The European Paediatric Non-alcoholic Fatty Liver Disease Registry (EU-PNAFLD): a Prospective, Longitudinal Follow-up of Children With Non-alcoholic Fatty Liver Disease
Conditions: Non-Alcoholic Fatty Liver Disease, Non-alcoholic Fatty Liver, Non-alcoholic Steatohepatitis
Sponsor: Cambridge University Hospitals NHS Foundation Trust

 
 
 
 
IRAS Project ID: 174534 
Study Title: European Paediatric NAFLD Registry (EU-PNAFLD) 
Principal Investigator: Dr. David B. Savage 
Revision Date: 22/08/2017 
Protocol Version: 3 
 
 
The European-Paediatric NAFLD (EU-PNAFLD) Registry 
Longitudinal follow-up of children with non-alcoholic fatty liver disease 
Mann JP1,2, Armstrong MJ3, Rajwal S5, Noble-Jamieson G6, van Mourik I7, Newsome PN3, 
O’Rahilly S1, Kelly D3, Nobili V9, Anstee QM10, Savage DB1 
 
Advisory board: Feldstein AE8. 
 
1University of Cambridge Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of 
Metabolic Science, Cambridge, UK 
2Department of Paediatrics, University of Cambridge, UK 
3NIHR Centre for Liver Research and Biomedical Research Unit, University of Birmingham, UK 
4Paediatric Liver, Gastrointestinal, and Nutrition Centre, King's College London School of Medicine, King's College 
Hospital, UK 
5Paediatric liver unit, Leeds General Infirmary, UK. 
6Paediatric gastroenterology department, Addenbrooke’s Hospital, Cambridge, UK 
7Liver Unit, Birmingham Children’s Hospital, UK 
8Division of gastroenterology, hepatology, and nutrition, UC San Diego, USA. 
9Hepato-metabolic unit, Bambino Gesu Hospital, Rome, Italy. 
10Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK 
 
Correspondence: jm2032@medschl.cam.ac.uk 
 
Vision 
We plan to develop a Europe-wide clinical research network and registry of children with 
non-alcoholic fatty liver disease (NAFLD) to facilitate international clinical and translational 
studies that will ultimately improve the outcome for affected patients. EU-PNAFLD 
(European Paediatric NALFD Registry) will be a network composed of European centres 
involved in the care of children with NAFLD, and will include Hepatologists, Endocrinologists, 
and Scientists supported by relevant international specialists. This collaboration will build on 
existing infrastructure (local databases and bio-repositories) and align with the adult 
European NAFLD Registry (“EPoS”, Elucidating Pathways of Steatohepatitis) to allow long-
term follow-up supported by translational studies. Through an international, well-
characterised large-scale cohort, we hope to: facilitate multi-centre clinical trials; extend our 
understanding of the key disease mechanisms of NAFLD; and establish the natural history of 
paediatric NAFLD. 
 
Background 
Obesity is one of the main public health issues for developed countries1 with a third of 9-
year olds in the UK now overweight or obese2. The rise in obesity has been mirrored by an 
increase in paediatric non-alcoholic fatty liver disease (NAFLD), a term that encompasses a 
spectrum of disease from steatosis (or non-alcoholic fatty liver (NAFL)) to non-alcoholic 
steatohepatitis (NASH) and cirrhosis3. 
 

 
 
 
 
IRAS Project ID: 174534 
Study Title: European Paediatric NAFLD Registry (EU-PNAFLD) 
Principal Investigator: Dr. David B. Savage 
Revision Date: 22/08/2017 
Protocol Version: 3 
 
 
 
 
There are several unanswered questions and important issues in the management of 
paediatric NAFLD. These include: 
1. What is the natural history of the condition? And, what is the prognostic significance 
of NAFL (‘simple steatosis’)? 
2. What treatments are effective for paediatric NAFLD? 
3. How can patients with paediatric NAFLD be non-invasively stratified into high-risk 
and low-risk groups? Are novel biomarkers or genetics helpful? 
4. Do monogenic disorders underlie a subset of patients with paediatric NAFLD? 
 
