ARTICLE

Redefining the Etiologic Landscape of Cerebellar Malformations

Kimberly A. Aldinger,1 Andrew E. Timms,2 Zachary Thomson,1 Ghayda M. Mirzaa,1,3
James T. Bennett,2,3 Alexander B. Rosenberg,4 Charles M. Roco,5 Matthew Hirano,4 Fatima Abidi,6
Parthiv Haldipur,1 Chi V. Cheng,1 Sarah Collins,1 Kaylee Park,1 Jordan Zeiger,1 Lynne M. Overmann,7
Fowzan S. Alkuraya,8 Leslie G. Biesecker,9 Stephen R. Braddock,10 Sara Cathey,6 Megan T. Cho,11
Brian H.Y. Chung,12 David B. Everman,6 Yuri A. Zarate,13 Julie R. Jones,6 Charles E. Schwartz,6
Amy Goldstein,14,15 Robert J. Hopkin,16 Ian D. Krantz,15,17 Roger L. Ladda,18,19 Kathleen A. Leppig,20
Barbara C. McGillivray,21 Susan Sell,18 Katherine Wusik,22 Joseph G. Gleeson,23
Deborah A. Nickerson,24,25 Michael J. Bamshad,3,24,25 Dianne Gerrelli,26 Steven N. Lisgo,7
Georg Seelig,4,27 Gisele E. Ishak,1,28 A. James Barkovich,29 Cynthia J. Curry,30 Ian A. Glass,1,3
Kathleen J. Millen,1,3 Dan Doherty,1,3 and William B. Dobyns1,3,31,*

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 var-
iants 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.

Introduction sistent classification, largely unknown pathogenesis, and

Cerebellar malformations, including Dandy-Walker mal-
formation (DWM) and several cerebellar hypoplasia
(CBLH) subtypes, are among the most common malforma-
tions recognized in utero, though their prevalence is un-
known.1 They have proven difficult to study due to incon-
diverse developmental outcomes that may include child-
hood epilepsy, intellectual disability (ID), cerebral palsy,
autism, and other neuropsychiatric disorders.2,3
Pathogenic variants in only a few genes have been impli-
cated in CBLH or DWM. After excluding rare, autosomal
recessive disorders such as a-dystroglycanopathies and

1
Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA 98101, USA; 2Center for Developmental Biology and Regenerative
Medicine, Seattle Children’s Research Institute, Seattle, WA 98101, USA; 3Department of Pediatrics, University of Washington, Seattle, WA 98105, USA;
4
Department of Electrical Engineering, University of Washington, Seattle, WA 98105, USA; 5Department of Bioengineering, University of Washington, Se-
attle, WA 98105, USA; 6Greenwood Genetic Center, Greenwood, SC 29646, USA; 7Institute of Genetic Medicine, Newcastle University, International Centre
for life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK; 8Department of Genetics, King Faisal Specialist Hospital Research Center, Riyadh, 11211,
Saudi Arabia; 9Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda,
MD, 20892 USA; 10Department of Pediatrics, Saint Louis University School of Medicine, St. Louis, MO 63104, USA; 11GeneDx, Gaithersburg, MD
20877, USA; 12Department of Pediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; 13Sec-
tion of Genetics and Metabolism, University of Arkansas for Medical Sciences, Little Rock, AR, 72202, USA; 14Mitochondrial Medicine Frontier Program,
Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA; 15The Department of Pediatrics,
University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA; 16Division of Human Genetics, Department of Pediatrics, Cincin-
nati Children’s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA; 17Division of Human Genetics and
Molecular Biology, The Children’s Hospital of Philadelphia, Philadelphia, PA, 19104 USA; 18Department of Pediatrics, Milton S Hershey Medical Center,
Hershey, PA 17033, USA; 19Departments of Pathology, Milton S Hershey Medical Center, Hershey, PA 17033, USA; 20Genetic Services, Kaiser Permanente
Washington, Seattle, WA 98112, USA; 21Department of Medical Genetics, Children’s and Women’s Health Centre of British Columbia, Vancouver, BC V6H
3N1, Canada; 22Division of Human Genetics, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA;
23
Department of Neurosciences, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093, USA; 24Department of Genome
Sciences, University of Washington, Seattle, WA 98195, USA; 25University of Washington Center for Mendelian Genomics, Seattle, WA 98195, USA; 26Uni-
versity College London Institute of Child Health, London WC1N 1EH, UK; 27Paul G. Allen School of Computer Science & Engineering, University of Wash-
ington, Seattle, WA 98195, USA; 28Department of Radiology, University of Washington, Seattle, WA 98195, USA; 29Departments of Radiology, Neurology,
Pediatrics, and Neurosurgery, University of California, San Francisco, San Francisco, CA 94143, USA; 30Genetic Medicine, Department of Pediatrics, Uni-
versity of California San Francisco, Fresno, CA, 93701, USA; 31Department of Neurology, University of Washington, Seattle, WA 98105, USA
*Correspondence: wbd@uw.edu
https://doi.org/10.1016/j.ajhg.2019.07.019.
Ó 2019 American Society of Human Genetics.

