The classical metabolic work-up, approved by the Ethics Committee of the Antwerp

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1 SUPPLEMENTARY MATERIALS METHODS Metabolic work-up The classical metabolic work-up, approved by the Ethics Committee of the Antwerp University Hospital and requiring written informed consent, included a detailed questionnaire on weight evolution, dietary habits, familial history, personal medical history, medication and alcohol use 1. It further included a clinical examination with anthropometry. Blood analysis included blood cell count, coagulation tests, electrolytes and kidney function tests, liver enzyme tests [aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), total bilirubin and fractions], creatinine kinase, total protein, protein electrophoresis, thyroid function, ferritin, vitamin B, folic acid; fasting glucose; fasting insulin; fasting C-peptide; insulin resistance estimation using the homeostasis model assessment (HOMA) calculated as [insulin (mu/l) x glucose (mmol/l)]/ and the quantitative assessment check index (QUICKI) score calculated as 1/[log(fasting plasma insulin (mu/l)) + log(fasting glucose (mg/dl))] 3. A CT-scan at L4-L5 level was performed to measure the cross-sectional area of total abdominal adipose tissue (TAT) area, visceral abdominal adipose tissue (VAT) and subcutaneous abdominal adipose tissue (SAT) according to previously described methods 4. Additional blood analysis S-choline-esterase, carcino-embryonic antigen, alpha-fetoprotein (AFP), anti-nuclear factor, anti-neutrophil cytoplasm antigen antibodies, anti-smooth muscle antibodies, antimitochondrial antibodies, anti liver-kidney microsome antibodies, serum copper and 1

2 ceruloplasmin, alpha-1-antitrypsin, Hepatitis B sag, anti Hepatitis B cab, anti Hepatitis C antibodies, carboxy-deficient transferrin. Additional tests SNP in PNPLA3: Taqman Pre-Designed Genotyping Assays (Applied Biosystems Inc., Foster City, CA, USA) were used to genotype the selected SNP, according to the manufacturer's protocol, on a Lightcycler 480 Real-Time PCR System (Roche, Penzberg, Germany). Published scores The NAFLD liver fat score was designed to diagnose NAFLD defined as a liver fat content of 5.56% of liver tissue weight using 1 H-Magnetic Resonance spectroscopy (MRS) as the gold standard 5. The score is calculated as follows: x MS (according to the IDF definition; yes = 1/no = 0) x type 2 diabetes (yes = 2/no = 0) x fasting insulin (mu/l) x AST (U/L) 0.94 x AST/ALT. The Fatty Liver Index (FLI) was designed to diagnose fatty liver (not discriminating between alcoholic and non-alcoholic fatty liver) using ultrasound as the gold standard 6. The FLI is calculated as follows: x BMI (kg/m²) x waist circumference (cm) x log e [TG (mg/dl)] x ln[ggt (U/L)]. The BARD score was designed to predict advanced fibrosis defined as fibrosis stage 3 according to Brunt et al using histology as the gold standard and is calculated as follows: BMI 28 kg/m² = 1 point, AST/ALT 0.8 = 2 points, diabetes = 1 point 7, 8. The AST-toplatelet index (APRI) was designed to predict significant fibrosis (defined as Ishak fibrosis score 3) and cirrhosis (Ishak fibrosis score 5-6) using histology as the gold standard and is calculated as follows: AST level (ULN)/platelet count (10 9 /L) 9, 10. The FIB-4 index was designed in patients with chronic hepatitis C to predict advanced fibrosis defined as F 4 2

