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Genetic basis of cerebellar and
brainstem malformations
Genetic basis of cerebellar and
brainstem malformations
Enza Maria ValenteEnza Maria Valente
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Cerebellar and brainstem congenital defectsCerebellar and brainstem congenital defects
Features
� defects of cerebellar and brainstem development in utero
� often detectable prenatally (II trimester of pregnancy)
� malformative and not degenerative (exception: PCH)
� already present at birth � neonatal phenotype
� usually non progressive (no worsening over time)
� imaging: cerebellar hypoplasia/dysplasia (but non progressive
atrophies are possible)
estimated incidence: > 1/5000 live births
estimated rate of pregnancy termination: up to 80%
estimated infant mortality rate: 10-66%
estimated rate of disability for survivals: 45-100%
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CBCDs: complex etiopatogenesis, simpler phenotypesCBCDs: complex etiopatogenesis, simpler phenotypes
Complex development
� 3 weeks gestat. to 20 months postnatal
� tightly regulated pathways
involving specific gene
cascades
� several transcription factors
and secreted signaling molecules
involved from Parisi et al, 2003
Shared clinical features of CBCDs
� hypotonia, ataxia
� psychomotor delay, mental retardation
� dysmetria, dysarthria, tremor
� abnormal ocular movements
� (epilepsy, cerebral palsy)
� cranial nerve abnormalities
� breathing and cardio-
vascular abnormalities
� variably associated CNS and
multiorgan defects
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MRI assessment is essential for diagnosisMRI assessment is essential for diagnosis
type of malformation: • hypoplasia
• dysplasia
• (atrophy) site of malformation:
• focal (i.e. vermis)
• generalised
extension of the malformation:• isolated to the cerebellum
• + other midbrain-hindbrain malformations
• + other CNS malformations
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normally formed cerebellar folia - compact
relative size of folia and fissures is normal
hypoplasia
shrunked cerebellar folia
very prominent fissures
atrophy
Boltshauser, AJMG 2004
Atrophy vs hypoplasiaAtrophy vs hypoplasia
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Ponto-cerebellar hypoplasia:
progressive, also atrophy!!!Congenital Defect
of Glycosilation IA:
both atrophy and hypoplasia
Non progressive
cerebellar ataxia:
mimicks atrophy!!
Atrophy vs hypoplasia: areas of confusionAtrophy vs hypoplasia: areas of confusion
Boltshauser, AJMG 2004
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- Joubert syndrome (and related ciliopathies)
- Pontocerebellar hypoplasias
- Poretti-Boltshauser syndrome
- Tubulin-related cerebellar malformations
- Other rare autosomal recessive disorders
- Cerebellar and pancreatic agenesis
- Chudley-McCollough syndrome
- Whole cerebellar hypoplasia in retarded males
- Horizontal gaze palsy and progressive scoliosis
Mendelian disorders (genetic testing available, ++ NGS)Mendelian disorders (genetic testing available, ++ NGS)
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Joubert syndrome and ciliopathies
