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Human Molecular Genetics Pages 763-769
DNA mismatch repair gene mutations in 55 kindreds with verified or putative hereditary non-polyposis colorectal cancer
Introduction
Results
   Pathogenic mutations
   Non-pathogenic sequence variations
Discussion
Materials And Methods
   Kindreds
   RT-PCR
   2-D DNA electrophoresis
   Sequence analysis
Acknowledgements
References

DNA mismatch repair gene mutations in 55 kindreds with verified or putative hereditary non-polyposis colorectal cancer

DNA mismatch repair gene mutations in 55 kindreds with verified or putative hereditary non-polyposis colorectal cancer Minna Nyström-Lahti1, Ying Wu2, Anu-Liisa Moisio1, Robert M. W. Hofstra2, Jan Osinga2, Jukka-Pekka Mecklin3, Heikki J. Järvinen4, Jaakko Leisti5, Charles H. C. M. Buys2, Albert de la Chapelle1 and Päivi Peltomäki1,*

1Department of Medical Genetics, Haartman Institute, P.O. Box 21 (Haartmaninkatu 3), University of Helsinki, FIN-00014 Helsinki, Finland, 2Department of Medical Genetics, Rijksuniversiteit Groningen, Antonius Deusinglaan 4, 9713 AW Groningen, The Netherlands, 3Jyväskylä Central Hospital, FIN-40620 Jyväskylä, Finland, 4Second Department of Surgery, Helsinki University Central Hospital, Haartmaninkatu 4, FIN-40620 Jyväskylä, Finland and 5Department of Clinical Genetics, Oulu University Central Hospital, FIN-90220 Oulu, Finland

Received January 3, 1996; Revised and Accepted March 19, 1996

The DNA mismatch repair genes MSH2 and MLH1 have been shown to account for a major share of hereditary non-polyposis colorectal cancer (HNPCC). We searched for germline mutations in these genes in 35 HNPCC kindreds fulfilling the Amsterdam diagnostic criteria and in a further 20 kindreds with an average of four affected members per family but not meeting the formal criteria. We first screened for truncations by reverse transcriptase (RT)-PCR. If no mutation was found, we screened genomic DNA by a novel application of two-dimensional (2-D) DNA electrophoresis that allows the simultaneous study of all exons of each gene. All abnormalities were followed up by sequencing. Eight different pathogenic germline mutations were found, two in MSH2 and six in MLH1. We report three major conclusions. First, these mutations together accounted for 86% (30/35) of the kindreds meeting the Amsterdam criteria, but only 30% (6/20) of the remaining kindreds, suggesting differences in etiology. Second, MLH1 was involved in >90% (34/36) of kindreds with a known predisposing mutation, suggesting that mutations in the MLH1 gene are responsible for most HNPCC kindreds in Finland. Third, our results indicate that the successive application of RT-PCR and 2-D DNA electrophoresis is a sensitive and efficient method for mutation screening in typical HNPCC.

INTRODUCTION

Hereditary non-polyposis colorectal cancer syndrome (HNPCC) is the most common cause of familial colorectal cancer and is associated with germline mutations in four different DNA mismatch repair genes, i.e. MSH2 on 2p (1 ,2 ), MLH1 on 3p (3 ,4 ), PMS1 on 2q and PMS2 on 7p (5 ). Based on linkage and mutation studies, MSH2 and MLH1 are responsible for a major share of HNPCC (6 -9 ). In all comprehensive studies published so far, a proportion of kindreds (~30%) show no germline mutations in DNA mismatch repair genes even when studied by the best methods presently available (9 ). These data raise several questions. Most studies focus on HNPCC kindreds meeting the stringent so-called Amsterdam criteria (10 ). Is the proportion of DNA mismatch repair gene mutations different in smaller cancer families not fulfilling these criteria? What causes the underlying predisposition in HNPCC families in which no mutation can be found? Do the proportions of mutations in different genes vary among ethnic or geographical groups? Are the methods for mutation detection efficient and specific?

To address some of these questions, we studied 55 Finnish HNPCC kindreds, 35 of which met the Amsterdam criteria. The detection rate of mutations was 65% overall but as high as 86% in families meeting the Amsterdam criteria, suggesting differences in etiology. The proportion of MLH1 mutations was high (>90%), suggesting that ethnic differences do occur. Finally, our findings lead us to suggest that under the present circumstances the employed detection system, a combination of RT-PCR-based transcript analysis and two-dimensional (2-D) electrophoresis of genomic DNA, is a useful one.

