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Hereditary Cancer in Clinical
Practice
Research
The contribution of
CHEK2
to the
TP53
-negative Li-Fraumeni
phenotype
Marielle WG Ruijs
1,3
, Annegien Broeks
2
, Fred H Menko
3
,
Margreet GEM Ausems
4
, Anja Wagner
5
, Rogier Oldenburg
5
, Hanne Meijers-
Heijboer
3,5
, Laura J van't Veer
2
and Senno Verhoef*
1
Address:
1
Family Cancer Clinic, The Netherlands Cancer Institute, Amsterdam, The Netherlands,
2
Department of Experimental Therapy, The
Netherlands Cancer Institute, Amsterdam, The Netherlands,
3
Department of Clinical Genetics and Human Genetics, VU University Medical
Centre, Amsterdam, The Netherlands,
4
Department of Medical Genetics, University Medical Centre, Utrecht, The Netherlands and
5
Department
of Clinical Genetics, Erasmus Medical Centre, Rotterdam, The Netherlands
Email: Marielle WG Ruijs - mwg.ruijs@vumc.nl; Annegien Broeks - a.broeks@nki.nl; Fred H Menko - fh.menko@vumc.nl;
Margreet GEM Ausems - M.G.E.M.Ausems@umcutrecht.nl; Anja Wagner - a.wagner@erasmusmc.nl;
Rogier Oldenburg - r.oldenburg@erasmusmc.nl; Hanne Meijers-Heijboer - H.Meijers@vumc.nl; Laura J van't Veer - l.vt.veer@nki.nl;
Senno Verhoef* - s.verhoef@nki.nl
* Corresponding author
Published: 17 February 2009
Hereditary Cancer in Clinical Practice
2009,
7
:4
Received: 14 November 2008
Accepted: 17 February 2009
doi:10.1186/1897-4287-7-4
This article is available from: http://www.hccpjournal.com/content/7/1/4
© 2009 Ruijs et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background:
CHEK2
has previously been excluded as a major cause of Li-Fraumeni syndrome
(LFS). One particular
CHEK2
germline mutation, c.1100delC, has been shown to be associated with
elevated breast cancer risk. The prevalence of
CHEK2
*1100delC differs between populations and
has been found to be relatively high in the Netherlands. The question remains nevertheless
whether
CHEK2
germline mutations contribute to the Li-Fraumeni phenotype.
Methods:
We have screened 65 Dutch
TP53
-negative LFS/LFL candidate patients for
CHEK2
germline mutations to determine their contribution to the LFS/LFL phenotype.
Results:
We identified six index patients with a
CHEK2
sequence variant, four with the c.1100delC
variant and two sequence variants of unknown significance, p.Phe328Ser and c.1096-?_1629+?del.
Conclusion:
Our data show that
CHEK2
is not a major LFS susceptibility gene in the Dutch
population. However,
CHEK2
might be a factor contributing to individual tumour development in
TP53
-negative cancer-prone families.
Background
Li-Fraumeni syndrome (LFS) is a rare autosomal domi-
nant cancer syndrome predisposing for bone and soft tis-
sue sarcoma, breast cancer, brain tumour, adrenocortical
carcinoma and leukaemia [1]. The classical LFS criteria
are: a proband with sarcoma aged under 45 years and a
first-degree relative with any cancer aged under 45 years,
plus a first or second-degree relative in the same lineage
with any cancer under the age of 45 years or sarcoma at
any age [2]. In addition, Li-Fraumeni-like syndrome (LFL)
criteria have been formulated as a proband with any child-
hood tumour or a sarcoma, brain tumour or adrenocorti-
cal tumour diagnosed under 45 years of age and a first or
second-degree relative in the same lineage with a typical
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Methods
All 65 affected index patients had been assessed and coun-
selled in various clinical genetics centres because of the
occurrence of different cancer types related to LFS and had
as a consequence been tested for
TP53
germline muta-
tions. On the basis of the available clinical data, the
TP53
-
negative families were classified into 3 groups: 1) classical
LFS [2], 2) LFL syndrome according to Birch [3] or Eeles
[4] and 3) LFS-suggestive, including childhood onset
(under 18 years) sarcoma or brain tumours, two or more
primary tumours at any age, two first-degree relatives with
a tumour at any age, of which at least one relative has a
typical LFS tumour or breast cancer under 30 years of age
(without
BRCA1
or
BRCA2
mutations) (Table 1). In fam-
ilies with multiple breast cancer cases and individuals
with breast and ovarian cancer,
BRCA1
or
BRCA2
muta-
tions were excluded, according to standard procedures.
Details are available on request.
