Portal de Periódicos da CAPES

APC Germline Mutations in Individuals Being Evaluated for Familial Adenomatous Polyposis

2012; Elsevier BV; Volume: 15; Issue: 1 Linguagem: Inglês

10.1016/j.jmoldx.2012.07.005

1943-7811

Sarah E. Kerr, Cheryl B. Thomas, Stephen N. Thibodeau, Matthew J. Ferber, Kevin C. Halling,

Colorectal Cancer Screening and Detection

Inactivating APC mutations cause familial adenomatous polyposis, classically characterized by hundreds to thousands of adenomatous colorectal polyps and cancer. Historically, 98% of pathogenic alterations in APC are nonsense or frameshift mutations; however, few reported series have used techniques that test for large deletions or duplications. Splice site mutations are only rarely documented. Consecutive cases (n = 1591) submitted for complete APC gene analysis during a 4-year period were reviewed. Testing included mutation screening (Sanger sequencing or conformation sensitive gel electrophoresis and protein truncation testing) with reflex confirmation sequencing. Gene deletion or duplication analysis was performed in 1421 cases by multiplex ligation-dependent probe amplification. Testing yielded 411 pathogenic, 20 likely pathogenic, 15 variant of uncertain significance, 140 likely benign, and 1005 negative reports. Identified were 168 novel variants (103 pathogenic, 5 likely pathogenic, 12 variant of uncertain significance, and 48 likely benign). Of the 431 pathogenic or likely pathogenic mutations, frameshift, nonsense, splice site, and large deletion or duplication mutations represented 43%, 42%, 9%, and 6% of cases, respectively. This is the largest report of clinical APC testing experience with concurrent multiplex ligation-dependent probe amplification. In addition to nonsense and frameshift mutations, large deletions or duplications and canonical splice site mutations are a significant cause of familial adenomatous polyposis. Despite technological advances, broad allelic, locus, and phenotypic heterogeneity continue to pose challenges for genetic testing of patients with colorectal adenomatous polyposis. Inactivating APC mutations cause familial adenomatous polyposis, classically characterized by hundreds to thousands of adenomatous colorectal polyps and cancer. Historically, 98% of pathogenic alterations in APC are nonsense or frameshift mutations; however, few reported series have used techniques that test for large deletions or duplications. Splice site mutations are only rarely documented. Consecutive cases (n = 1591) submitted for complete APC gene analysis during a 4-year period were reviewed. Testing included mutation screening (Sanger sequencing or conformation sensitive gel electrophoresis and protein truncation testing) with reflex confirmation sequencing. Gene deletion or duplication analysis was performed in 1421 cases by multiplex ligation-dependent probe amplification. Testing yielded 411 pathogenic, 20 likely pathogenic, 15 variant of uncertain significance, 140 likely benign, and 1005 negative reports. Identified were 168 novel variants (103 pathogenic, 5 likely pathogenic, 12 variant of uncertain significance, and 48 likely benign). Of the 431 pathogenic or likely pathogenic mutations, frameshift, nonsense, splice site, and large deletion or duplication mutations represented 43%, 42%, 9%, and 6% of cases, respectively. This is the largest report of clinical APC testing experience with concurrent multiplex ligation-dependent probe amplification. In addition to nonsense and frameshift mutations, large deletions or duplications and canonical splice site mutations are a significant cause of familial adenomatous polyposis. Despite technological advances, broad allelic, locus, and phenotypic heterogeneity continue to pose challenges for