Germline POLE and POLD1 variation in persons with colorectal cancer from the Colon Cancer Family

Registry Cohort

Khalid Mahmood

Melbourne Bioinformatics,

Colorectal Oncogenomics Group,

University of Melbourne

InSiGHT, 20191

Germline POLE and POLD1 in Colorectal Cancers (CRCs)

• Germline mutations in POLE and POLD1 are associated with CRCs

• Recurring germline exonuclease domain mutations (EDMs):• POLE: p.Leu424Val

• POLD1: p.Leu474Pro and p.Ser478Asn

• EDMs result in defective proofreading function• structural proximity to DNA binding/active sites likely perturbs function

• resulting in characteristic hypermutator phenotype

• also shown by functional assays in yeast and T4 bacteriophage

Palles et.al. (2013), Valle et.al. (2014) and Bellido et.al. (2016), Kokoska RJ et.al. (2000) 2

Aims

• Identify new germline mutations is POLE and POLD1 using a large cohort of persons with CRC

• Incorporate clinico-pathological and tumour molecular features to classify new POLE and POLD1 pathogenic variants

3

Study cohort: Colon Cancer Family Registry (CCFR)

Australian cohort

Incident CRC dx between 18-59 yrs (independent of FHx)

Cases = 861Controls = 247

Ontario cohort

Incident CRC dx between 20-74 yrs (weighted to FHx)

Cases = 633Controls = 575

Seattle cohort

Incident CRC dx between 18-74 yrs (independent of FHx)

Cases = 459Controls = 385

Cases = 1953Controls = 1207

4

Cases: recruited as population based probands diagnosed with CRC

Controls: recruited by random sampling from population without prior CRC

Results: germline POLE predicted pathogenic variantsProtein length 200 400 600 800 1000 1200 1400 1600 1800 2000 2200

200 400 600 800 1000 1200 1400 1600 1800 2000 2200

POLE

Gln180His 2

Lys2

80

Asn

Asp301Gly Lys398Arg

Met5

06V

al

Ala581Ser

Arg

680

Cys

His

68

4A

rg

Arg

759

Cys

Arg793Cys Arg1059His

Asp

1211

Ter

Arg1294Cys

Val1

444

Le

u

Arg1485His

Asn

1811

Ser

Glu

188

8Lys

Trp1980Cys

Gly

2179

Ala

67

47

+2T

>G

POLE_cases.txt

21 mutations

Ala

81T

hr

Asn143His Gln520Arg

Ile84

3V

al

Glu851SerfsTer7 2 Pro1157Ala Tyr1400His Glu2137Lys

Gly

228

5C

ys

POLE_controls.txt

10 mutations

Exonuclease

Polymerase

DUF

SPLICE

MISSENSE

NONSENSE

FRAMESHIFT

Somatic

Cases

Controls

5

POLE

POLE POLE_exo

Cases (n=1953) 21 (1.09%) 4 (0.21%)

Controls (n=1207) 10 (0.84%)OR 1.3 [0.61-2.75]P=5.8E-01

0 (0%)OR -p=3.1E-01

gnomAD (n=123134) 1168 (0.96%)OR 1.1 [0.74-1.74]P=5.6E-01

111(0.1%)OR 2.27 [0.84-6.16]p=1.1E-01

Results: germline POLD1 predicted pathogenic variantsProtein length 100 200 300 400 500 600 700 800 900 1000 1100

100 200 300 400 500 600 700 800 900 1000 1100

POLD1

Arg180GlyfsTer3 Arg311Cys

Gln

36

9H

is

Pro

39

0S

er

Asp40

2A

sn

Gln

411

His

Ser4

34C

ys

Arg

44

4G

ln

Lys486Glu Arg715Trp

Me

t798

Ile

Ala836Thr Arg881Gly

Arg881Pro

2

Ala

890S

er

Ser8

95Le

u

Ala

930S

er

Pro968Ser Arg1035Cys

POLD1_cases.txt

19 mutations

1 disease

Gly178Trp Arg409Trp Val455Met Arg561Trp Ile995Thr

POLD1_controls.txt

5 mutations

1 disease

Exonuclease

Polymerase

zf-C4pol

FRAMESHIFT

MISSENSE

Somatic

Cases

Controls

6

POLD1

POLD1 POLD1_exo

Cases (n=1953) 19 (0.98%) 8 (0.41%)