In adults uncomplicated steatosis often remains a benign condition, whereas steatohepatitis 
with fibrosis may progress to end-stage liver disease and decompensation4–8. However, the 
natural history of NAFLD in children is much less clear9 and based on a relatively small 
number of studies. The first prospective secondary care study of NASH in children 
demonstrated cirrhosis in one out of 36 children after 10 years follow-up10. In 2009, 
Feldstein et al reported a retrospective cohort study that found 2/66 patients required liver 
transplantation and 3 died (non-liver deaths) within 20 years11. In addition to a few selected 
follow-up studies12–14, there are also a few small case series of cirrhosis in children or young 
adults secondary to paediatric NAFLD15–17. 
 
However, these studies are relatively small in size and are predominantly retrospective in 
nature, thus limiting their value. There have been calls in the literature for longitudinal 
research into the natural history of paediatric NAFLD9,18–20, but as yet such studies have not 
been established. Such follow-up studies will be important in defining the natural history of 
such children, as well as allowing for an accurate description of the metabolic complications 
they may develop21,22. 
 
Currently, there are no approved pharmacological treatments for paediatric NAFLD23,24. 
Randomised controlled trials have failed to demonstrate any benefit from vitamin E25, 
metformin, or cysteamine on histology26. Polyunsaturated fatty acids have not been studied 
with validated outcomes (histology or non-invasive scores)27. Therefore, there is an urgent 
need for new, effective therapies that have been assessed in well-characterised cohorts28. 
 
Liver biopsy is the gold-standard investigation of paediatric NAFLD and the only fully 
validated method for assessing the presence of NASH or fibrosis3,29. Novel non-invasive 

 
 
 
 
IRAS Project ID: 174534 
Study Title: European Paediatric NAFLD Registry (EU-PNAFLD) 
Principal Investigator: Dr. David B. Savage 
Revision Date: 22/08/2017 
Protocol Version: 3 
 
 
markers (e.g. cytokeratin-18 and cathepsin D, or plasma lipidomics) may replace the need 
for liver biopsy over the next 5-10 years30–33. 
 
It is believed that NAFLD is polygenic (plus environmental) in the vast majority of affected 
patients, however a subset may be caused by rare genetic conditions34–36. These may be 
more likely to present with an abnormal phenotype (e.g. NAFLD with low BMI) but can lead 
to diagnoses with specific therapeutic options (e.g. lipodystrophy treated with leptin). 
 
 
Aims 
1. Describe natural history of paediatric NAFLD 
This observational study aims to describe the natural history of paediatric NAFLD in Europe, 
providing data on their survival, progression of liver disease and development of metabolic 
complications. Understanding the burden of metabolic disease in these patients will be 
important and will focus on aspects such as incidental rate of diabetes, diabetic 
complications, sleep apnoea, hypertension, and early-onset cardiovascular disease. 
This Registry will link with the already established adult EU-NAFLD Registry (PI: Prof. Quentin 
Anstee, Newcastle, UK). 
 
2. Facilitate translational basic science research 
Blood samples taken for genetic and metabolic analysis will allow additional insight into the 
mechanisms underlying NAFLD. These samples may also be used in future studies related to 
NAFLD. 
 
3. Backbone study for recruitment into further trials 
The study will provide a well characterised cohort of children with NAFLD who could be 
recruited into translational and interventional studies in the future. 
 
 
Methods 
Overview 
– Establish a multi-centre database of UK paediatric NAFLD patients with prospective 
follow-up and data collection from routine clinic appointments. 
– Diagnosis of NAFLD by liver biopsy, or non-invasive investigations 
– Prospective follow-up, as dictated by clinical care at individual centres. 
– Data will be collected as available, ideally at annual follow-up 
– Each centre outside the UK will be responsible for arranging their own ethical 
approval and study insurance 
 
Participant identification 
Retrospective: patient databases at defined institutions (see below) will be searched to 
screen for patients with biopsy-proven diagnosis of NAFLD between 2012 to-date. Data must 
be complete and good quality to allow retrospective enrollment. 
 
Prospective: Recruitment of patients from 2017 onwards from liver clinics for children with 
(invasive or non-invasive) diagnosis of NAFLD. 
 