3 according to Ishak et al using histology as the gold standard and is calculated as follows: age (years) x AST (U/L)/[platelet count (x10 9 /L) x (ALT (U/L))] The NAFLD fibrosis score was designed to predict advanced fibrosis (defined as fibrosis stage 3 according to Kleiner et al) using histology as the gold standard and is calculated as follows: x age (years) x BMI (kg/m²) x impaired fasting glucose or diabetes (yes = 1, no = 0) x AST/ALT x platelet count (x 10 9 /L) 0.66 x albumin (g/dl) 13, 14. Forns index was designed to predict significant fibrosis (defined as stage 2 according to Scheuer s classification) using histology as the gold standard and is calculated as follows: x ln[platelet count (10 9 /L) x ln[ggt (U/L)] x ln[age (year)] x serum cholesterol (mg/dl) 15, 16. Statistical analysis Values were expressed as mean ± standard deviation (SD) whenever applicable. The results were analyzed with an independent samples t-test (normally distributed continuous variables), Mann Whitney U test or Kruskall-Wallis test (non-normally distributed continuous variables, categorical variables, scores) and Chi square test (prevalences) for the comparison of different groups. Univariate logistic regression was performed to identify independent predictors of the presence of NASH, significant fibrosis and advanced fibrosis. Univariate linear regression was performed to identify factors independently predicting NAS. Factors were reported by the adjusted coefficient of determination (R²) and the significance (p-value). Multivariate logistic regression was used to build the different scores. All variables significantly associated with NASH, significant or advanced fibrosis or NAS in univariate logistic regression analysis were included in multivariate forward conditional analyses to identify variables that where independently associated. Subsequently the Area Under the Receiver Operating Curve (AUROC) was calculated and optimal cut-offs were determined with a sensitivity of 95% and 3

4 with a specificity of 95% for all scores. Calculations were made using SPSS 18.0 for Windows. A p value of < 0.05 was considered statistically significant. 4

5 RESULTS Liver biopsy The main characteristics of the patients who did undergo liver biopsy were compared to the patients with at least one criterion to propose liver biopsy, but who refused informed consent, and are listed in supplementary materials Table S1. The metabolic parameters were more pronounced in the patients who underwent a liver biopsy. Liver ultrasound parameters also were more pronounced in the group that underwent a biopsy, whereas liver biochemistry and ABT were not different between groups. Design and validation cohort The main characteristics, including histology, of the design and validation cohort and their comparison are shown in supplementary materials Table S2. Besides a higher mean arterial blood pressure (MABP) in the design cohort, no significant differences were noted (Table S2). CK 18 Mean CK 18 was 214 ± 21.1 U/L in the overall cohort, and not significantly different between design and validation cohort (Table S2). In univariate analysis, CK 18 was a predictor of the degree of fibrosis (adjusted R² 0.086, p < 0.001), but in multivariate analysis it was not significant anymore. There was no relation with steatosis, ballooning or lobular inflammation. CK 18 was not predictive for the presence of NASH or for the severity of NASH as expressed by the NAS. In univariate analysis it was predictive for the presence of significant (Table S7) and advanced (Table S8) fibrosis, but in multivariate analysis it was not significant anymore. 5

6 PNPLA3 The distribution according to PNPLA3 genotype for the overall and for the design and validation cohort are shown in Table 1 and Table S2 respectively. PNPLA 3 polymorphism is an independent predictor (multivariate analysis) of the severity of steatosis, ballooning and lobular inflammation, but not fibrosis (Table S3). PNPLA3 genotype is predictive for the presence of NASH (Table S4 and S5) and the severity of NASH as expressed by the NAS (Table S6) in univariate but not multivariate analysis. Features of NASH: steatosis, ballooning, lobular inflammation and fibrosis The factors that are significantly and independently (multivariate analysis) related to the severity of steatosis, ballooning, lobular inflammation and fibrosis, scored according to the NASH Clinical Research Scoring System 14 are listed in table S3 (design cohort). NASH according to the definition by Kleiner et al 14 The factors that were significantly related to the presence of NASH as defined according to Kleiner et al in a univariate analysis in the design cohort are listed in supplementary materials Table S5. In a multivariate analysis only ALT, the USS and the number of criteria of the MS according to the definition of the IDF appeared to be independent predictors of the presence of NASH (Table S5). The Antwerp NASH score 2 to predict the diagnosis of NASH according to the definition by Kleiner et al had the following formula: [0.029 x ALT (U/L)] + [1.394 x USS] + [1.223 x number of IDF criteria MS]. This score had a R² = The AUROC s of this score and the reference scores are shown in Table 2 (design and validation cohort) and Fig. 2B (design cohort). The other diagnostic accuracy indices are listed in Table 3. 6