Controls (n=1207) 5 (0.42%)OR 2.35 [0.88-6.27]P=9.3E-02

2 (0.17%)OR 2.47 [0.53-11.62]p=3.4E-01

gnomAD (n=123134) 438 (0.36%)OR 2.73 [1.73-4.32]P=1.4E-04

84 (0.07%)OR 6.0 [2.91-12.38]p=7.6E-05

Characterising POLE exonuclease domain mutations (EDMs)

Protein length 200 400 600 800 1000 1200 1400 1600 1800 2000 2200

200 400 600 800 1000 1200 1400 1600 1800 2000 2200

POLE

Gln180His 2

Lys2

80

Asn

Asp301Gly Lys398Arg

Met5

06V

al

Ala581Ser

Arg

680

Cys

His

68

4A

rg

Arg

759

Cys

Arg793Cys Arg1059His

Asp

1211

Ter

Arg1294Cys

Val1

444

Le

u

Arg1485His

Asn

1811

Ser

Glu

188

8Lys

Trp1980Cys

Gly

2179

Ala

67

47

+2T

>G

POLE_cases.txt

21 mutations

Ala

81T

hr

Asn143His Gln520Arg

Ile84

3V

al

Glu851SerfsTer7 2 Pro1157Ala Tyr1400His Glu2137Lys

Gly

228

5C

ys

POLE_controls.txt

10 mutations

Exonuclease

Polymerase

DUF

SPLICE

MISSENSE

NONSENSE

FRAMESHIFT

Somatic

Cases

Controls

7

Use matching tumour molecular features to characterise POLE EDMs

POLE:p.Lys280AsnPOLE:p.Asp301GlyPOLE:p.Lys398Arg

POLE

Results: Somatic mutational signature analysis

Deficient

Proficient

POLE EDMs

[TCT→A] and [TCG→T] mutations are enriched in tumours with POLE EDMs

8

Results: Tumour mutation burden analysis

9

206.36

3.51

213.41

124.98

70.26

3.4 2.44

0

50

100

150

200

POLE:p.Asp301Gly POLE:p.Lys280Asn POLE:p.Lys398Arg MMRd MMRd MMRp MMRp

Mutationburden(perMb)

Hypermutator>10 mut/Mb*

Ultra-hypermutators>100 mut/Mb*

*Campbell BB. et.al. Cell (2017)

Summary

POLE EDMs

In silico prediction

(CADD, REVEL)

gnomAD

Predicted structure stability

(DynaMut)*yeast POL2 structure

4m8o

Mutational Signature.10

Mutation burden

(mut/Mb)

ClinVarclassificatio

n

p.Asp301Gly 31.0, 0.78.12E-06

(1 in 100k)destabilising High

Ultra-hypermutator

VUS

p.Lys280Asn 27.7, 0.28 ultra-rare destabilising LowLow mutation

burdenVUS

p.Lys398Arg 17.8, 0.15 ultra-rare stabilising LowUltra-

hypermutatorVUS

10

*DynaMut, Rodrigues CHM et.al. NAR (2018)

Conclusions and Future directions

• Analysing matching tumour molecular

features can help characterise EDMs

• Future directions for EDM characterisation:

• incorporate structural and functional data

• perform segregation analysis for variant

characterisation

11

exonuclease domain

predicted pathogenic variants

Lys280Asn

Lys398ArgAsp301Gly

Leu424Val

4m80

12

Colorectal Oncogenomics Group, UoM

A/Prof Dan BuchananDr Mark ClendenningDr Bernard PopeDr Harindra JayasekaraDr Eric Ji-Hoon JooA/Prof. Christophe RostyDr Abi RagunathanSharelle JoselandSusan PrestonThomas GreenPeter GeorgesonRomy Walker

Colon Cancer Family Registry CollaboratorsOntario Institute of Cancer ResearchProf. Steven GallingerDr Ashton Connor

Melbourne BioinformaticsA/Prof Daniel Park

Precision Medicine, MonashProf Melissa SoutheyDr Tu Nguyen-DumontMembers of GEL

[ACCFR] Centre for Epidemiology & Biostatistics, UoMProf Mark JenkinsProf John HopperDr Aung Win

