American Journal of Obstetrics amp Gynecology 2015 Alastair MacLennan Emeritus Professor Obstetrics amp Gynaecology Suzanna Thompson Paediatric Neurology Jozef Gecz Professor of Neurogenetics Research ID: 935306
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Cerebral Palsy – Multiple pathways, outcomes and genetic variantsAmerican Journal of Obstetrics & Gynecology 2015Alastair MacLennan, Emeritus Professor Obstetrics & Gynaecology,Suzanna Thompson, Paediatric Neurology,Jozef Gecz, Professor of Neurogenetics ResearchThe University of Adelaide, South Australia
Slide21717 million affected worldwideNeed to look for causes well before birth
Slide3One third of cerebral palsy follows preterm delivery
Slide4Most cases of cerebral palsy have antenatal origins
Slide5Most causes of cerebral palsy are hard to identifyImage: Lennart Nilsson
Slide6Need to consider genetic variants at conception
Slide7Need to consider inherited genetic variants in GametesImage: Lennart Nilsson
Slide8“The Cerebral Palsies” - many typesMany clinical types (quadriplegia, diplegia, hemiplegia) Many co-morbiditiesMany pathways to a common motor dysfunctionMany causes (genetic, inflammatory, preterm etc.)Many neuropathology patterns on imagingMany putative causative mutations
Slide9Dispelling the assumption of “birth asphyxia”The incidence of acute severe hypoxia causing cerebral palsy is uncommon and has been identified in only 2% of cerebral palsy cases using objective international criteria MacLennan AH. BMJ 1999;319:1054-9Hankins GDV, Speer M. Obstet Gynecol 2003;102:628-36ACOG and AAP Task Force on Neonatal encephalopathy. 2014Most acute severe hypoxia follow intrapartum hypoxic events e.g. cord prolapse, uterine dehiscence, placental abruptionNormal arterial cord gases and placental histology often suggest longer standing pathology. Collect this evidence!- Wong L, MacLennan AH. Aust NZ J Obstet
Gynaecol 2011;51:17-21
Slide10Common Myth of Cerebral Palsy CausationRarely due to “Birth asphyxia”Most causes precede labour and birth Neurologically compromised may appear unwell at birthMedico-legal climate encourages blame of the insured20% of UK maternity budget is spent on litigation
Similar in Australia - $300+ million settlements/year
Major cause of caesarean escalation and adverse sequelae
Slide11Defensive obstetrics and EFM have contributed greatly to the escalation of caesarean delivery in AustraliaAustralian Bureau of Statistics; The Australian Cerebral Palsy Register Report 2013CP Rate per 1,000 births
Slide12Caesarean Delivery has had NO impact on cerebral palsy ratesSystematic review and meta-analysis of all 9 case-control and 4 cohort studies in literature O’Callaghan M, MacLennan AH. Obstet Gynecol 2013;122:1169-75No association of elective or emergency caesarean with CP outcomes OR 1.29 (0.92-1.79)
Slide13Known epidemiological risk factors and genetic evidence for CPPrematurityCongenital abnormalitiesFetal growth restrictionAltered fetal inflammatory responsePlacental pathology (chorioamnionitis, villitis)
Perinatal stroke and altered coagulation factors
Monozygotic twins higher than dizygotic twins. IVF further increases this risk
Tight nuchal cord and prolonged shoulder dystocia
2.5 times higher consanguineous families
Male
: Female 1.4:1
The Australian Cerebral Palsy Register Report 2013
Slide14Chromosome numberChromosome rearrangementGenomic copy number variations Individual gene mutationsEpigenetic adaption of function
Genomic changes
Slide15Investigating genetic contributions to cerebral palsyThe Australian Collaborative Cerebral Palsy Research Group Major funding from NHMRC, Cerebral Palsy Foundation and Tenix Foundation
Slide16Next Generation Sequencing TechnologyUsing older technologies single gene mutations were identified in 1-2% of cerebral palsy cases - mostly familialGenome wide association studies showed weak CP association with pro-inflammatory cytokines e.g. IL 6, IL 8, TNF and MBL Whole-exome sequencing (
WES)- better technology
WES sequences the protein coding regions of the genome 180,000 exons (~ 1% human genome)
Genome contains ~23,000
protein-coding genes
86
% of
disease
causing mutations
are
found in
protein
coding
genes
Slide17Where we look for disease causing mutationsAdapted from Manolio, T.A. et al (2009), Nature, 461(7265):747-53
Slide18Prevalence to date of potentially causative genetic alterations in other neurodevelopmental disordersRare variants – large effectNeurodevelopmentaldisordersWES Intellectual disability
16%
Autism spectrum
14%
Epilepsy
10%
Slide19Mutations are common in everyoneA mutation is a permanent change in the DNA sequence. Mutations range in size from a single DNA building block to a large segment of a chromosome (copy number variation).They can be inherited from a parent or acquired (de novo)Many have no effect on body functionSome are lethal or result in known disability syndromesMany are unsuspected but may cause various pathologies Exome/Whole G. Sequencing uncovers many new mutations
To prove causation many criteria must be satisfied
Slide20DNA variant and gene prioritisation for pathogenicityType of variant (i.e. stopgain, splice, missense) These are likely to disrupt functionIn silico prediction of functional effect
Disrupt normal protein production
Evolutionary conservation
Regions
of structural or functional
importance
