Update from Ashley Lab
`
` Publication update
` Major allele human reference
` Exome sequencing and HCM
` Whole genome sequencing for clinical medicine
` The intermediate step here now: targeted capture chip for
`clinical sequencing
` Whole genome sequencing for molecular autopsy
` Personalized medicine clinical trial
`
`Personalis EX2045.1
`
`

`

`
`
`Clinical assessment incorporating a personal genome
`Evan A Ashley, AtulJ Butte, MatthewTWheeler, Rong Chen,TeriEKlein, FrederickE Dewey,JoelT Dudley, Kelly E Ormond, Aleksandra Pavlovic,
`AlexanderA Morgan, Dmitry Pushkarev, NormaFNeff, Louanne Hudgins, LiGong, Laura M Hodges, DoritS Berlin, CarolineF Thorn,
`Katrin Sangkuhl,Joan M Hebert, MarkWoon, Hersh Sagreiya, RyanWhaley,Joshua WKnowles, MichaelF Chou,Joseph VThakuria,
`Abraham M Rosenbaum,AlexanderWaitZaranek, George M Church, HenryT Greely, Stephen R Quake, Russ BAltman
`
`
`
` ScienceWateh.com Data& Rankings. New Hot Papers [E)Fouowus EQ stare
`
`THOMSON REUTERS
`
`ihe LANCE |
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`
`CURRENT SPECIAL TOPIC
`
`
`SCIENCE WATCH
`HOME | ABOUTTHOMSON REUTERS | PRESSROOM | CONTACTUS|
`
`NEW HOT PAPERS
`MAIN MENU
`SCIENCEWATCH HOME
`INSIDE THIS MONTH
`from ThomsonReuters lists a new crop ofwhat it
`New Hot Papers Home
`NEW HOT PAPERS
`Every two months, Essential Science IndicatorsS
`INTERVIEWS
`calls hot papers in science. Hat papers are selected
`2011 Menu
`by virtueof beingcited amongthetopone-tenth of
`SPECIAL TOPICS
`‘onepercent (0.1%) in a current bimonthly period.
`ANALYSES
`Papers are selected in each of22 fields of science=MORE PAPERS
`DATA & RANKINGS
`and must bepublished within the last two years.
`
`Because the hot papers are updated every two
`Fast Breaking Papers
`‘Sci-Bytes
`months, new papers are added with every update,
`Current Classics
`Fast Breaking Papers
`and ScienceWatch.com tracks these new additions.
`NewHot Papers
`Here, ScienceWatch.com highlights the most-cited ofthese new entries, one
`Emerging Research Fronts
`from each field, which are,in addition, not more than oneyear old. Since new hot
`papers are very recent scientific contributionsthat are receiving recognition
`Fast MovingFronts
`during a current period, they may signal important new trends in research and
`Research Front Maps
`serve as leading indicators of scientific advancement. Full information,including
`‘CurrentClassics
`bimonthly citation histories, for these papers can befound in Essential Science
`Indicators. See also, Understanding Core Data; Hot Papers Methodology.
`TopTopics
`RisingStars
`‘Some papers have comments sentin by the author(s) ofthe paper which may
`New Entrants
`‘Country Profiles
`ABOUT SCIENCE WATCH
`
`include images and descriptionsoftheir work.
`
`esacd
`
`
`Choosethe yearmonth you wouldliketoviewfrom thetabs above.
`
`“As whole-genome sequencing
`becomesincreasingly
`widespread,availability of
`genomic information will no
`longer be the limiting factor
`CITETHIS PAGE: hitp:/sciencewatch.com/driahp/
`in application of genetics to
`ScienceWatch.com»Data & Rankings»New Hot Papers.
`clinical medicine.”
`See Articlespage 1525
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`G FOLLOWUSON
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`FEATURED IMAGE
`A figure from a New Hot Paper
`commentarytiled: "Liv Hornekzer
`Discusses Hydrogen Functionalization
`ofGraphene.”
`
` STANFORD
`q SCHOOL OF MEDICINE
`
`
`
`Personalis EX2045.2
`
`Personalis EX2045.2
`
`

`

`Invited revision – Plos Genetics
`
`Personalis EX2045.3
`
`

`

`Paper introduces:
`
` the development and application of ethnicity-specific major allele
`reference sequences
` the definition of meiotic crossover sites to a median resolution of
`<1000 base pairs
` the development of a computational algorithm to achieve genome-wide
`long-range haplotype phasing
` the application of this phasing to HLA typing and clinical interpretation
`of genomes from a family quartet using internally developed databases
`of all publicly accessible genotype-phenotype association data
` the demonstration and quantification of discordance of inherited
`disease risk within a nuclear family
` the successful prediction of the only manifest disease phenotype in this
`family.
`
`Personalis EX2045.4
`
`

`

`WHAT’S NEW?
`WHAT'S NEW?
`
`DPeR?
`
` STANFORD
`
`~ Personalis EX2045.5
`
`Personalis EX2045.5
`
`

`

`Variant identification with a major allele
`human reference sequence
`
`Personalis EX2045.6
`
`