Exclusion criteria: 

 
 
 
 
IRAS Project ID: 174534 
Study Title: European Paediatric NAFLD Registry (EU-PNAFLD) 
Principal Investigator: Dr. David B. Savage 
Revision Date: 22/08/2017 
Protocol Version: 3 
 
 
– Secondary fatty liver disease (e.g. glycogen storage diseases, Wilson disease, viral 
hepatitis, drug-related, autoimmune hepatitis, type 1 diabetes mellitus)1 
– Post-transplant fatty liver 
– >20g/day ethanol intake 
 
Inclusion criteria: 
– Diagnosis made under 18 years of age. 
– Diagnosis of NAFLD spectrum disease (simple steatosis (NAFL), steatosis with 
abnormal transaminases, NASH ± fibrosis or cirrhosis) 
– Diagnosis established by: 
o Radiological evidence of hepatic steatosis (e.g. increased hepatic 
echogenicity on ultrasound), with 
o Exclusion of secondary causes (negative serological liver screen for 
HBV/HCV, caeruloplasmin >0.20g/L, no history of excess alcohol 
consumption, no evidence of iron overload, and no clinically significant 
alpha-1 antitrypsin (A1AT) phenotype (i.e. SZ, ZZ, SS), with or without 
o Histology (>5% steatosis and histology consistent with paediatric NAFLD) 
 
Definitions: 
– NAFL: presence of hepatic steatosis with no evidence of hepatocellular injury in the 
form of ballooning of the hepatocytes. 
– NAFLD: presence of hepatic steatosis with either abnormal aminotransferases or 
evidence of hepatocellular injury on histology that doesn’t meet criteria for diagnosis 
of NASH. 
– NASH: presence of hepatic steatosis and inflammation on biopsy2 
o Type 1 NASH: centrilobular inflammation with hepatocyte injury in the form 
of ballooning (with Mallory-Denk bodies), with or without fibrosis, and 
without portal inflammation or fibrosis 
o Type 2 NASH: portal inflammation with or without periportal fibrosis, and 
absence of ballooning or centrilobular inflammation 
o Overlap NASH: features of both type 1 and type 2 NASH 
– Cirrhosis: histological evidence of cirrhosis, radiological evidence strongly suggestive 
of cirrhosis (e.g. CT or MRI), or intraoperative visualisation of macroscopic cirrhosis. 
 
Institutions involved 
– Centres included to date: 
o Birmingham Children’s Hospital – Dr. Indra van Mourik & Prof. D. Kelly 
(DAK) 
o Queen Elizabeth Hospital, Birmingham – Prof. P.N. Newsome & Dr. 
Matthew J. Armstrong 
o Leeds General Infirmary – Dr. S. Rajwal 
o Addenbrooke’s Hospital, Cambridge – Dr. Jake P. Mann (JPM), Dr. G. Noble-
Jamieson, Prof. David B. Savage (DBS), & Dr. Matthias Zilbauer (MZ) 
o Bambino Gesu Hospital, Rome – Prof. Valerio Nobili (VN) 
                                                 
1 NAFLD associated with insulin-resistance syndromes (e.g. lipodystrophy, Bardet-Biedl syndrome) are 
not to be excluded but will form a specific sub-groups 
2 At this stage, NASH can only be diagnosed on biopsy. However, if a validated method of non-
invasive diagnosis becomes available then this can be implemented. 

 
 
 
 
IRAS Project ID: 174534 
Study Title: European Paediatric NAFLD Registry (EU-PNAFLD) 
Principal Investigator: Dr. David B. Savage 
Revision Date: 22/08/2017 
Protocol Version: 3 
 
 
o Newcastle upon Tyne Hospitals NHS Foundation Trust (RVI & Freeman 
Hospitals), Newcastle – Prof Quentin M. Anstee (QMA) 
o Hanover, Germany – Dr. Ulrich Baumann 
o Warsaw, Poland – Prof. Piotr Socha (PS) 
 
Data collection 
Data will be collected from: 
– Electronic and paper patient records 
– Patient (and parent) questionnaires at follow-up 
– Telephone interviews 
– NHS data systems (e.g. HES data, Death registry) for long-term follow-up data of UK-
based patients 
 
Data will then be entered into a secure online database hosted by Newcastle University. The 
database will be an extension of the adult European NAFLD Registry (EPoS Study). 
 