genetic testing of patients with colorectal adenomatous polyposis. Familial adenomatous polyposis (FAP), in its classic form, is an autosomal, dominantly inherited, hereditary cancer syndrome characterized by the development of 100 to 1000s of colorectal adenomatous polyps. FAP is estimated to have a prevalence of 2 to 3 per 100,000 individuals1Burn J. Chapman P. Delhanty J. Wood C. Lalloo F. Cachon-Gonzalez M.B. Tsioupra K. Church W. Rhodes M. Gunn A. The UK Northern region genetic register for familial adenomatous polyposis coli: use of age of onset, congenital hypertrophy of the retinal pigment epithelium, and DNA markers in risk calculations.J Med Genet. 1991; 28: 289-296Crossref PubMed Scopus (91) Google Scholar, 2Jarvinen H.J. Epidemiology of familial adenomatous polyposis in Finland: impact of family screening on the colorectal cancer rate and survival.Gut. 1992; 33: 357-360Crossref PubMed Scopus (123) Google Scholar, 3Bulow S. Faurschou Nielsen T. Bulow C. Bisgaard M.L. Karlsen L. Moesgaard F. The incidence rate of familial adenomatous polyposis: results from the Danish Polyposis Register.Int J Colorectal Dis. 1996; 11: 88-91Crossref PubMed Google Scholar and is found in all ethnic groups. The untreated, classic form of FAP has nearly complete penetrance for colorectal cancer by the age of 50 years.4Bussey H. Familial Polyposis Coli: Family Studies, Histopathology, Differential Diagnosis, and Results of Treatment. Johns Hopkins University Press, Baltimore1975Google Scholar A truncating germline APC mutation can be detected in approximately 80% of classic cases; however, detection rates in patients with fewer colorectal polyps is lower,5Aretz S. Uhlhaas S. Goergens H. Siberg K. Vogel M. Pagenstecher C. Mangold E. Caspari R. Propping P. Friedl W. MUTYH-associated polyposis: 70 of 71 patients with biallelic mutations present with an attenuated or atypical phenotype.Int J Cancer. 2006; 119: 807-814Crossref PubMed Scopus (166) Google Scholar, 6Giardiello F.M. Brensinger J.D. Petersen G.M. Luce M.C. Hylind L.M. Bacon J.A. Booker S.V. Parker R.D. Hamilton S.R. The use and interpretation of commercial APC gene testing for familial adenomatous polyposis.N Engl J Med. 1997; 336: 823-827Crossref PubMed Scopus (413) Google Scholar suggesting that some FAP patients have mutations (such as deep intronic mutations, deletions or duplications,7Castellsague E, Gonzalez S, Guino E, Stevens KN, Borras E, Raymond VM, Lazaro C, Blanco I, Gruber SB, Capella G: Allele-specific expression of APC in adenomatous polyposis families. Gastroenterology 139:439-447, e431Google Scholar, 8Stekrova J. Sulova M. Kebrdlova V. Zidkova K. Kotlas J. Ilencikova D. Vesela K. Kohoutova M. Novel APC mutations in Czech and Slovak FAP families: clinical and genetic aspects.BMC Med Genet. 2007; 8: 16Crossref PubMed Scopus (25) Google Scholar, 9Friedl W. Aretz S. Familial adenomatous polyposis: experience from a study of 1164 unrelated German polyposis patients.Hered Cancer Clin Pract. 2005; 3: 95-114Crossref PubMed Scopus (79) Google Scholar, 10Nielsen M. Bik E. Hes F.J. Breuning M.H. Vasen H.F. Bakker E. Tops C.M. Weiss M.M. Genotype-phenotype correlations in 19 Dutch cases with APC gene deletions and a literature review.Eur J Hum Genet. 2007; 15: 1034-1042Crossref PubMed Scopus (33) Google Scholar, 11Aretz S. Stienen D. Uhlhaas S. Pagenstecher C. Mangold E. Caspari R. Propping P. Friedl W. Large submicroscopic genomic APC deletions are a common cause of typical familial adenomatous polyposis.J Med Genet. 2005; 42: 185-192Crossref PubMed Scopus (74) Google Scholar complex rearrangements, or somatic mosaicism12Aretz S. Stienen D. Friedrichs N. Stemmler S. Uhlhaas S. Rahner N. Propping P. Friedl W. Somatic APC mosaicism: a frequent cause of familial adenomatous polyposis (FAP).Hum Mutat. 