Usinglarge-scalegenomicdatasetstounderstandtheimpactofhuman

geneticvariation

DanielMacArthurMassachusettsGeneralHospital

BroadInstituteofMITandHarvardHarvardMedicalSchoolTwitter:@dgmacarthur

Makingsenseofonegenomerequiresplacingitinapopulationcontext

vs

• more than one million genomes and exomes have been sequenced worldwide

• …yet many are inaccessible for ethical, political and technical reasons

ExomeAggregationConsortium(ExAC):aggregatingandcalling92,000exomes

Subset of 60,706 “reference” samples:• high-quality exomes• unrelated individuals• consent for public data sharing• free of known severe pediatric disease

All data reprocessed

with BWA/Picard

Joint calling across all samples

with GATK 3 Haplotype

Caller

Laramie Duncan

1000 Genomes ESP ExAC

010000

20000

30000

40000

50000

60000

Sample Size (N) and Ancestral Diversity1000 Genomes, ESP, ExAC

Indi

vidu

als

with

Exo

me

Seq

uenc

e D

ata East Asian

South AsianEuropeanMiddle EasternAfricanNative American ancestryDiverse Other

World proportions

World PopulationScaled to ExAC height

1000 Genomes

ESP ExAC World

Biggerandmorediverse

LatinoAfricanEuropeanSouth AsianEast AsianOther

Catalogueofprotein-codingvariation• Largestevercollectionofhumanprotein-codinggeneticvariants– over10millionvariants:onevariantevery

6basepairs;mostarerareandnovel

singleton 2-10 alleles 0.01% 0.1% 1% ≥10%

0%

10%

20%

30%

40%

50%

60%

ExAC global allele frequency

Prop

ortio

n of

ExA

C a

llele

s

novelother dbSNPin 1kG or ESP

Publicdatarelease• Allvariantsandpopulationfrequenciesarepubliclyavailable:

exac.broadinstitute.org

KonradKarczewski

TheExAC browser

KonradKarczewskiexac.broadinstitute.org

Caveats

• ExACsampleinclusionisopportunistic:mostsampleshavelimitedphenotypedata,aren’tconsentedforrecontact

• Severepediatric diseasecasesaredepletedfromthedataset,butnotabsent

HowExACimprovesVUSanalysis

1. Filteringvariantsthataretoocommontobecausal

2. Comparisonswithlargecaseseriestoassesspenetrance

3. Identifyinggenes(orregions)thataredepleted forspecificclassesofvariation

Valueforrarediseasefiltering

• #variantsremaininginanexomeafterapplyinga0.1%filteracrossallpopulations

• Bothsizeandancestraldiversityincreasefilteringpower

Eric MinikelESP ExAC

East AsianSouth AsianLatinoAfricanEuropean

# va

riant

s re

mai

ning

afte

r filt

erin

g

• Manuallyreviewedsupportfor197reportedpathogenicvariantsat>1%inatleastoneExACpopulation– effectivelyallarespuriousclaims

• Therearehundreds ofExACindividualscarryingvariantsreportedtocausesevere,dominantpediatric disease

Applicationtoreportedpathogenicvariants

Anne O’Donnell Luria

UsingExACtoassesspenetrance• What explains these alleged dominant

disease variants in “healthy” people?– False positive assertions of pathogenicity– Undiagnosed disease– Somatic mosaicism– Incomplete penetrance?

• Prion diseases as a model– Severe, fatal adult diseases, lifetime risk ~1/10,000– 15% of cases are genetic, due to >60 known

dominant gain-of-function variants in PRNP– Virtually every case has PRNP sequenced– These mutations as a class are >30x more common

in ExAC than they should be given disease incidence!

Eric VallabhMinikel

Science Translational Medicine 8:322ra9 (2016)

Allelecountsin10,460sequencedcasesversus60,706controls

Cases reported to prion surveillance centers

Alle

le c

ount

in E

xAC

popu

latio

n co

ntro

ls

●

●●●●●●●●●●●●● ●●●●● ●●

●

●●●

●

●●●●●● ●

●

●●●●

●

●

●

●●●● ●●

●

●

●

●

●

●●

●

●●●●●

●

●●

0 100 200 300 400 500 600

0

1

2

3

4

5

6

7

8

9

10

P39L

P102LA117V

R148H

D178N

V180I

T188R

E196A

E200K

V203I

R208C

R208H

V210I

M232R ● reportedly pathogenic variantsegregation in multiple multigenerational families;spontaneous disease in mouse models

Allelecountsin10,460sequencedcasesversus60,706controls

• VariantswithbestevidenceforpathogenicityareindeedrareCases reported to prion surveillance centers

Alle

le c

ount

in E

xAC

popu

latio

n co

ntro

ls

●

●●●●●●●●●●●●● ●●●●● ●●

●

●●●

●

●●●●●● ●

●

●●●●

●

●

●

●●●● ●●

●

●

●

●

●

●●

●

●●●●●

●

●●

0 100 200 300 400 500 600

0

1

2

3

4

5

6

7

8

9

10

P39L

P102LA117V

R148H

D178N

V180I

T188R

E196A

E200K

V203I

R208C

R208H

V210I

M232R ● reportedly pathogenic variantsegregation in multiple multigenerational families;spontaneous disease in mouse models

high penetrance

Allelecountsin10,460sequencedcasesversus60,706controls

• Variantswithbestevidenceforpathogenicityareindeedrare• Variantsrareincases,commonincontrolsmaybebenign