LFS tumour at any age, plus a first or second-degree rela-
tive in the same lineage younger than 60 years with any
cancer [3]. Less stringent LFL criteria were formulated by
Eeles et al. as two first or second-degree relatives with typ-
ical LFS-extended tumours (classical LFS tumours plus
melanoma, prostate cancer and pancreatic cancer) at any
age [4]. The Chompret criteria for
TP53
germline muta-
tion testing have been updated in 2008 as: (1) a proband
with a tumour belonging to the LFS tumour spectrum
(sarcomas, brain tumours, pre-menopausal breast cancer,
adrenocortical carcinoma, leukaemia, lung bronchoalveo-
lar cancer) cancer before 46 years of age and at least one
first or second-degree relative with an LFS tumour before
56 years of age or multiple tumours; or (2) a proband with
multiple tumours two of which belong to the narrow LFS
tumour spectrum and the first of which occurred before
46 years of age; or (3) a patient with adrenocortical carci-
noma or a patient with breast cancer before 36 years of age
without
BRCA
mutation, irrespective of the family history
[5].
DNA from peripheral blood lymphocytes was isolated
according to standard procedures. Screening for
TP53
germline mutations was performed by sequence analysis
of all coding exons (2–11) including flanking intron-exon
boundaries (details are available on request) and multi-
plex ligation-dependent probe amplification (MLPA) [25]
(
TP53
MLPA KIT, MRC Holland). In 34
TP53
-negative
LFS, LFL, or LFS-suggestive families all exons and flanking
intron-exon boundaries of the
CHEK2
gene were investi-
gated using denaturing gradient gel electrophoresis
(DGGE, see Table 1) [26]. All possible candidate variants,
identified as aberrant DGGE fragments, were confirmed
by sequence analysis. To avoid amplification of pseudo-
genes, a long range PCR was performed first for exons 10
to 14, followed by a nested PCR. Data on exons 1–10 were
obtained for all patients, on exons 11–14 for 29 of the 34
individuals. All 65
TP53
-negative individuals were
screened for the c.1100delC
CHEK2
mutation and
CHEK2
DNA rearrangements by multiplex ligation-dependent
probe amplification (MLPA, see Table 1). Details are
available on request (
CHEK2
MLPA KIT, MRC Holland).
Mutation analysis was performed using the following ref-
erence sequence: CHEK2 (AF086904.1, GI:3982839,
).
In 1990 germline mutations in the
TP53
gene were
described in LFS [6]. So far, 419
TP53
-positive families
have been reported (IARC mutation database, R13,
November 2008 [7]). At present, in approximately 75% of
LFS and 40% of LFL families, a germline
TP53
mutation
can be detected [8]; i.e. 25% to 60% of LFS/LFL families
do not carry a detectable germline
TP53
mutation, imply-
ing the existence of alternative LFS susceptibility genes.
CHEK2 is a cell cycle checkpoint kinase involved in DNA
repair, cell death and cell cycle control by stabilizing the
p53 protein [9]. In 1999 Bell et al. first described the pos-
sible association of the
CHEK2
gene with Li-Fraumeni
syndrome [10]. Subsequent studies have addressed the
possible contribution of
CHEK2
germline mutations to
LFS and LFL syndrome, but could not confirm
CHEK2
as
a major gene involved in LFS [10-18].
In other studies, the specific
CHEK2
c.1100delC
frameshift mutation was shown to be associated with an
elevated breast cancer risk [19-22] and it has been sug-
gested that it contributes to a hereditary breast and color-
ectal cancer phenotype [23]. The prevalence of this
c.1100delC mutation seems to differ according to ethnic
backgrounds and populations and is relatively high in the
Netherlands [19,24]. We have investigated the
CHEK2
gene mutation status of 65 index patients from 65 Dutch
LFS/LFL families and families suggestive of LFS who had
tested negative for
TP53
germline mutations, to determine
the contribution of
CHEK2
germline mutations to the
phenotype in those families.
Sequence variants were weighted according to their poten-
tial pathogenicity. Three silent sequence variants were
seen and not further analysed: c.252A>G, p.Glu84Glu in
exon 1, a previously reported silent polymorphism [15],
found once, c.1566C>T, p.Pro522Pro and c.1608A>G,
p.Pro536Pro, both in exon 14, found in five and seven
families, respectively. For these three variants two splice
site prediction programs were used, NetGene2 Server
and BDGP
Splice Site Prediction/Neural Network
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Table 1: Number of
TP53
negative families available for
CHEK2
gene analysis divided into 3 groups: LFS, LFL, or LFS-suggestive family
history (n = 65), including the cancer type in tested individuals.
(family) history
Complete
CHEK2
mutation
analysis
(n = 34)
1100delC mutation analysis and
DNA rearrangements
(n = 31)
Cancer type in tested
individuals: B/S/other
1
LFS
1
0
1/0/0
2
LFL
20
15
18/7/10
3
LFS-suggestive
13
16
17/5/7
-childhood onset sarcoma or
brain tumour
1
1
0/1/1
-at least 2 primary tumours
3
7
5/1/4
-2 first degree relatives with
cancer
5
6
6/3/2
-breast cancer before 30 years
4
2
6/0/0
LFS = Li-Fraumeni syndrome
LFL = Li-Fraumeni-like syndrome according to Birch or Eeles
B = breast cancer
S = sarcoma
Other = other cancers, including adrenal cortical tumour, bladder cancer, brain tumour, colon cancer, kidney cancer, leukaemia, lung cancer,
melanoma, non-Hodgkin lymphoma, ovarian cancer and thyroid cancer.
