Identification of Concurrent Germ-Line Mutations in hMSH2 ......Vol. 6, 1057-1064, Decenther 1997 Cancer Epidendologj, Biomarkers & Prevention 1057 Identification of Concurrent Germ-Line

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

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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!



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,



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



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




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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



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.,




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.



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

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Identification of Concurrent Germ-Line Mutations in hMSH2 ......Vol. 6, 1057-1064, Decenther 1997 Cancer Epidendologj, Biomarkers & Prevention 1057 Identification of Concurrent Germ-Line