Comprehensive constitutional genetic and epigenetic characterization

of Lynch-like individuals

Estela Dámaso, Maribel González-Acosta, Gardenia Vargas-Parra, Matilde Navarro, JudithBalmaña, Teresa Ramon y Cajal, Noemí Tuset, Bryony Thompson, Fátima Marín, AnnaFernández, Carolina Gómez, Àngela Velasco, Ares Solanes, Sílvia Iglesias, Gisela Urgell,Consol López, Jesús del Valle, Olga Campos, Maria Santacana, Xavier Matias-Guiu, ConxiLázaro, Laura Valle, Joan Brunet, Marta Pineda*, Gabriel Capellá*

Catalan Institute of Oncology

High

High

Intensive

Intermediate

Intermediate

No optimal guidelinesScreening

Cancer risk of relative carriers

Risk of colorectal cancer

Low

Low

Specific

Adapted from Buchanan et al, The Application of Clinical Genetics, 2014

LS diagnostic yield and associated risk

SporadicLynch

syndromeLynch-like

MMR deficient CRC tumor

No identified MMR mut

(unknown)

MMR gene mut(inherited)

Somatic MLH1hypermethylation

(52%) (48%) ~57% 43%

Hampel 2006, Rodríguez-Soler 2013, Win 2015, Mas-Moya 2015, Kang 2015, Chika 2017, O’Kane 2017

Catalan Institute of Oncology

Our aim was to elucidate the constitutional basis of MMR-deficiency in Lynch-like patients throughout a comprehensivegenetic and epigenetic analysis

56 MLH1/PMS2-

27 MSH2/MSH6-

12 MSH6-

5 PMS2-

15 MSI/No loss

115 Lynch-like syndrome

MLH1 non-meth. / BRAF wt 61 Lynch syndrome

12 MLH1 epimutations

41 healthy controls

Catalan Institute of OncologyVall d’Hebron HospitalSanta Creu i Sant Pau HospitalArnau de Vilanova Hospital

Analysis of MMR genes according to the IHC pattern

(Sanger seq or DGGE)

10 MMR VUS

NGS custom panel of CRC-associated genes

1 MLH1

2 MSH2

3 MSH6

4 PMS2

5 EPCAM

6 MLH3

7 MSH3

8 PMS1

9 EXO1

MMR

pathway

Lynch

syndrome

10 APC

11 ENG

12 MUTYH

13 STK11

14 SMAD4

15 PTEN

16 AXIN2

17 POLE

18 POLD1

19 BMPR1A

Polyposic

syndromes

20 TP53

21 CDH1

22 CHEK2

23 BUB1B

24 BUB1

25 BUB3

26 FAN1

Other non-

polyposic

hereditary

syndromes

26 CRC-associated genes

Multifactorial likelihood analysis (in collaboration with B. Thompson)

cDNA analysis

Withoutpuromycin

Puromycin

Lymphocyteculture

RNA extractionReverse

transcriptionPCR with

specific primers

•Splicing analysis•Stability analysis

Infinium Human Methylation 450K beadchipIt includes 485.764 cytosines and 21.233 genes

Two pathogenic and 4 VUS variants in MMR genes were identified

1 MLH1

2 MSH2

3 MSH6

4 PMS2

5 EPCAM

6 MLH3

7 MSH3

8 PMS1

9 EXO1

MMR

pathway

Lynch

syndrome

10 APC

11 ENG

12 MUTYH

13 STK11

14 SMAD4

15 PTEN

16 AXIN2

17 POLE

18 POLD1

19 BMPR1A

Polyposic

syndromes

20 TP53

21 CDH1

22 CHEK2

23 BUB1B

24 BUB1

25 BUB3

26 FAN1

Other non-

polyposic

hereditary

syndromes

26 CRC-associated genes

115 Lynch-like syndrome

42 LLS cases selected

Previous analysis: DGGE MLH1NGS: MLH1 c.676C>T p.(R226*)

Previous analysis: Sanger MLH1+MSH2NGS: MSH6 c.2219T>A p.(L740*)

➢ Reanalysis of gross rearrangements by MLPA: MSH2 E8 duplication➢ 4 MMR VUS

•6

15 VUS in MMR genes

7 cases selected for cDNA/multifactorial

analysis(10 from previous analysisand 5 identified by NGS)

5 reclassified as pathogenic

Five VUSs were reclassified as pathogenic mutations

Case ID Gene Variant Predicted protein

RNA analyses

Multifactorial calculations

Final classificationcDNA splicing analysis

cDNA stabilityanalysis

35 MLH1 c.25C>T p.(Arg9Trp) r.25C>T; p.Arg9Trp Class 3

39 MSH2 c.1787A>G p.(Asn596Ser) r.1787A>G; p.Asn596SerNon allelicimbalance Class 3

57 MSH2 exon 8 duplication p.?r.1277_1387dup; p.Val463Glufs*11 Class 5

67 MSH6c.1153_1155delAG

Gp.(Arg385del)

r.1153_1155delAGG; p.Arg385del

Non allelic imbalance

>0,99 Class 5

70 & 75 MSH6 c.1618_1620delCTT p.(Leu540del)r.[1607_3172del;=];

p.[Ser536_Asp1058delinsAsn;Leu540del]

