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

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)]/22.5 2 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

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: -2.89 + 1.18 x MS (according to the IDF definition; yes = 1/no = 0) + 0.45 x type 2 diabetes (yes = 2/no = 0) + 0.15 x fasting insulin (mu/l) + 0.04 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: -15.745 + 0.139 x BMI (kg/m²) + 0.053 x waist circumference (cm) + 0.953 x log e [TG (mg/dl)] + 0.718 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

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))] 10-12. 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: -1.675 + 0.037 x age (years) + 0.094 x BMI (kg/m²) + 1.13 x impaired fasting glucose or diabetes (yes = 1, no = 0) + 0.99 x AST/ALT 0.013 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: 7.811 3.131 x ln[platelet count (10 9 /L) + 0.781 x ln[ggt (U/L)] + 3.467 x ln[age (year)] 0.014 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

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

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

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: -6.117 + [0.029 x ALT (U/L)] + [1.394 x USS] + [1.223 x number of IDF criteria MS]. This score had a R² = 0.508. 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

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: - 13.376 + [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 = 0.429. 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 ± 0.51. 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 < -3.860, 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 (-0.145 ± 1.877 vs. 0.413 ± 1.641, p = 0.004), the Antwerp NASH score 2 (-1.582 ± 2.151, p = 0.12), the Antwerp NAFLD significant fibrosis (-2.627 ± 1.343 vs. -2.011 ± 1.333, p = < 0.001) and the Antwerp NAFLD advanced fibrosis score (-4.222 ± 1.331 vs. -3.569 ± 1.449, p < 0.001) were significantly lower in those who did not have a liver biopsy compared to those 7

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 > -1.34 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 > -2.50 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 > -2.90 in the overall cohort. Based on the PPV, prevalence was estimated at 16.4%. Furthermore 87.5% had a value > -0.76 reliably excluding significant fibrosis without need for a biopsy. Concerning the diagnosis of advanced fibrosis, 54.1% had an Antwerp advanced fibrosis score > -4.11 in the overall cohort. Based on the PPV, prevalence was estimated at 5.4%. Furthermore 92.3% had a value > -2.14 reliably excluding advanced fibrosis without need for a biopsy. 8

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

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;34:899-907. 2. 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;28:412-419. 3. 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;85:2402-2410. 4. van der Kooy K, Seidell JC. Techniques for the measurement of visceral fat: a practical guide. Int J Obes Relat Metab Disord 1993;17:187-196. 5. 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;137:865-872. 6. 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;6:33. 7. 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;57:1441-1447. 10

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;94:2467-2474. 9. 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;38:518-526. 10. Ishak K, Baptista A, Bianchi L et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995;22:696-699. 11. 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;43:1317-1325. 12. Vallet-Pichard A, Mallet V et al. FIB-4: a simple, inexpensive and accurate marker of fibrosis in HCV-infected patients. Hepatology 2006;44:769-770. 13. 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;45:846-854. 14. Kleiner DE, Brunt EM, Van Natta M et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:1313-1321. 15. Forns X, Ampurdanes S, Llovet JM et al. Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model. Hepatology 2002;36:986-992. 16. Scheuer PJ. The nomenclature of chronic hepatitis: time for a change. J Hepatol 1995;22:112-114. 11

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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 %/% 21.3/78.7 33.7/66.3 0.013* Age y 41.9 ± 13.2 43.48 ± 12.8 0.278 BMI kg/m² 36.7 ± 5.4 38.9 ± 5.9 < 0.001* Waist cm 111.1 ± 13.2 118.1 ± 13.2 < 0.001* WHR 0.923 ± 0.108 0.972 ± 0.109 < 0.001* VAT cm² 178.5 ± 85.3 205.2 ± 90.9 0.007* MABP mm Hg 90.6 ± 10.8 93.9 ± 10.1 0.006* LDH U/L 539.5 ± 109.9 566.9 ± 101.6 0.020* AST U/L 29.4 ± 13.0 32.3 ± 14.9 0.070 ALT U/L 40.8 ± 20.9 45.2 ± 23.0 0.069 AST/ALT 0.77 ± 0.21 0.75 ± 0.19 0.335 GGT U/L 41.6 ± 53.9 40.9 ± 32.7 0.885 Total Cholesterol mg/dl 208.9 ± 39.4 204.8 ± 43.9 0.378 HDL-cholesterol mg/dl 54.0 ± 15.2 49.7 ± 14.2 0.008* TG mg/dl 157.4 ± 110.2 150.6 ± 73.5 0.490 Fasting glucose mg/dl 82.6 ± 11.7 85.7 ± 25.4 0.199 13

