AMIL Rev. GeMt. 1995. 29:329-48 Copyright @ 1995 by Annual Reviews Inc. All rights reserved

GENETICS OF HEREDITARY

COLON CANCER

Albert de la ChapeUe and Piiivi Peltomiiki Department of Medical Genetics, University of Helsinki, and Folkhalsan Institute of Genetics, 00290 Helsinki, Finland

KE YWO RDS: DNA mismatch repair genes, cancer susceptibility, microsatellite instability,

germline and somatic mutation. founder mutation

ABSTRACT

A new mechanism leading to cancer has been delineated in the last two years when genes whose mutations cause susceptibility to hereditary nonpolyposis colorectal cancer, HNPCC, have been mapped, cloned, and characterized. The genes involved belong to a family of DNA mismatch repair genes, and the homozygous effects of their mutations lead to a so-called mutator or replication error phenotype characterized by genome-wide mutations most readily detectable as lengthening or shortening of microsatellite repeats in tumor tissue as compared to normal tissue from the same individual. Germline mutations are inherited in a dominant Mendelian fashion causing the multiorgan cancer susceptibility syn­drome misnamed HNPCC. Clinically, the molecular characterization of these mutations in affected individuals now allows genotype-phenotype correlations, and a new view of the natural history of the disease may arise. In at risk individuals, it allows predictive testing for cancer susceptibility, enhanced clini­cal surveillance with the aim of early cancer detection and cure, and preventive measures.

CONTENTS

INTROD UCfION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 0

PHENO TYPE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 History and Designation. . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 0 Definition • • . • . . . . . . . . . . . . . . . . . • • • . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . 331 Clinical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

INC IDENCE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 2

HNPC C SUSCEP TIBILITY GENES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Human DNA Mismatch Repair Genes. . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . 333 Proportion of HNPCC Caused by the Different Mismatch Repair Genes. . . . . . . . . . 335 Types of Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 Founder Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 6 Other Genes in the CalLSation of HNPCC Susceptibility . . . . . . . . . . . . . . . . . . . . . . . 340

EFFECTS OF DNA MISMATCH REPA IR GENES ............................ 340 Bacteria and Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 Human . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Homozygosity verslLS Heterozygosity for Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

329

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330 de la CHAPELLE & PELTOMAKI

THE "MUTATOR" OR "REPLICATION ERROR" PHENOTYPE................ 342 Association with Different Types of Colorectal Cancer . . . . . . . . . . . . . . . . . . . . . . . . 342 Genetic Basis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Relation to HNPCC Tumor Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344

OUTLOOK. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345

INTRODUCTION

Colorectal cancer (CRC) is the second or third most common cancer in Western countries and the incidence is rising (13). It is rarer in the third world and e.g. in Japan. Up to 90% of aU CRCs are sporadic. Among the hereditary forms, Familial Adenomatous Polyposis is a rare dominantly inherited condition leading to cancer and caused by germline mutations of the APC gene. In this review we concentrate on hereditary CRCs that are not associated with multiple polyps or adenomas. We describe recent developments in the study of DNA mismatch repair genes whose mutations cause susceptibility to hereditary nonpolyposis colorectal cancer, HNPCC. An important question relates to the incidence of these mutations. In the light of the "mutator" phenotype of microsatellite instability, we discuss the putative role of these and other genes in the causation of "sporadic" tumors. Finally, we deal briefly with the impact that the genetic findings will have on our understanding of the natural history of this disorder, on early detection and prevention of cancer, and on predictive genetic testing.

PHENOTYPE

History and Designation

Colorectal cancer is one of the most common forms of cancer, with a cumu­lative lifetime incidence of between five and six percent (5); its occasional occurrence in two or more close relatives by chance is therefore relatively common. Familial clustering is often attributed to more than one weakly predisposing gene, "multifactorial inheritance." This translates into empirically derived figures for "heritability" and "risk estimates" for relatives of an af­fected individual. For colon cancer, empirical risk for first-degree relatives is of the order of 1.8- to 8.O-fold (17). These figures are useful in clinical practice and genetic counseling, but shed little light on the underlying genetic mecha­nisms.

