Risk factors for histological progression of nonalcoholic steatohepatitis analyzed from repeated biopsy cases

[1]

Hamaguchi M, Kojima T, Takeda N, et al. The metabolic syndrome as a predictor of

nonalcoholic fatty liver disease. Ann Intern Med. 2005; 143: 722-8.

[2]

Kojima S, Watanabe N, Numata M, Ogawa T, Matsuzaki S. Increase in the prevalence of

fatty liver in Japan over the past 12 years: analysis of clinical background. J Gastroenterol. 2003;

38: 954-61.

[3]

Amarapurkar DN, Hashimoto E, Lesmana LA, Sollano JD, Chen PJ, Goh KL. How

common is non-alcoholic fatty liver disease in the Asia-Pacific region and are there local

differences? J Gastroenterol Hepatol. 2007; 22: 788-93.

[4]

Teli MR, James OF, Burt AD, Bennett MK, Day CP. The natural history of nonalcoholic

fatty liver: a follow-up study. Hepatology. 1995; 22: 1714-9.

This article is protected by copyright. All rights reserved.

[5]

Tokushige K, Hashimoto E, Horie Y, Taniai M, Higuchi S. Hepatocellular carcinoma in

Japanese patients with nonalcoholic fatty liver disease, alcoholic liver disease, and chronic liver

disease of unknown etiology: report of the nationwide survey. J Gastroenterol. 2011; 46: 1230-7.

[6]

Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ.

Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology.

1999; 116: 1413-9.

[7]

James O, Day C. Non-alcoholic steatohepatitis: another disease of affluence. Lancet

(London, England). 1999; 353: 1634-6.

[8]

Seko Y, Sumida Y, Tanaka S, et al. Serum alanine aminotransferase predicts the

histological course of non-alcoholic steatohepatitis in Japanese patients. Hepatology research : the

official journal of the Japan Society of Hepatology. 2015; 45: E53-61.

[9]

Angulo P, Kleiner DE, Dam-Larsen S, et al. Liver Fibrosis, but No Other Histologic

Features, Is Associated With Long-term Outcomes of Patients With Nonalcoholic Fatty Liver

Disease. Gastroenterology. 2015; 149: 389-97.e10.

[10]

Nakahara T, Hyogo H, Yoneda M, et al. Type 2 diabetes mellitus is associated with the

fibrosis severity in patients with nonalcoholic fatty liver disease in a large retrospective cohort of

Japanese patients. J Gastroenterol. 2014; 49: 1477-84.

[11]

Rotman Y, Koh C, Zmuda JM, Kleiner DE, Liang TJ. The association of genetic

variability in patatin-like phospholipase domain-containing protein 3 (PNPLA3) with histological

severity of nonalcoholic fatty liver disease. Hepatology. 2010; 52: 894-903.

[12]

Tokushige K, Takakura M, Tsuchiya-Matsushita N, Taniai M, Hashimoto E, Shiratori K.

Influence of TNF gene polymorphisms in Japanese patients with NASH and simple steatosis. J

Hepatol. 2007; 46: 1104-10.

[13]

Hotta K, Yoneda M, Hyogo H, et al. Association of the rs738409 polymorphism in

PNPLA3 with liver damage and the development of nonalcoholic fatty liver disease. BMC medical

genetics. 2010; 11: 172.

This article is protected by copyright. All rights reserved.

[14]

Ricci C, Longo R, Gioulis E, et al. Noninvasive in vivo quantitative assessment of fat

content in human liver. J Hepatol. 1997; 27: 108-13.

[15]

Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR.

Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J

Gastroenterol. 1999; 94: 2467-74.

[16]

Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological

scoring system for nonalcoholic fatty liver disease. Hepatology. 2005; 41: 1313-21.

[17]

Hoofnagle JH, Van Natta ML, Kleiner DE, et al. Vitamin E and changes in serum

alanine aminotransferase levels in patients with non-alcoholic steatohepatitis. Alimentary

pharmacology & therapeutics. 2013; 38: 134-43.

