identification of concurrent germ-line mutations in hmsh2 ......vol. 6, 1057-1064, decenther 1997...
TRANSCRIPT
Vol. 6, 1057-1064, Decenther 1997 Cancer Epidendologj�, Biomarkers & Prevention 1057
Identification of Concurrent Germ-Line Mutations in hMSH2 and/or
hMLHJ in Japanese Hereditary Nonpolyposis Colorectal
Cancer Kindreds1
Masahiro Nakahara, Hiroshi Yokozaki, Wataru Yasui,Kiyohiko Dohi, and Elichi Tahara2
First Department of Pathology EM. N., H. Y., W. Y., E. T.] and Second
Department of Surgery [M. N., K. Dl, Hiroshima University School of
Medicine, Minami-ku, Hiroshima 734, Japan
Abstract
We analyzed microsatellite instability, alterations of thepolyadenine tract in TGF-fl RI! (transforming growthfactor 13 type II receptor gene), and mutations of hMSH2and hMLHJ in 32 patients with familial colorectal cancer(29 kindreds) fulfilling the clinical criteria for hereditary
nonpolyposis colorectal cancer (IINPCC), defined at the34th Annual Meeting of Japanese Society for Cancer ofthe Colon and Rectum (Tokushima, Japan, 1991),including five kindreds fulfilling the Amsterdam criteria.Eighteen of 32 (56%) cases were replication error positive(RER�) at two or more microsatellite loci analyzed. Theclinicopathological characteristics of RER� cases
corresponded well with those reported previously. Elevenof 18 RER� cases showed RER� at most of themicrosatellite loci examined. Among these 11 cases (10kindreds), 3 kindreds fulfilled the Amsterdam criteriaand 7 kindreds did not. For these 10 kindreds, germ-linemutations in hMSH2 and hMLHJ were detected for 6kindreds by PCR-SSCP analysis and direct sequencing.Only two of these six fulfilled the Amsterdam criteria;more than one germ-line mutation was detected inhMSH2 and/or hMLHJ. Specifically, two point mutationsof hMSH2 were detected in two kindreds, one pointmutation of both hMSH2 and hMLHJ was detected inone kindred, two point mutations of hMSH2 and onepoint mutation of hMLHJ were detected in one kindred,and two point mutations of hMLHJ and one pointmutation of hMSH2 were detected in one kindred. Inaddition, 19 of 26 (74%) cancer lesions of these 11 caseswith the RER phenotype showed alterations of thepolyadenine tract in TGF-�3 Ru. From our data, althoughseven kindreds did not fulfill the Amsterdam criteria, weconsidered them as HNPCC. Therefore, we suggest that
Received 5/2/97; revised 8/14/97; accepted 8/18/97.
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.
I This work was supported by a Grant-in-Aid for Cancer Research from the
Ministry of Education, Science, Culture and Sports of Japan and from theMinistry of Health and Welfare of Japan.2 To whom requests for reprints should be addressed, at First Department of
Pathology, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku,
Hiroshima 734, Japan. Phone: 8 1 -82-257-5 147; Fax: 81-82-257-5149.
the “Japanese criteria” have the advantage of being ableto detect more HNPCC kindreds from borderlineHNPCC kindreds.
Introduction
HNPCC3 is a clinically defined autosomally dominant disorderthat accounts for approximately 6-10% of colon cancers (1, 2).It is characterized by the early-onset, syn- and metachronous
colorectal cancer, and an increased frequency of other cancers,including cancers of the endometrium, stomach, small intestine,hepatobibiary tract, and urobogical tract (3-5). Pedigree analysis
is a key for the diagnosis of HNPCC. In 1990, this disease wasdefined at the International Collaborative Group on HNPCC by
the following criteria (Amsterdam criteria): (a) at least threerelatives should have histologically verified coborectal cancer,with at beast two of them being first-degree relatives; (b) at least
two successive generations should be affected; (c) in one of therelatives, coborectab cancer should be diagnosed at under 50
years of age; and (d) the diagnosis of familial adenomatouspolyposis, a distinct autosomal dominant coborectal cancer pre-
disposition syndrome, is excluded (6). Although the clinicaldefinition of this syndrome was facilitated greatly by the es-
tablishment of the Amsterdam criteria, there remains a greatdeal of phenotypical variability among families. Therefore, a
new set of clinical diagnostic criteria was proposed at the 34th
Annual Meeting of the JSCCR (1991, Tokushima, Japan) andwas reported by Kunitomo et a!. at the 5th International Sym-
posium on Coborectal Cancer (1991, Turin, Italy; Ref. 7). The
“Japanese criteria” were as follows: (a) a case with three ormore colorectal cancers among the first-degree relatives, or (b)
a case with two or more coborectal cancers among the first-degree relatives and with any of the following: age at onset ofcolorectal cancer(s) less than 50 years old; right colon involve-ment; synchronous or metachronous multiple coborectal can-cers; or association with extracolorectal malignancy. These
criteria have two conditions, and cases are diagnosed with
HNPCC by fulfilling either condition (a) or (b). In addition,condition (b) has four additional independent criteria. If twocoborectal patients are in the family and at least one patientfulfills any four additional eligible criteria, this family is diag-nosed as having HNPCC by fulfilling condition (b) of the
Japanese criteria. Using these criteria, many HNPCC patientshave been identified from borderline HNPCC patients in Japan.
(7).
Recent studies have shown that cancer DNA of affected
3 The abbreviations used are: HNPCC, hereditary nonpolyposis colorectal cancer;
JSCCR, Japanese Society for Cancer of the Colon and Rectum; MIN, microsat-
ellite instability; RER, replication error; MMR, mismatch repair; poly(A), polya-
denine; SSCP, single-strand conformational polymorphism.