; no alternative splice sites
were predicted.
breast cancer susceptibility allele [19]. Relatives with a
50% chance of being a c.1100delC carrier in this family
who had developed breast cancer were not available for
testing. However, it is not likely to be the LFS-causing
mutation in this family, considering the absence of the
c.1100delC in the patient's son who developed a sarcoma
at 15 years of age. In an LFL and LFS-suggestive family, the
patients identified as carrying the c.1100delC had breast
cancer (Figure 1B and 1D); in a fourth family, a LFS-sug-
gestive family, the patient identified with the c.1100delC
sequence variant had both breast and colorectal cancer
(Figure 1C). No additional material was available for test-
ing to see if and how the mutation segregates in these fam-
ilies. In all four of the c.1100delC families, this sequence
variant seemed to be associated with breast cancer or
breast and colorectal cancer, rather than LFS. The reported
frequency of the
CHEK
c.1100delC in Dutch controls is
1.4%, in Dutch breast cancer patients not selected for fam-
ily history 2.5% and in Dutch
BRCA1/2
-negative families
with breast cancer 4.9%[19]. In our sample the frequency
was 6.2% (4/65), significantly different from that for
healthy controls (p = 0.006).
When possible, the presence of a sequence variant
detected in an index patient was investigated in other
affected relatives. A control group of 150 anonymous
Dutch (male and female) blood donors was analysed by
DGGE to determine the prevalence of the sequence vari-
ants in a general population sample.
A chi-square test was used to determine the statistical sig-
nificance of the proportion of
CHEK2
mutation carriers in
our study group compared to healthy controls.
Results and discussion
Sixty-five
TP53
-negative individuals from 65 families were
screened for the
CHEK2
1100delC germline mutation and
DNA rearrangements. Thirty-four of these individuals
were screened comprehensively by DGGE for
CHEK2
mutations. Six index patients were found to carry a possi-
bly pathogenic germline
CHEK2
sequence variant.
The c.
1100delC in exon 10 of the
CHEK2
gene
, a muta-
tion located in the kinase domain of the gene and abolish-
ing the kinase activity of the protein, was detected in four
index patients. In one family, a classical LFS family, the
c.1100delC was detected in a patient who developed
breast cancer at the age of 48 years (Figure 1A), which is
in line with the c.1100delC acting as a low penetrance
Another sequence variant, c.
983T>C, p.Phe328Ser in
exon 8
, localised in the kinase domain of the gene, was
detected in a female patient who had developed a leiomy-
osarcoma at 2 years of age and a schwannoma at 27 years
of age (Figure 1E). The family of the index patient fulfilled
the LFL criteria (Eeles [4]). The parents of the index
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B.
LFL family,
c.1100delC
C.
LFS-suggestive family,
c.1100delC
A.
LFS family, c.1100delC
I
1
Bl70
1
C71
d76
4
cancer
I
7
5
2
B43
B48
mut +
B47
B71
d82
4
St72
B67
13
B43
14
L70
15
C61
B42, B71
B67
d74
L
II
3
2
B42
4
OS15
mut -
5
Leu21
6
End33
2
DFS17
1
B37, C42
mut +
7
OS
d21
D.
LFS-suggestive family,
c.1100delC
E.
LFL family, p.Phe328Ser
F. LFL family,
c.1096-?_1629+?del
I
2
cancer
3
Ki
4
Abd
2
cancer
2
3
I
2
5
B, O
2
B70
d72
6
Br45
4
St55
6
C>60
II
3
3
4
2
1
B38, B43
2
Sk
3
B48, SG
B43
d52
B53
I
1
Ho17
2
Ho23
1
LMS2, Schw27
mut +
2
RMS22
Pedigrees of germline
CHEK2
sequence variation families
Figure 1
Pedigrees of germline
CHEK2
sequence variation families
. Square symbols indicate males, round symbols indicate
females, line across symbol means deceased individual. Filled symbols indicate affected individuals with diagnosis confirmed by
pathology reports. A quarterly filled symbol indicate affected individuals with diagnosis by family history. Tumour type and age
at diagnosis of the tumours are indicated below the individual identifiers, d = age of death. The index patient is indicated with
an arrow. Abd = abdominal cancer, B = breast cancer, Bl = bladder cancer, Br = brain tumour, C = colorectal cancer, cancer =
cancer of unknown origin, DFS = dermatofibrosarcoma, End = endometrial cancer, Ho = Hodgkin lymphoma, Ki = kidney can-
cer, L = lung cancer, Leu = leukaemia, LMS = Leiomyosarcoma, O = ovarian cancer, Oes = oesophagus carcinoma, OS = oste-
osarcoma, RMS = rhabdomyosarcoma, Schw = schwannoma, SG = salivary gland cancer, Sk = skin cancer, St = stomach cancer,
mut + = mutation detected, mut - = mutation excluded.
patient are both healthy and over 60 years of age. A mater-
nal aunt died of breast cancer at 45 years of age and a sister
of the index patient's maternal grandmother died of a
brain tumour; no material was available for testing. Under
the assumption that these two affected family members
were carriers of the mutation, penetrance would be
incomplete with non-penetrance exhibited by two older
healthy obligate mutation carriers. The hypothesis that
the exon 8 mutation caused the complete LFL phenotype
in this family is unlikely although a de novo mutation,
contributing to the disease in the index patient, cannot be
excluded. The p.Phe328Ser missense mutation has not
been described in the literature before and was not found
in 150 healthy Dutch controls. The phenylalanine in this
position is conserved in mice and frogs but not in either
zebrafish or C. Elegans (Ensembl, v39-Jun 2006 [27]).
