It is important to determine the noninvasive parameters of histological features in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the value of genetic variations as surrogate markers of histological features.
The parameters that affected the histological features of NAFLD were investigated in 211 Japanese patients with biopsy-proven NAFLD. The relationships between genetic variations in
The fibrosis stage of
In Japanese patients with biopsy-proven NAFLD,
It is important to determine the noninvasive parameters of histological features in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the value of genetic variations as surrogate markers of histological features.
The parameters that affected the histological features of NAFLD were investigated in 211 Japanese patients with biopsy-proven NAFLD. The relationships between genetic variations in
The fibrosis stage of
In Japanese patients with biopsy-proven NAFLD,
Nonalcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide across different ethnicities,1–6 and results in serious health-care issue. NAFLD includes a wide spectrum of liver pathologies ranging from nonalcoholic fatty liver, which is usually benign, to nonalcoholic steatohepatitis (NASH), which may lead to liver cirrhosis, hepatocellular carcinoma, and liver failure without excessive alcohol intake.7 Vitamin E8 and Farnesoid X nuclear receptor ligand obeticholic acid9 practically had improved the histological features. NASH can only be diagnosed by histological components, such as steatosis, lobular inflammation, ballooning, and fibrosis. Although histological diagnosis (such as steatosis, lobular inflammation, ballooning, and fibrosis) is currently the gold standard for diagnosing progressive NASH, liver biopsy has many drawbacks, such as cost, sampling error, and risk of complications.6 It is important to determine the noninvasive parameters as surrogate markers of histological features.
The severity and progression of NAFLD is influenced by a complex of multiple factors, including environmental factors and genetic variations.10 Especially, one of the most significant genetic risk factor for NAFLD is a variant located in the
The present study included 211 patients with biopsy-proven NAFLD. The aims of the study were to investigate the relationships between genetic variations (
A single-center retrospective cohort study was performed based on the patients of biopsy-proven NAFLD. Two hundred eleven Japanese patients were diagnosed with NAFLD by liver biopsy from 1980 to 2015 at Toranomon Hospital. NAFLD was diagnosed based on liver biopsy findings of steatosis in 5% or more of hepatocytes and the exclusion of other liver diseases (such as primary biliary cirrhosis, autoimmune hepatitis, drug induced liver disease, viral hepatitis, hemochromatosis, biliary obstruction, α-1-antitrypsin deficiency-associated liver disease, and Wilson disease). Patients consuming more than 20 g/day alcohol were excluded. The study protocol was in compliance with the Good Clinical Practice Guidelines and the 1975 Declaration of Helsinki, and was approved by the Institutional Review Board at Toranomon Hospital. All patients provided written informed consent at the time of liver biopsy.
Liver biopsy specimens were obtained using a 14-gauge modified Vim Silverman needle (Tohoku University style; Kakinuma Factory, Tokyo, Japan), a 16-gauge core tissue biopsy needle (Bard Peripheral Vascular Inc., Tempe, AZ, USA) or surgical resection. Tissue was fixed in 10% formalin, and sections were stained with hematoxylin and eosin, Masson trichrome, silver impregnation, and periodic acid-Schiff after diastase digestion. The specimens were evaluated by three pathologists (M.I., T.F., and T.F.) who were blinded to the clinical findings. An adequate liver biopsy sample was defined as a specimen of length of more than 1.5 cm and/or having more than 11 portal tracts. Specimen with steatosis of <5%, 5%–33%, 34%–66%, and >66% was scored as having steatosis grade of 0, 1, 2, and 3, respectively. Lobular inflammation of no foci, <2 foci, 2–4 foci, and >4 foci per 200× field was scored as 0, 1, 2, and 3, respectively. Hepatocyte ballooning of none, few cells, and many cells was scored as 0, 1, and 2, respectively. NAFLD activity score was the sum of steatosis, lobular inflammation, and hepatocyte ballooning scores (range, 0–8 points; 5–8 points as definition of NASH). Fibrosis stage of none, zone 3 perisinusoidal fibrosis (stage 1), zone 3 perisinusoidal fibrosis with portal fibrosis (stage 2), zone 3 perisinusoidal fibrosis and portal fibrosis with bridging fibrosis (stage 3), and cirrhosis (stage 4) was scored as 0, 1, 2, 3, and 4, respectively.19,20 Patients were also classified into four categories by histology according to the classification by Matteoni