[18]

Ohnishi Y, Tanaka T, Ozaki K, Yamada R, Suzuki H, Nakamura Y. A high-throughput

SNP typing system for genome-wide association studies. Journal of human genetics. 2001; 46:

471-7.

[19]

Kovalic AJ, Banerjee P, Tran QT, Singal AK, Satapathy SK. Genetic and Epigenetic

Culprits in the Pathogenesis of Nonalcoholic Fatty Liver Disease. Journal of clinical and

experimental hepatology. 2018; 8: 390-402.

[20]

Sharma M, Mitnala S, Vishnubhotla RK, Mukherjee R, Reddy DN, Rao PN. The Riddle of

Nonalcoholic Fatty Liver Disease: Progression From Nonalcoholic Fatty Liver to Nonalcoholic

Steatohepatitis. Journal of clinical and experimental hepatology. 2015; 5: 147-58.

[21]

Kawaguchi T, Shima T, Mizuno M, et al. Risk estimation model for nonalcoholic fatty

liver disease in the Japanese using multiple genetic markers. PLoS One. 2018; 13: e0185490.

[22]

Macaluso FS, Maida M, Petta S. Genetic background in nonalcoholic fatty liver disease:

A comprehensive review. World J Gastroenterol. 2015; 21: 11088-111.

[23]

Dongiovanni P, Anstee QM, Valenti L. Genetic predisposition in NAFLD and NASH:

impact on severity of liver disease and response to treatment. Curr Pharm Des. 2013; 19: 5219-38.

[24]

Seko Y, Yamaguchi K, Itoh Y. The genetic backgrounds in nonalcoholic fatty liver disease.

This article is protected by copyright. All rights reserved.

Clin J Gastroenterol. 2018; 11: 97-102.

[25]

Danford CJ, Yao ZM, Jiang ZG. Non-alcoholic fatty liver disease: a narrative review of

genetics. J Biomed Res. 2018; 32: 389-400.

[26]

Liu J, Xing J, Wang B, et al. Correlation Between Adiponectin Gene rs1501299

Polymorphism and Nonalcoholic Fatty Liver Disease Susceptibility: A Systematic Review and

Meta-Analysis. Med Sci Monit. 2019; 25: 1078-86.

[27]

Vespasiani-Gentilucci U, Gallo P, Dell'Unto C, Volpentesta M, Antonelli-Incalzi R,

Picardi A. Promoting genetics in non-alcoholic fatty liver disease: Combined risk score through

polymorphisms and clinical variables. World J Gastroenterol. 2018; 24: 4835-45.

[28]

Eguchi Y, Mizuta T, Sumida Y, et al. The pathological role of visceral fat accumulation in

steatosis, inflammation, and progression of nonalcoholic fatty liver disease. J Gastroenterol. 2011;

46 Suppl 1: 70-8.

[29]

Chen Z, Han CK, Pan LL, et al. Serum alanine aminotransferase independently

correlates with intrahepatic triglyceride contents in obese subjects. Digestive diseases and

sciences. 2014; 59: 2470-6.

[30]

Yoneda M, Imajo K, Eguchi Y, et al. Noninvasive scoring systems in patients with

nonalcoholic fatty liver disease with normal alanine aminotransferase levels. J Gastroenterol.

2013; 48: 1051-60.

[31]

Pais R, Charlotte F, Fedchuk L, et al. A systematic review of follow-up biopsies reveals

disease progression in patients with non-alcoholic fatty liver. J Hepatol. 2013; 59: 550-6.

[32]

Wong VW, Wong GL, Choi PC, et al. Disease progression of non-alcoholic fatty liver

disease: a prospective study with paired liver biopsies at 3 years. Gut. 2010; 59: 969-74.

[33]

Adams LA, Sanderson S, Lindor KD, Angulo P. The histological course of nonalcoholic

fatty liver disease: a longitudinal study of 103 patients with sequential liver biopsies. J Hepatol.

2005; 42: 132-8.

[34]

Ekstedt M, Franzen LE, Mathiesen UL, et al. Long-term follow-up of patients with

This article is protected by copyright. All rights reserved.