606 The American Journal of Human Genetics 105, 606–615, September 5, 2019
ciliopathies, the list includes variants of CASK (MIM:
300172), OPHN1 (MIM: 300127), ZIC1-ZIC4 (MIM:
220200), and FOXC1 (MIM: 601090).4 Cerebellar abnor-
malities have also been linked to non-genetic mechanisms,
especially prematurity, twinning, and prenatal cerebellar
hemorrhage.5–7 To fundamentally advance our under-
standing of these disorders, we analyzed neuroimaging
and other phenotypic data, and we performed exome
sequencing in 100 families with CBLH or DWM. We
further performed single-cell RNA sequencing of human
fetal cerebellar tissue to evaluate whether cerebellar
malformation genes map onto specific cell types.
Material and Methods
Discovery Cohort
From our cohort of >4,200 individuals with developmental brain
disorders, we used specific inclusion and exclusion criteria for
CBLH and DWM (Table S1) to select 100 individuals with
cerebellar imaging abnormalities. We included individuals with
available DNA and clinical data, including neuroimaging studies,
and we excluded individuals with prior genetic diagnoses or recog-
nizable disorders such as ciliopathies. Written informed consent
was obtained under protocols approved by institutional review
boards at the University of Chicago, Seattle Children’s Hospital,
and the University of Washington.
Exome Sequencing and Variant Analysis
We performed exome sequencing on genomic DNA from 105
affected individuals and 177 parents (Table S2). Variants were
filtered for >103 coverage, <0.001 minor allele frequency in
public databases, and CADD score >10,8 as well as for de novo,
autosomal recessive, or X-linked inheritance. Filtered variants
were visually inspected using IGV,9 and frameshift mutations
were validated by Sanger sequencing. The criteria used to deter-
mine pathogenicity are described in Supplemental Methods (see
Supplemental Data).
Confirmation Cohort
The cerebellar malformation confirmation cohort included 131 in-
dividuals who were identified through our developmental brain
disorders cohort, colleagues, or GeneMatcher10 and who had
previously received a genetic diagnosis for one of the monogenic
disorders detected in the discovery cohort. We obtained original
neuroimaging studies for all 131 individuals.
Single-Cell RNA Sequencing and Analysis of Human
Cerebellar Tissue
We obtained cerebellar tissue from the Birth Defects Research
Laboratory at the University of Washington and from the Human
Developmental Biology Resource11 with ethics board approval and
maternal written consent. Specimens were dissociated to obtain
whole cells (n ¼ 4) or flash frozen in liquid nitrogen and stored
at -80 C until use (n ¼ 1). Whole-cell dissociation and fixation
were performed as described.12 Nuclei were isolated using a pub-
lished protocol modified by fixation conditions specified in the
SPLiT-seq protocol.13,14 We used SPLiT-Seq for single-cell or sin-
gle-nucleus RNA sequencing14 as described in the Supplemental
Methods (see Supplemental Data).
The American Journal of Human Genetics 105, 606–615, September 5, 2019 607
Statistical Analyses
For clinical data, pairwise comparisons were performed using
Fisher’s exact test (with any number of cells less than five) or
two-tailed Chi-square test. Detailed analyses of gene expression
and single-cell datasets are described in the Supplemental
Methods (see Supplemental Data).
Results
Of the 100 probands, 88 had European ancestry, 60 were
male, and 67% were younger than seven years of age; 94
had cognitive function data available (Figure S1 and Ta-
bles S3 and S4). All probands had neuroimaging features
of CBLH (57 individuals) or DWM (43 individuals).
Examples of the variability among cerebellar malforma-
tions are shown in Figures 1, S2, and S3. The rate of
ID was significantly higher in CBLH (48 of 57 [84%])
than in DWM (20 of 37 [54%]; c2 ¼ 10.2, df ¼ 1, p ¼
0.001).
Cerebellar Malformation Gene Discovery
We identified 41 pathogenic or likely pathogenic (diag-
nostic) variants in 36 families; these variants included 27
de novo (66%; 19 autosomal, five X-linked heterozygous,
and three X-linked hemizygous), three inherited X-linked
hemizygous (7%), one autosomal homozygous (2%), and
10 autosomal compound heterozygous (24%) variants
involving 27 genes (Figure 2A and Table S5). The 41 diag-
nostic variants included 30 missense, two in-frame dele-
tion, seven protein truncating, and two splice-acceptor
mutation variants; 14 variants (34%) were previously re-
ported as pathogenic.
Among the 27 distinct monogenic disorders detected,
seven genes accounted for 16 (44.4%) of the 36 genetic
diagnoses: BCL11A (MIM: 606557), FOXP1 (MIM:
605515), SETD2 (MIM: 612778), STXBP1 (MIM:
602926), TUBA1A (MIM: 602529), CASK, and DDX3X
(MIM: 300160). However, the majority of genetic disor-
ders identified (20 of 27 [74%]) were unique to a single
family in the discovery cohort. Diagnostic variants
in SETD2 and TUBA1A were each detected in two in-
dividuals, one with CBLH and another with DWM. We
found diagnostic variants in three genes associated with
disorders excluded by our selection criteria, two with Jou-
bert syndrome (ARMC9 [MIM: 617612], PIBF1 [MIM:
607532]) and one with pontocerebellar hypoplasia
(RARS2 [MIM: 611524]); the neuroimaging features
were atypical and only recognized on re-review. Five
genes were initially characterized as novel but were re-
ported during the course of our study (ARMC9, MACF1
[MIM: 608271], PPP1CB [MIM: 600590], TUBB2A [MIM:
615101], WDR37) and one had not previously been asso-
ciated with disease in humans (FZD3). We also identified
heterozygous de novo variants of uncertain significance in
three candidate genes (BACH1, BAG6, DPYSL5), and two
genes previously associated with different phenotypes