In contrast to familial occurrence, the existence of hereditary forms of colorectal cancer has been suggested. The first description is usually ascribed to Warthin (77); however, the tumor spectrum of the largest family that he described was notably different from that seen in most HNPCC families reported later. Some 50 years later, studies by Lynch began to draw attention

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GENETICS OF HEREDITARY COLORECfAL CANCER 331

to and define an entity that is now designated Hereditary Nonpolyposis Col­orectal Cancer or HNPCC (38-42). This is a misnomer because tumors in organs other than the colorectum are a hallmark of the condition. Alternative terms used are Lynch syndrome-subdivided into Lynch I syndrome, where all tumors are colorectal, and Lynch II syndrome, where tumors in other organs

occur as well-and Cancer Family Syndrome.

Definition

Until now, the definition of HNPCC has been exclusively based on pedigree structure and age at onset. The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer suggested three obligate criteria known as the Amsterdam criteria (76): At least three relatives have histologically verified colo rectal cancer; one individual must be a first-degree relative to the other two; at least two successive generations are affected; and in at least one individual the diagnosis is made at <50 years of age. These criteria serve to eliminate most, but not all, cases of chance clustering. They clearly favor a "classical" form of the disease. For instance, the requirement of two successive generations excludes families with low penetrance and de novo germline mutations. Significantly, the definition ignores tumors other than colorectal and thus potentially excludes families in which tumors in other organs are frequent. Finally, the age requirement obviously excludes families in which penetrance occurs at higher age. Thus the Amsterdam criteria favor "classical" features and could ignore atypical, late-onset, multiorgan, or otherwise differ­ent forms.

Clinical Features

For comprehensive descriptions of the clinical features of HNPCC, readers are referred to previous reviews (9, 42, 46). The high susceptibility to cancer often leads to the occurrence of more than one tumor at the same time (synchronous tumors), and to repeated occurrences (metachronous tumors). Further charac­teristics of HNPCC are: younger age at onset (average 40--45 years) than in sporadic CRC (over 60 years); pronouncedly more often right-sided tumors (approximately two thirds) than in sporadic CRCs (approximately one third), with no explanation thus far of the difference in sidedness (9); a generally

unfavorable histology (poor differentiation) with a prognosis that is paradoxi­cally better than in sporadic CRe. Very few chromosome studies have been reported, although DNA flow cytometry shows that HNPCC tumors display less deviation from diploidy than do sporadic tumors. Thus neither loss nor addition of chromosomes or major parts thereof is common.

Tumors in HNPCC, as in sporadic CRC, develop via a precancerous growth called an adenoma. Sporadic CRC can be prevented by colonoscopic screening

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332 de la CHAPELLE & PELTOMAKI

and removal of adenomas (81); recently, the same has been shown for HNPCC (23).

Much attention has been paid to the spectrum of tumors that occur in HNPCC. We are not aware of any explanation of the fact that some organs are, and others clearly are not, susceptible to cancer in this condition. As shown in Table 1, the data from Finland and the United States are similar. Approxi­mately two thirds of the tumors seen in members of the HNPCC families under study were in the colorectum, with the rest in other organs. Among these, the endometrium, stomach, pancreas and bile ducts, and kidney and ureters are most clearly involved, whereas other tumors in, for example, breast, sarcoma, skin, and lung may or may not arise as a result of the inherited susceptibility. Clarification of this point is obviously of major interest and can only be done by determining who has and who does not have the predisposing mismatch repair gene mutation that occurs in each family. We note with interest the statement by Lynch and colleagues (38) that lung cancer is underrepresented in the families studied, which implies a protective effect of these gene muta­tions against lung cancer. Finally, in the large family frrst reported by Warthin (77), endometrial and stomach cancer predominated, whereas later generations of the same family display a predominance of CRC (37,75). If it can be proven that in this family the mutation is indeed the same in all generations, then the changed pattern of organ involvement is likely to be caused by nongenetic factors.

INCIDENCE

The incidence of HNPCC is actively debated but not known. Estimates vary widely; whether this reflects real differences or differences in methodology remains to be determined. The highest estimates could imply a frequency of I in 200 or higher (11, 21); if this were correct, HNPCC would be the commonest Mendelian disorder so far described. A more commonly quoted estimate, I in 2000, would still rank HNPCC among the commonest heritable disorders, in the same order as cystic fibrosis and the fragile X syndrome, for example. Other estimates are lower. such as that recently made by Bodmer et

al (6) who claimed that HNPCC is rarer than familial adenomatous polyposis, in which the incidence is of the order of I in 8000 to 1 in 15000.