Data collection and entry onto the online database will be performed by local collaborators 
and research assistants, where available. 
 
Participants who are lost to follow-up after initial diagnosis will remain in the database and 
can contribute to long-term morbidity/mortality analysis. 
 
If participants are still diagnosed with NAFLD or under the care of a liver clinic when they 
turn 18-years, they will be invited to join the adult European NAFLD Registry. This would be 
a separate consent process. 
 
Genetic and metabolic analysis 
Participants who consent to having blood taken for genetic and metabolic analysis, samples 
will be taken when they have their routine blood tests at the liver clinic. Blood may also be 
taken from parents to facilitate genetic trios analysis. 
For DNA: 10ml blood collected into plain tubes, which can be sent without further 
processing. 
For metabolites: 5ml blood collected into serum gel activator tubes, which will be spun and 
separated at the local laboratory. Serum will be frozen prior to transport. 
Blood samples will be sent to the Metabolic Research Laboratories, Biomedical campus, 
Cambridge, UK, for processing and storage. Samples may also be stored at the National 
BioResource in Milton Keynes, if necessary. 
Ethical approval will also be sought for gut microbiome analysis. 
 
Natural history outcomes: 
– Primary: Survival (liver- and non-liver related mortality) 
– Secondary: Transplant-free survival; mortality due to cardiovascular disease; 
progression of liver histology3; development of decompensated liver disease; 
development of type 2 diabetes mellitus; development of clinical cardiovascular 
disease; liver disease variables, including: ALT, AST, cholesterol, triglycerides, waist 
circumference, Fibroscan values 
– A sub-analysis of only children with liver biopsies will be performed 
                                                 
3 Fibrosis staged according to the system proposed by Kleiner et al. (2005). 

 
 
 
 
IRAS Project ID: 174534 
Study Title: European Paediatric NAFLD Registry (EU-PNAFLD) 
Principal Investigator: Dr. David B. Savage 
Revision Date: 22/08/2017 
Protocol Version: 3 
 
 
– Data will be analysed in conjunction with natural history data from the adult 
European NAFLD Registry (EPoS) study 
 
Data will be collected at presentation and at approximately annual-yearly intervals alongside 
routine care. More frequent follow-up data may be available for those patients under 
regular review. (Minimum dataset in bold.) 
– Name, NHS number (for UK participants), date of birth, gender, post code 
– Date of visit (initial, or follow-up) 
– Demographics (age, gender, ethnicity, country of origin) 
– Height, weight and BMI (with centiles4), waist  circumference, hip circumference, 
blood pressure 
– Method of presentation (abnormal USS, abnormal LFTs, other) 
– Self-reported 24-hour food diary 
– Alcohol consumption 
– Past medical history (diabetes, psychological, other) 
– Complications of liver disease, metabolic syndrome or insulin resistance, polycystic 
ovarian syndrome, sleep apnoea, transplantation, renal impairment, number of 
hospital admissions in last 6 months 
– Family history (obesity, diabetes, ischaemic heart disease, thyroid disease, other) 
– Medications 
– Clinical evidence of portal hypertension or chronic liver disease 
– Liver function tests, to include: AST, ALT, ALP, Bil, GGT, Alb, PT/INR, platelet count 
– HbA1c, TSH, lipid profile (ideally fasting), ferritin, CRP, urate, creatinine/eGFR, 
Immunoglobulins (IgA, IgG, IgM).  
– Quantification of insulin resistance (fasting insulin measured at OGTT, HOMA-IR, or 
ADIPO-IR) 
– On initial visit only: caeruloplasmin, A1AT, serum immunoglobulins, 
autoantibodies, ferritin, transferrin saturation, viral screen (HBV/HCV)5 
– Ultrasound scan (USS) results: echogenic liver, spleen size5 
– Liver histology: degree and distribution of steatosis, portal/centrilobular 
inflammation, fibrosis stage3,5 
o Biopsy may have been performed, as clinically indicated, prior to entry the 
study, but it is not an absolute requirement for prospectively recruited 
participants 
o Patients may be offered a repeat biopsy within 10-years, according to 
clinical need 
– Diagnosis: simple steatosis / NAFL, NAFLD, NASH, or cirrhosis (and invasive or non-
invasive diagnosis) 
– Management (new medications, participation in clinical trials of lifestyle 
change/pharmacological agents) 
– Referrals to other specialties (e.g. endocrine & diabetes, cardiology, adult services) 
 