7 Advanced fibrosis The factors that were significantly related to the presence of advanced fibrosis (7.3% of the patients) in a univariate analysis in the design cohort are listed in supplementary materials Table S8. In a multivariate analysis only AST, AST > 40 U/L and waist appeared to be independent predictors of the presence of advanced fibrosis (Table S8). The Antwerp NAFLD advanced fibrosis score to predict the diagnosis of advanced fibrosis had the following formula: [0.057 x waist (cm)] + [0.112 x AST (U/L)] [3.078 x (AST > 40 U/L: no = 0, yes = 1). This score had a R² = The AUROC s of this score and the reference scores are shown in Table 4 (design and validation cohort) and Fig. 2D (design cohort). The other diagnostic accuracy indices are listed in Table 3. Application of the scores to the overall cohort The patients in whom none of the criteria for liver biopsy were met, the mean calculated NAS was 0.16 ± They all had an Antwerp NASH score 1 < -1.34, an Antwerp NASH score 2 < -2.50, an Antwerp NAFLD significant fibrosis score < -2.9 and an Antwerp NAFLD advanced fibrosis score of < , confirming there was no need for a liver biopsy. In the patients meeting at least one of the criteria for liver biopsy, the Antwerp NASH score 1 ( ± vs ± 1.641, p = 0.004), the Antwerp NASH score 2 ( ± 2.151, p = 0.12), the Antwerp NAFLD significant fibrosis ( ± vs ± 1.333, p = < 0.001) and the Antwerp NAFLD advanced fibrosis score ( ± vs ± 1.449, p < 0.001) were significantly lower in those who did not have a liver biopsy compared to those 7

8 who underwent a biopsy, suggesting that the prevalences and severity of the histological lesions were lower in the overall cohort compared to the group that had a histological assessment. Concerning the diagnosis of NASH according to Brunt et al, 71.6% had an Antwerp NASH score 1 > in the overall cohort, allowing to reliably exclude NASH in 28.4%. Based on the PPV, prevalence was estimated at 48.7%. Furthermore 26.1% has a value > 1.34 compatible with a reliable diagnose of NASH without need for a biopsy. Concerning the diagnosis of NASH according to Kleiner et al, 88.1% had an Antwerp NASH score 2 > in the overall cohort. Based on the PPV, prevalence was estimated at 45.8%. Furthermore 42.7% has a value > 0.70 compatible with a reliable diagnosis of NASH without need for a biopsy. Based on the calculated NAS, 37.2% had a NAS > 4. Concerning the diagnosis of significant fibrosis, 74.6% had an Antwerp significant fibrosis score > in the overall cohort. Based on the PPV, prevalence was estimated at 16.4%. Furthermore 87.5% had a value > reliably excluding significant fibrosis without need for a biopsy. Concerning the diagnosis of advanced fibrosis, 54.1% had an Antwerp advanced fibrosis score > in the overall cohort. Based on the PPV, prevalence was estimated at 5.4%. Furthermore 92.3% had a value > reliably excluding advanced fibrosis without need for a biopsy. 8

9 FIGURES Fig. S1: Antwerp NASH severity score. Scatter plot and linear fit of the Antwerp NASH severity score and the NAS in the index cohort. NASH = non-alcoholic steatohepatitis; NAS = NASH Activity Score. 9

10 REFERENCES 1. Verrijken A, Francque S, Mertens I et al. Visceral adipose tissue and inflammation correlate with elevated liver tests in a cohort of overweight and obese patients. Int J Obes (Lond) 2010;34: Matthews DR, Hosker JP, Rudenski AS et al. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28: Katz A, Nambi SS, Mather K et al. Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. J Clin Endocrinol Metab 2000;85: van der Kooy K, Seidell JC. Techniques for the measurement of visceral fat: a practical guide. Int J Obes Relat Metab Disord 1993;17: Kotronen A, Peltonen M, Hakkarainen A et al. Prediction of non-alcoholic fatty liver disease and liver fat using metabolic and genetic factors. Gastroenterology 2009;137: Bedogni G, Bellentani S, Miglioli L et al. The Fatty Liver Index: a simple and accurate predictor of hepatic steatosis in the general population. BMC Gastroenterol 2006;6: Harrison SA, Oliver D, Arnold HL et al. Development and validation of a simple NAFLD clinical scoring system for identifying patients without advanced disease. Gut 2008;57:

11 8. Brunt EM, Janney CG, Di Bisceglie AM et al. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 1999;94: Wai CT, Greenson JK, Fontana RJ et al. A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C. Hepatology 2003;38: Ishak K, Baptista A, Bianchi L et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995;22: Sterling RK, Lissen E, Clumeck N et al. Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection. Hepatology 2006;43: Vallet-Pichard A, Mallet V et al. FIB-4: a simple, inexpensive and accurate marker of fibrosis in HCV-infected patients. Hepatology 2006;44: Angulo P, Hui JM, Marchesini G et al. The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD. Hepatology 2007;45: 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: Forns X, Ampurdanes S, Llovet JM et al. Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model. Hepatology 2002;36: Scheuer PJ. The nomenclature of chronic hepatitis: time for a change. J Hepatol 1995;22:

