APC gene testing

SIGNIFICANCE:APC gene testing is used to identify individuals at extreme risk for colon cancer caused by a germline mutation in the adenomatous polyposis coli gene. Introduced in 1994, APC gene testing was the first service commercially developed for presymptomatic diagnosis of an adult-onset disease; it illustrates well the utility and limitations of such testing.

APC Structure and Function

First identified (cloned) in 1991, the APC gene is located on the long arm of human chromosome 5 (megabase position 112.1). The gene encompasses 91 kilobases of DNA, making it a relatively large target for mutations. Its fifteen exons produce an transcript of 10.4 kilobases that encodes a protein of 2,843 amino acids (309 kilodaltons). This huge protein, consists of binding sites for a variety of proteins including APC itself, axin, beta-catenin, conductin, EB1, tubulin, and protein kinases.

APC functions in a variety of tissues as part of a large protein complex that targets beta-catenin for degradation. In the absence of this degradation, beta-catenin translocates to the nucleus to promote cell division. APC also interacts with proteins necessary for chromosome segregation, cell adhesion, and cytoskeletal structure; the absence of these interactions could potentially promote tumor growth as well.

APC Mutations

IAccording to the American Society of Clinical Oncology (ASCO), about 6 percent of colorectal cancer cases were believed to be caused by germline APC mutations; the remainder are somatic mutations present only in the tumor. A mutation in the germline, because it is present in all cells, leads uniquely to hundreds or thousands of colon polyps. This occurs because individuals with only one functional copy of APC are highly susceptible to losing all normal APC activity in a cell by chance mutation of the second copy, especially in tissues with a high turnover rate such as the colorectal epithelium—loss of APC function in a single cell is sufficient to initiate polyp development. Consequently, inheriting a nonfunctional or dysfunctional APC allele results inevitably in polyps. According to the ASCO, approximately 30 percent of familial adenomatous polyposis (FAP) cases are not inherited; they are caused by a new (de novo) germline mutation of the APC gene.

More than 1,500 APC mutations causing FAP had been identified in the international Human Gene Mutation Database, with the majority producing a truncated protein. Forty-six percent are small deletions, 28 percent are nonsense mutations, 13 percent are large deletions or duplications, 10 percent are small insertions, and about 3 percent are missense mutations, as reported by Madhuri Hedge et al. Two of the most common germline mutations occur at codons 1061 and 1309, accounting for approximately 30 percent of all mutations, as reported by Hedge et al. All these mutations lead to polyposis, and several additional somatic mutations of other genes have to occur for the polyps to become cancerous. When there are many polyps, however, the lifetime risk of malignancy is 100 percent, with cancer typically occurring between thirty-four and forty-three years of age, as reported by Kory W. Jasperson and Randall W. Burt.

Mutation Testing

Individuals with a high familial risk for FAP may undergo APC gene testing by ten years of age, as Jasperson and Burt noted. In cases where the APC mutation of relatives has not already been identified, using the RNA-based protein truncation test, or in vitro synthesized protein assay, for APC testing has a 70 to 90 percent chance of discovering a causal mutation, while DNA sequencing can detect 87 percent of point and truncating mutations, according to Hedge et al. The location of the mutation also provides important diagnostic information: Classic FAP is typically associated with mutations between codons (amino acid positions) 1309, 168, and 1580, whereas mutations outside this region are often associated with attenuated FAP (AFAP; fewer than one hundred polyps and older age of onset). A study conducted in 1992 found that severe polyposis is more likely when mutations occur between codons 1250 and 1464, and a number of mutations tend to be associated with additional pathologies outside the colon and rectum.

A variety of methods are used for APC mutation testing, usually conducted on DNA from a peripheral blood sample. The most thorough method, used in high-throughput laboratories, is to sequence the entire coding region and splice junctions. Another method used by low- to medium-throughput laboratories is denaturing high-performance liquid (DHPLC). This method uses DNA fragments generated by the polymerase chain reaction (PCR) and hybridizes them to complementary fragments with no mutations. Sequence mismatches alter the migration rate of the hybrid DNA through an HPLC column. A limitation of this method is that large deletions or rearrangements may prevent PCR amplification. A third procedure is real-time reverse transcription polymerase chain reaction (RT-PCR), which measures the relative RNA copy number of each exon, with a reduced copy number indicating a deletion or truncation. A similar approach is the RNA-based protein truncation test (also called an in vitro synthesized-protein assay).

