CHD3 helicase domain mutations cause a neurodevelopmental syndrome with macrocephaly and impaired speech and language

Abstract

Chromatin remodeling is of crucial importance during brain development. Pathogenic alterations of several chromatin remodeling ATPases have been implicated in neurodevelopmental disorders. We describe an index case with a de novo missense mutation in CHD3, identified during whole genome sequencing of a cohort of children with rare speech disorders. To gain a comprehensive view of features associated with disruption of this gene, we use a genotype-driven approach, collecting and characterizing 35 individuals with de novo CHD3 mutations and overlapping phenotypes. Most mutations cluster within the ATPase/helicase domain of the encoded protein. Modeling their impact on the three-dimensional structure demonstrates disturbance of critical binding and interaction motifs. Experimental assays with six of the identified mutations show that a subset directly affects ATPase activity, and all but one yield alterations in chromatin remodeling. We implicate de novo CHD3 mutations in a syndrome characterized by intellectual disability, macrocephaly, and impaired speech and language.

Fig. 1

Photographs of affected individuals. Facial photographs showing dysmorphisms in 18 individuals with de novo CHD3 mutations. The majority of individuals have macrocephaly with a prominent or bossing forehead, individual 5 has microcephaly. Hypertelorism or telecanthus is common, often accompanied by narrow palpebral fissures, deep-set eyes, peri-orbital fullness, and/or epicanthal folds. The combination of macrocephaly and deep-set eyes leads to a more prominent supra-orbital ridge. Some individuals show midface hypoplasia. Many individuals have low-set ears that can be posteriorly rotated, and sometimes simple with thick helices. A broad nasal base, prominent nose, a bifid nasal tip, and characteristic pointy chin is also frequently seen, as well as laterally sparse eyebrows

Fig. 2

Schematic view of CHD3 transcript and protein with de novo mutations. a Schematic view of CHD3 exons (transcript 1, NM_001005273.2) with the splice site mutation c.4073-2A>G shown that most likely leads to skipping of exon 27 (22 amino acids), while preserving the reading frame. Exon 27 is part of the beginning of the second DUF domain (DUF 1086). Colors of the domains in a match with colors of domains in b and c. Five different types of domains are specified: plant homeodomains (PHD), chromodomains (Chromo), a Helicase domain consisting of two parts (Helicase ATP-binding and Helicase C-terminal), domains of unknown function (DUF), and a C-terminal 2 domain. b Schematic view of linear CHD3 protein (transcript 1, NM_001005273.2) with all mutations, except for the splice site mutation that is shown in a, found in our cohort. Almost all missense mutations cluster in or around the Helicase domain of the CHD3 protein. c Overview of one of the two CHD3-models used in this study, based on the 3MWY protein structure. This figure shows the different domains of the protein in their three-dimensional conformation: chromo domain 1 494–595 (magenta), chromo domain 2 631–673 (red), helicase ATP binding domain (yellow), helicase C-terminal domain (green), ATP binding residues 761–768 (cyan). ATP is orange, and gray residues do not belong to an indicated domain. Colors of the domains in c match with colors of domains in a and b. d The same structure as c, but in this figure the positions of the mutated residues are indicated in red, the sidechains of these residues are shown as red balls. The ATP molecule is shown in yellow. This figure illustrates the clustering of mutations on specific sites within the Helicase ATP-binding domain and Helicase C-terminal domain. A more detailed analysis of the different missense mutations in our cohort can be found in Supplementary Note 1

Fig. 3

ATPase assays. Radiometric ATPase assays were performed to assess the activity of the mutant proteins relative to wild-type, in the presence of recombinant nucleosomes (blue), dsDNA (green), or in the absence of DNA substrates as a control (Supplementary Fig. 4). Released phosphate was separated from unhydrolyzed ATP by thin layer chromatography, and detected by exposure to a phosphorimager. The experimental values (percentage hydrolyzed ATP) for the different mutant conditions were normalized to values for the wild-type condition within the experiment, to derive a normalized ATPase activity. The experimental data are presented as means ± standard deviation, individual data points are shown as red triangles. Three independent experiments from two individual purifications (wild-type, p.Leu915Phe, p.Arg1121Pro, p.Asn1159Lys, p.Arg1172Gln, and p.Arg1187Pro) (N = 6) or one purification (p.Trp1158Arg) (N = 3) were performed. Raw values from the individual experiments can be found in Supplementary Data 2. Asterisk (*) indicates significant difference for mutant values compared to wild-type values (unpaired t-test, P < 0.05) within the same substrate condition

Fig. 4

Restriction enzyme accessibility assay. a Restriction enzyme accessibility analysis of CHD3 wild-type and mutant proteins. 3.125, 6.25, or 12.5 nM of CHD3 proteins were incubated with 347 bp mono-nucleosomes. Digested fragments were analyzed by native polyacrylamide gel. b Quantitative analysis of restriction enzyme accessibility. Three individual experiments from two individual purifications (wild-type, p.Leu915Phe, p.Arg1121Pro, p.Asn1159Lys, p.Arg1172Gln, and p.Arg1187Pro) (N = 6) or one purification (p.Trp1158Arg) (N = 3) were conducted. The experimental data are presented as means with standard deviations