NAFLD and elevated liver enzymes. Hepatology. 2006; 44: 865-73.

[35]

Fassio E, Alvarez E, Dominguez N, Landeira G, Longo C. Natural history of nonalcoholic

steatohepatitis: a longitudinal study of repeat liver biopsies. Hepatology. 2004; 40: 820-6.

[36]

Argo CK, Northup PG, Al-Osaimi AM, Caldwell SH. Systematic review of risk factors for

fibrosis progression in non-alcoholic steatohepatitis. J Hepatol. 2009; 51: 371-9.

[37]

Romeo S, Kozlitina J, Xing C, et al. Genetic variation in PNPLA3 confers susceptibility

to nonalcoholic fatty liver disease. Nat Genet. 2008; 40: 1461-5.

[38]

Kawaguchi T, Sumida Y, Umemura A, et al. Genetic polymorphisms of the human

PNPLA3 gene are strongly associated with severity of non-alcoholic fatty liver disease in

Japanese. PLoS One. 2012; 7: e38322.

[39]

Wang JZ, Cao HX, Chen JN, Pan Q. PNPLA3 rs738409 underlies treatment response in

nonalcoholic fatty liver disease. World journal of clinical cases. 2018; 6: 167-75.

[40]

Kan H, Hyogo H, Ochi H, et al. Influence of the rs738409 polymorphism in patatin-like

phospholipase 3 on the treatment efficacy of non-alcoholic fatty liver disease with type 2 diabetes

mellitus. Hepatology research : the official journal of the Japan Society of Hepatology . 2016; 46:

E146-53.

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Figure 1. Histological changes between the first and second biopsies. The horizontal axis

shows the percentage of progress, improve or no change. The striped bars denote progression

cases, the filled bars denote no change, and the diagonal bars denote improvement. (a)

Fibrosis, (b) NAFLD activity score

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Figure 2 Comparison of histological progression and non-progression cases (a) Body mass

index (BMI), subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and

liver/spleen ratio (LS ratio) in computed tomography.

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Figure 2 Comparison of histological progression and non-progression cases (b) Aspartate

aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyltranspeptidase (GGT),

uric acid (UA), LDL cholesterol, HbA1c, fasting blood sugar (FBS), and homeostasis model

assessment ratio (HOMA-IR). In each figure, the horizontal axis shows the first and second

biopsies and the longitudinal axis shows the means. The solid lines show progression cases

and the dotted line shows non-progression cases.

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Figure 3. Relationship between histological exacerbation and rs1799964 allele, ALT response,

and HbA1c elevation.

The horizontal axis shows HbA1c increase or non-increase and non-TT or TT in rs1799964.

The dark bars show ALT responders and the light bars show ALT non-responders. The

longitudinal axis shows percentage of patients who experienced histological exacerbation.

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Figure 4. Risk estimation of NAFLD using genetic variation and biochemical markers. The

receiver Operating Characteristic (ROC) curves shows the potential to predict histological

exacerbation based on ALT response, HbA1c levels, and allele type of rs1799964. The

probability for correct diagnosis rate is 90.0% with AUC=0.884. Using leave one out

cross-validation, we obtained a probability for correct diagnosis rate of 78.6% with

AUC=0.827.

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Table 1 Patient background

median

min

Age, years

61

Sex, (Male/Female)

(45/35)

observation period, days

931

NAS (first, 2/3/4/5/6/7/8)

(2/29/21/9/7/10/2)

Fibrosis stage(first, 1/2/3)

(17/37/26)

BMI, kg/m2

27.4

Platelet count, x104

21.9

AST, IU/L

32

( 328

max

90

2640

19.6

38.3

9.2

58.9

45.5

17

163

ALT, IU/L

79

23

324

γ-GTP, IU/L

61

18

481

Total cholesterol, mg/dL

222

139

372

Triglyceride, mg/dL

142.5

65

634

HDL cholesterol, mg/dL

49.5

29

100

LDL cholesterol, mg/dL

141.5

53

294

Fasting blood sugar, mg/dL

108

71

239

HbA1c, %

5.9

3.8

11.7

UA, mg/dL

5.8

2.9

8.9

Ferritin, ng/mL

179.4

8.2

1598.9 )