2007; 28: 985-992Crossref PubMed Scopus (107) Google Scholar, 13Rohlin A. Wernersson J. Engwall Y. Wiklund L. Bjork J. Nordling M. Parallel sequencing used in detection of mosaic mutations: comparison with four diagnostic DNA screening techniques.Hum Mutat. 2009; 30: 1012-1020Crossref PubMed Scopus (124) Google Scholar, 14Hes F.J. Nielsen M. Bik E.C. Konvalinka D. Wijnen J.T. Bakker E. Vasen H.F. Breuning M.H. Tops C.M. Somatic APC mosaicism: an underestimated cause of polyposis coli.Gut. 2008; 57: 71-76Crossref PubMed Scopus (117) Google Scholar) that are not detected by routine methods or are FAP phenocopies. Biallelic germline mutations in MUTYH are known to cause a less severe form of adenomatous polyposis similar to attenuated FAP5Aretz S. Uhlhaas S. Goergens H. Siberg K. Vogel M. Pagenstecher C. Mangold E. Caspari R. Propping P. Friedl W. MUTYH-associated polyposis: 70 of 71 patients with biallelic mutations present with an attenuated or atypical phenotype.Int J Cancer. 2006; 119: 807-814Crossref PubMed Scopus (166) Google Scholar, 15Sieber O.M. Lipton L. Crabtree M. Heinimann K. Fidalgo P. Phillips R.K. Bisgaard M.L. Orntoft T.F. Aaltonen L.A. Hodgson S.V. Thomas H.J. Tomlinson I.P. Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH.N Engl J Med. 2003; 348: 791-799Crossref PubMed Scopus (736) Google Scholar in which the patient typically develops fewer than 100 adenomatous polyps. Approximately 15% to 20% of FAP patients have de novo germline mutations.16Bisgaard M.L. Fenger K. Bulow S. Niebuhr E. Mohr J. Familial adenomatous polyposis (FAP): frequency, penetrance, and mutation rate.Hum Mutat. 1994; 3: 121-125Crossref PubMed Scopus (363) Google Scholar The disease spectrum for individuals with FAP includes not only colorectal neoplasia but also other premalignant and malignant lesions throughout the gastrointestinal tract, such as fundic gland polyps in the stomach, adenomatous polyps of the stomach and small bowel, and periampullary carcinoma. In addition, the Gardner syndrome subtype of FAP includes desmoid tumors, epithelial inclusion cysts, osteoid osteomas, and supernumerary teeth; congenital hypertrophy of the retinal pigmented epithelium can be seen with or without the Gardner syndrome. Other less common manifestations reported in FAP include embryonal tumors (hepatoblastoma and medulloblastoma), pancreatobiliary carcinoma, papillary thyroid carcinoma (especially cribriform-morular variant),17Chikkamuniyappa S. Jagirdar J. Cribriform-morular variant of papillary carcinoma: association with familial adenomatous polyposis: report of three cases and review of literature.Int J Med Sci. 2004; 1: 43-49Crossref PubMed Google Scholar and adrenal cortical tumors. Genotype-phenotype correlation studies suggest that mutations in exons 3 and 4 are associated with an attenuated phenotype, whereas the Gardner phenotype has been primarily associated with mutations in the distal half of the gene.9Friedl W. Aretz S. Familial adenomatous polyposis: experience from a study of 1164 unrelated German polyposis patients.Hered Cancer Clin Pract. 2005; 3: 95-114Crossref PubMed Scopus (79) Google Scholar, 10Nielsen M. Bik E. Hes F.J. Breuning M.H. Vasen H.F. Bakker E. Tops C.M. Weiss M.M. Genotype-phenotype correlations in 19 Dutch cases with APC gene deletions and a literature review.Eur J Hum Genet. 2007; 15: 1034-1042Crossref PubMed Scopus (33) Google Scholar, 18Wallis Y.L. Macdonald F. Hulten M. Morton J.E. McKeown C.M. Neoptolemos J.P. Keighley M. Morton D.G. Genotype-phenotype correlation between position of constitutional APC gene mutation and CHRPE expression in familial adenomatous polyposis.Hum Genet. 