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13 SUPPLEMENTARY TABLES Table S1. Comparison of the main characteristics of the patients who underwent a liver biopsy and those who had an indication for biopsy but refused informed consent. Units No biopsy Biopsy p-value n = 165 n = 313 Gender M/F %/% 21.3/ / * Age y 41.9 ± ± BMI kg/m² 36.7 ± ± 5.9 < 0.001* Waist cm ± ± 13.2 < 0.001* WHR ± ± < 0.001* VAT cm² ± ± * MABP mm Hg 90.6 ± ± * LDH U/L ± ± * AST U/L 29.4 ± ± ALT U/L 40.8 ± ± AST/ALT 0.77 ± ± GGT U/L 41.6 ± ± Total Cholesterol mg/dl ± ± HDL-cholesterol mg/dl 54.0 ± ± * TG mg/dl ± ± Fasting glucose mg/dl 82.6 ± ±

14 Fasting insulin μu/ml 13.9 ± ± * Fasting C-peptide nmol/l 0.99 ± ± * HBA1c % 5.61 ± ± QUICKI index ± ± * HOMA IR 2.93 ± ± * USS 1.6 ± ± * Diabetes absent/present %/% 90.4/ / Liver right craniocaudal diameter mm 15.1 ± ± * Spleen diameter mm 10.4 ± ± ABT peak excretion % 9.76 ± ± ABT cumulative excretion % ± ± Number criteria MS NCEP ATP III 2.5 ± ± Number criteria MS IDF 2.6 ± ± MS NCEP ATP III absent/present %/% 54.1/ / MS IDF absent/present %/% 53.3/ / Number criteria liver biopsy 1.9 ± ± * The main characteristics of the patients who had an indication for biopsy are compared between those who refused a liver biopsy and those who underwent a biopsy. Continuous variables are compared using Student t-test if normally distributed. Not normally distributed continuous variables and categorical variables are compared using Mann-Whitney U test. A p- value of < 0.05 is considered statistically significant (*). M = male; F= female; WHR = waistto-hip ratio; VAT = visceral adipose tissue; MABP = mean arterial blood pressure; LDH = lactate dehydrogenase; AST = aspartate aminotransferase; ALT = alanine aminotransferase; 14

15 GGT = gamma glutamyl transpeptidase; HDL = high density lipoprotein; TG = triglycerides; HBA1c = glycosylated haemoglobin; QUICKI = quantitative assessment check index calculated as 1/[log(fasting insulin) + log(fasting glucose)]; HOMA IR = homeostasis model of assessment insulin resistance calculated as [fasting insulin (mu/l) x fasting glucose (mmol/l)]/22.5; USS = ultrasound steatosis score; ABT = aminopyrine breath test; y/n = yes or no; MS = metabolic syndrome; NCEP ATP III = US Third Adult Treatment Panel of the National Cholesterol Education Program; IDF = International Diabetes Federation. 15

16 Table S2. Main characteristics of the design and validation cohort and their comparison, including histology. Units Design cohort Validation cohort p-value n = 200 n = 113 Gender M/F %/% 31.5/ / Age y 44.0 ± ± BMI kg/m² 39.1 ± ± Waist cm ± ± WHR ± ± VAT cm² ± ± MABP mm Hg 96.0 ± ± 9.3 < 0.001* LDH U/L ± ± AST U/L 32.0 ± ± ALT U/L 45.3 ± ± AST/ALT 0.74 ± ± GGT U/L 37.7 ± ± Total Cholesterol mg/dl ± ± HDL-cholesterol mg/dl 50.4 ± ± TG mg/dl ± ± Fasting glucose mg/dl 87.4 ± ± Fasting insulin μu/ml 17.2 ± ± Fasting C-peptide nmol/l 1.14 ± ± HBA1c % 5.69 ± ± QUICKI index ± ±

