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© 2003 American Society for Clinical Oncology Altered Expression of MLH1, MSH2, and MSH6 in Predisposition to Hereditary Nonpolyposis Colorectal Cancer
From the Departments of Medical Genetics and Pathology, University of Helsinki; the Department of Clinical Genetics and the Second Department of Surgery, Helsinki University Hospital, Helsinki; and the Department of Surgery, Jyväskylä Central Hospital, Jyväskylä, Finland; the Division of Human Cancer Genetics, The Ohio State University, Columbus, OH; the Department of Oncology, University Hospital, Lund; and the Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden. Address reprint requests to Päivi Peltomäki, MD, Department of Medical Genetics, Biomedicum Helsinki, PO Box 63 (Haartmaninkatu 8), FIN-00014 University of Helsinki, Finland; e-mail: paivi.peltomaki{at}helsinki.fi.
Purpose: A considerable fraction (30% to 70%) of families with verified or putative hereditary nonpolyposis colorectal cancer fails to show mutations in DNA mismatch repair (MMR) genes. Our purpose was to address the genetic etiology of such families. Materials and Methods: We scrutinized a population-based cohort of 26 families from Finland that had screened mutation-negative by previous techniques. Blood was tested for allelic messenger RNA (mRNA) expression of MLH1, MSH2, and MSH6 by single nucleotide primer extension (SNuPE), and tumor tissue for MMR protein expression by immunohistochemistry (IHC) as well as for microsatellite instability (MSI). Full-length cDNAs of genes implicated by SNuPE or IHC were cloned and sequenced. Results: Unbalanced mRNA expression of MLH1 alleles was evident in two families. An inherited nonsense mutation was subsequently identified in one family, and complete silencing of the mutated allele was identified in the other family. Extinct protein expression by IHC implicated MLH1 in these two and in four other families, MSH2 in four families, and MSH6 in one family. Although no unequivocal genomic mutations were detected in the latter families, haplotype and other findings provided support for heritable defects. With one exception, all tumors with IHC alterations showed MSI, in contrast to the remaining families, which showed neither IHC changes nor MSI. Conclusion: Our expression-based strategy stratified the present "mutation-negative" cohort into two discrete categories: families linked to the major MMR genes MLH1, MSH2, and MSH6 (11 [42%] of 26) and those likely to be associated with other, as yet unknown susceptibility genes (15 [58%] of 26).
GERMLINE MUTATIONS in the DNA mismatch repair (MMR) genes MSH2 and MLH1, which are the two "major" genes associated with hereditary nonpolyposis colon cancer (HNPCC), occur in 22% to 86% of families meeting the international diagnostic (Amsterdam I) criteria1 for the disorder.2–4 Among families not meeting these criteria, less than 30% show mutations.2,3 Importantly, even among families that meet the Amsterdam I criteria and show microsatellite instability (MSI) in tumor tissue, a third fails to display any structural changes in the MMR genes.5 This suggests either that other as yet unknown genes are involved or, alternatively, that the presently known HNPCC-associated MMR genes have alterations that escape detection by conventional techniques. Mutations leading to protein truncation constitute the majority of inactivating mutations for the cancer susceptibility genes BRCA1,6 BRCA2,7 APC,8 and the MMR genes MSH2 and MLH1.9 Transcripts containing premature termination codons as a result of frameshift or nonsense mutations are subject to nonsense-mediated RNA decay, the function of which is to eliminate the formation of truncated proteins with deleterious dominant-negative or gain-of-function effects.10 Because of the instability of the gene products, such mutations may remain undetected by RNA-based techniques, whereas a majority would be detected by DNA-based methods. Conversely, the latter techniques mostly rely on the amplification of individual exons by flanking intronic primers, and would miss large genomic deletions and changes in the more distant noncoding regions. Presumable alterations in the promoter areas or in other noncoding regions that might result in lost messenger RNA (mRNA) expression from one allele, without any structural coding changes, are among the most challenging types of mutations to diagnose. Evidence of the existence of such mutations may be revealed by techniques that allow the individual assessment of the paternal and maternal allele, such as monoallelic mutation analysis11,12 or the more recently developed Conversion technology.13 These methods are, however, relatively complicated and/or require special equipment; therefore, they are not yet part of routine diagnostic practice. In the present investigation, relatively simple expression-based methods, mRNA analysis by single nucleotide primer extension (SNuPE) and protein analysis by immunohistochemistry (IHC), combined with MSI analysis, were used to address the genetic etiology of a population-based cohort of HNPCC or HNPCC-like families that had screened MMR gene mutation negative. Our findings allowed us to stratify these families into two major categories according to the presence or absence of MLH1, MSH2, or MSH6 gene involvement.
