Biallelic Mutations in NBAS Cause Recurrent Acute Liver Failure with Onset in Infancy

Abstract

Acute liver failure (ALF) in infancy and childhood is a life-threatening emergency. Few conditions are known to cause recurrent acute liver failure (RALF), and in about 50% of cases, the underlying molecular cause remains unresolved. Exome sequencing in five unrelated individuals with fever-dependent RALF revealed biallelic mutations in NBAS. Subsequent Sanger sequencing of NBAS in 15 additional unrelated individuals with RALF or ALF identified compound heterozygous mutations in an additional six individuals from five families. Immunoblot analysis of mutant fibroblasts showed reduced protein levels of NBAS and its proposed interaction partner p31, both involved in retrograde transport between endoplasmic reticulum and Golgi. We recommend NBAS analysis in individuals with acute infantile liver failure, especially if triggered by fever.

Figure 1

Pedigrees of Investigated Families Pedigrees of ten families affected by RALF and mutations in NBAS. Mutation status of affected (closed symbols) and healthy (open symbols) family members.

Figure 2

NBAS Structure and Conservation of Identified Mutations Gene structure of NBAS with known protein domains of the gene product and localization and conservation of amino acid residues affected by mutations. Mutation c.5741G>A (p.Arg1914His) in exon 45 in the C-terminal domain of unknown function is associated with SOPH syndrome. Amino acids 90–371 form a quinoprotein aminedehydrogenase, beta chain like domain (IPR011044) and amino acids 725–1,376 form a secretory pathway sec39 domain (IPR013244). Intronic regions are not drawn to scale.

Figure 3

Quantification of NBAS and p31 Protein Levels Mutant and control fibroblast cell lines were cultivated at 37°C. 10 μg protein of collected cells were separated on a 4%–12% acrylamide gradient gel, transferred to a PVDF membrane, and immunodecorated with antibodies against NBAS, p31, and β-actin. The antibody for NBAS was detecting a single protein band at approximately 270 kDa corresponding to the predicted molecular weight of NBAS. β-actin was used as a loading control. The quantified protein levels are based on three independent experiments and expressed as percentages of a control cell line, corrected for β-actin. Five of the eight investigated subject cell lines are shown. NBAS and p31 protein levels were severely reduced in fibroblast cell lines of RALF-affected individuals to 21% and 33% of control subjects, respectively. Error bar indicates 1 SD. Protein amount in control 1 was set as 100%.

Figure 4

Increased Expression of ER Stress Response Genes Quartiles (boxes) and 1.5 times the interquartile range (whiskers) of the fold change of normalized RNA-seq read counts from fibroblasts are displayed. Three individuals with pathogenic variants in NBAS (F1:II.1, F2:II.1, and F5:II.2) are compared against the median per gene over 12 control samples. We obtained interaction partners of the NBAS protein from the BioGrid database (v.3.3). The genes associated with the corresponding gene ontology terms were downloaded from UniProt (accessed March 2015). For every box, we computed a two-sided Wilcoxon test whether it is symmetric about 1. Single data points are shown for boxes with less than 50 genes. Gene ontology (GO) terms: GO 0034976, response to endoplasmic reticulum stress; GO 0001889, liver development; GO 0006259, DNA metabolic process; GO 0006119, oxidative phosphorylation. ns, p > 0.1.