17 HOMA IR 3.88 ± ± USS 1.9 ± ± Diabetes absent/present %/% 92.0/ / Liver right craniocaudal diameter mm 15.6 ± ± Spleen diameter mm 10.7 ± ± ABT peak excretion % 8.89 ± ± ABT cumulative excretion % ± ± Number criteria MS NCEP ATP III 2.8 ± ± Number criteria MS IDF 2.8 ± ± MS NCEP ATP III absent/present %/% 43.0/ / MS IDF absent/present %/% 40.5/ / Number criteria liver biopsy 2.1 ± ± Steatosis score 1.2 ± ± NAS 3.2 ± ± Fibrosis score 0.6 ± ± PNPLA3 polymorphism n (%) 125 (62.5)/68 (34)/7 60 (53.1)/43 (38.1)/ (CC/CG/GG) (3.5) (8.8) CK 18 U/L ± ± The main characteristics of the patients in the design and validation cohort are compared, including histology. Continuous variables are compared using Student t-test if normally distributed. Not normally distributed continuous variables and categorical variables including histological scores are compared using Mann-Whitney U test. A p-value of < 0.05 is considered statistically significant (*). M = male; F= female; WHR = waist-to-hip ratio; VAT = visceral adipose tissue; MABP = mean arterial blood pressure; LDH = lactate dehydrogenase; AST = aspartate aminotransferase; ALT = alanine aminotransferase; GGT = gamma glutamyl transpeptidase; HDL = high density lipoprotein; TG = triglycerides; 17

18 HBA1c = glycosylated haemoglobin; QUICKI = quantitative assessment check index calculated as 1/[log(fasting insulin) + log(fasting glucose)]; HOMA IR = homeostasis model of assessment insulin resistance calculated as [fasting insulin (mu/l) x fasting glucose (mmol/l)]/22.5; USS = ultrasound steatosis score; ABT = aminopyrine breath test; y/n = yes or no; y/n = yes or no; MS = metabolic syndrome; NCEP ATP III = US Third Adult Treatment Panel of the National Cholesterol Education Program; IDF = International Diabetes Federation; NAS = NASH Activity Score; CK 18 = cytokeratin 18; PNPLA3 = patatin like phospholipase domain-containing protein 3. 18

19 Table S3. Factors independently related to the severity of steatosis, ballooning, lobular inflammation and fibrosis in a multivariate analysis. Steatosis Ballooning Lobular inflammation Fibrosis Range of score B1 p B1 p B1 p B1 p Waist VAT < AST < ALT < TG USS < < < HBA1c Fasting C-peptide < PNPLA < The factors independently related (multivariate analysis) to the severity of the steatosis, ballooning, lobular inflammation and fibrosis (scored according to the NASH Clinical Research Network Scoring System 14 ) are shown with the corresponding B and p value. A p- value of < 0.05 is considered statistically significant. VAT = visceral adipose tissue; AST = aspartate aminotransferase; ALT = alanine aminotransferase; TG = triglycerides; HBA1c = glycosylated haemoglobin; USS = ultrasound steatosis score; PNPLA3 = patatin like phospholipase domain-containing protein 3. 19

20 Table S4. Factors that are significantly related to the presence of NASH defined according to Brunt et al in a univariate and multivariate analysis in the design cohort (binary logistic regression). R² p 1 -value B p 2 -value Waist WHR < VAT < VAT/SAT < VAT/TAT < SBP DBP MABP BP criterion MS AST < AST > 40 U/L < ALT < ALT > 56 U/L < ALT > 40 U/L < < AST/ALT GGT < GGT > 30 U/L < S-choline-esterase HDL-cholesterol

21 TG TG criterion MS Uric acid < Albumin Ferritin < Fasting glucose Fasting insulin Fasting C-peptide < HBA1c QUICKI < HOMA IR Diabetes absent/present < USS < < Spleen diameter Portal flow velocity ABT peak excretion ABT cumulative excretion Number of criteria MS NCEP ATP III < MS NCEP ATP III yes/no < Number of criteria MS IDF < MS IDF yes/no < PNPLA3 polymorphism < R² = adjusted R square of the binary logistic regression; p 1 = p-value of univariate analysis; p 2 = p-value of the multivariate analysis; VAT = visceral adipose tissue; SAT = subcutaneous 21