(MACF1, SEMA6B) as summarized in the Supplemental
Note (see Supplemental Data).
The diagnostic yield was highest among individuals with
CBLH (29 of 57 [51%]) and with ID (33 of 68 [49%];
Figure 2B and Table S6). Only seven out of 43 individuals
with DWM (16%) received a genetic diagnosis, which was
significantly lower than for CBLH (c2 ¼ 12.73, df ¼ 1, P ¼
0.0004). Among the 43 individuals with DWM, 36 had neu-
roimaging with sufficient resolution to assess for the pres-
ence of an unpaired caudal lobule or ‘‘DW-tail’’ of the cere-
bellar vermis.15 The rate of genetic diagnosis was lowest
608 The American Journal of Human Genetics 105, 606–615, September 5, 2019
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 bot-
tom 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 communi-
cating 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 periven-
tricular nodular heterotopia (white arrow),
and small cerebellar hemispheres (h). The
child in the second row has cerebellar ver-
mis 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 fea-
tures 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 cere-
bellar hemisphere hypoplasia more severe
on the right (H). In the bottom row, the
axial image (K) shows posterior predomi-
nant 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).
among individuals with the DW-tail (4 of 30 [13%]) and
higher in those without the DW-tail (3 of 6 [50%]).
Cerebellar Malformations Co-Occur with Distinct
Monogenic Disorders
Most of the genes identified in our discovery cohort (21
of 27 [78%]; Figure 2A) were previously associated with
known neurodevelopmental disorders; these included
11 genes associated with non-syndromic early childhood
epilepsy, ID, or autism. We reviewed original neuroimag-
ing studies from another 122 individuals with
 A C

