Abstract
The link between bile acid (BA) metabolism and glucose and lipid metabolism has been increasingly recognised with particular interest in the field of non-alcoholic fatty liver disease (NAFLD). BA homeostasis is regulated by a negative feedback loop which relies on the activity of the farnesoid X receptor (FXR) which modulates the synthesis and uptake of BA through many pathways in enterocytes and hepatocytes. Levels of BA and FGF19 have been found to be elevated in patients with NAFLD, especially in patients with progressive steatofibrosis. An association between elevated levels of BA with steatosis, inflammation and fibrosis has been observed in adults and children. Moreover, an altered expression of mRNA levels of genes belonging to the bile acids synthesis and transport has been described in cohorts of patients with NAFLD. Environmental factors, as well as genetic factors, contribute to NAFLD, and define its multifactorial pathogenicity. The genetic predisposition in patients with NAFLD is well recognized and several studies have proven the association between PNPLA3, TM6SF2, GCKR and MBOAT7 by genome-wide association studies (GWAS). However, these studies give partial information on the genetic contribution to a disease, as they only evaluated common variants. The gap left by GWAS could be filled using more inclusive approaches that also evaluate rare variants. The aim of this study was to investigate the contribution of genes belonging to the BA metabolism and transport to disease in a paediatric cohort of patients with NAFLD.A next-generation sequencing (NGS) candidate-gene approach was employed to investigate the presence of variants in a panel of 135 genes and 42 additional SNPs. The cohort, selected from the King’s Paediatric Liver Centre, was comprised of 99 children with a biopsy-proven diagnosis of NAFLD with no co-existent liver diseases. Clinical data was recorded, and blood samples were processed for extraction and preparation of DNA for the sequencing. In addition, the paediatric cohort plasma levels of BA were evaluated along with FGF19 plasma levels. The changes in mRNA expression of genes belonging to the BA synthesis and transport were investigated in primary human hepatocytes and two cell lines, HepG2 and IHH after 24h treatment with palmitic acid (PA) and oleic acid (OA).
The paediatric cohort included 99 patients (13 years old; m = 62) with a diagnosis of NAFLD. Seventy-two patients (72.7%) had a steatosis grade greater than 2 and 66 patients (66.7%) had fibrosis stage greater than 2, indicating significant disease. The presence of previously identified variants was confirmed for the cohort, alongside with rare variants found in genes involved in pathways central to the pathogenesis of NAFLD. In analysis of genes involved in the bile acid pathway, both common and rare variants in NR1H4, NR0B2, HNF4A and SLC10A2 have been identified among children with severe NAFLD and steatofibrosis. Plasma levels of CA and CDCA were increased in 99 (100%) and 74 (74.8%) patients respectively, as well as unconjugated levels (n=77, 77.8%). Reduced FGF19 levels were found in 72 (72.7%) patients confirming results from previous studies. The treatment with PA and OA induced changes in the gene expression in all three cell models treated with FA, showing an overall disruption of BA metabolism and transport as a consequence of lipid accumulation and toxicity.
Overall, this study has demonstrated the presence of common and rare variants in genes encoding proteins involved in BA homeostasis, although no single statistically significant association with NAFLD histology staging or grading was found. An interlink between BA and lipid accumulation has been confirmed in vitro.
Further studies in larger cohorts are needed to define the association of variants in genes involved with BA metabolism and transport with NAFLD.
| Date of Award | 1 Feb 2023 |
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| Original language | English |
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| Supervisor | Emer Fitzpatrick (Supervisor) & Richard Thompson (Supervisor) |