One
CHEK2
DNA rearrangement was found,
c.1096-
?_1629+?del
, a deletion of exons 10–14 of the gene. The
family fulfilled the LFL criteria according to Birch[3] and
the index patient developed breast cancer at the age of 55
years. A deletion of this size in the kinase domain of the
gene will probably abolish the kinase activity. This dele-
tion has not been described in the literature before. A
deletion of exon 9–10 predicting protein truncation at
codon 381 was discovered as a founder mutation among
patients of Czecholovakian ancestry with breast cancer
[17]. Unfortunately, no material from the daughter who
developed a rhabdomyosarcoma at 22 years of age was
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available. Four sibs of the index patient were healthy; her
father developed stomach cancer at 55 years of age.
families. Our data are in line with the hypothesis that the
CHEK2
c.1100delC might be associated with an elevated
breast cancer risk [19,20], and possibly with a breast and
colorectal cancer phenotype [23] or more generally a
multi-organ cancer susceptibility [32]. We propose that
the germline
CHEK2
sequence variants contribute to
tumour development in the index patients. Without these
tumours, the families would not have fulfilled the estab-
lished LFS/LFL criteria and
TP53
germline mutation test-
ing would not have been indicated. In this way, the
individual
CHEK2
sequence variants may contribute to
the Li-Fraumeni phenotype seen in these families.
So far, 8 studies have been published on
CHEK2
analysis
of a total of 196
TP53
-negative LFS families or families
suggestive of LFS (Table 2) [10-17]. Of the seven variants
presented, only the c.1100delC, the p.Ile157Thr and the
p.Arg145Trp mutation are of reported functional signifi-
cance. Bell et al [10] found the p.Ile157Thr in an index
patient with three primary cancers; no other family mem-
bers were tested. Allinen et al. [11] screened the
CHEK2
gene in 21 LFS/LFL families and only found the
p.Ile157Thr mutation; since it was found in healthy con-
trols as well, they concluded that it does not contribute to
an LFL-associated breast cancer risk. Some authors found
an association between the p.Ile157Thr mutation and risk
of female breast cancer [28-30], others found no associa-
tion [31]. The p.Arg145Trp, leading to a destabilised pro-
tein, was described in a Li-Fraumeni-like kindred; it was
only tested in one family-member with a sarcoma at 20
years and breast cancer at 42 years. It was not found in 200
controls [13].
Because only 75% of classical LFS families and 40% of LFL
families have germline
TP53
mutations, research groups
have looked at candidate genes like
Bcl10
[33],
CDKN2
[34,35],
TP63
[12],
PTEN
[34,36],
CHEK1
[10,14] and
BAX
[37]; no possible alternative LFS genes were found.
Two polymorphisms, p.Arg72Pro (
TP53
gene) and
SNP309 T>G (
MDM2
gene), have been shown to have a
modifying effect, resulting in an earlier age of onset of
cancer in
TP53
mutation carriers [38,39]; there is even a
synergistic effect when both polymorphisms are present.
