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Research article
Mutation analysis of the CHK2 gene in breast carcinoma and
other cancers
Sigurdur Ingvarsson
1
, Bjarnveig I Sigbjornsdottir
2
, Chen Huiping
2
, Sigridur H Hafsteinsdottir
2
,
Gisli Ragnarsson
2
, Rosa B Barkardottir
2
, Adalgeir Arason
2
, Valgardur Egilsson
2
and Jon Th Bergthorsson
2
1
Institute for Experimental Pathology, University of Iceland, Reykjavik, Iceland
2
Department of Pathology, University Hospital, Reykjavik, Iceland
Correspondence: Sigurdur Ingvarsson, Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, 112 Reykjavik, Iceland.
Tel: +354 5674700; fax: +354 5673979; e-mail: siguring@hi.is
Introduction
Chk2 (Cds1) is a protein kinase that is involved in cell-
cycle checkpoint control by phosphorylating Cdc25 phos-
phatases, which subsequently results in their inhibition (i.e.
degradation or export from the nucleus) [1–4]. Other sub-
strates of Chk2 are p53 and Brca1, which are involved in
cell-cycle control, apoptosis, and DNA repair [4–7]. The
serine 20 of p53 is phosphorylated by Chk2, and thereby
interrupts the binding of p53 to Mdm2 and interrupts p53
ubiquitination, resulting in greater stability of p53 [8].
Chk2 is activated on DNA damage by phosphorylation
signaling from the Atm kinase [8–13].
LOH = loss of heterozygosity; PCR = polymerase chain reaction; RT = reverse transcriptase; SSCP = single-strand conformation polymorphism.
Available online http://breast-cancer-research.com/content/4/3/R4
Abstract
Background: Mutations in the CHK2 gene at chromosome 22q12.1 have been reported in families
with Li–Fraumeni syndrome. Chk2 is an effector kinase that is activated in response to DNA damage
and is involved in cell-cycle pathways and p53 pathways.
Methods: We screened 139 breast tumors for loss of heterozygosity at chromosome 22q, using
seven microsatellite markers, and screened 119 breast tumors with single-strand conformation
polymorphism and DNA sequencing for mutations in the CHK2 gene.
Results: Seventy-four of 139 sporadic breast tumors (53%) show loss of heterozygosity with at least
one marker. These samples and 45 tumors from individuals carrying the BRCA2 999del5 mutation
were screened for mutations in the CHK2 gene. In addition to putative polymorphic regions in short
mononucleotide repeats in a non-coding exon and intron 2, a germ line variant (T59K) in the first
coding exon was detected. On screening 1172 cancer patients for the T59K sequence variant, it was
detected in a total of four breast-cancer patients, two colon-cancer patients, one stomach-cancer
patient and one ovary-cancer patient, but not in 452 healthy individuals. A tumor-specific 5′ splice site
mutation at site +3 in intron 8 (TTgt[a → c]atg) was also detected.
Conclusion: We conclude that somatic CHK2 mutations are rare in breast cancer, but our results
suggest a tumor suppressor function for CHK2 in a small proportion of breast tumors. Furthermore, our
results suggest that the T59K CHK2 sequence variant is a low-penetrance allele with respect to tumor
growth.
Keywords: breast cancer, CHK2, chromosome 22q, mutation, polymorphism
Received: 21 September 2001
Revisions requested: 6 November 2001
Revisions received: 3 January 2002
Accepted: 26 February 2002
Published: 20 March 2002
Breast Cancer Res 2002, 4:R4
This article may contain supplementary data which can only be found
online at http://breast-cancer-research.com/content/4/3/R4
© 2002 Ingvarsson et al., licensee BioMed Central Ltd
(Print ISSN 1465-5411; Online ISSN 1465-542X)
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Breast Cancer Research Vol 4 No 3 Ingvarsson et al.