RESULTS

Pathogenic mutations

To screen for truncating mutations, we performed polymerase chain reaction coupled with reverse transcription (RT-PCR) analyses on lymphoblastoid cell RNA. In MLH1, three such mutations were detected and shown to delete exons 6, 14 and 16, respectively, by sequencing of the RT-PCR products. To define the mutations at the genomic level, the coding regions and the intron-exon boundaries of the relevant exons were sequenced (Table 1 ). Six kindreds carried an identical point mutation that destroys the splice acceptor site of exon 6 (11 ). One kindred showed a mutation at the splice acceptor site of exon 14 resulting in the deletion of this exon from the transcript and a frameshift with premature termination. A further 22 kindreds shared a 3.5 kb genomic deletion affecting exon 16 and flanking introns (11 ).

Moreover, in three kindreds, two further heterozygous alterations in MLH1 were detected at the cDNA level, both appearing as exon skipping, but no genomic change could be detected by the methods we have used so far. In two of these kindreds (Nos 8 and 39) exon 12 was deleted. Both of these families show unequivocal linkage to the MLH1 gene based on conventional linkage analysis and segregation of a common haplotype in each family (data not shown). In the third kindred (No. 4) exons 3, 4, and 5 were deleted. If these cDNA alterations are indeed mutations that predispose to cancer, genomic changes located further into the flanking introns or in the promoter region of the gene might be responsible. These changes could also be due to alternative splicing in analogy to what has been described for MLH1 exons 9-11 (12 ), suggesting that this gene might be prone to splicing defects.

Two-dimensional electrophoretic separation of exon fragments according to size and sequence was performed to screen genomic DNAs from all kindreds in which no alteration was detectable by RT-PCR. Five different germline mutations (Table 1 ) were detected. In the MSH2 gene, two different mutations causing frameshifts and premature termination resulted from a 2 bp deletion in exon 10 in one kindred (No. 38), and a 2 bp insertion in exon 12 in the other kindred (No. 25). In the MLH1 gene, two different missense mutations and one nonsense mutation were found. Three families (Nos 28, 51 and 67) showed an isoleucine to arginine substitution in codon 107 of exon 4 (Table 1 , Fig 1 ). One family (No. 7) had an arginine to proline substitution in codon 659 of exon 17, while another kindred (No. 83) had an arginine to stop change in the same codon (Table 1 ).

It is of major importance to be able to distinguish missense mutations that lead to cancer susceptibility from polymorphisms without clinical significance. We consider the two missense mutations (in MLH1 exons 4 and 17) pathogenic for four reasons: (i) they result in changes of amino acids belonging to different polarity groups; (ii) the involved codons are evolutionarily conserved; (iii) the mutations were not found in a sample of 100 additional independent chromosomes, suggesting that they were not likely to be merely polymorphic variants in the population; and (iv) segregation with the disease phenotype could be demonstrated in family 67, in which samples were available from three affected members and all showed the mutation (in the remaining kindreds, segregation analysis was not possible due to unavailability of samples).

Non-pathogenic sequence variations

Additionally, 2-D DNA electrophoresis revealed five variants (two in MSH2 and three in MLH1), two of which have not been published before (Table 2 ). We consider these variants as polymorphisms since, unlike the two missense mutations described above, they resulted in no significant amino acid changes and showed allele frequencies of 4-26% as estimated by the study of 54 chromosomes from independent HNPCC family members.

Table 1 . Germline mutations identified and fully characterized at the genomic DNA level
Family

 Exon

 Codon

 Nucleotide change

 Predicted coding change
MSH2
38

 10

 518

 CACAGT -> CAGT

 Frameshift; termination
 

  

  

 (deletion CA)

 28 bp downstream
25

 12

 619-620

 ATGT -> ATGTGT

 Frameshift; termination
 

  

  

 (insertion TG)

 195 bp downstream
MLH1
28, 51,67

 4

 107

 ATA -> AGA

 Isoleucine to arginine
 

  

  

 (T -> G substitution)
13,26,27,29,55,91

 6

 Splice

 5'-cagGTG-3' -> 5'-caaGTG-3'

 Out-of-frame 92 bp deletion;
 

  

 acceptor

  

 termination 24 bp downstream
40

 14

 Splice

 5'-cagTTC-3' -> 5'-catTTC-3'

Out-of-frame 109 bp deletion;
 

  

 acceptor

  

 termination 101 bp downstream
1,2,3,6,9,10,11,12,19,43,

 16

  

 3.5 kb genomic deletion

In-frame 165 bp deletion
50,54,58,59,60,63,66,69,77,82
7

 17

 659

 CGA -> CCA

 Arginine to proline

 

  

  

 (G -> C substitution)
83

 17

 659

 CGA -> TGA

 Arginine to stop
 

  

  

 (C -> T substitution)
The identification numbers of kindreds fulfilling the Amsterdam criteria are underlined.

