Editor: Jian-Gao Fan, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, China
17β-hydroxysteroid dehydrogenase 13 (
Consecutive biopsy-proven NAFLD patients and controls without fatty liver were recruited for this study between 2009 and 2014. Genotyping for
This study is unique in that it evaluates the association of
An adverse liver outcome is one of the leading causes of death in non-alcoholic fatty liver disease (NAFLD) [
Several studies hypothesised that patatin-like phospholipase domain-containing 3 (
This study aimed to evaluate the clinical impact of
Consecutive biopsy-proven NAFLD patients were recruited from the Gastroenterology and Hepatology Clinic, University of Malaya Medical Centre (UMMC) from 2009 to 2014. Patients with viral hepatitis B or C infection, autoimmune liver diseases, significant alcohol intake (>21 units per week for men and >14 units per week for women), and use of medication that can cause steatosis or other causes of chronic liver diseases were excluded from this study [
Demographic, anthropometric, clinical, and laboratory data were obtained using a standard protocol. Obesity was defined as BMI ≥25 kg/m2 [
Ultrasound-guided percutaneous liver biopsy was performed using 18G TERUMO® Hypodermic Needle (Cardinal Health, Dublin, OH, USA). Liver biopsy slides were examined by an experienced histopathologist and reported according to the NASH clinical research network scoring system [
The QiAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used to extract genomic DNA from the concentrated white blood cells of the buffy coat with the quality ranging from 1.8–2.0 (OD260/OD280).
Only biopsy-proven NAFLD patients with a minimum follow-up of 5 years were included in the longitudinal study. These patients were on a regular 6-monthly follow-up at the gastroenterology and hepatology clinic with blood test ± ultrasound performed prior to the clinic visit. Their medical records were also carefully studied from the date of the liver biopsy until 31st August 2019 to record any cardiovascular events, liver-related complications, malignancy, or mortality. Cardiovascular events included a history of acute myocardial infarction, congestive cardiac failure, history of coronary revascularisation, and stroke. Liver-related complications included ascites, hepatic encephalopathy, spontaneous bacterial peritonitis, history of gastro-oesophageal varices or variceal bleeding, HCC, and hepatorenal syndrome. The cause of malignancy or cause of death was recorded for patients who developed malignancy or died.
Power analysis was carried out using Quanto to calculate the sample size [
A total of 223 biopsy-proven NAFLD patients and 205 controls were included in the case-control study. The characteristics of the subjects are shown in
The characteristics of biopsy-proven NAFLD patients with different
One hundred and sixty-five biopsy-proven NAFLD patients had follow-up data for at least 5 years and were included in the outcome analysis. A total of 58 patients were excluded from the outcome analysis (49 patients who were lost to follow-up and nine patients had follow-up of <5 years). The characteristics of the subjects are summarised in
The mean duration of follow-up for the study subjects was 89 months (range, 60–118), corresponding to a total follow-up of 1,227 person-years. During follow-up, 32 patients (19.4%) experienced at least one clinical outcome. Cardiovascular events topped the list with 22 patients (13.3%) followed by liver-related complications in six patients (3.6%). Five patients (3.0%) developed extrahepatic malignancy. Six patients (3.6%) died during the follow-up period; three were due to extrahepatic malignancy, two were liver-related deaths, and one was due to infection. A detailed breakdown of the clinical outcomes according to the genetic variants is shown in
The log-rank test revealed no difference in the survival probability of patients with different
The rs72613567 T/TA genotype was associated with a higher incidence of cardiovascular outcome (hazard ratio [HR], 3.57; 95% CI, 1.34–9.48;
Serum GGT level was the only independent predictor of extrahepatic malignancy and all-cause mortality.