The great variation in incidence estimates is due to the lack of objective

diagnostic criteria other than pedigree structure. Most attempts to determine the incidence of HNPCC begin by pedigree analysis to determine the propor­tion, in a given study population of colorectal cancer patients, of those who are HNPCC. Once this figure (e.g. 5%) is known. one can extrapolate the incidence of HNPCC from the cumulative lifetime risk of acquiring CRC (e.g.

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GENETICS OF HEREDITARY COLORECfAL CANCER 333

5%). Using the above figures as an example, an incidence of HNPCC of 1 in 400 can be deduced.

Early attempts to use this method in Finland resulted in the conclusion that HNPCC accounted for between 3.8 and 5.5 percent of all CRCs (45). This was a retrospective study limited to one small district in Finland with a population of 0.25 million. In a recent similar study comprising several geo­graphic regions with a total population of -1 million people, the corresponding figures were 0.7 to 2.2 percent (48). A third recent study conducted in Finland took advantage of computerized automatic searches of two sets of records (3) and identified a number of candidate HNPCC families whose pedigrees and cancer status were further refined by a variety of methods. As a result, 0.5-0.9% of all CRCs were calculated to be HNPCC (3).

In a geographical region in Italy with 0.26 million inhabitants, consecutive CRC patients were screened for a family history of cancer, with an estimated proportion of 3.4% HNPCC (64,65). The proportion of HNPCC among CRC patients aged <50 years in Alberta, Canada, was 3.1 % (80) and similarly, in Northern Ireland the proportion of HNPCC was 6% in patients under 55 years; these figures calculate the overall proportion at between 1 and 2.6 percent of CRCs (24). By analyzing segregation in a series of consecutive CRCs, Houlston et al (21) concluded that at least 13% of the total CRC burden fits the model of dominant inheritance. Other methods were used by Burt et al (10) and Cannon-Albright et al (11) to arrive at even higher estimates.

HNPCC SUSCEPTIBILITY GENES

Human DNA Mismatch Repair Genes

After the first HNPCC susceptibility locus was mapped and assigned to chro­mosome 2p by linkage analysis (62) and the replication error (RER) phenome­non associated with HNPCC ( I ), it became plausible that the gene was involved in DNA mismatch repair (71). Thus in attempting to clone the gene, positional cloning strategies were combined with the search for a candidate gene with the presumed function. A human homologue of the mutS gene in bacteria (51) and MSH2 gene in yeast (67) was cloned (16, 30) and named human MSH2.

Its role in causing cancer susceptibility in HNPCC was proven based on heritable germJine mutations that segregated with the disease in several HNPCC kindreds (30). The cloning of MSH2 was preceded by the genetic mapping of another locus for HNPCC in chromosome 3p (32); this gene, MLHI, was soon cloned based on its presumed homology with bacterial mulL

and yeast MLHI (8, 58) and likewise shown to cause HNPCC (58). In the search for MLH I, a repository of expressed sequence tags and sequenced

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Table 1 Tumor distribution in HNPCC kindreds from Finlanda and the United Statesb

Number of tumors (%)

Site of cancer Mecklin & Jarvinen" Watson & Lynchb

Colorectum 300 (63) 287 (64) Endometrium 40 (8)C 53 (12) Stomach 29 (6) 17 (4) Biliopancreatic 18 (4) 13 (3) Urinary tract 10 (2) 20 (4) Breast 8 (2) 19 (4) Sarcoma 8 (2) Skin 8 (2) 3 (004) Small bowel 5 (I) 10 (2) Lung 4 (I) 5 (I) Ovary 4 (I) 13 (3) Other/not specified 38 (8) 8 (2)

Total 472 (100) 448 (100)

'Reference 47 b Reference 78 'Tumor site in the uterus not specified in all cases.

cDNAs (4) was screened for sequences homologous to yeast MLH1; not only was human MLHl found, but two further mutL-like genes were also uncovered (58). These genes were later cloned and named PMSl (chromosome 2q) and PMS2 (chromosome 7p) and shown to harbor germline mutations causing

susceptibility to HNPCC (54). Table 2 summarizes relevant data on the four human DNA mismatch repair genes whose germline mutations have been implicated in HNPCC.