on July 20, 2021. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from
1058 Germ-Line Mutations in Japanese HNPCC
individuals in HNPCC kindreds showed frequent [80-90%
(8 - 1 1 ) of casesi alterations in microsatellite sequences, termed
as MIN or RER, which reflect the malfunction in DNA repair.The presence of tumor MIN has offered a potential marker forthe identification of individuals who were at high risk for
possessing germ-line mutations in DNA MMR genes, such ashMSH2, hMLHJ, hPMSJ, and hPMS2 (12-15). Indeed, themutation of these genes has been reported to account for 50%
of total HNPCC kindreds (1 1). Among them, 90% of the
kindreds display alterations of hMSH2 or hMLHJ, and only afew patients contain mutations in hPMSJ and hPMS2 (11).Germ-line mutations of these genes are responsible for thehereditary susceptibility to coborectal and rebated cancers, anddetection of germ-line mutation(s) of a MMR gene in a patient
with colorectal cancer confirms on a molecular basis the diag-
nosis of HNPCC. The presence of tumor MIN does not neces-sarily define which one of the four MMR genes is involved, and
they have not detected germ-line or somatic mutations in a
substantial number of MIN� sporadic colon cancer and MIN�familial colorectal cancer (10, 16-18). However, hypermethy-
lation of the hMLHJ promotor region has recently been re-ported in sporadic colorectal tumors, and DNA methylation is
likely to be a common mode of MMR gene inactivation (19).Recently, it was reported that inactivation of the TGF-/3 RI!occurred in colon cancer cell lines with MIN (20). The TGF-flRI! has two sites of repetitive sequences, poly(A) (nucleotides
709-7 18) and GT repeat (nucleotides 193 1-1936). Mutationswere found at only the poby(A) tract of these two repetitive
sequences in TGF-� RI! (21, 22). Therefore, the poly(A) tractin TGF-f3 RI! may be one of the target genes of the defective
DNA repair and may play an important robe in the carcinogen-esis of HNPCC (20-22).
In this study, we analyzed MIN, including poly(A) tractalteration, in TGF-3 RI! as well as hMSH2 and hMLHJ muta-
tions in Japanese HNPCC patients, who fulfilled criteria de-
fined at the 34th Annual Meeting of the JSCCR.
Materials and Methods
Patient Population. Twenty-nine kindreds, including 32 pa-tients of 498 patients, who were treated at Hiroshima University
School of Medicine from 1980 to 1995, were selected for thisstudy using the Japanese registry’s clinical diagnostic criteria
for HNPCC proposed at the 34th Annual Meeting of the JSCCR(7). In these kindreds, five fulfilled the strict Amsterdam cri-
teria for HNPCC. Clinicopathobogical features of these patientswere shown in Table 1 . Normal and neoplastic tissues used for
these studies included mostly paraffin-embedded tissues and afew fresh-frozen tissues.
DNA Extraction. DNA extraction from fresh-frozen tissueswas performed using the phenol-chloroform method after treat-
ment with SDS and proteinase K. DNA from paraffin-embed-
ded tissues was obtained as described by Shibata et a!. (23),with some modifications. Tissues were incubated at 55#{176}Cover-
night in each 50-sb DNA extraction solution [100 m’vi Tris-HC1,2 msi EDTA (pH 8.0), and 400 mg/mb proteinase K]. After
extraction, proteinase K was inactivated by boiling for 10 mm.Samples were cooled rapidly, and DNA was stored at 4#{176}C.
Microsatellite Assay. To assess the RER status, we used oh-
gonucleotide primer sets for 12 microsatellite markers.D2S123, D2S136, D3S1067, D3S161 1, D5S505, D7S486, and
D17S855 were used as a CA repeat marker, and BAT-13,
BAT-25, BAT-26, BAT-40, and BAT-Ril were used as apoly(A) marker (22, 24, 25). BAT-RIl alteration indicatedmutation of poly(A) tract in TGF-f3 RI!, because this primer set
was established to contain the poly(A) tract of TGF-� RI! (22).PCR was performed as described by Semba et a!. (26). Briefly,
each l5-�d reaction mixture containing about 10-20 ng ofDNA, 6.7 mM Tris-HCI (pH 8.8), 6.7 mrsi EDTA, 6.7 mM
MgCl2, 0.33 �.LM of labeled primer with [y-32P]ATP, 0.175 �.LM
unlabeled primer, 1.5 m’vi deoxynucleotide triphosphates, and0.75 units of recombinant Taq DNA polymerase, was amplified
for 40 cycles with the following regime: denaturation at 94#{176}Cfor 30 5, annealing at 55#{176}Cfor 30 s, and extension at 72#{176}Cfor
30 s. PCR products were electrophoresed in 6% polyacryb-amide-8 M urea-32% formamide gels and autoradiographed
overnight at -80#{176}C.
PCR-SSCP Analysis. To screen the hMSH2 and hMLHJ forvariant sequences, PCR-SSCP analysis was performed accord-
ing to Orita et a!. (27, 28), with some modifications. PCR
primer sets for amplification of each exon of hMSH2 andhMLHJ were described previously (29-32). In this study, each
25-pi reaction mixture contained 1 X PCR buffer II [8.0 mM
Tris-HC1 (pH 8.3), 40 mM KC1] (Perkin-Elmer, Branchburg,NJ), 4 mM MgCl2, 0.3 mu of each deoxynucleotide triphos-phate, 50 pmol of each primer, 10-20 ng of genomic DNA, 2.5
mCi of [a-32P]dCTP (3000 Ci/mmob, 10 mCi/mi), 1 .25 units ofTaq DNA polymerase (Perkin-Elmer). Reaction mixtures were
heated to 94#{176}Cfor 2 mm, followed by 35 cycles of denaturationat 94#{176}Cfor 1 mm, annealing at 55#{176}Cfor 1 mm, and strandelongation at 72#{176}Cfor 2 mm. After PCR, the samples were
electrophoresed using 6% polyacrybamide gel (ratio of acryl-amide:bis-acrybamide, 19:1) with 10% glycerol at 4#{176}C.Thengels were subjected to autoradiography overnight at room tem-
perature.
DNA Sequencing. To identify mutations in hMSH2 andhMLHJ, direct sequencing was performed according to Fujii eta!. (33), with some modifications. At first, the aberrant migra-
tion band on the SSCP gel was cut out and amplified again
using the same PCR protocol as in SSCP. The amplified PCRproducts were separated by electrophoresis with low meltingpoint agarose, purified using Wizard PCR Prep (Promega,Madison, WI) and directly sequenced on both strands using the
PRISM AmphiTaq DNA pobymerase FS Ready Reaction DyePrimer sequencing kits (Applied Biosystems and Perkin-Elmer)and the Applied Biosystems model 310 automated sequencer.