Table 1 summarizes the patients’ characteristics at the biopsy of 211 patients, and these factors were investigated to determine the parameters that affected to histological features of NAFLD. Normal range of aspartate aminotransferase (AST) was evaluated as 13 to 33 IU/L. Normal range of alanine aminotransferase (ALT) was evaluated as 8 to 42 IU/L for male, and 6 to 27 IU/L for female. Obesity was defined as a body mass index (BMI) of more than 25.0 kg/m2. Cardiovascular disease (CVD) risk was investigated by exploring the total cholesterol (TC)/high-density lipoprotein cholesterol (HDL-C) ratio.22
Nonparametric tests (chi-squared test, Fisher exact probability test, and Mann-Whitney U test) were used to compare the characteristics of the groups. Multiple comparisons were examined by the Bonferroni test. Univariate and multivariate logistic regression analyses were used to determine those factors that significantly affected to histological features. The odds ratios (ORs) and 95% confidence intervals were also calculated. All p-values less than 0.05 by the two-tailed test were considered significant. Variables that achieved statistical significance (p<0.05) on univariate analysis were entered into multiple logistic regression analysis to identify significant independent predictive factors. Each variable was transformed into categorical data consisting of two simple ordinal numbers for univariate and multivariate analyses. Statistical analyses were performed using the SPSS software version 2 (SPSS Inc., Chicago, IL, USA).
Among 211 patients, 140 could be evaluated
Fig. 1A shows the distribution of
Table 3 indicated the factors associated with fibrosis stage, according to severity of fibrosis. Multivariate analysis identified four parameters that independently influenced fibrosis stage 1 or more: AST (≥1.5×ULN; OR, 20.0; p=0.001), high-density lipoprotein cholesterol (<41 mg/dL; OR, 9.17; p=0.012), ferritin (≥191 μg/L; OR, 4.92; p=0.023), and age (≥55 years; OR, 4.35; p=0.030). Multivariate analysis identified six parameters that independently influenced fibrosis stage 2 or more: GGT (<219 IU/L; OR, 71.4; p=0.007),
Thus,
Fig. 2A shows the distribution of
Fig. 3A shows the distribution of
The impact of
The impact of
Consistent with data previously reported,15 this preliminary study based on the small number of
Although histological diagnosis is currently the gold standard for diagnosing progressive NASH, liver biopsy has many drawbacks, such as cost, sampling error, and risk of complications.6 It is important to determine the clinical parameters as surrogate markers of histological features. Furthermore, inter- and intraobserver variability, and pathological diagnosis also presents the serious problems for the histological diagnosis of NASH.6 To minimize these shortfalls, the impact of genetic variations that affected to pathological criteria for the diagnosis of NASH (Matteoni classification and NAFLD activity score) was evaluated. Kawaguchi and coworkers14 reported that Matteoni type 4 NAFLD was both a genetically and clinically different subset from the other spectrums of the disease and that the
In conclusion, genetic variations might partly affect histological changes.
Author contributions: N.A., Y.K., Y.A., F.S., H.S., T.H., M.K., M.K., S.S., Y.S., K.I., H.K. contributed to this work. N.A., Y.K. analyzed the data. N.A. wrote the paper. N.A., Y.K., Y.A., F.S., H.S., T.H., M.K., S.S., Y.S., K.I., H.K. provided the sample.
The authors thank the following individuals for assistance in pathological diagnosis: Masafumi Inoue, M.D., Department of Pathology, Toranomon Hospital; Toshio Fukusato, M.D., Department of Pathology, Teikyo University School of Medicine; and Takeshi Fujii, M.D., Department of Pathology, Toranomon Hospital.