Haploinsufficiency index
When a single functional copy of a gene is insufficient to maintain normal function
Brain expression pattern
RNA disruption in brain
Known disease association (OMIM)
Registered on
Online
Mendelian Inheritance in Man
regist
er
Then prioritisation with
Residual
Variation Intolerance
and
Combined
Annotation-Dependent
Depletion Scores
Slide21Results of WES in 98 case-parent CP trios McMichael et al. Mol Psych 2015;20:176-8257 of cases had validated genetic variants14% were deemed likely to be pathogenic by strict bioinformatic criteria8 were novel genes in CP 5 were known disease genes with CP as a new phenotypeAnother 44% had variants of lesser bioinformatic priorityAll of these variants require function tests to help determine pathogenicity
Slide22GeneTypeProtein changeFunctionDisease associationKDM5CDN
P480L
Histone demethylase
ID; spasticity
SCN8A
DN
G1050S
Sodium
i
on channel
Cognitive impairment; epilepsy
TUBA1A
(x2)
DN
R123C;
L152Q
Neuronal
migration
ID;
Lissencephaly
L1CAM
X
Q161A
Neurite
outgrowth
ID;
Hydrocephalus
PAK3
X
R493C
Synapse formation
ID; epilepsy
AGAP1
DN
Splice
Adaptor-protein 3
JHDM1D
DN
S727W
Histone demethylase
MAST1
DN
P500L
NAA35
DN
W532C
N-terminal acetyltransferase C complex
RFX2
DN
Y91C
Encodes transcription factors
WIPI2
DN
Y246C
Component
of autophagy machinery
ODZ1
X
G2533S
Cellular signal transducer
CD99L2
X
Stopgain
Homophilic
adhesion molecule
Slide23Copy Number Variations at cell division
Deletion
Duplication
Inversion
Figure modified from Eichlerlab.gs.washington.edu/research.html
Slide24Novel Copy Number Variations In Cerebral PalsyBoth de novo and inherited CNVs found in cerebral palsyof likely pathogenicity require function studies for confirmationInherited genetic variants where no parental CP phenotype may be susceptibility genes triggered by environmental factorsThe Australian CP Research group in 2013 found CNVs in 20% of unselected CP cases using arrays and WES (McMichael et al)
Segel
et al (Israel) in 2015 found “clinically significant” CNVs in 31% of selected CP cases of unknown aetiology using array
Slide25CNVs and exome sequencing are discovering a genetic basis for many neurodevelopmental disordersDisorderCNVs
Exome sequencing
Total
to date
Intellectual disability
10 -15%
15%
30%
Autism
10 -15%
15%
30%
Epilepsy
8%
10%
18%
Cerebral Palsy
20-31%
14%
34-45%
Slide26From mutation to clinical outcomeIdentified 127 genetic variants thus far: how can we tell if they are causative or coincidental?Studying functional outcomes of mutations:in patient cell linesin animal models
WES - genetic variation
Cellular phenotype e.g. RNAseq
Animal model – phenotype in organism
Slide271. Functional studies in patient cell lines RNA sequencing – tells us about the functionality of a gene and effects on the cell as a whole.
Also stem cell and neuronal function studies
These help prioritise mutations for animal function studies
Slide282. Functional studies in animal modelsLooking at effects of genetic variation on whole organism (mice, flies, zebrafish)Zebrafish– small, fast breeding, embryos develop rapidly, optically transparent embryos, vertebrate therefore shares majority of genes with humans24h
Slide292. Functional studies in animal modelsInjection of modified RNA molecule into zebrafish embryo at 1 cell stage used to reduce gene expression – look for phenotypei.e. swim defect. Rescue of motility with normal RNA
CTRL injected –
72h
Swims off when touched
ZC4H2 mo injected –
72h
Little response to stimulus
Slide30Genetic Susceptibility and Environmental Triggers?Epigenetic interaction likely with known risk factors for CPe.g. Prematurity, IUGR, infection, acute and chronic hypoxiaScreening, outside research projects, for CP mutations is not yet recommended due to their heterogeneity, required confirmatory function studies and complex bioinformatic interpretationThe future challenge will be early recognition of CP susceptibility perhaps by early antenatal diagnosis using fetal DNA in the maternal circulation
Then early intervention may be possible with gene therapy or treatment/prevention of the environmental trigger.
Slide31Gene silencing, gene insertion, gene modification
Slide32Robinson Research Institute
The University of Adelaide
Prof Alastair MacLennan
Jessica Broadbent
Dr Clare Van Eyk
Gai McMichael
Kelly Harper
Josh
Woenig
Bregje
van Bon
Dept
of Neurogenetics
The University of Adelaide
Prof Jozef Gecz
Dr Mark Corbett
Dr Alison Gardner
Dr Morgan Newman
Dr Michael Lardelli
Western Australia
Princess Margaret Hospital,
Dr
Jane Valentine
Peta Watts
New South Wales
SCH
Dr Kevin Lowe
Dr Michael
Stenning
Amana
Walkaden
Nisha
Berthon
-Jones
CHW
Dr Mary-Clare Waugh
Dr Matthias
Axt
Dr Brian Martin
Twinkle Bahaduri
Queensland (LCCH)
Dr Lisa Copeland
Dr Theresa Carroll
Megan Kentish
Rebecca
Kratchman
Vicky
Witherford
Genetics and Molecular
Pathology, SA Pathology,
WCH, Adelaide, SA
Prof Eric Haan
Dept
of
Paediatric
Rehabilitation, WCH, Adelaide, SA
Dr
Ray Russo
Dr
James Rice
Dr
Andrew
Tidemann
Baylor College of Medicine,
HGSC, Houston, Texas
Dr
Richard Gibbs
Dr
Matthew Bainbridge
Acknowledgements
Australian
Collaborative Cerebral Palsy
Research
Group
Slide33Our Funders
Australian NHMRC
Research Foundation, Cerebral Palsy Alliance
Tenix Foundation
Adelaide
Women’s and Children’s Hospital
Many many thanks
Acknowledgements