Redefining the Etiologic Landscape of Cerebellar Malformations

Abstract

Cerebellar malformations are diverse congenital anomalies frequently associated with developmental disability. Although genetic and prenatal non-genetic causes have been described, no systematic analysis has been performed. Here, we present a large-exome sequencing study of Dandy-Walker malformation (DWM) and cerebellar hypoplasia (CBLH). We performed exome sequencing in 282 individuals from 100 families with DWM or CBLH, and we established a molecular diagnosis in 36 of 100 families, with a significantly higher yield for CBLH (51%) than for DWM (16%). The 41 variants impact 27 neurodevelopmental-disorder-associated genes, thus demonstrating that CBLH and DWM are often features of monogenic neurodevelopmental disorders. Though only seven monogenic causes (19%) were identified in more than one individual, neuroimaging review of 131 additional individuals confirmed cerebellar abnormalities in 23 of 27 genetic disorders (85%). Prenatal risk factors were frequently found among individuals without a genetic diagnosis (30 of 64 individuals [47%]). Single-cell RNA sequencing of prenatal human cerebellar tissue revealed gene enrichment in neuronal and vascular cell types; this suggests that defective vasculogenesis may disrupt cerebellar development. Further, de novo gain-of-function variants in PDGFRB, a tyrosine kinase receptor essential for vascular progenitor signaling, were associated with CBLH, and this discovery links genetic and non-genetic etiologies. Our results suggest that genetic defects impact specific cerebellar cell types and implicate abnormal vascular development as a mechanism for cerebellar malformations. We also confirmed a major contribution for non-genetic prenatal factors in individuals with cerebellar abnormalities, substantially influencing diagnostic evaluation and counseling regarding recurrence risk and prognosis.

Keywords: Dandy-Walker malformation; autism; cerebellar hypoplasia; cerebellum; epilepsy; exome; genes; heterotopia; intellectual disability; twins.

Figure 1

Neuroimaging of Cerebellar Malformations Four cerebellar malformation patterns are shown with midline sagittal (left column), axial (middle column), and axial or coronal (right column) images using T2-weighted (A–C, H–I, N–O), T1-weighted (D, G), or volumetric (E–F, J–L) sequences. The white horizontal bars in the left column mark the obex, which approximates the normal lower limit of the vermis. The top and bottom rows demonstrate features of DWM in individuals LR05-354 at 1 day (A–C) and LR05-265 at 5 years (J–L). Midline sagittal images (A, J) demonstrate small to very small and upwardly rotated vermis (v), widely open fourth ventricle communicating with large posterior fossa fluid spaces (^∗∗) or cystic dilatation of the fourth ventricle (4V), and elevated tentorium. The newborn in the top row (A–C) has an easily detected unpaired caudal lobule or Dandy-Walker tail (black arrow), a single periventricular nodular heterotopia (white arrow), and small cerebellar hemispheres (h). The child in the second row has cerebellar vermis hypoplasia (v), mega-cisterna magna (^∗∗), and a mildly small right cerebellar hemisphere (h), which is associated with a FOXP1 mutation (from confirmation cohort). The bottom two rows show features associated with prenatal risk factors in individuals LR05-398 at 1 year (G–I; discordant monozygotic twin) and LR05-265 at 5 years (J–L). Midline sagittal image from the affected twin (G) shows a small cerebellar vermis (v) and posterior fossa, while the second individual (J) has classic DWM with small upwardly rotated vermis (v), Dandy-Walker tail (white arrow in d), cystic enlargement of the fourth ventricle (^∗∗), and elevated tentorium. In the third row, the axial image (H) shows bilateral cerebellar clefts (white arrows), while the coronal image (I) shows asymmetric cerebellar hemisphere hypoplasia more severe on the right (H). In the bottom row, the axial image (K) shows posterior predominant periventricular nodular heterotopia larger on the left (black arrows; the right side of axial images shows the left side of the brain). The coronal image (F) shows small asymmetric cerebellar hemispheres, smaller on the left (H), and a large cleft removing most of the left middle cerebellar peduncle (F, white arrow).