`

`All positions
`
`Disease-associated positions
`
`Towards an
`(improved)
`human reference
`sequence
`
`Personalis EX2045.7
`
`

`

`Disease risk alleles on the human
`reference sequence
`
`Rong Chen
`
`Personalis EX2045.8
`
`

`

`Disease
`risk
`alleles on
`the
`human
`reference
`sequence
`
`Rong Chen
`
`Personalis EX2045.9
`
`

`

`Alignment to a major allele
`reference sequence
`
`One lane of data from the Illumina GA II was aligned to each reference
`sequence using BWA
`
`Personalis EX2045.10
`
`

`

`Variant identification using a
`major allele reference sequence
`
`CEU-specific calls
`
`HG19-specific calls
`
`MIE rate per 10 kbp
`
`CEU reference resulted in ~40%
`lower error rate at disease-
`associated loci
`85% of CEU-specific calls validated
`with chip
`36% of HG19-specific calls
`validated with chip
`
`Personalis EX2045.11
`
`

`

`Exome sequencing and hypertrophic
`cardiomyopathy
`
`Personalis EX2045.12
`
`

`

`Study subjects
`Pedigree ascertained
`through 52 y/o proband
`4 affected individuals,
`including one severely
`affected individual in
`generation III
`One obligate carrier in
`generation II
`One unaffected spouse in
`generation I
`
`Personalis EX2045.13
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`

`

`Alignment and variant identification
`
`Personalis EX2045.14
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`

`

`Sequencing coverage and depth
`
`1
`
`2
`
`4
`
`3
`Subject
`
`5
`
`6
`
`Total reads: 2.4B
`Bases called: 73 M
`Ti/Tv: 2.62
`
`180
`160
`140
`120
`100
`80
`60
`40
`20
`0
`
`Read depth, x
`
`% of targeted positions covered
`
`Personalis EX2045.15
`
`

`

`Variant statistics
`
`No putative loss of function variants were seen in 29 genes previously associated with
`hypertrophic cardiomyopathy
`
`Personalis EX2045.16
`
`

`

`Variant filters
`High confidence “damaging” SNVs/indels were
`filtered by:
`Novelty (not found in dbSNP 132 or 1KG):
`415
`Cosegregation with phenotype: 38
`Variants passing these filters were annotated
`by:
`
`Polyphen2/SIFT
`GERP/Phastcons scores
`Association with known CV disease genes in
`OMIM
`
`Personalis EX2045.17
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`

`

`Top hits - SNVs
`
`Personalis EX2045.18
`
`

`

`Nesprin 1 modulates atrial and ventricular refractory
`times and contractility
`
`J Mol Cell
`Cardiol. 2010
`Apr;48(4):600-8.
`Epub 2009 Nov
`24.
`
`Personalis EX2045.19
`
`

`

`Top hits - indels
`
`Personalis EX2045.20
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`

`

`Myomegalin is an A-kinase associated protein necessary for
`adrenergically-mediated phosphorylation of MYBPC
`
`BMC Cell Biol. 2011 May 10;12:18.
`
`Personalis EX2045.21
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`

`

`Follow-up work
`Functional assay of myomegalin indel and nesprin-1 SNV
`IBD analysis to identify possible modifier loci
`
`Personalis EX2045.22
`
`

`

`Whole genome sequencing for clinical
`diagnosis
`
`Personalis EX2045.23
`
`

`

`Complex diseases and de novo mutations
` de novo mutation is often overlooked in complex diseases
`
`No real analogue on cardiac
`h
`t
`
`Personalis EX2045.24
`
`

`

`Hunting for de novo mutations using WGS
`
` Hypertrophic Cardiomyopathy manifestation at
`young age
`
`No family history
`
`Parents have no related phenotype
`
` Genotyping for common associated variants gave
`negative results
`
`Personalis EX2045.25
`
`

`

`Whole genome
`Sequencing
`(Illumina)
`
`SNPs
`(CASAVA + GATK)
`
`Structural Variants
`(Breakdancer + GATK+ Pindel)
`
`Convservation filters
`(44 way mammalian
`alignment)
`
`In house annotations
`(Cardiac specific) Public DB annotations
`
`De novo variant
`Assessment
`(SIFT + Synopype)
`
`Personalis EX2045.26
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`

`

`SNPs
`
`Description
`
`Coordinates
`
`Symbol
`
`GLDN
`GLDN
`KCTD2
`KCTD13
`TTN
`
`chr15,51692419,C/T
`chr15,51689763,G/A
`chr16,29922353,G/C
`chr17,73043403,G/T
`chr2,179634421,T/G
`
`Both variations on gliomedin are
`scored as damaging. It is
`expressed in the heart, but not in
`cardiac myocytes.
`These are heart-expressed
`potassium channel
`tetramerization domains.
`Mutations in titin are causal of
`hypertrophic cardiomyopathies.
`Note that there is a discrepancy
`between the protein and DNA
`sequences annot+ated in the
`UCSC browser in this region.
`Structural Variants
`chr1,236899900,frameshift
`ACTN2
`Mutations in actinin alpha 2 have
`deletion:1
`been previously related with
`sigmoidal and dilated
`cardiomyopathies
`chr5,1800464931, frameshift
`FLT4
`Encodes a tyrosine kinase,
`deletion:2
`receptor for endothelial growth
`factors, which have been
`associated with hypertrophy
`
`Coordinates
`
`Symbol
`
`Description
`
`Personalis EX2045.27
`
`