Destabilization >0,99 Class 5

82 MSH6 c.3150_3161dup p.(Val1051_Ile1054dup)r.3150_3161dup;

p.Val1051_Ile1054dupNon allelic imbalance

>0,99 Class 5

77 MSH6 c.3226C>T p.(Arg1076Cys) r.3226C>T*; p.Arg1076Cys Class 4

Variants identified in other CRC-associated genes

1 MLH1

2 MSH2

3 MSH6

4 PMS2

5 EPCAM

6 MLH3

7 MSH3

8 PMS1

9 EXO1

MMR

pathway

Lynch

syndrome

10 APC

11 ENG

12 MUTYH

13 STK11

14 SMAD4

15 PTEN

16 AXIN2

17 POLE

18 POLD1

19 BMPR1A

Polyposic

syndromes

20 TP53

21 CDH1

22 CHEK2

23 BUB1B

24 BUB1

25 BUB3

26 FAN1

Other non-

polyposic

hereditary

syndromes

26 CRC-associated genes

42 LLS cases selected

13 variants predicted as pathogenic

32 carriers of variants

Case ID

Variant calling MAFIn silico prediction

Splicing

Protein function

Gene cDNA changePredicted protein

changeExAC/ESP SIFT

MutationTaster

Polyphen2 /HumDiv

Polyphen2 /HumVar

Provean

10 EPCAM c.811G>T p.(Val271Phe) NR/NR No changes D (0) D (1) PrD (1.000) PrD (0.989) NP29 POLD1 c.2275G>A p.(Val759Ile) 0.002/0.001 No changes D (0) D (1) PrD (1.000) PrD (0.988) NP30 APC c.7936C>G p.(Gln2646Glu) NR/NR No changes D (0.02) D (1) PsD (0.688) B (0.182) NP39 FAN1 c.149T>G p.(Met50Arg) 0.002/0.002 No changes T (0.08) D (1) PrD (0.991) PsD (0.690) NP55 PMS1 c.497A>C p.(Lys166Thr) NR/NR No changes D (0) D (1) PsD (0.757) PsD (0.599) NP

58 EXO1 c.2212-1G>A p.Val738_Lys743del 0.0019/0.0028 Loss of ASS NA NA NA NA NA

59 APC c.1966C>G p.(Leu656Val) NR/NR Gain of DSS D (0) D (1) PrD(0.999) PrD (0.998) NP62 & 74 MSH3 c.2732T>G p.(Leu911Trp) 0.002/0.004 No changes D (0) D (0.999) PrD (1.000) PrD (0.978) NP

65 MUTYH c.1437_1439del p.Glu480del NR/0.000 No changes NA NA NA NA D (-7.78)74 MSH3 c.685T>C p.(Tyr229His) NR/NR No changes D (0.01) D (0.999) PrD (1.000) PrD (0.973) NP81 BUB1 c.2473C>T p.(Pro825Ser) NR/NR No changes D (0) D(1) PrD (1.000) PrD (0.997) NP

85MSH3 c.3072G>C p.(Gln1024His) 0.000/0.000

Loss of DSS / Inconclusive ASS

T (0.39) P (0.996) B (0.007) B (0.013) NP

96 APC c.7514G>A p.(Arg2505Gln) 0.001/0.001 No changes D (0.04) D (1) PrD (1.000) PrD (0.961) NP

MLH1 epimutation carriers (n=12)Lynch syndrome patients (n=51)

Healthy controls (n=42)

Identification of a constitutional MLH1 epimutation

1,6Kb

BRAF wtMLH1 methyl. non-evaluable

+

9 Lynch syndrome

2 previously identified MMR mutations

6 reclassified MMR VUS

1 MLH1 constitutional epimutation

13 predicted pathogenic variants

3 in MSH3

3 in APC

7 in others

Epigenetic analysisGenetic analysis

NGS CRC-associated gene panelVUS assessment Global methylome

Reclassification rate to LS:9+5 (Vargas-Parra 2017)

14/120 (12%)

115 LLScases

Lynch syndrome research group

Hereditary Cancer Program

G. Vargas

E. Dámaso

A. Fernández

M. González G. Capellá

M. Pineda

Thank you!Collaborators:

Members of the Hereditary Cancer Program at ICOJudith Balmaña, Hospital Vall d’Hebron, BarcelonaTeresa Ramon y Cajal and Consol López, Hospital de SantPau, BarcelonaNoemí Tuset and Gisela Urgell, Genetic Counselling Unit, Hospital Arnau de Vilanova, LleidaBryony Thompson, University of MelbourneMaria Santacana, Xavier Matias-Guiu and Maria Santacana, PathologyDepartment, Hospital Arnau de Vilanova, Lleida