Download Clinical Genetics - Marcus Autism Center

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Can be syndromic or non-syndromic
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Environmental and Genetic Factors
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Pre and perinatal factors, infections, family histories,
parental age, pesticides, drugs and chemicals
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Observed in all ethnic groups
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More than 600 genes described in literature
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Majority of cases are non-syndromic with no other
features to assist in diagnosis
Most have not be replicated
Many individuals with autism are still unresolved –
more genes/loci?
Genetic testing is recommended for all children with ASD
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~25-30% have an identified genetic syndrome or variant
This means that ~70% have no mechanism identified as yet
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Comorbidity with ID, epilepsy, motor impairment, certain
dysmorphic features supports a likely underlying genetic
etiology
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Future Goal: genetic characterization of etiology will
facilitate targeted treatments based on the underlying
mechanism of the disease
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Concordance in monozygotic twins
approaches 70%
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Recurrence rates in siblings of children with
ASD range from 5% to 20%
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Recurrence rate increases to 33% if a family
has 2 children with ASD
Pediatr Clin N Am 2015;62:607-18
Nature 2012;485:242-5
Nature 2012;485:246-50
The Double Helix – April 1953
James Watson Francis Crick
Chromosomes: 46, XX or 46, XY.
Rosalind
Rosalind Franklin
Franklin
Mitochondrial DNA
23 chromosomes from mother, 23 from father.
Genes arranged on chromosomes which code for proteins
(enzymes, transporters, etc).
Maurice Wilkins
X-ray diffraction photographs of DNA - 1951
2007
 50
J. Craig Venter
Decoded a full diploid genome –
his and James Watson’s
years later
It took 3 months and $300,000 each
It took 13 years and 3 billion dollars
Currently it takes ~3 months and $5,000 to $7,000
Goal is under $1000 for genome sequencing
Chromosome Technology Progress
Technology
Resolution
Sample Diagnosis
Karyotype
(1970’s)
Whole
Chromosome
Down syndrome
Fluorescent In
Situ Hybridization
- FISH
Large Deletions or duplications (> 4 Mb)
Looks for small specific
sections of DNA (30-50
genes) that would be
missed by routine
chromosome analysis
Fluorescence in situ
~ 100 kb
22q11.2 syndrome
Hybridization (FISH)
VCFS
(1990’s)
Tests a single locus at a time
Need Prior knowledge of region
Array CGH
(2000’s)
Flexible, only
limited by probe
spacing (> 1 kb)
Submicroscopic
deletions/duplications
anywhere in the
genome
Detects microdeletion
syndromes like:
22q11.2 deletion
7q11 del Williams
Can test whole-genome simultaneously
Slide courtesy of Jennifer Mulle PhD
22q11.2 Region and FISH
Fish for Williams syndrome 7q11.23 deletion
Incidence 1:10 to 15,000
Normal
Deleted
Array-based
Comparative Genomic Hybridization
What is array CGH
Comparative Genomic Hybridization?
Patient DNA
Genomic
Clones
(aka - Chromosomal Microarray)
NORMAL
Only detects
unbalanced
rearrangements
What can it tell me?
Control DNA
Pinkel et al., Nat Genet (1998), 20(2):207-11
Slide courtesy of Christa Martin, PhD
Array-based CGH
Patient DNA
Turner synd. (45,X)
Normal (46,XX)
47,XXX
Genomic
Clones
Loss: ratio < 0.8
Normal: ratio 0.8 - 1.2
Gain: ratio > 1.2
Control DNA
Diagnostic Yield for ID, ASD, DD and MCA:
Conventional Karyotype - 5%
oligo probe
coverage on
EmArray
FISH probe
(~100 kb)
used for
testing only
covers this
gene
3 Mb DGS
region
Microarray – 20% positive for CNVs
Yield for ASD alone – 10% positive for CNVs
Recommendation: order a microarrays a first tier test for
1. Intellectual Disability
2. Multiple Congenital Anomalies
3. Developmental Delays
4. Autism Spectrum Disorders
American Journal of Human Genetics 86, 749-764, May 13, 2010
Genetics in Medicine 15 (7) July 2013
DNA Primer
DNA Testing
Transcription
mRNA (messenger)
tRNA (transfer)
rRNA (ribosomal)
Translation
Protein
Production
mRNA
(U instead of T
Single Stranded)
Gene – String of A’s, T’s
C’s and G’s
ATG GGG TTT TCT CCA CAC
TAC CCC AAA AGA GGT GTG
AUG GGG UUU UCU CCA CAC
Enzyme
Transporter etc.