`

`SNVs
`
`Description
`
`Coordinates
`
`Symbol
`
`GLDN
`GLDN
`KCTD2
`KCTD13
`TTN
`
`chr15,51692419,C/T
`chr15,51689763,G/A
`chr16,29922353,G/C
`chr17,73043403,G/T
`chr2,179634421,T/G
`
`Both variations on gliomedin are
`scored as damaging. It is
`expressed in the heart, but not in
`cardiac myocytes.
`These are heart-expressed
`potassium channel
`tetramerization domains.
`Mutations in titin are causal of
`hypertrophic cardiomyopathies.
`Note that there is a discrepancy
`between the protein and DNA
`sequences annot+ated in the
`UCSC browser in this region.
`Structural Variants
`chr1,236899900,frameshift
`ACTN2
`Mutations in actinin alpha 2 have
`deletion:1
`been previously related with
`sigmoidal and dilated
`cardiomyopathies
`chr5,1800464931, frameshift
`FLT4
`Encodes a tyrosine kinase,
`deletion:2
`receptor for endothelial growth
`factors, which have been
`associated with hypertrophy
`
`Coordinates
`
`Symbol
`
`Description
`
`Personalis EX2045.28
`
`

`

`Targeted high-throughput sequencing in
`familial cardiomyopathy
`
`Personalis EX2045.29
`
`

`

`Prior expectation – CV disease genes
`
`Personalis EX2045.30
`
`

`

`Prior expectation – CV disease genes
`Custom Agilent SureSelect in solution hybridization
`selected regions associated with 251 target genes implicated
`in myocardial disease and/or function:
`Exons +/- 100 base pairs
`TFBS within 1.5 kbp
`Enhancers/repressors within 1.5 Mbp
`Conserved regions from MSA within 2kbp of coding
`regions and in introns
`miRNA target sites
`miRNA coding regions
`Targets 3.5 Mbp of genomic sequence
`
`Personalis EX2045.31
`
`

`

`Predicted coverage
`
`% coverage
`
`Frequency
`
`% coverage
`
`Target length
`
`Personalis EX2045.32
`
`

`

`Study subjects and follow-up work
`80 subjects with dilated and hypertrophic cardiomyopathy
`in whom clinical genetic testing has not identified a
`pathogenic mutation
`65 subjects have DNA already/15 subjects require DNA
`collection
` Plan for sequencing on Hi-Seq using indexed primers
`and sample pooling for maximum sequencing efficiency
`Ethnicity-specific variant identification as prior
`Targeted genotyping/re-sequencing/re-phenotyping
`contingent on results of discovery sequencing
`Potential clinical tool and tool for investigating modifier
`loci in other subjects
`
`Personalis EX2045.33
`
`

`

`Molecular autopsy using whole
`genome sequencing
`
`Personalis EX2045.34
`
`

`

`Pedigree
`
`DNA from paraffin-
`embedded formalin
`fixed myocardial tissue
`was sequenced on
`Helicos Heliscope
`
`Personalis EX2045.35
`
`

`

`Compound heterozygous variants in the
`inward rectifying potassium channel
`Kir2.2
`
`Personalis EX2045.36
`
`

`

`Sanger sequence confirmation
`
`G T A C T T G C A C
`
`T A C A A G A A C
`
`G T A C T C G C A C
`
`T A C G A G A A C
`
`Variant
`
`c.1028C>T
`
`c.1132G>A
`
`Personalis EX2045.37
`
`

`

`Evidence for purifying selection at both
`nucleotide positions
`
`Personalis EX2045.38
`
`

`

`Control chromosomes
`Neither KCNJ12 variant was seen in 629 control subjects
`genotyped using low coverage sequencing in the 1000
`genomes project
`Neither KCNJ12 variant was seen in 60 full (high coverage)
`genomes released by Complete Genomics
`Overall, the variant was absent in 1400 control
`chromosomes
`However, both variants were present in 4/6 healthy control
`samples genotyped using a bacterial cloning strategy, one
`variant was seen in 27/30 separate controls, and the second
`was seen in 1/30 separate controls
` Both variants fall into a recently annotated pseudogene
`region, which may mask variation in short-read WGS
`
`Personalis EX2045.39
`
`

`

`Follow-up work
`Sequence read alignment re-processed using GATK for
`local re-alignment and variant identification
`iPS cells currently are being made from the father and
`mother of the proband
`
`Personalis EX2045.40
`
`

`

`PERSONALIZED MEDICINE
`CLINICAL TRIAL
`
`Personalis EX2045.41
`
`

`

`Utility of adding genotype risk data to
`management of coronary artery disease risk
`
` Well validated CAD SNPs
` Genotype data given to patient,
`not to doctor
` Follow up for hard end points
` Lipid panel
` Follow up for behavior
`endpoints
`
`Personalis EX2045.42
`
`