De Novo Variants in CNOT1, a Central Component of the CCR4-NOT Complex Involved in Gene Expression and RNA and Protein Stability, Cause Neurodevelopmental Delay

Abstract

CNOT1 is a member of the CCR4-NOT complex, which is a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. We report on 39 individuals with heterozygous de novo CNOT1 variants, including missense, splice site, and nonsense variants, who present with a clinical spectrum of intellectual disability, motor delay, speech delay, seizures, hypotonia, and behavioral problems. To link CNOT1 dysfunction to the neurodevelopmental phenotype observed, we generated variant-specific Drosophila models, which showed learning and memory defects upon CNOT1 knockdown. Introduction of human wild-type CNOT1 was able to rescue this phenotype, whereas mutants could not or only partially, supporting our hypothesis that CNOT1 impairment results in neurodevelopmental delay. Furthermore, the genetic interaction with autism-spectrum genes, such as ASH1L, DYRK1A, MED13, and SHANK3, was impaired in our Drosophila models. Molecular characterization of CNOT1 variants revealed normal CNOT1 expression levels, with both mutant and wild-type alleles expressed at similar levels. Analysis of protein-protein interactions with other members indicated that the CCR4-NOT complex remained intact. An integrated omics approach of patient-derived genomics and transcriptomics data suggested only minimal effects on endonucleolytic nonsense-mediated mRNA decay components, suggesting that de novo CNOT1 variants are likely haploinsufficient hypomorph or neomorph, rather than dominant negative. In summary, we provide strong evidence that de novo CNOT1 variants cause neurodevelopmental delay with a wide range of additional co-morbidities. Whereas the underlying pathophysiological mechanism warrants further analysis, our data demonstrate an essential and central role of the CCR4-NOT complex in human brain development.

Keywords: CCR4-NOT complex; CNOT1; Drosophila; de novo mutations; developmental delay; exome sequencing; genomics; intellectual disability; neurodevelopment.

Figure 1

Schematic Representation of CNOT1 and Facial Photos of Individuals Carrying a (Likely) Pathogenic CNOT1 Variant (A) Graphical representation of the linear protein structure of CNOT1 with the functional domains indicated by colored boxes (blue, HEAT; pink, TTP; green, CAF; purple, DUF3819; orange, Not1). Variants observed in this study are indicated by circles, in which the size of the circle corresponds to the number of recurrences and the color of the circle to the variant type. Note that the variant in individual 16, affecting the last base of exon 34, creates a splice site (indicated by an asterisk). The variants in individual 28, who was shown to have two de novo missense variants, are outlined in bold. (B) Facial photos of individuals with a (likely) pathogenic CNOT1 variant, categorized by the type of variant observed. The colored lines underneath the individuals with missense variants correspond to the functional domains indicated in (A). Although shared facial features can be observed between individuals in the cohort, e.g., straight eyebrows (individuals 2, 5, 6, 12, 18, 19, 27, 33), hypertelorism (individuals 18, 25, 29, 33, 39), epicanthus inversus (individuals 8, 18, 30, 33), low nasal bridge (individuals 2, 14, 18, 29, 39), large ears (individuals 15, 18, 25, 39), thickened helix (individuals 10, 14, 18, 25, 29, 35), and protruding helix (individuals 5, 7, 10, 14, 33, 39), a syndromic facial phenotype is lacking. Also, no evident facial resemblance is observed when comparing between individuals based on variant type. Clinical photos of hands and feet are provided in Figure S2 and additional clinical information for all individuals in this study is provided in Table S2 with a summary in Table 1.

Figure 2

Disease-Associated Variants of Human CNOT1 Induce Memory Deficits in Drosophila Model (A) Depletion of Drosophila Not1 function in all neurons by inducible RNAi knockdown in adults only by using two different UAS-RNAi lines – Not1^GD1 and Not1^KK with elav^GS-Gal4 and exposure to RU (GeneSwitch²⁴), or specifically in the mushroom body with OK107-Gal4, produces memory deficits. (B and C) RU-induced expression of disease-associated human CNOT1 variant cDNAs (UAS-CNOT1^{p.X or WT}) in a pan-neuronal Not1^GD1–RNAi knockdown background (using elav^GS-Gal4) induces memory deficits both during adult (B) and larval (C) stages. One-way ANOVA, ^∗∗p < 0.01, ^∗∗∗p < 0.0001.

Figure 3

Genetic Interactions between Not1 and Known ID/ASD Genes and Rescue of Neuronal Not1 Knockdown-Induced Neurodevelopmental Defects with Transcriptional Modifiers (A) Neuronal RNAi knockdown of known ID/ASD genes (ASH1, DYRK1A, MED13, SHANK3; in flies ash1, mnb, skd, Prosap, respectively) exacerbates Not1 knockdown-induced memory defects in adults. (B) Overexpression of MED13 or knockdown of PRC2 components E(z) (EZH2 in humans) and esc (EED in humans) ameliorates Not1 knockdown-induced memory deficits in adults. One-way ANOVA, ^∗∗p < 0.01, ^∗∗∗p < 0.0001.