SUPPORTING MATERIAL
Supporting Statistics.
In the subgroups of subjects who underwent a subcutaneous fat biopsy to address
potential ethnic differences and assess statistical significance, we performed a withinethnic-group permutaion test as follows: for each permutation, we permuted the trait
values within each ethnic group and then recalculated the mean difference between
genotype groups across different ethnic groups. Because this procedure only
permuted within each ethnic group, the potential contribution from ethnicity is
controlled. We repeated the permutation steps 10,000 times and the statistical
significance level was assessed by comparing the observed mean difference between
the two genotype groups and the empirical distribution through permutations. Based
on this procedure, the statistical significance for the % small cell was estimated to be
0.004 where that for the median was estimated to be 0.0142.
Liver Biopsy (Supporting Figure 1)
was performed in 6 subjects (5F) because of persistent elevation in Alanine
transaminase (ALT) (mean ALT 130; 95%CI 80-213). Biopsies were formalin-fixed,
paraffin embedded, stained with Hematoxyllin & Eosin, trichrome and Gordon’s
reticulin techniques. All biopsies were 2 cm or more in length and were reviewed by a
pediatric pathologist according to the Brunt approach. Four Hispanics and 2
Caucasians (mean age was 13.1, 95%CI 10.4 – 15.8; mean BMI z-score was 2.30,
95%CI 20.1 – 2.63) underwent a liver biopsy, all of them had hepatic steatosis (mean
% HFF 27.3, 95%CI 12. 5 – 36.9). Of these, 3 were heterozygous (CG) and 3
homozygous (GG). In both the heterozygotes and homozygotes carrier varying grades
of steatosis with inflammation and fibrosis were present (Figure 4). These histological
data not only corroborate the association found between the SNP and the hepatic
steatosis measured by MRI, but also it illustrates the clear presence of more advance
NAFLD disease.
Quantitative real-time PCR.
Total RNA was isolated using TRIzol reagent and further purified using an RNeasy
kit (Qiagen, Valencia, CA). The quality of total RNA was evaluated by A260/A280
ratio which had to be at least 1.8, and by electrophoresis on an Agilent Bioanalyzer.
Quantitative RT-PCR was performed using an ABI 7000 Sequence Detection System
(Applied Biosystems, Foster City, CA). The nucleotide sequences of the primers and
PCR conditions are available on request. Samples were run in duplicates for both the
gene of interest and 18S. Quantitative analysis was determined by Δ/ΔCT method
normalized to both a control and 18S message.
Manuscript number: HEP-10-0345