Molecular and cellular basis of hyperassembly and protein aggregation driven by a rare pathogenic mutation in DDX3X

Abstract

Summary Current studies estimate that 1–3% of females with unexplained intellectual disability (ID) present de novo splice site, nonsense, frameshift, or missense mutations in the DDX3X protein (DEAD-Box Helicase 3 X-Linked). However, the cellular and molecular mechanisms by which DDX3X mutations impair brain development are not fully comprehended. Here, we show that the ID-linked missense mutation L556S renders DDX3X prone to aggregation. By using a combination of biophysical assays and imaging approaches, we demonstrate that this mutant assembles solid-like condensates and amyloid-like fibrils. Although we observed greatly reduced expression of the mutant allele in a patient who exhibits skewed X inactivation, this appears to be enough to sequestrate healthy proteins into solid-like ectopic granules, compromising cell function. Therefore, our data suggest ID-linked DDX3X L556S mutation as a disorder arising from protein misfolding and aggregation.

Saturation genome editing of DDX3X clarifies pathogenicity of germline and somatic variation

“…al. proposed that the L556S and R376C de novo variants observed in female patients render DDX3X prone to protein aggregation 22 .…”

“…Alternatively, we may lack power to discern such effects, as most phenotyping studies remain small, employ disparate neuropsychometric tests and not all patients are uniformly assessed 11,20,35,36 . It is also notable that DDX3X escapes X-inactivation to a variable degree between individuals [56][57][58] , and a high incidence of severe skewing of X-inactivation has been observed in patients with DDX3X-related neurodevelopmental disorder 11,22 . Such factors will affect variant expressivity and may obfuscate identification of genotype-phenotype relationships.…”

“…al. proposed that the L556S and R376C de novo variants observed in female patients render DDX3X prone to protein aggregation 22 . Both variants are functionally abnormal depleting variants in our assay.…”

“…Several studies have functionally characterised DDX3X pathogenic variants observed in individuals with NDDs, using a variety of model systems 11,15,[20][21][22] . The largest of these involved quantifying the impact of eight variants on helicase activity in vitro 20 .…”

“…Functional experiments in zebrafish have suggested one missense variant seen in an affected male might result in partial loss-of-function 21 . In vitro functional characterisation of one pathogenic missense variant seen in an affected female suggests that it might operate via a toxic protein aggregation mechanism 22 .…”