Results
MIN. MIN was demonstrated by the presence of new frag-ments of variable size in tumor DNA. Eighteen of 32 (56%)cases fulfilling the Japanese criteria for HNPCC (Table 1 ) were
RER� at two or more microsatelbite loci analyzed. The clini-copathological characteristics of R.ER � cases were the follow-ing: (a) the patients had a tendency to be affected by coborectalcancer at less than 60 years of age (average age, 56.5); (b)coborectal cancers preferentially arose in the proximal colon
(63%); (c) the patients tended to have multiple colonic and/or
extracobonic cancers (56%); and (d) most (90%) of the carci-nomas in kindreds were moderately or well-differentiated ad-enocarcinomas, histologically. Of the 18 RER� cases, 1 1 (cases4, 7, 8, 10, 11, 12, 15, 17, 22, 27, and 28) revealed RER� incancer lesions at most of the microsatellite loci examined(Table 2). Therefore, these 1 1 cases were considered as RER
phenotype cases (Table 1). Case 1 1 was a brother of case 17 inthe same kindred. Of these 10 kindreds, only 3 fulfilled the
Amsterdam criteria, and the remaining 7 did not. On the otherhand, two kindreds of case 24 and 25 who fulfilled the Am-
sterdam criteria did not show RER at any microsatellite locianalyzed (Table 1). In addition, alterations of the BAT-RI!
on July 20, 2021. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from
Cancer Epidemiology, Biomarkers & Prevention 1059
Table I Clinico pathological features of HNPCC c ases which fulfilled the J apanese criteria
aCase SexAge at
.operation
. bSite
. .Histological type
Multiple colorectaldcancers
Cancer in other
organ. .
Familial history of cancei��
RER�
45 M 42 R, I Moderate (2) +
�g F 36 A Well Uterus
8 M 53 D, R Muc., well +
10 F 85 T Moderate Uterus
I l� M 41 C Well Stomach Uterus, ovary
12 F 57 T, S Moderate, well + Uterus
I5� M 43 R Well
17� F 55 T (2), D Moderate, well (2) + Uterus Stomach, ovary
22 M 57 T (2) Moderate (2) +
27 F 54 A (5), 5 (2) Well (4), moderate (2), poor + Uterus
28 F 57 A (3) Well (2), moderate + Stomach, uterus
I F 44 S Ad-sq. Uterus Esophagus
3 F 65 5 Well Lung
9 M 63 T Moderate
I 3 F 63 R Well Uterus
19 F 66 A,D WelI(2) +
20 M 58 R Moderate Stomach
23 M 78 R Well Breast?
RER
2 F 39 R Moderate Stomach
5 M 76 C Well
6 F 56 T Well Liver
14 M 57 5, D Moderate (2) + Larynx
16 F 51 A Well Liver
18 F 64 C Well
21 M 69 T Moderate Stomach
24� F 66 R Well
25g F 7 1 5 Moderate
26 F 72 R Well
29 F 61 A Well Esophagus, stomach
30 M 43 R Well Stomach
31 F 60 5, D Well, moderate + Esophagus, breast
32 M 76 D (2) Moderate (2)
a Case 7, 11, 15, 17, 24, and 25 fulfilled Amsterdam criteria defined ICG-HNPCC. Case 11 is a brother of case 17, case 20 is a brother of case 21 and ca.se 22 is a son
of case 23. Cases 4, 7, 8, 10, 1 1, 12, 15, 17, 22, 27, and 28 indicated RER� at most of the microsatellite loci examined, and cases I, 3, 9, 13, 19, 20, and 23 indicated
RER� at two to four microsatellite loci examined.
b Site of carcinoma in colon. C, cecum; A, ascending colon; T, transverse colon; D, descending colon; S. sigmoid colon; R, rectum.
‘ Well, well-differentiated adenocarcinoma; Moderate, moderately differentiated adenocarcinoma; Poor, poorly differentiated adenocarcinoma; Muc., rnucinous carcinoma;
Ad-sq., adenosquamous cell carcinoma.
d .� existence of multiple colorectal cancers.
� Synchronous or metachronous carcinoma in other organ(s).
1Cancer(s) in other organ(s) of member(s) in the kindred.
a Cases fulfilling the Amsterdam criteria.
sequence were detected in 19 of 26 (74%) cancer lesions in all
1 1 RER phenotype cases (Table 2).
Mutations of hMSH2 and hMLHJ. SSCP analysis was per-formed for the entire coding regions of hMSH2 and hMLHJ inDNA samples from 1 1 RER phenotype cases (28 cancer be-sions; 25 in the colon, 2 in the uterus, and 1 in the stomach). Allcases that showed aberrant mobihities on the gels were se-
quenced using an automated sequencer, and representative re-sults are shown in Figs. 1 and 2. Fig. la shows the results of
SSCP analysis of exon 5 of hMLHJ in cases 1 1 and 17. In case1 1, an altered mobility in both normal and cancer DNA was
detected, which suggested a germ-line mutation. By direct
sequencing, a nonsense mutation at codon 133 (GGA to TGA;Gly to stop) was confirmed in both normal and cancer DNA(Fig. lb). In case 17, we also detected the same nonsense
mutation in hMLHJ. Fig. 2a reveals the results of SSCP anal-ysis of exon 2 of hMLHI in case 12. An altered mobility was
seen only in cancer DNA but not in normal DNA, indicating asomatic mutation. By direct sequencing, a missense mutation at
codon 69 (AGG to GGG; Mg to Gly) in hMLHJ was detected
only in cancer DNA (Fig. 2b). On the whole, we found hMSH2
and hMLHJ germ-line variants in six kindreds. Among them,
only two kindreds fulfilled the Amsterdam criteria. However,
we could not detect mutations of these MMR genes in theremaining four families, i.e., cases 7, 22, 27, and 28, including
one family that fulfilled the Amsterdam criteria.
As for the nature of the germ-line variants, we detected
one nonsense mutation of hMLHJ, two missense mutations ofhMSH2, and two missense mutations of hMLHI. We found no
frameshift mutations in our kindreds. Interestingly, these germ-line variants were found at more than one locus of two MMR
genes in five kindreds. For example, in case 12, one missensemutation at codon 639 (CAT to CGT; His to Arg) in hMSH2,
one nonsense mutation at codon 133 (GGA to TGA) in hMLHJ,
and another missense mutation at codon 219 (ATC to GTC; Ileto Val) in hMLHJ were detected. In addition, several mutations
were confirmed in more than one kindred. A missense mutationat codon 639 of hMSH2 was detected in cases 4, 10, 11 (17), 12,
on July 20, 2021. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from
1060 Germ-LIne Mutations in Japanese HNPCC
Table 2 MIN in cancer lesions of RER phenotype kindreds
Microsate llite assay on c hromosome 1, 2, 3, 5, 7 and 17 in c olorectal and extracolorectal tissues. +, RER positive; - , RE R negativ e; NI, not informative.