Table 2 Non-pathogenic sequence variations detected in the present study
Exon

 Codon

 Change

 Allele frequency

Reference
MSH2
6

 322

 GGC -> GAC

 2/54 chromosomes

 (21)
 

  

 Gly -> Asp
13

 713

 GGG -> GGC

 2/54 chromosomes

This study
 

  

 Gly -> Gly
MLH1
Intron 5

 -51 of 5'-exon 6

 t -> c

 2/54 chromosomes

 This study
8

 219

 ATC -> GTC

 13/54 chromosomes

 (21)
 

  

 Ile -> Val
Intron 14

 -19 of 5'-exon 15

 a -> g

 14/54 chromosomes

 (21)
Allele frequencies determined in independent, unrelated members of the Finnish HNPCC pedigrees not including families with either of the two common founding mutations.

DISCUSSION


Figure 1. (A) Two-dimensional DNA electrophoresis separation patterns of 20 PCR-amplified fragments representing all 19 exons of the MLH1 gene (exon 13 was amplified in two fragments). Left, affected member of family 67 showing an altered pattern with four bands (two homoduplexes and two heteroduplexes) for exon 4 (vertical line); right, normal control. (B) The aberrant 2-D DNA electrophoresis pattern in family 67 results from a heterozygous change of T to G in MLH1 codon 107, as shown by sequence analysis (the mutated base is indicated by an arrow).We found germline mutations in either MSH2 or MLH1 in a high proportion (30/35, 86%) of the kindreds meeting the Amsterdam criteria. This relatively high figure reflects in part the fact that two of eight mutations described in the present study are widespread among Finnish kindreds due to founder effects (11 ). In contrast, the proportion of kindreds not fulfilling the Amsterdam criteria that had a detectable mutation in MSH2 or MLH1 was much lower (6/20, 30%). This suggests that, at least in part, different etiologies are involved. The International Collaborative Group (`Amsterdam') criteria were originally developed as minimum criteria for families to be included in collaborative studies (10 ). Our results show that the clinical definition of HNPCC based on the Amsterdam criteria is in fact more stringent than a definition based on involvement of DNA mismatch repair genes. Nineteen kindreds, 14 of which did not meet the international diagnostic criteria, revealed no MSH2 or MLH1 mutations. Interestingly, a majority of tumors available from the above-mentioned 19 kindreds were later found to show no microsatellite instability (Aaltonen et al., unpublished, and unpublished data from the authors), suggesting that genes other than those responsible for DNA mismatch repair, might predominantly be involved.

To date, some 90 verified or putative HNPCC kindreds are known to us in Finland, and 55 were included in this study, with the availability of blood samples being the only basis for selection. Forty three of the above-mentioned 90 families fulfil the Amsterdam criteria, and 35 were among the kindreds studied here. The distribution of mutations that we describe can thus be considered representative of all Finnish HNPCC kindreds. Our results emphasize that the proportion of mutations in different HNPCC genes may show considerable inter-ethnic variation. Most studies published previously focus on one mismatch repair gene only (MSH2 or MLH1) and, therefore, comparisons between different series are not yet meaningful. MLH1 was reported to account for 24% (8/34) of HNPCC kindreds mainly from Japan (13 ), while the proportion of kindreds attributable to mutations in MSH2 was 21% (7/34) in a Dutch series (14 ) and 34% (10/29) in a series mainly from North America (8 ). Three studies (two dealing with North American kindreds and one with European families) are available in which the role of MSH2 and MLH1 was investigated simultaneously and suggest that MSH2 and MLH1 account for roughly equal proportions of HNPCC (7 ,9 ,15 ). In view of these results, our finding of MLH1 being involved in 83% (29/35) and MSH2 in 3% (1/35) of kindreds meeting the Amsterdam criteria is exceptional. Although the presence of two relatively widespread founding mutations in MLH1 emphasizes the relative significance of MLH1 mutations in Finland, it is important to note that in our series, an identical approach to mutation detection resulted in the identification of only two different pathogenic mutations in MSH2 as compared with six in MLH1. Given that a variety of polymorphic variants was detected in both genes, it is unlikely that the unequal distribution of pathogenic mutations among the two genes is due to biased selection or technical shortcomings.

