Introducing the Cerebellum Mid-Hindbrain Morphogenesis

Home , Cerebellar hemisphere, Cerebellar vermis, Cerebellum

1/19/2017

Introducing the cerebellum

• Highly conserved structure and function – Foliation conserved across evolution • Structure is “simple” – Only 9 principle types of neurons – All morphologically distinct – Layers and circuitry are stereotyped • Contains more neurons than rest of brain – In mouse, 59/71 million neurons (83%) – In human, 69/86 billion neurons (80%) • Malformations in mice and in humans – Window into developmental neurogenetics Canary in the coal mine: the cerebellum as a –“Canary in the coal mine” for rest of brain sentinel for developmental brain disorders

Mid-hindbrain morphogenesis Famous paper towel model - CBL

Mouse 9 days 9.5 days 10 days 10.5 days Human 25 days 30 days 36 days 39 days

V H V H V H H

Rostro-caudal  medio-lateral

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Isthmic organizer CBL from dorsal anterior hindbrain

Glutaminergic

GABAergic

Late development of cerebellum Late development GW37

CBL GW7.5

In mice GW9 and man

2mm

2mm GW17

2mm GW23

GW26

2mm

2mm Bayer and Altman 2004: Human CNS Development atlas

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Foxc1 is a mesenchymal TF Mesenchymal signaling in the PF

+/- • Foxc1 transcription factor is expressed in overlying mesenchyme NOT cerebellum WT Foxc1 -/- ▫ directly regulates mesenchyme development (posterior fossa) Foxc1 ▫ indirectly regulates cerebellar development via Foxc1 cbl cbl transcriptional control of secreted factors SDF1α e12.5 ▫ Loss of function causes major midline rhombic  SDF1α lip problem Glu 4V

+/+ -/- Foxc1–/– GABAergic e14.5

RL Normal CBL CBVH – CBVH/MCM - DWM

Normal mid-hindbrain structure in Mid-hindbrain malformations humans • Incidence – Relatively common ~1/5000 live births •Outcome – Most cause DEV delay ± ID ± motor abnormalities – Represent “tip of the iceberg” for DEV disorders • ID and ID syndromes, autism, early life epilepsy • Causes – Can occur in isolation or part of syndrome – Genes identified for only a few rare forms • Ciliopathies (JS/MGS), CDG, PCH, and a few others • Prenatal issues – Most (not all) are visible by GW20 but – Difficult to distinguish by fetal ultrasound/MRI

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Cerebellar hypoplasia (CBLH) is a common finding associated with a highly heterogeneous group of disorders. Causes include prenatal infections and Mid-hindbrain … exposure to teratogens, chromosomal anomalies, metabolic disorders, genetic syndromes, and more. • Early patterning defects – CBL agenesis – Altered segmentation (GTAV) – Rhombencephalosynapsis • Common imaging patterns –CBLHdiffuse – CBVH isolated – DWM-MCM-CBVH spectrum • Special classes of MHM – Cobblestone malformations – Molar tooth malformation (JS) – Pontocerebellar “hypoplasia”

Dandy-Walker malformation DWM and intellectual disability

50 • The 800-pound gorilla in the room . . . . . 45 percent • Diagnostic criteria • Data from 104 DWM – cerebellar vermis hypoplasia and upward rotation 40 patients – Cystic enlargement 4V (wide outflow tract) 35 – 6 references – Enlarged posterior fossa with elevated torcula • Common associated malformations 30 – 1981 – 1995 – Cerebellar hemisphere hypoplasia 25 – Mortality 24% – Brainstem hypoplasia autism 20 – Hydrocephalus 20-80% • Most from Neurosurgery • Phenotype 15 clinics –Ataxia 10 – Intellectual disability 40% 5 – Motor, visual problems – Epilepsy 0 IQ DWM LR09-094 LR09-094 >80 56-79 <55

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CBLH subtypes and outcome Cerebellar hypoplasia variations