Germ line mutations have been detected in the TP53 and
CHK2 genes in patients with Li–Fraumeni syndrome
[14–16]. These mutated forms of the Chk2 may have dis-
abilities in protein–protein interactions and in being phos-
phorylated by Atm [17]. Characteristic tumor types in
patients with Li–Fraumeni syndrome are breast cancer,
sarcoma, brain carcinoma and adrenal cortex carcinoma,
and multiple primary tumors can be observed in the same
individuals. There are few reports on somatic mutations of
CHK2 in tumors, but mutations have been found in a
colon carcinoma cell line and in a case of primary small-
cell lung cancer, lymphoma and myelodysplastic syn-
drome, respectively [14,18–20]. We used microsatellite
markers to analyse the loss of heterozygosity (LOH) at
chromosome region 22q, where the CHK2 gene is
located, and screened breast and other tumors for muta-
tions in the CHK2 gene.
Materials and methods
Primary breast carcinoma tissue was obtained on the day of
surgery. Blood samples from the patients were collected in
EDTA and, if not processed immediately, tumor and blood
were quick-frozen at –70°C. LOH at chromosome 22q was
analysed using seven microsatellite markers on 139 spo-
radic breast tumors. DNA was analysed by PCR primers
that amplify markers D22S277, D22S283, D22S1177,
D22S272, D22S423, D22S1179 and D22S282. These
markers map telomeric to CHK2 (at position 25,750 kb), or
at positions 32,830 kb, 33,300 kb, 33,800 kb, 35,600 kb,
36,900 kb, 40,100 kb and 40,350 kb, respectively.
DNA samples (25 ng) were subjected to PCR analysis in a
total volume of 25 µl using DynaZyme™ polymerase
(Finnzymes Oy, Espoo, Finland), in 120 µM of each
deoxynucleotide triphosphate and 0.24 µM primers. After
5 min of denaturation at 94°C, samples were subjected to
35 cycles of amplification, consisting of 30 s at 94°C, 30 s
at 55°C and 1 min at 72°C. The PCR products were
denatured in formamide buffer, separated on 6.5% poly-
acrylamide denaturing gels, and transferred to a Hybond-
N
+
nylon membrane (Amersham, Aylesbury, UK).
Hybridisation of a peroxidase-labeled probe to the PCR
products was visualised using the enhanced chemilumi-
nescence labeling method (ECL kit; Amersham).
LOH was evaluated visually by comparing the intensity of
alleles from normal and tumor DNA. The absence or
decrease in the intensity of one allele relative to the other
was considered as LOH. Tumor samples were scored for
LOH at chromosome 22q if at least one informative marker
showed LOH. One sample that was homozygous for all
tested markers was excluded from the study, while other
samples tested heterozygous with two to seven markers.
Sporadic tumors (BRCA1 and BRCA2 mutation carriers
were excluded) showing LOH at chromosome 22q (74
cases) and 45 breast tumors from carriers of the BRCA2
999del5 mutation [21], not analysed for LOH at 22q, were
analysed with single-strand conformation polymorphism
(SSCP) and DNA sequencing, using 17 primers for all of
the 15 exons of the CHK2 gene. The set of BRCA2
samples was screened to address the possibility that
germ line variants could modify BRCA2 risk. The primers
were ordered from TAG Copenhagen A/S and information
on them is presented in Table 1. The SSCP and DNA
sequencing conditions were as described earlier [22].
An additional 1098 tumors of various origins were
analysed for the detected T59K sequence variant using
the primers from exon 2 and DNA sequencing. RNA was
isolated from a tumor showing a splice site mutation and
analysed with RT-PCR using primers from exon 7
(forward, 5′-CCCAGCTCTCAATGTTGAAACAG-3′) and
exon 9 (reverse, 5′-CTGCACAGCCAAGAGCATCTGG-3′).
Abnormally sized PCR products were cut from 4%
agarose gels and DNA sequenced.
The chi-squared test and Fisher’s exact test were used to
compare differences of the frequency of sequence vari-
ants, between controls, individuals with sporadic breast
cancer and individuals with breast cancer carrying the
BRCA2 999del5 germ line mutation. The research plan
was approved by the National Bioethics Committee.