Patients and Samples This study included 26 families from Finland (Table 1  ) that had screened negative for MSH2, MLH1, MSH6, PMS1, and PMS2 mutations by a combination of DNA and RNA-based techniques (denaturing gradient gel electrophoresis, direct exon-specific sequencing, reverse transcriptase polymerase chain reaction [RT-PCR], and protein truncation test).2,14–16 Additionally, Mutation 1, a common founder mutation in the Finnish population that consists of a 3.5-kb genomic deletion of exon 16 of MLH1, was excluded using a specific test.17 These kindreds formed a population-specific series comprising all available families with verified or putative HNPCC from the Hereditary Colon Cancer Registry of Finland that had originally been included in the mutation analyses based on clinical and family criteria and screened negative.2 We also studied four families from Sweden that had screened mutation-negative18 (Table 1). The Swedish families represented a series originally selected for mutation analyses based on the presence of MSI in tumor tissue, and the particular four were chosen for this study based on their known informativeness for the polymorphisms utilized in the SNuPE analysis. With the exception of two families, all fulfilled the Amsterdam 1,1 Amsterdam 2,19 or Bethesda criteria.20
To validate the SNuPE method, 10 individuals who were known carriers of seven different germline mutations were investigated. MLH1 mutations (consequences in parentheses) included the following: G > A at nucleotide 454 - 1, splice acceptor of exon 6 (frameshift); G > A at nucleotide 1,039 - 1, splice acceptor of exon 12 (frameshift); A > C at nucleotide 1,559 - 2, splice acceptor of exon 14 (frameshift); 3.5-kb genomic deletion of exon 16 (in frame deletion); delAAG at nucleotide 1,846 in exon 16 (in frame deletion); and R659P in exon 17 (missense). We also studied a MSH6 mutation consisting of delCT at nucleotide 3,052 in exon 4 (frameshift). Samples of blood or paraffin-derived tissues were used. All human specimens were obtained after informed consent according to the guidelines of the institutional review boards.
Polymorphisms Studied
SNuPE Primer extension analysis relies on the incorporation of a single ddNTP that is selected to allow the differential extension of a primer annealed next to the polymorphic site.22,23 The design of the present SNuPE assays is shown in Table 2  , along with primer sequences. Fragments of genomic DNA (gDNA) or cDNA encompassing the polymorphic site are PCR-amplified and used as templates for primer extensions. The extension reaction uses a fluorescently labeled extension primer and a combination of three dNTPs (eg, dGTP, dCTP, and dTTP) and a ddNTP (ddATP); the latter represents either one of the two alternative bases at the polymorphic site. Using the MLH1 exon 8 A/G polymorphism as an example, primer extension continues until the polymorphic site for alleles having A in this position (T in the template strand), given that ddATP is incorporated causing chain-termination. For alleles with G, dGTP is incorporated and strand elongation proceeds until the next A (located 8 bp downstream). Hence, two fragments of different length are produced according to the base present at the polymorphic site. As allele discrimination depends on constitutional heterozygosity for a polymorphism, individuals with identical bases in both alleles will not be informative in the analysis. SNuPE analyses were performed on RT-PCR products of RNA extracted from Epstein-Barr virus transformed lymphoblastoid cells, and the results were compared with amplification products obtained from gDNA samples of the same individuals. The products of the primer extension reaction were analyzed on an ABI377 sequencer with 9% denaturing polyacrylamide gels. The allelic ratios were quantified with the Genotyper 2.0 program (Applied Biosystems, Foster City, CA) and were calculated as follows, using an A/G polymorphism as an example: Allelic ratio = (peak area of allele A:peak area of allele G) in cDNA/(peak area of allele A:peak area of allele G) in gDNA
As a worked example for F73/1 in Figure 1
cDNA Cloning RNA extracted from lymphoblastoid cells was reverse-transcribed with oligo-dT and SuperScript II according to the manufacturer’s instructions (SuperScript First-Strand Synthesis System for RT-PCR; Invitrogen, Carlsbad, CA). Full-length cDNA of MLH1 (2.3 kb) and MSH2 (2.8 kb) was amplified with primers RT-Lf1 and RT-Lr1 (for MLH1), and RT-Sf1 and RT-Sr1 (for MSH2),25 using Expand Long Template PCR System (Roche, Basel, Switzerland). The products were cloned into pCRII-TOPO vector taking advantage of TOPO TA Cloning system (Invitrogen). The resulting plasmids were used to transform XL1-Blue (Stratagene, La Jolla, CA) cells, and recombinant clones were identified based on white color on Xgal-containing plates.