(1) Norio Akuta has received speakers’ bureau from MSD K.K., Mitsubishi Tanabe Pharma, Janssen Pharmaceutical K.K., Bristol-Myers Squibb, and holds a right to get some loyalty from SRL, Inc. (2) Hiromitsu Kumada has received speakers’ bureau from MSD K.K., Mitsubishi Tanabe Pharma, Dainippon Sumitomo Pharma, Bristol-Myers Squibb, Janssen Pharmaceutical K.K., GlaxoSmithKline K.K., and holds a right to get some loyalty from SRL., Inc. (3) Fumitaka Suzuki has received speakers’ bureau from Bristol-Myers Squibb. (4) Yoshiyuki Suzuki has received speakers’ bureau from Bristol-Myers Squibb. (5) Yasuji Arase has received speakers’ bureau from MSD K.K. (5) Kenji Ikeda has received speakers’ bureau from Dainippon Sumitomo Pharma, Eisai Co., Ltd., Olympus Co. The other authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Biopsy Characteristics of 211 Patients Diagnosed with Nonalcoholic Fatty Liver Disease
Characteristic | Value |
---|---|
Demographic data | |
No. of patients | 211 |
Gender, male/female | 122/89 |
Age, yr* | 52 (20–85) |
Body mass index, kg/m2* | 25.9 (18.1–40.4) |
Histological findings | |
Steatosis, 5%–33%/33%–66%/>66% | 80/80/47 |
Lobular inflammation, no foci/<2 foci/2–4 foci/>4 foci per 200× field | 25/113/58/11 |
Ballooning, none/few cells/many cells | 28/113/66 |
Stage, 0/1/2/3/4 | 30/82/25/56/18 |
Matteoni classification, type 1/2/3/4 | 15/11/7/174 |
NAFLD activity score, ≤2/3,4/≥5 | 31/81/99 |
Genetic variation | |
| 21/60/59/71 |
| 104/33/2/72 |
Laboratory data* | |
Serum aspartate aminotransferase, IU/L | 51 (12–312) |
Serum alanine aminotransferase, IU/L | 78 (15–338) |
γ-Glutamyl transpeptidase, IU/L | 68 (11–605) |
Serum albumin, g/dL | 4.1 (2.8–5.8) |
Platelet count, ×104/mm3 | 21.0 (4.5–38.9) |
Fasting plasma glucose, mg/dL | 100 (65–273) |
Uric acid, mg/dL | 5.9 (2.7–10.6) |
Total cholesterol, mg/dL | 206 (101–370) |
Triglycerides, mg/dL | 134 (31–610) |
High-density lipoprotein cholesterol, mg/dL | 45 (14–82) |
Low-density lipoprotein cholesterol, mg/dL | 125 (28–243) |
Total cholesterol/high density lipoprotein cholesterol | 4.6 (1.7–10.3) |
Serum ferritin, μg/L | 231 (10–1,474) |
Hyaluronic acid, μg/L | 35 (1–814) |
High sensitive C-reactive protein, mg/dL | 0.097 (0.006–2.240) |
Type IV collagen 7S, ng/mL | 4.2 (2.0–11.0) |
The data displayed represent the number of patients, except those denoted by *, which represent the median (range) values.
NAFLD, nonalcoholic fatty liver disease.