1994; 94: 543-548Crossref PubMed Scopus (101) Google Scholar, 19Soravia C. Berk T. Madlensky L. Mitri A. Cheng H. Gallinger S. Cohen Z. Bapat B. Genotype-phenotype correlations in attenuated adenomatous polyposis coli.Am J Hum Genet. 1998; 62: 1290-1301Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 20Wallis Y.L. Morton D.G. McKeown C.M. Macdonald F. Molecular analysis of the APC gene in 205 families: extended genotype-phenotype correlations in FAP and evidence for the role of APC amino acid changes in colorectal cancer predisposition.J Med Genet. 1999; 36: 14-20PubMed Google Scholar, 21Friedl W. Caspari R. Sengteller M. Uhlhaas S. Lamberti C. Jungck M. Kadmon M. Wolf M. Fahnenstich J. Gebert J. Moslein G. Mangold E. Propping P. Can APC mutation analysis contribute to therapeutic decisions in familial adenomatous polyposis? experience from 680 FAP families.Gut. 2001; 48: 515-521Crossref PubMed Scopus (248) Google Scholar The APC gene spans 108 kb of genomic DNA (8532 coding bp), is located at chromosome 5q22, and contains 15 coding exons and 3 upstream noncoding exons. The gene encodes a protein that functions as a tumor suppressor by negatively regulating the β-catenin oncoprotein. The APC protein contains multiple domains that bind and facilitate phosphorylation of β-catenin, targeting β-catenin for ubiquitination and degradation. In the absence of the APC protein, β-catenin accumulates in the nucleus and interacts with factors that up-regulate the transcription of genes involved in cell cycle entry and progression.22Sieber O.M. Tomlinson I.P. Lamlum H. The adenomatous polyposis coli (APC) tumour suppressor–genetics, function and disease.Mol Med Today. 2000; 6: 462-469Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Clinical laboratory testing for germline mutations in APC has been challenging, primarily because of the large size of the gene and a high level of allelic heterogeneity. In addition, whole or partial gene deletions are known to cause disease in a small percentage of patients, and PCR-based techniques to easily assess for these types of mutations have only recently become available. Because detection of pathogenic mutations in probands is critical to identifying at risk family members for screening, the ideal testing method would have as high sensitivity as possible for the detection of alterations in APC. Novel mutations are commonly detected in clinical testing; therefore, laboratories must be proficient with the interpretation of private mutations using a variety of resources (in-house and online databases, in silico mutation analysis software, literature sources, linkage analysis interpretation, and possibly functional studies). We describe the Mayo Clinic experience with 1591 consecutive clinical cases submitted for full APC gene analysis during a 4-year period. Clinical laboratory reports from consecutive cases submitted for full APC gene analysis from January 1, 2006, through December 31, 2009, were reviewed. APC alteration nomenclature was based on RefSeq NM_000038. Some patients also had concurrent targeted MUTYH (RefSeq NM_001048171.1) gene testing, and these results were also reviewed where available. In addition, all of the APC result data during this period were reviewed to confirm the method of testing used [eg, conformation sensitive gel electrophoresis (CSGE) versus sequencing for exons 1 through 14] and to look for alterations that were interpreted as benign that would have not been included in the final clinical report. Reported and unreported alterations were updated to current Human Genome Variation Society nomenclature recommendations, including a review of the original sequence in some cases to clarify the recorded alteration. In silico analysis of all variants not definitively pathogenic was performed using Alamut version 