Exome sequencing implicates genetic disruption of prenatal neuro-gliogenesis in sporadic congenital hydrocephalus

Abstract

Congenital hydrocephalus (CH), characterized by enlarged brain ventricles, is considered a disease of excessive cerebrospinal fluid (CSF) accumulation and thereby treated with neurosurgical CSF diversion with high morbidity and failure rates. The poor neurodevelopmental outcomes and persistence of ventriculomegaly in some post-surgical patients highlight our limited knowledge of disease mechanisms. Through whole-exome sequencing of 381 patients (232 trios) with sporadic, neurosurgically treated CH, we found that damaging de novo mutations account for >17% of cases, with five different genes exhibiting a significant de novo mutation burden. In all, rare, damaging mutations with large effect contributed to ~22% of sporadic CH cases. Multiple CH genes are key regulators of neural stem cell biology and converge in human transcriptional networks and cell types pertinent for fetal neuro-gliogenesis. These data implicate genetic disruption of early brain development, not impaired CSF dynamics, as the primary pathomechanism of a significant number of patients with sporadic CH.

Extended Data Fig. 1 |. De novo, transmitted, and unphased mutations in TRIM71.

a, Pedigrees and sequencing electropherograms of Sanger sequencing depict all TRIM71 mutations in genomic DNA from CH probands. b, representative T1 or T2-weighted brain magnetic resonance images for all available probands.

Extended Data Fig. 2 |. De novo, transmitted, and unphased mutations in SMARCC1.

a, Pedigrees and sequencing electropherograms of Sanger sequencing depict all SMARCC1 mutations in genomic DNA from CH probands. b, representative T1 or T2-weighted brain magnetic resonance images or head CTs for all available probands.

Extended Data Fig. 3 |. De novo, transmitted, and unphased mutations in PIK3CA.

a, Pedigrees and sequencing electropherograms of Sanger sequencing depict all PIK3CA mutations in genomic DNA from CH probands. b, representative T1 or T2-weighted brain magnetic resonance images for all available probands.

Extended Data Fig. 4 |. De novo, transmitted, and unphased mutations in PTEN.

a, Pedigrees and sequencing electropherograms of Sanger sequencing depict all PTEN mutations in genomic DNA from CH probands. b, representative T1 or T2-weighted brain magnetic resonance images or head CTs for all available probands.

Extended Data Fig. 5 |. De novo, transmitted, and unphased mutations in MTOR.

a, Pedigrees and sequencing electropherograms of Sanger sequencing depict all MTOR mutations in genomic DNA from CH probands. b, representative T1 or T2-weighted brain magnetic resonance images or head CTs for all available probands.

Extended Data Fig. 6 |. De novo and transmitted mutations in FOXJ1.

a, Pedigrees and sequencing electropherograms of Sanger sequencing depict all FOXJ1 mutations in genomic DNA from CH probands. b, representative T1 or T2-weighted brain magnetic resonance images for all available probands.

Extended Data Fig. 7 |. De novo and transmitted mutations in FMN2.

a, Pedigrees and sequencing electropherograms of Sanger sequencing depict all FMN2 mutations in genomic DNA from CH probands. b, representative T1 or T2-weighted brain magnetic resonance images for all available probands. c, The CrYP-SKIP algorithm prediction on splicing defects for FMN2: c.2137−2 A > G.

Extended Data Fig. 8 |. De novo, transmitted, and unphased mutations in PTCH1.

a, Pedigrees and sequencing electropherograms of Sanger sequencing depict all PTCH1 mutations in genomic DNA from CH probands. b, representative T1 or T2-weighted brain magnetic resonance images or head CTs for all available probands.

Extended Data Fig. 9 |. Transmitted and unphased mutations in FXYD2.

a, Pedigrees and sequencing electropherograms of Sanger sequencing depict all FXYD2 mutations in genomic DNA from CH probands. b, representative T1 or T2-weighted brain magnetic resonance images for all available probands. c, The CrYP-SKIP algorithm prediction on splicing defects for FXYD2: c.299−1 G > A. d, The CrYP-SKIP algorithm prediction on splicing defects for FXYD2: c.410 + 1 G > A.

Extended Data Fig. 10 |. Damaging recessive genotypes in human dystroglycanopathy genes and homologs of mouse hydrocephalus genes.

Available clinical-neuroimaging phenotypes of CH probands with damaging recessive mutations.

Fig. 1 |. TRIM71 and SMARCC1 are bona fide CH risk genes.

a, Quantile-quantile plot comparing observed versus expected P values for DNMs in each gene in 225 cases. TRIM71 and SMARCC1 exhibit genome-wide significant enrichment of DNMs in CH cases and TRIM71 is the overall most commonly mutated CH gene. b, Representative T1- or T2-weighted axial and sagittal brain magnetic resonance images (MRIs) or head computed tomography (CT) images of neurosurgically treated CH probands with the indicated TRIM71 and SMARCC1 mutations (Supplementary Tables 6 and 7 and Extended Data Figs. 1 and 2 contain clinical and neuroradiographic details for available patients). c, Locations of identified TRIM71 and SMARCC1 mutations in relation to critical functional domains. The recurrent TRIM71 p.Arg608His and p.Arg796His mutations impact conserved residues in the 16th position of the respective first and fifth blades of NHL domain, which mediates the binding to target mRNAs (Supplementary Fig. 2). p.Asn701Lys localizes to the third NHL domain and is predicted to destabilize protein–RNA interactions (Supplementary Fig. 2). RF, ring finger domain; CC, coiled-coil domain. The identified SMARCC1 mutations were mapped in relation to its SWIRM and Myb-DNA-binding domains, which mediate SMARCC1 interaction with BAF47 and its in vivo function in the SWI-SNF and ADA complexes, respectively.

