TRIM71 mutations cause a neurodevelopmental syndrome featuring ventriculomegaly and hydrocephalus

Abstract

Congenital hydrocephalus, characterized by cerebral ventriculomegaly, is one of the most common reasons for paediatric brain surgery. Recent studies have implicated lin-41 (lineage variant 41)/TRIM71 (tripartite motif 71) as a candidate congenital hydrocephalus risk gene; however, TRIM71 variants have not been systematically examined in a large patient cohort or conclusively linked with an OMIM syndrome. Through cross-sectional analysis of the largest assembled cohort of patients with cerebral ventriculomegaly, including neurosurgically-treated congenital hydrocephalus (totalling 2697 parent-proband trios and 8091 total exomes), we identified 13 protein-altering de novo variants (DNVs) in TRIM71 in unrelated children exhibiting variable ventriculomegaly, congenital hydrocephalus, developmental delay, dysmorphic features and other structural brain defects, including corpus callosum dysgenesis and white matter hypoplasia. Eight unrelated patients were found to harbour arginine variants, including two recurrent missense DNVs, at homologous positions in RPXGV motifs of different NHL domains. Seven patients with rare, damaging, unphased or transmitted variants of uncertain significance were also identified. NHL-domain variants of TRIM71 exhibited impaired binding to the canonical TRIM71 target CDKN1A; other variants failed to direct the subcellular localization of TRIM71 to processing bodies. Single-cell transcriptomic analysis of human embryos revealed expression of TRIM71 in early first-trimester neural stem cells of the brain. These data show TRIM71 is essential for human brain morphogenesis and that TRIM71 mutations cause a novel neurodevelopmental syndrome that we term 'TRIM71-associated developmental disorders (TADD)', featuring variable ventriculomegaly, congenital hydrocephalus and other structural brain defects.

Keywords: de novo variants; TRIM71; brain development; hydrocephalus; neural stem cells; structural brain disorders.

Figure 1

De novo and transmitted variants in TRIM71 are highly enriched in patients with congenital hydrocephalus. (A) Quantile–quantile (Q-Q) plot of observed versus expected P-values for de novo mutations (DNMs) in each gene in 2697 trio cases. P-values were calculated by one-sided Poisson test (see the ‘Materials and methods’ section). For protein-damaging TRIM71 de novo variants (DNVs) [loss-of-function (LoF), meta-analytic support vector machine (MetaSVM) = deleterious and/or Missense badness, PolyPhen-2 and Constraint (MPC) > 2, P = 5.07 × 10⁻²²]. (B) Damaging DNVs in TRIM71 mapped to highly conserved protein domains across different species. Locations of variants (p.Arg608His, p.Asp624Asn, p.Arg649Gln, p.Arg655Gln, p.Arg796His) are indicated in red. RPXGV motifs are highlighted in yellow. (C) Schematic diagram of variant locations in TRIM71 protein domains. Each filled circle represents a patient expressing a variant. DNVs are indicated with red circles. Transmitted and unphased variants are indicated with grey circles. (D) In silico modelling predicts modified protein structure of missense variants in TRIM71 (p.C15G, p.D624N p.R655Q and p.N701K), suggesting impaired function.

Figure 2

TRIM71 variants cause a novel form of syndromic congenital hydrocephalus. Representative brain MRIs of patients with de novo and transmitted TRIM71 mutations showing ventriculomegaly and other structural brain defects, including dysgenesis of the corpus callosum (yellow arrow), absence/dehiscence of the septum pellucidum (red arrow), and interhemispheric cysts (blue asterisk). Sagittal (A–D), axial (E–) and coronal (I–L) MRIs are shown of proband Patients KCHYD85-1 (A, E and I), KCHYD79-1 (B, F and J), KCHYD154-1 (C, G and K) and KCHYD419-1 (D, H and L).

Figure 3

TRIM71 variants disrupt the RNA-binding capacity of its NHL domain to CDKN1A and EGR1 or fail to localize TRIM71 to P-bodies. (A) Representative Flag immunoblot of HEK-293T total cell lysates showing the expression levels of the different constructs, i.e. Flag-control, Flag-TRIM71-WT (human) and Flag-TRIM71 variants ΔNHL6, -DN701 K, -R655Q, -D624N, -R608C, -K403N, -Q334R, -Q228Ter, -C15G. Predicted relative molecular mass (Mr) of TRIM71 Q228Ter is 24 kDa; Mr of wild-type (WT) and other variants is ∼80 kDa (predicted Mr 93 kDa). (B) Representative Flag immunoblot of anti-FLAG immunoprecipitates. Cross-linking and immunoprecipitation-quantitative real-time PCR enrichment of CDKN1A (C) and EGR1 (D) normalized to input and enrichment of housekeeping gene 18S RNA (means ± standard deviation; n ≥ 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. (not significant): P > 0.05. One-sample t-test versus WT. (E) P-body localization of congenital hydrocephalus (CH)-associated TRIM71 variants was analysed by co-expression of an RFP empty vector, RFP-tagged human Trim71-WT or CH-associated mutations (TRIM71-p.Asp624Asn, TRIM71-p.Lys403Asn, TRIM71-p.Gln334Arg and TRIM71-p.Gln228Ter) (red) in HEK293T cells together with GFP-tagged P-body marker DCP1A (green). Shown are representative confocal images where some P-bodies with co-localized TRIM71 are marked with an arrow, while cells without co-localization are labelled with a number sign. (F) Percentage of cells exhibiting TRIM71-DCP1A co-localization. The number of cells counted for each condition was ≥ 50 (29 cells for RFP empty vector as negative control). Nuclei were stained with DAPI (blue).

Figure 4

TRIM71 is expressed in neural stem cells during human fetal brain development. (A) A t-distributed stochastic neighbour embedding (t-SNE) plot of transcriptional dataset of developing human brain [postconceptual weeks (PCW) 3–12] coloured by main cell type: neural stem cells (NSC), intermediate progenitor cells (IPC), glutamatergic neurons (Glu N), GABAergic neurons (GABA N), dopaminergic neurons (DA N), cholinergic neurons (Cholin N), neuromesodermal progenitors (NMP), lateral plate mesoderm (LPM) and proliferating mesoderm (MesoPro). (B) Temporal gene expression profile of TRIM71 from PCW 3–12. (C) Differential TRIM71 expression across main cell type sub-clusters. Inset: Temporal distribution across PCW 3–12 of the top three significantly differentially expressed NSC clusters. Shading depicts the percentage of single cells in clusters found at each PCW.