The Histological Features of Patients Diagnosed with Nonalcoholic Fatty Liver Disease according to
p-value* | ||||
---|---|---|---|---|
CC | CG | GG | ||
Steatosis, 5%–33%/>33%–66%/>66% | 8/11/2 | 23/26/10 | 27/17/14 | 0.270 |
Lobular inflammation, no foci/<2 foci/2–4 foci/>4 foci per 200× field | 1/14/6/0 | 7/33/18/1 | 6/24/20/8 | 0.074 |
Ballooning, none/few cells/many cells | 3/11/7 | 8/38/13 | 5/25/28 | 0.060 |
Stage, 0/1/2/3/4 | 2/12/0/3/4 | 10/27/7/14/2 | 5/11/11/24/8† | 0.001 |
Matteoni classification, type 1/2/3/4 | 1/2/0/18 | 4/3/3/49 | 2/2/2/52 | 0.805 |
NAFLD activity score, ≤2/3,4/≥5 | 2/12/7 | 9/26/25 | 6/17/36 | 0.100 |
p-value* | ||||
CC | CT | TT | ||
Steatosis, 5%–33%/>33%–66%/>66% | 42/41/19 | 15/12/6 | 1/0/1 | 0.727 |
Lobular inflammation, no foci/<2 foci/2–4 foci/>4 foci per 200× field | 9/53/35/5 | 5/17/8/3 | 0/1/1/0 | 0.803 |
Ballooning, none/few cells/many cells | 11/57/34 | 5/15/13 | 0/2/0 | 0.574 |
Stage, 0/1/2/3/4 | 13/38/12/31/10 | 4/11/5/9/4 | 0/1/0/1/0 | 0.990 |
Matteoni classification, type 1/2/3/4 | 5/5/4/88 | 2/2/1/28 | 0/0/0/2 | 0.998 |
NAFLD activity score, ≤2/3,4/≥5 | 13/40/51 | 4/14/15 | 0/1/1 | 0.976 |
NAFLD, nonalcoholic fatty liver disease.
†p=0.001, compared with the CG genotype by Bonferroni test.
Multivariate Analysis of Factors Associated with Fibrosis Stage, according to the Severity of Fibrosis in 211 Patients Diagnosed with Nonalcoholic Fatty Liver Disease
Factor | Category | OR | 95% CI | p-value |
---|---|---|---|---|
Factors associated with stage 1 or more | ||||
Serum aspartate aminotransferase, IU/L | <1.5×ULN | 1 | ||
≥1.5×ULN | 20.0 | 3.48–115 | 0.001 | |
High-density lipoprotein cholesterol, mg/dL | ≥41 | 1 | ||
<41 | 9.17 | 1.63–52.6 | 0.012 | |
Serum ferritin, μg/L | <191 | 1 | ||
≥191 | 4.92 | 1.25–19.4 | 0.023 | |
Age, yr | <55 | 1 | ||
≥55 | 4.35 | 1.15–16.4 | 0.030 | |
Factors associated with stage 2 or more | ||||
γ-Glutamyl transpeptidase, IU/L | ≥219 | 1 | ||
<219 | 71.4 | 3.17–1,000 | 0.007 | |
| CC | 1 | ||
CG | 18.8 | 2.11–166 | 0.008 | |
GG | 38.2 | 4.29–341 | 0.001 | |
Low-density lipoprotein cholesterol, mg/dL | ≥86 | 1 | ||
<86 | 28.6 | 1.66–500 | 0.021 | |
Platelet count, ×104/mm3 | ≥15.0 | 1 | ||
<15.0 | 21.7 | 1.39–333 | 0.028 | |
Hyaluronic acid, μg/L | <51 | 1 | ||
≥51 | 7.40 | 1.51–36.4 | 0.014 | |
Serum aspartate aminotransferase, IU/L | <1.5×ULN | 1 | ||
≥1.5×ULN | 5.26 | 1.38–20.1 | 0.015 | |
Factors associated with stage 3 or more | ||||
Serum aspartate aminotransferase, IU/L | <1.5×ULN | 1 | ||
≥1.5×ULN | 14.3 | 2.64–77.6 | 0.002 | |
Fasting plasma glucose, mg/dL | <126 | 1 | ||
≥126 | 11.9 | 1.77–80.1 | 0.011 | |
Low-density lipoprotein cholesterol, mg/dL | ≥86 | 1 | ||
<86 | 11.9 | 1.11–125 | 0.040 | |
Hyaluronic acid, μg/L | <51 | 1 | ||
≥51 | 8.52 | 2.19–33.2 | 0.002 | |
Factors associated with stage 4 | ||||
Low-density lipoprotein cholesterol, mg/dL | ≥86 | 1 | ||
<86 | 41.7 | 3.55–500 | 0.003 | |
Platelet count, ×104/mm3 | ≥15.0 | 1 | ||
<15.0 | 20.4 | 3.05–143 | 0.002 |
OR, odds ratio; CI, confidence interval; ULN, the upper limit of normal.
