2 diabetes mellitus; HCC: hepatocellular carcinoma; BMI: body mass index; ALT: alanine aminotransferase; AST: aspartate aminotransferase; FIB4 fibrosis 4 index;
Carriage of rs328:G in LPL was also associated with a reduced risk of HCC and metabolic traits. However, although the effect sizes were generally comparable to rs13702, the statistical significance was weaker, likely reflecting the lower allele frequency of rs328:G. Neither rs13702:C (beta: -0.1; p=0.21) nor rs328:G (beta: -0.01; p=0.38) were associated with hepatic fat content assessed by MRI on the population level.
Discussion
Genetic case control studies are useful to investigate mechanisms for disease whose development spans several decades. This is in particular true for cirrhosis and HCC, in which many years of ongoing liver damage and genetic predisposition contribute to occurrence of disease.
. Knowledge of such risk factors may lead to better surveillance strategies in patients at risk and elucidate the molecular basis of HCC development.
Analysing different cohorts of cases with HCC and controls with alcohol-associated cirrhosis, alcohol-abusers without liver disease and healthy controls regarding the frequency of the LPL rs13702 genotypes, we found that the CC genotype was protective against development of HCC in two independent cohorts and after correction for age, sex, PNPLA3 I148M genotype and diabetes. Interestingly, we noted that carriage of the CC genotype at the LPL rs13702 locus did not mitigate the risk for HCC in the high risk group of homozygous PNPLA3 148 M carriers, underlining the complex interplay of different genetic risk factors and hinting at a shared molecular pathway. International guidelines recommend regular screening by ultrasound in all patients affected by ALD and advanced fibrosis.
European Association For The Study Of The Liver, European Organisation For Research And Treatment Of Cancer EASL-EORTC clinical practice guidelines: management of hepatocellular carcinoma.
. Since the number of patients with alcohol-associated liver disease is high – amounting to 60.000 in England and Wales in 2012, for example - and the incidence of alcohol abuse is rising,
Addressing liver disease in the UK: a blueprint for attaining excellence in health care and reducing premature mortality from lifestyle issues of excess consumption of alcohol, obesity, and viral hepatitis.
, it will be essential to narrow the number of patients to screen down to make efficient surveillance feasible. In NAFLD, a polygenic risk score comprising 5 different genetic risk loci showed a high negative predictive value of 94% for the development of HCC with at the same time limiting the number of patients at risk to only 23% of the cirrhotic study population.
. The discovery of new genetic variants such as at the LPL rs13702 locus may help to develop similar genetic risk scores for ALD patients with an even better predictive capacity when interactions such as between PNPLA3 and LPL variants are considered. With an odds ratio of 0.49, the LPL rs13702 C C genotype has a major impact on the individual risk of HCC. In addition, we investigated theLPLrs328 variant, which has a similar effect on lipid levels
Associations of Lipoprotein Lipase Gene Polymorphisms With Longitudinal Plasma Lipid Trends in Young Adults: The Coronary Artery Risk Development in Young Adults (CARDIA) Study.
. While we observed a numerically similar effect to theLPLrs13702 minor variant, it failed statistical significance for HCC risk. This fits in with reports from a previous study on lipid levels which indicated that rs328 is in linkage disequilibrium with rs13702 and exerts weaker effects
MicroRNA-410 regulated lipoprotein lipase variant rs13702 is associated with stroke incidence and modulated by diet in the randomized controlled PREDIMED trial.
From a mechanistic point of view, the association of LPL genetic variation to the risk of HCC highlights the importance of lipid metabolism for HCC development in ALD. With PNPLA3,
and apolipoprotein E(6), other prominent genetic loci for HCC have been implicated in hepatic lipid accumulation. While the underlying mechanisms leading to hepatic lipid accumulation have been partly unravelled, the molecular background behind the increased hepatocarcinogenesis is only incompletely understood. Among the factors supposed to facilitate HCC development and potentially linked to genetic variation are oxidative stress by overload with fatty acids and chronic inflammation.
. Concerning PNPLA3, we have reported that hepatocytes carrying PNPLA3 148 M secrete more pro-inflammatory chemokines when exposed to increased lipid levels, creating a tumorigenic environment.