Additional data, if available: 
– Bioimpedance 
– Non-invasive markers (e.g. ELF, CK-18, NEFA, cathepsin D) 
– Fibroscan values 
                                                 
4 Obesity will be defined as BMI >95% centile for age and sex. 
5 If diagnosis is not established using biopsy, then the full panel of tests for exclusion of secondary 
causes must be recorded, in addition to ultrasound results. 

 
 
 
 
IRAS Project ID: 174534 
Study Title: European Paediatric NAFLD Registry (EU-PNAFLD) 
Principal Investigator: Dr. David B. Savage 
Revision Date: 22/08/2017 
Protocol Version: 3 
 
 
 
Research Sample Collection 
Participants will be invited to give samples for research purposes that can be used in future 
translational science studies. Samples to include: 
– Blood for DNA / RNA extraction 
– Serum & plasma 
– Biopsy (liver, adipose, muscle) samples 
– Stool samples 
 
Follow-up 
Data will be collected as available from follow-up clinics, ideally annually. If patients have 
been discharged, then follow-up will be via letter/telephone or GP. Total follow-up will 
continue for up to 10-years in the first instance. This will require interaction between 
paediatric and adult services. Data will be analysed in conjuction with the adult EU-NAFLD 
Registry but will not be transferred between the two. If funding allows, data collection will 
continue as long as patients remain under hepatology follow-up. At 20-years from entry into 
the trial we will search pseudoanonymised data systems (e.g. HES, GP database, and 
European-equivalents) to look for cardiovascular and metabolic outcomes. In addition, we 
may contact participants and their GPs to ask for details if our data is incomplete. 
If participants have NAFLD as adults, they may be separately invited to formally join the 
adult NAFLD Registry. 
 
Statistical analysis 
The majority of this analysis will be descriptive so as to define the natural history of the 
patient group. Survival data from study cohort will be compared to an age- and sex-matched 
cohort from the national GP database. 
 
Cox multiple variable regression will facilitate survival analysis to assess the primary 
outcome. This will allow identification of the factors that significantly influence survival. 
However, due to a lack of published data any power calculation would be entirely 
speculative. 
 
Ethical approval 
Informed consent will be sought prior to starting data collection. National Research Ethics 
Committee approval will be sought to allow for the collection and analysis of patient data for 
the purposes of this research study. 
 
Funding 
The study is supported by the European Union H2020 programme through the Elucidating 
Pathways of Steatohepatitis (EPoS) consortium grant and an EASL Registry Grant (to Prof 
Quentin Anstee). Additional support is from the NIHR Cambridge Biomedical Resource 
Centre, the NIHR Newcastle Biomedical Research Centre and by Wellcome Trust grants to 
Prof. David B. Savage and Prof. Sir Steve O’Rahilly. 
 
Plan of work: phases & work streams 
To manage the implementation of the above protocol, we have split the plan of work into 
two phases. The direction of the registry will be lead by a steering committee, comprising: 
VN, DAK, PS, JPM, and QMA. Each Work stream will have a lead clinician responsible for its 
implementation. 
 

 
 
 
 
IRAS Project ID: 174534 
Study Title: European Paediatric NAFLD Registry (EU-PNAFLD) 
Principal Investigator: Dr. David B. Savage 
Revision Date: 22/08/2017 
Protocol Version: 3 
 
 
Phase 1 –  
Phase 1 has been divided into 2 work streams and will focus on establishing the EU-PNAFLD 
network, including the set-up of a core database and bio-bank. This phase forms the area of 
support applied for through the ESPGHAN networking grant. 
 