1.5 (Interactive Biosoftware, Rouen, France) integrated software tools GeneSplicer, MaxEntScan, NNSPLICE, and SpliceSiteFinder-like with default settings. Missense mutations were also analyzed using online tools accessed through Alamut: Align GVGD, PolyPhen, and SIFT. In silico analysis interpretation was reviewed by two of the authors (S.E.K. and C.B.T.) for consensus on contribution to predicted pathogenicity. All alterations were searched for in the online databases Human Gene Mutation Database and Leiden Open Variation Database via references to reports regarding the clinical significance of the alteration. Finally, clinical reports from family members tested by targeted APC mutation analysis were also reviewed when available to aid in classification of variants of uncertain clinical significance (VUSs). For example, if a proband had a pathogenic mutation and a VUS, the affected father had only the pathogenic mutation, and the unaffected mother had the VUS, we assumed that the pathogenic mutation and VUS were in trans (on different chromosomes) in the proband and that the VUS could be reclassified to a likely benign variant (see classification levels below). Alterations were classified based on a five-level classification system similar to that outlined by the recommendations of the International Agency for Research on Cancer Unclassified Genetic Variants Working Group23Plon S.E. Eccles D.M. Easton D. Foulkes W.D. Genuardi M. Greenblatt M.S. Hogervorst F.B. Hoogerbrugge N. Spurdle A.B. Tavtigian S.V. Sequence variant classification and reporting: recommendations for improving the interpretation of cancer susceptibility genetic test results.Hum Mutat. 2008; 29: 1282-1291Crossref PubMed Scopus (660) Google Scholar and applied to BRCA1 and BRCA2 variant interpretation,24Spearman A.D. Sweet K. Zhou X.P. McLennan J. Couch F.J. Toland A.E. Clinically applicable models to characterize BRCA1 and BRCA2 variants of uncertain significance.J Clin Oncol. 2008; 26: 5393-5400Crossref PubMed Scopus (71) Google Scholar, 25Vallee M.P. Francy T.C. Judkins M.K. Babikyan D. Lesueur F. Gammon A. Goldgar D.E. Couch F.J. Tavtigian S.V. Classification of missense substitutions in the BRCA genes: a database dedicated to Ex-UVs.Hum Mutat. 2012; 33: 22-28Crossref PubMed Scopus (56) Google Scholar using concepts for analyzing sequence information only without further Bayesian calculation of probability. Small frameshift insertions and/or deletions, nonsense point mutations, large deletions, and mutations involving splice donor (intron + 1 G or + 2 T) or splice acceptor (intron − 1 G or − 2 A) were categorized as unequivocally pathogenic. If not unequivocally pathogenic, alterations were considered likely pathogenic if there was a functional study in the literature supporting that a change altered splicing and/or in silico analysis strongly suggested that a change altered splicing by consensus of two reviewers (S.E.K. and C.B.T.) (see previously described methods) or if there was an out-of-frame (presumably tandem) exon duplication. Alterations were interpreted as likely benign given one or more of the following: alteration seen in the same patient as an unequivocally pathogenic mutation, alteration seen in the homozygous state, functional study suggesting benignity, and/or synonymous substitution without in silico evidence of splicing alteration. Variants were considered benign polymorphisms if they were found to have an allele frequency of >1% in two or more reported normal population entries in National Center for Biotechnology Information's Single Nucleotide Polymorphism database (build 131). The remaining unclassified alterations were considered VUSs. Genomic DNA was extracted from whole blood. Mutations in coding exons 1 