Case” Site”Microsatellite loci
DJSI9I D2S123 D2S136 D3S1067 D3S1611 D5S505 D7S486 D17S855 BATI3 BAT25 BAT26 BAT-RIT
4-1 Colon (T) + + - - - + - + + + + +
4-2 Colon (In.) - + + - - - + + - + + +
4-3 Colon (R) - + + - - - - + + + + +
7 Colon(A) + - - - - - - - + + + +
8-1 Colon(D) - + + - + NI + + - + + -
8-2 Colon (R) + + + - - + + + - + + +
10 Colon (T) + + + - NI - + + + + + +
1 1-1 Colon (C) + NI - + + + + + + + + +
11-2 Stomach NI NI - - NI + - NI - + NI -
12-1 Colon (T) + + + + + + + + + + + +
12-2 Colon (5) - - - - - - - + - + - -
I 2-3 Uterus + + - - + - + + + + . + +
15 Colon (R) + + + + + + + + + + + +
17-I Colon (TI) + NI + + + + + + + + + -
1 7-2 Colon (T2) NI NI + - + + + - - + + -
17-3 Colon(S) + NI - + - - + + - + + +
17-4 Uterus NI NI + - - - - - - + + -
22-1 Colon (Tl) + - + - + - + - + 4- + +
22-2 Colon (T2) + + + - - - + + + + + +
27-I Colon (A) - - - - - + - + - + - -
27-2 Colon (A) + + 4- + + + - + - + + NI
27-3 Colon (A) + - + + - NI - NI - + - -
27-4 Colon (A) + - - - - - + + - + + +
27-5 Colon (5) + + + - - - + NI + + + +
27-6 Colon (5) NI + + - - + + - - + + +
28-I Colon (A) NI - - + - + - - + + + +
28-2 Colon (A) + + + + + + + - + + - +
28-3 Colon (A) + - - NI - NI - + + + - +
a Patient and tumor number. If a patient has multiple tumors, tumor number is added after patient number. Cases 7, 1 1, 15, and 17 fulfilled Amsterdam criteria; case 11
is a brother of case 17 in the same kindred.b Site of carcinoma. C, cecum; A, ascending colon; T, transverse colon; D, descending colon; S. sigmoid colon; R. rectum; In., invasion of transverse colon cancer.
C Oligonucleotide primer set was established to contain poly(A) tract of TGF-gS RI! (22).
and 15. All these germ-line variants are summarized in Table 3.These PCR-SSCP analyses and direct sequencing reactions
were repeated at least twice and performed on both sense and
antisense strands. Nineteen somatic mutations were found incancer DNA of these families. Eleven of 19 somatic mutationswere detected in hMLHJ, and the remaining 8 were seen inhMSH2. We observed 12 missense mutations and 6 silent
mutations. All of these somatic mutations are summarized inTables 4 and 5. In these somatic mutations of hMSH2, missense
mutations at codon 647 were seen in cases 11, 12, and 17. Wealso detected missense mutations at codon 679 (ACT to AT!’;Thr to lie) in hMSH2 in cases 10 and 11. Four somatic muta-
tions (three missense mutations and one silent mutation) in thetwo MMR genes were detected in a sigmoid colon cancer ofcase 17.
Discussion
HNPCC is a common dominantly inherited cancer susceptibil-ity syndrome, and diagnostic criteria, called the Amsterdamcriteria, were defined in 1990 (4-6). But these criteria were too
strictly defined for HNPCC, and there have been several reportson the kindreds defined as “non-HNPCC,” although they havebeen proven to share germ-line mutations in one or more of theMMR genes (34, 35). Therefore, new clinical criteria to detect
borderline HNPCC kindreds were proposed at the 34th Meetingof the JSCCR in Japan (7). At first, we analyzed MIN of 29
kindreds (32 cases) fulfulling these criteria. Eighteen of 32
cases (56%) revealed RER� at two or more microsatelbite boci
examined. Although MIN has been reported to occur in 80-
90% of HNPCC cancers, our results were lower than those
described previously (8-11). “Non-HNPCC” kindreds might
be included in these 29 kindreds, because the Japanese criteriawere defined to detect putative HNPCC for screening. The
clinicopathobogical characteristics of 18 RER� cases were the
following: (a) early-onset coborectal cancer (average age, 56.5years); (b) colorectal cancer in a proximal site (63%); (c)
multiple colonic and/or extracolonic cancers (56%); and (d)mainly moderate and well-differentiated adenocarcinoma his-
tologicalby. These data corresponded well to those that have
been reported previously on the HNPCCs defined by the Am-
sterdam criteria (2-5). Eleven (10 kindreds; cases 1 1 and 17 are
members in the same kindred) of 18 RER� cases showedRER� at most of the microsatellite boci examined (Tables 1 and
2). MIN in these kindreds must be the phenotype of a profound
genomic instability caused by deficiency of MMR genes. In
addition, 19 of 26 cancer lesions in these 1 1 RER phenotypecases revealed alteration of poly(A) tract in TGF-(3 RI! (Table
2). Poly(A) tract alteration of TGF-(3 RI! is frequently found inHNPCC and is suggested to be one of the target genes of the
defective DNA repair (20-22). Among these 10 kindreds, only
3 fulfulbed the Amsterdam criteria, and the remaining 7 did not.It was reported that approximately 10% of total HNPCCs
fulfilling the Amsterdam criteria did not show RER (8, 10, 11).
We also detected two RER-negative kindreds in five kindreds
on July 20, 2021. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from
a exon5
11C -
Ni N
17
i 234
of hMLHI in one kindred, and two point mutations of /IMLHIand one point mutation of hMSH2 in one kindred were found in
our study. Moreover, we detected the same mutations in bothIZMSH2 and hMLHI in cases 1 1 and 17, who were brothers inthe same kindred. These mutations were sure to be germ-linemutations of this kindred, and we will be abbe to use thesemutations as a “genetic marker” to detect at-risk young mdi-
viduals of the family in the future; (b) in our study, germ-line
variants were either missense mutations (four cases) or non-
sense mutation (one case). In the previous studies, germ-linemutations resulting in truncation of the predicted protein prod-
uct, such as frameshift, nonsense mutations, and splice-site
mutations, as well as large intragenic deletions, shared 70% of
a exon2N123
-‘-�-�
b11-N
A I T 0 A A A A C I 0 A A A 0 C CCC T C C
* 70 as
11-1
..--�--...
