coupling sequencing and functional studies in neonates · 2020. 1. 21. · plp1 and gjc2 sequencing...
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Coupling sequencing and functional studies in neonates
Nicholas Katsanis, Ph.D.
Center for Human Disease Modeling
Duke University Medical Center
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1. Birth defects: 21% of infant deaths
2. Mortality rates plateaued
3. 5-10% of repeat admissions
Why babies (apart from the obvious!)
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1. Early diagnosis/prognosis
2. High-value information content (families)
3. Focused clinical investment
4. Maximum therapeutic window
Why (part II)
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1. Communication across disciplines
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2. Information management
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3. Ethical transmission of information
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A bold new activity
Duke University Task Force for Neonatal Genomics
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synthesis of disciplines around patient needs
clinics genomics
ethics function
patient
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The process 1. Trio-based recruitment 2. WES/WGS 3. Data filtering 4. Systematic functionalization of ALL
candidate alleles
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Heavily reliant on inferred data and predictions…
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Interpretation
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Neuroanatomical
Golzio et al, Nature 485:
363-367 (2012)
Muscular dystrophy
Sarparanta et al, Nat Genet 44: 450-455 (2012)
Craniofacial anomalies
Chassaing et al, J Med Genet 49:
373-379 (2012)
Vascular integrity
Lee et al, Mol Biol Cell 23: 2412-2423
(2012)
cm
lc2:e
GF
P
L R L R L R
Cardiac malformations
In vivo models in human disease modeling
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In vivo functional testing of missense variants
Loss of function Dominant negative Gain of function
Morpholino (MO) injection
into 1-4 cell stage zebrafish
embryos
Phenotype (1-7 days post-fertilization)
Functional null
Hypomorph
Benign
Dominant negative
Gain of function
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to date:
~350 genes modeled
~1,200 human non-synonymous variants
98% sensitivity
84% specificity
suite of models for testing
collapse into recurrent pathways
tools for lead compound discovery
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-1000 -1001
-0001 -0100 -0101
Clinical features
intractable seizures
severe developmental delay
irritability
cortical visual impairment
gastroesophageal reflux
Clinical testing
normal female karyotype
normal female microarray
metabolic testing – negative
genetic testing – negative
Referred for research studies
Marie McDonald (Med Genet)
Consented for research studies
proband age 21 months
Vignette1: An opportunity to learn new biology- SCN2A and congenital seizures
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The candidacy of SCN2A p.S229P
S229P
Arguments for causality:
• Novel, de novo, protein-altering variant
• SCN2A is a known epilepsy gene
Arguments against causality:
• Patient phenotype more severe than most
other documented SCN2A cases
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SCN2A S229P: gain-of-function
209D
209N
hyper polarizing shift of voltage-dependent activation
Haidun Yan
Geoff Pitt
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Clinical features
severe developmental delay
hypotonia
optic nerve disorder/nystagmus
auditory neuropathy spectrum disorder
demyelination on brain MRI
Clinical testing
normal female microarray
metabolic testing – negative
extensive genetic testing – negative
deafness panel
mitochondrial disease panel
PLP1 and GJC2 sequencing
-1000 -1001
-0001
T793M
T793M
H883Y
H883Y
Vignette 2: Surprising diagnoses
IKBKAP: Elongator protein complex 1. Causes familial dysautonomia
MGAM: Maltase-glucoamylase
RGS22: Regulator of G-protein signaling
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A variant form of familial dysautonomia
Disorganization of vascular networks rescuable by WT human mRNA
Reduced size
of midbrain and
cerebellar
structures
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Clinical features
Global developmental delay
microcephaly
feeding issues
failure to thrive
abnormal muscle tone
low immunoglobulins
frequent respiratory infections
Clinical testing
normal female microarray
metabolic testing – negative
extensive genetic testing – negative
-001 -002
-004 -003 -004
Vignette 3: Gene identification when genetic
resolution reaches an impasse
BTG2: Involved in the G1/S transition of the cell cycle
NOS2: Nitric oxide synthase 2, inducible
TTN: Titin
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BTG2 is responsible for the microcephaly