Fasting insulin μu/ml 13.9 ± 8.5 17.2 ± 11.5 0.005* Fasting C-peptide nmol/l 0.99 ± 0.37 1.11 ± 0.40 0.010* HBA1c % 5.61 ± 0.48 5.67 ± 0.61 0.359 QUICKI index 0.339 ± 0.036 0.329 ± 0.039 0.012* HOMA IR 2.93 ± 2.06 3.73 ± 3.19 0.019* USS 1.6 ± 1.1 1.9 ± 1.0 0.024* Diabetes absent/present %/% 90.4/9.6 92.1/7.9 0.583 Liver right craniocaudal diameter mm 15.1 ± 2.6 15.8 ± 2.8 0.019* Spleen diameter mm 10.4 ± 1.4 10.7 ± 1.7 0.062 ABT peak excretion % 9.76 ± 4.40 9.16 ± 4.59 0.232 ABT cumulative excretion % 14.33 ± 6.27 13.16 ± 6.19 0.087 Number criteria MS NCEP ATP III 2.5 ± 1.1 2.7 ± 1.0 0.067 Number criteria MS IDF 2.6 ± 1.2 2.7 ± 1.0 0.098 MS NCEP ATP III absent/present %/% 54.1/45.9 43.6/56.4 0.062 MS IDF absent/present %/% 53.3/46.7 42.4/57.6 0.051 Number criteria liver biopsy 1.9 ± 0.7 2.1 ± 0.7 0.001* 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

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

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 %/% 31.5/68.5 33.6/66.4 0.812 Age y 44.0 ± 12.2 42.9 ± 12.9 0.421 BMI kg/m² 39.1 ± 6.7 39.7 ± 6.3 0.466 Waist cm 116.9 ± 12.6 119.6 ± 14.6 0.098 WHR 0.963 ± 0.104 0.978 ± 0.110 0.226 VAT cm² 202.6 ± 89.8 214.4 ± 91.3 0.271 MABP mm Hg 96.0 ± 10.0 89.7 ± 9.3 < 0.001* LDH U/L 561.9 ± 105.0 586.1 ± 111.7 0.062 AST U/L 32.0 ± 16.8 33.5 ± 14.8 0.432 ALT U/L 45.3 ± 23.5 45.2 ± 23.1 0.959 AST/ALT 0.74 ± 0.17 0.78 ± 0.23 0.056 GGT U/L 37.7 ± 27.4 44.4 ± 23.1 0.073 Total Cholesterol mg/dl 201.0 ± 40.0 208.4 ± 47.1 0.144 HDL-cholesterol mg/dl 50.4 ± 14.7 48.9 ± 13.7 0.494 TG mg/dl 151.4 ± 81.5 149.6 ± 64.6 0.841 Fasting glucose mg/dl 87.4 ± 27.4 82.0 ± 12.1 0.053 Fasting insulin μu/ml 17.2 ± 10.9 17.3 ± 13.1 0.881 Fasting C-peptide nmol/l 1.14 ± 0.43 1.10 ± 0.37 0.470 HBA1c % 5.69 ± 0.66 5.62 ± 0.46 0.286 QUICKI index 0.328 ± 0.039 0.331 ± 0.039 0.495 16

HOMA IR 3.88 ± 4.46 3.62 ± 3.07 0.514 USS 1.9 ± 1.1 1.9 ± 1.0 0.661 Diabetes absent/present %/% 92.0/8.0 89.4/10.6 0.684 Liver right craniocaudal diameter mm 15.6 ± 3.1 16.0 ± 2.2 0.222 Spleen diameter mm 10.7 ± 1.7 10.6 ± 1.6 0.529 ABT peak excretion % 8.89 ± 4.45 9.17 ± 4.64 0.615 ABT cumulative excretion % 12.76 ± 6.15 13.23 ± 6.13 0.536 Number criteria MS NCEP ATP III 2.8 ± 1.1 2.6 ± 1.1 0.116 Number criteria MS IDF 2.8 ± 1.1 2.7 ± 1.1 0.200 MS NCEP ATP III absent/present %/% 43.0/57.0 50.0/50.0 0.240 MS IDF absent/present %/% 40.5/59.5 50.0/50.0 0.109 Number criteria liver biopsy 2.1 ± 0.8 2.1 ± 0.8 0.654 Steatosis score 1.2 ± 1.0 1.4 ± 1.1 0.066 NAS 3.2 ± 2.3 3.4 ± 2.4 0.527 Fibrosis score 0.6 ± 0.9 0.6 ± 0.9 0.353 PNPLA3 polymorphism n (%) 125 (62.5)/68 (34)/7 60 (53.1)/43 (38.1)/10 0.065 (CC/CG/GG) (3.5) (8.8) CK 18 U/L 207.3 ± 18.8 218.9 ± 32.9 0.852 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