�Ii
bWild type
12-3
Fig. 1. Results of PCR-SSCP analysis and direct sequencing of exon 5 in
hMLHI. a. the mutant bands (arrow) were found in normal mucosa (N) and in a
cancer lesion (1). Also, in case 17, the mutant bands were seen in normal rnucosa
(N) and in cancer lesions (1-4). C, normal mucosa without colorectal cancer. b.
in case 1 1 , a nonsense mutation was detected at codon 133 (*; GGA to TGA: Glv
to stop). In case 17. the same nonsense mutations were detected in both normal
mucosa and cancer lesions (data not shown).
fulfilling the Amsterdam criteria. These kindreds with no RERmight be excluded from HNPCC.
In our study, among these 10 kindreds, 6 (60%) displayed
germ-line variants in hMSH2 and/or hMLHJ by PCR-SSCPanalysis and direct sequencing. No alteration in hMSH2 orhMLHI was detected in the remaining four kindreds. However,three interesting points stood out regarding germ-line variants
of MMR genes in our kindreds: (a) all kindreds except onekindred revealed more than one mutation in hMSH2 and/orhMLHJ. In detail, two point mutations of hMSH2 in two kin-
dreds, one point mutation of both hMSH2 and hMLHJ in onekindred, two point mutations of hMSH2 and one point mutation
Fig. 2. Results of PCR-SSCP analysis and direct sequencing of exon 2 in
hMLHI in case 12. a, the mutant band (arrow) was seen in only one cancer lesion
(3) but not in normal mucosa (N) and cancer lesions (I and 2). b. missense
mutation at codon 69 (*) was detected in cancer lesions (AGG to GGG: Ar.g to
Glv).
Cancer Epidemiology, Biomarkers & Prevention /061
on July 20, 2021. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from
1062 Germ-Line Mutations in Japanese HNPCC
4 Internet address: http://www.nfdht.nll.
Table 3 hMSH2/hMLHI alterations in HNPCC patients
Case Gene Exon affectedGenomic DNA alteration
Designation of mutations” Predicted effectsNormal DNA Cancer DNA
4 /IMSH2 Exon I 2 CAT to CGT CAT to COT M His (639) Arg
hMSH2 Exon 1 2 GAA to AAA GAA to AAA M Glu (647) Lys
8 hMSH2 Exon I 2 CAT to CGT CAT to CGT M His (639) Arg
hMSH2 Exon I 2 GAA to AAA GAA to AAA M Glu (647) Lys
10 hMLHJ Exon 5 GGA to TGA GGA to TGA N Gly (133) Stop
II hMSH2 Exon I 2 CAT to CGT CAT to CGT M His (639) Arg
IiMLHJ Exon 5 GGA to TGA GGA to TGA N Gly (I 33) Stop
12 hMSH2 Exon I 2 CAT to CGT CAT to CGT M His (639) Arg
hMLHI Exon 5 GGA to TGA GGA to TGA N Gly (133) Stop
hMLHJ Exon 8 ATC to GTC ATC to GTC P lIe (219) Val
I 5 hMSH2 Exon I 2 CAT to COT NI” M His (639) Arg
/JMSH2 Exon I 2 GAA to AAA NI M Gly (647) Lys
hMLHI Exon 8 CGC to TGC CGC to TGC M Arg (217) Cys
17 6MSH2 Exon 1 2 CAT to COT CAT to COT M His (639) Arg
6MLHI Exon 5 OGA to TGA OGA to TGA N Gly (133) Stop
(‘ M. missense mutation; N, nonsense mutation; P. polymorphism.
/, NI. not informative.
Table 4 Somatic mutations of hMS H2 in HNPCC patients
Oenomic DNAExon Case-tumor
change
Designation of.
mutations
Predicted
effects
Exon 2 8- 1 AAG to ACO
Exon 9 12-2 CAG to CAA
Exon I 2 1 1 - 1 GAA to AAA
I 1-2 GAA to AAA
12-1 OAAtoAAA
12-2 GAA to AAA
17-4 GAA to AAA
TAC to CAC
Exon 13 1 1-2 AlT to ACT
I 7-3 ATO to GIG
8- 1 AlT to ACT
8-2 AlT to ACT
ACT to AlT
Exon 14 17-2 ACA to ACO
Lys ( I 10) Thr
GIn (493) GIn
Olu (647) Lys
Olu (647) Lys
OIu(647)Lys
Glu (647) Lys
Olu (647) Lys
Thr (656) His
lIe (679) Thr
Met (729) Val
lie (679) Thr
lie (679) Thr
Thr (732) lIe
Arg (772) Arg
M
S
M
M
M
M
M
M
M
M
M
M
M
S
a M, missense mutation; 5, silent mutation.
MMR genes in HNPCCs (36). However, in our kindreds, non-sense mutations made up only 20% of all of the mutations, and
the remaining 80% were missense mutations. We could notdetect frameshift or splice-site mutations in the two MMR
genes; and (c) these germ-line variants were found in more than
one kindred. For example, a missense mutation at codon 639 ofhMSH2 in five kindreds, a missense mutation at codon 647 ofhMSH2 in three kindreds, and a nonsense mutation at codon133 of hMLHJ in three kindreds were detected. These 10kindreds had no relation between each other from the familial
analysis. Although a missense mutation at codon 639 (CAT toTAT; His to Tyr) in hMSH2 was already reported in theWestern world ( 14), no report was found on point mutations at
codon 647 in hMSH2 and at codon 133 in hMLHJ. A missensemutation at codon 647 in hMSH2 was confirmed not only as agerm-line variant in three kindreds but also as a somatic mu-
tation in three kindreds. From our data, it is suggested that point
mutations at codons 639 and 647 in hMSH2 and codon 133 inhMLHI are frequent mutational points, so-called “hot spots,” inMMR genes in Japanese putative HNPCC kindreds. As to these
four missense mutations of germ-line variants, it is important todecide whether these mutations are polymorphisms or not.
