The Hereditary Nonpolyposis Colorectal Cancer Syndrome: Genetics and Clinical Implications

In the United States, an individual's lifetime risk for developing some form of cancer is estimated to be as high as 40% (1). Cancer is fundamentally a genetic disorder, meaning that mutations in specific genes confer a selective growth advantage for tumor cells. Such mutations may develop sporadically or may be inherited through the germline, which results in a hereditary predisposition to multiple cases of early-onset cancer. Identification of these cancer-susceptibility genes has revealed the cellular processes that ordinarily guard against tumor formation. One particularly instructive example is the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. Defining the genetic basis for HNPCC has demonstrated a fascinating connection between the cellular machinery that regulates the replication and repair of DNA and cancer formation. Replication is the process by which DNA is copied in preparation for cell division. Errors of replication are inevitable, but specialized repair systems have evolved to prevent the accumulation of harmful mutations in the genome. The DNA mismatch repair system functions as a critical spell checker that identifies and then corrects mismatched base pairs of DNA. A germline mutation in a DNA mismatch repair gene results in the HNPCC syndrome, which is inherited as an autosomal dominant disorder. The 1990 Amsterdam criteria strictly define HNPCC as occurring when colon cancer is diagnosed in at least three family members, one a first-degree relative of the other two. The cases must span two generations, and one case must be diagnosed before 50 years of age (2). Less stringent criteria, including the Amsterdam II, modified Amsterdam, and Bethesda criteria, have also been developed. This review first highlights the key features of normal DNA replication and the mechanisms that ensure the fidelity of this process. The next section focuses on the genes involved in DNA mismatch repair and illustrates the biological consequences that result when mutations arise in these genes. Finally, we discuss the clinical implications of these genetic insights in the management of HNPCC families as well as persons with sporadic colon cancer. DNA Replication Replication of DNA takes place in preparation for mitosis. Consequently, each daughter cell carries an exact replica of the entire parental genome. Many nuclear proteins work in concert to execute the three phases of initiation, elongation, and termination. These mechanisms of DNA replication have been defined primarily from studies in bacteria and yeast, but the fundamental processes are relevant to higher eukaryotic cells. Replication is always initiated at specific points of origin in a chromosome when a cell enters the S (or DNA Synthesis) phase of the cell cycle (3). The helicase enzyme separates the double strands of DNA at these replication origin points. This unwinding of DNA results in an open loop structure. Each of the two parental strands can then serve as a template for the synthesis of a new complementary strand. Because each daughter chromosome contains one parental strand and one newly synthesized strand, the replication process is termed semi-conservative. The DNA polymerase enzyme carries out new DNA synthesis, but it cannot begin until a primer is available. Most commonly, a specific RNA polymerase called primase synthesizes a short RNA primer at the replication origin. Two replication forks are created as DNA is synthesized in a bi-directional manner away from the point of origin (4). A multicomponent complex containing DNA polymerase carries out the elongation process that adds each complementary nucleotide to the primer. Replication always proceeds in a 5 to 3 direction. This results in continuous DNA synthesis along one strand (the leading strand), but it is necessarily discontinuous along the other template strand (the lagging strand) (5). The lagging strand is thus composed of many Okazaki fragments that are initiated from discontinuous primers (Figure 1), and DNA ligase joins each of these fragments to form one continuous strand. Figure 1. Events at the replication fork. Replication is terminated when the entire length of a chromosome has been synthesized, resulting in two complete copies of each chromosome. In circular bacterial chromosomes, termination occurs simply when the origin of replication has been reached. The mechanisms that control termination of linear eukaryotic chromosomes are far more complex. DNA Damage: Errors of DNA Replication Maintaining the integrity of DNA is essential for normal cellular function. Chemical carcinogens or ultraviolet radiation can directly induce structural alterations, such as thymidine dimers, single- or double-stranded breaks, and covalent cross-linking. The DNA excision repair system recognizes and excises such structurally altered nucleotides; specific details are available in a recent excellent review (6). The current review