Table 2 Characteristics of four DNA mismatch repair genes implicated in HNPCC

Number of Gene Chromosome amino acids Number of References for symbol location in protein exons structure

MSH2 2p 934 16 16,27,30,34 MLHI 3p 756 19 8, 18, 33, 58 PMSI 2q 932 NK 54 PMS2 7p 862 NK S4

NK, Not known

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GENETICS OF HEREDITARY COLORECTAL CANCER 335

Proportion of HNPCC Caused by the Different Mismatch Repair Genes

In a comprehensive study of 29 mainly North American kindreds with HNPCC, 12 had evidence of involvement of MSH2 (34). This result was based in part on linkage analysis, but also on reverse transcriptase-polymerase chain reaction (RT-PCR) analysis followed by in vitro transcription and translation into protein, and, if no mutation was found by the above methods, by direct sequencing of the RT-PCR products. The observed figure of 12 kindreds out of 29 (41 %) involving MSH2 could be an underestimate, because mutations leading to very unstable transcripts might escape detection, and the promoter region was not analyzed. This led the authors to suggest that as many as 50% of all HNPCC families might be caused by MSH2 mutations. In a comparable

but less comprehensive study, Han et al (18) found 8 of 34 HNPCC patients

(24%) to have germline mutations of MLHI. Of the 34 patients, 28 were from Asia and 6 from the United States. A study of 13 kindreds of Caucasian ethnicity based on linkage analysis and mutation detection by sequencing indicated the following involvement of genes: MSH2, 6; MLH I, 4; neither, I ; inconclusive, 2 (55). These early data suggest, therefore, that MSH2 and MLH 1 together account for a majority, perhaps 80%, of all HNPCC . Of note, however.

is that the families studied in detail so far are likely to represent a sample biased in several ways. First, they are "classical" in that they fulfil the Am­

sterdam criteria. Second, the ones chosen for linkage studies are generally large. Third, most are Caucasians, probably predominantly Anglo-Saxon. Is there evidence of ethnic or geographical differences in this regard? There have

been no systematic studies thus far, but preliminary evidence from Finland

suggests a very low proportion of MSH2 involvement (1; M Nystrom-Lahti et aI, unpublished data). MLHI mutations are common in Finland due to founder

mutations (see below). These data are suggestive of, but do not yet prove, differences between populations. If confirmed. one implication would be that most HNPCC mutations are inherited rather than de novo, allowing different

mutations to predominate in different populations through founder effects

and/or genetic drift. The proportion of kindreds due to mutations in PMSI or PMS2 has not yet been determined conclusively.

Types of Mutations

Among the MSH2 gene germline mutations initially detected in HNPCC kin­dreds (3�}, there was a CCA to CTA change predicting a substitution of leucine

for proline in a highly conserved position. Other mutations included a point

mutation leading to a stop codon and a point mutation in a donor splice site

leading to the skipping of an exon (30). A small number of further mutations have been described so far (27.34.44); these support the notion that germline

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336 de la CHAPELLE & PEL TOMAKI

mutations in MSH2 are distributed over the entire coding region, not clearly clustered, and consist of both point mutations (transitions and transversions), and small deletions and insertions. Most are predicted to result in a truncated protein product.

For MLH 1 the same appears to be true. A graphic representation of mutations published so far is shown in Figure 1. As can be seen, mutations are distributed over the coding region and, possibly with the exception of the region around exon 16, do not show predilection spots. Among 14 different mutations, 5 are of missense type leading to single amino acid changes, whereas 6 are truncating and 3 frameshift mutations without reported stop codons.

In PMS1, a C to T transition converting a glutamine to a stop codon at position 233 has been identified as a germline mutation, whereas in PMS2

a genomic deletion appears to be a germJine change (54). Reports of systematic searches for mutations in PMSl and PMS2 have not yet been published.

Thus, in summary, mutations of diverse types and of seemingly random distribution have apparently been found in the germJine of HNPCC patients. Clearly, it is too early to exclude the possibility that the mutations described so far represent a biased set of HNPCC and that changes in a certain gene or in certain domains of a gene product, for example, give rise to clinical features that do not meet the Amsterdam criteria, and that have therefore not yet been systematically studied. An intriguing possibility is that heterozy­gosity for certain mutations might have phenotypic consequences (see be­low).