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9q34 INPP5E Bielas et al, Nat Genet 2009
11 TMEM216 Valente et al, Nat Genet 2010
6q23 AHI1
2q13 NPHP1
12q21 CEP290 Valente et al, Nat Genet 2006
8q24 TMEM67 Brancati et al, Hum Mutat 2008
16q RPGRIP1L
3q11 ARL13B Cantagrel et al, AJHG 2008
4p15 CC2D2A
Xp OFD1
2q24 TTC21B
15q26 KIF7
12q24 TCTN1
12q24 TCTN2
2q33 TMEM237
7q32 CEP41 Lee et al, Nat Genet 2012
11 TMEM138 Lee et al, Science 2012
5p13 C5Orf42
10q24 TCTN3
16q12 ZNF423
16q23 TMEM231
8q13 CSSP1
2q37 PDE6D Thomas et al, Hum Mutat 2013
1q42 EXOC8
17q22 MKS1 Romani et al, OJRD 2014
17p11 B9D1 Romani et al, OJRD 2014
19q13 B9D2
12q21 POC1B Beck et al, Hum Mutat 2014
16p12 KIAA0556
17p13 TMEM107
14q23 KIAA0586 Roosing et al, Elife 2015
2p15 TMEM17 Li et al, PLoS Biol 2015
1p36 CEP104
1p36 NPHP4
5q23 CEP120 Roosing et al, J Med Genet 2016
13q21 PIBF1
11q13 C2CD3
17p13 KIAA0753
all JS-causative genes encode for proteins
implicated in the formation, maintainance or
functioning of the primary cilium
Gene identification in JSGene identification in JS
Am J Hum Genet 2003
Primary cilia control neural and limb patterning, by modulating:
- Sonic Hedgehog pathway
- Wnt / beta-catenin pathway
- planar cell polarity pathway
- (and others…)
Primary cilia regulates the major developmental pathwaysPrimary cilia regulates the major developmental pathways
Valente et al, Nat Rev Neurol 2013
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A novel JS gene links ciliopathies with SHH pathwayA novel JS gene links ciliopathies with SHH pathway
- macrocephaly, macrosomia
- hypertelorism
- frontal bossing
- deep nasal bridge
- polydactyly
- PM delay, intellectual deficit
- mild molar tooth sign
- (polymicrogyria)
De Mori et al, AJHG 2017
WES
SUFU p.Ile406Thr
SUFU p.His176Arg
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SUFU is an essential repressor of the SHH pathwaySUFU is an essential repressor of the SHH pathway
• Oncosuppressor gene
• Biallelic mutations NEVER reported
• Germline and somatic heterozygous loss of
function mutations overactivate Shh pathway and
predispose to cancer (++ medulloblastoma)
Sufu KO is lethal
Conditional KO (limbs,
cerebellum) � polydactyly,
cerebellar hypoplasia
De Mori et al, AJHG 2017
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Mutant Sufu is unstable and more rapidly degraded by the UPSMutant Sufu is unstable and more rapidly degraded by the UPS
IMCD3 cells
overexpressing Sufu-HA
Patients’ fibroblasts
treated with Mg132
(proteasomal
inhibition)
Patients’
fibroblasts
treated with
cycloeximyde
(block of
protein
synthesis)
De Mori et al, AJHG 2017
Missense variants impair the repressor role of SUFUMissense variants impair the repressor role of SUFU
Levels of GLI3 and GLI3R are reduced in basal conditions in mutant SUFU fibroblasts
Several target genes are deregulated in basal conditions and do not
adequately respond to pathway activation
De Mori et al, AJHG 2017
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Do SUFU heterozygous LOF variants predispose to JS?Do SUFU heterozygous LOF variants predispose to JS?
