Reinitiation of mRNA translation in a patient with X-linked infantile spasms with a protein-truncating variant in ARX

Abstract

Mutations in the Aristaless-related homeobox gene (ARX) lead to a range of X-linked intellectual disability phenotypes, with truncating variants generally resulting in severe X-linked lissencephaly with ambiguous genitalia (XLAG), and polyalanine expansions and missense variants resulting in infantile spasms. We report two male patients with early-onset infantile spasms in whom a novel c.34G4T (p.(E12*)) variant was identified in the ARX gene. A similar variant c.81C4G (p.(Y27*)), has previously been described in two affected cousins with early-onset infantile spasms, leading to reinitiation of ARX mRNA translation resulting in an N-terminal truncated protein. We show that the novel c.34G4T (p.(E12*)) variant also reinitiated mRNA translation at the next AUG codon (c.121-123 (p.M41)), producing the same N-terminally truncated protein. The production of both of these truncated proteins was demonstrated to be at markedly reduced levels using in vitro cell assays. Using luciferase reporter assays, we demonstrate that transcriptional repression capacity of ARX was diminished by both the loss of the N-terminal corepressor octapeptide domain, as a consequence of truncation, and the marked reduction in mutant protein expression. Our study indicates that premature termination mutations very early in ARX lead to reinitiation of translation to produce N-terminally truncated protein at markedly reduced levels of expression. We conclude that even low levels of N-terminally truncated ARX is sufficient to improve the patient's phenotype compared with the severe phenotype of XLAG that includes malformations of the brain and genitalia normally seen in complete loss-of-function mutations in ARX.

Regulating transcriptional activity by phosphorylation: A new mechanism for the ARX homeodomain transcription factor

“…To determine the impact of serine phosphorylation on the function of the ARX protein, phospho-null substitutions were generated in which the serine residues identifed to be phosphorylated were replaced with alanines. Using a dual-luciferase reporter assay described in our previous work [ 20 , 21 ] we measured the impact of these mutations on the interaction with an orthologous Arx-binding site in the enhancer region of Lmo1 [ 22 ] inserted upstream of an SV40 promoter driving luciferase expression. Using this assay, we demonstrate that phosphorylation of serine residues identified in this study does not affect ARX transcriptional activity in HEK293T cells ( S6 Fig ).…”

Nonsense pathogenic variants in exon 1 of PHOX2B lead to translational reinitiation in congenital central hypoventilation syndrome

“…Generally, patients with PT variants leading to total loss-of-function (LOF) have more severe XLAG, whereas patients with polyalanine expansions generally have ISSX or a form of intellectual disability [Kato et al, 2004]. Recently, cousins with c.81C>G (p.Tyr27*) and two brothers with c.34G>T (p.Glu12*) in ARX were reported with ISSX [Fullston et al, 2010; Moey et al, 2016]; none had the clinical features of XLAG as would be expected from a total loss of functional protein. Both, the p.Tyr27* and the p.Glu12* pathogenic variants, reinitiated translation at p.M41 suggesting that an N-terminally truncated ARX partially rescued the more severe brain malformation phenotype.…”

Genetics and mechanisms leading to human cortical malformations

“…Moey and col. [129] and Fullston and col. [121] have shown that certain missense variants early on in the ARX gene induce a premature termination of the protein (amino acid residue 12 or 27) in patients with early-onset infantile spasms. In these cases they show that a reinitiation of translation is possible (e.g.…”