• In the MFM literature • Dandy-Walker malformation (DWM) – Classic DWM with tail – DWM is “bad” – Classic DWM no tail – CBVH – vermis – DW “variant” is not so bad 1978 – CBVH with mega-cisterna magna (MCM) • Where did this come from? • Posterior fossa cysts – Blake’s pouch cyst • In Lisdb database and DEV – Mega-cisterna magna only literature – Arachnoid cysts – CBLH and PCH are bad • Cerebellar hypoplasia (CBLH) – Cerebellar agenesis (CBAG) – CBVH is bad when it is real – CBLH – diffuse – CBVH – vermis – DWM is bad only half the time – CBLH unilateral-asymmetric • So DW “variant” has proven to • CBLH other specific types be a terrible name – Molar tooth malformation (Joubert) – Pontocerebellar hypoplasia (PCH)

LR01-276

Cerebellar hypoplasia variations Cerebellar hypoplasia variations

• Dandy-Walker malformation (DWM) • Dandy-Walker malformation (DWM) – Classic DWM with tail – Classic DWM with tail – Classic DWM no tail – Classic DWM no tail – CBVH – vermis – CBVH – vermis – CBVH with mega-cisterna magna (MCM) – CBVH with mega-cisterna magna

• Posterior fossa cysts • Posterior fossa cysts – Blake’s pouch cyst – Blake’s pouch cyst – Mega-cisterna magna only – Mega-cisterna magna only – Arachnoid cysts – Arachnoid cysts

• Cerebellar hypoplasia (CBLH) • Cerebellar hypoplasia (CBLH) – Cerebellar agenesis (CBAG) – Cerebellar agenesis (CBAG) – CBLH – diffuse – CBLH – diffuse – CBVH – vermis – CBLH unilateral-asymmetric – CBLH unilateral-asymmetric – CBVH – vermis

• CBLH other specific types • CBLH other specific types – Molar tooth malformation (Joubert) – Molar tooth malformation (Joubert) – Pontocerebellar hypoplasia (PCH) – Pontocerebellar hypoplasia (PCH)

LR02-196 LR13-199

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Cerebellar hypoplasia variations Cerebellar hypoplasia variations

• Dandy-Walker malformation (DWM) • Dandy-Walker malformation (DWM) – Classic DWM with tail – Classic DWM with tail – Classic DWM no tail – Classic DWM no tail – CBVH – vermis Dup 6p25.3 including FOXC1 – CBVH with mega-cisterna magna – CBVH with mega-cisterna magna – CBVH – vermis

• Posterior fossa cysts • Posterior fossa cysts – Blake’s pouch cyst – Blake’s pouch cyst – Mega-cisterna magna only – Mega-cisterna magna only – Arachnoid cysts – Arachnoid cysts

• Cerebellar hypoplasia (CBLH) • Cerebellar hypoplasia (CBLH) – Cerebellar agenesis (CBAG) – Cerebellar agenesis (CBAG) – CBLH – diffuse – CBLH – diffuse – CBLH unilateral-asymmetric – CBLH unilateral-asymmetric – CBVH – vermis – CBVH – vermis

LR06-253a1 unknownAJB

Cerebellar hypoplasia variations Cerebellar hypoplasia variations

• Dandy-Walker malformation (DWM) • Dandy-Walker malformation (DWM) – Classic DWM with tail – Classic DWM with tail – Classic DWM no tail – Classic DWM no tail – CBVH with mega-cisterna magna – CBVH with mega-cisterna magna – CBVH – vermis – CBVH – vermis

• Posterior fossa cysts • Posterior fossa cysts – Blake’s pouch cyst – Blake’s pouch cyst – Mega-cisterna magna only – Mega-cisterna magna only – Arachnoid cysts – Arachnoid cysts

LR13-153 LR06-201

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Cerebellar hypoplasia variations Cerebellar hypoplasia variations

• Posterior fossa cysts • Posterior fossa cysts – Blake’s pouch cyst – Blake’s pouch cyst – Mega-cisterna magna only – Mega-cisterna magna only – Arachnoid cysts – Arachnoid cysts

LR09-114 LR03-279

Cerebellar hypoplasia variations Cerebellar hypoplasia variations

• Posterior fossa cysts • Posterior fossa cysts – Blake’s pouch cyst – Blake’s pouch cyst DP99-044a2 LR01-303 – Mega-cisterna magna only – Mega-cisterna magna only – Arachnoid cysts – Arachnoid cysts

• Cerebellar hypoplasia (CBLH) • Cerebellar hypoplasia (CBLH) – Cerebellar agenesis (CBAG) – Cerebellar agenesis (CBAG) – CBLH – diffuse – CBLH – diffuse – CBLH unilateral-asymmetric-clefts – CBLH unilateral-asymmetric – CBVH – vermis – CBVH – vermis