heterozygous rare variants of SUFU found in 12/731 (1.6%) JS probands
cDNA variant protein change proba
nds
frequency
in gnomAD
segregation other
mutated
gene#
c.56G>T p.Gly19Val 1 - healthy father het -
c.109A>G p.Ile37Val 1 2.47e-5 healthy mother het -
c.229G>A p.Val77Met 1 - healthy mother het C5Orf42
c.598-1G>C p.? 1 - healthy mother het -
c.846dupC## p.(Glu283Argfs*3) 1 - healthy mother and
affected sib het
-
c.866G>A p.Arg289Gln 1 1.219e-5 healthy father and
healthy sib het
INPP5E
c.877A>G p.Ile293Val 1 8.127e-6 healthy mother het,
affected sib WT
CEP290
c.895C>T p.(Arg299X) 1 - healthy mother het -
c. 1145C>T p.Pro382Leu 1 4.062e-6 healthy father and
healthy sib het,
affected sib WT
TMEM17
c.1192_1193_delAA p.(Ser399Tyrfs*3) 1 - de novo -
c.1325A>G p.Lys442Arg 1 8.121e-6 healthy father het -
c.1442G>A p.Ser481Asn 1 - healthy father het CEP290
het LOF variants:
3/761 Joubert (0.4%) versus 4/138.629 gnomAD (0.003%)
p<2-16
- SUFU het variants
reported in several
children with
medulloblastoma
- some have
macrocephaly and/or
hypertelorism
- no signs of Joubert
reported in these
children
negative43%
C5orf429%
CEP2908%
AHI16%
CC2D2A6%
TMEM676%
KIAA05863%
RPGRIP1L3%
OFD12%
INPP5E2%
MKS12%
CSPP12%
TMEM2161%
B9D11%
TMEM2371%
other genes6%
Prevalence of known genes and phenotypic correlatesPrevalence of known genes and phenotypic correlates
Cerebello-
oculo-renal
Pure JS (++
retinopathy)
Variable
phenotypes
JS + liver
JS + kidney
- Pure JS
- JS + JATD + OFD
• 416 JS probands
• 120 ciliary genes
- Pure JS (++
polydactyly)
- OFDVI
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Heterogeneous group of autosomal recessive neurodegenerative
disorders with prenatal onset, characterized by cerebellar
hypoplasia/atrophy and variable pontine hypoplasia
Ponto-Cerebellar HypoplasiasPonto-Cerebellar Hypoplasias
Namavar et al, 2011
Clinical features
Hypotonia at birth (severe cases: onset in utero, contractures )
feeding difficulties, failure to thrive
abnormal ocular movements
developmental delay, intellectual disability
spasticity, hyperreflexia
breathing abnormalities
epilepsy
progressive microcephaly
Variable multiorgan involvement (retina, skeleton, others…)
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PCH GENE LOCUS CLINICAL FEATURES
PCH1EXOSC3, VRK1
TSEN54, RARS2
9p13
14q32
Onset at birth
axonal motor neuropathy
PCH2
TSEN54
TSEN2
TSEN34
17q25
3p25
19q13
Onset at birth, dyskinesias / chorea
vermis less hypoplastic than hemispheres
(dragonfly appearance)
PCH3 Not known 7q11-q21Onset at birth, optic atrophy
hearing impairement
PCH4/PCH5 TSEN54 17q25 Fetal onset, myoclonus
PCH6 RARS2 6q15Onset at birth
Reduced activity of mito respiratory chains
PCH8 CHMP1A 16q24 Onset at birth
CASK CASK Xp11progressive microcephaly, ID, deafness
Dragonfly/butterfly, normal CC
PCH9 AMPD2 1p13unspecific clinical features, death by age 10
CC hypoplasia, «8» shape of vermis (axial cut)
PCH10 CPL1 11q12Slow onset, progressive spasticity/seizures
Absent or delay speech
Ponto-Cerebellar Hypoplasias: clinical and genetic classification
Navamar 2010; Burglen 2012
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PCH screening (CBCD Italian group)PCH screening (CBCD Italian group)
• unique inclusion criterion: PCH
confirmed on neuroimaging
• 63 probands analyzed
• 44 genes
CASK mutations
Males:
missense or splice
Males:
mosaic loss of function
severe missense
Females:
het loss of function
Males:
hemiz. loss of function
progressive microcephaly
variable PCH
moderate to severe DD/ID
very limited language
seizures, short stature, scoliosis
sensory-neural hearing loss
butterfly (also dragonfly)
normal corpus callosum
DD/ID
nystagmus
seizures (not severe)
(short stature)
(tremor, hypotonia)
(dysmorphism)
most severe clinical presentation
severe to profound DD
rapidly progressive microcephaly
cortical atrophy / hypomielination
severe intractable seizures
(congenital heart defects, contractures)
often lethal
Burglen 2012, Saitsu 2012, Moog 2015