Figure 2

Summary of Genetic and Prenatal Risk Factor Analyses in Cerebellar Malformations (A) The left panel shows the counts of individuals with genetic diagnosis per gene in the discovery cohort. Counts for DWM are represented below zero in orange and counts for CBLH are above zero in blue. Novel gene is indicated with an asterisk. The right panel shows the counts for individuals with cerebellar malformations in the discovery cohort, confirmation cohort, and literature combined per gene. The counts for three genes (TUBA1A, CASK, OPHN1) extend beyond the chart. (B) Clinical diagnoses and genetic diagnostic yield. The left panel shows the frequency of genetic diagnosis per cerebellar malformation diagnosis. The right panel shows the frequency of genetic diagnosis per cognitive function. The rate of ID is higher in CBLH than in DWM. The rate of genetic diagnosis was highest in CBLH and ID. Plus or minus sign indicates presence or absence, respectively. (C) Rates of prenatal risk factors, genetic diagnosis, and cerebellar malformation group. The relative proportions of individuals with prenatal risk factors, genetic diagnosis, and cerebellar malformation diagnosis. Plus or minus sign indicate presence or absence, respectively. Only 3% of individuals with genetic diagnoses also had prenatal risk factors, while 30% of individuals without genetic diagnoses had prenatal risk factors for any cerebellar malformation. The genetic diagnostic yield was highest among individuals with CBLH who lacked prenatal risk factors. The genetic diagnostic yield was lowest among individuals with DWM who also lacked prenatal risk factors. Abbreviations: CBLH, cerebellar hypoplasia; DWM, Dandy-Walker malformation; GDX, genetic diagnosis; ID, intellectual disability; PRF, prenatal risk factors.

Figure 3

Cell Types in the Prenatal Cerebellum and Enrichment of Cerebellar Malformation Genes (A) Workflow for single-cell transcriptome profiling of prenatal cerebellum cells or nuclei. (B) Cell clusters from SPLiT-seq analysis visualized by t-stochastic neighbor embedding (tSNE). Colors indicate cell type. (C) Heatmap of differential gene expression per cell type for each of the 27 genes identified in the discovery cohort. Genes with significant differential expression (FDR < 0.05) per cluster are indicated (∗). (D) The same tSNE scatter plot as in (B) but cells are colored according to three broad cell classes. (E) Heatmap of differential gene expression per broad cell class for each of the 27 genes identified in the discovery cohort. Genes with significant differential expression (FDR < 0.05) per cluster are indicated (^∗).

Figure 4

PDGFRB Neuroimaging Phenotype and Prenatal Expression in Human Cerebellum (A–B) Midline sagittal MRI in two individuals with PDGFRB (GenBank: NM_002609.3) variant c.1696T>C (p.Trp566Arg) show large head with prominent occiput, thin and stretched corpus callosum either diffusely (white arrow in [A]) or posteriorly (black arrow in [B]), massively enlarged cisterna magna (^∗∗), and severe cerebellar vermis hypoplasia. (C–D) Midline sagittal MRI in two individuals with PDGFRB (GenBank: NM_002609.3) c.1994T>C (p.Val665Ala) variant show normal head contour and corpus callosum, mildly enlarged cisterna magna (^∗) and mild cerebellar vermis hypoplasia. The horizontal white or black lines to the right of the lower brainstem mark the typical inferior limit of the vermis. The images shown are for LR05-118 in the discovery cohort (A), an individual reported by Zarate et al. (B), and patients 1 (C) and 3 (D) reported by Johnston et al. (E–G) PDGFRB expression as detected by in situ hybridization in the human cerebellum at Carnegie stage 20 (E), 14 pcw (F), and 18 pcw (G) localizes to pericytes and mesenchyme. At 14 pcw, PDGFRB expression is also detected in the residual ventricular zone ([F], arrows in inset), the stem cell niche that gives rise to Purkinje cells.