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

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 0-3 0-2 0-3 0-4 B1 p B1 p B1 p B1 p Waist 0.022 0.001 VAT 0.002 < 0.001 AST 0.014 < 0.001 0.011 0.002 ALT 0.009 < 0.001 0.004 0.029 TG 0.001 0.026 USS 0.460 <0.001 0.184 < 0.001 0.267 < 0.001 HBA1c 0.387 0.002 Fasting C-peptide 0.288 0.018 0.246 < 0.001 PNPLA3 0.395 < 0.001 0.075 0.025 0.236 0.004 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

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 0.035 0.002 WHR 0.117 < 0.001 VAT 0.089 < 0.001 VAT/SAT 0.103 < 0.001 VAT/TAT 0.092 < 0.001 SBP 0.030 0.012 DBP 0.024 0.024 MABP 0.031 0.010 BP criterion MS 0.055 0.001 AST 0.221 < 0.001 AST > 40 U/L 0.139 < 0.001 ALT 0.228 < 0.001 ALT > 56 U/L 0.152 < 0.001 ALT > 40 U/L 0.190 < 0.001 1.315 < 0.001 AST/ALT 0.028 0.015 GGT 0.041 < 0.001 GGT > 30 U/L 0.061 < 0.001 S-choline-esterase 0.031 0.010 HDL-cholesterol 0.021 0.032 20

TG 0.042 0.004 TG criterion MS 0.051 0.001 Uric acid 0.071 < 0.001 Albumin 0.038 0.005 Ferritin 0.077 < 0.001 Fasting glucose 0.052 0.004 Fasting insulin 0.045 0.004 Fasting C-peptide 0.095 < 0.001 1.223 0.014 HBA1c 0.034 0.013 QUICKI 0.076 < 0.001 HOMA IR 0.066 0.001 Diabetes absent/present 0.056 < 0.001 USS 0.306 < 0.001 1.084 < 0.001 Spleen diameter 0.047 0.002 Portal flow velocity 0.027 0.042 ABT peak excretion 0.025 0.023 ABT cumulative excretion 0.026 0.019 Number of criteria MS NCEP ATP III 0.092 < 0.001 MS NCEP ATP III yes/no 0.086 < 0.001 Number of criteria MS IDF 0.098 < 0.001 MS IDF yes/no 0.084 < 0.001 PNPLA3 polymorphism 0.121 < 0.001 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

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

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 0.041 0.043 Waist 0.072 0.007 WHR 0.078 < 0.001 TAT 0.043 0.036 VAT 0.067 0.010 VAT/SAT 0.055 0.015 LDH 0.046 0.027 AST 0.273 < 0.001 AST > 40 U/L 0.152 < 0.001 ALT 0.301 < 0.001 0.029 < 0.001 ALT > 56 U/L 0.179 < 0.001 ALT > 40 U/L 0.186 < 0.001 GGT 0.068 0.014 GGT > 30 U/L 0.072 0.006 S-choline-esterase 0.047 0.030 HDL-cholesterol 0.101 0.006 TG criterion MS 0.038 0.045 Uric acid 0.047 0.028 Alpha foetoprotein 0.038 0.046 23

Ferritin 0.043 0.033 Fasting insulin 0.079 0.001 Fasting C-peptide 0.124 < 0.001 HBA1c 0.066 0.021 QUICKI 0.074 < 0.001 HOMA IR 0.091 0.001 Diabetes absent/present 0.030 0.025 USS 0.408 < 0.001 1.394 < 0.001 Liver right craniocaudal diameter 0.064 0.003 Spleen diameter 0.029 0.032 ABT peak excretion 0.049 0.007 ABT peak excretion > 5.4 0.025 0.042 ABT cumulative excretion 0.058 0.003 ABT cumulative excretion > 8.1 0.037 0.014 ABT abnormal 0.039 0.011 Number of criteria MS NCEP ATP III 0.097 < 0.001 MS NCEP ATP III yes/no 0.043 0.007 Number of criteria MS IDF 0.114 < 0.001 0.423 0.001 MS IDF yes/no 0.058 0.002 PNPLA3 polymorphism 0.095 < 0.001 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

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

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 0.021 0.015 Age 0.014 0.043 Waist 0.074 < 0.001 WHR 0.118 < 0.001 VAT 0.100 < 0.001 VAT/SAT 0.021 0.017 BP criterion MS 0.026 0.009 LDH 0.051 < 0.001 AST 0.178 < 0.001 AST > 40 U/L 0.114 < 0.001 ALT 0.190 < 0.001 0.021 < 0.001 ALT > 56 U/L 0.132 < 0.001 ALT > 40 U/L 0.145 < 0.001 Alkaline phophatase 0.021 0.016 GGT 0.047 0.001 GGT > 30 U/L 0.087 < 0.001 S-choline-esterase 0.044 0.001 HDL-cholesterol 0.021 0.016 HDL-cholesterol criterion MS 0.018 0.023 26

