Clinical Cancer Research Vol. 6, 536-540, February 2000
© 2000 American Association for Cancer Research
Molecular Oncology, Markers, Clinical Correlates |
Mutational Analysis of the Transforming Growth Factor ß Receptor Type II Gene in Hereditary Nonpolyposis Colorectal Cancer and Early-onset Colorectal Cancer Patients1
Ki-Hyuk Shin,
Young Jin Park and
Jae-Gahb Park2
Korean Hereditary Tumor Registry, Laboratory of Cell Biology, Cancer Research Center, and Cancer Research Institute, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea
 |
ABSTRACT
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Somatic
mutations in the transforming growth factor ß receptor type II
(TGF-ßRII) gene have been observed in various human
cancers showing microsatellite instability. Most of the mutations
observed were additions or deletions of the mononucleotide repeat
sequence present in TGF-ßRII coding region, suggesting
that the TGF-ßRII may be a target gene of genomic
instability in tumorigenesis. Recently, we reported germ-line
frameshift mutations in the mononucleotide repeat sequence of the
hMSH6 gene, which is believed to be one of the target
genes of genomic instability in tumorigenesis, suggesting the
possibility of germ-line mutation in mononucleotide repeat sequences.
Moreover, one case of germ-line mutation in the
TGF-ßRII gene was identified in a hereditary
nonpolyposis colorectal cancer (HNPCC) kindred, indicating the
involvement of TGF-ßRII inactivation in tumorigenesis
of HNPCC. However, germ-line mutation analysis of all of the
coding sequences and the mononucleotide repeat sequence of the
TGF-ßRII in HNPCC patients has not yet been fully
elucidated. Therefore, to further investigate the presence of
germ-line mutations, we screened all of the coding region
sequences and mononucleotide repeat sequence of
TGF-ßRII from 35 HNPCC, 44 suspected HNPCC, and 45
sporadic early-onset colorectal cancer patients. However, no pathogenic
mutations other than silent mutations, introgenic mutation, and
polymorphisms were identified. Two silent mutations at codons 309 (ACG
to ACA) and 340 (CAT to CAC) in the kinase domain located in exon 4
were detected. A 1-bp cytidine deletion was observed 6 bases
from the 3' end of intron. Two polymorphisms were identified at codon
389 (AAC to AAT) and at the fourth-to-last base in intron 3. The
polymorphism at codon 389 was more frequent in HNPCC (20%; 7 of 35)
and suspected HNPCC patients (18%; 8 of 44) than in nonmalignant
control group (10%; 5 of 50). Moreover, the frequency was
significantly higher in early-onset colorectal cancer patients (31%;
14 of 45). This is the first report of a different frequency of
polymorphism in HNPCC, suspected HNPCC, early-onset colorectal cancer
patients, and healthy normal individuals. This result suggests that:
(a) germ-line mutation of the TGF-ßRII
gene may be a rare event during tumorigenesis in HNPCC and sporadic
early-onset colorectal cancer; (b) the mononucleotide
repeat sequence of the TGF-ßRII gene is an apparent
target of genomic instability but not of germ-line mutation; and
(c) the polymorphism of codon 389 (AAC to AAT) is
frequent, especially in early-onset colorectal cancer patients, in
which it is more frequent than in control group.
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INTRODUCTION
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HNPCC3
is the most
common hereditary condition predisposing patients to the development of
colorectal cancer. Germ-line mutations in genes of the mismatch repair
system, namely hMSH2, hMLH1, hPMS1,
hPMS2, and hMSH6, have been identified in
patients with HNPCC (1, 2, 3, 4, 5, 6, 7, 8)
. Cells with defective mismatch
repair genes display an elevated instability of microsatellite
sequences, indicating genomic instability.
Somatic mutations in the TGF-ßRII gene have been
identified in various human cancers, including HNPCC, sporadic
colorectal, gastric, and ovarian carcinomas (9, 10, 11, 12, 13)
. Most
of the mutations found were additions or deletions of the
mononucleotide repeat sequence present in the TGF-ßRII
coding region (9
, 14, 15, 16)
, suggesting that
TGF-ßRII may be a target gene of genomic instability in
tumorigenesis. However, the recent observation of a germ-line mutation
of the TGF-ßRII gene in an HNPCC patient indicated a
involvement of TGF-ßRII inactivation in the tumorigenesis
of HNPCC (17)
.
Therefore, to clarify whether or not germ-line mutation in the entire
coding region of TGF-ßRII is implicated in tumorigenesis,
we screened 35 HNPCC patients satisfying the ICG-HNPCC criteria and 44
patients suspected of having HNPCC who did not fulfill the criteria of
the ICG-HNPCC (4
, 18)
. Forty-five early-onset colorectal
cancer patients (who developed colorectal cancer before the age of 40
years) without any family history of colorectal cancer were also
examined.