IHC
MSI Analysis
MLH1 Promoter Methylation Analysis
Haplotype and Genetic Linkage Analyses
MLH1 and MSH2 Promoter Mutation Analysis
Allelic mRNA Expression of MMR Genes by SNuPE To validate the SNuPE method, we first tested mutation-positive HNPCC families available to us.14,15,30 Among those, carriers of six different MLH1 mutations and one MSH6 mutation turned out informative (heterozygous) for the polymorphisms used (see Materials and Methods). Comparison of paired cDNA and gDNA samples indicated that all frameshift mutations studied were associated with decreased mRNA expression of one allele of MLH1 or MSH6. Moreover, the allele with reduced expression was always the one that was linked to the cancer phenotype when gDNA samples from several family members were genotyped relative to the same polymorphisms. Conversely, mutations that did not alter the reading frame (a missense change and two in-frame genomic deletions) resulted in balanced allelic expression. As proof of specificity, MMR genes not involved in genetic predisposition in a particular family (eg, MSH6 in families from our actual study series that turned out to be MLH1-linked; Table 3 ) invariably showed balanced expression. Based on our results, the threshold values for allelic ratios considered abnormal were set to be  0.5, corresponding to allelic ratios of 1:2, or  2 (2:1; see Materials and Methods).
Next, the SNuPE method was applied to 26 HNPCC or HNPCC-like families from Finland that originally screened negative for MMR gene mutations. Eleven families (42%) were informative for the MLH1 codon 219 A/G polymorphism, and two (F36 and F73) showed unbalanced mRNA expression (Tables 1
Among four mutation-negative families from Sweden, two (S4 and S346) showed unbalanced allelic expression of MLH1 mRNA. None was associated with unbalanced expression of MSH6, and MSH2 was uninformative (Tables 1 and 3, Figs 1 and 2).
MMR Protein Expression by IHC
MSI results correlated well with IHC findings: With the exception of F44 (a metastatic colon tumor), all tumors with abnormal protein expression showed MSI, whereas those with normal protein expression were microsatellite-stable (Table 1
Among the four Swedish families that had undergone preselection for MSI and heterozygosity for polymorphisms, three (75%) showed the loss of MLH1 protein, and one (25%) the loss of MSH2 and MSH6 proteins in tumor tissue (Table 1
Mutation Analyses Analysis of the remaining families was hampered by homozygosity for coding polymorphisms, making it impossible for us to verify whether both alleles were represented among the cDNA clones. Sequencing of randomly picked clones did not reveal alterations compatible with pathogenic mutations. All four families with MSH2 implicated by IHC (F70, F71, F81, and F88) showed a recurrent deletion of exon 4, whose frequency varied between 1/24 to 5/24 among MSH2 cDNA clones tested. This deletion was absent in a healthy control individual, and it is not included among alternative splice products reported for MSH2.34,35 However, sequencing of exon 4 (including exon/intron junctions) detected no point mutations in gDNA, and long-range genomic PCR from exon 3 to exon 5 failed to reveal any large rearrangements either. The significance of the cDNA deletion in MSH2 exon 4 for cancer susceptibility in our families, therefore, remains unknown. A different approach (quantitative multiplex PCR of short fluorescent fragments36) was applied to the Swedish families, and S4 showed an out-of-frame genomic deletion of exon 11 of MLH1 (our unpublished data), consistent with the present finding of unbalanced expression of MLH1 alleles in this family.