Work stream 1: Mapping the natural history of paediatric NAFLD and provide a cohort for 
trial recruitment 
Lead: QMA, PS 
The objective of this work stream is to describe the long-term outcomes of children with 
NAFLD, including clinical liver events (e.g. HCC, variceal bleeds), subclinical progression (e.g. 
progression of fibrosis), and metabolic outcomes (e.g. development of type 2 diabetes). The 
initial goal will be to develop an international, multicenter, registry of children with biopsy-
proven NAFLD for a retrospective analysis and which will also facilitate recruitment into 
interventional trials. Local databases at participating institutions will be used to 
retrospectively identify potentially eligible participants with a previous biopsy-proven 
diagnosis of NAFLD-spectrum disease. 
The network database will be paediatric-specific extension of the existing adult European 
NAFLD Registry database and will be managed by the University of Newcastle, UK. Clinical, 
anthropometric, and biochemical pseudoanonymised data will be uploaded locally. 
All participants will be asked give consent to be contacted by other researchers (including 
those internationally) regarding recruitment into further clinical trials. The EU-PNAFLD 
Network database will serve as a central point of contact for researchers wishing to recruit a 
cohort of children with NAFLD. 
 
Work stream 2: Establishing a bio-bank of samples for translational research 
Lead: DBS, JPM 
The objective of work stream 2 is to develop a bio-bank of samples from children with 
NAFLD that can be used for translational laboratory science project, including those 
described in Phase 2, below. 
We have funding and infrastructure in place for the storage of blood (DNA), serum, stool, 
and liver biopsy specimens at the Institute of Metabolic Science in Cambridge, UK. 
Consenting participants will be asked to give an additional sample of blood during their next 
routine tests, which can be processed by protocolled procedures and sent to Cambridge, UK. 
Parents of participants will also be invited to give a sample for DNA storage for future 
genetic trio analysis. Stool samples will be obtained to facilitate microbiome studies. 
 
Phase 2 
Phase 2 comprises three work streams, aimed at prospective recruitment/follow-up and 
translational basic science. 
Work streams 3: Non-invasive biomarkers of paediatric NAFLD 
Lead: VN 
Work stream 3 will employ the following key strategies: 
I. 
Validation of existing biomarkers that have previously been assessed in single 
cohorts only, including: Enhanced Liver Fibrosis (ELF) test, CK-18, cathepsin-D, miR-122. 
II. 
Exploration of novel serum biomarkers, including: targeted and untargeted lipidomic 
analyses, serum proteome analysis by mass spectrometry, exosomes, intestinal microbiome, 
and microRNAs. 
 
Work stream 4: Genetics of paediatric NAFLD 
Leads: JPM, UB 

 
 
 
 
IRAS Project ID: 174534 
Study Title: European Paediatric NAFLD Registry (EU-PNAFLD) 
Principal Investigator: Dr. David B. Savage 
Revision Date: 22/08/2017 
Protocol Version: 3 
 
 
Work stream 4 will focus on three main strategies: 
I. 
Whole exome sequencing of children with suspected monogenic disorders, by 
selecting children with ‘lean’ NAFLD, severe insulin resistance, or severe fibrosis, particularly 
in those with a family history of NAFLD. 
II. 
Assessment of epigenetics in paediatric NAFLD by characterisation of hepatic and 
circulating methylation profiles, combined with RNA sequencing to  
III. 
Single-cell sequencing of liver biopsy specimens from patients with simple steatosis, 
NASH, and all stages of fibrosis. 
 
Work stream 5: Prospective recruitment and follow-up for long-term natural history data 
Lead: DAK 
Work stream 5 will be a prospective continuation of Work stream 2, aiming to provide long-
term clinical outcome data and recruitment into interventional trials. Prospectively recruited 
participants will not require a biopsy diagnosis of NAFLD, this is for 4 reasons. Firstly, to 
include participants who would not meet criteria for biopsy, giving a less biased assessment 
of natural history of the condition. Secondly, to account for variation in practice between 
centres; this is necessary to facilitate a multi-centre trial. It will be possible to perform sub-
analyses of only participants with a biopsy-diagnosis. Finally, developments in non-invasive 
assessment of NAFLD may reduce the need for biopsy in the next 5-10 years. 
 