focuses instead on a different type of error that occurs during replication. Errors of DNA replication are observed primarily during the elongation phase. The most common error is a simple mispairing of nucleotides. Patterns of hydrogen bonding govern the pairings of nucleotides. An adenine (A) is always paired with a thymidine (T), and a cytosine (C) is always paired with a guanine (G). The DNA polymerase may incorrectly pair an adenine with a guanine, for example, and such mispairings are predicted to occur once every 103 to 104 base pairs. Repair mechanisms keep the actual error rate much lower. In bacteria, de novo mutations occur only once every 1010 base pairs (7). A second type of error can occur during DNA replication. There can be slippage of the DNA polymerase complex, a process analogous to a loose bicycle chain transiently slipping but then reengaging. This slippage occurs during the replication of microsatellite DNA sequences, which are defined as short dinucleotide (5-TCAATGCCACACACACACACACCTGAGGC-) or mononucleotide (5-TCTAGGCTAAAAAAAAATGCCGAGT-) repeats. The daughter strands may then contain either too many or too few copies of these repeated sequences. With intact mismatch repair mechanisms, these errors of slippage are quickly corrected, and microsatellite DNA sequences are considered stable. However, in the presence of deficient mismatch repair function, these errors are not corrected. This phenomenon is called microsatellite instability (MSI) (Figure 2). Figure 2. Slippage during DNA replication. MSS MSI The DNA polymerase complex itself provides the first line of defense against errors of replication. Through poorly defined mechanisms, DNA polymerase can immediately recognize a mismatched pair of nucleotides. An intrinsic enzyme subunit with 3 to 5 exonuclease activity excises the mispaired nucleotide from the newly synthesized strand, and the excised sequences are replaced with the correct nucleotides. The DNA Mismatch Repair System For errors that are not immediately corrected by DNA polymerase, the DNA mismatch repair system provides a secondary system of proofreading. This system corrects not only single base-pair mismatches but also small mispaired loops of DNA that result from replication errors of microsatellite tracts. Studies in yeast have demonstrated that defective mismatch repair specifically leads to a 100- to 700-fold increase in the instability of these microsatellite tracts (8). The DNA mismatch repair system requires the cooperation of many genes from the mutS (hMSH2, hMSH3, hMSH6) and mutL (hMLH1, hMLH3, hPMS1, and hPMS2) families. Briefly, hMSH2 serves as the scout that recognizes and binds directly to the mismatched DNA sequence (9, 10). It forms a heterodimeric complex with hMSH6 if a single base-pair mismatch is recognized or with hMSH3 if there is a larger two- to eight-nucleotide insertion or deletion (Figure 3). A second heterodimeric complex of hMLH1 and hPMS2 is then recruited to excise the mismatched nucleotides. Although heterodimers of hMLH1/hPMS1 and hMLH1/hMLH3 also form, their specific roles remain to be defined. Figure 3. Components of the DNA mismatch repair system. Mutations in DNA Mismatch Repair Genes A mutation in one of several mismatch repair genes results in deficient DNA mismatch repair activity. The extent of the mismatch repair deficiency depends on the specific gene that is altered. If hMSH2 or hMLH1 is inactivated, then a high level of MSI is typically observed, the so-called MSI-H phenotype (11). Mutations in genes such as hMSH6 result in only a partial deficiency of mismatch repair and low levels of MSI (the MSI-L phenotype) (12, 13). The U.S. National Cancer Institute defines the MSI-H phenotype as present when two of five microsatellite markers from a standard panel display instability and the MSI-L phenotype as present when one of the five markers is unstable. Mutations in mismatch repair genes may occur in germline or somatic DNA. Of interest, an inherited germline alteration does not result in widespread developmental anomalies. One possible explanation for this is that the second wild-type allele may provide sufficient DNA mismatch repair function. The observed phenotype is the predisposition to early-onset tumors, primarily of the colon and endometrium. The biological basis for this organ specificity is unknown. In each tumor that forms, the second copy of the affected DNA mismatch repair gene has been somatically mutated, thereby leading to bi-allelic gene inactivation (14, 15). The direct consequence of defective DNA mismatch repair is the so-called mutator or replication-error phenotype. Rather than directly causing malignant transformation, DNA mismatch repair deficiency creates the milieu that permits mutations to accumulate in other growth-regulatory genes. Colon tumors that develop in persons with germline mutations in hMSH2 or hMLH1 characteristically display the MSI-H phenotype (16, 17). Most microsatellite sequences in the genome are located within noncoding, or intr