Hotspots of missense mutation identify neurodevelopmental disorder genes and functional domains

Abstract

Although de novo missense mutations have been predicted to account for more cases of autism than gene-truncating mutations, most research has focused on the latter. We identified the properties of de novo missense mutations in patients with neurodevelopmental disorders (NDDs) and highlight 35 genes with excess missense mutations. Additionally, 40 amino acid sites were recurrently mutated in 36 genes, and targeted sequencing of 20 sites in 17,688 patients with NDD identified 21 new patients with identical missense mutations. One recurrent site substitution (p.A636T) occurs in a glutamate receptor subunit, GRIA1. This same amino acid substitution in the homologous but distinct mouse glutamate receptor subunit Grid2 is associated with Lurcher ataxia. Phenotypic follow-up in five individuals with GRIA1 mutations shows evidence of specific learning disabilities and autism. Overall, we find significant clustering of de novo mutations in 200 genes, highlighting specific functional domains and synaptic candidate genes important in NDD pathology.

Figure 1. Burden and recurrence of de novo missense mutations

Bars for cases and controls represent observed data and error bars indicate the 95% confidence interval (CI) for the observed proportions (Clopper-Pearson method). Box-and-whisker plots for downsampled cases represent the distribution of one million permutations. Boxes show interquartile range (IQR) with lines at the median and whiskers are 1.5 times the IQR. Asterisks indicate p < 0.05. a) 4,301 out of 8,477 cases (50.7%) and 1,042 out of 2,178 controls (47.8%) have one or more de novo missense mutations (denovo-db v.0.9) that are rare in the general population (MAF < 0.1% in ESP). The fraction of individuals with one or more de novo missense mutation is significantly higher in cases (p = 0.016, OR = 1.12, two-sided Fisher’s exact test) even after downsampling (empirical p = 9.22×10⁻⁴, OR = 1.12 [1.06–1.19]). b) The number of genes with two or more mutations in downsampled cases is significantly greater than controls (empirical p = 0.011, OR = 1.26 [1.10–1.42]), as is the number of genes with three or more mutations (empirical p = 3.1×10⁻⁵, OR = 3.13 [2.22–4.03]).

Figure 2. Severity of de novo missense mutations

a) De novo missense mutations are more likely to be deleterious in cases (n = 5,807 mutations) versus controls (n = 1,475 mutations) and the positive likelihood ratio increases as severity increases (as measured by Combined Annotation Dependent Depletion (CADD) score). b) The distribution of CADD scores skews significantly as the number of de novo missense mutations per gene in cases increases (p = 5.87×10⁻²⁹, one-way ANOVA) indicating an enrichment for genes with pathogenic mutations. Boxes show IQR with notches representing the 95% CI of the median; whiskers are 1.5 times the IQR. Circles are outliers.

Figure 3. Recurrent mutations fall in or near functional domains

Published mutations in NDD patients are above the protein and new mutations identified by targeted sequencing are below the protein. De novo mutations (lightning bolt) and paternally inherited mutations (blue) are indicated. Inheritance is unknown for the remaining mutations. Protein domains are from UniProt. a) PACS1, NP_060496.2. b) ALG13, NP_060936.1. c) SMAD4, NP_005350.1. d) SATB2, NP_001165988.1.

Figure 4. Functional effect of recurrent GRIA1 missense mutations

a) Linear representation of annotated domains in the protein GRIA1, a.k.a. GluA1 (NP_001244950.1) as defined in UniProt. b) A recurrent mutation observed only in NDD cases (n = 6 patients) falls within a highly conserved M3 transmembrane domain, important in channel gating. The alanine mutated in these patients (red) is homologous to the one that causes severe ataxia in Lurcher mice in the delta-2 subunit of this receptor (GRID2). c–e) Example current traces from a 1.8s voltage-ramp from −100mV to +80mV for c) WT, d) A636T, and e) heteromeric WT/A636T transfected HEK cells. The three current traces per panel correspond to voltage-ramp currents in the presence of normal extracellular solution (NaCl), extracellular solution supplemented with 50 uM NBQX (NBQX), and the isolated GluA1 dependent current determined by subtracting the NBQX current from the NaCl current (NaCl – NBQX). f) Average leak current at −60mV. The GluA1-mediated current (NaCl – NBQX) was determined at −60mV and averaged across cells (n = 5 (WT), 7 (A636T), and 5 (heteromeric); p = 0.024, F = 4.91, 2 degrees of freedom, one-way ANOVA). Data are mean +/− S.E.M.

Figure 5. Proteins with excessive clustering of missense mutations in NDD cases

The pattern of de novo missense mutations in cases with NDDs is contrasted with rare missense variants from the 1000 Genomes Project (1KG) and private missense mutations from the Exome Aggregation Consortium (ExAC) excluding neuropsychiatric cases. Missense mutations are colored by severity (CADD heatmap) and recurrent de novo mutations at a specific amino acid position are indicated (triangle). Significance of clustering was calculated based on comparison to ExAC using CLUMP. a) GRIN1 (NP_001172019.1) shows greater missense mutation clustering in NDD patients (CLUMP = 1.68, p = 0.013) with region-specific significance corresponding to the transmembrane domains (amino acids 550–845; Fisher’s exact test p = 5.6×10⁻⁸). b) Similarly, missense mutations cluster for GRIN2B (NP_000825.2; CLUMP = 1.34, p = 0.003) in particular between the second and fourth transmembrane domains (amino acids 600–820; p = 2.0×10⁻⁹). c) KCNK3 (NP_002237.1) patient missense mutations cluster (CLUMP = 0.54, p = 0.036) near the first transmembrane domain (amino acids 120–140, p = 9.4×10⁻⁵, OR = Inf). The average per-base rate of ExAC samples with ≥10× coverage across the exon harboring mutations in cases was 79.1%. d) KCNQ2 (NP_742105.1) shows several missense mutation hotspots (CLUMP = 0.36, p < 1×10⁻³) corresponding to the pore and voltage sensor of the channel (amino acids 200–350, p = 2.0×10⁻¹⁴). e) Finally, patients show more severe CTCF (NP_006556.1) missense mutations that cluster (CLUMP = 1.0, p = 0.007) at two locations between the fourth and seventh C2H2 zinc finger motifs (amino acids 350–490, p = 9.1×10⁻⁸).