Multivariate Cox regression analysis revealed that the rs72613567 homozygous TA allele and the rs6834314 G/G genotype were associated with a reduced risk of liver-related complications (HR, 0.004; 95% CI, 0–0.64;
In this study of a well-characterised cohort of multi-ethnic Asian biopsy-proven NAFLD patients, we found loss-of-function
These novel findings highlight the different impacts of the same genetic variants in persons of different ethnic backgrounds and complements previous interesting observations in genetic studies of NAFLD. Browning and colleagues were the first to report on ethnic differences in the prevalence of significant hepatic steatosis in their landmark paper [
We set out to determine if the loss-of-function
rs72613567 is an insertion of an adenine adjacent to the donor splice site of exon 6 of
Another highlight of our study is that the loss-of-function
There was a lack of observed association between the TA allele of rs72613567 and steatosis, which is consistent with the findings from previous studies [
A recent genetic association study in Japan reported that the carriage of the rs6834314 G allele attenuates the effect of
In this study, we also found that serum GGT level is significantly associated with various clinical outcomes. Serum GGT level is a reliable marker of oxidative stress. Haring et al. [
An interesting finding from our study showed that the rs72613567 heterozygous mutant genotype is associated with a higher incidence of cardiovascular outcome, but not in the homozygous mutant. We speculate that phenotypic consequences of a dominant mutation can be observed in a heterozygous individual carrying one mutant and one wild-type allele. In 1987, Herskowitz [
To our knowledge, this study is the first longitudinal cohort study among the Asian population to report a potential protective effect of the protein-truncating
In conclusion, in this two-part study on a cohort of biopsy-proven multi-ethnic Asian NAFLD patients, the loss-of-function variants
The authors thank all the participants and staff of the UMMC for the assistance during patient’s recruitment. This study was supported by University Malaya Research Grant BK077-2016.
RM, SMZ and ZM designed the research study. YWT, SMZ, WKC, HLT established cohort and collected the clinical data for patients. YWT, ASYK, SMZ and HLT carried out genotyping. YWT carried out data analysis and documented the findings. YWT, WKC and SMZ wrote the manuscript. RM, SMZ, WKC and YFP provided critical inputs to the manuscripts. The final version of the manuscript has been read and approved by all the authors.
The authors have no conflicts to disclose.
Supplementary material is available at Clinical and Molecular Hepatology website (
Additional methodological details
The
Comparison of clinical characteristics and baseline histological parameters between
Comparison of clinical characteristics and baseline histological parameters between
Association of
Clinical outcomes of the 165 biopsy-proven NAFLD patients with genotype and outcome data
Association of
Association of
Association of
Survival curve of (A) CVS outcome, (B) liver-related complications, (C) extrahepatic malignancy, and (D) overall mortality, stratified by
Survival curve of (A) CVS outcome, (B) liver-related complications, (C) extrahepatic malignancy, and (D) overall mortality, stratified by
body mass index
confidence interval
fasting blood glucose
fatty liver index
gamma-glutamyl transferase
genome-wide association study
hepatocellular carcinoma
high-density lipoprotein
hazard ratio
17β-hydroxysteroid dehydrogenase 13
HardyWeinberg equilibrium
insertion and deletion
low-density lipoprotein
non-alcoholic fatty liver
non-alcoholic fatty liver disease
non-alcoholic steatohepatitis
odds ratio
principal component analysis
patatin-like phospholipase domain-containing 3
single nucleotide polymorphism
University of Malaya Medical Centre
Characteristics of biopsy-proven NAFLD patients compared with controls
Biopsy-proven NAFLD patients (n=223) | Controls (n=205) | ||
---|---|---|---|
Age (years) | 55.6±12.5 | 51.0±12.5 | <0.001 |
Male | 117 (52.5) | 87 (42.4) | 0.038 |
Ethnicity | 0.021 | ||
Malay | 113 (50.7) | 71 (34.6) | |
Chinese | 61 (27.4) | 86 (42.0) | |
Indian | 49 (22.0) | 48 (23.4) | |
BMI (kg/m2) | 29.0±4.7 | 22.0±2.2 | <0.001 |
HbA1c (%) | 6.5±1.5 | 5.5±0.5 | <0.001 |
Total cholesterol (mmol/L) | 5.1±1.1 | 4.6±0.7 | <0.001 |
Triglyceride (mmol/L) | 1.7±0.7 | 1.2±0.4 | <0.001 |
HDL (mmol/L) | 1.2±0.3 | 1.3±0.4 | <0.001 |
LDL (mmol/L) | 3.2±1.0 | 2.3±0.5 | <0.001 |
ALT (U/L) | 81.4±46.8 | 33.8±17.4 | <0.001 |
AST (U/L) | 45.6±25.9 | 22.1±9.3 | <0.001 |
GGT (U/L) | 106.8±106.3 | 41.2±26.0 | <0.001 |
Liver histological features | |||
Steatosis | |||
0 | 0 (0.0) | ||
1 | 65 (29.1) | ||
2 | 103 (46.2) | ||
3 | 55 (24.7) | ||
Lobular inflammation | |||
0 | 7 (3.1) | ||
1 | 126 (56.5) | ||
2 | 86 (38.6) | ||
3 | 4 (1.8) | ||
Hepatocyte ballooning | |||
0 | 37 (16.6) | ||
1 | 125 (56.1) | ||
2 | 61 (27.4) | ||
NASH | 175 (78.5) | ||
Fibrosis | |||
0 | 50 (22.4) | ||
1 | 89 (39.9) | ||
2 | 43 (19.3) | ||
3 | 36 (16.1) | ||
4 | 5 (2.2) |
Values are presented as mean±standard deviation or number (%).