Results
Seventy-four out of 139 (53%) sporadic breast tumors were
detected with LOH at chromosome 22q. Only 11 (16%) of
the tumors with 22q LOH showed an almost complete loss
of the alleles, while other tumors showed less decrease in
allele intensity, presumably due to contamination of DNA
from non-malignant cells. To evaluate whether CHK2
sequence variants could have modifying effects on the phe-
notype of BRCA2 mutation carriers, these 74 samples and
45 breast tumors from individuals carrying the BRCA2
999del5 mutation were screened for mutations in the
CHK2 gene using SSCP and DNA sequencing.
Five sequence variants were detected: four in the group of
74 sporadic tumors and three in the BRCA2 999del5
group, with two variants common to both groups (Tables 2
and 3). Four of these five sequence variants were also
detected in the blood of the patients, indicating a germ line
variant, and one mutation was tumor specific, a +3TTgt(a
→ c)agt in the 5′ splice site of intron 8, detected in the
group of sporadic breast cancer. This somatic mutation
was detected in the tumor of an individual diagnosed with
breast cancer at the age of 45 years. LOH at 22q in this
tumor was evident from the microsatellite marker analysis.
This position in the splice site at the exon 8–intron 8
boundary does not include the part with the highest con-
servation, but we analysed the RNA from the tumor to see
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whether splicing was altered. RT-PCR analysis of RNA
from this tumor showed one extra band of lower molecular
weight and one band of higher molecular weight than the
wild-type transcript, not detected in five nonmalignant and
five breast cancer tissues (data not shown). DNA
sequencing of the RT-PCR product of the smaller sized
transcript showed that the exon 8 sequence was missing.
One of the sequence variants was a clear polymorphism
of A → G at nucleotide 252, not affecting the correspond-
ing codon 84 for glutamic acid. We also detected a
sequence variant in a non-coding exon of the CHK2 gene,
a deletion of a thymine. This occurred in a short stretch of
Available online http://breast-cancer-research.com/content/4/3/R4
Table 1
Information on the primers used for single-strand conformation polymorphism and DNA sequencing of the CHK2 gene
Exon number Forward primer Reverse primer
15′-GGGTTTTGATTGGCTGAGG-3′ 5′-GCTCAAAACTACAGACAAAGC-3′
25′-CTCACCTTTGTTGTTGGACAC-3′ 5′-GGACACTGTCTCTAAGGAGC-3′
25′-AGTCCTCTCACTCCAGC-3′ 5′-ATCAGAACCTTCCACCTGG-3′
35′-ATTCAACAGCCCTCTGATGC-3′ 5′-CAGCTCTCCTAGATACATGG-3′
45′-TCTGCTATTCAAAGTCTG-3′ 5′-TCCTCCTATGAGAGAGTGG-3′
55′-GAAATGAGAAACCACCAATCAC-3′ 5′-TCAGTGATCGCCTCTTGTG-3′
65′-TACTTGAAGTGGACCCAGG-3′ 5′-GGGAAGTTATGAAGACGTG-3′
75′-CAAAGTGCTAGGGTTACAGG-3′ 5′-CAGCCTTGAGTCAACTGAG-3′
85′-GCTCTTGTGGTTTTCCTCTTGG-′ 5′-CCTACATTAGATTCTTTGGTGG-3′
95′-CTGTCCAAGTGCGTTTTCC-3′ 5′-CGATTTCTGCCTAATTCAGGG-3′
10 5′-ACGGCTTACGGTTTCACC-3′ 5′-CAAGAATCTACAGGAATAGCC-3′
11 5′-CTTGGACTGGCAGACTATG-3′ 5′-CTCCTACCAGTCTGTGCAG-3′
12 5′-ATGCCACTGAGAATGCCAC-3′ 5′-CTCCCACCACAGCACATAC-3′
13 5′-CCTTTTCACTGTGATTTGCCC-3′ 5′-CATGTTTCTGTCCTCTGTCTC-3′
14 5′-CTAGCCCTGTCATTCTAGG-3′ 5′-CTCCTTAAGCCCAGACTAC-3′
15 5′-TGTGTTGTGAACTCCGTGG-3′ 5′-CAGAGTGAGACTCCATCTC-3′
15 5′-CTTTACTGGAAGCATATTGAGG–3′ 5′-AGATGACAGAGTGAAAGAAGG-3′
Table 2
CHK2 sequence variations in breast cancer
Sequence variation Codon Nucleotide change and position Effect on protein
Germ line 59 C176A T59K
Non-coding delT (exon 1) None
Non-coding insA (intron 2) None
Somatic 303 +3TTgt(a → c)agt (intron 8) Splice site
Polymorphism 84 A252G None
All mutations were heterozygous. The total number of patients was 119 (74 sporadic and 45 BRCA2 999del5).