The high detection rate of mutations in this study can be interpreted to indicate that a combination of RT-PCR and 2-D DNA electrophoresis was a useful approach to mutation detection. The former technique reveals truncations resulting from splice site mutations and many genomic deletions, while the latter detects most base pair substitutions, small deletions and insertions in exons and flanking introns (16 ,17 ). As compared with most conventional techniques used for mutation detection, 2-D DNA electrophoresis has two particular advantages: all exons of a mismatch repair gene can be analyzed simultaneously, and no RNA is needed. In the present study, RT-PCR was necessary to screen for splice site mutations since, due to limited availability of sequence information, all intron-exon borders could not be covered by the primers we used for 2D-DNA electrophoresis (see Materials and Methods). Now that full sequence information is available, it is possible to design primers that cover the complete exons and every splice site (Wu et al., in preparation) and, consequently, 2-D DNA electrophoresis alone should be very suitable as a primary screening technique in HNPCC where most predisposing mutations identified to date are point mutations (9 ,13 ,14 ).

MATERIALS AND METHODS

Kindreds

Mutation screening was performed in 55 Finnish HNPCC kindreds from which appropriate samples were available. Of the kindreds, 35 fulfilled the Amsterdam diagnostic criteria (10 ). The mean number of affected individuals was 10 (range 3-33) in kindreds meeting the criteria and four (range 2-6) in the remaining kindreds. The average age of onset was 42.3 years and 46.2 years in the former and latter kindreds, respectively. Extracolonic cancer was present in most kindreds from both groups.

RT-PCR

Total cellular RNA was extracted from lymphoblasts by the guanidinium thiocyanate method (18 ), and an aliquot (1 [mu]g) was reverse-transcribed with Moloney murine leukemia virus (M-MLV) reverse transcriptase. For PCR amplification, MSH2 cDNA (2.8 kb) was divided into six, and MLH1 cDNA (2.3 kb) into five overlapping fragments, each covering two to seven exons (Table 3 ).

2-D DNA electrophoresis

DNA amplification. All primers, fragment sizes, PCR annealing temperatures, and melting temperatures used for MSH2 and MLH1 are listed in Tables 4 and 5 5. For MSH2, all exon fragments, except exon 1, were amplified by multiplex reactions. Combination A included exons 4, 6, 7, combination B exons 2, 8, 9, 11, combination C exons 3, 13, 14, 15 and combination D exons 5, 10, 12, 16. PCR was carried out on 400 ng of DNA in a total volume of 50 [mu]l for 40 cycles at 94oC for 1 min, at the specified annealing temperature for 1 min and at 72oC for 2 min. The PCR mixture contained 1* Super Taq DNA polymerase (HT Biotechnology LTD, Cambridge, UK), 600 [mu]M dNTP, and 25 pmol of each primer. For MLH1, exons were amplified separately in a 50 [mu]l reaction mixture containing 100 ng of genomic DNA, 200 [mu]M dNTP, 25 pmol of each primer, and 0.125 U of Taq DNA polymerase. To enhance the formation of heteroduplex molecules before 2-D analysis, samples were denatured for 10 min at 96oC followed by renaturation for 1 h at the annealing temperature of the primers used.

Table 3 . Sequences of primers used for RT-PCR, the sizes of PCR products and annealing temperatures
Exon

 Sense primer sequence

 Antisense primer sequence

Size

 Annealing
 

 (5' -> 3')

 (5' -> 3')

 (bp)

 temp. (oC)
MSH2
1-3

 GCATTTTCTTCAACCAGGAG

 ATGGAATCCACATACCCAAC

 542

 55
3-6

 GCCAGAGACAGGTTGGAG

 GTTTTACACTTATTCAGCAAGGCAG

 535

 56
8-12

 CTTTAGATATGGATCAGGTGG

 ATCTTGAACTTCAACACAAGC

 569

 55
12-14

 TTGGAGAAAGGACAAGGAAG

 CAGTTGGTATCTGATTGGC

 515

 56
5-9

 CAGGGTTCTGTTGAAGATAC

 TGAGATTAGGATCAAATGAAGG

 496

 53
14-16

 TACGATGGATTTGGGTTAGC

 ACCTTCATTCCATTACTGGG

 562

 55
MLH1
1-6

 GTTTCCTTGGCTCTTCTG

 TTCTCCTCGTGGCTATGTTG

 516

 56
5-11

 AAGGGACCCAGATCACGG

 CTCCAGGATGCTCTCCTC

 537

 59
11-14

 CGGGTGCAGCAGCACATCG

 TGCCAAGGCCCACTGAGGATTC

 651

 63
14-18

 CTTGGCACAGCATCAAACC

 CGCATTCTTTACTGAGGCT

 425

 54
18-19

 GCTATGTTCTATTCCATCCG

 GAGAAAGAAGAACACATCCC

 272

 51

Table 4 . Experimental conditions for 2-D DNA analysis of the MSH2 gene AnnealingMelting
Exon