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 repre-
sented 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 malfor-
mation 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, respec-
tively. 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.

monogenic disorders involving the 21 known neurodeve-
lopmental disorder-associated genes, and we verified
CBLH or DWM in 99 of 122 (81%; Tables S7, S8, S9,
and S10). We found reports supporting cerebellar malfor-
mations for 13 genes; these included reports for four
genes that were not in our confirmation cohort
(AHDC1 [MIM: 615790], FGFR1 [MIM: 136350], TUBB2A,
DKC1 [MIM: 200126]). These results confirm a cerebellar
phenotype for 23 of 27 (85%) genes (Tables S7 and S11).
Notably, DWM was an inconsistent feature of any genetic
disorder.
Differences in Brain Gene Expression
The disparate rates of ID and genetic diagnosis in CBLH
versus DWM (Figure 2B) led us to examine whether
genes found in each neuroimaging group were differen-
tially expressed in prenatal brains. We used the Brain-
Span Atlas of the Developing Human Brain to examine
expression of the genes found in our discovery cohort
between 8 and 37 postconceptual weeks (pcw;
Figure S5).16 All 27 genes were expressed in the cere-
bellum and neocortex, and we found significant correla-
tion between the two regions (Spearman’s r ¼ 0.976,

The American Journal of Human Genetics 105, 606–615, September 5, 2019 609
 A C E

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 signif-
icant 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 (*).

p ¼ 1.26 3 10-7). Neuroimaging group was a significant
predictor of brain gene expression with lower brain
expression for genes with diagnostic variants in individ-
uals with DWM compared those with CBLH [F(2, 3882)
¼ 5.443, p ¼ 0.028].
CBLH and DWM Genes Have Cell-Type Specificity
To examine how differences in gene expression between
neuroimaging groups extended to cerebellar cell types,
we used SPLiT-seq to generate single-cell RNA sequencing
data from five fetal cerebellar samples (12–21 pcw), and
we obtained 4,306 single-cell transcriptomes (Figure 3A
and Table S12). We used unsupervised clustering to
define distinct cell clusters and applied known gene
expression patterns to define 13 identities encompassing
multiple neuronal, glial, and vascular cell types
(Figure 3B, Figure S6, and Table S13). We examined
expression for the 27 discovery cohort genes among
developmental cerebellar cell types and found enrich-
ment in Purkinje cells (nine of 27 [33%]; Figure 3C and
Table S14). We then grouped the 13 cerebellar cell clus-
ters into broad categories (neurons, glia, and vascular
cell types), and we found that genes mutated in individ-
uals with cerebellar malformations were specifically
enriched in neurons and vascular cells (Figure 3D–E and
Table S15).
Cerebellar Abnormalities Are Associated with Prenatal
Factors
Our data left 64% of individuals without genetic diagno-
ses. We therefore reviewed prenatal clinical history and
neuroimaging studies (Table S16) to identify individuals
with high risk factors for prenatal brain injury. We found
four extremely low gestational age newborns (ELGAN,
defined as 22–27 weeks gestation) and 12 discordant twins
(one ELGAN). The four individuals with ELGAN all had
CBLH. The twins included six who were discordant for
DWM (four of six were monozygotic), five who were
discordant for CBLH (four of five were dizygotic), and
one with intrauterine demise of her co-twin. The overall
rate of molecular diagnosis was lower in individuals with
clinical prenatal risk factors (two of 15 [13%]) versus
without (21 of 52 [40%]; Figure 2C and Table S17).
We also re-examined neuroimaging studies for features
associated with prenatal brain injury including (1) unilat-
eral or highly asymmetric CBLH and (2) cerebellar
clefts.17–19 We also considered (3) posterior-predominant
periventricular nodular heterotopia (PNH, Figure 1B, 1K)