phenotype
BTG2: de novo
NOS2: de novo
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Unbiased analysis of WES data CRITICAL
Chan et al, N Eng J Med in press
Vignette 4:The case for unbiased analysis of
exomes
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In vivo testing of epistasis
Chan et al, N Eng J Med in press
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Acknowledgements Duke Center for Human
Disease Modeling:
Erica Davis
Jason Willer
Natalie Mola
Azita Sadeghpour
Christelle Golzio
Christine Oien
Dustin Dowless
Jeremiah Savage
Nicholas Katsanis
Duke Neonatal Perinatal
Research Institute:
C. Michael Cotten
Kimberley Fisher
Rebecca Jones
Cynthia Ross
Laura Stern
Mandy Marion
Margarita Bidegain
Ronald Goldberg
Duke Fetal Diagnostic
Center:
Kristin Weaver
Amanda French
Brita Boyd
Amy Murtha
Duke Pediatric Specialty Clinics:
Medical Genetics
Katie Sheets
Courtney Downtain
Marie McDonald
Blood and Marrow Transplant:
Jessica Sun
Kristin Page
Rebecca Lewis
Sloan Kojis
June Allison Thacker
Joanne Kurtzburg
Urology/Nephrology:
Jennifer Stout
Rasheed Gbadegesin
John Wiener
Neurology:
William Gallentine
Cardiology:
Jennifer Li
Duke Clinical Laboratory:
Erica Owens
Catherine Rehder
Baylor HGSC:
Aniko Sabo
Shannon-Dugan Rocha
Donna Muzny
Richard Gibbs
Duke Center for Human
Genetics:
Kristin McDonald
Heidi Cope
Allison Ashley-Koch
Michael Hauser
Elizabeth Hauser
Duke Institute for Genome
Sciences & Policy:
Sara Katsanis
Joyce Kim
Misha Angrist
Duke Ion Channel Group:
Haidun Yan
Geoffrey Pitt
U Lausanne:
Alex Raymond
Jacqui Beckmann
Mass General/Harvard:
Susan Slaugenhapt Funding: NIH-NIDDK P50 DK096415
www.dukegenes.org
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Can also model copy number variants
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deletion duplication normal dose
AUTISM
HYPERPHAGIA
OBESITY
MACROCEPHALY
…
SCHIZOPHRENIA
AUTISM
ANOREXIA
UNDERWEIGHT
MICROCEPHALY
…
16p11.2 CNV: Complex Mirrored Neurodevelopmental Phenotypes
600 kb
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Strategy to Identify Which Gene(s) Cause(s) the Complex Mirror Phenotype at the 16p11.2 Locus
Screening for microcephaly at 5 dpf
Candidate gene(s) identified
Screening for reciprocal phenotype: macrocephaly
Identification of the causal gene(s)
Microinjection morpholinos into Zebrafish embryos 1- to 2-cell stage
Design Morpholino(s) against Zebrafish ortholog(s)
Overexpression human mRNAs
Microinjection mRNAs into Zebrafish embryos 1- to 2-cell stage
29 genes in the CNV 26 Zebrafish orthologs
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50 70 90 110 130 150
Width (μm)
KCTD13+CDIPT
KCTD13+CDIPT Overexpression
leads to Microcephaly
Golzio et al, Nature 2012
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KCTD13 Dosage Changes lead to Head Size Defects
Microcephaly
Macrocephaly
Golzio et al, Nature 2012
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Complex Mirror Phenotypes at the 16p11.2 CNV
Crespi et al. (2010) PNAS 107 :1736-1741
Diametric model
Dysregulated over-development of social brain
Under-development of social brain
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deletion duplication normal dose
Macrocephaly
Microcephaly
Normal
Shinawi M et al, JMG, 2010
Autism Schizophenia Sociability
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Etiology: Gene(s) Within the 16p11.2 CNV but Which One(s)?
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Strategy to Identify Which Gene(s) Cause(s) the Complex Mirror Phenotype at the 16p11.2 Locus
Overexpression human mRNAs
Microinjection morpholinos into Zebrafish embryos 1- to 4-cell stage
Screening for microcephaly
Candidate gene(s) identified Design Morpholino(s) against Zebrafish ortholog(s)
Screening for reciprocal phenotype: macrocephaly
Identification of c ontributory gene(s)
Microinjection mRNAs into Zebrafish embryos 1- to 4-cell stage
29 genes in the region 26 Zebrafish orthologs
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50 70 90 110 130 150
Width (μm)
KCTD13+CDIPT
KCTD13+CDIPT Overexpression leads to Microcephaly
Golzio et al, Nature 2012
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KCTD13 Dosage Changes lead to Head Size Defects
Microcephaly
Macrocephaly
Golzio et al, Nature 2012
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KCTD13 Dosage Changes lead to Proliferation
and Apoptosis Defects at 2-3 dpf
Golzio et al, Nature 2012
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De Novo deletion of exon 4 of KCTD13 in a Patient with Autism
c
d
a
b
Golzio et al, Nature 2012
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Summary
Accelerated molecular diagnosis, early diagnosis
Complementation of WES data with functional assays aid
interpretation significantly
Identify genetic phenomena such as synergistic effects,
modifiers in trans, etc
can be used to model CNVs with anatomical surrogates