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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