Founder Mutations

Founder mutations have arisen once and subsequently been passed on through successive generations to present-day carriers and patients. Some are so wide­spread that they account for major proportions of today's disease-bearing chromosomes, such as the delta F508 mutation of the eFTR gene giving rise to cystic fibrosis (53). Somewhat surprisingly, three founder mutations have already been detected in HNPCC. As shown in Table 3, each of the three mutations is expected to lead to truncation of the protein through the loss of one exon. On the genomic level, two are point mutations at splice sites, while one is a large genomic deletion with breakpoints in the introns flanking the missing exon.

In the exon 16 mutation of MLHl in Finland, genealogical analyses have

..

Figure 1 Spectrum of germline mutations of MLH1. The 1gexons are shown in the middle; above

are germline DNA changes, below are predicted protein changes. Cases F2 to RA are from Reference 58, case 2 is from Reference 8, case I is from Reference 34, cases SNUH-H7 to US-6 are from Reference 18, and case F36 is from unpublished data by Nystrom-Lahti et aI.

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338 de la CHAPELLE & PELTOMA.KI

100 km 1-----1

Polar circle

•

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.. e!.. Jyvaskyla· ...

Figure 2 Ancestral geographic origin of Finnish kindreds with founding mutations in MLH 1. Five

families share a mutation in exon 6 (circles with diagonal stripes), and 14 kindreds share a mutation in exon 16 (shaded circles).

established that the mutation is indeed ancestral, as most families carrying it come from the same geographical region, and five families share a common ancestor, born in 1505 (56). Moreover, other ostensibly unrelated families

carrying the same mutation share a large haplotype of alleles around MLHI.

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GENETICS OF HEREDITARY COLORECfAL CANCER 339

Table 3 Founder mutations in HNPCC

cDNA Number of Gene Mutation change families Population References

MSH2 Point mutation at Exon 5 3 Anglo-Saxon 30 splice donor deleted

MLHI Genomic deletion Exon 16 14 Finnish 56, 58, M Nystrom-deleted Lahti et ai, un-

published data MLHI Point mutation at Exon 6 5 Finnish M Nystrom-Lahti et

splice acceptor deleted ai, unpublished data

Indeed, for (CA)n marker D3S1611, which is located in an intron of MLH1,

all affected families share an allele hitherto not seen in Finns unaffected with HNPCC (56); M Nystrom-Lahti et ai, unpublished observations). It is pos­sible that the mutation is even older than 500 years. Its origins, if outside of Finland, should be traceable by screening HNPCC populations in different locations worldwide. The exon 6 mutation of MLHl has so far been found in five ostensibly unrelated families; notably, their geographical origins are confined to a small region in southern Finland and perhaps suggest a more recent origin (Figure 2). The MSH2 founder mutation that results in the skipping of exon 5 has been observed in three families in North America, all of whom report Anglo-Saxon heritage (30). The largest family has its roots in Newfoundland, whereas the other two live in different parts of the United States. As no polymorphic intragenic or immediately adjacent marker has been available for MSH2 haplotype studies, the likelihood that all these families descend from a common founding ancestor, perhaps in Britain, remains to be determined.

The existence of putative founder mutations raises several issues. How can a mutation whose phenotypic effect is an almost 100% susceptibility to cancer survive in the population for many generations? The average age at diagnosis of first cancer in HNPCC is 40--45 years (41, 49) so the average effect on reproduction should perhaps not be significant even though many patients acquire cancer (and die of it) in their 20s and 30s. Could it be that the age of onset has changed with time and that it was higher previously? Or might heterozygosity for a mismatch repair mutation, perhaps of a specific type, confer a selective advantage or have done so previously? These questions await answers through the meticulous study of the inci­dence, epidemiology, and phenotypic consequences of these particular mutations.