Table 3
Frequency of the detected sequence variants of the CHK2 gene
in breast cancer and controls
Sequence variant Controls Sporadic BRCA2 999del5
T59K 0/904 1/146 0/74
Exon 1 (delT) 5/328 1/148 2/90
Intron 2 (insA) 38/344 21/130 14/78
A252G 5/344 0/130 1/74
The numbers stated refer to the number of chromosomes analysed.
mononucleotide repeats of four thymines. An additional
sequence variant was detected in intron 2, an insertion of
an adenine in a mononucleotide repeat of five adenines. A
missense mutation at codon 59 was also detected, which
substituted lysine for threonine.
The frequency of these sequence variants was estimated
in individuals without previous diagnosis of cancer, in indi-
viduals with sporadic breast cancer, and in individuals with
breast cancer carrying the BRCA2 999del5 mutation
(Table 3). Chi-squared analysis and Fisher’s exact tests
did not result in significant difference between the three
groups for any of the sequence variants.
The only variant allele not appearing in normal control was
the T59K mutation, and this was analysed further in addi-
tional controls (in total, 452 individuals) and in individuals
diagnosed with cancer in the breast and other tissues (in
total, 1172 individuals diagnosed with cancer) (Table 4).
The T59K sequence variant was found in additional
patients with cancer of the breast (four individuals, one
thereof bilateral), cancer of the colon and ovary (one indi-
vidual), cancer of the colon (one individual), and cancer of
the stomach (one individual). All individuals who were
detected with the CHK2 T59K mutation in this screening
are females.
Four of the CHK2 T59K carriers were found to be
members of two cancer families. One of the two individu-
als with colorectal cancer is a sister of the individual with
gastric cancer. A third member of this family (a nephew of
the two aforementioned cases) also developed colon
cancer, but was not a carrier of the CHK2 T59K sequence
variant. However, a distant relative with prostate cancer
was a carrier of the T59K sequence variant.
Two of the breast-cancer cases (diagnosed at 29 years
and 45 years, respectively) were first cousins in a previ-
ously reported cancer family [23]. The third cancer case in
this family (stomach cancer diagnosed at the age of
55 years and thyroid cancer diagnosed at the age of
58 years in the same individual), a brother of the breast-
cancer case diagnosed at age 45 years, is a T59K carrier,
but two individuals within this family (with breast cancer
and thyroid cancer, respectively) were not T59K carriers.
In addition, six healthy individuals of this family did not
carry the T59K sequence variant. The two breast-cancer
cases with the T59K sequence variant, but no obvious
family history of cancer, were diagnosed at 83 years (a
bilateral case) and 42 years of age, respectively.
In summary, the CHK2 T59K sequence variant was
detected in seven individuals by the screening (including
the original carrier from Table 2) and in an additional two
individuals in the family analysis, producing a total of nine
individuals. Three out of these nine individuals were diag-
nosed with two tumors (i.e. in total, 12 tumors in nine indi-
viduals). Eleven out of the 12 tumors were available for
LOH analysis at chromosome 22q, using markers
D22S277, D22S1177, D22S272, D22S423 and
D221179. Eight of these 11 tumors showed LOH with at
least one marker. The three tumors not showing LOH
derived from breast cancer (an individual with bilateral
disease had LOH at chromosome 22q in only one of the
two primary tumors).
Discussion
This screening of a large number of tumor samples sug-
gests that CHK2 gene inactivation does not play a major
role in the pathogenesis of cancer growth. We identified a
somatic mutation at a splice site in one tumor sample,
resulting in abnormal splicing of the gene, in an individual
diagnosed with breast cancer at the age of 45 years. The
wild-type copy of the CHK2 gene was lost in this individ-
ual, suggesting a typical two-hit mechanism of a tumor
suppressor gene.