Biopsy Characteristics of 211 Patients Diagnosed with Nonalcoholic Fatty Liver Disease
Characteristic | Value |
---|---|
Demographic data | |
No. of patients | 211 |
Gender, male/female | 122/89 |
Age, yr* | 52 (20–85) |
Body mass index, kg/m2* | 25.9 (18.1–40.4) |
Histological findings | |
Steatosis, 5%–33%/33%–66%/>66% | 80/80/47 |
Lobular inflammation, no foci/<2 foci/2–4 foci/>4 foci per 200× field | 25/113/58/11 |
Ballooning, none/few cells/many cells | 28/113/66 |
Stage, 0/1/2/3/4 | 30/82/25/56/18 |
Matteoni classification, type 1/2/3/4 | 15/11/7/174 |
NAFLD activity score, ≤2/3,4/≥5 | 31/81/99 |
Genetic variation | |
| 21/60/59/71 |
| 104/33/2/72 |
Laboratory data* | |
Serum aspartate aminotransferase, IU/L | 51 (12–312) |
Serum alanine aminotransferase, IU/L | 78 (15–338) |
γ-Glutamyl transpeptidase, IU/L | 68 (11–605) |
Serum albumin, g/dL | 4.1 (2.8–5.8) |
Platelet count, ×104/mm3 | 21.0 (4.5–38.9) |
Fasting plasma glucose, mg/dL | 100 (65–273) |
Uric acid, mg/dL | 5.9 (2.7–10.6) |
Total cholesterol, mg/dL | 206 (101–370) |
Triglycerides, mg/dL | 134 (31–610) |
High-density lipoprotein cholesterol, mg/dL | 45 (14–82) |
Low-density lipoprotein cholesterol, mg/dL | 125 (28–243) |
Total cholesterol/high density lipoprotein cholesterol | 4.6 (1.7–10.3) |
Serum ferritin, μg/L | 231 (10–1,474) |
Hyaluronic acid, μg/L | 35 (1–814) |
High sensitive C-reactive protein, mg/dL | 0.097 (0.006–2.240) |
Type IV collagen 7S, ng/mL | 4.2 (2.0–11.0) |
The data displayed represent the number of patients, except those denoted by *, which represent the median (range) values.
NAFLD, nonalcoholic fatty liver disease.
The Histological Features of Patients Diagnosed with Nonalcoholic Fatty Liver Disease according to
p-value* | ||||
---|---|---|---|---|
CC | CG | GG | ||
Steatosis, 5%–33%/>33%–66%/>66% | 8/11/2 | 23/26/10 | 27/17/14 | 0.270 |
Lobular inflammation, no foci/<2 foci/2–4 foci/>4 foci per 200× field | 1/14/6/0 | 7/33/18/1 | 6/24/20/8 | 0.074 |
Ballooning, none/few cells/many cells | 3/11/7 | 8/38/13 | 5/25/28 | 0.060 |
Stage, 0/1/2/3/4 | 2/12/0/3/4 | 10/27/7/14/2 | 5/11/11/24/8† | 0.001 |
Matteoni classification, type 1/2/3/4 | 1/2/0/18 | 4/3/3/49 | 2/2/2/52 | 0.805 |
NAFLD activity score, ≤2/3,4/≥5 | 2/12/7 | 9/26/25 | 6/17/36 | 0.100 |
p-value* | ||||
CC | CT | TT | ||
Steatosis, 5%–33%/>33%–66%/>66% | 42/41/19 | 15/12/6 | 1/0/1 | 0.727 |
Lobular inflammation, no foci/<2 foci/2–4 foci/>4 foci per 200× field | 9/53/35/5 | 5/17/8/3 | 0/1/1/0 | 0.803 |
Ballooning, none/few cells/many cells | 11/57/34 | 5/15/13 | 0/2/0 | 0.574 |
Stage, 0/1/2/3/4 | 13/38/12/31/10 | 4/11/5/9/4 | 0/1/0/1/0 | 0.990 |
Matteoni classification, type 1/2/3/4 | 5/5/4/88 | 2/2/1/28 | 0/0/0/2 | 0.998 |
NAFLD activity score, ≤2/3,4/≥5 | 13/40/51 | 4/14/15 | 0/1/1 | 0.976 |
NAFLD, nonalcoholic fatty liver disease.