Royal Melbourne HospitalProf Ingrid WinshipProf Finlay Macrae

University of Virginia, School of MedicineProf. Graham Casey

http://www.buchananlab.org/

Translational Genomics Research Institute (TGEN)Assoc. Prof. David Duggan

Mayo ClinicProf. Stephen ThibodeauProf. Laney Lindor

Australasian Colorectal Cancer Family Registry

Colon Cancer Family Registry Cohort

Genetics of Colonic Polyposis Study

Acknowledgments

Structural analysis

13

exonuclease domain

predicted pathogenic variants

Lys280Asn

Lys398Arg

Asp301Gly

Leu424Val

Somatic POLE and POLD1 mutations (COSMIC)

Protein length 100 200 300 400 500 600 700 800 900 1000 1100

100 200 300 400 500 600 700 800 900 1000 1100

POLD1

G10R G12R V13G

3R17W

L22F D25N R30Q E35K D37G M44I

2E54K

E57K Q59K D67G G68_E2splice Q69H

2P72L

S73L D76H

R78CR78H

2

W79* A86V

P89SP89A

2

T91_E92fs L94F Q98P D102N G106_E3splice P110S

5

G11

3fs

3P115fs

R126HR126C

2

A127T D132N V137I H142R F144L A145D

3P153fs

G162A R166W R174Q

D175YD175G

2

3 G178W

2 G178fs

G178V

6

G184E P185L

V190LV190M

2

R195fsR195LR195Q

3

M198I S207F

3 P208L

P208S

4

R211HR211L

2

V214M

2A215VA215G

3

R218C

2P222L

A223T

4 R224H

2 R224C

6

3R225H

L226F A242V E245* N247I D249Y F250S E251Q R253_E6splice W265* E267V

2A274T

R276S T282M L291M V295M A308V

2R311C

D316N A320S

G324_E9spliceG324_E8splice

2

F326L P333T I335V S339L

2R343HR343C

3

2W344fs

P347L E348D

2P349S2R352C

L357_C360fs R358W

2C360R

A361VA361fs

2

P362S A366D S370I D376N

A380VA380_E10splice

2

S382P T383A

4V392M

Q399R D402N

3R409Q

T412I K414*

K414_E10spliceV415_E11splice

2

P419S G422S

2R423HR423C

3

A425D G426S L427I

3 R432W

R432Q

4

2F436L

Q437*

2T441M

R443WR443Q

2

R444W K447E G453D

2R454CR454H

3

2V455L

Q461L R470H

2T473M

V477M

S478RS478N

2

D488Y I494F N499_E13splice D502N R505C

2R507H

A509P V510M D515N A516V R525W E534K A536E Q552*

2V556I

L559R

2R561QR561W

3

L568P

P571LP571S

2

V572L

2E579K

T582R A584S E588K P589S

L590PL590F

2

V596I S605A L606V

2Y607CY607H

3

P608LP608S

2

A613T T620M R623Q Q636* F637L I638T R639S T640fs G643R K653fs

4 G654W

4 G654E

G654R

9

L656_P657fs I659M L660P

3 R667Q

R667L

4

R669_E17splice K671M D679fs

P680LP680T

2

3 R682W

R682Q

4

R683H Q684H

6 R689W

A692V S696N

3A697T

2S699F

3V700I

G702D

2P712L

S717L

2Q718KQ718H

3

V720I R725H E729K S746R A749S V751_E19splice M760I

R762*R762Q

2

G764V E770K

A771VA771T

2

R776Q W781C P787S S788L R791W E795D

F799VF799Y

2

2S805N

R808C A810T S815_D822fs R817Q D825Y E830G V840M N842Y

L843VL843M

2

L852R R855_E20splice

2A860V

V861A

3A864T

V867I

3S869L

L871P

C873RC873W

2

2R875H

3D877N

I878V S879Y L881M T888I

2R889CR889H

3

D893N Q898E H900N V901M

D911YD911N

2

2P912S

A915V V925L

K931fsA930T

2

A935T M937T S939L E940K V945L P951S A963V

2R968L

E971KE971G

2