Table 5 Somatic mutations of hMLHI in HNPCC patients
Case (-tumor)Exon Genomic DNA
affected change
Designation of.
mutations
Predicted
effects
15
12-3
28-4
15
I 7-2
17-3
17-2
17- 1
8-I
I 7-3
17-3
Exon 2 CAG to CAA
AGO to 006
CAG to OCO
Exon 3 ACT to AlT
TCC to TO’
‘fl’OtoCTG
Exon 9 AAG to AAA
Exon 14 CAT to TAT
Exon 15 cagTGA to tagTGA
Exon I 7 GGG to AGO
Exon 19 OAT to OTT
GIn (60) GIn
Arg (69) Gly
Gin (53) Ala
Thr (8 1) lIe
5cr (87) Ser
Leu(85)Leu
Lys (255) Lys
His (526) Tyr
Splice site
Gly (634) Arg
Asp (737) Val
S
M
M
M
S
S
S
M
M
M
a 5 silent mutatio n; M, missense mutation.
Because of the insufficient establishment of a genetic counsel-ing system for hereditary diseases in Japan, we could notconduct medical surveillance of these six kindreds. Therefore,we consulted the MMR gene mutation database of the Interna-
tional Collaborative Group on HNPCC.4 According to this
database, three missense mutations, except for one mutation atcodon 219 in hMLHJ (ATC to GTC; Ile to Vab), were found not
to be polymorphisms. However, more investigations, includingmedical surveillance, would be necessary to clarify the nature
of these germ-line mutations, although we could not detectgerm-line variants in hMSH2 or hMLHJ in four kindreds withMIN at most of the microsatellite loci examined with ourmethod. The reasons why we could not detect germ-line van-
ants in these families were considered to be the following: (a)the aberrant migration band was not indicated on the SSCP gel;
otherwise, there would be a mutation in hMSH2 or hMLHJ; and(b) the alterations in other MMR genes, such as hPMSJ,/iPMS2, GTBP, hMSH3, or genes as yet unidentified, might be
involved in these kindreds. Investigations for alterations inhPMSJ, hPMS2, GTBP, and hMSH3 should be required.
Presently, HNPCC is identified according to the number of
individuals affected by coborectal cancer. However, several
on July 20, 2021. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from
Case 12 Case 8
Cancer Epidemiology, Biomarkers & Prevention 1063
Fig. 3. Partial pedigrees of the families with germ-line mutations in hMSH2
and/or hMLHI. #{149}and #{149},affected subjects; 0 and 0, asymptomatic individuals;
cross-line, deceased; arrow, proband. For each subject, the tumor and age of
diagnosis are indicated. In case 12, HER was revealed in both colon and uterine
cancer lesions, and three germ-line mutations were detected at codon 639 inhMSH2, at codon 133 in hMLIII, and at codon 219 in hMLHI. In case 8, RER
was shown in a cancer lesion, and a germ-line mutation at codon 639 and 647 in
hMSH2 was found.
kindreds with MIN in cancer lesions, as well as germ-lineMMR gene mutations, have been reported not to fulfill theAmsterdam criteria (17, 34, 35). In our six kindreds with MMR
gene germ-line mutation, only two fulfilled the Amsterdam
criteria, and remaining four did not. For example, we presenttwo families with germ-line mutations in MMR genes detectedin this study (Fig. 3). These kindreds fulfill the Japanese criteria
for HNPCC but do not satisfy the Amsterdam criteria. How-ever, these families not only revealed MIN at most of themicrosateblite boci examined but also displayed germ-line mu-
tations in hMSH2 and/or hMLHJ. Therefore, we viewed thesefamilies as HNPCC and we are going to undergo surveillance
carefully for the occurrence of carcinoma in the patient andyoung individuals that are not affected yet by malignancy in the
same kindreds. From these observations, the Amsterdam crite-
ria may be too strict to detect whole HNPCC kindreds. On the
other hand, the Japanese criteria have the advantage of detect-ing borderline HNPCC kindreds, because hMSH2 or hMLHI
germ-line mutations were also found in putative HNPCC kin-dreds in our study. Moreover, we should manage carefully“HNPCC-like” kindreds, detected by “the Japanese criteria”without the RER phenotype, such as cases 2, 5, 6, 14, 16, 18,
21 , 24, 25, 26, 29, 30, 3 1 and 32. The RER phenotype reflectingthe deficiency of the MMR gene has been found in approxi-
mateby 90% of cancer lesions in HNPCC kindreds (8-1 1).Therefore, those 14 of 32 cases who did not indicate RER
phenotype may not be at risk for HNPCC. However, RER has
not been detected in 10% ofHNPCCs (8-11), and we must alsoconduct medical surveillance carefully for these 14 HNPCC-bike cases. More HNPCC will be found from borderlineHNPCC by investigating MIN in cancer lesions of the patientin the kindred whose cancer was first detected according to the
Japanese criteria.Finally, we propose that a microsatellite assay is necessary
to detect more “true-HNPCC kindreds” from borderlineHNPCC kindreds, and molecular findings of RER phenotypeshould be added to the criteria for HNPCC.
Acknowledgments
We are grateful to Masayoshi Takatani and Tomoyuki Nomi for their skillful
technical assistance.
References
I . RUschoff, J., Bocker, T., Schlegel, J., Stumm, 0., and Hofstaedter, F. Micro-satellite instability: new aspects in the carcinogenesis of colorectal carcinoma.
Virchows Arch., 426: 215-222, 1995.
2. Lynch, H. T., Smyrk, T., and Lynch, J. F. Overview of natural history.pathology, molecular genetics and management of HNPCC (Lynch syndrome).
Int. J. Cancer (Pred. Oncol.), 69: 38-43, 1996.
3. Vasen, H. F. A., Mecklin, J-P., Watson, P., Utsunomiya, J., Bertario, L.,
Lynch, P., Svendsen, L. B., Cristofaro, 0., MUller, H., Khan P. M., and Lynch,
H. T. The International Collaborative Group on HNPCC: surveillance in hered-
itary nonpolyposis colorectal cancer. Dis. Colon Rectum, 36: 1-4, 1993.
4. Mecklin, J-P., and JSrvinen, H-J. Tumor spectrum in cancer family syndrome
(hereditary nonpolyposis colorectal cancer). Cancer (Phila.), 68: 1 1 09-1 1 12,
I 991.
5. Lynch, H. T, Smyrk, T. C., and Watson, P. Genetics, natural history, tumor
spectrum, and pathology of hereditary nonpolyposis colorectal cancer: an updated
review. Gastroenterology, 104: 1535-1549, 1993.