LR14-232 LR01-340

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Figure 1. Patterns of cerebellar malformations in Venn diagram

Cerebellar hypoplasia variations

• Dandy-Walker malformation (DWM) – Classic DWM with tail – Classic DWM no tail – CBVH with mega-cisterna magna – CBVH – vermis

• Posterior fossa cysts – Blake’s pouch cyst – Mega-cisterna magna only – Arachnoid cysts

• Cerebellar hypoplasia (CBLH) – Cerebellar agenesis (CBAG) – CBLH – diffuse – CBLH unilateral-asymmetric – CBVH – vermis • CBAG, cerebellar agenesis (total) • SCA, spinocerebellar ataxia {degeneration) • CBLH other specific types • MIC-CBLH, severe congenital microcephaly with • RES, rhombencephalosynapsis – Molar tooth malformation (Joubert) disproportionate cerebellar hypoplasia • CBVH, cerebellar vermis hypoplasia – Pontocerebellar hypoplasia (PCH) • CBLH, cerebellar hypoplasia {diffuse) • MTM, molar tooth malformation (Joubert) • PCH, pontocerebellar hypoplasia • DWM, Dandy-Walker malformation LR08-085a1 • ICCA, infantile cerebral and cerebellar atrophy • MCM, mega-cisterna magna

Causes of cerebellar hypoplasia Causes of DWM and CBLH

Extrinsic (non-genetic) Genetic syndromes – estimates – • Prenatal hemorrhage • Copy number variants (CNV) – Chromosome abnormalities – Cerebellar Other – Posterior fossa – Small CNV • Genetic syndromes with CBLH • Prenatal HIE – Metabolic disorders • Prenatal infections MFM – Brain malformations MFM SEQ CNV –CMV – Recognizable syndromes SEQ –Zika – Epileptic encephalopathies and Other • Teratogens similar DEV disorders CNV – Anticonvulsants (Dil, VP) • Complex NDEV disorders – Retinoic acid – Autism, schizophrenia and psychosis, bipolar disorder – Alcohol, cocaine

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Mechanisms Extrinsic causes of CBLH

– Prenatal hemorrhage • MFM • PF and CBL – Prenatal infections • CNV •CMV • Zika virus – Teratogens • SEQ • Antiepileptic drugs – Phenytoin, • Other valproate • Alcohol; cocaine • Retinoic acid

Retinoic acid exposure at 6 weeks gestation; no cardiac defects LR13-178

Limperopoulos et al. 2005 in PEDIATRICS CBL bleeds premies 35 of 1242 infants <1500 g CBL bleed and DWM

Prenatal 19 wkg

9 1/19/2017

CBL bleed and DWM CBL bleed and DWM

26 weeks 32 weeks 5 days 1 year

• Prenatal 27 wkg – AV canal, diffuse CBLH • Prenatal 31 wkg – CBLH, enlarged 4V but normal PF, hemosiderin on CBL • Postnatal 5 days – CBLH, enlarged 4V but normal PF, hemosiderin on CBL • Postnatal 1 year –CBLH  DWM, enlarged 4V and enlarged PF, no hemosiderin 26 weeks 5 days 1 year

Mechanisms Copy number variants (CMV)

N DX CYTO Copy Genome position hg19 Genes • MFM ~10 DWM 3q24 1 chr3:142960932-149959022 ZIC1, ZIC4 ~20 DWM 6p25.3 1 chr6:1-2300000 FOXC1, FOXF2 • CNV ~02 DWM 7p21.3 1 chr7:10,818,761-11,268,476 ~02 DWM 7p21.3 3 chr7:10,358,477-11,060,11 • SEQ ~01 DWM 8p21.2p21.3 1 chr8:23061616-25293157 FGF17 (?) ~15 DWM 9p24.2p21.3 3 chr9:2424485-24111280 • Other ~17 DWM 13q32.2q33.1 1 chr13:98685966-104622258 ZIC2 (?) ~07 CBVH 17p13.3 3 chr17:1235314-2183796 HIC1 (?) NDWMTrisomy133 NDWMTrisomy183 NDWMTrisomy213