These are proposed examples of modifying factors or low
penetrance gene mutations that play a role in age of onset
and tumour clustering in cancer-prone families [40]. In
In our study six index patients were found to carry a
CHEK2
sequence variant by screening 65
TP53
-negative
index patients, with no evidence that the sequence vari-
ants found caused the complete LFS phenotype in their
Table 2: Literature on
CHEK2
analysis in LFS and LFS-related families
number of families tested: LFS/
LFL/suggestive
total
CHEK2
analysis
CHEK2 1100delC analysis +
DNA rearrangements
mutations found
Allinen et al
. [11]
1/20/0
21
0
p.Ile157Thr
Bell et al
. [10]
4/18*
22
0
c.1100delC
p.Ile157Thr
Bougeard et al
. [12]
0/4/0
4
0
-
Lee et al
. [13]
10/49*
59
0
p.Arg145Trp
p.Arg3Trp
p.Ile157Thr
Siddiqui et al
. [16]
1/13/1
0
15
-
Sodha et al
. [15]
5/21/0
26
0
IVS5-11G>A
c.483-485delAGA
Vahteristo et al
. [14]
1/6/32
39
0
c.1100delC
Walsh et al
. [16]
3/7/0
10
-
our results
1/35/29
34
31
p.Phe328Ser
c.1100delC
c.1081-?_1771+?del
* = LFL and LFS-suggestive combined, subdivision not further mentioned
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zanotowane.pl doc.pisz.pl pdf.pisz.pl hannaeva.xlx.pl
Practice
Research
The contribution of
CHEK2
to the
TP53
-negative Li-Fraumeni
phenotype
Marielle WG Ruijs
1,3
, Annegien Broeks
2
, Fred H Menko
3
,
Margreet GEM Ausems
4
, Anja Wagner
5
, Rogier Oldenburg
5
, Hanne Meijers-
Heijboer
3,5
, Laura J van't Veer
2
and Senno Verhoef*
1
Address:
1
Family Cancer Clinic, The Netherlands Cancer Institute, Amsterdam, The Netherlands,
2
Department of Experimental Therapy, The
Netherlands Cancer Institute, Amsterdam, The Netherlands,
3
Department of Clinical Genetics and Human Genetics, VU University Medical
Centre, Amsterdam, The Netherlands,
4
Department of Medical Genetics, University Medical Centre, Utrecht, The Netherlands and
5
Department
of Clinical Genetics, Erasmus Medical Centre, Rotterdam, The Netherlands
Email: Marielle WG Ruijs - mwg.ruijs@vumc.nl; Annegien Broeks - a.broeks@nki.nl; Fred H Menko - fh.menko@vumc.nl;
Margreet GEM Ausems - M.G.E.M.Ausems@umcutrecht.nl; Anja Wagner - a.wagner@erasmusmc.nl;
Rogier Oldenburg - r.oldenburg@erasmusmc.nl; Hanne Meijers-Heijboer - H.Meijers@vumc.nl; Laura J van't Veer - l.vt.veer@nki.nl;
Senno Verhoef* - s.verhoef@nki.nl
* Corresponding author
Published: 17 February 2009
Hereditary Cancer in Clinical Practice
2009,
7
:4
Received: 14 November 2008
Accepted: 17 February 2009
doi:10.1186/1897-4287-7-4
This article is available from: http://www.hccpjournal.com/content/7/1/4
© 2009 Ruijs et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background:
CHEK2
has previously been excluded as a major cause of Li-Fraumeni syndrome
(LFS). One particular
CHEK2
germline mutation, c.1100delC, has been shown to be associated with
elevated breast cancer risk. The prevalence of
CHEK2
*1100delC differs between populations and
has been found to be relatively high in the Netherlands. The question remains nevertheless
whether
CHEK2
germline mutations contribute to the Li-Fraumeni phenotype.
Methods:
We have screened 65 Dutch
TP53
-negative LFS/LFL candidate patients for
CHEK2
germline mutations to determine their contribution to the LFS/LFL phenotype.
Results:
We identified six index patients with a
CHEK2
sequence variant, four with the c.1100delC
variant and two sequence variants of unknown significance, p.Phe328Ser and c.1096-?_1629+?del.
Conclusion:
Our data show that
CHEK2
is not a major LFS susceptibility gene in the Dutch
population. However,
CHEK2
might be a factor contributing to individual tumour development in
TP53
-negative cancer-prone families.
Background
Li-Fraumeni syndrome (LFS) is a rare autosomal domi-
nant cancer syndrome predisposing for bone and soft tis-
sue sarcoma, breast cancer, brain tumour, adrenocortical
carcinoma and leukaemia [1]. The classical LFS criteria
are: a proband with sarcoma aged under 45 years and a
first-degree relative with any cancer aged under 45 years,
plus a first or second-degree relative in the same lineage
with any cancer under the age of 45 years or sarcoma at
any age [2]. In addition, Li-Fraumeni-like syndrome (LFL)
criteria have been formulated as a proband with any child-
hood tumour or a sarcoma, brain tumour or adrenocorti-
cal tumour diagnosed under 45 years of age and a first or
second-degree relative in the same lineage with a typical
Page 1 of 7
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2009,
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:4
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Methods
All 65 affected index patients had been assessed and coun-
selled in various clinical genetics centres because of the
occurrence of different cancer types related to LFS and had
as a consequence been tested for
TP53
germline muta-
tions. On the basis of the available clinical data, the
TP53
-
negative families were classified into 3 groups: 1) classical
LFS [2], 2) LFL syndrome according to Birch [3] or Eeles
[4] and 3) LFS-suggestive, including childhood onset
(under 18 years) sarcoma or brain tumours, two or more
primary tumours at any age, two first-degree relatives with
a tumour at any age, of which at least one relative has a
typical LFS tumour or breast cancer under 30 years of age
(without
BRCA1
or
BRCA2
mutations) (Table 1). In fam-
ilies with multiple breast cancer cases and individuals
with breast and ovarian cancer,
BRCA1
or
BRCA2
muta-
tions were excluded, according to standard procedures.
Details are available on request.