neg
34%
CASK
37%
TSEN54
17%
EXOSC3
5%
RARS2
2%
VLDLR
2%
PPM2/ATP2B
3
2%
TOE1
2%
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The importance of deep phenotyping: TOE1The importance of deep phenotyping: TOE1
Lardelli et al, Nat Genet 2016
12 families collected worldwide with PCH and ambiguous genitalia
biallelic loss of function mutations in TOE1 (role in RNA splicing)
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Cerebellar dysplasias: sporadic or autosomal recessive Cerebellar dysplasias: sporadic or autosomal recessive
from small focal portions of a single hemisphere to
abnormal foliation of the entire cerebellum
High resolution MRI
and DTI-Tractography studies
Extremely variable clinical presentation:
• Incidental finding with no clinical relevance
• Severe neurological impairment
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Cerebellar dysplasia with cerebellar cystsCerebellar dysplasia with cerebellar cystsAicardi syndrome
PMG (GPR56)
Congenital muscular dystrophies
- PMG (A>P gradient)
- Patchy / diffuse white matter abnormalities
- Hypoplastic pons
- Females with infantile spasms and
ophthalmological abnormalities
- agenesis/dysgenesis CC
- wide areas of PMG, heterotopias
- abnomal myelination
- Muscle weakness, hypotonia, contractures, �CK
- Seizures, ophthalmological abnormalities
- Typical supratentorial malformations (e.g.
cobblestone cortex, PMG, white matter changes)
- Hypoplastic pons, other brainstem defects
Boltshauser et al, 2014
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Autosomal recessive cerebellar dysplasia with cystsAutosomal recessive cerebellar dysplasia with cysts
�Ataxia, Intellectual disability
�Language impairment
�High myopia or retinal dystrophy
�Diffuse dysplasia with cysts
�Square-like IV ventricle
�Splayed SCP
Report of 15 patients
Mutations in ~100% cases
1 founder mutation (~40%)
No genotype-phenotype
correl.
Aldinger et al, 2012; Micalizzi et al, 2015
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Cerebellar dysplasia in tubulinopathiesCerebellar dysplasia in tubulinopathies
Tubulin gene mutations:
• Autosomal dominant inheritance (++de novo)
• Malformations involving:
• Cerebral cortex (Lys, pachigyria, PMG…)
• Commissural tracts
• Basal ganglia
• Pons
• Cerebellum
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Cerebellar dysplasia in tubulinopathiesCerebellar dysplasia in tubulinopathies
Our data (22 patients):
Cerebellar involvement found in 18 patients (82%)
Most cases (16/18) showed a typical pattern:
- unilateral hemispheric dysplasia with right prevalence
- mostly localized in postero-superior regions
- regular aspect of the cerebellar cortex
- frequently associated vermian dysplasia or hypoplasia
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cerebellar agenesis + pancreatic agenesis
• severe IUGR, microcephaly, death in infancy
• dysmorphisms
• marked cerebellar hypoplasia / agenesis
• neonatal diabetes, near absence of subcutaneous fat
PTF1A (recessive)
Rare and peculiar cerebellar/brainstem malformationsRare and peculiar cerebellar/brainstem malformations
Chudley-McCullough Syndrome
• cerebellar dysplasia
• hydrocephalus, partial CC agenesis
• frontal PMG, gray matter
heterotopias
• profound sensorineural hearing loss
•(mild motor/cognitive delay)
GPSM2 (recessive)
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Whole cerebellar hypoplasia in retarded males
• moderate-severe intellectual impairment
• hypotonia, developmental delay, no clear ataxia
• (seizures, tall stature, hypoplastic genitalia, macrocephaly)
• moderate whole cerebellar hypoplasia
• ventriculomegaly, mild frontotemporal atrophy
OPHN1 (X-linked recessive)
Rare and peculiar cerebellar/brainstem malformationsRare and peculiar cerebellar/brainstem malformations
horizontal gaze palsy +
progressive scoliosis
• congenital absence of horizontal gaze
• severe progressive scoliosis
• absence of decussation of corticospinal and