. By abolishing the inhibitory effect of miR-410 on its gene expression, the minor variant of the rs13702 polymorphism leads to decreased circulating levels of triglycerides.
. Given the protective effect of the minor variant on hyperlipidaemia, it is not surprising that its presence is associated with a decreased risk for stroke, depending on the diet of the patient,
MicroRNA-410 regulated lipoprotein lipase variant rs13702 is associated with stroke incidence and modulated by diet in the randomized controlled PREDIMED trial.
. Nevertheless, liver LPL mRNA levels were increased 3.6 fold in 16 patients with NAFLD compared to 8 patients without steatosis undergoing bariatric surgery and correlated closely with liver fat content.
Genes involved in fatty acid partitioning and binding, lipolysis, monocyte/macrophage recruitment, and inflammation are overexpressed in the human fatty liver of insulin-resistant subjects.
. Similarly, hepatic mRNA expression was reported as very low in 14 healthy controls, increased in 21 patients with NAFLD but highest in 54 patients with NASH(17). Interestingly, immunofluorescence staining attributed the LPL expression in the liver not to hepatocytes, but to hepatic stellate cells (HSC).
MicroRNA-410 regulated lipoprotein lipase variant rs13702 is associated with stroke incidence and modulated by diet in the randomized controlled PREDIMED trial.
, it is possible that the effect of the LPL rs13702 minor variant on HCC development might not have its mechanistical origin in liver tissue, but that the lipid content of lipoproteins taken up by the liver might be responsible for the differences in HCC risk. However, while impact of the rs13702 LPL genotype on serum lipid levels in our patients with alcohol-associated cirrhosis was only mild, we noted lower liver enzymes and lower hepatic fat content among patients carrying the rs13702 C C genotype, supporting an impact on the liver metabolism. Since that the effect on liver fat missed statistical significance on a population level, dietary habits such as alcohol abuse may be an important precondition.</u>
Additionally, our study expands the knowledge on hepatic LPL expression by showing that LPL upregulation in the human liver is dependent on the number of C alleles at the rs13702 locus in alcohol-associated cirrhosis, highlighting the potential hepatic functional relevance of this genetic variant for metabolic liver disease. Finally, comparison of LPL expression in different liver derived cell lines supported the finding by Teratani et al. that HSCs are a major source of LPL(17). Although an important role for HSCs not only in hepatic fibrosis, but also in creating a tumorigenic microenvironment has been suspected based on their property to produce growth factors and respective cytokines,
, evidence from clinical human studies has still been scarce. Our study now provides sound evidence that HSCs may be crucially involved in HCC development in humans. In line, the important role of cell metabolism for activation of HSCs has been reviewed recently.
. Because high LPL expression was not limited to HSC, but also found in LSEC, they might contribute to the reduced risk for HCC as well.
The role of LPL in advanced liver disease and the mechanism by which LPL upregulation decreases hepatic tumorigenesis are not known. Similar to PNPLA3, which differs functionally in mice and humans, findings on LPL function in mice and humans are contradictory. Human LPL high producers are protected from NAFLD(9). Similarly, our data suggest that overexpression of LPL is beneficial regarding HCC development in human alcoholic liver disease. However, knock-out of LPL in HSC in a murine NASH model reduced hepatic fibrosis
. The major differences in the experimental design concerning disease – NASH versus ALD, species – murine versus human; and stage of liver disease – fibrosis versus HCC might account for these apparently contradictory results. Most importantly, experimental knock-out of a protein which results in complete absence might lead to different effects compared to differences in the level of expression. LPL knock-out mice survive birth only for hours.
Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impaired very low density lipoprotein clearance in heterozygotes.
. In support of the concept that LPL expression in hepatocytes is irrelevant to liver disease, knock-out of LPL specifically in hepatocytes did not change hepatic fat content.
. Genetic LPL deficiency in humans is a very rare and often severe condition, that leads to high blood triglyceride levels and recurrent pancreatitis, with fatty liver being clinically not relevant in these patients.
. Thus, knock-out of LPL only in HSCs may not reflect the complex interplay involving several tissues in humans. It seems plausible that LPL upregulation in human NAFLD and ALD occurs in compensation to pathological changes in lipid metabolism, as shown for chronic alcohol abuse,
, which would explain why genetic high producers are to some extent protected from hepatic liver overload, NAFLD and HCC. Intrahepatic fat content is recognised as a risk factor for HCC in humans,
. However, the hypothesis that LPL is upregulated in compensation to hepatic fat overload, which results in reduction of hepatic fat, lipotoxicity and subsequently HCC development, has still to be proven.