6. Vasen, H. F. A., Mecklin, J-P., Meera Khan, P., and Lynch, H. T. The
International Collaborative group on Hereditary Non-polyposis Colorectal Can-
cer. Dis. Colon Rectum, 34: 424-425, 1991.
7. Kunitomo, K., Terashima, Y., Sasaki, K., Komi, N., Yosikawa, R.,
Utsunomiya, J., and Yasutomi, M. HNPCC in Japan. Anticancer Res., 12:
1856-1857, 1992.
8. Aaltonen, L. A., Peltom#{228}ki, P., Leach, F. S., Sistonen, P., Pylkkanen. L.,
Mecklin, J-P., Jarvinen, H., Powell, S. M., Jen, J., Hamilton, S. R., Petersen,
0. M., Kinzler, K. W., Vogelstein, B., and de Ia Chapelle, A. Clues to the
pathogenesis of familial colorectal cancer. Science (Washington DC), 260: 812-
860, 1993.
9. Lathe, R. A., Peltomaki, P., Meling, 0. I., Aaltonen, L. A., Nystrom-Lahti, M..
Pylkkanen, L., Heimdal, K., Andersen, T. I., Moller, P., Rornum, T. 0., Fossa,
S. D., Haldorsen, T., Langmark, F., Brogger, A., de Ia Chapelle, A., and Borresen,
A. L. Genomic instability in colorectal cancer: relationship to clinicopathological
variables and family history. Cancer Res., 53: 5849-5852, 1993.
10. Aaltonen, L. A., Peltomaki, P., Mecklin, J-P., Jarvinen, H., Jass, J. R., Green,
J. S., Lynch, H. T., Watoson, P., Tallqvist, 0., Juhola, M., Sistonen, P., Hamilton,
S. R., Kinzler, K. W., Vogelstein, B., and de Ia Chapelle, A. Replication errors in
benign and malignant tumors from hereditary nonpolyposis colorectal cancer
patients. Cancer Res., 54: 1645-1648, 1994.
11. Liu, B., Parsons, R., Papadopoulos, N., Nicolaides, N. C., Lynch, H. T.,
Watson, P., Jass, J. R., Dunlop, M., Wyllie, A., Peltomaki, P., de Ia Chapelle, A.,
Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. Analysis of mismatch repair
genes in hereditary non-polyposis colorectal cancer patients. Nat. Med., 2: 169-
174, 1996.
12. Bronner, C. E., Baker, S. M., Morrison, P. T., Warren, 0., Smith, L. 0.,
Lescoe, M. K., Kane, M., Earabino, C., Lipford, J., Lindblom, A., Tannergrand,
P., BoIlag, R. J., Godwin, A. R., Ward, D. C., Nordenskjoid, M., Fishel, R.,
Kolodner, R., and Liskay, R. M. Mutation in the DNA mismatch repair gene
homologue hMLH1 is associated with hereditary nonpolyposis colon cancer.Nature (Land.), 368: 258-261, 1994.
13. Nicolaides, N. C., Papadopoulos, N., Liu, B., Wei, Y-F., Carter, K. C.,Ruben, S. M., Rosen, C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C. M.,
Adams, M. D., Venter, J. C., Dunlop, M. 0., Hamilton, S. R., Petersen, 0. M., de
Ia Chapelle, A., Vogelstein. B., and Kinzler, K. W. Mutations of two PMS
homologues in hereditary nonpolyposis colon cancer. Nature (Lond.), 371: 75-
80, 1994.
14. Leach, F. S., Nicolaides, N. C., Papadopoulos, N., Liu, B., Jen, J., Parsons,
R., Peltomaki, P., Sistonen, P., Aaltonen, L. A., Nystrom-Lahti, M., Guan, X-Y.,
Zang, J., Meltzler, P. S., Yu, J-W., Kao, F-T., Chen, D. J., Cerosaletti, K. M.,
Fournier, R. E. K., Todd, S., Lewis, T., Leach, R. J., Naylor, S. L., Weissenbach,
J., Mecklin, J-P., Jarvinen, H., Petersen, 0. M., Hamilton, S. R., Green, J., Jass,J., Watson, P., Lynch, H. T., Trent, J. M., de Ia Chapelle, A., Kinzler, K. W., and
Vogelstein, B. Mutations of a mutS homolog in hereditary nonpolyposis colorec-
tal cancer. Cell, 75: 1215-1225, 1993.
15. Papadopoulos, N., Nicolaides, N. C., Wei, Y-F., Ruben, S. M., Carter, K. C.,
Rosen, C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C. M., Adams, M. D..
Venter, J. C., Hamilton, S. R., Petersen, 0. M., Watson, P., Lynch, H. T.,
Peltomaki, P., Mecklin, J-P., de Ia Chapelle, A., Kinzler, K. W., and Vogelstein,B. Mutation of a mutL homolog in hereditary nonpolyposis colon cancer. Science
(Washington DC), 263: 1625-1629, 1994.
16. Moslem, 0., Tester, D. J., Lindor, N. M., Honchel, R., Cunningham, J. M.,
French, A. J., Hailing, K. C., Schwab, M., Goretzki, P., and Thibodeau, S. N.
Microsatellite instability and mutation analysis of hMSH2 and hMLH1 in patientswith sporadic, familial and hereditary colorectal cancer. Hum. Mol. Genet., 5:
1245-1252, 1996.
17. Liu, B., Nicolaides, N. C., Markowitz, S., Willson, J. K. V., Parsons, R. E.,
Jen, J., Papadopoulos, N., Peltomkki, P., de la Chapelle, A., Hamilton, S. R.,
on July 20, 2021. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from
1064 Germ-Line Mutations in Japanese HNPCC
Kinzler, K. W., and Vogelstein, B. Mismatch repair gene defects in sporadic
colorectal cancers with microsatellite instability. Nat. Genet., 9: 48-55, 1995.
18. Muta, H., Noguchi, M., Perucho, M., Ushio, K., Sugihara, K., OChIai, A.,
Nawata, H., and Hirohashi, S. Clinical implications of microsatellite instability in
colorectal cancers. Cancer (Phila.), 77: 265-270, 1996.
