Detailed Molecular Genetics of the APC*E1317Q Mutation in Tumor Tissue Suggest it May Not Be Pathologically Significant : Diagnostic Molecular Pathology

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00019606-201309000-00006ReportDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2013 by Lippincott Williams & Wilkins.22September 2013 p 161–163Detailed Molecular Genetics of the APC*E1317Q Mutation in Tumor Tissue Suggest it May Not Be Pathologically SignificantCase ReportZauber, Peter MD*; Marotta, Stephen P. PhD†; Sabbath-Solitare, Marlene PhD†Departments of *Medicine†Pathology, Saint Barnabas Medical Center, Livingston, NJThe authors declare no conflict of interest.Supported by the Nussbaum Foundation of Saint Barnabas Medical Center.Reprints: Peter Zauber, MD, Department of Medicine, Saint Barnabas Medical Center, 100 Old Short Hills Road, Livingston, NJ 070399 (e-mail: [email protected]).AbstractAPC*E1317Q is a low-penetrance variant of the APC gene suggested as a risk for the development of colorectal adenomas and carcinomas. There is very little in the literature describing the molecular details of APC*E1317Q in tumor tissue. We provide information about the molecular genetics of 3 patients with APC*E1317Q. For 1 patient, we show linkage to a specific APC allele. We further show that loss of heterozygosity of the APC gene in tumors from carriers of the APC*E1317Q mutation may involve the mutated allele, not just the wild-type allele, suggesting the APC*E1317Q missense mutation may not be pathologically significant in the development of colorectal tumors.Germline mutations of the APC gene (5q21-22) cause the syndrome of familial adenomatous polyposis and are truncating, resulting in a shortened or absent protein. APC*E1317Q (c.3949G>C) is a low-penetrance variant of APC, resulting from a point mutation affecting the first position of codon 1317 in exon 15. A change from guanine to cytosine results in substitution of glutamine for glutamic acid, GAA to CAA. APC*E1317Q is a missense mutation, and it is therefore difficult to predict the effect upon protein function. Since the first description,1 several recent articles have reported conflicting results with regard to cancer and adenoma risk. In 1 publication, 3 of 124 patients with multiple adenomatous polyps were found to be carriers.2 A larger study reported that 3.3% of patients with colorectal cancer and adenomas were carriers, while only 0.8% of controls were carriers. After adjustment, APC*E1317Q was considered to be a significant predictor for colorectal adenomas but not for colorectal cancers.3 Three other large studies concluded it was unlikely this variant is associated with an increased risk for colorectal cancer.4–6However, very little has been published on the molecular details of APC*E1317Q. In the original description, 2 colon carcinomas were described with loss of heterozygosity (LOH), and both showed loss of the wild-type allele.1 All other reports primarily limit molecular analysis to just detection of the germline variant. We report more detailed molecular genetics findings in several of our patients with the APC*E1317Q germline mutation.MATERIALS AND METHODSPatients were identified during a prospective study of germline mutations in Jewish patients undergoing colonoscopies for colorectal cancer screening. The study was approved by the hospital Institutional Review Board. Written informed consent was obtained from the patients.Paraffin-embedded tissue was the source for both normal and tumor tissue. DNA was extracted using the QIAamp Tissue Kit (Qiagen, Valencia, CA). Purified DNA was reconstituted in 10 mM of Tris (pH of 8.0) buffer and placed in storage at 4°C. A Beckman DU-20 spectrophotometer (Beckman Coulter Inc., Fullerton, CA) was used to quantify the DNA. The region of codon 1307 was sequenced to screen for mutations. This method is preferred because of the number of other possible mutations that can occur in this region of the APC gene. Purified DNA was amplified using the primer set 5-CCAATATGTTTTTCAAGATGTAGTTC-3_ (sense) and 5-AATTCAACAGCTTTGTGCCT-3 (antisense). This primer set flanks the codon 1307 region of exon 15 of the APC gene and produces a 262-base product that effectively screens codons 1277-1348. Post polymerase chain reaction (PCR) product was purified for sequencing using the QIAquick PCR Purification Kit (Qiagen) and an aliquot was run on an agarose minigel to determine quality and estimate quantity.PCR-based DyeDeoxy Terminator sequencing was performed using an ABI Prism 377 DNA