 
 
 
References 
1. 
Bhatt, D. L. et al. International prevalence, recognition, and treatment of 
cardiovascular risk factors in outpatients with atherothrombosis. JAMA 295, 180–9 
(2006). 
2. 
Jackson, A. A., Wiseman, M. & Wootton, S. A. Tackling the obesity crisis: how do we 
‘measure up’? Arch. Dis. Child. 99, 95 (2014). 
3. 
Vajro, P. et al. Diagnosis of nonalcoholic fatty liver disease in children and 
adolescents: position paper of the ESPGHAN Hepatology Committee. J. Pediatr. 
Gastroenterol. Nutr. 54, 700–13 (2012). 
4. 
Ekstedt, M. et al. Long-term follow-up of patients with NAFLD and elevated liver 
enzymes. Hepatology 44, 865–73 (2006). 
5. 
Ekstedt, M. et al. Fibrosis stage is the strongest predictor for disease-specific 
mortality in NAFLD after up to 33 years of follow-up. Hepatology 61, 1547–1554 
(2015). 
6. 
Bhala, N. et al. The natural history of nonalcoholic fatty liver disease with advanced 
fibrosis or cirrhosis: an international collaborative study. Hepatology 54, 1208–16 
(2011). 
7. 
Younossi, Z. M. et al. Global epidemiology of nonalcoholic fatty liver disease—Meta-
analytic assessment of prevalence, incidence, and outcomes. Hepatology 64, 73–84 
(2016). 
8. 
Musso, G., Gambino, R., Maurizio, C. & Pagano, G. Meta-analysis: Natural history of 
non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive 
tests for liver disease severity. Ann. Med. 43, 617–649 (2011). 
9. 
Nobili, V. et al. A 360-degree overview of paediatric NAFLD: Recent insights. J. 
Hepatol. 58, 1218–1229 (2013). 
10. 
Rashid, M. & Roberts, E. A. Non-alcoholic Steatohepatitis in Children. J. Pediatr. 
Gastroenterol. Nutr. 30, 48–53 (2000). 

 
 
 
 
IRAS Project ID: 174534 
Study Title: European Paediatric NAFLD Registry (EU-PNAFLD) 
Principal Investigator: Dr. David B. Savage 
Revision Date: 22/08/2017 
Protocol Version: 3 
 
