Whole Exome Sequencing in Children With Autoimmune Hepatitis Identified Mutations in Genes Involved in the mTORC1 Signaling Pathway

Abstract

Background and aims: Autoimmune hepatitis (AIH), a severe immune-mediated liver disease resulting from defective immune tolerance, was thought to be caused by monogenic predisposition with possible external triggers. The development of AIH in monogenic primary immune deficiencies prompted us to search for causative genetic defects in AIH.

Methods: Twenty-two children with AIH were included for whole exome sequencing analysis. Data were analyzed with an in-house software, combined with in silico tools and confirmed by Sanger sequencing. Mechanistic target of rapamycin (mTOR) pathway activation was assessed by phosphoS6-RP expression by fluorescence activated cells sorting.

Results: In six children with type 1 or type 2 AIH, seven rare missense candidate variants with damaging predictive scores were identified in five genes involved in the regulation of the mTOR complex 1 (mTORC1) signaling pathway (RRAGC, LAMTOR3, MTOR, TSC2, and PRKAG1). Excess of phosphorylated-S6 ribosomal protein expression both ex vivo by monocytes and in vitro by activated T cells demonstrated an abnormal activation of mTORC1 in five out of six tested patients. Persistent mTORC1 activation in starved activated T cells could be abolished by mTOR inhibitor.

Conclusion: These data showed that pediatric patients with AIH-1 or AIH-2, especially when burdened with poly-autoimmunity, may carry mutations in key partners of the mTORC1 signaling pathway. Moreover, even without identified genetic defect, we observed a deregulation of the mTOR pathway in five out of six tested patients. These results shed new light on the pathogenesis of AIH. Moreover, they suggested that the patients could benefit from specific targeted agents such as mTOR inhibitors.

Keywords: Autoimmune Hepatitis; Genetics Pediatric Auto-Immunity Rapamycin; mTOR Pathway.

Graphical abstract

Figure 1

Segregation analysis in six families with variants identified in genes involved in the mTORC1 pathway. Circles: females; squares: males; vertical bar: carriers of the variants. The arrow points at the proband. Nomenclature is given for each canonical transcript. LAMTOR, late endosomal/lysosomal adaptor MAPK and MTOR activator; MTOR, mechanistic target of rapamycin; NA, not assessed (because no available DNA); PRKAG1, protein kinase AMP-activated non-catalytic subunit gamma 1; RRAGC, Ras-related GTP binding C; TSC, tuberous sclerosis complex; WT, wild type.

Figure 2

Phosphorylation of S6 ribosomal protein expression on patients’ peripheral blood mononuclear cells (PBMCs). FACS analysis of phosphorylation of S6 ribosomal protein (PS6RP) ex vivo on thawed PBMC has been performed for two AIH-m(+) patients: patient B (PRKAG1-compound heterozygous p.V65L and p.V317M) and patient C (LAMTOR3-p.Y74C) on 2 independent experiments at two different times on different samples collected at more than 2 years apart. Three AIH-m(−) patients (patients 2, 10, and 15) have been tested in parallel in one of these two experiments. Eleven untreated ALPS-FAS patients (ALPS-FAS standard of care is rapamycin (mTOR inhibitor) treatment) have been used as positive controls for mTOR pathway overactivation. At least 2 healthy controls (HC) and 2 ALPS-FAS patients were analyzed in each experiment. The relative expression of PS6RP was calculated in the subpopulations of PBMCs by analyzing the CD3+/CD4+ cells (CD4 lymphocytes); the CD3+/CD4 cells (CD8 lymphocytes); the CD3-/CD19+ cells (B lymphocytes); and the CD3-/CD14+ cells (Monocytes) for each individual as compared to 100% corresponding to the mean of the mean of fluorescence intensity (MFI) obtained for the 2 healthy controls included in each experiment. Statistical significance was calculated using GraphPad Prism v.9.1.0. Ordinary one-way ANOVA test was used to determine relevant overexpression of PS6RP in each group, and significant results are indicated: ∗P < .05; ∗∗P < .01. AIH, autoimmune hepatitis; ALP-FAS, autoimmune lymphoproliferative syndrome carrying FAS mutations; NS, not significant.

Figure 3

Phosphorylation of S6 ribosomal protein expression in vitro on patients’ activated T cells. FACS analysis of phosphorylated S6 ribosomal protein (PS6RP) on activated T cells for the same AIH patients analyzed in Figure 2. The two AIH-m(+) (patients B and C) and the three AIH-m(−) patients (patients 2, 10, and 15) have been tested in parallel, and 2 healthy controls were used in the experiment. The median of fluorescence intensity (MFI) for PS6RP staining is depicted for each individual after 6 days of culture either in complete medium (A); after amino acids (AA) starvation for 1 hour (B); or after starvation then TCR stimulation by OKT3 (0.5 μg/mL) (C). The mTOR pathway activation specificity is assessed by using rapamycin as mTOR inhibitor at 50 nM during the stimulation by OKT3 (D). Ctrls, controls; MFI, median of fluorescence intensity.