Background Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited colorectal cancer predisposition caused by germ line mutations in the APC (adenomatous polyposis coli) gene. Current recommendations for APC mutation analysis advise full gene sequencing to identify point mutations and small insertions/deletions as well as the multiplex ligation dependent probe amplification (MLPA) technique to detect gene dosage alterations. Use of the protein truncation test (PTT) as a pre-screening tool has thus been largely replaced with direct end-to-end sequencing, mainly because of its limited sensitivity and failure to identify APC missense alterations.
Methods and results This report describes two unrelated patients with classical polyposis coli and unremarkable family history in whom neither full sequencing nor MLPA on leucocyte derived DNA could identify a pathogenic APC mutation. Applying the PTT, however, provided evidence of aberrant bands in both patients. Subsequent targeted mutation analysis of their tumour derived DNA allowed the identification of two novel, pathogenic APC alterations present in a mosaic state, at blood levels (1–15%) below the detection limits of conventional Sanger sequencing.
Conclusion The findings demonstrate the value of the PTT in identifying mosaic mutations in apparently APC mutation negative FAP patients with de novo classical polyposis and the need to keep the PTT within the diagnostic repertoire for APC mutation analysis.
- Clinical genetics
- genetic screening/counselling
- molecular genetics
- cancer: colon
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Familial adenomatous polyposis (FAP, MIM #175100) is an autosomal dominantly inherited colorectal cancer predisposition caused by germ line mutations in the tumour suppressor gene APC (adenomatous polyposis coli). In classical FAP, APC mutation carriers develop hundreds to thousands of colorectal polyps which, if left untreated, will progress to colorectal cancer in the third or forth decade of life.1 Attenuated FAP (AFAP), also referred to as ‘multiple’ polyps, is characterised by the presence of 5–99 colorectal adenomas and later age at diagnosis compared with classical FAP.2 3 The detection rate of APC mutations in FAP patients depends heavily on the phenotype and the methods used. In classical FAP patients, truncating point mutations in APC have been identified in 50–80% of cases.4–6 Large genomic deletions were found in around 7–12% of patients displaying a typical FAP phenotype.7–10 In AFAP patients, however, only 20–30% are found to harbour APC mutations.2 4
Recommendations for APC mutation analysis by the American Cancer Society (2008) and the National Cancer Institute (2009) encompass full gene sequencing to identify point mutations and small insertions/deletions as well as the multiplex ligation dependent probe amplification (MLPA) technique to detect gene dosage alterations. The protein truncation test (PTT), formerly used as a pre-screening tool for protein truncating mutations, has thus been largely replaced with direct end-to-end sequencing because of its limited sensitivity (around 80%) and failure to identify APC missense alterations (about 4% of all APC germ line mutations described, http://www.hgmd.cf.ac.uk).
Mosaicism denotes the presence of two or more genetically different cell lines within the body (somatic mosaicism) and/or the precursor cells of egg or sperm (germ line mosaicism, formerly called gonadal mosaicism). The clinical relevance of mosaicism has been reported for many genetic conditions such as Cornelia de Lange syndrome and Duchenne muscular dystrophy.11–13
Mosaicism for APC gene alterations has been rarely described but is becoming increasingly recognised.14 15 Whereas current literature mainly deals with somatic APC mosaicism, the incidence of truly germ line mosaicism in FAP is largely unknown.16 Here, we report two unrelated patients with classical polyposis coli in whose leucocyte derived DNA no pathogenic APC germ line alteration could be detected by conventional Sanger sequencing and MLPA analysis. The subsequent application of the PTT followed by mutation analysis of the patients' tumour derived DNA finally allowed the identification of two novel, pathogenic APC alterations present in a mosaic state and at low levels in both patients' leucocyte derived DNA.
DNA from peripheral blood was isolated by applying the salting-out method described by Miller et al.17 To obtain DNA from hair and saliva samples, QIAamp DNA Mini Kit (Qiagen, Basel, Switzerland) and Oragene Kit (DNA Genotek, Ontario, Canada) were applied according to the respective manufacturers' protocols. DNA from formalin fixed, paraffin embedded tumour tissue blocks and from sperm samples was extracted using the QIAamp tissue kit according to the manufacturer's protocol.
APC mutation analysis
Sequencing analyses and PTT were performed in duplicate as described previously.18 Sequencing of PCR products covering all 15 APC coding exons as well as the exon/intron boundaries was performed on PCR products purified with ExoSAP-IT (USB, Cleveland, Ohio, USA), using the ABI Prism Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, California, USA). Products were then analysed on the ABI Prism 310 Genetic Analyser (Applied Biosystems).
Mutation specific fragment analysis
To determine the degree and distribution of mutated alleles in different tissues, mutation specific fragment analysis (MSFA) was carried out. PCR was performed using primers flanking the mutations with the forward primer being FAM labelled. Primer sequences and PCR conditions are available from the authors upon request. Fragment analysis was carried out on an ABI Prism 310 Genetic Analyser (Applied Biosystems). The proportion of mutant allele was calculated using the peak areas of the mutant and the wild-type allele; usage of peak height led to similar results (data not shown).