SAT

199.6

68.57 -

420.8

VAT

136.5

58.2

340.6

waist

96.59

79.27 -

124.20 )

CTLSratio

0.70

0.05

1.40

HOMA-IR

3.158

0.933 -

NGT/IGT/DM

14/27/39

Hyper tension

42/80

Dyslipidemia

71/80

Hyper uremia

23/80

12.039 )

Values are median( range )

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Table 2 (a) Comparison of histological progression cases and non-progression cases.

Progression

mean

Non-progression

sd

14.9

mean

60.06

sd

age

63.38

11.8

sex (M/F)

13/14

BMI

27.95

4.2

27.64

3.8

0.890

BMI (second)

26.05

6.8

25.66

5.2

0.339

SAT

219.58

98.9

208.65

95.4

0.662

SAT (second)

231.16

103.7 )

189.15

86.5

0.113

VAT

148.03

68.1

141.63

42.1

0.924

VAT (second)

130.35

53.1

113.58

48.3

0.193

LSratio

0.76

0.3

0.68

0.3

0.304

LSratio_ (second)

0.96

0.2

1.00

0.3

0.231

34/21

0.329

0.212

date are expressed as mean values and standard deviation.

p-values associated with continuous variables via a Mann-whitney U-test.

p-values associated with categorical values were caculated with t-test.

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Table 2 (b) Clinical parameters contribute to the histological progression.

Progression

Non--progression

mean

mean

sd

Mulltivariate analysis

sd

AST

50.4

( 28.0 )

57.7

35.9 )

0.538

AST (second)

41.0

( 23.8 )

30.1

21.5 )

0.031

AST_nonresponder 10/26

15/54

78.8

( 39.1 )

97.8

63.2 )

0.361

ALT_ (second)

57.3

( 40.9 )

45.6

63.7 )

0.041

12/54

3.3.E-04

rGTP

119.3

( 102.6 )

89.0

85.5 )

0.070

rGTP_ (second)

83.1

( 84.1 )

38.7

29.8 )

0.002

rGTP nonresponder 8/26

95%CI lo

95%CI hi

0.230

0.071

0.748

0.015

1.014

1.000

1.028

0.053

4.651

1.453

14.883

0.010

0.593

ALT

ALT_nonresponder 13/26

OR

10/54

0.010

LDL

134.5

( 34.9 )

146.0

42.7 )

0.423

LDL (second)

95.3

( 25.3 )

106.7

28.5 )

0.083

LDL increase

4/26

FBS

109.5

( 22.4 )

118.9

33.6 )

0.314

FBS_ (second)

115.1

( 26.4 )

110.9

33.0 )

0.700

HbA1c

6.31

( 1.4

6.37

1.2 )

0.382

HbA1c (second)

6.40

( 0.9

6.08

0.9 )

0.113

HbA1c increase

18/26

UA

5.95

( 1.5

5.89

1.3 )

0.933

UA_ (second)

5.39

( 1.4

5.47

1.2 )

0.890

UA increase

10 / 26

HOMA_IR

3.50

( 2.0

4.14

2.7 )

0.420

HOMA_IR_(second) 3.50

( 2.2

3.54

2.4 )

0.861

10/54

0.975

18/54

0.005

19 /54

0.970

responder ʂdecrease of more than 30% of the value, or decrease to the normal range

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Table 3 Risk factors contributing to the histological progression.

Univariate

analysis

Multivariate季 analysis季

OR

ALT_nonresponder

3.3.E-04

0.035

4.073

1.100

- 15.078

γ – GTP_nonresponder

0.010

HbA1c increase

0.005

0.003

11.902

2.264

- 62.555

rs1799964(CC vs nonCC)

0.006

0.009

9.558

1.755

- 52.045

95%CI lo

95%CI hi

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