Benefits and Caveats

Colorectal cancer is highly preventable when polyps are detected and removed as soon as possible. In the case of FAP, treatment ordinarily means removing the entire colon and perhaps rectum. APC gene testing helps physicians decide how soon and frequently patients should be monitored for polyps and how aggressively to intervene surgically. Testing also provides the important benefit of identifying family members who are not carrying the APC mutation.

Like all genetic tests, APC testing has limitations. One is that a negative result does not rule out a mutation in families where the mutation has not previously been identified. A second is that even with surgery and subsequent monitoring, patients still have an above-normal risk of cancer. Testing for an APC mutation can also cause anxiety and depression regardless of test outcome: those testing positive because of the emotional burden and those testing negative because of possible survivor’s guilt.

Impact

In the United States, colorectal cancer is the leading cause of cancer among nonsmokers; according to the American Cancer Society, the death toll from colorectal cancer in the United States was estimated at 53,010 in 2024. APC was the first colorectal cancer gene identified, and APC testing has contributed to a better understanding of colorectal cancer and saved many lives.

Key Terms

• adenomabenign tumor originating from secretory (glandular) epithelial cells
• adenomatous polyposis coli (APC)a familial adenomatous polyposis caused by mutations in the APC gene; APC can also refer to the protein encoded by the APC gene; when italicized, APC refers to the APC gene
• APC genea tumor-suppressor gene; mutations of this gene cause several closely related colorectal polyposis syndromes including FAP, Gardner syndrome, and Turcot syndrome
• autosomal dominantmutation on a nonsex chromosome in which only one mutant allele is sufficient to produce the phenotype (disease)
• familial adenomatous polyposis (FAP)a precancerous, genetically inherited polyposis of the epithelial lining of the large intestine (colon) or rectum; subclassified as classic, attenuated, or autosomal recessive, with classic and attenuated FAP caused by mutations of the APC gene and autosomal recessive FAP caused by mutations in the DNA repair gene MUTYH
• missense mutationa single base substitution resulting in a codon specifying an incorrect amino acid
• nonsense mutationa single base substitution producing a premature stop codon
• polypabnormal growth or tumor projecting from a mucous membrane
• polyposisthe presence of many adenoma polyps, generally more than one hundred
• tumor-suppressor genea gene in which functional loss of both alleles in a somatic cell causes tumorogenesis

Bibliography

"APC Gene." Medline Plus, 1 Mar. 2013, medlineplus.gov/genetics/gene/apc/. Accessed 6 Sept. 2024.

Chapman, P. D., and J. Burn. “Colorectal Cancer.” Molecular Genetics of Cancer. Ed. J. K. Cowell. 2nd ed. Oxford: BIOS Scientific, 2001. Print.

"Colorectal Cancer Risk Factors." American Cancer Society, 29 Jan. 2024, www.cancer.org/cancer/types/colon-rectal-cancer/causes-risks-prevention/risk-factors.html. Accessed 6 Sept. 2024.

Harrington, Susan M., and Malcolm G. Dunlop. “Familial Colon Cancer Syndromes and Their Genetics.” Genetic Predisposition to Cancer. Ed. R. A. Eeles, D. F. Easton, B. A. J. Ponder, and C. Eng. 2nd ed. New York: Oxford UP, 2004. Print.

Hegde, Madhuri, and Benjamin Roa. “Detecting Mutations in the APC Gene in Familial Adenomatous Polyposis (FAP).” Current Protocols in Human Genetics. New York: Wiley, 2006. Print.

Hedge, Madhuri, et al. "ACMG Technical Standards and Guidelines for Genetic Testing for Inherited Colorectal Cancer (Lynch Syndrome, Familial Adenomatous Polyposis, and MYH-Associated Polyposis)." Genetics in Medicine, vol. 16, no. 1, 2014, pp. 101–16. DOI: 10.1038/gim.2013.166. Accessed 6 Sept. 2024.

Yen, Timothy, Peter P Stanich, Lisen Axell, and Swati G Patel. "APC-Associated Polyposis Conditions." GeneReviews, 12 May 2022, www.ncbi.nlm.nih.gov/books/NBK1345/. Accessed 6 Sept. 2024.