22 adipose tissue; TAT = total adipose tissue; SBP = systolic blood pressure; DBP = diastolic blood pressure; MABP = mean arterial blood pressure; BP = blood pressure; MS = metabolic syndrome; AST = aspartate aminotransferase; ALT = alanine aminotransferase; GGT = gamma glutamyl transpeptidase; HDL = high density lipoprotein; TG = triglycerides; HBA1c = glycosylated haemoglobin; QUICKI = quantitative assessment check index calculated as 1/[log(fasting insulin) + log(fasting glucose)]; HOMA IR = homeostasis model of assessment insulin resistance calculated as [fasting insulin (mu/l) x fasting glucose (mmol/l)]/22.5; USS = ultrasound steatosis score; ABT = aminopyrine breath test; NCEP ATP III = US Third Adult Treatment Panel of the National Cholesterol Education Program; IDF = International Diabetes Federation; PNPLA3 = patatin like phospholipase domain-containing protein 3. 22

23 Table S5. Factors that are significantly related to the presence of NASH defined according to Kleiner et al in a univariate and multivariate analysis in the design cohort (binary logistic regression). R² p 1 -value B p 2 -value BMI Waist WHR < TAT VAT VAT/SAT LDH AST < AST > 40 U/L < ALT < < ALT > 56 U/L < ALT > 40 U/L < GGT GGT > 30 U/L S-choline-esterase HDL-cholesterol TG criterion MS Uric acid Alpha foetoprotein

24 Ferritin Fasting insulin Fasting C-peptide < HBA1c QUICKI < HOMA IR Diabetes absent/present USS < < Liver right craniocaudal diameter Spleen diameter ABT peak excretion ABT peak excretion > ABT cumulative excretion ABT cumulative excretion > ABT abnormal Number of criteria MS NCEP ATP III < MS NCEP ATP III yes/no Number of criteria MS IDF < MS IDF yes/no PNPLA3 polymorphism < R² = adjusted R square of the binary logistic regression; p 1 = p-value of univariate analysis; p 2 = p-value of the multivariate analysis; BMI = body mass index; VAT = visceral adipose tissue; SAT = subcutaneous adipose tissue; TAT = total adipose tissue; LDH = lactate dehydrogenase; AST = aspartate aminotransferase; ALT = alanine aminotransferase; GGT = 24

25 gamma glutamyl transpeptidase; HDL = high density lipoprotein; TG = triglycerides; MS = metabolic syndrome; HBA1c = glycosylated haemoglobin; QUICKI = quantitative assessment check index calculated as 1/[log(fasting insulin) + log(fasting glucose)]; HOMA IR = homeostasis model of assessment insulin resistance calculated as [fasting insulin (mu/l) x fasting glucose (mmol/l)]/22.5; USS = ultrasound steatosis score; ABT = aminopyrine breath test; NCEP ATP III = US Third Adult Treatment Panel of the National Cholesterol Education Program; IDF = International Diabetes Federation; PNPLA3 = patatin like phospholipase domain-containing protein 3. 25

26 Table S6. Factors that significantly correlate with the severity of NASH as expressed by the NASH Activity Score according to Kleiner et al in a univariate and multivariate analysis in the design cohort (linear regression analysis). R² p 1 -value B p 2 -value Gender Age Waist < WHR < VAT < VAT/SAT BP criterion MS LDH < AST < AST > 40 U/L < ALT < < ALT > 56 U/L < ALT > 40 U/L < Alkaline phophatase GGT GGT > 30 U/L < S-choline-esterase HDL-cholesterol HDL-cholesterol criterion MS

27 TG < TG criterion MS < Uric acid < Albumin Ferritin < Haemoglobin Fibrinogen Fasting glucose Fasting insulin < Fasting C-peptide < HBA1c < QUICKI < HOMA IR < Diabetes absent/present USS < < Liver right craniocaudal diameter < Spleen diameter < ABT peak excretion ABT peak excretion > ABT cumulative excretion < ABT cumulative excretion > ABT abnormal Number of criteria MS NCEP ATP III < MS NCEP ATP III yes/no <