NAFLD, non-alcoholic fatty liver disease; BMI, body mass index; HbA1c, haemoglobin A1c; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl transferase; NASH, non-alcoholic steatohepatitis.
The
Allele frequency | OR (95% CI) | Adjusted OR (95% CI) | |||
---|---|---|---|---|---|
rs72613567 TA allele | |||||
NAFLD vs. control | |||||
Overall | 0.24 vs. 0.34 | 0.58 (0.40–0.86) | 0.006 | 0.59 (0.40–0.88) | 0.009 |
Malays | 0.28 vs. 0.40 | 0.60 (0.33–1.09) | 0.092 | 0.62 (0.34–1.13) | 0.117 |
Chinese | 0.26 vs. 0.41 | 0.41 (0.21–0.81) | 0.010 | 0.31 (0.15–0.67) | 0.003 |
Indians | 0.11 vs. 0.14 | 0.78 (0.31–1.97) | 0.597 | 0.71 (0.27–1.87) | 0.492 |
NAFL vs. control | |||||
Overall | 0.27 vs. 0.34 | 0.73 (0.39–1.37) | 0.330 | 0.72 (0.38–1.38) | 0.326 |
Malays | 0.22 vs. 0.40 | 0.45 (0.18–1.11) | 0.081 | 0.49 (0.20–1.24) | 0.132 |
Chinese | 0.50 vs. 0.41 | 1.98 (0.51–7.71) | 0.327 | 1.81 (0.44–7.53) | 0.414 |
Indians | 0.06 vs. 0.14 | 0.39 (0.04–3.44) | 0.392 | 0.30 (0.03–2.98) | 0.303 |
NASH vs. control | |||||
Overall | 0.23 vs. 0.34 | 0.55 (0.36–0.83) | 0.004 | 0.55 (0.36–0.83) | 0.005 |
Malays | 0.31 vs. 0.40 | 0.65 (0.35–1.23) | 0.187 | 0.67 (0.35–1.28) | 0.224 |
Chinese | 0.20 vs. 0.41 | 0.27 (0.13–0.57) | 0.001 | 0.19 (0.08–0.45) | <0.001 |
Indians | 0.12 vs. 0.14 | 0.87 (0.33–2.26) | 0.772 | 0.83 (0.31–2.24) | 0.708 |
NASH vs. NAFL | |||||
Overall | 0.23 vs. 0.27 | 0.75 (0.39–1.43) | 0.384 | 0.75 (0.39–1.44) | 0.386 |
Malays | 0.31 vs. 0.22 | 1.46 (0.61–3.49) | 0.402 | 1.51 (0.62–3.67) | 0.368 |
Chinese | 0.20 vs. 0.50 | 0.14 (0.03–0.57) | 0.006 | 0.13 (0.03–0.57) | 0.007 |
Indians | 0.12 vs. 0.06 | 2.26 (0.25–20.65) | 0.471 | 2.25 (0.23–22.13) | 0.488 |
rs6834314 G allele | |||||
NAFLD vs. control | |||||
Overall | 0.26 vs. 0.39 | 0.59 (0.40–0.86) | 0.006 | 0.59 (0.40–0.87) | 0.009 |
