De novo coding variants in the AGO1 gene cause a neurodevelopmental disorder with intellectual disability

Abstract

Background: High-impact pathogenic variants in more than a thousand genes are involved in Mendelian forms of neurodevelopmental disorders (NDD).

Methods: This study describes the molecular and clinical characterisation of 28 probands with NDD harbouring heterozygous AGO1 coding variants, occurring de novo for all those whose transmission could have been verified (26/28).

Results: A total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Recurrently identified variants were present in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del). AGO1 encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linker domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behaviour and additional behavioural manifestations.

Conclusion: Our study establishes that de novo coding variants in AGO1 are involved in a novel monogenic form of NDD, highly similar to the recently reported AGO2-related NDD.

Keywords: genetics; medical; microRNA; missense; mutation; nervous system diseases.

Figure 1:. Facial characteristics of individuals with AGO1 variants.

(A) Front faces, (B) profile faces from families who consented for photographs publication (C) Face2Gene Facial analysis using Face2Gene Research application (FDNA Inc. Boston, MA) of unrelated individuals with AGO1-associated disorder (n = 14) compared to controls matched (n= 14) for sex, age, and ethnicity.

Figure 2:. Schematic representation of AGO1 protein with its functional domains showing the locations of the coding variants identified in individuals with ID

(A) AGO1 protein (NP_036331.1) with its functional domains: N-terminal domain (34–164), Linker 1 (L1) domain (173–225), PAZ domain (226–368), Liker 2 (372–418), Mid domain (427–508), and PIWI domain (515–816). PAZ and PIWI domains have motifs of interaction with RNA guide and PIWI domain has motif of RNA blocked access to the active site. The arrows show de novo variants identified in individuals from this cohort or previously reported (P: previously reported in literature); T: found in monozygotic twins, and in underlined those that are recurrent. (B) Protein structure of AGO1 colored by protein domain and with the sites of variants indicated by spheres. The linker domains, designated L1 and L2, are separated in sequence by the PAZ domain, but intertwine in 3D, forming common interfaces between the N-terminal, PAZ, and PIWI domains (C) Many of the variants of interest are within the first linker domain. This domain forms a narrow beta sheet with three strands. Variants occur in the middle of these strands at the closest point between domain L1 and the guide RNA backbone, and towards the end of L1 and near the PAZ domain interface (D) D216 is within L1 and makes specific contacts with R712 in the PIWI domain. R712 also interacts directly with the guide RNA.

Figure 3:. Simulations reveal changes in domain orientation associated with de novo missense AGO1 variants

We used MD simulations to assess how the native structure of AGO1 would respond to the introduction of a subset of identified genomic variants. (A) Variability of each amino acid was quantified using RMSF after aligning each trajectory to the initial WT conformation of the PIWI domain and averaging across replicates. Domains are colored as in Figure 1 and each variant colored according to the domain it is within. (B-E) We monitored selected distances and angles as a simple way to assess conformational changes between the (B) linker and PIWI domains, (C) linker and MID domains, (D) the orientation of the PAZ domain, and (E) the openness of the RNA binding region. (F) We show the free energy landscape across molecular dynamics (MD) simulations as a color gradient from high-energy to low energy. Above, we show one-dimensional PC samplings as a combined violin and boxplot. The left- and right-hand panels summarize all data from the WT and from our novel variants, respectively. Selected variant’s PC1 sampling is shown above the panel, and all variant’s (that underwent MD) PC2 sampling to the side of the panel. Four regions of low energy are indicated by letters A-D. Representative images of AGO1 structure from the simulations taken from these points of low energy are shown below and labeled by the corresponding letters with the WT shown for comparison. To summarize one aspect of the difference between these four regions, we show the angle between the centers of mass of the MID, PIWI, and PAZ domains.