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Other Genes in the Causation of HNPCC Susceptibility

If MSH2 and MLH 1 together account for some 80% of all classical HNPCC, and if the proportion ascribed to PMSI and PMS2 is quite small, then as yet undiscovered genes could account for a minority of cases. As the proportion of HNPCC tumors that show microsatellite instability (the RER phenotype) approaches 100% (2), such genes would be expected to affect DNA stability. As the mutator gene family comprises several members in Escherichia coli (51), further homologous human genes could exist and might cause HNPCC. Both the mutS and mutL families could comprise more human homologues than those described to date.

Finally, mutations or variants in the proofreading domain of DNA polym­erase 0 have recently been found at the DNA level in RER-positive tumors. In at least one case the mutation was of germline distribution and altered an amino acid at a highly conserved codon (12). Of note, these tumors appeared to have a somewhat lower than average proportion of microsatellites showing instability. These results are preliminary but provocative. If it can be shown that mutations of a polymerase causes cancer susceptibility and a mild RER phenotype, other types of genes affecting DNA replication, DNA repair, or RNA-processing, for example, could be implicated in these types of cancer.

EFFECTS OF DNA MISMATCH REPAIR GENES

In studies that followed the mapping of the first HNPCC susceptibility gene, instability at multiple random microsatellite sequences throughout the genome was found to characterize tumors from HNPCC patients (1). A similar pheno­type had previously been observed in bacterial and yeast strains with DNA mismatch repair gene mutations (31, 71). The work by Strand et al (71) demonstrated that mutations in any of three genes, PMSl, MLHI or MSH2,

resulted in 100- to 700-fold increases in tract instability, whereas mutations affecting the proofreading function of DNA polymerases had little effect. These data together provided a functional clue that ultimately led to the identification of the four human homologues of mutS and mulL genes that are presently known to be associated with HNPCC, as described above.

Bacteria and Yeast

The DNA mismatch repair system in bacteria and yeast is well characterized (51, 52). In E. coli, mutS and mutL proteins participate in two main repair pathways, the methyl-directed long-patch and the very short patch (YSP) pathway. The methyl-directed pathway functions by correcting base-base mispairs, small insertions, and deletions resulting from errors in DNA replication. The specific function of the VSP pathway is to correct G-T mispairs in nonreplicating DNA that arise as a consequence of deamination of 5-methylcytosine residues. The

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GENETICS OF HEREDITARY COLORECfAL CANCER 341

mechanism of methyl-directed mismatch repair is complex and in E. coli depends on ten activities. Repair is initiated by binding of mutS to the mismatch, followed by the addition of mutL. This complex activates mutH, an endonuclease, which makes a nick at an unmethylated GATC site located 1-2 kb away on either side of the mismatch. Subsequently, the portion containing the mismatch is excised by a 3'-5' or 5'-3' exonuclease (depending on the location of the nick), and a new tract is synthesized by a DNA polymerase to replace the excised segment. In yeast, after recognition of the mismatch by MSH2, a heterodimer is formed by MLHl and PMS1, followed by a ternary complex formation by MLH1, PMS1, and MSH2 (66). This complex then recruits additional proteins that carry out the actual repair as in the bacterial system.

Human Less is known about the mechanisms of DNA mismatch repair in human cells than in bacteria and yeast. Biochemical analyses have demonstrated that the human MSH2 protein binds to DNA containing mismatched nucleotides, provid­ing a target for the repair process (15,57). In analogy to bacteria and yeast, repair is strand-specific and is directed by a nick located 5' or 3' to the mismatch (60, 74). Human colorectal and endometrial cell lines displaying microsatellite instability have revealed a profound defect in DNA mismatch repair in biochemi­cal assays (60, 73, 74). H6 cells with no wild-type MLHl gene product were shown to be hypermutable and deficient in repair of both base-base mismatches and small displaced loops (60). Unlike E. coli, whose methyl-directed mismatch repair is unable to correct DNA heteroduplexes with more than four unpaired bases (59), human cells may be capable of repairing loops of up to 14 nucIeotides (14). This is important since human DNA contains numerous microsatellites with many consecutive repeats and some with rather long repeat units, which may generate large loops as a consequence of strand slippage during replication. Recent evidence suggests that while mutations in human MSH2 and MLH 1 result in comparable deficiencies in single base mismatch repair, MSH2 may play a more important role than MLH 1 in loop repair (73).

