Deep Sequencing Reveals Novel Genetic Variants in Children with Acute Liver Failure and Tissue Evidence of Impaired Energy Metabolism

Abstract

Background & aims: The etiology of acute liver failure (ALF) remains elusive in almost half of affected children. We hypothesized that inherited mitochondrial and fatty acid oxidation disorders were occult etiological factors in patients with idiopathic ALF and impaired energy metabolism.

Methods: Twelve patients with elevated blood molar lactate/pyruvate ratio and indeterminate etiology were selected from a retrospective cohort of 74 subjects with ALF because their fixed and frozen liver samples were available for histological, ultrastructural, molecular and biochemical analysis.

Results: A customized next-generation sequencing panel for 26 genes associated with mitochondrial and fatty acid oxidation defects revealed mutations and sequence variants in five subjects. Variants involved the genes ACAD9, POLG, POLG2, DGUOK, and RRM2B; the latter not previously reported in subjects with ALF. The explanted livers of the patients with heterozygous, truncating insertion mutations in RRM2B showed patchy micro- and macrovesicular steatosis, decreased mitochondrial DNA (mtDNA) content <30% of controls, and reduced respiratory chain complex activity; both patients had good post-transplant outcome. One infant with severe lactic acidosis was found to carry two heterozygous variants in ACAD9, which was associated with isolated complex I deficiency and diffuse hypergranular hepatocytes. The two subjects with heterozygous variants of unknown clinical significance in POLG and DGUOK developed ALF following drug exposure. Their hepatocytes displayed abnormal mitochondria by electron microscopy.

Conclusion: Targeted next generation sequencing and correlation with histological, ultrastructural and functional studies on liver tissue in children with elevated lactate/pyruvate ratio expand the spectrum of genes associated with pediatric ALF.

Fig 1. Liver histology, ultrastructure, mtDNA copy number and respiratory complex activity of subjects with heterozygous mutations in RRM2B.

(A) Liver biopsy following the second episode of ALF in subject ID#11 showed mild steatosis. H&E stain. (B) Mitochondria at that time were normal in size with pale matrix and subtle haphazard arrangement of cristae. (C and D) Random, non-zonal macro- and microvesicular steatosis was prevalent in H&E- stained sections from the explanted livers of both subjects, confirmed with Oil red O in a frozen section (C, insert). Both explanted livers contained unusual necrotic/apoptotic hepatocytes with distorted contours, granular eosinophilic cytoplasm, and fat droplets. (E) Copy numbers of mtDNA in liver tissues from age-matched control subjects without ALF and from subjects #3 and #11 were determined by qPCR for the mitochondrial genes CytB and Cox2, normalized to the nuclear gene B2M. (F) Respiratory chain complex activities were simultaneously determined on frozen samples from explanted livers of ID#3 and ID#11 and from liver tissue of control subjects without ALF. (G)Total protein extracts from liver tissues from ID#11and from two age-matched controls without ALF were analyzed by SDS-PAGE and immunoblotting against p53R2, the 39kDa gene product of RRM2B.

Fig 2. Isolated complex 1 deficiency in an infant with compound heterozygous mutations in ACAD9.

Subject #12 harboring the variant L314P and the mutation E63X in ACAD9 succumbed to ALF and multi-organ failure within the first 24 hours of life. Post-mortem, liver architecture was preserved without significant collapse or inflammation and with minimal steatosis (H&E, A). However, abnormally granular hepatocytes (H&E, B) and mitochondrial hyperplasia detected by immunohistochemistry against mitochondrial antigens (C) suggest a mitochondrial hepatopathy. Respiratory chain complex assay demonstrates reduced activity of complex 1 in frozen liver, compared to liver from controls without ALF (D).

Fig 3. Heterozygous variants in POLG and DGUOK may predispose to drug-induced ALF with a histological phenotype of impaired OXPHOS.

Subject # 2 with Y831C in POLG developed fatal ALF following exposure to dantrolene. (A) Hepatocytes at time of diagnosis revealed mild random steatosis, and prominent oncocytic changes in isolated and clustered hepatocytes (insert). H&E stain. (B). Concurrent ultrastructure study demonstrated numerical excess of mitochondria accompanied by mild pleomorphism (insert). Subject #6 with Q170R in DGUOK developed ALF following exposure to acetaminophen. (C) Liver showed lobular cholestasis, prominent pseudoacinar transformation, hepatocyte swelling, and moderate non-zonal macro- and microvesicular steatosis. (D-F) Concurrent ultrastructure study revealed several populations of mitochondria in many hepatocytes of which three are illustrated. Intermixed with normal mitochondria were numerous mitochondria that displayed “Reye-syndrome”- like changes, including ameboid shape and watery degenerative matrix.