Our findings are based on a case control study, with the well-known limitations of this approach. Most importantly, we cannot exclude that some of the controls will develop HCC in the future or would have developed HCC if they had not died before from another complication of liver cirrhosis, such as bacterial sepsis. Such issues could be only resolved in a prospective study including only patients at diagnosis of cirrhosis. However, although such studies are highly welcome to assess the so fare known risk factors for HCC, this approach would likely need decades, because HCC formation in cirrhosis is a process spanning many years. Next, we cannot prove an additive effect, meaning that patients carrying only oneLPLrS13702 C allele have a HCC risk in between wildtype and homozygous CC allele carriers in our cohort. This might be due to a threshold effect: LPL mRNA expression in the liver was much more increased in homozygous compared to heterozygous CC allele carriers (figure 4C) and the effect on liver enzyme levels and hepatic fat was much more pronounced in homozygous CC allele carriers (Figure 2A-C). LPL activity is tightly controlled also on a posttranscriptional level
, so that the effect of a single C allele might be diminished considerably. In addition, our study may be underpowered to show an additive effect: studies showing an additive effect on lipid levels in the blood included more than 27.000
MicroRNA-410 regulated lipoprotein lipase variant rs13702 is associated with stroke incidence and modulated by diet in the randomized controlled PREDIMED trial.
individuals. Nevertheless, as illustrated infigure 5of the revised manuscript, analysis of the UK Biobank cohort showed an additive effect.
The correlation of hepaticLPLmRNA expression with the number of rs13702 C alleles in patients with alcohol-associated cirrhosis is limited by the rather small number of specimens which we analysed. However, higherLPLexpression in carriers of a C allele has been described before in non-liver tissue
. Given that patients with alcoholic-associated cirrhosis carry two C alleles in only 10%, analysis of more liver specimen from those patients is challenging.
Next, we cannot exclude that genetic variation atLPLrs13702 is not causally related to HCC, but only in linkage with the real associated factor. Mechanistic studies in human are nearly impossible, andLPLmice models do not fully reflect human phenotype. Since LPL was highly upregulated in liver tissue of patients with ALD and expression levels correlated to the number of rs13702 C alleles, it is highly likely that LPL is crucially involved in advanced ALD. Measurement of intrahepatic LPL activity might linkLPLrs13702 genotypes mechanistically more closely to HCC development, but we did not have enough patient specimen to analyse LPL activity.
In summary, we report a novel genetic variant inLPLassociated with decreased occurrence of HCC in ALD.
Acknowledgments
The authors would like to thank all study participants, clinicians and administrative staff who contributed to this study. We also acknowledge the UK biobank resource: application number 8764.
Appendix A. Supplementary data
The following is/are the supplementary data to this article:
Stickel F, Lutz P, Buch S, Nischalke HD, Silva I, Rausch V, et al. Genetic variation in HSD17B13 reduces the risk of developing cirrhosis and hepatocellular carcinoma in alcohol misusers. Hepatol. Baltim. Md. 2019;
Genes involved in fatty acid partitioning and binding, lipolysis, monocyte/macrophage recruitment, and inflammation are overexpressed in the human fatty liver of insulin-resistant subjects.
MicroRNA-410 regulated lipoprotein lipase variant rs13702 is associated with stroke incidence and modulated by diet in the randomized controlled PREDIMED trial.
Common Variants in Lipid Metabolism-Related Genes Associate with Fat Mass Changes in Response to Dietary Monounsaturated Fatty Acids in Adults with Abdominal Obesity.
Associations of Lipoprotein Lipase Gene Polymorphisms With Longitudinal Plasma Lipid Trends in Young Adults: The Coronary Artery Risk Development in Young Adults (CARDIA) Study.
Addressing liver disease in the UK: a blueprint for attaining excellence in health care and reducing premature mortality from lifestyle issues of excess consumption of alcohol, obesity, and viral hepatitis.
Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impaired very low density lipoprotein clearance in heterozygotes.
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