LFS tumour at any age, plus a first or second-degree rela-
tive in the same lineage younger than 60 years with any
cancer [3]. Less stringent LFL criteria were formulated by
Eeles et al. as two first or second-degree relatives with typ-
ical LFS-extended tumours (classical LFS tumours plus
melanoma, prostate cancer and pancreatic cancer) at any
age [4]. The Chompret criteria for
TP53
germline muta-
tion testing have been updated in 2008 as: (1) a proband
with a tumour belonging to the LFS tumour spectrum
(sarcomas, brain tumours, pre-menopausal breast cancer,
adrenocortical carcinoma, leukaemia, lung bronchoalveo-
lar cancer) cancer before 46 years of age and at least one
first or second-degree relative with an LFS tumour before
56 years of age or multiple tumours; or (2) a proband with
multiple tumours two of which belong to the narrow LFS
tumour spectrum and the first of which occurred before
46 years of age; or (3) a patient with adrenocortical carci-
noma or a patient with breast cancer before 36 years of age
without
BRCA
mutation, irrespective of the family history
[5].
DNA from peripheral blood lymphocytes was isolated
according to standard procedures. Screening for
TP53
germline mutations was performed by sequence analysis
of all coding exons (2–11) including flanking intron-exon
boundaries (details are available on request) and multi-
plex ligation-dependent probe amplification (MLPA) [25]
(
TP53
MLPA KIT, MRC Holland). In 34
TP53
-negative
LFS, LFL, or LFS-suggestive families all exons and flanking
intron-exon boundaries of the
CHEK2
gene were investi-
gated using denaturing gradient gel electrophoresis
(DGGE, see Table 1) [26]. All possible candidate variants,
identified as aberrant DGGE fragments, were confirmed
by sequence analysis. To avoid amplification of pseudo-
genes, a long range PCR was performed first for exons 10
to 14, followed by a nested PCR. Data on exons 1–10 were
obtained for all patients, on exons 11–14 for 29 of the 34
individuals. All 65
TP53
-negative individuals were
screened for the c.1100delC
CHEK2
mutation and
CHEK2
DNA rearrangements by multiplex ligation-dependent
probe amplification (MLPA, see Table 1). Details are
available on request (
CHEK2
MLPA KIT, MRC Holland).
Mutation analysis was performed using the following ref-
erence sequence: CHEK2 (AF086904.1, GI:3982839,
).
In 1990 germline mutations in the
TP53
gene were
described in LFS [6]. So far, 419
TP53
-positive families
have been reported (IARC mutation database, R13,
November 2008 [7]). At present, in approximately 75% of
LFS and 40% of LFL families, a germline
TP53
mutation
can be detected [8]; i.e. 25% to 60% of LFS/LFL families
do not carry a detectable germline
TP53
mutation, imply-
ing the existence of alternative LFS susceptibility genes.
CHEK2 is a cell cycle checkpoint kinase involved in DNA
repair, cell death and cell cycle control by stabilizing the
p53 protein [9]. In 1999 Bell et al. first described the pos-
sible association of the
CHEK2
gene with Li-Fraumeni
syndrome [10]. Subsequent studies have addressed the
possible contribution of
CHEK2
germline mutations to
LFS and LFL syndrome, but could not confirm
CHEK2
as
a major gene involved in LFS [10-18].
In other studies, the specific
CHEK2
c.1100delC
frameshift mutation was shown to be associated with an
elevated breast cancer risk [19-22] and it has been sug-
gested that it contributes to a hereditary breast and color-
ectal cancer phenotype [23]. The prevalence of this
c.1100delC mutation seems to differ according to ethnic
backgrounds and populations and is relatively high in the
Netherlands [19,24]. We have investigated the
CHEK2
gene mutation status of 65 index patients from 65 Dutch
LFS/LFL families and families suggestive of LFS who had
tested negative for
TP53
germline mutations, to determine
the contribution of
CHEK2
germline mutations to the
phenotype in those families.
Sequence variants were weighted according to their poten-
tial pathogenicity. Three silent sequence variants were
seen and not further analysed: c.252A>G, p.Glu84Glu in
exon 1, a previously reported silent polymorphism [15],
found once, c.1566C>T, p.Pro522Pro and c.1608A>G,
p.Pro536Pro, both in exon 14, found in five and seven
families, respectively. For these three variants two splice
site prediction programs were used, NetGene2 Server
and BDGP
Splice Site Prediction/Neural Network
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Table 1: Number of
TP53
negative families available for
CHEK2
gene analysis divided into 3 groups: LFS, LFL, or LFS-suggestive family
history (n = 65), including the cancer type in tested individuals.
(family) history
Complete
CHEK2
mutation
analysis
(n = 34)
1100delC mutation analysis and
DNA rearrangements
(n = 31)
Cancer type in tested
individuals: B/S/other
1
LFS
1
0
1/0/0
2
LFL
20
15
18/7/10
3
LFS-suggestive
13
16
17/5/7
-childhood onset sarcoma or
brain tumour
1
1
0/1/1
-at least 2 primary tumours
3
7
5/1/4
-2 first degree relatives with
cancer
5
6
6/3/2
-breast cancer before 30 years
4
2
6/0/0
LFS = Li-Fraumeni syndrome
LFL = Li-Fraumeni-like syndrome according to Birch or Eeles
B = breast cancer
S = sarcoma
Other = other cancers, including adrenal cortical tumour, bladder cancer, brain tumour, colon cancer, kidney cancer, leukaemia, lung cancer,
melanoma, non-Hodgkin lymphoma, ovarian cancer and thyroid cancer.