Homozygosity versus Heterozygosity for Mutation While tumor cells with a homozygous MLHl mutation were highly deficient in DNA mismatch repair, as reported by Parsons et al (60), lymphoblasts from an HNPCC patient (presumed to be heterozygous for a mismatch repair gene mutation) appeared repair proficient. This implies a classical tumor-suppressor mechanism in that two hits are required to cause a phenotypic effect (26). A two-hit mechanism was further supported by the demonstration of homozygous mutations in DNA mismatch repair genes in colorectal tumors from both HNPCC and sporadic cases (33). Furthermore, Hemminki et al (20) showed that loss of heterozygosity at MLH 1 or adjacent loci is a nonrandom event in tumors from MLH1-linked kindreds and that the loss consistently affects the

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wild-type allele, which is in agreement with a tumor-suppressor function of MLH1. Unexpectedly, a subset of HNPCC patients have recently been found to show hypermutability associated with a profound defect in mismatch repair not only in their tumors but also in their nonneoplastic cells (61). Two of these patients had a germline mutation in PMS2 and one in MLH 1. It was hypothe­sized that certain PMS2 and MLH1 mutations may have a dominant negative effect resulting from inability of the mutated proteins to interact with other proteins involved in DNA mismatch repair (61). Surprisingly, these patients had relatively few tumors even though their mismatch repair capacities were greatly impaired. This suggests that mismatch repair deficiency is compatible with normal human development (61).

THE "MUTATOR" OR "REPLICA nON ERROR"

PHENOTYPE

Association with Different Types of Colorectal Cancer

Instability at short tandem repeat sequences (microsatellites) reflects m alfunc­tion in the replication or repair of DNA. For this reason, it is also referred to as the replication error (RER) phenomenon (I). The abnormality appears as extra alleles that typically are observed in tumor DNA when compared to normal DNA from the same individual, indicating that a gain or loss of short repeat units [e.g. CA dinucleotides in a (CA)n repeat] has occurred (Figure 3). Some 100,000 microsatellite repeats are scattered throughout the human genome (79). About 90% of colorectal cancers from HNPCC patients show microsatellite instability and a majority of microsatellite loci are apparently involved (1, 2). Thus, in HNPCC tumors, the total number of mutations at microsatellite loci alone could be as high as 100,000 per cell. The underlying defect (mutation in a DNA mismatch repair gene) can lead to a cascade of secondary mutations in different oncogenes and tumor-suppressor genes and, hence, contribute to tumor develop­ment in HNPCC patients (29). Therefore, even though instability at microsatel­lite sequences may not primarily affect the phenotype of the cell, it serves as a useful marker of a mutator phenotype (35).

Approximately 15% of apparently sporadic colorectal carcinomas exhibit microsatellite instability (1, 22, 36, 72). Interestingly, RER+ colorectal tumors, whether representative of HNPCC or sporadic, are characterized by unique clinical and pathological features, including proximal tumor location, diploid DNA content, and a more favorable prognosis as compared to RER- tumors (1, 22, 28, 36, 41, 72). Histopathologically, RER+ tumors are often poorly differentiated, mucinous, and show a heavy lymphoid infiltration (25, 41). There is an apparent discrepancy between the more favorable prognosis and the aggressive histological features. It has been argued that a high mutation burden caused by DNA replication or repair deficiency may be detrimental to

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GENETICS OF HEREDITARY COLORECfAL CANCER 343

1 2 3

NTNTNT

DSS404

D17S787

Figure 3 Microsatellite analysis of normal (N) and tumor (T) samples from three patients (1-3). DNA samples were amplified by PeR and the products separated by electrophoresis in

polyacrylamide gels. Microsatellite repeat patterns at D5S404 and D17S787 are shown. Amplification products of altered length indicating microsatellite instability are marked with arrowheads.

the tumor cells (69). Alternatively, mutations involving proteins that are ex­

pressed on the cell surface (such as the HLA system) may provoke a strong immune response against tumor cells (6), and there is a correlation between the presence of microsatellite instability and the absence ofbeta-2-microglobu­

lin expression in colorectal carcinoma cell lines (7).