through the intron/exon boundary of exon 15 were detected by either CSGE or Sanger sequencing. Protein truncation testing (PTT) was used to screen for truncating mutations in exon 15. All mutations detected by the initial screen (CSGE or PTT) were confirmed by follow-up Sanger sequencing. In addition, multiplex ligation-dependent probe amplification (MLPA) was used to detect deletions or duplications in the APC 5′ untranslated region, promoter, and all coding exons (Figure 1). For some cases, targeted testing by restriction fragment length polymorphism analysis for the MUTYH c.494A>G (p.Y165C) and c.1145G>A (p.G382D) mutations had also been performed. Patient DNA was PCR amplified in multiplex reactions to produce amplicons, including coding exons 1 through 14 and the first 300 nucleotides of exon 15. 33P-labeled products were then denatured, reannealed, and separated on a 1,4-bis(acrolyl) piperazine, ethylene glycol, and formamide–enhanced polyacrylamide gel, which was then radioimaged and analyzed for variant mobility bands. Sanger sequencing was performed on samples with variant mobility bands to determine the DNA alteration. Patient DNA was PCR amplified in multiplexed reactions to produce template amplicons for exons 1 through 14 and the first 300 nucleotides of exon 15. If sequencing were being performed to follow up a positive CSGE or PTT result, the template PCR included the amplicon covering the appropriate gene segment. Using the template PCR, products were assessed with an agarose check gel to confirm amplification of the expected products and absence of contamination. The PCR template was then treated with an Exonuclease I/Shrimp Alkaline Phosphatase mix to purify the PCR products for sequencing. The template was then mixed with internal sequencing primers and BigDye Terminator (Applied Biosystems/Life Technologies, Carlsbad, CA) dilution master mix and thermocycled under standardized conditions. The resulting sequencing products were desalted and separated by capillary electrophoresis. Results were analyzed using Mutation Surveyor software version 3.24 (SoftGenetics, LLC, State College, PA) with forced alignment against a normal control. For the first few months of 2006, PCR templates were sequenced by incorporation of 33Armstrong J.G. Davies D.R. Guy S.P. Frayling I.M. Evans D.G. APC mutations in familial adenomatous polyposis families in the Northwest of England.Hum Mutat. 1997; 10: 376-380Crossref PubMed Scopus (37) Google ScholarP-labeled nucleotides with analysis on polyacrylamide gel, often in parallel with fluorescent sequencing. A standardized concentration of patient DNA was denatured and then hybridized with MLPA probes (MRC Holland, Amsterdam, Netherlands) for 16 to 24 hours. The MLPA probe pairs were then ligated and amplified by limited cycle PCR. Fluorescently labeled products were separated by capillary electrophoresis and analyzed against internal and external dosage controls using GeneMarker software version 1.80 (SoftGenetics). Patient DNA was amplified in multiplexed reactions to produce four overlapping template amplicons covering all of exon 15.26Powell S.M. Petersen G.M. Krush A.J. Booker S. Jen J. Giardiello F.M. Hamilton S.R. Vogelstein B. Kinzler K.W. Molecular diagnosis of familial adenomatous polyposis.N Engl J Med. 1993; 329: 1982-1987Crossref PubMed Scopus (627) Google Scholar The template was then transcribed and translated using a T7 TnT-coupled transcription-translation mix (Promega, Madison, WI). 35Nilbert M. Kristoffersson U. Ericsson M. Johannsson O. Rambech E. Mangell P. Broad phenotypic spectrum in familial adenomatous polyposis; from early onset and severe phenotypes to late onset of attenuated polyposis with the first manifestation at age 72.BMC Med Genet. 