DELEZIONE DI TACSTD1 E METILAZIONE DI MSH2 NELLA SINDROME DI LYNCH: 2 NUOVI

CASI DA UNA CASISTICA ITALIANA

Alessandra Viel,Elisa Pin, Michele Quaia, Marco Agostini, Mara Fornasarig, Vincenzo Canzonieri, Emanuele D. Urso, Salvatore Pucciarelli, Donato Nitti,

Isabella Mammi, Chiara Pastrello

This gene encodes a carcinoma-associated antigen that is expressed on the surface of most normal epithelial cells and gastrointestinal carcinomas but not on mesodermal or neural cell membranes.

This protein functions as a cell adhesion molecule.

May act as a physical interaction molecule between intestinal epithelial cells and intraepithelial lymphocytes at the mucosal epithelium for providing immunological barrier as a first line of defense against mucosal infection

EPCAM antigen is being used as a target for immunotherapy.

EPCAM overexpression is associated with proliferation, migration, and invasiveness of tumour cell lines and is a negative prognostic marker for proliferation.

EPCAM has oncogenic potential and is activated by release of its intracellular domain, which can signal into the cell nucleus by engagement of elements of the wnt pathway

EPCAM has also a role in stem cell fate and is a possible marker for cancer-initiating cells.

Mutations in this gene result in congenital tufting enteropathy (Aut. Rec.).

TACSTD1 (Tumor-associated calcium signal transducer 1)=

EPCAM (Epithelial cell adhesion molecule)

Genomic deletion does not eliminate exons of MLH1, MSH2, MSH6 genes, but….

Deletion of a sequence upstream the MSH2 gene (–16kb from ATG) involving the TACSTD1 gene

CFS363FamA-AV107

control

CFS394

FamA-PD31

Del 5’

CFS396

FamA-AV114

Del 5’

CFS395

FamA-PD31

Del 5’

-29kb -16kb MSH2

Ex 1

-18 kb and –16 kb: deleted -29kb, -2.5kb, -173b: retained

-29kb

TACSTD1

-18kb

TACSTD1

-2.5kb

5’ MSH2

-173b

5’ MSH2

CFS363FamA-AV107

control

CFS396

FamA-AV114

Del 5’

CFS488

FamA-AV114

Del 5’

The deletion involves the 3’ portion of TACSTD1 (3’UTR, ex 9, ex8 ,….) but does not involve the promoter of MSH2

TACSTD1 del and diseaseco-segregationin the families

del

deldel

(del)

del del

A-AV114

A-PD31

MLPA and DNA sequencing

LOH with loss of the other allele

CFS396A

BLOOD

CFS396T

TUMOR

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 4 7 10 13 16 19 22 25 28 31 34 37 40

A

T

MS

H2 3

39G

>A

(K

113K

)

These large germline deletions encompassing the last exons of the TACSTD1 gene, by removing the transcriptional termination sequences, give rise to TACSTD1/MSH2 fusion transcripts with premature stop codon.

Transcription of TACSTD1 extends into MSH2

The MSH2 promoter in cis with the deletion is methylated

Silencing of MSH2 in TACSTD1 expressing tissues with mosaic pattern

MMR gene promoter methylation analysis by MLPA

BLOODControl

UNDIG

UNDIG

HhaI

HhaI

HhaI

HhaI

TUMORControl

CFS396ABLOOD

EPCAM Del

CFS396T

TUMOR

EPCAM Del

TUMOR

CONTROLMLH1 met

MSH2 peaksMLH1 peaks

IHC MSH2: cytoplasmic staining ?

Osservazioni

In data 29.04.2010; E' stata eseguita un'indagine

immunoistochimica per la verifica dell’espressione

delle proteine di MMR.

L'esame ha dato il seguente esito:

• MLH1 positiva (debole 70%)

• MSH2 apparentemente negativa, ma presenza di

"staining" citoplasmatico.

• MSH6 negativa.

• PMS2 positiva (50%)

FUSION TRANSCRIPTS in INTESTINAL MUCOSA

m.w

.

CF

S396N

Wild

typ

e

TACSTD1 exon 5 MSH2 exon 2

TACSTD1 exon 5 intergenic criptic 111bp exon (Alu seq) MSH2 exon 2

Published TACSTD1 deletions (4.9-22.8 kb)

Due to Alu repeats, our first attempts to amplify and sequence the breakpoint region from gDNA were unsuccessful

TACSTD1 MSH2

2 5 6 7 8 93 41 1 2

Guarinos et al

SUMMARY of DATA

A new molecular mechanism of tumorigenesis: Gene silencing by transcriptional read-through

of a neighboring gene.

Deletion upstream the MSH2 gene in 2 Italian families

No other MMR clear-cut pathogenic mutations Del. involves TACSTD1 gene (exons 6–9 and stop

codon). We do not know the exact 5’ and 3’ extension at gDNA level

TACSTD1 del. produces fusion TACSTD1/MSH2 transcripts

TACSTD1 del. cosegregates with disease in both families

LOH in one tumor Cytoplasmic MSH2 protein expression in one tumor Partial methylation of MSH2 promoter in one tumor NO methylation of MSH2 promoter in blood of 3

carriers.