Genetic Basis

Although sporadic RER+ tumors and verified HNPCC tumors have many clinicopathological features in common, these two groups are not identical. For example, among RER+ tumors, right-sidedness is even more prominent in sporadic than in HNPCC tumors, which may suggest site-specific differences

in the pathogenetic mechanisms (2). The genetic background of microsatellite instability may not be the same in HNPCC and sporadic cases. In HNPCC, microsatellite instability is believed to be due to a germline mutation plus a

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Table 4 Genetic causes of microsatellite instability in colorectal cancer

Type of colo­rectal cancer

HNPCC

Sporadic (subset) Early age of onset Distal tumor location

Other sporadic

% of all col- Defective gene (mutation) orectal cancers

2-5

5

5-10

DNA mismatch repair gene (germ­line + somatic)

DNA mismatch repair gene (germ­line + somatic, or 2 somatic)

Unknown: DNA polymerase 0 in some

somatic event inactivating the second allele of the mismatch repair gene involved. Unselected RER+ tumors may comprise unverified cases ofHNPCC, but alternatively, they might have de novo germline mutations of known

HNPCC genes, or be true sporadic cases with two somatic mutations in DNA mismatch repair or other genes. Liu et al (33) found that only one in ten patients with RER+ sporadic colorectal tumor had a detectable germline mutation in a mismatch repair gene, suggesting that hereditary cases may not be very com­mon in this group. Moreover, only three of seven sporadic tumor cell lines with microsatellite instability had mutations in MSH2, MLH1, PMS1, or PMS2, implying that a significant fraction of sporadic RER+ cancers arise from mu­tations in other genes (33). A proofreading defect resulting from a mutation in the 3'-5' exonuclease domain of DNA polymerase B may cause microsatel­lite instability in some tumors (12). Table 4 summarizes probable causes of genetic instability in different types of colon cancer.

Relation to HNPCC Tumor Spectrum

Apart from colorectal cancer, microsatellite instability occurs in other sporadic and hereditary cancers, especially those that are part of the HNPCC tumor spectrum (2, 19,63, 68). The RER abnormality was detected in all cases of extracolonic cancer from HNPCC patients studied by Aaltonen et al (2), and cancer of the endometrium, kidney, stomach, duodenum, and ovary were represented. Risinger et al (68) found that the proportion ofRER+ endometrial tumors in HNPCC vs sporadic cases is quite similar to that described above for colorectal cancer. However, microsatellite instability is not restricted to cancers belonging to the HNPCC tumor spectrum but occurs with variable frequencies also in cancers not occurring in excess in HNPCC, such as lung cancer (50, 70). In the latter cases, the pattern of microsatellite instability may be different (43, 82). The genetic basis for the tissue distribution of microsatel­lite instability or HNPCC tumor spectrum is not known. Northern blot and

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GENETICS OF HEREDITARY COLORECfAL CANCER 345

reverse transcriptase-PCR analyses of different human tissues have shown that DNA mismatch repair genes are widely expressed, in keeping with their presumptive housekeeping function (30, 58). Refined expression and mutation analyses both of genes involved in DNA replication and repair, and of other genes known to be important in the development of particular tumors, are necessary before the underlying mechanisms can be understood.

OUTLOOK

Two years after the frrst HNPCC susceptibility gene was mapped and just over a year since defective DNA mismatch repair was associated with cancer susceptibility it is too early to assess the full impact of these discoveries. Obviously, the emergence of a totally new mechanism leading to cancer has prompted many basic researchers to begin to test a variety of mechanisms by which mismatch repair and its abnormalities affect previously known pathways to cancer. Such studies are only now beginning to come to fruition. The mutator or replication error phenotype is seen in many tumors, both with and without gerrnline mutations of mismatch repair genes, and the underlying mechanisms are yet to be explored. As a result the tumor spectrum of HNPCC will finally be both fully defined and fully explained. Clinically, HNPCC represents the frrst common cancer that provides an opportunity to offer predictive testing; such testing and counseling has already started. A Pandora's box of ethical, psychological, social, and legal issues has thus been opened. Large groups of patients, counselees, and health care personnel are facing these issues. There is considerable optimism that in HNPCC and related cancers, early detection and/or prevention will now be readily available, yet critical scientific data steering our procedures may take years to assemble.

Any Annual Review chapter, as well as any article cited in an Annual Review chapter, may be purchased from the Annual Reviews Preprints and Reprints service.

1-800-347-8007; 415-259-5017; email: arpr@class.org

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