somatosensory axonal tracts in the medulla
ROBO3 (recessive)
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- Isolated cerebellar vermis hypoplasia
- Dandy-Walker syndrome
- Romboencephalosynapsis
- Pontine Tegmental Cap Dysplasia
«Sporadic» disorders (genetic testing mostly unavailable)«Sporadic» disorders (genetic testing mostly unavailable)
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Isolated CVH still remains without genetic definitionIsolated CVH still remains without genetic definition
High resolution SNP-Array in 110 pazients:
� Isolated vermis hypoplasia
�Whole cerebellar hypoplasia
�Cerebellar (hypo)dysplasia
�PTCD ( Pontine Tegmental Cap Displasia)
�Romboencefalosynapsis
�Dandy Walker Malformation (DWM)
10% of CNV “de novo”, all in
syndromic cases
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• hypoplasia and upward rotation of the
cerebellar vermis, hemispheres less affected
• cystic dilatation of the fourth ventricle
• tentorium displaced
• (CC agenesis, other CNS defects)
Common clinical features: • Obstructive Hydrocephalus (50-80%)
• Hypotonia evolving into spasticity
• Ataxia, abnormal ocular movements, seizures
• DD, intellectual impairment
• Associated organ malformations (e.g. heart)
Dandy Walker syndromeDandy Walker syndrome
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Del3q contiguous gene syndromeDel3q contiguous gene syndrome
Ferraris et al, 2013; Willemsen et al, 2011
BPES
blepharophimosis
ptosis, epicantus inversus
upslanting palpebral fissures,
unusually shaped eyebrows,
coarse nose, large mouth with
full lower lip, umbilical hernia
digital abnormalities: short 4th
metatarsals, recessed 4th toes
Wisconsin syndrome
del3q22.q25 del3q23.q25
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Rare sporadic malformations with genetic defect unknownRare sporadic malformations with genetic defect unknown
missing cerebellar vermis with apparent
fusion of hemispheres
1) Isolated
2) Gomez-Lopez-Hernandez
3) VACTERL
± Other SNC anomalies (aqueductal stenosis,
absent olfactory bulbs, CC dysgenesis…)
romboencephalosynapsis
Pontine tegmental cap dysplasia• cerebellar vermis hypo-dysplasia
• near absence of MCP, lateralized SCP
• flattened ventral pons with tegmental CAP
- multiple cranial nerve anomalies (neurosensorial
deafness, corneal opacities, oculomotor anomalies,
facial paralysis, difficulties in chewing and swallowing)
- cardiac, gastrointestinal, genitourinary and skeletal
defects (++ribs, vertebrae)
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WES of the two probands �
pathogenic recessive mutations in GSX2
Toresson et al,
Development 2000
2 unrelated sporadic patients with unspecific clinical features (DD, tetraparesis,
hyperkinetic movements) but identical and highly peculiar MRI pattern:
control patient 1
hypothalamic-
midbrain fusion
absence of
putamina
absence of
olfactory tracts
KO mouse model
fully recapitulates
CNS malformations
seen in patients
control Gsh2 ko mouse (E16.5)
De Mori et al, manuscript in preparation
GSX2
α-Actin
38 -
42 -
The importance of deep phenotyping: GSX2The importance of deep phenotyping: GSX2
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- Mild psychomotor delay
- Mild cerebellar signs
- Bilateral congenital
cataract
Seeking for novel genes…Seeking for novel genes…
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Take home messagesTake home messages
• Diagnosis must be seached by combining clinical and imaging features:
deep phenotyping is essential!!!
• Genetic testing now available for a subset of malformations (++
autosomal recessive or X-linked), but high genetic heterogeneity
• Many causative genes still have to be identified: share patients!
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III CBCD meeting
Rome, 29 Apr 2011
AcknowledgementsAcknowledgements
Eugen Boltshauser (Zurich)
Andrea Poretti (Baltimore)
Joe Gleeson (San Diego)
All clinical collaborators
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