 Sense primer sequence

Antisense primer sequence

 Sizea

 

 (5' -> 3')

 (5' -> 3')

 (bp)

 temp. (oC)

 temp. (oC)
1

 GC-clamp-CTTCAACCAGGAGGTGAGGAGG

 TCCCCAGCACGCGCCGTC

 269

 62

 82
2

 TTTAAGGAGCAAAGAATCTGC

 GC-clamp-CCTTATATGCCAAATACCAATC

 177

 50

 65
3

 GC-clamp-GCTTCTCCTGGCAATCTCTC

 GAATCTCCTCTATCACTAGACTC

 313

 50

 71
4

 GC-clamp-TTTCTTATTCCTTTTCTCATAGTAG

 ATGTACCTGATTCTCCATTTC

 201

 50

 67
5

 GTTGCAGTTTCATCACTGTC

 GC-clamp-CTGAAAAAGGTTAAGGGCTC

 150

 52

 67
6

 AGGGTTCTGTTGAAGATACCAC

 GC-clamp-CTCTCCTCTATTCTGTTCTTATC

 136

 50

 70
7

 CAGATTGAATTTAGTGGAAGC

 GC-clamp-CTTCATGTTTTTCCAGAGCC

 218

 50

 69
8

 TAGGAAAACACCAGAAATTATTG

 GC-clamp-CCTGATCCATATCTAAAGTTGT

 114

 50

 68
9

 TTGTCACTTTGTTCTGTTTGC

 GC-clamp-CCTCCAATGACCCATTCT

 180

 50

 67
10

 GCTTGGACCCTGGCAAAC

 GC-clamp-CGACTTGCAAACCTGTTGG

 163

 54

 68
11

 GC-clamp-GATTTGCAGCAAATTGACTTCTT

 CCAGGTGACATTCAGAAC

 146

 50

 64
12

 GC-clamp-GCTATGTAGAACCAATGCAG

 CCACAAAGCCCAAAAACCAG

 290

 54

 67
13

 GC-clamp-GCCCCAATATGGGAGGTA

 CTGAGGATAGAAGCAGTTTC

 205

 50

 72
14

 GC-clamp-GGTCTGCAACCAAAGATTC

 CTTTCTTCACCTGATAAAGC

 248

 50

 69
15

 GC-clamp-ATAGGTGTCTGTGATCAAAG

 CTCTCTTTCCAGATAGCAC

 180

 50

 70
16

 ACATGTGTTTCAGCAAGGTG

 GC-clamp-TACCTTCATTCCATTACTGGG

 198

 52

 65
GC-clamp: CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCG.GC-clamp: GCCGCCTGCAGCCCGCGCCCCCCGTGCCCCCGCCCCGCCCCCGGCCCGGGCGCCG.aWithout GC-clamp. Electrophoresis. Two-dimensional DNA electrophoresis according to Fischer and Lerman (19 ) was performed as described elsewhere (14 ). The PCR products were mixed in equal amounts, ethanol-precipitated with tRNA and re-dissolved. A 30 [mu]l mixture of the amplified exons was first subjected to size separation in a 0.75 mm thick 9% polyacrylamide (PAA) gel (acrylamide:bisacrylamide = 37.5:1) in 0.5* TAE (1* TAE = 40 mM Tris, HAC pH 8.0; 20 mM NaAc; 1 mM NA2EDTA) at 11 V/cm and 45oC for 5 h. The separation pattern was visualized by ethidium bromide (EtBr) staining for 10 min and UV transillumination of the gel. The 500-100 bp region in the middle part of the lane was cut out, and applied to a 1 mm thick 9% PAA gel containing a 30-65% UF (100% UF = 7 M urea and 40% deionized formamide) and 0-10% glycerol gradient. After electrophoresis at 6 V/cm at 56oC for 14 h, the gel was again stained by EtBr and documented as described above.