19. Kane, M. F., Lads, M., Gaida, 0. M., Lipman, J., Mishra, R., Goldman, H.,
Jessup, J. M., and Kolodner, R. Methylation of the hMLHJ promotor correlates
with lack of expression of hMLH1 in sporadic colon tumors and mismatch
repair-defective human tumor cell lines. Cancer Res., 57: 808-811, 1997.
20. Markowitz, S., Wang, J., Myerhoff, L., Parsons, R., Sun, L., Lutterbaugh,
J., Fan, R. S., Zborowska, E., Kinzler, K. W., Vogelstein, B., Brattain, M., andWillson, J. K. V. Inactivation of the type II TGF-� receptor in colon cancer
cells with microsatellite instability. Science (Washington DC), 268: 1336-
1338, 1995.
21. Lu, S-L., Akiyama, Y., Nagasaki, H., Saiyo, K., and Yuasa, Y. Mutation of
the transforming growth factor-fl type II receptor gene and genomic instability in
hereditary nonpolyposis colorectal cancer. Biochem. Biophys. Res. Commun.,
216: 452-457, 1995.
22. Parsons, R., Myerhoff, L. L., Liu, B., Willson, J. K. V., Markowitz, S. D.,
Kinzler, K. W., and Vogelstein, B. Microsatellite instability and mutations of the
transforming growth factor f3 receptor type II gene in colorectal cancer. Cancer
Res., 55: 5548-5550, 1995.
23. Shibata, D., Hawes, D., Li, Z-H., Hernandez, A. M., Spnsck, C. H., and
Nichols, P. W. Specific genetic analysis of microscopic tissue after selective
ultraviolet radiation fractionation and the polymerase chain reaction. Am. J.
Pathol., 141: 539-543, 1992.
24. Hon. A., Han, H-J., Shimada, M., Yanagisawa, A., Kato, A., Ohta, H.,
Yasui, W., Tahara, E., and Nakamura, Y. Frequent replication errors at micro-
satellite loci in tumors of patients with multiple primary cancers. Cancer Rca., 54:
3373-3375, 1994.
25. Hemminki, A., Peltomaki, P., Mecklin, J-P., Jarvinen, H., Salovaara, R.,
NystrOrn-Lahti, M., de Ia Chapelle, A., and Aaltonen, L. A. Loss of the wild typeMLHI gene is a feature of hereditary nonpolyposis colorectal cancer. Nat. Genet.,
8: 405-410, 1994.
26. Semba, S., Yokozaki, H., Yamamoto, S., Yasui, W., and Tahara, E. Micro-
satellite instability in precancerous lesions and adenocarcinomas of the stomach.
Cancer (Phila.), 77: 1620-1627, 1996.
27. Grits, M., Suzuki, Y., Sekiya, T., and Hayashi, K. A rapid and sensitive
detection of point mutations and genetic polymorphism using the polymerase
chain reaction. Genomics, 5: 874-879, 1989.
28. Orita, M., Iwahara, H., Kanazawa, H., and Sekiya, T. Detection of polymor-phism of human DNA by gel electrophoresis as single strand conformation
polymorphism. Proc. Nail. Acad. Sci. USA, 86: 2766-2770, 1989.
29. Kolodner, R. D., Hall, N. R., Lipford, J., Kane, M. F., Rao, M. R. S.,
Morrison, P., Wirth, L, Floats, P. J., Burn, J., Chapman, P., Earabino, C.,
Merchant, E., and Bishop, D. T. Structure of the human MSH2 locus and analysis
of two Muir-Torre kindreds for msh2 mutations. Genomics, 24: 516-526, 1994.
30. Han, H-J., Maruyama. M., Baba, S., Park, J-G., and Nakamura, Y. Genomic
structure of a human mismatch repair gene, hMLHJ, and its mutation analysis in
patients with hereditary nonpolyposis colorectal cancer (HNPCC). Hum. Mol.
Genet., 4: 237-242, 1995.
31. Kobayashi, K., Matsusima, M., Koi, S., Saito, H., Sagae, S., Kudo, R., and
Nakamura, Y. Mutation analysis of mismatch repair genes, hMLJIJ and IZMSH2,
in sporadic endometrial carcinomas with microsatellite instability. Jpn. J. Cancer
Res., 87: 141-145, 1996.
32. Lu, S-L., Akiyama, Y., Nagasaki, H., Nomizu, T., Ikeda, E., Baba, S., Ushio,
K., Iwaina, T., Maruyama, K., and Yuasa, Y. Loss or somatic mutations of
hMSH2 occur in hereditary nonpolyposis colorectal cancers with IZMSH2 germ-
line mutations. Jpn. J. Cancer Res., 87: 279-287, 1996.
33. Fujii, K., Yokozaki, H., Yasui, W., Kuniyasu, H., Hirata, M., Kajiyama, 0.,
and Tahara, E. High frequency of p53 gene mutation in adenocarcinoma of the
gallbladder. Cancer Epidemiol. Biomarkers Prey., 5: 461-466, 1996.
34. Mauillon, J. L, Michel, P., Limacher, J-M., Latouche, i-B., Dechelotte, P.,
Charbonnier, F., Martin, C., Moreau, V., Metayer, I., Paillot, B., and Frebourg, 1.Identification of novel germline hMLHJ mutations including a 22 kb AIu-
mediated deletion in patients with familial colorectal cancer. Cancer Res., 56:5728-5733, 1996.
35. Nystrom-Lahti, M., Wu, Y., Moisio, A-L., Hofstra, R. M. W., Osinga, J.,
Mecklin, i-P., Jarvinen, H. i., Leisti, i., Buys, C. H. C., de la Chapelle, A., andPeltomSki, P. DNA mismatch repair gene mutations in 55 kindreds with verified
or putative hereditary non-polyposis colorectal cancer. Hum. Mol. Genet., 6:763-769, 1996.
36. Kinzier, K. W., and Vogelstein, B. Lessons from hereditary colorectal cancer.
Cell, 87: 159-170, 1996.
on July 20, 2021. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from
1997;6:1057-1064. Cancer Epidemiol Biomarkers Prev M Nakahara, H Yokozaki, W Yasui, et al. cancer kindreds.and/or hMLH1 in Japanese hereditary nonpolyposis colorectal Identification of concurrent germ-line mutations in hMSH2
Updated version
http://cebp.aacrjournals.org/content/6/12/1057
Access the most recent version of this article at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://cebp.aacrjournals.org/content/6/12/1057To request permission to re-use all or part of this article, use this link
on July 20, 2021. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from