Raine Study Gen2-17: NAFLD phenotype prevalence and characteristics
Table 1 summarizes characteristics of the 707 adolescents who had liver assessments, genome, and epigenome-wide profiling at the Raine Study Gen2-17 follow-up. Overall prevalence of NAFLD was 14.5%, with a higher prevalence in females than males (17.4% vs 11.8%, p-value=0.02). NAFLD was associated with higher adiposity, HOMA-IR, serum ALT, GGT and hsCRP, and with lower adiponectin (Table 1).
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Epigenome-wide DNA methylation association with adolescent NAFLD
DNA samples from 707 (52.1% males) adolescents who had undergone ultrasound assessment for NAFLD were analysed for EWAS. Our criteria identified eight dmCpGs in three genes: three dmCpGs (cg19537719, cg27650870, and cg18614735) in ankyrin-1 (ANK1, chromosome 8p11), three dmCpGs (cg04514255, cg01572694, and cg15649236) in microRNA 10a (MIR10A, chromosome 17q21), and two dmCpGs (cg22676516 and cg05821571) in protein tyrosine phosphatase receptor type N2 (PTPRN2, chromosome 7q36) (Table 2). Supplementary Table 3 shows the fully annotated results for EWAS analysis for all four models.
Validation by Pyrosequencing of NAFLD-associated CpG loci in adolescence
Four dmCpGs were selected for validation by pyrosequencing; cg19537719 and cg27650870 located in the gene body of ANK1, cg01572694 located near MIR10A, and cg05821571 in the gene body of PTPRN2. Supplementary Figure 1 shows Bland-Altman plots comparing DNA methylation levels measured by the EWAS and pyrosequencing at these CpG loci.
Association results for 16 pyrosequenced dmCpGs from the three genes identified during EWAS are shown in Figure 2 and Table 2 for steatosis score and NAFLD. Accounting for sex and age, consistent with EWAS results 13 pyrosequenced dmCpGs were associated with steatosis score and 12 dmCpGs with NAFLD. When adjusted for estimated cell type, 8 dmCpGs remained associated with steatosis score and 5 dmCpGs with NAFLD. When waist circumference was included as a measure of adiposity, 6 dmCpGs remained significant for steatosis score and 5 dmCpGs with NAFLD.
ANK1 CpGs were the most consistent across all outcomes and models, with 5 dmCpGs associated with NAFLD. When waist circumference was included in the model, the ANK1 CpGs become more significant (Figure 2). The dmCpG 8:41583512 (ANK1 CpG_10) was the most significantly associated with NAFLD across all pyrosequencing models.
For MIR10A, 3 dmCpGs (cg01572694 (MIR10A CpG_10), MIR10A CpG_7, MIR10A CpG_9) were associated with steatosis score and one CpG (MIR10A CpG_7) associated with NAFLD after cell count adjustment. When waist circumference was included in the steatosis score and NAFLD models no MIR10A dmCpGs remained significant. None of the three PTPRN2 dmCpGs that were measured by pyrosequencing were associated with NAFLD. Full results are shown in Table 2.
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Association of CpGs identified by pyrosequencing with additional biochemical markers of liver function
We investigated the association of sixteen pyrosequenced CpGs with three liver biochemical markers (GGT, ALT, AST) using linear regression (Supplementary Table 4 and Figure 3). All seven MIR10A CpGs were associated with ALT and GGT when accounting for age and sex. When further adjusted for cell count, four MIR10A CpGs demonstrated associations with ALT and six CpGs with GGT. Three ANK1 CpGs were significant for ALT and AST in both statistical models. For GGT five ANK1 CpGs were significant for age and sex and three remained associated after cell count adjustment.
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Overlap with adult CpGs identified in adult NAFLD meta-analysis EWAS
We investigated if 22 dmCpGs associated with liver fat in adults7 were associated with NAFLD in adolescence (Table 3). After correction for multiple testing (Bonferroni correction p-value <0.05/2=2.3x10-3), we identified one adult dmCpG (cg11024682) for steatosis score and three dmCpGs (cg14476101, cg26894079, cg11024682) for NAFLD. The dmCpG, cg11024682, was associated with both steatosis score and NAFLD. In addition, dmCpGs cg14476101 and cg26894079 were associated with NAFLD and nominal significance (p<0.05) with steatosis score.
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