2008; 9: 101Crossref PubMed Scopus (15) Google ScholarS-labeled protein products were separated on a polyacrylamide gel, radioimaged, and analyzed for variant protein bands. Sanger sequencing was performed on samples with positive PTT results to determine the DNA alteration. Patient DNA was PCR amplified with primers flanking coding exons 7 and 13 of MUTYH. Products were digested with BbvI or BglII, respectively, and separated on a polyacrylamide gel, which was then fluorescently stained and analyzed for the expected fragment sizes to assess for Y165C and G382D mutations at nucleotides 494 and 1145. During the 4-year period, 1591 cases were analyzed; 215 cases were screened by CSGE of exons 1 through 14 and PTT of exon 15, and the remaining 1376 cases were screened by DNA sequencing of exons 1 through 14 and PTT of exon 15. MLPA was performed on 1421 of the 1591 cases. The germline APC alterations that were detected in these patients are listed in Table 1, Table 2, Table 3, Table 4 (benign polymorphic variants, likely benign variants, VUSs, and likely pathogenic mutations) and Supplemental Table S1.Table 1Benign Polymorphic APC VariantsDNAProteinCoding exon/intronNo. of cases detectedMutant allele frequency in this studyRange of mutant allele frequency in populations∗Both the mutation frequencies found in this study and the range of frequencies reported in the Single Nucleotide Polymorphism database (build 131) populations of >100 chromosomes are given.Referencec.136-53T>Cp.?11250.040.01–0.31rs2304793c.645 + 32C>Tp.?52310.080.03–0.08rs2909961c.1458T>Cp. =1110480.510.09–0.73rs2229992c.1635G>Ap. =1311140.560.52–0.84rs351771c.5034G>Ap. =151U0.52–0.84rs42427c.5268T>Gp. =152U0.51–0.83rs866006c.5465T>Ap.Val1822Asp152U0.01–0.24rs459552c.5880G>Ap. =151U0.48–0.90rs465899Note that the common c.1458T>C and c.1635G>A were not detected by CSGE; therefore, allele frequencies were calculated using only those individuals who underwent exon 11 and 13 sequencing. Polymorphic variants detected in exon 15 were found during PTT follow-up sequencing; therefore, frequencies were not calculated. Standard Human Genome Variation Society nomenclature was used to describe the protein level alteration.p. =, a synonymous substitution not altering the amino acid sequence; p.?, an intronic alteration or deletion/duplication with intronic breakpoints; U, unknown.∗ Both the mutation frequencies found in this study and the range of frequencies reported in the Single Nucleotide Polymorphism database (build 131) populations of >100 chromosomes are given. Open table in a new tab Table 2Likely Benign APC VariantsDNAProteinCoding exon/intronEvidence for benign diagnosisNo. of cases detectedMutant allele frequencyReferencec.120G>Ap. =1pt40.0013This studyc.135 + 81_+85delTACTTp.?1p3UThis studyc.136-64A>Gp.?1p30.0009This studyc.220 + 18G>Ap.?2p20.0006This studyc.220 + 44A>Gp.?2i10.0003This studyc.221-29G>Cp.?2p30.0009This studyc.221-27A>Cp.?2i20.0006This studyc.295C>Tp.Arg99Trp3pt30.000927Dobbie Z. Spycher M. Hurliman R. Ammann R. Ammann T. Roth J. Muller A. Muller H. Scott R.J. Mutational analysis of the first 14 exons of the adenomatous polyposis coli (APC) gene.Eur J Cancer. 1994; 30A: 1709-1713Abstract Full Text PDF PubMed Scopus (39) Google Scholarc.422 + 75T>Cp.?3p5UThis studyc.423-32T>Cp.?3p10.0003This studyc.423-16A>Tp.?3i1Urs78919815∗The c.423-17 position falls at the 3′ end of a poly 7T sequence, followed by a poly 13A sequence near the forward sequencing primer. Variation at this position was seen clearly by radioactive sequencing but is not reliably detected by our capillary electrophoresis method.c.423-17dupTp.?3i4UThis study∗The c.423-17 position falls at the 3′ end of a poly 7T sequence, followed by a poly 13A sequence near the forward