Genotype–phenotype correlations and novel molecular insights into the DHX30-associated neurodevelopmental disorders

Abstract

Background We aimed to define the clinical and variant spectrum and to provide novel molecular insights into the DHX30-associated neurodevelopmental disorder. Methods Clinical and genetic data from affected individuals were collected through Facebook-based family support group, GeneMatcher, and our network of collaborators. We investigated the impact of novel missense variants with respect to ATPase and helicase activity, stress granule (SG) formation, global translation, and their effect on embryonic development in zebrafish. SG formation was additionally analyzed in CRISPR/Cas9-mediated DHX30-deficient HEK293T and zebrafish models, along with in vivo behavioral assays. Results We identified 25 previously unreported individuals, ten of whom carry novel variants, two of which are recurrent, and provide evidence of gonadal mosaicism in one family. All 19 individuals harboring heterozygous missense variants within helicase core motifs (HCMs) have global developmental delay, intellectual disability, severe speech impairment, and gait abnormalities. These variants impair the ATPase and helicase activity of DHX30, trigger SG formation, interfere with global translation, and cause developmental defects in a zebrafish model. Notably, 4 individuals harboring heterozygous variants resulting either in haploinsufficiency or truncated proteins presented with a milder clinical course, similar to an individual harboring a de novo mosaic HCM missense variant. Functionally, we established DHX30 as an ATP-dependent RNA helicase and as an evolutionary conserved factor in SG assembly. Based on the clinical course, the variant location, and type we establish two distinct clinical subtypes. DHX30 loss-of-function variants cause a milder phenotype whereas a severe phenotype is caused by HCM missense variants that, in addition to the loss of ATPase and helicase activity, lead to a detrimental gain-of-function with respect to SG formation. Behavioral characterization of dhx30-deficient zebrafish revealed altered sleep-wake activity and social interaction, partially resembling the human phenotype. Conclusions Our study highlights the usefulness of social media to define novel Mendelian disorders and exemplifies how functional analyses accompanied by clinical and genetic findings can define clinically distinct subtypes for ultra-rare disorders. Such approaches require close interdisciplinary collaboration between families/legal representatives of the affected individuals, clinicians, molecular genetics diagnostic laboratories, and research laboratories.

Genetic subtypes, allelic effects, and convergent neurodevelopmental mechanisms

“…The authors further used cell culture and zebrafish models to demonstrate that endogenous DHX30 leads to stress granule aggregation under heat stress conditions, while DHX30 deletion contributes to a reduction in the number of stress granules in these models. In addition, behavioral assessment of DHX30 knockouts in zebrafish showed altered sleep-wake behavior, which is consistent with patterns of sleep disturbance observed in several individuals carrying DHX30 variants [ 1 ].…”

“…For instance, loss-of-function variants, such as frameshift and nonsense mutations or whole gene deletions, only resulted in milder phenotypes, suggesting distinct effects on protein function due to missense variants in specific locations. Although in vitro experiments did not paint a clear picture, overall trends suggested that missense mutations led to a significant loss of ATPase and helicase activity, reduction in global translation, and formation of stress granules [ 1 ].…”

“…Mannucci and colleagues validated the role of DHX30 and its protein variants towards formation of stress granules [ 1 ]. Stress granules are an aggregation of multiple ribonucleoprotein complexes that pause translation in response to cellular stress [ 10 ].…”

“…Using this strategy, several genes have been reported, with each gene causing a subtype of neurodevelopmental disorder. Writing in Genome Medicine , Mannucci and colleagues describe a rare subtype of neurodevelopmental disorder caused by mutations in the RNA helicase encoding DHX30 and characterize location-specific functional effects of different mutations in this gene using molecular assays and behavioral studies in zebrafish [ 1 ]. DHX30 belongs to the Super Family 2 (SF2) of RNA helicases with integral roles in mRNA regulation, transport, storage, and decay—all of which contribute to neuronal differentiation, development, and maturation.…”

“…DHX30 belongs to the Super Family 2 (SF2) of RNA helicases with integral roles in mRNA regulation, transport, storage, and decay—all of which contribute to neuronal differentiation, development, and maturation. DHX30, along with other SF2 members such as DDX3X, DDX6, and DDX59, have been strongly implicated in neurodevelopmental disorders [ 1 ]. Based on the concepts in this study, we highlight general themes uncovered when dissecting genetic heterogeneity as well as overlapping and converging mechanisms of genes contributing to neurodevelopmental disorders.…”