Malays | 0.31 vs. 0.44 | 0.65 (0.36–1.20) | 0.171 | 0.66 (0.36–1.22) | 0.189 |
Chinese | 0.29 vs. 0.45 | 0.39 (0.20–0.77) | 0.006 | 0.31 (0.14–0.66) | 0.002 |
Indians | 0.12 vs. 0.19 | 0.71 (0.29–1.74) | 0.458 | 0.64 (0.25–1.64) | 0.355 |
NAFL vs. control | |||||
Overall | 0.29 vs. 0.39 | 0.79 (0.42–1.48) | 0.453 | 0.74 (0.38–1.47) | 0.391 |
Malays | 0.26 vs. 0.44 | 0.54 (0.22–1.31) | 0.173 | 0.60 (0.24–1.49) | 0.267 |
Chinese | 0.50 vs. 0.45 | 2.52 (0.52–12.15) | 0.250 | 2.26 (0.44–11.61) | 0.329 |
Indians | 0.06 vs. 0.19 | 0.31 (0.04–2.79) | 0.299 | 0.20 (0.02–1.90) | 0.160 |
NASH vs. control | |||||
Overall | 0.26 vs. 0.39 | 0.54 (0.36–0.82) | 0.003 | 0.48 (0.31–0.75) | 0.001 |
Malays | 0.33 vs. 0.44 | 0.70 (0.37–1.32) | 0.270 | 0.71 (0.37–1.35) | 0.291 |
Chinese | 0.23 vs. 0.45 | 0.25 (0.12–0.53) | <0.001 | 0.20 (0.09–0.45) | <0.001 |
Indians | 0.13 vs. 0.19 | 0.81 (0.32–2.03) | 0.648 | 0.76 (0.29–2.00) | 0.582 |
NASH vs. NAFL | |||||
Overall | 0.26 vs. 0.29 | 0.69 (0.36–1.31) | 0.256 | 0.69 (0.36–1.32) | 0.263 |
Malays | 0.33 vs. 0.26 | 1.30 (0.55–3.09) | 0.555 | 1.37 (0.57–3.30) | 0.485 |
Chinese | 0.23 vs. 0.50 | 0.10 (0.02–0.50) | 0.005 | 0.10 (0.02–0.50) | 0.006 |
Indians | 0.13 vs. 0.06 | 2.57 (0.28–23.31) | 0.402 | 2.29 (0.23–22.48) | 0.476 |
NAFL was defined as hepatic steatosis with no or only mild lobular inflammation and no hepatocyte ballooning. NASH was defined as the presence of hepatic steatosis, lobular inflammation and hepatocyte ballooning (at least grade 1 each) with or without fibrosis.
Logistic regression was used to obtain OR. The adjusted OR was adjusted for age and gender.
Control was the reference group in analysis NAFLD vs. control, NAFL vs. control and NASH vs. control. NAFL was used as the reference group in analysis for NASH vs. NAFL.
