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.