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MATERIALS AND METHODS
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Subjects and DNA Isolation from Blood Samples.
The previously reported samples, 35 HNPCC, 44 suspected HNPCC, and 45
sporadic early-onset colorectal cancer patients registered in the
Korean Hereditary Colorectal Cancer Registry were used in this study
(8)
. All patients enrolled in this study were Korean and
were the proband case of (suspected) HNPCC family or patients of
early-onset colorectal cancer, and thus all of them originated from
unrelated families. Twenty ml of peripheral blood from each patient
were used to prepare genomic DNA from WBCs, as described elsewhere
(19)
.
Fifty blood samples for control were taken from patients with benign
proctological diseases (48 patients with hemorrhoids and 2 patients
with anal fissure). None of individuals in the control group had family
history suggesting the HNPCC or development of colon cancer in earlier
age. There were few differences in demographic findings between control
and patient groups. The only significantly different finding was that
patients with early-onset colorectal cancer were approximately 10 years
younger than the other groups.
PCR and SSCP Analysis.
The PCR-SSCP method was used to screen for mutations of all of
the exons of the TGF-ßRII gene. The sequences of
the primers and the detailed reaction conditions for amplification have
been described previously (10)
.
Analysis of the Mononucleotide Repeat Sequence in TGF-ßRII.
We analyzed frameshift mutations in the repeat sequence by modified
PCR-SSCP. The PCR primers for the 10-bp polyadenine repeat in exon 3 of
TGF-ßRII were as follows: sense, 5'-TGA CTG ATA CTT CTA
CCA GC-3'; antisense, 5'-AAC ATT TGT TCC TCA CCT GC-3'. The repeat
sequences were amplified from 100 ng of genomic DNA using the sense
primer of each gene labeled with [
-32P]dATP
using T4 polynucleotide kinase and the unlabeled antisense primer of
each gene. The PCR conditions consisted of 35 cycles at 95°C for
30 s, 45°C for 1 min, and 70°C for 1 min. The PCR products
were denatured, separated on 7% polyacrylamide gels at a constant 60
W, and visualized by autoradiography.
DNA Sequencing Analysis.
When abnormal patterns were detected by PCR-SSCP analysis, the PCR
products were purified with the QIAquick PCR purification kit (Qiagen,
Inc., Chatsworth, CA) and then sequenced directly with a Taq dideoxy
terminator cycle sequencing kit on an ABI 377 automatic DNA sequencer
(Perkin-Elmer, Foster City, CA).
Statistical Analysis.
To compare the differences in the portion of cases harboring mutation
of specific type of TGF-ßRII gene polymorphism,
statistical analysis was performed using Fisher’s exact test.
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RESULTS AND DISCUSSION
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We examined all of the exons of the TGF-ßRII
gene by PCR-SSCP analysis. An abnormal band pattern was revealed in
exon 4 of a suspected HNPCC patient (patient SNU-H1033), in which
germ-line mutations of hMSH2 and hMLH1 were not
detected (Fig. 1A)
. Subsequent
sequencing analysis of the TGF-ßRII gene revealed that
SNU-H1033 has two silent mutations at codon 309 (ACG to ACA) and codon
340 (CAT to CAC) that reside on different alleles (Fig. 1, B and C)
. In a sporadic early-onset colorectal cancer patient
(patient SNU-YC44), a 1-bp cytosine deletion 6 bases from the 3'
end of intron 3 was found (Fig. 2)
.
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Fig. 1. Silent mutations of the
TGF-ßRII gene in suspected HNPCC patient (SNU-H1033)
A, PCR-SSCP analysis of exon 4-3 of
TGF-ßRII. An abnormal band pattern was detected in
patient SNU-H1033. B, a silent mutation at codon 309
from ACG (Thr) to ACA (Thr). C, a silent mutation at
codon 340 from CAT (His) to CAC (His).
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Fig. 2. Genetic alterations in intron 3 of the
TGF-ßRII gene from a sporadic early-onset colorectal
cancer patient (patient SNU-YC44) A, an abnormal band
pattern was detected in patient SNU-YC44 by PCR-SSCP analysis.
B, a 1-bp cytosine deletion at the sixth-to-last
base and A/T polymorphism at the fourth-to-last base were identified.
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Furthermore, we found a previously reported polymorphism at the third
nucleotide of codon 389 (AAC to AAT) in exon 4 (13)
. Three
types of PCR-SSCP patterns were identified and revealed AAC/AAC (type
A), AAC/AAT (type B), and AAT/AAT (type C; Fig. 3
). Among the 35 HNPCC patients, 28 were
of the AAC/AAC type (80%), and 7 were of the AAC/AAT type (20%).