TG 0.058 < 0.001 TG criterion MS 0.048 < 0.001 Uric acid 0.052 < 0.001 Albumin 0.020 0.019 Ferritin 0.055 < 0.001 Haemoglobin 0.019 0.021 Fibrinogen 0.016 0.030 Fasting glucose 0.030 0.005 Fasting insulin 0.093 < 0.001 Fasting C-peptide 0.153 < 0.001 0.936 0.005 HBA1c 0.064 < 0.001 QUICKI 0.082 < 0.001 HOMA IR 0.113 < 0.001 Diabetes absent/present 0.040 0.001 USS 0.396 < 0.001 1.200 < 0.001 Liver right craniocaudal diameter 0.058 < 0.001 Spleen diameter 0.045 < 0.001 ABT peak excretion 0.046 0.001 ABT peak excretion > 5.4 0.017 0.028 ABT cumulative excretion 0.055 < 0.001 ABT cumulative excretion > 8.1 0.029 0.006 ABT abnormal 0.021 0.016 Number of criteria MS NCEP ATP III 0.093 < 0.001 MS NCEP ATP III yes/no 0.061 < 0.001 27

Number of criteria MS IDF 0.103 < 0.001 MS IDF yes/no 0.064 < 0.001 PNPLA3 polymorphism 0.098 < 0.001 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

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 0.080 0.001 Waist 0.213 < 0.001 0.048 0.016 WHR 0.156 < 0.001 TAT 0.130 < 0.001 VAT 0.140 < 0.001 SAT 0.032 0.039 LDH 0.087 0.001 AST 0.202 < 0.001 0.043 < 0.001 AST > 40 U/L 0.130 < 0.001 ALT 0.153 < 0.001 ALT > 56 U/L 0.144 < 0.001 ALT > 40 U/L 0.083 0.001 GGT 0.059 0.005 GGT > 30 U/L 0.047 0.014 S-choline-esterase 0.042 0.019 HDL-cholesterol 0.077 0.004 TG 0.033 0.030 Uric acid 0.068 0.003 Ferritin 0.040 0.018 29

Fasting glucose 0.137 < 0.001 Fasting insulin 0.144 < 0.001 Fasting C-peptide 0.211 < 0.001 1.373 0.019 HBA1c 0.196 < 0.001 QUICKI 0.186 < 0.001 HOMA IR 0.205 < 0.001 Diabetes absent/present 0.060 0.003 USS 0.067 0.006 Liver right craniocaudal diameter 0.037 0.021 Portal flow velocity 0.052 0.049 ABT peak excretion 0.061 0.008 ABT peak excretion normal y/n 0.044 0.012 ABT cumulative excretion 0.065 0.005 ABT cumulative excretion normal y/n 0.046 0.010 ABT normal y/n 0.034 0.027 Weight at age 32 0.088 0.006 Number of criteria MS NCEP ATP III 0.097 < 0.001 MS NCEP ATP III yes/no 0.069 0.003 Number of criteria MS IDF 0.046 0.013 MS IDF yes/no 0.030 0.047 CK 18 0.145 0.008 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

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 18. 31

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 0.037 0.036 Waist 0.138 < 0.001 0.057 0.012 WHR 0.120 0.001 TAT 0.052 0.018 VAT 0.064 0.007 LDH 0.176 < 0.001 AST 0.331 < 0.001 0.112 0.001 AST > 40 U/L 0.096 0.001-3.078 0.047 ALT 0.131 < 0.001 ALT > 56 U/L 0.085 0.002 ALT > 40 U/L 0.042 0.044 AST/ALT 0.075 0.004 GGT 0.041 0.023 TG 0.040 0.027 Uric acid 0.095 0.002 Total bilirubin 0.035 0.045 Ferritin 0.056 0.009 Fasting glucose 0.036 0.046 Fasting insulin 0.049 0.014 32

Fasting C-peptide 0.091 0.001 HBA1c 0.127 0.009 QUICKI 0.096 0.003 Diabetes absent/present 0.064 0.004 ABT peak excretion 0.046 0.043 ABT peak excretion normal y/n 0.062 0.008 ABT cumulative excretion 0.064 0.016 ABT cumulative excretion normal y/n 0.076 0.004 ABT normal y/n 0.064 0.009 Weight at age 32 0.125 0.004 Number of criteria MS NCEP ATP III 0.041 0.041 CK 18 0.180 0.005 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 18. 33

FIGURES Fig. 1S 34