Sequence variants in HECTD1 result in a variable neurodevelopmental disorder

Abstract

Dysregulation of genes encoding the homologous to E6AP C-terminus (HECT) E3 ubiquitin ligases has been linked to cancer and structural birth defects. One member of this family, the HECT-domain-containing protein 1 (HECTD1), mediates developmental pathways, including cell signaling, gene expression, and embryogenesis. Through GeneMatcher, we identified 14 unrelated individuals with 15 different variants in HECTD1 (10 missense, 3 frameshift, 1 nonsense, and 1 splicing variant) with neurodevelopmental disorders (NDDs), including autism, attention-deficit/hyperactivity disorder, and epilepsy. Of these 15 HECTD1 variants, 10 occurred de novo, 3 had unknown inheritance, and 2 were compound heterozygous. While all individuals in this cohort displayed NDDs, no genotype-phenotype correlation was apparent. Conditional knockout of Hectd1 in the neural lineage in mice resulted in microcephaly, severe hippocampal malformations, and complete agenesis of the corpus callosum, supporting a role for Hectd1 in embryonic brain development. Functional studies of select variants in C. elegans revealed dominant effects, including either change-of-function or loss-of-function/haploinsufficient mechanisms, which may explain phenotypic heterogeneity. Significant enrichment of de novo variants in HECTD1 was also shown in an independent cohort of 53,305 published trios with NDDs or congenital heart disease. Thus, our clinical and functional data support a critical requirement of HECTD1 for human brain development.

Keywords: HECTD1; autism; epilepsy; neurodevelopmental disorders; ubiquitin-proteasome system.

Figure 1

Location of HECTD1 variants (A) Variant subtypes and their locations within protein domains. (B) Three-dimensional model showing location of variants near protein domains. (C) Multiple sequence alignment for missense variants obtained through taxonomic filters on blastp with MUSCLE. Variant p.His159Arg is a compound heterozygote with p.Ala2179Vfs^∗35 in individual 14.

Figure 2

Cortical abnormalities in temporal lobe resected for drug-resistant epilepsy in individual 2 with de novo HECTD1 p.Leu237Ser variant (A) Magnetic resonance and (B) computed tomographic brain images in an axial section comparing control (left) to individual 2 (right). (C and D) Hematoxylin and eosin-stained sections demonstrating dysmorphic and bi- to multinucleate balloon neurons with moderate to abundant glassy eosinophilic cytoplasm (arrows). (E–G) Immunostaining highlights architectural disarray, i.e., cortical dyslamination (NeuN) and weak labeling of dysmorphic/balloon neurons with glial fibrillary acidic protein (GFAP) and synaptophysin (SYN).

Figure 3

Sox1-Cre-mediated conditional knockout of Hectd1 results in microcephaly (A and B) Representative wild-type (WT) and conditional KO brains showing gross microcephaly. (C) P30 cKO mice have significantly smaller brain mass compared to WT (n = 8, p < 1e−4). (D) P30 cKO mice have variable but significantly diminished body weight compared to WT (n = 8, p = 0.01). (E) No correlation between brain and body mass as all cKO brains are smaller, but only half of the mice had reduced body mass.

Figure 4

Sox1-Cre-mediated conditional knockout of Hectd1 results in brain morphological phenotypes (A–E) Nissl staining of a P30 wild-type (WT) littermate brain shown in serial sections from rostral to caudal. (F–J) cKO sections are also shown from rostral to caudal. cKO brains appear smaller and exhibit several morphological abnormalities. Complete agenesis of the corpus callosum and ventricular dilation in cKO brain are shown by red arrow and star in (F′), (G′), (H′), and (I′). The CA3 hippocampal layer exhibits dysmorphology with abnormal bending and dispersion (black triple arrow in J′). Data are representative of seven cKO and three WT sectioned brains from 11 litters, respectively. CTX, cerebral cortex: STR, striatum; TH, thalamus; HY, hypothalamus; PAL, pallidum; CC, corpus callosum; HC, hippocampal commissure; MB, midbrain; DG, dentate gyrus; CA1, CA2, and CA3 subfields.

Figure 5

Functional analysis in C. elegans indicates that missense and nonsense variants are damaging to HECD-1 function (A) Crawl speed of homozygous wild-type (WT, purple), null (Δ) mutants (orange), control edit (light blue), and two independent CRISPR-edited variant lines with the p.Gly1345Ter variant (dark blue) worms on agar plates determined by WormLab (MBF Bioscience). Each dot represents an animal. n = 61–83 for each genotype. (B) Schematic showing how the Ub^G76V-GFP (abbreviated as UbGFP) reporter assay works. HECD-1 mediates the polyubiquitylation of the mutant, UbGFP. The mutation targets GFP for proteasomal degradation but not mRFP protein, which serves as a normalization control. (C) Representative bright-field, GFP, and RFP images of UbGFP animals captured on the CX7 high-content imager (Thermo Fisher Scientific). (D–F) Quantification of UbGFP accumulation (expressed as a ratio of GFP/RFP) in homozygous hecd-1 p.Gly1345Ter (D), p.Asp1239Asn (E), and p.Arg350Gly (F) variants. Each dot represents a well containing approximately ten animals. n = 9 wells for (D); n = 30–31 wells for (E); n = 35 wells for (F). (G and H) Quantification of UbGFP accumulation in heterozygous p.Asp1239Asn (G) and p.Arg350Gly (H) variants. n = 25–27 wells for (G); n = 19–32 wells for (H). Error bars display the mean plus 95% confidence interval in (A), (D), (F), and (G) or the median plus interquartile range in (E) and (H).