 
11. 
Feldstein, A. E. et al. The natural history of nonalcholic fatty liver disease in children: 
a follow-up study for up to 20-years. Gut 58, 1538–1544 (2010). 
12. 
Manton, N. D. et al. Non-alcoholic steatohepatitis in children and adolescents. Med. J. 
Aust. 173, 476–479 (2000). 
13. 
Schwimmer, J. B. et al. Histopathology of pediatric nonalcoholic fatty liver disease. 
Hepatology 42, 641–649 (2005). 
14. 
Nobili, V. et al. NAFLD in children: a prospective clinical-pathological study and effect 
of lifestyle advice. Hepatology 44, 458–65 (2006). 
15. 
Kinugasa, A. et al. Fatty liver and its fibrous changes found in simple obesity of 
children. J Pediatr Gastroenterol Nutr 3, 408–414 (1984). 
16. 
Molleston, J. P., White, F., Teckman, J. & Fitzgerald, J. F. Obese children with 
steatohepatitis can develop cirrhosis in childhood. Am. J. Gastroenterol. 97, 2460–
2462 (2002). 
17. 
Adams, L. A., Feldstein, A., Lindor, K. D. & Angulo, P. Nonalcoholic Fatty Liver Disease 
among Patients with Hypothalamic and Pituitary Dysfunction. Hepatology 39, 909–
914 (2004). 
18. 
Marion,  a W., Baker,  a J. & Dhawan,  a. Fatty liver disease in children. Arch. Dis. 
Child. 89, 648–52 (2004). 
19. 
Patton, H. M. et al. Pediatric nonalcoholic fatty liver disease: a critical appraisal of 
current data and implications for future research. J. Pediatr. Gastroenterol. Nutr. 43, 
413–427 (2006). 
20. 
Roberts, E. a. Pediatric nonalcoholic fatty liver disease (NAFLD): a ‘growing’ problem? 
J. Hepatol. 46, 1133–42 (2007). 
21. 
Pacifico, L., Nobili, V., Anania, C., Verdecchia, P. & Chiesa, C. Pediatric nonalcoholic 
fatty liver disease, metabolic syndrome and cardiovascular risk. World J. 
Gastroenterol. 17, 3082–3091 (2011). 
22. 
Nobili, V. et al. The potential role of fatty liver in paediatric metabolic syndrome: A 
distinct phenotype with high metabolic risk? Pediatric Obesity 7, (2012). 
23. 
Vajro, P. et al. Therapeutic options in pediatric non alcoholic fatty liver disease: 
current status and future directions. Ital J Pediatr 38, 55 (2012). 
24. 
Mitchel, E. & Lavine, J. Review article: the management of paediatric nonalcoholic 
fatty liver disease. Aliment. Pharmacol. … 40, 1155–70 (2014). 
25. 
Lavine, J. E. et al. Effect of vitamin E or metformin for treatment of nonalcoholic fatty 
liver disease in children and adolescents: the TONIC randomized controlled trial. 
JAMA 305, 1659–68 (2011). 
26. 
Schwimmer, J. B. et al. In Children with Nonalcoholic Fatty Liver Disease, Cysteamine 
Bitartrate Delayed  Release Improves Liver Enzymes but does not Reduce Disease 
Activity Scores. Gastroenterology (2016). doi:10.1053/j.gastro.2016.08.027 
27. 
Janczyk, W. et al. Omega-3 Fatty acids therapy in children with nonalcoholic Fatty 
liver disease: a randomized controlled trial. J. Pediatr. 166, 1358–1363 (2015). 
28. 
Rotman, Y. & Sanyal, A. J. Current and upcoming pharmacotherapy for non-alcoholic 
fatty liver disease. Gut gutjnl-2016-312431 (2016). doi:10.1136/gutjnl-2016-312431 
29. 
Manco, M. et al. Metabolic syndrome and liver histology in paediatric non-alcoholic 
steatohepatitis. Int. J. Obes. (Lond). 32, 381–7 (2008). 
30. 
Fitzpatrick, E. & Dhawan, A. Noninvasive biomarkers in non-alcoholic fatty liver 
disease: Current status and a glimpse of the future. World J. Gastroenterol. 20, 
10851–10863 (2014). 
31. 
Feldstein, A. E. et al. Serum cytokeratin-18 fragment levels are useful biomarkers for 
nonalcoholic steatohepatitis in children. Am. J. Gastroenterol. 108, 1526–31 (2013). 
32. 
Mansoor, S., Collyer, E. & Alkhouri, N. A comprehensive review of noninvasive liver 

 
 
 
 
IRAS Project ID: 174534 
Study Title: European Paediatric NAFLD Registry (EU-PNAFLD) 
Principal Investigator: Dr. David B. Savage 
Revision Date: 22/08/2017 
Protocol Version: 3 
 
 
fibrosis tests in pediatric nonalcoholic Fatty liver disease. Curr. Gastroenterol. Rep. 
17, 23 (2015). 
33. 
Gorden, D. L. et al. Biomarkers of NAFLD progression: a lipidomics approach to an 
epidemic. J. Lipid Res. 56, 722–736 (2015). 
34. 
Dongiovanni, P. & Valenti, L. Genetics of nonalcoholic fatty liver disease. Metabolism 
1–12 (2015). doi:10.1016/j.metabol.2015.08.018 
35. 
Anstee, Q. M. & Day, C. P. The genetics of NAFLD. Nat. Rev. Gastroenterol. Hepatol. 
10, 645–55 (2013). 
36. 
Parker, V. E. R. & Semple, R. K. Genetics in endocrinology: genetic forms of severe 
insulin resistance: what endocrinologists should know. Eur. J. Endocrinol. 169, R71-80 
(2013).