Context Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease, affecting approximately 3 in 10 obese children worldwide. Objective We aimed to investigate the potential relationship between gut microbiota and NAFLD in obese youth, while considering the role of PNPLA3 rs738409, a strong genetic contributor to NAFLD. Design In this cross-sectional study, participants completed abdominal MRI to measure hepatic fat fraction (HFF), oral glucose tolerance test, and PNPLA3 rs738409 genotyping. Fecal samples were collected to analyze the V4 region of the 16S rRNA gene for intestinal bacteria characterization. Setting Yale Pediatric Obesity Clinic. Participants Obese youth (BMI > 95th percentile) with NAFLD (HFF ≥ 5.5%; n=44) and without NAFLD (HFF < 5.5%; n=29). Main Outcome Measure Shannon-Wiener diversity index values and proportional bacterial abundance by NAFLD status and PNPLA3 genotype. Results Subjects with NAFLD had decreased bacterial alpha-diversity compared to those without NAFLD (p=0.013). Subjects with NAFLD showed a higher Firmicutes to Bacteroidetes (F/B) ratio (p=0.019) and lower abundance of Bacteroidetes (p=0.010), Prevotella (p=0.019), Gemmiger (p=0.003), and Oscillospira (p=0.036). F/B ratio, Bacteroidetes, Gemmiger, and Oscillospira were associated with HFF when controlling for group variations. We also observed an additive effect on HFF by PNPLA3 rs738409 and Gemmiger, and PNPLA3 rs738409 and Oscillospira. Conclusions Obese youth with NAFLD have a different gut microbiota composition than those without NAFLD. These differences were still statistically significant when controlling for factors associated with NAFLD, including PNPLA3 rs738409.

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Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease. Since gut microbiota has been associated with pediatric obesity, we explored the potential relationship between gut microbiota and NAFLD in obese youth, while considering the role of PNPLA3 rs738409, a genetic contributor to NAFLD. Seventy-three obese youth (BMI > 95th percentile) underwent an MRI to measure hepatic fat fraction (HFF), an oral glucose tolerance test, and fasting blood sample collection. PNPLA3 rs738409 was assessed by automatic sequencing. A stool sample from each subject was collected to analyze the V4 region of the 16S rRNA gene and characterize intestinal bacteria. Shannon-Wiener index values were calculated for all samples and were compared between participants without NAFLD (HFF < 5.5%; n= 29) and with NAFLD (HFF ≥ 5.5%; n= 44). We observed that subjects with NAFLD had decreased (p=0.013) bacterial diversity compared to those without NAFLD. When comparing the abundance of each bacterial phylum (n=8) and genus (n=51) between the two groups, we observed a higher Firmicutes to Bacteroidetes (F/B) ratio (p=0.019) and lower abundance of Bacteroidetes (p=0.010), Prevotella (p=0.019), Gemmiger (p=0.003), and Oscillospira (p=0.036) in those with NAFLD, compared to those without NAFLD. Multiple linear regression was conducted to determine if each phylum and genus that was different (p<0.05) in abundance between the two groups was found to be predictive of HFF, while controlling for PNPLA3 genotype, race, and BMI z-score. Regression results showed that F/B ratio, Bacteroidetes, Gemmiger, and Oscillospira were associated with HFF when controlling for group variations. We also observed an additive effect of PNPLA3 rs738409 and Gemmiger, and PNPLA3 rs738409 and Oscillospira on HFF. We demonstrate that obese youth with NAFLD have different gut microbiota composition than those without NAFLD and identified individual phyla and genera that are associated with intrahepatic fat content. Disclosure A. Monga Kravetz: None. T. Testerman: None. B. Galuppo: None. J. Graf: None. S. Siebel: None. R. Feinn: None. N. Santoro: None. Funding National Institutes of Health (R01DK114504)