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DWM and FOXC1

CNV are rare CNV are rare in CBLH-DWM

• Compare – Agenesis corpus callosum – CBLH N=220 – Polymicrogyria

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Mechanisms CBVH and OPHN1

• MFM • CNV • SEQ OPHN1–/Y LR02-469a1 OPHN1–/Y LR02-469a2 • Other

OPHN1–/Y LR02-469a3 OPHN1–/Y LR02-469a4

CBLH and CASK CBLH and CASK

CASK+/– or CASK–/Y

LR04-131 LR01-361 LR04-131 LR07-206 LR07-135

LR06-130 NORMAL

LR07-040 LP99-156 LR01-361 LR07-040 LP99-156

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WES in 71 families with CBLH WES in 71 families with CBLH

• CBLH all types including DWM • DEV disorder – known genes – N=13/71 (18%) solved probable – BCL11A (x2), DDX3X (x2) – N=21/71 (30%) solved probable or possible – AHDC1, AUTS2, CASK, FOXP1, SETD2, STXBP1 • CBLH excluding DWM – N=10/42 (24%) solved probable • DEV disorder – brain malformation genes – N=16/42 (38%) solved probable or possible – FGFR1, TUBA1A

• DWM classic • DEV disorder – candidate genes – N=3/29 (10%) solved probable – ADCY1, BRAF, BRAT1, KCTD1, KLHL17, TRIO, – N=5/29 (17%) solved probable or possible WDR37 p=0.2150

CBLH and AUTS2 CBLH and AUTS2

LR05-007

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DWM and SETD2 CBLH with AD or XL inheritance

• Developmental encephalopathies – AHDC1, AUTS2, BCL11A, FOXP1, PITX2, SETD2 • Developmental epilepsies – KPNA7, STXBP1 • Cerebellar hypoplasia (relatively pure) – FGFR1, FOXC1-6p25.3, ZIC1-ZIC4, NID1, LAMC1 • Malformations of cortical development – TUBA1A, TUBB2B, TUBB, TUBB3, DYNC1H1 • CBLH with X-linked inheritance – AP1S2, CASK, DDX3X, OFD1, OPHN

LR12-434

CBLH with AR inheritance CBLH and WNT1

• Transcription factors – PTF1A, WNT1 • Ciliopathies and MTM – AHI1, CEP290, RPGRIP1L and ~30 others • Dystroglycanopathies and lamininopathies – POMT1, POMT2, POMGnT1, ~15 others, LAMA2, LAMB1 • Pontocerebellar hypoplasia (tRNA splicing) – TSEN54, AMPD2, CHMP1A, RARS2, TSEN2, TSEN34, EXOSC3, VRK1 • Congenital disorders of glycosylation – B3GALTL, B4GALT1, PMM2 and others

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Mechanisms DWM in BWS

Is DWM due to an • MFM imprinting defect? • CNV • SEQ • Other

DWM in BWS DWM in BWS (11p15.4 imprint)

Patient Mutation • BWS and DWM • Bui 2009LOM at IC2 – Loss of methylation IC2 6 • Patient_1 LOM at IC2 – Mutations CDKN1C 3 • Patient_2 LOM at IC2 • Patient_3 LOM at IC2 – Unknown 1 • Patient_4 • Patient_5 CDKN1C p.Gly261fsX • Patient_6 CDKN1C p.Gln58X • Patient_7 LOM at IC2, del 11p15.4 • LR13-168 LOM at IC2 • LR13-313 CDKN1C p.Ala211GlyfsX26

Gardiner pt1 Gardiner pt2 LR13-313

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MZ twins DWM in MZ twin DWM in MZ twins

• Complicated monochorionic twin pregnancies – DWM in 10/660 (1.5%) – But NONE had confirmed DWM!

• Growth abnormalities are common – Non-DWM twins 217/650 (33.4%) • p = 0.03 significant – DWM twins 7/10 (70%) LR12-308 LR12-308 • Either DONOR twin or just IUGR – Twin-twin transfusion syndrome (TTTS) N=4 – Intrauterine growth retardation (IUGR) N=5

• Summary from Kontopoulos et al – Incidence of DWM in complicated monochorionic twins is 200 times expected for singletons • DWM more likely in smallertwin • Mechanism could be related to either TTTS orIUGR H and H from MN DWM LR12-308