; no alternative splice sites
were predicted.
breast cancer susceptibility allele [19]. Relatives with a
50% chance of being a c.1100delC carrier in this family
who had developed breast cancer were not available for
testing. However, it is not likely to be the LFS-causing
mutation in this family, considering the absence of the
c.1100delC in the patient's son who developed a sarcoma
at 15 years of age. In an LFL and LFS-suggestive family, the
patients identified as carrying the c.1100delC had breast
cancer (Figure 1B and 1D); in a fourth family, a LFS-sug-
gestive family, the patient identified with the c.1100delC
sequence variant had both breast and colorectal cancer
(Figure 1C). No additional material was available for test-
ing to see if and how the mutation segregates in these fam-
ilies. In all four of the c.1100delC families, this sequence
variant seemed to be associated with breast cancer or
breast and colorectal cancer, rather than LFS. The reported
frequency of the
CHEK
c.1100delC in Dutch controls is
1.4%, in Dutch breast cancer patients not selected for fam-
ily history 2.5% and in Dutch
BRCA1/2
-negative families
with breast cancer 4.9%[19]. In our sample the frequency
was 6.2% (4/65), significantly different from that for
healthy controls (p = 0.006).
When possible, the presence of a sequence variant
detected in an index patient was investigated in other
affected relatives. A control group of 150 anonymous
Dutch (male and female) blood donors was analysed by
DGGE to determine the prevalence of the sequence vari-
ants in a general population sample.
A chi-square test was used to determine the statistical sig-
nificance of the proportion of
CHEK2
mutation carriers in
our study group compared to healthy controls.
Results and discussion
Sixty-five
TP53
-negative individuals from 65 families were
screened for the
CHEK2
1100delC germline mutation and
DNA rearrangements. Thirty-four of these individuals
were screened comprehensively by DGGE for
CHEK2
mutations. Six index patients were found to carry a possi-
bly pathogenic germline
CHEK2
sequence variant.
The c.
1100delC in exon 10 of the
CHEK2
gene
, a muta-
tion located in the kinase domain of the gene and abolish-
ing the kinase activity of the protein, was detected in four
index patients. In one family, a classical LFS family, the
c.1100delC was detected in a patient who developed
breast cancer at the age of 48 years (Figure 1A), which is
in line with the c.1100delC acting as a low penetrance
Another sequence variant, c.
983T>C, p.Phe328Ser in
exon 8
, localised in the kinase domain of the gene, was
detected in a female patient who had developed a leiomy-
osarcoma at 2 years of age and a schwannoma at 27 years
of age (Figure 1E). The family of the index patient fulfilled
the LFL criteria (Eeles [4]). The parents of the index
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B.
LFL family,
c.1100delC
C.
LFS-suggestive family,
c.1100delC
A.
LFS family, c.1100delC
I
1
Bl70
1
C71
d76
4
cancer
I
7
5
2
B43
B48
mut +
B47
B71
d82
4
St72
B67
13
B43
14
L70
15
C61
B42, B71
B67
d74
L
II
3
2
B42
4
OS15
mut -
5
Leu21
6
End33
2
DFS17
1
B37, C42
mut +
7
OS
d21
D.
LFS-suggestive family,
c.1100delC
E.
LFL family, p.Phe328Ser
F. LFL family,
c.1096-?_1629+?del
I
2
cancer
3
Ki
4
Abd
2
cancer
2
3
I
2
5
B, O
2
B70
d72
6
Br45
4
St55
6
C>60
II
3
3
4
2
1
B38, B43
2
Sk
3
B48, SG
B43
d52
B53
I
1
Ho17
2
Ho23
1
LMS2, Schw27
mut +
2
RMS22
Pedigrees of germline
CHEK2
sequence variation families
Figure 1
Pedigrees of germline
CHEK2
sequence variation families
. Square symbols indicate males, round symbols indicate
females, line across symbol means deceased individual. Filled symbols indicate affected individuals with diagnosis confirmed by
pathology reports. A quarterly filled symbol indicate affected individuals with diagnosis by family history. Tumour type and age
at diagnosis of the tumours are indicated below the individual identifiers, d = age of death. The index patient is indicated with
an arrow. Abd = abdominal cancer, B = breast cancer, Bl = bladder cancer, Br = brain tumour, C = colorectal cancer, cancer =
cancer of unknown origin, DFS = dermatofibrosarcoma, End = endometrial cancer, Ho = Hodgkin lymphoma, Ki = kidney can-
cer, L = lung cancer, Leu = leukaemia, LMS = Leiomyosarcoma, O = ovarian cancer, Oes = oesophagus carcinoma, OS = oste-
osarcoma, RMS = rhabdomyosarcoma, Schw = schwannoma, SG = salivary gland cancer, Sk = skin cancer, St = stomach cancer,
mut + = mutation detected, mut - = mutation excluded.
patient are both healthy and over 60 years of age. A mater-
nal aunt died of breast cancer at 45 years of age and a sister
of the index patient's maternal grandmother died of a
brain tumour; no material was available for testing. Under
the assumption that these two affected family members
were carriers of the mutation, penetrance would be
incomplete with non-penetrance exhibited by two older
healthy obligate mutation carriers. The hypothesis that
the exon 8 mutation caused the complete LFL phenotype
in this family is unlikely although a de novo mutation,
contributing to the disease in the index patient, cannot be
excluded. The p.Phe328Ser missense mutation has not
been described in the literature before and was not found
in 150 healthy Dutch controls. The phenylalanine in this
position is conserved in mice and frogs but not in either
zebrafish or C. Elegans (Ensembl, v39-Jun 2006 [27]).