Sequencing System (Applied Biosystems, Foster City, CA). The sequencing reaction was performed with the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and 5 pmol of either the sense or antisense primer. Sequence reaction products were cleaned using AutoSeq G-50 spin columns (Amershan Pharmacia, Piscataway, NJ) and separated on a 5% Long Ranger acrylamide gel (Cambrex Bio Science Rockland Inc., Rockland, ME).LOH of the APC gene was determined through the PCR amplification of a CA-repeat marker within the D5S346 locus of the DP1 gene. The alleles are designated A1 through A15. PCR products were analyzed on an ABI Prism 377 DNA Sequencer with GeneScan collection software (Applied Biosystems). For LOH analysis, neoplastic tissue was evaluated simultaneously with normal colonic mucosal tissue from the same patient. The ratio of the height of the band intensities for the 2 alleles from neoplastic tissue was divided by the corresponding ratio from normal mucosal tissue. LOH was defined operationally as a resultant ratio of ≤0.5. LOH of D5S346 was qualitatively confirmed on the I1307K sequence analysis by a reduction in intensity of 1 peak at the 1307 position (data not shown). The complete linkage disequilibrium therefore permitted us to assess LOH.CASE PRESENTATIONS AND RESULTSPatient B.R. was found to have a rectal carcinoma at age 67 years. There was no family history of colorectal cancer or adenomas. B.R. is heterozygous for both APC*E1317Q and APC*I1307K. APC*I1307K (c.3920T>A) is an inherited variant associated with colorectal tumor risk found almost exclusively in those of Ashkenazi Jewish ancestry. A single nucleotide substitution in the second position of codon 1307 results in an adenine replacing a thymine and creating an oligo-adenine tract (A8). Using a CA repeat microsatellite marker within the DP-1 locus (D5S346) near the APC gene, we previously showed that APC*I1307K is in linkage disequilibrium with the A10 (by our designation) allele.7 We determined this patient’s 2 DP-1 alleles to be A10 and A11. The patient’s tubular adenoma demonstrated LOH of the A10 allele. A sequencing scan showed a normal thymine at the second position of codon 1307, confirming the loss of the A10 allele. Further, there was a markedly diminished wild-type guanine peak at the first position of codon 1317 in the tumor sample, relative to a scan of the patient’s normal tissue. We therefore concluded that the remaining cytosine peak at the first position of codon 1317 corresponds to the allele with the APC*E1317Q mutation, the A11 allele (Fig. 1).JOURNAL/dimp/04.03/00019606-201309000-00006/figure1-6/v/2021-02-17T200100Z/r/image-jpegDNA sequence analysis (patient B.R.) from codons 1305 through 1318. A, Normal colon tissue. The white arrow highlights 2 peaks, thymine and adenine, at the second position of codon 1307, indicating heterozygosity for the APC*I1307K mutation. The black arrow highlights 2 peaks, guanine and cytosine, at the first position of codon 1317, indicating heterozygosity for the APC*E1317Q mutation. B, Tubular adenoma DNA with known allelic loss. The white arrow indicates only wild-type thymine at the second position of codon 1307. The black arrow indicates cytosine at the first position of codon 1317.Patient S.G. carried the DP-1 alleles A11 and A12. A transverse carcinoma was removed from this patient at age 57 years. The patient’s father had colon cancer at age 80, and the patient’s mother had breast cancer at age 78 years. The carcinoma from S.G. demonstrated LOH for the A11 allele. Sequencing of the tumor DNA indicated 2 peaks at the first position of codon 1317, with the mutant allele carrying the cytosine diminished relative to that in the sequence of normal tissue DNA. Thus, the missing allele is the mutant, or All, allele.Patient D.S. carries the DP-1 alleles A11 and A15. Sequencing of the patient’s normal colonic mucosa showed 2 peaks, guanine and cytosine, at the first position of codon 1317. Two left-sided tubular adenomas were removed at age 53 years; neither showed LOH, but both adenomas demonstrated the 2 peaks guanine and cytosine at the first position of codon 1317 consistent with the APC*E1317Q mutation.DISCUSSIONThe 3 patients of our study demonstrated both guanine and cytosine peaks at the first position of codon 1317 in their normal tissue, thereby confirming they were heterozygous carriers for the APC*E1317Q mutation. The mutation is directly linked with the A11 allele of DP-1 in our first case, and our other patients also carried the A11 allele, suggesting that perhaps