610 The American Journal of Human Genetics 105, 606–615, September 5, 2019
and curvilinear subcortical heterotopia based on low yield
in exome studies and occurrence in discordant monozy-
gotic twins including one twin (LR12-313a2) in this
study.20–23 We identified 24 individuals with neuroimag-
ing evidence supporting prenatal brain injury; these indi-
viduals included 20 with prominent cerebellar asymmetry,
seven with cerebellar clefts, and 15 with cerebral heteroto-
pia. The rate of molecular diagnosis was significantly lower
among individuals with disruptive neuroimaging patterns
(one of 24 [4%]) versus individuals without (35 of 76
[46%]; Figure 2C and Table S17).
We next combined clinical and neuroimaging prenatal
risk factors and found that 24 of 33 (73%) had two or
more risk factors. Overall, prenatal risk factors had similar
rates in CBLH (21 of 57 [37%]) and DWM (12 of 43 [28%];
Figure 2C). And as expected, the rate of molecular diag-
nosis was significantly lower in individuals with any pre-
natal risk factor (3 of 33 [9%]) versus those without (33
of 67 [49%];Figure 2C and Table S17).
Discussion
The scientific literature contains reports of cerebellar mal-
formations dating from the 1800s, and DWM was clearly
defined by 1942.24–26 Many distinct cerebellar malforma-
tions have since been described.4 In this study, we report
(1) a high rate (36%) of molecular diagnosis in individuals
with cerebellar malformations that genetically overlap
with neurodevelopmental disorders, (2) significantly lower
rates of ID and molecular diagnosis in DWM versus CBLH,
(3) a high rate of prenatal risk and neuroimaging features
associated with injury that implicate frequent non-genetic
causes of cerebellar malformations including DWM,
and (4) evidence suggesting that genetic defects of vasculo-
genesis may underlie some cerebellar malformations,
providing an important biological link between genetic
and non-genetic causes.
Genetic Basis for Cerebellar Malformations
The molecular diagnostic rate in our discovery cohort
(36%) is similar to those for childhood epilepsy (25%), ID
(28%), and autism (28%).27–29 However, after removing
the 33 individuals with any prenatal risk factor, the diag-
nostic yield in our cohort rises to 33 of 67 (49%). If we
then partition our cohort by cerebellar malformation, the
diagnostic yield is 72% (26 of 36) in CBLH and 23% (7 of
31) in DWM. These data support a ‘‘genotype-first’’ testing
approach for affected individuals without these defined
prenatal risk factors.
Cerebellar Malformation in Neurodevelopmental
Disorders
Though cerebellar malformations have historically been
used as primary diagnoses, we show that CBLH often oc-
curs and DWM sometimes occurs as a co-morbidity of
monogenic neurodevelopmental disorders with variable
The American Journal of Human Genetics 105, 606–615, September 5, 2019 611
expressivity. These include at least 11 genes associated
with childhood epilepsy, ID, or autism without syndromic
features: AHDC1, AUTS2 (MIM: 607270), BCL11A,
DDX3X, FOXP1, KIF4A (MIM: 300521), PUS3 (MIM:
616283), SETD2, SPTAN1 (MIM: 182810), STXBP1, and
TMLHE (MIM: 300777). We also identified several individ-
uals with diagnostic variants in these genes with expanded
phenotypes which included other congenital anomalies,
for example, individuals with AUTS2, KIF4A, and SETD2.
Our results might also explain the small reductions in
regional cerebellar volume reported in autism, schizo-
phrenia, and bipolar disorder, as well as anecdotal reports
of CBLH associated with these disorders (Table S18).
Finally, our data combined with prior reports identified
only four genes associated with non-syndromic (and
inconsistently syndromic) ID that have a high penetrance
of CBLH—BCL11A, CASK, FOXP1, and OPHN1 (Tables S7
and S8).
DWM Differs from CBLH
DWM has long been viewed as a distinct malformation,
although neuroimaging studies in large cohorts have
suggested that it may represent one end of a wide CBLH–
DWM spectrum,30,31 and several genes have been associ-
ated both with CBLH subtypes and with DWM, including
FOXP1, SETD2, PUS3 (all mentioned previously in this
article), ZIC1-ZIC4, and FOXC1.32,33 However, we report
a higher frequency of typical cognitive function and a
lower rate of molecular diagnosis in DWM compared to
CBLH; this supports important clinical distinctions be-
tween them. Further, the unpaired caudal lobule or
Dandy-Walker ‘‘tail’’ was recently proposed as an impor-
tant diagnostic sign.15 Our data support this observation,
showing that the molecular diagnostic rate is even lower
in DWM with the tail compared to DWM without the
tail, although the numbers are small. If future studies
confirm this difference, the tail should become an addi-
tional criterion for classic DWM. We also show that
DWM-associated genes have lower expression in the pre-
natal brain, which provides additional support for DWM
being mechanistically distinct from CBLH despite the
overlap in underlying genes and prenatal risk factors.
Prenatal Risk Factors Contribute to Both CBLH and
DWM
Prior studies have shown that the risk for cerebellar lesions
with prematurity is greater at earlier gestational ages and
rises to 16% in ELGAN.7,34–36 Observations of cerebellar
malformations detected by prenatal ultrasound in twins
have been attributed to twin-twin transfusion syndrome
(TTTS) in monozygotic twins.5,37 Several reports have sug-
gested that prenatal cerebellar disruptions such as hemor-
rhage can cause cerebellar asymmetry and clefts,17–19 and
a few reports have documented evidence of prenatal cere-
bellar hemorrhage preceding a postnatal DWM diag-
nosis.38–40
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.44 (B), and patients 1 (C) and 3 (D)
reported by Johnston et al.42
(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.