Thirty-six of the AAC/AAC type (82%) and 8 of the AAC/AAT type (18%)
were identified in the 44 suspected HNPCC patients. Among the 45
sporadic early-onset colorectal cancer patients, 31 were of the AAC/AAC
type (69%), 10 were of the AAC/AAT type (22%), and 4 were of
the AAT/AAT type (9%). Furthermore, to determine the existence of any
association between the polymorphism and an altered risk for colorectal
cancer, 50 samples from individuals with nonmalignant tumors
were genotyped by means of PCR-SSCP. As shown in Table 1
, this type of polymorphism was
especially frequent at sporadic early-onset colorectal cancer patients
(31%; P = 0.02). The polymorphism was also more
frequent in the ICG-HNPCC (20%; P = 0.22) and
suspected HNPCC (18%; P = 0.37) groups than in the
control group (10%), although it was not statistically significant.
The polymorphism, however, was not associated with the age of patients,
tumor site, or differentiation of tumor, as shown Table 2
. Although the biological significance
of the polymorphism is still unclear, we report the first description
of the prevalence and genotype of the AAC/AAT polymorphism in HNPCC,
suspected HNPCC, sporadic early-onset colorectal cancer patients, and
normal controls. A previously reported introgenic polymorphism was also
found at the fourth-to-last base in intron 3 (Ref. 20
;
Fig. 2
).
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Fig. 3. Polymorphism analysis of the
TGF-ßRII gene at codon 389 A, PCR-SSCP
analysis of exon 4-4 shows three types of band pattern.
B, sequencing analysis reveals that type A is AAC/AAC,
type B is AAC/AAT, and type C is AAT/AAT.
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Table 1 Frequencies of polymorphism at codon 389 (AAC to
AAT) in patients with ICG-HNPCC, suspected HNPCC, and sporadic early
onset colorectal cancer
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Table 2 Clinical characteristics of colorectal cancer
patients with or without polymorphism at codon 389 (AAC to AAT)
No parameters had statistical significance according to the presence or
absence of the AAT type polymorphism (Student’s t test).
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Compelling evidence has indicated that TGF-ßRII is a tumor
suppressor gene and is mutationally inactivated in several types of
human cancers exhibiting MSI (10, 11, 12, 13, 14, 15)
. The predominant
region of mutations of the TGF-ßRII gene is within a 10-bp
polyadenine repeat at exon 3. MSI frequently occurs when the DNA
mismatch repair genes hMSH2 and hMLH1 are
defective. Although MSI has been detected in a variety human cancers,
the role of MSI in tumorigenesis remains unclear. Several studies have
shown a frequent frameshift mutation in simple repeat sequences within
the open reading frame of the TGF-ßRII, BAX, hMSH3, or
insulin-like growth factor II receptor (IGFIIR) genes in
cancer tissues and cell lines exhibiting MSI, indicating that they may
be the target genes of genomic instability in tumorigenesis (14
, 21, 22, 23)
. Recently, we reported germ-line frameshift mutations in
a polycytidine repeat sequence of hMSH6, which is also one
of the target genes of genomic instability in tumorigenesis, suggesting
a possibility of germ-line mutation in a mononucleotide repeat sequence
(8)
. In this study, one of our aims was to investigate
whether a germ-line frameshift mutation exists in the 10-bp polyadenine
repeat of the TGF-ßRII gene. However, no such alteration
was observed in any of the tested HNPCC, suspected HNPCC, and sporadic
early-onset colorectal cancer patients.
In summary, our results suggest that germ-line mutation of the
TGF-ßRII gene may be a rare event in the tumorigenesis of
HNPCC and sporadic early-onset colorectal cancer, and the 10-bp
polyadenine repeat sequence of the TGF-ßRII gene is an
apparent target of genomic instability but not of germ-line mutation.
Although the biological significance of the polymorphism at codon 398
(AAC to AAT) remains unknown, the frequency of polymorphism is higher
in patients with HNPCC and suspected HNPCC and especially in sporadic
early-onset colorectal cancer patients than in normal, healthy
individuals. Therefore, study of the biological significance of
the polymorphism and further rigorous statistical investigation are
warranted.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by grants from the 1997 Good Health
R & D Project, the Ministry of Health and Welfare of the Republic of
Korea, and the Korea Science and Engineering Foundation (KOSEF-CRC97-8)
through the Cancer Research Center at Seoul National University. 
2 To whom requests for reprints should be
addressed, at the Laboratory of Cell Biology, Cancer Research Center
and Cancer Research Institute, Seoul National University College of
Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea. Phone:
82-2-760-3380; Fax: 82-2-742-4727; E-mail: jgpark{at}plaza.snu.ac.kr
3 The abbreviations used are: HNPCC, hereditary
nonpolyposis colorectal cancer; ICG, International Collaborative Group;
MSI, microsatellite instability; SSCP, single-strand conformation
polymorphism; TGF, transforming growth factor; TGF-ßRII, TGF-ß
receptor type II.
Received 9/15/99;
revised 11/ 9/99;
accepted 11/10/99.
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ABSTRACT
INTRODUCTION