Histological features were compared among the three genotypes of
p=0.001, compared with the CG genotype by Bonferroni test.
Multivariate Analysis of Factors Associated with Fibrosis Stage, according to the Severity of Fibrosis in 211 Patients Diagnosed with Nonalcoholic Fatty Liver Disease
Factor | Category | OR | 95% CI | p-value |
---|---|---|---|---|
Factors associated with stage 1 or more | ||||
Serum aspartate aminotransferase, IU/L | <1.5×ULN | 1 | ||
≥1.5×ULN | 20.0 | 3.48–115 | 0.001 | |
High-density lipoprotein cholesterol, mg/dL | ≥41 | 1 | ||
<41 | 9.17 | 1.63–52.6 | 0.012 | |
Serum ferritin, μg/L | <191 | 1 | ||
≥191 | 4.92 | 1.25–19.4 | 0.023 | |
Age, yr | <55 | 1 | ||
≥55 | 4.35 | 1.15–16.4 | 0.030 | |
Factors associated with stage 2 or more | ||||
γ-Glutamyl transpeptidase, IU/L | ≥219 | 1 | ||
<219 | 71.4 | 3.17–1,000 | 0.007 | |
| CC | 1 | ||
CG | 18.8 | 2.11–166 | 0.008 | |
GG | 38.2 | 4.29–341 | 0.001 | |
Low-density lipoprotein cholesterol, mg/dL | ≥86 | 1 | ||
<86 | 28.6 | 1.66–500 | 0.021 | |
Platelet count, ×104/mm3 | ≥15.0 | 1 | ||
<15.0 | 21.7 | 1.39–333 | 0.028 | |
Hyaluronic acid, μg/L | <51 | 1 | ||
≥51 | 7.40 | 1.51–36.4 | 0.014 | |
Serum aspartate aminotransferase, IU/L | <1.5×ULN | 1 | ||
≥1.5×ULN | 5.26 | 1.38–20.1 | 0.015 | |
Factors associated with stage 3 or more | ||||
Serum aspartate aminotransferase, IU/L | <1.5×ULN | 1 | ||
≥1.5×ULN | 14.3 | 2.64–77.6 | 0.002 | |
Fasting plasma glucose, mg/dL | <126 | 1 | ||
≥126 | 11.9 | 1.77–80.1 | 0.011 | |
Low-density lipoprotein cholesterol, mg/dL | ≥86 | 1 | ||
<86 | 11.9 | 1.11–125 | 0.040 | |
Hyaluronic acid, μg/L | <51 | 1 | ||
≥51 | 8.52 | 2.19–33.2 | 0.002 | |
Factors associated with stage 4 | ||||
Low-density lipoprotein cholesterol, mg/dL | ≥86 | 1 | ||
<86 | 41.7 | 3.55–500 | 0.003 | |
Platelet count, ×104/mm3 | ≥15.0 | 1 | ||
<15.0 | 20.4 | 3.05–143 | 0.002 |
OR, odds ratio; CI, confidence interval; ULN, the upper limit of normal.