Our data strongly support these prior studies by showing
that prenatal risk factors contribute to CBLH generally,
providing a systematic analysis demonstrating a non-
genetic basis for three co-occurring brain abnormalities
(asymmetric CBLH, cerebellar clefts, and cerebral heteroto-
pia—especially posterior predominant PNH), and pro-
viding further evidence that the same prenatal risk factors
contribute to DWM. However, our cohort includes DWM
and CBLH in both monozygotic and dizygotic twins,
which suggests that factors other than TTTS may be
involved. In one of the original reports of DWM in 1954,
Benda et al. reported an infant with DWM whose co-
twin died at 22–25 weeks gestation.26
CBLH-DWM and Vasculogenesis
We a priori expected genes identified in our discovery
cohort to be highly expressed in prenatal human cerebellar
tissue. However, several genes expressed at low levels in
bulk cerebellar tissue had enriched expression in devel-
oping neuronal and vascular cell types (Figures 3C and
3E). This led us to hypothesize that disruption of genes
functioning in vasculogenesis might lead to the same final
common pathway as prenatal risk factors by predisposing
the individual to prenatal cerebellar hemorrhage or altered
vascular perfusion.
The best example involves PDGFRB (MIM: 173410), a
gene which is expressed primarily in vascular pericytes
and radial glia and which has previously been associated
with a spectrum of developmental disorders reported as
infantile myofibromatosis and as Penttinen and Kosaki
overgrowth syndromes.41–44 We identified a pathogenic
variant in PDGFRB in a girl who had infantile myofibroma-
tosis as well as severe CBLH and mega-cisterna magna.
Although we initially found no reports of CBLH for this
disorder, reports of Penttinen syndrome described poste-
rior fossa cysts.42,44,45 We obtained original neuroimaging
studies for two individuals with Penttinen syndrome, and
we found CBLH in addition to posterior fossa cysts
(Figure 4A-D and Figure S3). Then a recent report described
Kosaki syndrome and DWM in an individual with the
same gain-of-function PDGFRB mutation found in our pro-
band.44 Our single-cell transcriptomic analysis showed
that PDGFRB expression is highly enriched in pericytes
within the prenatal cerebellum. PDGFRB in situ hybridiza-
tion in human cerebellar tissue confirmed pericyte expres-
sion throughout development and also transient expres-
sion in the residual ventricular zone that gives rise to
Purkinje cells (Figure 4E-G).
Several other genes with diagnostic variants in our
discovery cohort were also highly expressed in vascular
cell types; these genes included BRAF (MIM: 164757),
DDX3X, FGFR1, FOXP1, PPP1CB, and SETD2. Adding
FOXC1, HRAS (MIM: 190020), PTPN11 (MIM: 176876),
and WNT1 (MIM: 164820) from prior reports establishes