One
CHEK2
DNA rearrangement was found,
c.1096-
?_1629+?del
, a deletion of exons 10–14 of the gene. The
family fulfilled the LFL criteria according to Birch[3] and
the index patient developed breast cancer at the age of 55
years. A deletion of this size in the kinase domain of the
gene will probably abolish the kinase activity. This dele-
tion has not been described in the literature before. A
deletion of exon 9–10 predicting protein truncation at
codon 381 was discovered as a founder mutation among
patients of Czecholovakian ancestry with breast cancer
[17]. Unfortunately, no material from the daughter who
developed a rhabdomyosarcoma at 22 years of age was
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available. Four sibs of the index patient were healthy; her
father developed stomach cancer at 55 years of age.
families. Our data are in line with the hypothesis that the
CHEK2
c.1100delC might be associated with an elevated
breast cancer risk [19,20], and possibly with a breast and
colorectal cancer phenotype [23] or more generally a
multi-organ cancer susceptibility [32]. We propose that
the germline
CHEK2
sequence variants contribute to
tumour development in the index patients. Without these
tumours, the families would not have fulfilled the estab-
lished LFS/LFL criteria and
TP53
germline mutation test-
ing would not have been indicated. In this way, the
individual
CHEK2
sequence variants may contribute to
the Li-Fraumeni phenotype seen in these families.
So far, 8 studies have been published on
CHEK2
analysis
of a total of 196
TP53
-negative LFS families or families
suggestive of LFS (Table 2) [10-17]. Of the seven variants
presented, only the c.1100delC, the p.Ile157Thr and the
p.Arg145Trp mutation are of reported functional signifi-
cance. Bell et al [10] found the p.Ile157Thr in an index
patient with three primary cancers; no other family mem-
bers were tested. Allinen et al. [11] screened the
CHEK2
gene in 21 LFS/LFL families and only found the
p.Ile157Thr mutation; since it was found in healthy con-
trols as well, they concluded that it does not contribute to
an LFL-associated breast cancer risk. Some authors found
an association between the p.Ile157Thr mutation and risk
of female breast cancer [28-30], others found no associa-
tion [31]. The p.Arg145Trp, leading to a destabilised pro-
tein, was described in a Li-Fraumeni-like kindred; it was
only tested in one family-member with a sarcoma at 20
years and breast cancer at 42 years. It was not found in 200
controls [13].
Because only 75% of classical LFS families and 40% of LFL
families have germline
TP53
mutations, research groups
have looked at candidate genes like
Bcl10
[33],
CDKN2
[34,35],
TP63
[12],
PTEN
[34,36],
CHEK1
[10,14] and
BAX
[37]; no possible alternative LFS genes were found.
Two polymorphisms, p.Arg72Pro (
TP53
gene) and
SNP309 T>G (
MDM2
gene), have been shown to have a
modifying effect, resulting in an earlier age of onset of
cancer in
TP53
mutation carriers [38,39]; there is even a
synergistic effect when both polymorphisms are present.
These are proposed examples of modifying factors or low
penetrance gene mutations that play a role in age of onset
and tumour clustering in cancer-prone families [40]. In
In our study six index patients were found to carry a
CHEK2
sequence variant by screening 65
TP53
-negative
index patients, with no evidence that the sequence vari-
ants found caused the complete LFS phenotype in their
Table 2: Literature on
CHEK2
analysis in LFS and LFS-related families
number of families tested: LFS/
LFL/suggestive
total
CHEK2
analysis
CHEK2 1100delC analysis +
DNA rearrangements
mutations found
Allinen et al
. [11]
1/20/0
21
0
p.Ile157Thr
Bell et al
. [10]
4/18*
22
0
c.1100delC
p.Ile157Thr
Bougeard et al
. [12]
0/4/0
4
0
-
Lee et al
. [13]
10/49*
59
0
p.Arg145Trp
p.Arg3Trp
p.Ile157Thr
Siddiqui et al
. [16]
1/13/1
0
15
-
Sodha et al
. [15]
5/21/0
26
0
IVS5-11G>A
c.483-485delAGA
Vahteristo et al
. [14]
1/6/32
39
0
c.1100delC
Walsh et al
. [16]
3/7/0
10
-
our results
1/35/29
34
31
p.Phe328Ser
c.1100delC
c.1081-?_1771+?del
* = LFL and LFS-suggestive combined, subdivision not further mentioned
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