V982L

11 R985_E24splice

2 R985_E23splice

13

V994E G997S V999M G1000D

2 G1001D

2 G1001S

4

A1004G

2R1008CR1008H

3

Q1022H G1023_E25splice C1026F E1040G S1042F

S1052LS1052W

2

R1053HR1053C

2

T1056A R1060C L1065R I1070M F1079L R1082C E1093* Q1094* R1097Q R1098C F1099L

2P1101fs

P1102A G1103R E1105D

COSMIC

431 of 546 mutations

22 tissue types

Exonuclease

Polymerase

zf-C4pol

MISSENSE

FRAMESHIFT

NONSENSE

SPLICE

Somatic

Protein length 200 400 600 800 1000 1200 1400 1600 1800 2000 2200

200 400 600 800 1000 1200 1400 1600 1800 2000 2200

POLE

M1L

S2PS2Y

2

G6fs2G7VG7W

3

8

E1

8K

R21_E2splice D22G

G24SG24RG24fs

3

V29F L32R2R34C2E36KE36*

3

D42N K43E E58Q N65K P68H E70K D75N R77C

R93IR93K

2

2V96_E4splice

Y103H 2G111_E5splice

C112F

4 R114*

R114Q

5

K122N T133I V134D P135S P142_E6splice2H144R

L145F R150* 2S156A2T159A

V164D2E169D

V174M A186V A189T

G198AG198D

2

2T202I

D203N T207A D213N

R222CR222H

2

D225N2R231HR231CR231L

4

H239Y2V240M

A241_E8splice R249Q E256D R266Q

D275GD275V

2

E277Q2T278M

T279N

L283PP282_L283>PF

2

91 P286R

2 P286S

P286T

94

2E289K

M295RM295I

2

14 S297F

S297A

15

3D301N

G302D Q303K G304D I312F2E315*

E318K D319E T323A P324fs P331T E337A

P338SP338T

2

D339Y2E340_E10splice

H342R2Q345*

W347G N363D3G364RG364W

4

2F367SF367C

3

D368Y2W369*W369_E12spliceW369_E11splice

4

R375Q S382R E386K F389L D392G Q394*3E396fsE396G

4

2A399V

I403T D406E

46

V4

11

L

D414G Y416H2P418S

H422N

5 L424V

2 L424I

7

3A426V

A427T D435N

3 P436R

2 P436H

5

V437M3P441L2M444K2R446W

Q453*

14 A456P

2 A456V

16

3T457MT457S

4

Y458H

11

S4

59F

3A463VA463S

4

3A465V

M471L

Y473CY473H

2

3V474I

I478M M487V R494W S497T M506V 2L527P

D529Y D530V G531*

V533LV533M

2

E537DE537G

2

T538I

V544MV544L

2

2R553C

R559P A566T L571P2E575K

K576N

R579CR579H

2

2 A581T

A581P

A581D

4

V599_E17splice S605N L607F R616H2C619FC619Y

3

D626E G628R M630L R639C 2E648K

A652VA652S

2

A661E2R665W

K666M

W669*W669G

2

R672M S681R2R685WR685Q

3

Q687* Q689* K694E2P696S

5 P697fs

P697A

6

P697fs A704T R705W Q715H A716V E719K R721T A724T2D725_E20splice

K733N H735L K738N2R742H

V755A V758L

4 R759C

R759H

5

R762QR762W

2

E767K F768S G770W V774_E21splice

K777TK777N

2

S780L A781V A782V D787N A788V E790G K792R

R793PR793C

2

N796S

5

Y8

01C

D802H S814F 3R821C2G823_E21splice

A824V R825C M831I T844I A846P R847W E851Q G854_E22splice R855G P856H L857S D860N G863C N875T N882I K884R K887Q S891F P893L2G894S

A895T M896V2M900T

K902NK902R

2

2E903_E24splice

E903_D908fs A914V2E915K

T920N2V922I

R924CR924H

2

E932KF931fs

2

G935E M940V P943S A944V K954N R955_E25splice Y956F A957V V958A G963R A966V E967Q E972Q3R975C

2 R976C

R976S

R976H

4

G977WG977R

2

Q980P Q986*

A992VA992T

2

G996S T998M E1001K Y1003C W1013* A1021_E26splice S1027C L1032F E1035K N1036K2R1037C