this mutation is on the A11 allele. A previous study of 7 patients with this mutation found no APC variants in linkage disequilibrium with APC*E1317Q.8 However, it would require a much larger sample to prove linkage disequilibrium and that the APC*E1317Q mutation is truly a founder mutation.The majority of mutations in APC encode a truncated version of the protein product, which would be nonfunctional. A missense mutation such as APC*E1317Q produces a full-length protein, and its impact upon protein function can be quite variable. Additional support for APC*E1317Q as a pathologic germline mutation was suggested,8 based on the reports of the mutation occurring as a somatic change in an adenoma (GAA to CAA) and carcinoma (GAA to TAA).1,9 We also identified a somatic mutation of GAA to TAA at codon 1317 in a tubular adenoma of another patient (data not shown). These somatic mutations may be a reflection of the mutability of this region of the APC gene, rather than proof of the causative role of germline mutations in codon 1317.Our results show that LOH of the APC gene in tumors from carriers of the APC*E1317Q mutation may involve the mutated allele, not just the wild-type allele. Because a truly pathologic mutation would most likely be paired with LOH of the wild-type allele, we suggest that the APC*E1317Q missense mutation may not be pathologically significant in the development of colorectal tumors.We therefore conclude that the presence of the APC*E1317Q mutation in patients with multiple adenomas or with colorectal carcinoma may be coincidental, and the actual predisposition for the neoplasms may reside elsewhere in the germline. APC*E1317Q may simply be a polymorphism that does not significantly influence APC protein function.REFERENCES1. White S, Bubb VJ, Wylllie AH.Germline APC mutation (Gln 1317) in a cancer-prone family that does not result in familial adenomatous polyposis.Genes, Chromosomes Cancer.1996;15:122–128.[Context Link]2. Fearnhead NS, Wilding JL, Winney B, et al..Multiple rare variants in different genes account for multifactorial inherited susceptibility to colorectal adenomas.Proc Natl Acad Sci USA.2004;101:15992–15997.[Context Link]3. Hall MJ, Liberman E, Dulkart O, et al..Risk of colorectal neoplasia associated with the adenomatous polyposis coli E1317Q variant.Ann Oncol.2009;20:1517–1521.[Context Link]4. Rozek LS, Rennert G, Gruber SB.APC*E1317Q is not associated with colorectal cancer in a population-based case-control study in northern Israel.Can Epidemiol Biomarkers Prev.2006;15:2325–2327.[Context Link]5. Popat S, Stone J, Coleman G, et al..Prevalence of the APC E1317Q variant in colorectal cancer patients.Cancer Lett.2000;149:203–206.[Context Link]6. Cleary SP, Kim H, Croitoru ME, et al..Missense polymorphisms in the adenomatous polyposis coli gene and colorectal cancer risk.Dis Colon Rectum.2008;51:1467–1474.[Context Link]7. Zauber NP, Sabbath-Solitare M, Marotta SP, et al..The characterization of somatic APC mutations in colonic adenomas and carcinomas in ashkenazi jews with the APC* I1307K variant using linkage disequilibrium.J Pathol.2003;199:146–151.[Context Link]8. Lamlum H, Al Tassan N, Jaeger E, et al..Germline APC variants in patients with multiple colorectal adenomas, with evidence for the particular importance of E1317Q.Hum Mol Genet.2000;9:2215–2221.[Context Link]9. Laken SJ, Petersen GM, Gruber SB, et al..Familial colorectal cancer in ashkenazim due to a hypermutable tract in APC.Nature Genet.1997;17:79–83.[Context Link]APC gene; APC*E1317Q mutation; APC*I1307K mutation; linkage disequilibrium; colon tumors; loss of heterozygosityDNA sequence analysis (patient B.R.) from codons 1305 through 1318. A, Normal colon tissue. The white arrow highlights 2 peaks, thymine and adenine, at the second position of codon 1307, indicating heterozygosity for the APC*I1307K mutation. The black arrow highlights 2 peaks, guanine and cytosine, at the first position of codon 1317, indicating heterozygosity for the APC*E1317Q mutation. B, Tubular adenoma DNA with known allelic loss. The white arrow indicates only wild-type thymine at the second position of codon 1307. The black arrow indicates cytosine at the first position of codon 1317.Detailed Molecular Genetics of the <em xmlns:mrws="http://webservices.ovid.com/mrws/1.0">APC*E1317Q</em> Mutation in Tumor Tissue Suggest it May Not Be Pathologically SignificantZauber Peter MD; Marotta, Stephen P. PhD; Sabbath-Solitare, Marlene PhDCase ReportCase Report322p 161-163

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