612 The American Journal of Human Genetics 105, 606–615, September 5, 2019
10 cerebellar malformation genes with high expression
during vasculogenesis, including two Forkhead box tran-
scription factors and four RAS pathway genes.33,46–48 These
data support our hypothesis that disruption of vascular-
expressed genes alters cerebellar development via second-
ary prenatal perfusion defects or hemorrhage, and predict
that DWM and a subset of CBLH will be variable features
of associated genetic syndromes.
Summary
We report that cerebellar malformations most often repre-
sent either a variable feature of diverse neurodevelopmen-
tal disorders or the sequela of prenatal risk factors that
predispose the fetal brain to cerebellar hemorrhage or
vascular injury. Beyond providing insight into the biolog-
ical mechanisms leading to CBLH and DWM, we expect
our results to change clinical care in several ways. First,
CBLH and DWM should only rarely serve as primary
diagnoses: when either is detected, the diagnostic journey
is not over. Second, CBLH in the absence of clinical or
neuroimaging evidence of prenatal injury is associated
with a high yield for genetic testing, supporting a ‘‘geno-
type first’’ approach. Finally, our results stress the need
for careful phenotypic assessment (prenatal history and
postnatal neuroimaging) to predict the yield from genetic
testing and most likely developmental outcome, thereby
improving pre- and postnatal counseling.
Accession Numbers
The accession numbers for the disease-associated variants described
in this report are ClinVar: SCV000916307.1–SCV000916347.1.
Supplemental Data
Supplemental Data can be found online at https://doi.org/10.
1016/j.ajhg.2019.07.019.
Acknowledgements
We thank the children as well as their families and referring phy-
sicians for their important contributions to our ongoing work on
cerebellar disorders. This study was funded by the National Insti-
tutes of Health under National Institute of Neurological Disorders
and Stroke (NINDS) or National Institute of Child Health and
Human Development (NICHD) grant numbers 5R01NS050375
to W.B.D, R01NS095733 to K.J.M., K08NS092898 to G.M.M.,
and R24HD000836 to I.A.G. The University of Washington Center
for Mendelian Genomics provided sequencing and data analysis,
supported by National Human Genome Research Institute
(NHGRI) grant number U54HG006493 to D.A.N. and M.J.B., and
the University of Washington Intellectual and Developmental
Disabilities Research Center (IDDRC) Genetics Core, supported
by NICHD grant number U54HG006493, provided support to
D.D. Additional funding was provided by The Dandy-Walker Alli-
ance and The Philly Baer Foundation. L.G.B. was supported by
grant number HG200328 from the Intramural Research Program
of the NHGRI. Human fetal material was provided, in part, by
The American Journal of Human Genetics 105, 606–615, September 5, 2019 613
the Joint Medical Research Council and Wellcome (MR/
R006237/1) Human Developmental Biology Resource. The con-
tent is solely the responsibility of the authors and does not
necessarily represent the official views of the funding sources.
Please note that the authors cite OMIM according to journal edito-
rial policy but do not endorse the referenced OMIM data.
Declaration of Interests
G.S., A.B.R., and C.R. have filed a patent related to the SPLiT-seq
method. All other authors declare no competing interests.
Received: June 3, 2019
Accepted: July 26, 2019
Published: August 29, 2019
Web Resources
BrainSpan Atlas of the Developing Human Brain, http://www.
brainspan.org/
CADD version 1.3, https://cadd.gs.washington.edu/home
Dandy-Walker Alliance, http://dandy-walker.org/
ExAC Browser, http://exac.broadinstitute.org
gnomAD database, https://gnomad.broadinstitute.org
Human Developmental Biology Resource, http://www.hdbr.org
OMIM, https://omim.org/
Philly Baer Foundation, https://www.phillybaerfoundation.org/
RefSeq, http://www.ncbi.nlm.nih.gov/RefSeq
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