Cases reported to prion surveillance centers

Alle

le c

ount

in E

xAC

popu

latio

n co

ntro

ls

●

●●●●●●●●●●●●● ●●●●● ●●

●

●●●

●

●●●●●● ●

●

●●●●

●

●

●

●●●● ●●

●

●

●

●

●

●●

●

●●●●●

●

●●

0 100 200 300 400 500 600

0

1

2

3

4

5

6

7

8

9

10

P39L

P102LA117V

R148H

D178N

V180I

T188R

E196A

E200K

V203I

R208C

R208H

V210I

M232R ● reportedly pathogenic variantsegregation in multiple multigenerational families;spontaneous disease in mouse models

high penetrance

benign or low risk

Allelecountsin10,460sequencedcasesversus60,706controls

• Variantswithbestevidenceforpathogenicityareindeedrare• Variantsrareincases,commonincontrolsmaybebenign• VariantsappeartobeneitherMendeliannorbenign

Cases reported to prion surveillance centers

Alle

le c

ount

in E

xAC

popu

latio

n co

ntro

ls

●

●●●●●●●●●●●●● ●●●●● ●●

●

●●●

●

●●●●●● ●

●

●●●●

●

●

●

●●●● ●●

●

●

●

●

●

●●

●

●●●●●

●

●●

0 100 200 300 400 500 600

0

1

2

3

4

5

6

7

8

9

10

P39L

P102LA117V

R148H

D178N

V180I

T188R

E196A

E200K

V203I

R208C

R208H

V210I

M232R ● reportedly pathogenic variantsegregation in multiple multigenerational families;spontaneous disease in mouse models

high penetrance

benign or low risk

incompletepenetrance

<0.1%

0.1-1%

1-10%

Identifyinggeneswithsignificantdepletionofvariation

• Usingamutationalmodelwecanpredictthenumberofvariantsinagivenfunctionalclassweshouldexpecttoseeineachgene inagivennumberofpeople(Samocha etal.2014NatGenet 46:944–950)

Obs

erve

d

Expected

Synonymous Missense Loss-of-function

0 500 1000 1500

050

010

0015

00

Expected Number of Synonymous Variants

Obs

erve

d N

umbe

r of S

ynon

ymou

s Va

riant

s

R = 0.9778

0 500 1000 1500 2000 2500 3000

050

010

0015

0020

0025

0030

00

Expected Number of Missense Variants

Obs

erve

d N

umbe

r of M

isse

nse

Varia

nts

R = 0.9482

0 50 100 150 200 250 300 350

050

100

150

200

250

300

350

Expected Number of Loss−of−Function Variants

Obs

erve

d N

umbe

r of L

oss−

of−F

unct

ion

Varia

nts

R = 0.5866

Expected Expected

Kaitlin Samocha

Empiricalidentificationofgenessubjecttostronghumanconstraint

0.97

0.33

0.03

1.02

0.55

0.01

• Overall we discover 2,651 genes with a high probability of LoF-intolerance (pLI > 0.95)

• Contains almost all known HI genes, but >75% have no known disease phenotype

Movingbeyondthegene:identifyingregionalmissenseconstraint

1 100 200 300 400 500 600 700 800 900 1000amino acid

protein kinase domain ATP binding site serine/threonine kinase domain signal peptidase serine active site

******** ***

*******

******

*******

******** *** *

constrained

• CDKL5mutationscausesevereinfantileseizures• ConstraintwouldallowustocorrectlyprioritizetheN-terminalregion,evenwithnopriorcausalmutations

• Overall,81% ofClinVar missenseinsevereHIgenesfallwithinthe14% mostconstrainedsequence

Whatnext?• Moresamples:willhavedatafrom~120Kexomesinnextrelease

• Movingtogenomes: testrunon5,500genomes;aimingfor20Kthisyear

• Genotype-basedrecall:movingfromgenotypetophenotypeinasubsetofExACsamples+othercohorts

What’sneeded?• Bigger,bettersamples:harmonized,centralizedrepositoriesofvariantslinkedethicallytophenotypes

• Regulatorysupportfordataaggregationandreuse – commondiseasesamplesaregreatcontrolsforrare

• Increasedfocusonsequencingsamplesconsentedforrecontact,deeperphenotypinganddatasharing

• Large,uniformlyascertainedcaseseriesforrarediseases toassesspenetrance

Daniel MacArthurDavid AltshulerDiego ArdissinoMichael BoehnkeMark DalyJohn DaneshRoberto ElosuaGad GetzChristina HultmanSekar KathiresanMarkku LaaksoSteven McCarroll

Mark McCarthyRuth McPhersonBenjamin NealeAarno PalotieShaun PurcellDanish SaleheenJeremiah ScharfPamela SklarPatrick SullivanJaakko TuomilehtoHugh WatkinsJames Wilson

ExAC Principal Investigators

Monkol LekEric MinikelKonrad KarczewskiAnne O’Donnell LuriaAndrew HillJames WareBeryl CummingsTaru TukiainenKarol EstradaDaniel BirnbaumBen WeisburdBrett ThomasIrina Armean

My Lab

Kaitlin SamochaLaramie DuncanMark DalyBen Neale

ATGU

exac.broadinstitute.orgBroad Genomics and Data Sciences Platforms

Menachem FromerDoug Ruderfer

ExAC analystsEric BanksTim FennellKathleen TibbettsNamrata GuptaStacey Donnelly

Broad Institute