Fig. 2 |. PI3K signaling genes PIK3CA, PTEN and MTOR are frequently mutated in sporadic CH.

a, Depiction of the PI3K signaling pathway with genes mutated in sporadic CH indicated by a red-filled circle. PIK3CA and PTEN mutations are anticipated to lead to increased PIP₃ production and mTOr activation, effects mimicked by CH-associated MTOR mutations. PI3K signaling regulates growth, proliferation and differentiation of embryonic and early postnatal NSCs. GF, growth factor; rTK, receptor tyrosine kinase. b, representative T1 or T2-weighted axial brain mrIs or head CT images of neurosurgically treated CH probands with the indicated PIK3CA, PTEN and MTOR mutations (Supplementary Table 11 and extended Data Figs. 3–5 contain clinical and neuroradiographic details for each patient). c, mutation mapping of PIK3CA, PTEN and MTOR mutations in relation to critical functional domains in each molecule. Frb, FKbP12-rapamycin-binding domain; FATC, C-terminal FAT domain. AbD, adaptor-binding domain; rbD, ras-binding domain; PDZ, PSD95, DLG1 and ZO1 domain.

Fig. 3 |. Multiple damaging DNMs in FOXJ1, FMN2, PTCH1 and an excess burden of rare LoF heterozygous mutations in FXYD2 in sporadic CH.

a, Quantile-quantile plot of observed versus expected P value of rare LoF heterozygous mutations. A one-tailed binomial test was conducted by comparing the observed number of LoF heterozygous mutations to the expected count. The genome-wide significant cutoff was 2.6 × 10⁻⁶ (0.05 of 19,347). FXYD2 was the single gene showing enrichment of rare, LoF heterozygous mutations. b, representative T1- or T2-weighted axial brain mrIs or axial head CT images of neurosurgically treated CH probands with the indicated FOXJ1, FMN2, PTCH1 and FXYD2 mutations (Supplementary Table 13 and extended Data Figs. 6–9 contain clinical and neuroradiographical details for each patient). c, mutation mapping of the FOXJ1, FMN2, PTCH1 and FXYD2 mutations in relation to critical functional domains in each molecule. FH, formin homology; Atp1G1_plm_mat8, ATP1G1/PLm/mAT8 family domain.

Fig. 4 |. CH risk genes are enriched in a coexpression network pertinent to other neurodevelopmental disorders and in cell types of early fetal neurogenesis.

a, enrichment analysis across weighted gene coexpression network analysis (WGCNA) modules of the midgestational human brain for genes with rare risk variation in CH (high confidence, probable risk and known human genes), ASD and DD (methods contains details of gene set definitions). Only seven modules are shown (labeled by color in line with Walker et al.); other modules demonstrated no significant enrichment for tested gene sets. Tiles labeled with −log₁₀(P value) and an asterisk represent statistically significant enrichment at the bonferroni multiple-testing cutoff (α = 0.05/17 = 2.94 × 10⁻³). b, Top 20 GO biological process terms and top 20 HP ontology terms enriched for the yellow module. The x axis depicts −log (adjusted P value) and the dotted line represents the α = 0.05 significance threshold. (P values are adjustted according to the g:SCS algorithm from g:Profiler). c, enrichment analysis across cell type markers of the midgestational human brain for genes with rare risk variation in CH (high confidence, probable risk and known human genes), ASD and DD. Tiles labeled with −log₁₀(P value) and an asterisk represent significant enrichment at the bonferroni multiple-testing cutoff (α = 0.05/16 = 3.13 × 10⁻³).

Fig. 5 |. A neural stem cell model of sporadic CH.

Schematic of the normal developing brain with the ventricular system surrounded by parenchyma consisting of neurons, astrocytes and components of neurogenesis at the cellular level (top). embryonic and fetal NSC populations, including neuroepithelial cells and radial glia cells (rGCs), together generate virtually all neuronal and glial cells that populate the brain, including multiciliated ependymal cells that line the ventricular system thought to participate in CSF circulation and maintenance of ventricular integrity. Defects in embryonic and fetal NSCs secondary to genetic mutations can thus drive CH via multiple pathogenic mechanisms that impact development and maturation of different cell types. Schematic of two possible developmental mechanisms of NSC alteration that may lead to ventriculomegaly (bottom). In one hypothesized scenario (left), ventriculomegaly results from impaired neurogenesis and an associated decrease in cortical cell mass that reflects a reduction in NSC proliferation. Continued CSF production from the unaffected choroid plexus would further expand the already enlarged ventricular compartment and even at low hydrostatic pressure push the thin, low-resistance cortical ribbon to the dural–bone interface. Ventricular enlargement and dysmorphology could then promote further ventricular expansion through secondary disruption of normal linear CSF laminar flow, eliciting fluid turbulence and current reversal. In another hypothesized scenario (right) that is not necessarily mutually exclusive from the former, altered NSC regulation leads to malformation of ependymal cells and their motile cilia, leading to impaired intraventricular CSF circulation and attendant CSF accumulation responsible for progressive ventricular dilation. Furthermore, defects in cilia-related genes may cause hydrocephalus not only by impairing motile cilia-driven CSF flow, but also by affecting development of primary cilia, which are nonmotile sensory organelles present on embryonic and fetal NSCs, crucial for multiple developmental processes, including patterning, neurogenesis, migration and survival. A combination of defects in NSC patterning and/or the proliferation–differentiation balance can also introduce anatomical defects, resulting in physical obstruction to CSF flow, such as aqueductal stenosis.