S1040Y D1045H

G1047RG1047W

2

T1052M A1057T K1058N R1059H A1061T K1070N A1072V R1077H P1084L T1090M

4 E1091K

E1091D

E1091*

6

A1093T

A1097PA1097D

2

Q1100* P1103L R1106K

R1111QR1111W

2

L1119F R1125* A1126E A1126_E27splice L1137M G1138R3A1140T

P1148S2A1149V

L1151M Q1153HV1154I K1155M N1156K P1157Q P1159S R1160H P1164L D1165N H1168Q K1170fsK1170fs D1176G K1181E L1189M

Q1194_E29spliceV1195A

2

2E1199K

V1195_S1201fs

P1205LP1205S

2

2P1207S

M1212T E1213Q G1216S L1217F V1218L L1220P A1224T A1225P 2R1233*3L1235I2S1238N

E1241D L1245H P1247L2W1251*W1251C

3

L1255V G1256fs P1259H A1260S G1262* E1267*2L1269H

V1270L R1284W R1286C A1288T R1289L E1299Q I1307T R1308W G1310V T1313M R1320*

P1330TP1330L

2

W1331C S1337G G1343D H1356D S1361R P1363L R1364C Y1367*2R1371*

K1374T2A1375V

E1377*E1377K

2

G1378E S1380L Y1381C R1382C V1384L2R1386W

L1388I P1389S R1390C L1397_Y1398fs V1402M P1403T E1404G D1405N E1413K E1417K P1421S D1422H E1424G

5 V1426I

V1426A

6

Q1430* P1432L2R1436QR1436W

3

19

V1

44

6fs

V1452L R1453G L1455F S1456L G1457D F1463S L1465V R1471C

2 Q1475P

Q1475R

Q1475H

4

P1481S S1483N

R1485CR1485H

2

2H1486Y

I1487M P1505L3R1508C

A1510V3V1514A2D1516E

T1517_E35splice

5 R1519C

2 R1519H

R1519L

8

E1533* G1535S K1540E G1542C

P1547TP1547S

2

P1549SP1549fs

2

R1556WR1556L

2

R1566I R1570Q

R1579HR1579C

2

R1580Q G1581E A1586T S1589F E1599fs2P1601S

P1610H 2K1616N

N1618S D1623Y

R1626CR1626H

2

R1634HR1634C

2

T1641I2S1644L

Q1645* Q1645_A1646>QT S1650C F1672L R1675H S1687F

P1692SP1692H

2

2E1698KE1698D

3

D1700N C1703Y M1706T 2E1715KE1715Q

3

T1724A V1725_E38splice L1729M D1747N E1749D2A1751S

D1752N

G1755EG1755R

2

Q1763* D1768G T1771M

S1777GS1777C

2

P1779L R1793_E39splice V1800M D1815N H1820N R1823C4R1826W

H1833NH1833Y

2

R1839C F1849V Q1851_E40splice L1852R2R1858C

V1863I

Y1865CY1865*

2

2A1866T

N1869SN1869K

2

2R1878C2R1879C

D1882H A1883V2Y1889C

H1895Y K1897NE1898* I1905N H1901_S1906fs F1907L2S1908A

E1912K S1930L2R1932C2R1932H

4

H1934QH1934Y

2

2G1936EG1936VG1936A

4

D1939YD1939N

2

2K1942E

E1947DD1948Y D1955N E1956G G1961V2A1967V

S1970T V1972G L1986F

C1992*C1992S

2

Q1993L Y1995C F1996S M1998V2V2000I2A2002_E44splice

Y2003C

D2013VD2013N

2

R2017HR2017C

2

S2018R P2020S P2024L

R2026SR2026G

2

5 A2030fs

Q2035H A2037V2E2038K

A2040SA2040V

2

G2042R M2047I T2049A Q2052E N2057fs L2059F Q2061* N2079T E2082G2P2088S

P2091fs E2103D2V2108L

S2113F V2122M2R2127*

D2128Y R2131C G2136S2E2137K

S2139C A2142V Q2143L R2145* D2146A C2148F3R2149H

Y2151C2V2152M

P2154R

D2166ND2166Y

2

D2168ED2168N

2

D2172ND2172H

2

S2173Y S2174Y

D2178NE2177_E46splice

2

3A2180V

L2186F A2192T S2196F2I2199F

E2200D2M2201I2T2202M

Q2208R

A2213TA2213S

2

Q2217* V2220G C2224R E2229A Y2235C C2238A A2239T

A2243TA2243S

2

L2244F T2245N

T2248IT2248S

2

V2250_F2251fs 3R2259W

A2262S

S2268WS2268L

2

Y2269C L2270H G2285D

P286R91

List

V411L46

List

COSMIC

1039 of 1195 mutations

30 tissue types

Exonuclease

Polymerase

DUF

MISSENSE

SPLICE

FRAMESHIFT

NONSENSE

Somatic

POLE

POLD1

EndometrialColorectal

14