sequencing primer. Variation at this position was seen clearly by radioactive sequencing but is not reliably detected by our capillary electrophoresis method.c.450A>Gp. =4i10.0003This studyc.564A>Gp. =5i10.0003This studyc.588C>Tp. =5i10.0003This studyc.645 + 33G>Ap.?5i10.0003This studyc.645 + 61C>Tp.?5p200.0063rs56328836c.705A>Gp. =6p, h170.0054LOVD†Leiden Open Variation Database (http://chromium.liacs.nl/LOVD2/colon_cancer/home.php?select_db=APC), last accessed August 12, 2010.c.729 + 23T>Cp.?6i10.0003rs75111475c.729 + 31A>Gp.?6i10.0003This studyc.729 + 69A>Gp.?6i1UThis studyc.729 + 88T>Cp.?6h2Urs79627325c.729 + 136C>Tp.?6i2UThis studyc.729 + 138T>Cp.?6i1UThis studyc.729 + 141A>Gp.?6p1UThis studyc.730-29A>Tp.?6p80.0025rs75083764c.777G>Tp. =7i10.0003This studyc.835-24A>Tp.?7i20.0006This studyc.879T>Cp. =8i10.0003This studyc.885T>Cp. =8i10.0003This studyc.933 + 24G>Cp.?8i10.0003This studyc.933 + 30A>Gp.?8p180.0057This studyc.934-17delAp.?8i10.0003This studyc.1302C>Gp.Asp434Glu9p10.0003This studyc.1312 + 27G>Ap.?9p150.0047rs74975092c.1312 + 31A>Gp.?9p10.0003This studyc.1312 + 136A>Gp.?9i8UThis studyc.1313-61A>Gp.?9p1UThis studyc.1409-48_-44del5insAATCp.?10p9UThis studyc.1419G>Ap. =11i10.0003This studyc.1548 + 17T>Cp.?11i10.0003This studyc.1548 + 110A>Gp.?11p3UThis studyc.1549-13A>Tp.?11p10.0003This studyc.1626 + 19C>Ap.?12i10.0003This studyc.1627-93A>Cp.?12i3UThis studyc.1627-52G>Ap.?12i1UThis studyc.1674T>Cp. =13i10.0003This studyc.1686G>Ap. =13i10.0003This studyc.1695A>Gp. =13p100.0031This studyc.1705G>Ap.Val569Met13p10.0003This studyc.1958 + 8T>Cp.?14pc90.0028rs62626346c.1958 + 27T>Gp.?14i10.0003This studyc.1959G>Ap. =15pt, f100.003628Aretz S. Uhlhaas S. Sun Y. Pagenstecher C. Mangold E. Caspari R. Moslein G. Schulmann K. Propping P. Friedl W. Familial adenomatous polyposis: aberrant splicing due to missense or silent mutations in the APC gene.Hum Mutat. 2004; 24: 370-380Crossref PubMed Scopus (88) Google Scholarc.2031C>Tp. =15i10.0003This studyc.2091A>Gp. =15p10.0003This studyc.2204C>Tp.Ala735Val15pt10.0003This studyc.2205G>Ap. =15i20.0006LOVD†Leiden Open Variation Database (http://chromium.liacs.nl/LOVD2/colon_cancer/home.php?select_db=APC), last accessed August 12, 2010.c.2232T>Gp. =15i10.0003This studyc.3165A>Tp. =15p1U29Kanter-Smoler G. Bjork J. Fritzell K. Engwall Y. Hallberg B. Karlsson G. Gronberg H. Karlsson P. Wallgren A. Wahlstrom J. Hultcrantz R. Nordling M. Novel findings in Swedish patients with MYH-associated polyposis: mutation detection and clinical characterization.Clin Gastroenterol Hepatol. 2006; 4: 499-506Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholarc.3173A>Gp.Asp1058Gly15pc2UThis studyc.3937A>Gp.Thr1313Ala15p1U30Nagase H. Horii A. Aoki T. Petersen G.M. Vogelstein B. Maher E. Ogawa M. Maruyama M. Utsunomiya J. Baba S. Nakamura Y. Mutations of the APC (adenomatous polyposis coli) gene.Hum Mutat. 1993; 2: 425-434Crossref PubMed Scopus (355) Google Scholar‡The reference states that the alteration was found in a colorectal cancer (unknown if in germline).c.3949G>Cp.Glu1317Gln15pt1Urs1801166, 31Fearnhead N.S. Wilding J.L. Winney B. Tonks S. Bartlett S. Bicknell D.C. Tomlinson I.P. Mortensen N.J. Bodmer W.F. Multiple rare variants in different genes account for multifactorial inherited susceptibility to colorectal adenomas.Proc Natl Acad Sci U S A. 2004; 101: 15992-15997Crossref PubMed Scopus (166) Google ScholarVariants were considered likely benign based on a published functional study, occurring in the same individual as a pathogenic allele, being seen in a homozygous state, or based on in silico analysis. Some deep intronic variants do not have calculated frequencies because the sequence read is not always interpretable out to that position from run to run.f, functional; h,