Download Could distal MSH2 upstream deletions cause HNPCC?

Could distal MSH2 upstream
deletions cause HNPCC?
… another cancer susceptibility factor.
John Taylor ([email protected])
CMGS Spring Conference 2009
Hereditary non-polyposis colorectal cancer.

HNPCC (Lynch syndrome)

Mutations in the Mismatch repair (MMR) pathway.


Four genes
 MLH1
 PMS2
 MSH2
 MSH6
Colorectal cancer (right hand side).

Extra-colonic carcinomas (MSH2 & MSH6).
 Endometrial
 Stomach
 Small bowel
 Hepatobiliary tract
Hereditary non-polyposis colorectal cancer.
U
A
T
G
T
A
T
MSH2
MSH6
G
C
G
C
Patient referrals.
“A”
Patient Initials
Previous molecular
analysis
“Z”
MSH2 / MLH1
–ve mutation
screen
MSH2 / MLH1
–ve mutation
screen
IHC analysis
Loss of MSH2
Loss of MSH2
MSI
MSI-H
MSI-H
Other affected family
members
Mother [“B”]
None
n
a
l
Interrogation of the MSH6 gene.
4 0 0 0 0
A
i g
Patients “A” and “Z” were referred for an MSH6 mutation
screen.
S

3 5 0 0 0
3 0 0 0 0
y
e
2 5 0 0 0
2 0 0 0 0
D
1 5 0 0 0
1 0 0 0 0

5 0 0 0
Direct sequence analysis .
MLPA analysis (MRC Holland kit P008)
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
4 5 0
5 0 0
S iz e ( n t)
n
a
l

0
i g
4 5 0 0 0
4 0 0 0 0
Both patients were normal and had no mutations other
than neutral polymorphisms in MSH6 and no deletions
were identified.
S
3 0 0 0 0
e

B
3 5 0 0 0
y
2 5 0 0 0
D
2 0 0 0 0
1 5 0 0 0
1 0 0 0 0
5 0 0 0
0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
4 5 0
5 0 0
a
l
S iz e ( n t)
n
4 5 0 0 0
i g
4 0 0 0 0
C
3 5 0 0 0
S
However, the MLPA kit contained additional probes:
3 0 0 0 0
y
e
2 5 0 0 0
2 0 0 0 0

D

1 5 0 0 0
1 0 0 0 0
5 0 0 0

TACSTD1 exon 7
TACSTD1 exon 9
0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
4 5 0
5 0 0
S iz e ( n t)
Figure 3.1. Multiplex ligation-dependant probe amplification
(MLPA) electropherogram traces for; A) a normal
control; B) patient “A”; C) patient “Z”.
SNPs excluded by direct sequence analysis of exon 9
TACSTD1 exon 9 deletion.

This region chromosome 2p is very repetitive; Alu-rich
sequence.

Previous studies have shown deletions within the coding
region of MSH2 can extend into the promoter region and
as far as the TACSTD1 gene. Francoise Charbonnier et al., (2005) Human
Mutation 26(3), 255-261.

Recombination between Alu elements have also been
shown (ex1-ex6). Anja Wager et al., (2003) Am J. Hum. Genet.72:1088-1100.

Could the apparent deletion in TACSTD1 ex9 extend into
the promoter of MSH2?
QF-PCR analysis. Charbonnier et al., (2002) Cancer Research 62: 848-853.
MSH2 start
- 23.4 kb
- 16.5 kb
Ex 7
P6
P5
+ 6.9 kb
P4
PZ
P3
PH1
TACSTD1
exon 7
TACSTD1
exon 8
TACSTD1
exon 9
P2
P1
Ex 1
MSH2
exon 1
Ex 3
MSH2
exon 2
MSH2 promoter
~4.4Kb*
Msh2 Ex 6
Normal
Msh2 Ex 6
Msh2 Ex 6
Msh2 Ex 16
“Z”
Msh2 Ex 6
Msh2 Ex 6
“A”
Map of the exons within chromosome 2 (positions 47,483,612 - 47,642,954)
*promoter region as determined by Iwahashi Y et al.,(1998) Gene 213:141–147.
MSH2
exon 3
QF-PCR analysis. Charbonnier et al., cancer Research 62, 848-853
MSH2 start
- 23.4 kb
- 16.5 kb
Ex 7
P6
P5
+ 6.9 kb
P4
PZ
P3
PH1
TACSTD1
exon 7
TACSTD1
exon 8
TACSTD1
exon 9
P2
P1
Ex 1
MSH2
exon 1
Ex 3
MSH2
exon 2
MSH2 promoter
~4.4Kb*
Msh2 Ex 6
Msh2 Ex 6
Msh2 Ex 6
Msh2 Ex 16
Msh2 Ex 6
Msh2 Ex 6
“A”
“B”
“Z”
Map of the exons within chromosome 2 (positions 47,483,612 - 47,642,954)
*promoter region as determined by Iwahashi Y et al.,(1998) Gene 213:141–147.
MSH2
exon 3
Long-Range PCR.
Standard curve using λ DNA ladder
0
λ
KB
23.1
9.4
6.6
2.3
2
1
2
3
4
5
6
7
8
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
9
10
y = -0.1821x + 1.8923
R2 = 0.9669
Series1
Series2
Linear (Series2)
Linear (Series1)
NC Z
A
B -VE
sample
nc
"Z" 1
"Z" 2
"A" 1
"A" 2
"B" 1
"B" 2
distance (cm) size estimation lambda ladder
4.2
4.2
5.7
4.2
4.45
4.2
4.45
1.12748
1.12748
0.85433
1.12748
1.081955
1.12748
1.081955
The primer set for MSH2P4 is
10 kb upstream of MSH2
estimated
size in Kb
13.4
13.4
7.2
13.4
12.1
13.4
12.1
What was the significance of these deletions?