Demographic and clinical characteristics of NAFLD patients with a minimum follow-up of 5 years (n=165)
Variable | NAFLD cases (n=165) |
---|---|
Age (years) | 58.9±10.4 |
Gender, male | 81 (49.1) |
Ethnicity | |
Malay | 80 (48.5) |
Chinese | 47 (28.5) |
Indian | 38 (23.0) |
Diabetes mellitus | 108 (65.5) |
Hypertension | 117 (70.9) |
Dyslipidaemia | 153 (92.7) |
Metabolic syndrome | 131 (79.4) |
BMI (kg/m2) | 28.9±4.9 |
Waist circumference (cm) | 98.1±11.1 |
Male | 99.9±10.7 |
Female | 95.4±10.2 |
Blood pressure | |
Systolic (mmHg) | 135.7±14.4 |
Diastolic (mmHg) | 78.5±10.4 |
Fasting blood glucose (mmol/L) | 6.6±2.1 |
HbA1c (%) | 7.1±1.6 |
Total Cholesterol (mmol/L) | 4.6±1.1 |
Triglyceride (mmol/L) | 1.7±0.9 |
HDL (mmol/L) | 1.3±0.3 |
LDL (mmol/L) | 2.6±1.0 |
Albumin (mg/L) | 38.8±5.5 |
Total bilirubin (µmol/L) | 13.3±8.7 |
ALT (U/L) | 47.8±33.5 |
AST (U/L) | 44.7±54.1 |
ALP (U/L) | 87.8±77.5 |
GGT (U/L) | 94.0±149.3 |
Platelet (109/L) | 293.0±216.9 |
Baseline liver histological features | |
Steatosis | |
0 | 0 (0.0) |
1 | 40 (24.3) |
2 | 84 (50.9) |
3 | 41 (24.8) |
Lobular inflammation | |
0 | 4 (2.4) |
1 | 90 (54.5) |
2 | 68 (41.2) |
3 | 3 (1.8) |
Hepatocyte ballooning | |
0 | 28 (17.0) |
1 | 89 (53.9) |
2 | 48 (29.1) |
NASH | 129 (78.2) |
Fibrosis | |
0 | 34 (20.6) |
1 | 68 (41.2) |
2 | 29 (17.6) |
3 | 30 (18.2) |
4 | 4 (2.4) |
|
|
T/T | 94 (57.0) |
T/TA | 55 (33.3) |
TA/TA | 16 (9.7) |
|
|
A/A | 89 (53.9) |
A/G | 63 (38.2) |
G/G | 13 (7.9) |
Values are presented as mean±standard deviation or number (%).
NAFLD, non-alcoholic fatty liver disease; BMI, body mass index; HbA1c, haemoglobin A1c; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, gamma-glutamyl transferase; NASH, non-alcoholic steatohepatitis;
Association of
Variable | Univariate analysis |
Multivariate analysis |
||
---|---|---|---|---|
HR (95% CI) | HR (95% CI) | |||
rs72613567 | ||||
T/T (reference) | – | – | – | – |
T/TA | 0.42 (0.05–3.73) | 0.434 | 0.12 (0.01–2.17) | 0.152 |
TA/TA | 1.11 (0.12–10.01) | 0.926 | 0.004 (0.00–0.64) | 0.033 |
Age | 1.04 (0.95–1.13) | 0.418 | – | – |
Gender | 0.47 (0.09–2.58) | 0.387 | – | – |
Advanced fibrosis | 8.94 (1.63–48.94) | 0.012 | 7.98 (1.54–41.50) | 0.014 |
Serum GGT | 1.003 (1.002–1.005) | <0.001 | 1.01 (1.00–1.02) | 0.010 |
Diabetes mellitus | Inf (NA) | 0.315 | – | – |
Hypertension | 1.77 (0.21–15.18) | 0.603 | – | – |
Dyslipidaemia | 0.35 (0.04–3.04) | 0.344 | – | – |
BMI | 1.05 (0.89–1.23) | 0.550 | – | – |
rs6834314 | ||||
A/A (reference) | – | – | – | – |
A/G | 0.97 (0.16–5.83) | 0.977 | 0.46 (0.06–3.66) | 0.462 |
G/G | 1.60 (0.17–15.49) | 0.685 | 0.01 (0.00–0.97) | 0.048 |
Age | 1.04 (0.95–1.13) | 0.418 | – | – |
Gender | 0.47 (0.09–2.58) | 0.387 | – | – |
Advanced fibrosis | 8.94 (1.63–48.94) | 0.012 | 8.93 (1.66–47.90) | 0.011 |
Serum GGT | 1.003 (1.002–1.005) | <0.001 | 1.01 (1.00–1.02) | 0.003 |
Diabetes mellitus | Inf (NA) | 0.315 | – | – |
Hypertension | 1.77 (0.21–15.18) | 0.603 | – | – |
Dyslipidaemia | 0.35 (0.04–3.04) | 0.344 | – | – |
BMI | 1.05 (0.89–1.23) | 0.550 | – | – |
Inf (NA) represented number was too large to be recorded.