Methods
NAFLD, NASH, muscle antibody, autoimmune hepatitis
AUTOIMMUNE HEPATITIS, EVENT, INSULIN RESISTANCE, LIVER
The Treatment of NAFLD in Children Trial (TONIC) was a NIH sponsored randomized, double-blind, placebo-controlled trial that was conducted at 10 research centers in the US with 173 patients (age 8–17) with biopsy confirmed NAFLD to assess the impact of vitamin E (400 IU twice daily) compared with 500 mg metformin twice daily versus placebo over 96 weeks (ClinicalTrials.gov identifier: NCT00063635, 03/07/2003). The primary outcome was reduction in serum alanine aminotransferase (ALT) levelsAs part of the TONIC trial, whole blood was collected from children to collect DNA samples for potential genetic testing. DNA was extracted using the Qiagen Autopure LS DNA extractors using PUREGENE reagents using a modified salting out procedure. DNA was rehydrated by incubating at 56 °C for 1 h in a shaking incubator and placed on orbital shakers for 36–48 h prior to stock transfer. In the event a sample had a 260/280 ratio less than 1.7 (indicative of protein contamination), it was re-precipitated to improve its purity. DNA extraction was performed as soon as samples were received by the lab. Samples that were processed for DNA extraction and passed quality control were assigned storage locations by RUCDR STARLIMS in a − 80 °C freezer until LTL assay.Liver biopsies were obtained at study entry and subsequently after 96 weeks of therapy for all children enrolled in the TONIC trial. For all biopsies, the NASH Clinical Research Network (CRN) recommended a 16-gauge biopsy needle and a specimen length of at least 1.5 cm. The liver tissue was prepared locally for light microscopy with stains including hematoxylin and eosin, Masson’s trichrome and iron stain. All slides were sent to the data coordinating center for central reading by the study pathologistsAt baseline and 96 weeks of follow-up liver function tests and biomarkers of metabolic health including lipids and insulin resistance were also evaluated in the children. Anthropometrics including body mass index (BMI) and waist circumference were also assessed. Additionally, at baseline, autoantibodies associated with autoimmune hepatitis were assessed including anti-nuclear antibody (ANA), anti-smooth muscle antibody (ASMA) and anti-mitochondrial antibody (AMA).