28 Number of criteria MS IDF < MS IDF yes/no < PNPLA3 polymorphism < R² = adjusted R square of the binary logistic regression; p 1 = p-value of univariate analysis; p 2 = p-value of the multivariate analysis; WHR = waist-to-hip ratio; VAT = visceral adipose tissue; SAT = subcutaneous adipose tissue; BP = blood pressure; MS = metabolic syndrome; LDH = lactate dehydrogenase; AST = aspartate aminotransferase; ALT = alanine aminotransferase; GGT = gamma glutamyl transpeptidase; HDL = high density lipoprotein; TG = triglycerides; HBA1c = glycosylated haemoglobin; QUICKI = quantitative assessment check index calculated as 1/[log(fasting insulin) + log(fasting glucose)]; HOMA IR = homeostasis model of assessment insulin resistance calculated as [fasting insulin (mu/l) x fasting glucose (mmol/l)]/22.5; USS = ultrasound steatosis score; ABT = aminopyrine breath test; NCEP ATP III = US Third Adult Treatment Panel of the National Cholesterol Education Program; IDF = International Diabetes Federation; PNPLA3 = patatin like phospholipase domain-containing protein 3. 28

29 Table S7. Factors that are significantly related to the presence of significant fibrosis defined as a fibrosis score 2 according to Kleiner et al in a univariate and multivariate analysis in the design cohort (binary logistic regression) R² p 1 -value B p 2 -value BMI Waist < WHR < TAT < VAT < SAT LDH AST < < AST > 40 U/L < ALT < ALT > 56 U/L < ALT > 40 U/L GGT GGT > 30 U/L S-choline-esterase HDL-cholesterol TG Uric acid Ferritin

30 Fasting glucose < Fasting insulin < Fasting C-peptide < HBA1c < QUICKI < HOMA IR < Diabetes absent/present USS Liver right craniocaudal diameter Portal flow velocity ABT peak excretion ABT peak excretion normal y/n ABT cumulative excretion ABT cumulative excretion normal y/n ABT normal y/n Weight at age Number of criteria MS NCEP ATP III < MS NCEP ATP III yes/no Number of criteria MS IDF MS IDF yes/no CK R² = adjusted R square of the binary logistic regression; p 1 = p-value of univariate analysis; p 2 = p-value of the multivariate analysis; BMI = body mass index; WHR = waist-to-hip ratio; TAT = total adipose tissue; VAT = visceral adipose tissue; SAT = subcutaneous adipose 30

31 tissue; LDH = lactate dehydrogenase; AST = aspartate aminotransferase; ALT = alanine aminotransferase; GGT = gamma glutamyl transpeptidase; HDL = high density lipoprotein; TG = triglycerides; HBA1c = glycosylated haemoglobin; QUICKI = quantitative assessment check index calculated as 1/[log(fasting insulin) + log(fasting glucose)]; HOMA IR = homeostasis model of assessment insulin resistance calculated as [fasting insulin (mu/l) x fasting glucose (mmol/l)]/22.5; USS = ultrasound steatosis score; ABT = aminopyrine breath test; y/n = yes or no; MS = metabolic syndrome NCEP ATP III = US Third Adult Treatment Panel of the National Cholesterol Education Program; IDF = International Diabetes Federation; CK 18 = cytokeratin

32 Table S8. Factors that are significantly related to the presence of advanced fibrosis defined as a fibrosis score 3 according to Kleiner et al in a univariate and multivariate analysis in the design cohort (binary logistic regression). R² p 1 -value B p 2 -value BMI Waist < WHR TAT VAT LDH < AST < AST > 40 U/L ALT < ALT > 56 U/L ALT > 40 U/L AST/ALT GGT TG Uric acid Total bilirubin Ferritin Fasting glucose Fasting insulin

33 Fasting C-peptide HBA1c QUICKI Diabetes absent/present ABT peak excretion ABT peak excretion normal y/n ABT cumulative excretion ABT cumulative excretion normal y/n ABT normal y/n Weight at age Number of criteria MS NCEP ATP III CK R² = adjusted R square of the binary logistic regression; p 1 = p-value of univariate analysis; p 2 = p-value of the multivariate analysis; BMI = body mass index; WHR = waist-to-hip ratio; TAT = total adipose tissue; VAT = visceral adipose tissue; LDH = lactate dehydrogenase; AST = aspartate aminotransferase; ALT = alanine aminotransferase; GGT = gamma glutamyl transpeptidase; TG = triglycerides; HBA1c = glycosylated haemoglobin; QUICKI = quantitative assessment check index calculated as 1/[log(fasting insulin) + log(fasting glucose)]; ABT = aminopyrine breath test; y/n = yes or no; MS = metabolic syndrome NCEP ATP III = US Third Adult Treatment Panel of the National Cholesterol Education Program; CK 18 = cytokeratin

34 FIGURES Fig. 1S 34