Results and discussion
Patient I/1, a 38-year-old woman, was diagnosed with profuse polyposis throughout the colorectum, and rectal adenocarcinoma. Upper gastrointestinal endoscopy further revealed the presence of duodenal polyps. Following conventional DNA sequencing of the entire coding region of the APC gene, as well as gene dosage analysis by MLPA, no pathogenic alteration could be detected. The subsequent PTT on the patient's DNA revealed a faint, novel band in segment 2 encompassing APC amino acids 687 to 1584 (figure 1A). Only sequencing analysis of the respective exonic part (exons 15a to 15i) of APC in DNA extracted from the rectal cancer finally led to the identification of a novel, yet undescribed, heterozygous five base pair insertion (c.2715_2716insGAAGT) leading to a frame shift and a premature stop codon (p.Ser906GlufsX11) (figure 2A). MSFA showed that the quantity of mutant allele amounted to about 3% in leucocyte derived DNA and to 42% in DNA derived from the rectal cancer (supplementary figure 3A). Since the patient died shortly after the initial diagnosis, no additional tissues could be investigated. The parents and siblings refused genetic testing but agreed to endoscopic surveillance which was uneventful in all family members investigated (parents and one brother).
Patient II/1, a 40-year-old man, was diagnosed with rectal adenocarcinoma; several hundred colorectal adenomas were present throughout the large intestine, which were removed by total colectomy. Leucocyte derived DNA was screened for APC point mutations and gene dosage alterations by conventional DNA sequencing and MLPA, respectively. PTT analysis on the patient's DNA identified a novel, though faint, band in segment 2 encompassing APC exons 15a to 15i (figure 1B). Subsequently, the index patient's daughter (patient II/2) was diagnosed with profuse polyposis at the age of 18 years. The PTT revealed the same, albeit much stronger, band previously identified in her father (figure 1B). Sequencing analysis of the corresponding region revealed a novel, yet undescribed heterozygous four base pair deletion in APC exon 15c (c.2802_2805delTTAC; p.Thr934ThrfsX19), only detectable in DNA extracted from adenoma tissue of patient II/1 (in contrast to leucocyte derived DNA from his daughter, patient II/2; figure 2B). The family history of patient II/1 was negative for polyposis or colorectal cancer: his father had died at the age of 80 years from aortic dissection and his mother, age 74 years, was mutation negative (based on assessment of leucocyte derived DNA) and had undergone an uneventful lower endoscopy.
In order to investigate the degree and distribution of mosaicism in the three germ layers and the germ line of patient II/1, MSFA was performed on DNA extracted from tumour, saliva (endoderm), leucocytes (mesoderm), hair (ectoderm), and sperm samples. The investigation showed that the amount of mutant allele differed significantly between the various germ layers (about 1.5% to 14.8%) and the adenoma tissues (about 42% to 43.7%; supplementary figure 3B), but was similar among soma- as well as the germ line-derived (about 14.2%) cell populations. Since both of our index patients had no clinically affected parents or siblings, the APC mutations most likely occurred de novo in them. Because all three germ cell layers (ecto-, meso- and endoderm) as well as the germ line were affected, the mutational event probably occurred during the second week of gestation, before the primordial germ cells presumably separated from the ectodermal layer of the embryo.19
Initial observations by Farrington and Dunlop20 in 1999 reported somatic mosaicism in two out of five FAP families with evidence for a de novo mutation. Aretz et al14 were able to identify somatic mosaicism in eight (11%) out of 75 cases with suspected or confirmed de novo mutation. Based on molecular data from our FAP registry, we similarly estimated the minimal frequency for germ line mosaicism in our study population to be about 11% (two out of 18 APC mutation positive FAP patients with confirmed de novo mutation).
Our findings impressively highlight the importance of keeping the PTT within the analytical repertoire of genetic diagnostic services offering screening for APC mutation analysis. This stands in contrast to current recommendations for APC mutation analysis (American Cancer Society 2008, National Cancer Institute 2009) which largely favour direct end-to-end sequencing (and gene dosage analysis) and omit the PTT method because of its limited sensitivity.21
As exemplified in our study, the detection of faint aberrant bands in the PTT allowed targeted mutation screening of the corresponding part of the APC gene in DNA from tumour tissue, and led to the identification of as yet undescribed pathogenic APC mutations in both patients, enabling presymptomatic carrier testing in their offspring. Despite its advantages described in our study, the PTT clearly has its limitations—for example, an inability to detect missense alterations, the requirement of mRNA for the analysis of exons 1 to 14, and the presence of background signals potentially masking faint bands on autoradiograms. Alternatively, as also demonstrated in this study, direct mutation analysis of tumour DNA may constitute another valuable strategy for the detection of APC somatic mosaicism.
In conclusion, APC mosaicism has to be kept in mind when dealing with FAP patients with apparently de novo classical polyposis and no detectable APC germ line alteration, as assessed by conventional Sanger sequencing and gene dosage analysis. In these situations we propose to use the PTT as an additional screening tool which may subsequently allow targeted mutation analysis of the respective gene part in tumour derived DNA. Despite its limitations, the protein truncation assay remains a powerful analytical tool to identify (mosaic) APC germ line mutations and should remain in the diagnostic repertoire of any molecular genetics laboratory offering APC mutation analysis.
The authors would like to thank the patients and their families for participating in this study and the physicians for providing pertinent clinical information.
Funding The study was supported by grants from the Krebsliga beider Basel and Oncosuisse.
Competing interests None to declare.
Patient consent Obtained.
Ethics approval This study was conducted with the approval of the Ethikkommission beider Basel (“Basler Studie über familiaere Tumorkrankheiten”, Nr. 258/05).
Provenance and peer review Not commissioned; externally peer reviewed.
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