Met
Gly
Met
Gly Phe Ser Pro His
Ser
Phe
Pro
His
Codons
Room for Normal Variation
Single base variants
Silent mutation: codes for
same amino acid (AA)
Conservative missense:
codes for similar AA –
protein works
Altered Biological Function = Disease
Known Genes with Autism as a feature:
Autosomal Dominant – TSC1 and TSC2 Tuberous Sclerosis
Autosomal Recessive – BCKDK - Branched Chain Ketoacid Dehydrogenase Kinase
X-Linked – Fragile X syndrome
Nonconservative missense:
codes for different AA –
protein may lose function
Nonsense: STOP codon
reduced or no protein made
Frame Shift: insertion or
deletion shifts reading frame
First Generation (Sanger Sequencing):
• Single base variants
Other Mechanisms:
•
•
•
•
•
•
Small Deletions
Whole Gene Deletions
Splice Mutations
Chromosomal deletions
Rearrangements
Epigenetic changes
Benefits:
- All nucleotides interrogated in a specific gene
- Analyst reviews quality at every basepair
- Gold standard
Limitations:
- Large deletions or duplications cannot be detected
- Relatively high cost, labor intensive
- Low through put
- Limited automation in data review
- Potentially complicated interpretation and reporting
Next Generation Sequencing Second Generation (NextGeneration Sequencing):
New sequencing Techniques
Sometime we know what gene to sequence
Benefits:
- Sophisticated bioinformatics
- Highly automated
- Large amount of sequence
Limitations:
- High cost for infrastructure
- Sophisticated bioinformatics
- Reagent cost
- Complicated interpretation
and reporting
Autism and Macrocephaly – sequence the PTEN
tumor suppressor gene on chromosome 10q
Gene Panels
Utility of Panels: autism; cardiomyopathy; seizures etc
Cost wise: panels are frequently close to the same cost
as sequencing one single gene
Cardiomyopathy Panel
• 63-gene cardiomyopathy NGS panel
Emory Autism Panel
• 62 genes:
• ADSL, AFF2, AP1S2, ARX, ATRX, BCKDK, BRAF, CACNA1C,
CASK, CDKL5, CHD7, CHD8, CNTNAP2, CREBBP, CYP27A1,
DHCR7, DMD, EHMT1, FGD1, FMR1, FOLR1, FOXG1, FOXP1,
FOXP2, HPRT1, KDM5C, L1CAM, MAGEL2, MBD5, MECP2,
MED12, MEF2C, MID1, NHS, NIPBL, NLGN3, NLGN4X, NR1I3,
NRXN1, NSD1, OPHN1, PAFAH1B1, PCDH19, PHF6, PNKP,
PQBP1, PTCHD1, PTEN, PTPN11, RAB39B, RAI1, RELN,
SCN1A, SLC2A1, SLC9A6, SMARCB1, SMC1A, TCF4, UBE2A,
UBE3A, VPS13B, ZEB2
• all except 3 of these genes are associated with
known genetic syndromes or intellectual disability
Venn diagram of the overlap of genes affected by hot zone
de novo mutations across four neuropsychiatric disorders
Slide courtesy of: David B. Goldstein, Institute for Genomic Medicine, Columbia University
Baker, Elizabeth and Jeste, Shafali: Pediatr Clin N Am 62 (2015) 607-618
Exome sequencing
The Next Test?
WES – Whole Exome Sequencing
WGS – Whole Genome Sequencing
Panels vs Exome
Next Generation Sequencing
Genome vs. Exome
exon
Cost
Turn-around
time
Analytical
sensitivity
Clinical
sensitivity
Gene coverage
Parental testing
Potential
Results
Panel
Exome
$2,500-$3,200
8-12 weeks
$6,700 trios
16 weeks
99%; All coding exons of all genes on
panel are analyzed; Del/Dup included
All genes are associated with specific
phenotype of panel
Only genes included on panel are
analyzed
Not required; parental follow up may be
useful
Mutations and VOUS identified in genes
associated with specific phenotype
92%; all exons of all genes are not
covered; no del/dup
No specific phenotype needed; not all
exons/genes covered
Captures exomes indiscriminately
Recommended; can help with
interpretation and classification
Mutations, VUS, and carrier status can
be identified in any gene, including
adult onset, cancer and non-medically
actionable genes
intron