Long-range PCR excluded the possibility of
rearrangements and inversions within this region.
chromosomal

All deletions are in excess of 10kb upstream of the MSH2
transcriptional start and are unlikely to affect transcription factor
binding sites. Farré et al., (2007) Genome Biology 8:R140.

The deletion co-segregates with the apparent loss of MSH2 by IHC
(although only through one generation)

Two additional patients from the Salisbury lab were tested and found
to have the same deletion size and haplotype (two markers [D2S123
and D2S391]) as patients “A” and “B”.
Findings up until Nov 2008
Recent publications
MSH2 start
- 23.4 kb
- 16.5 kb
Ex 7
P6
P5
+ 6.9 kb
P4
PZ
P3
PH1
TACSTD1
exon 7
TACSTD1
exon 8
TACSTD1
exon 9
Publications since January 2009:
P2
P1
Ex 1
MSH2
exon 1
Ex 3
MSH2
exon 2
MSH2
exon 3
MSH2 promoter
~4.4Kb*
1. Heritable somatic methylation and inactivation of MSH2 in families with Lynch
syndrome due to deletion of the 3’ exons of TACSTD1. Ligtenberg et al., 2009. Nat. Gen.
Vol 41 (1) p112-117.
2. Deletions removing the last exon of TACSTD1 constitute a distinct class of mutations
predisposing to lynch syndrome. Kovacs et al., 2009. Hum. Mut. Vol. 30, No. 2, 197–203.
Both publications agree that the removal of the TACSTD1 polyadenylation signal in
exon 9 resulted in the continuation of the transcriptional elongation from the TACSTD1
gene into the downstream MSH2 gene.
Concluding remarks.

What started of as a coincidental finding appears to a novel cancer
susceptibility factor associated with HNPCC.

Upstream deletions within exon 9 of the TACSTD1 gene can lead to
the loss of MSH2 in colonic adenoma tissue, detectable by IHC, and
is consistent with a diagnosis of HNPCC.

It is unlikely that these deletions alter the expression on MSH2
directly (i.e. removing transcription factor binding sites), but rather
indirectly by transcriptional silencing due to the strength of the
TACSTD1 promoter and loss of the polyadenylation signal.

One further consideration:

Could this type of mutation influence the spectrum of cancers associated
with MSH2 traditionally associated with extra-colonic carcinomas?
Acknowledgements.

Jennie Bell

Fiona Macdonald

Matthew Smith

Dave Bunyan (Salisbury)
Appendix 1 – Functions of TACSTD1/EpCAM
Trzpis et al., (2007) The American Journal of Pathology, Vol. 171, p386-395
Appendix 2 – Signalling and CC-ICs
Ricci-Vitiani et al., Gut
(2008);57;538-548
The morphological unit of the small intestine is the crypt–villus, lined with Paneth cells at the bottom of the crypt
(yellow). Stem cells are located on top of Paneth cells (violet) and also give rise to transit amplifying (TA)
precursors in the remainder of the crypt. TA cells undergo terminal differentiation under the influence of gradients
of morphogenetic ligands while migrating to the villus, before being shed in the lumen. Right: a schematic
description of the differentiation pathways in the gut epithelium. BMP, bone morphogeneic protein; TGFβ,
transforming growth factor β.
Appendix 3 - Congenital Tufting Enteropathy (CTE)
Taken from Sivagnanam et al., Gastroenterology (2008);135:429–437
Congenital tufting enteropathy (CTE) is a rare autosomal recessive diarrheal disorder presenting in the neonatal
period. CTE is characterized by intestinal epithelial cell dysplasia leading to severe malabsorption and significant
morbidity and mortality.
Patients P1 and P2 were found to be homozygous G>A
substitution in the affected patients at the donor splice site
(c.491+1G>A) of exon 4 of TACSDT1/EpCAM
EpCAM function may be important for the development of the
crypt villus axis, where epithelial cells originate from stem
cells in the crypt and migrate distally to the tip of the villus
prior to shedding.
Schematic of duodenal mucosa showing histology of (A)
normal intestinal villus and (B) congenital tufting
enteropathy villus with crowded epithelial cells forming
tufts, villus atrophy. (C) H&E-stained duodenal tissue
(original magnification, 20x )from affected patients (P1
and P2) exhibiting tufting and crowding of epithelial
cells.