Only one of the four detected germ line variants affects
the Chk2 protein sequence, by substituting lysine for
threonine at amino acid position 59. This region of the
protein is poorly characterised and the functional aspects
of the threonine in this position are unclear. This position
is close to an Atm phosphorylation site at T68, but there is
no evidence so far for a phosphorylation at T59 [9,13].
The T59K sequence variant is not likely to be highly pene-
trant with respect to tumor growth. Seven of the nine indi-
Breast Cancer Research Vol 4 No 3 Ingvarsson et al.
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Table 4
Frequency of the CHK2 T59K mutation in nine Icelandic
groups
a
Patient group Number tested Carriers %
Breast cancer 685
b
4
c
0.6
Colorectal cancer 119 2
d
1.6
Lung cancer 74 0 0
Renal cell carcinomas 71 0 0
Stomach cancer 37 1 2.7
Ovarian cancer 43 1
d
2.3
Testis cancer 31 0 0
Other cancers 112 0 0
Controls 452 0 0
a
The different groups consist of unselected patients, except in the
case of breast cancer where patients with BRCA2 999del5 have been
omitted.
b
Including samples from Table 3.
c
One of these individuals is
a bilateral case.
d
The total number of carriers in this screening is seven,
but one individual is a carrier of both colon and ovarian cancer and
therefore appears twice in the table.
viduals with the T59K sequence variant were diagnosed
with more than one primary tumor and/or are members of
cancer families. The remaining two individuals carrying the
CHK2 T59K mutation were diagnosed with breast cancer
at the age of 42 years and colon cancer at the age of
66 years, respectively. The absence of complete segrega-
tion in one family with a history of colon cancer and a
second family with breast cancer (in addition to other
tumor types), and the low frequency of mutation in individ-
uals with various tumor types, do not support the idea of a
highly penetrant germ line variant, but the modifying effect
on tumor pathogenesis may be of relevance.
We suggest that the T59K germ line variant is a low-pene-
trance allele with respect to tumor growth, but additional
genetic variations of unknown origin may enhance the
family history of cancer. Even though an amino acid is
changed as a result of the sequence variation, this study
does not clearly show, but does support, a dysfunctional
variant. As suggested by the absence of sequence vari-
ants in the CHK2 gene in individuals carrying the BRCA2
999del5 mutation, there is no evidence of CHK2 acting as
a modifying genetic factor on the tumor phenotype in
these individuals. The remaining three germ line variants of
CHK2 are also detected in the normal population and are
therefore putative polymorphisms. The silent polymor-
phism at codon 84 has been reported earlier in other pop-
ulations [14,18], while the other two polymorphisms at a
short mononucleotide stretch are novel.
The CHK2 gene is known to have several genomic copies
through the genome that have a high sequence conserva-
tion [24]. Our sequence comparison indicates that the
first 10 exons do not give problems due to DNA sequence
homology between genes, but homology is detected for
exon 11 to exon 14 of the CHK2 gene. In our primer set,
only primers from exon 11 give a perfect match with an
additional CHK2 copy from chromosome 10. Other
primers for exons 12 to 14 have one to three nucleotide
mismatches. It is therefore unlikely that the detected vari-
ants are from other copies of the CHK2 gene, since they
are not from the highly conserved part of the gene. There
is a risk that we are missing sequence variants from exons
11 to 14, although they could be detected as bands with
weak intensity. It is therefore difficult to rely on this work
being a full screen of CHK2 sequence variants.
In conclusion, CHK2 germ line mutations and somatic
mutations are detected in breast cancer, but CHK2 inacti-
vation does not play a major role in the cancer growth.
Somatic mutations in the CHK2 gene are rare in breast
tumors. The detected CHK2 T59K germ line allele is prob-
ably of low penetrance with respect to cancer growth. In
breast cancer, mutations in TP53 and CHK2 genes proba-
bly explain only a part of the genetic instability detected in
breast tumors.
Acknowledgements
This work was funded by the Icelandic Cancer Society and the Univer-
sity of Iceland Research Fund.
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