Table 5 . Experimental conditions for 2-D DNA analysis of the MLH1 gene AnnealingMelting
Exon

 Sense primer sequence

 Antisense primer sequence

 Sizea

 

 (5' -> 3')

 (5' -> 3')

 (bp)

 temp. (oC)

 temp. (oC)
1

 GC-clamp-GTTTCCTTGGCTCTTCTG

 CCGTTAAGTCGTAGCCC

 191

 56

 73
2

 GC-clamp-CTGTTTGATTTGCCAGTTTAG

 GCACAAACATCCTGCTAC

 144

 52

 70
3

 GC-clamp-TTCAAAGAGATTTGGAAAAATGAG

 TCAACAGGAGGATATTTTACAC

 224

 52

 67
4

 AACCTTTCCCTTTGGTGAGG

 GC-clamp-TGTTGAGACAGGATTACTCTGAGAC

 262

 56

 70
5

 GC-clamp-AGTATCTATCTCTCTACTGG

 GCTTCAACAATTTACTCTCC

 171

 52

 69
6

 GC-clamp-CTTTTGCCAGGACATCTTG

 ACAAATCTCAGAGACCCAC

 206

 58

 68
7

 TAGTGTGTGTTTTTGGCAAC

 GC-clamp-CATAAAACAAAACCATCCCC

 133

 52

 66
8

 CCTTGTGTCTTCTGCTGTTTG

 GC-clamp-ATAGGTTATCGACATACCGAC

 135

 60

 71
9

 GC-clamp-GAATCTCTTTTCTAATAGAGAACTG

 CCCTGTGGGTGTTTCCTG

 184

 58

 69
10

 GC-clamp-AGTTTTGAACTGGTTGCTTTC

 TGGTTGAGGAGTTTGGTG

 156

 50

 70
11

 GC-clamp-AGGTAATTGTTCTCTCTTAT

 ATCTGGGCTCTCACGTCTG

 262

 54

 75
12

 ACAGACTTTGCTACCAGGAC

 GC-clamp-AGAATAAAGGAGGTAGGCTG

 417

 54

 72
13I

 GC-clamp-CCTCCAAAATGCAACCCAC

 GTCATTTCCTTTCGGGAATC

 145

 54

 68
13II

 GATTCCCGAAAGGAAATGAC

 GC-clamp-GCAGTTGAGCCCTATCATC

 148

 52

 72
14

 TGGTAGGATTCTATTACTTACCTG

 GC-clamp-GCTCTGCTTGTTCACACAC

 190

 50

 64
15

 GC-clamp-CCAACTGGTTGTATCTCAAG

 TCAGAAGTGAAAAGGATCTAAAC

 152

 54

 67
16

 CTTGCTCCTTCATGTTCTTG

 GC-clamp-AGAAGTATAAGAATGGCTGTC

 221

 52

 72
17

 GC-clamp-TTGTTCCCTTGTCCTTTTTCC

 GCATGTACCGAAATGCTTAGTATC

 161

 56

 72
18

 GC-clamp-CCTATTTTGAGGTATTGAATTTCTTTG

 ACCTGCTGGCCTGAGAG

 173

 56

 72
19

 CAGGGAGGCTTATGACATC

 GC-clamp-GAGAAAGAAGAACACATCCC

 243

 54

 70
For GC clamp sequences, see Table 4.aWithout GC-clamp.

Sequence analysis

All abnormal products from RT-PCR or 2-D DNA electrophoresis experiments were sequenced. The fragment or exon in question was amplified by PCR, run in 0.8% low-melting-point agarose (NuSieve FMC BioProducts), cut out and cycle sequenced by a modified rapid PCR method (19 ). The sequencing primers were either primers used for RT-PCR, or primers used for 2-D DNA electrophoresis but without the GC-clamp (Tables 4 , 4 and 4 ).

ACKNOWLEDGEMENTS

We thank Siv Lindroos, Sinikka Lindh, Marilotta Turunen, Kirsi Hopponen, Tuula Lehtinen, Liisa Ukkola and Kaija Collin for expert assistance at various steps of the work. We are grateful to the members of the HNPCC families for their willingness to participate in this study. This work was financially supported by the Academy of Finland; the Finnish Cancer Foundation; the Sigrid Juselius Foundation; the Federation of the Finnish Insurance Companies; the Maud Kuistila Foundation; Ingeny BV, Leiden; Stichting voor Erfelijkheidsvoorlichting, Groningen; and the Nijbakker-Morra Stichting, Laren.

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*To whom correspondence should be addressed

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