Clinical Characteristics Versus Etiology
A major problem in the diagnosis of human mutations is that alterations in one copy of a chromosome pair may be masked by the normal sequence present on the other copy. Both Conversion technology13 and the approach we used (SNuPE and sequencing of cloned full-length RT-PCR products) aim to circumvent this problem by analyzing the maternal and paternal alleles separately. In Conversion technology, the genomic copies of the alleles are physically separated by converting the human chromosome complement to a haploid state through fusion to a rodent recipient cell line. In our SNuPE experiments, coding polymorphisms were used as allele-specific markers to distinguish the genomic copies of the paternal and maternal alleles as well as their corresponding mRNA transcripts, and subsequent cloning of the full-length RT-PCR products allowed the physical separation of maternal and paternal cDNAs for sequencing. By the present expression-based approach, an MMR gene-linked basis of cancer predisposition was suggested in as many as 42% of families (11 of 26) that had screened MMR gene mutation-negative by standard mutation detection methods. Based on our test cases and a panel of mutation-positive controls, allelic expression analysis by SNuPE provides a rapid method to screen blood mRNA samples for the existence of mutations associated with premature termination codon. Among four families with unbalanced MLH1 mRNA expression, one (F73) showed a nonsense mutation (known to occur in some other HNPCC families as well),37 another (F36) showed complete silencing of one MLH1 allele for an unknown cause, and a third (S4) showed an out-of-frame genomic deletion. A disadvantage of the SNuPE method is that it requires heterozygosity for coding polymorphisms, which, at least in the present Scandinavian populations, limited the MSH2 analyses in particular. Furthermore, the ensuing lack of allele-specific markers reduced the efficiency of mutation detection by cDNA cloning. Among families uninformative by SNuPE, a heritable defect was supported by additional evidence, such as the presence of an MLH1- or MSH2-linked haplotype cosegregating with disease (F53, F71) and concordant IHC and/or MSI findings in two or more family members (F53, F71, F81, F88) (data not shown). Based on literature reports, large genomic rearrangements may occur in as many as 23% of HNPCC families without detectable mutations in MSH2 or MLH1,36,38 and occasional kindreds show promoter mutations.39 Except for one family (S4, with a genomic deletion), such changes were not identified in the present families. Conversions may improve the detection rates of some types of genomic rearrangements and splicing alterations,40 which, at least in principle, should be identifiable by other techniques, too, such as those we used (cDNA cloning and quantitative multiplex PCR of short fluorescent fragments). Conversions are particularly valuable in the detection of large deletions that involve the promoter region, as shown for certain MSH2 alterations.13 Such changes cannot be caught by our present cDNA cloning approach, since the region complementary to the forward primer would be deleted, resulting in the absence of any RT-PCR product from the mutated allele. Importantly, however, heterozygosity for promoter polymorphisms (C>T 118 bp upstream of exon 141 and T>G 433 bp upstream of exon 1 in this study) in our MSH2-linked cases made the possible existence of such changes unlikely. Even with the best available methods, structural changes are not identified in all cases with apparent hereditary defects.42 Some patients with familial adenomatous polyposis (FAP) show decreased constitutional mRNA expression from one allele of the APC gene, without any detectable sequence changes, suggesting that this expression reduction is associated with FAP predisposition. The mutations underlying the expression alterations in such cases may reside deep within introns43 or at a considerable distance upstream or downstream of the respective genes,44 which also remains a possibility in our mutation-negative HNPCC cases. While MSI and IHC analyses have turned out most valuable (as in this study and those of Thibodeau et al,45 Salahshor et al,46 and Wahlberg et al47) as prescreening methods for MMR gene alterations, these techniques—unlike SNuPE—are unable to distinguish between hereditary versus acquired defects. MLH1 promoter methylation should be considered as a plausible mechanism for the loss of MLH1 protein expression in tumors, especially in the absence of any significant family history.48 We addressed this possibility in our small families F44, F48, and F57, but no promoter hypermethylation was present (data not shown), thus failing to explain the observed MLH1 inactivation in these families. Finally, the fact that in all cases with MSI the "major" MMR genes MLH1, MSH2, and MSH6 were involved suggests that such families are mostly associated with "hidden" mutations in the existing genes; hence, the discovery of new major HNPCC predisposition genes from the MMR pathway is unlikely.
Besides implicating the MMR genes in the etiology of cancer predisposition in the families described, our results are equally important in providing cumulative evidence against MMR gene involvement in the remaining families (15 [58%] of 26). In a previous investigation, MSI status was used to stratify MMR gene mutation–negative families into two clinically distinct groups: those with presumable "hidden" MMR gene mutations and those truly MMR gene mutation–negative.49 In addition to the lack of MSI (and the absence of any identifiable germline mutations, as reported earlier), the mutation-negativity of our families was also supported by balanced allelic mRNA expression and normal MMR protein expression by IHC, combined with distinct clinical features (Table 4
The authors indicated no potential conflicts of interest.
We thank Saila Saarinen and Mariana Loto Peña for expert technical assistance; Jinmin Miao for cell cultures; Michael Stevens, Hidewaki Nakagawa, and Maria Curia for sharing experimental information; Kirsi Pylvänäinen, Tuula Lehtinen, and Henna Väistö for assistance with sample collection; and Tuomo Kukkola for computer support.
Supported by the Sigrid Juselius Foundation, the Academy of Finland, the Finnish Cancer Foundation, the Science Foundation of Helsinki University, the Paulo Foundation, the National Institutes of Health (grant CA82282), and the Swedish Cancer Society.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
), carriers of R100X mutation.
