next generation mendelian genetics by exome sequencing
TRANSCRIPT
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Next Generation Mendelian Genetics by Exome Sequencing
Jay Shendure, MD, PhDDept. of Genome SciencesUniversity of Washington
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Second generation sequencing
• 10,000-fold drop in the cost of DNA sequencing since 2005 (& continuing to fall)
• How are these technologies best applied to key problems in human genetics?
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Exome = Protein Coding Genome
• split amongst >160,000 exons
• total length of ~30 megabases
• include canonical splice-sites, miRNAs
• exclude 5’ & 3’ UTRs (untranslated)
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No structural variation!No non-coding variation!
Exome Resequencing
~1% of human genome~20x cheaper
nonsynonymous variation
implicated= causal
functionalvalidation
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Technical challenge
humangenome
PCR conventionalsequencing
next-generation sequencing?
one exon
>160,000 exons
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Many ways to skin a cat…
• Array hybridization
• In solution hybridization
• Molecular inversion probes
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Exome capture by hybridization
exomemicroarray
shotgun genomic library
massively parallel sequencing
of enriched library
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Enrichment for reads overlapping exons
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Consistency by population
Ng et al., Nature (2009)
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Next-generation DNA sequencing
• Sequencing of exomes & genomes has become a very practical thing to do
• But what is it actually useful for?
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TA Manolio et al. Nature 461, 747-753 (2009) doi:10.1038/nature08494
Manolio et al. (2009)
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• To genotype for common variants
• To profile yourself (or a patient) for GWAS risk alleles for common diseases
• much cheaper by genotyping and not very medically informative anyways
Bad reasons to sequence exomes & genomes
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• Rare germline variants rare diseases• Rare germline variants common diseases
• Somatic mutations cancer
• Disease gene discovery• Clinical or molecular diagnostics
Good reasons to sequence exomes & genomes
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• Rare, monogenic diseases
• Monogenic subsets of common diseases
• >2,000 solved• >2,000 unsolved
Mendelian disorders
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Lupski et al. 2010
How does one
pinpoint a causal
variant?
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1. Sequence exomes of several unrelated, affected individuals
2. Remove “common” variants• 8 HapMap ‘control’ exomes• public SNP databases
3. Find genes that contain “uncommon”variants in all affected individuals
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Freeman-Sheldon syndrome (FSS)• congenital contracture syndrome• autosomal dominant• caused by mutations in MYH3
Toydemir et al., Nature Genetics (2006)
Can exome sequencing be applied to identify the cause of a Mendelian disorder?
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Can exome sequencing be applied to identify the cause of a Mendelian disorder?
1MYH3838360…not in either dbSNP or 8
HapMap exomes
1/1 2/2 3/3 4/4…any nsSNP, splice-site,
or indel4,510 3,284 2,765 2,479
…not in dbSNP 513 128 71 53
…not in 8 HapMap exomes 799 168 53 21
Freeman-Sheldon probandsHow many genes in
genome with….
Ng et al. Nature (2009)
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What about an unknown Mendelian?
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Miller syndrome
posterior upper limb defects
pectus abnormalities, scoliosis
supernumerary nipplesposterior lower limb
defects
mandibularhypoplasia
hearing loss, cupped ears
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• Postaxial acrofacial dysostosis
• Presumed autosomal recessive
• Sequenced 4 exomes from 3 kindreds
• 2 siblings from kindred #1 also had CF-like lung phenotype (but not in other Miller cases)
Miller syndrome
Ng et al. Nature Genetics (2010)
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Exome analysis of Miller syndrome
1-A 1-B 1-A+B 2/2 3/3…any 2 nsSNP,
splice-site, or indel2,789 2,777 2,278 1,740 1,461
…not in dbSNP 93 80 40 16 12
…not in 8 HapMap exomes 127 120 47 8 3
…not in either dbSNP or 8
HapMap exomes31 26 8 1
DHODH1
DHODH
Ng et al. Nature Genetics (2010)
How many genes in genome with…
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DHODH = dihydroorotate dehydrogenase
de novo pyrimidine biosynthesis
inborn error of metabolism
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Connection to rudimentary
specific wing anomalies, defective oogenesis, and malformed legs(T.H. Morgan, 1912)
Caused by mutations in de novo pyrimidinebiosynthesis (Rawls & Fristom, 1975)
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Similarity to methotrexate embryopathy
Bawle et al. Teratology 57:51-55 (1978)
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What about the lung phenotype?
• Sibling kindred (only)
• Chronic sinopulmonary infections (CF-like)
• Compound heterozygotes for rare, damaging mutations in DNAH5
• Primary cilliary dyskinesia
Ng et al. Nature Genetics (2010)
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Strategies to Identify Causal Variation
• Genetics alone may be insufficient to identify causal variation
• Purifying selection (i.e. evolutionary ‘constraint’) reduces rates of evolution at functional sites
ArendSidow
GregCooper
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Genomic Evolutionary Rate Profiling
Calculate GERP score at each site based on expected versus observed # of substitutions
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Constraint-Based Identification of Disease Genes
Cooper et al.Nature Methods (2010)
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TA Manolio et al. Nature 461, 747-753 (2009) doi:10.1038/nature08494
Manolio et al. (2009)
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Rare, coding alleles common diseases
• BRCA1/2 (& 11 other genes) – breast cancer• NRXN1, SHANK3, others – autism spectrum disorders• APP, PS1/2, UBQLN1 – early-onset Alzheimer’s• ANGPTL3/4/5, NPC1L1, PCSK9 – cholesterol, triglycerides
1) Exome sequencing of many, many unrelated individuals, all affected by the same common disease (or phenotypic extreme)
2) Focused analyses on monogenic subsets of common disease
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exomes, exomes, exomes!
LOW LDLLOW LDL HIGH LDLHIGH LDLLOW LDLLOW LDL HIGH LDLHIGH LDL
NHGRI Medical Sequencing Project
NHLBI Exome Sequecing Project
(“ESP-GO”)
20+ mendelian diseases
~200 exomes
Extremes of phenotypic distribution for NHLBI-
relevant traits
~7,000 exomes
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• ‘High-yield’ genetics– Rare, monogenic diseases
– Monogenic subsets of common diseases
• Transition to whole genomes as costs evolve
Mendelian Genetics by Exome
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Clinical diagnosis by exome / genome
Molecular diagnosis by exome / genome
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What do we need?
• Technology is there (exomes & genomes)• Software for sequencing is pretty much there• We need better tools for:
• annotating variants• manipulating variant data• prioritizing variants
(both coding & non-coding)• prioritizing candidate genes • pathway analysis for genetic heterogeneity• methods for functional analysis
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U. WashingtonAnita BeckAbby BighamKati BuckinghamZoran BrkanacGreg CooperEvan EichlerRenee GeorgeHeidi GildersleevePhil GreenMark HannibalCharlie LeeAlex McKenzieMargaret McMillinBrian O’RoakWendy RaskindPeggy Robertson Mark RiederJerrod SchwartzJosh SmithWillie SwansonJim ThomasHolly Tabor
Bamshad LabNickerson / Reider LabShendure Lab
Michael Knowles (UNC)Maimoona Zariwala (UNC)Karin Dent (U. Utah)Ethylin Jabs (Mt. Sinai)Koh-ichiro Yoshiura (Nagasaki Univ.)Naomichi Matsumoto (Yokohama Univ.)Tohru Ohta (Univ. of Hokkaido)Norio Niikawa (Univ. of Hokkaido)1000 Genomes Consortium
Michael Bamshad
EmilyTurner
DebbieNickerson
SarahNg
Northwest Genomics
Center