Germline hypermethylation of the APC promoter is not a frequent cause of familial adenomatous polyposis in APC/MUTYH mutation negative families

Authors

  • Jordi Romero-Giménez,

    1. Molecular Oncology Group, Molecular Biology and Biochemistry Research Center (CIBBIM), Nanomedicine Research Program, Vall d'Hebron Hospital Research Institute, Barcelona, Spain
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    • The first two authors contributed equally to this paper.

  • Higinio Dopeso,

    1. Molecular Oncology Group, Molecular Biology and Biochemistry Research Center (CIBBIM), Nanomedicine Research Program, Vall d'Hebron Hospital Research Institute, Barcelona, Spain
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    • The first two authors contributed equally to this paper.

  • Ignacio Blanco,

    1. Prevention and Cancer Control Department, Genetic Counseling Unit and Translational Research Laboratory, IDIBELL, Catalan Institute of Oncology, L'Hospitalet de Llobregat, Barcelona, Spain
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  • Angel Guerra-Moreno,

    1. Nanomedicine Research Program, Molecular Biology and Biochemistry Research Center (CIBBIM), Vall d'Hebron Hospital Research Institute, Barcelona, Spain
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  • Sara Gonzalez,

    1. Prevention and Cancer Control Department, Genetic Counseling Unit and Translational Research Laboratory, IDIBELL, Catalan Institute of Oncology, L'Hospitalet de Llobregat, Barcelona, Spain
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  • Stefanie Vogt,

    1. Institute of Human Genetics, University of Bonn, Bonn, Germany
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  • Stefan Aretz,

    1. Institute of Human Genetics, University of Bonn, Bonn, Germany
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  • Simo Schwartz Jr,

    1. Nanomedicine Research Program, Molecular Biology and Biochemistry Research Center (CIBBIM), Vall d'Hebron Hospital Research Institute, Barcelona, Spain
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  • Gabriel Capella,

    1. Prevention and Cancer Control Department, Genetic Counseling Unit and Translational Research Laboratory, IDIBELL, Catalan Institute of Oncology, L'Hospitalet de Llobregat, Barcelona, Spain
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  • Diego Arango

    Corresponding author
    1. Molecular Oncology Group, Molecular Biology and Biochemistry Research Center (CIBBIM), Nanomedicine Research Program, Vall d'Hebron Hospital Research Institute, Barcelona, Spain
    • Molecular Oncology Group, Molecular Biology and Biochemistry Research Center (CIBBIM), Valle Hebron Hospital Research Institute, Passeig Vall d'Hebron 119-129, Barcelona 08035, Spain

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    • Fax: +34-93-489-4040.


Abstract

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome predisposing to colorectal cancer and affects 1 in 5–10,000 births. Inheritance of a mutant allele of the adenomatous polyposis coli (APC) gene is the cause of ∼80% of FAP and 20–30% of an attenuated form of FAP (AFAP), whereas mutations in MUTYH account for a small proportion of the remaining cases. However, the genetic cause of FAP/AFAP in a significant number of families is not known, and cancer risk for individual members of these families cannot be assessed. There is, therefore, an acute need to identify the underlying genetic cause responsible for FAP/AFAP in APC/MUTYH mutation negative families. Hypermethylation of CpG islands in the promoter of tumor suppressor genes can result in gene silencing, has been shown to be functionally equivalent to genetic mutations and can be inherited. Moreover, APC promoter hypermethylation is observed in ∼20% of sporadic colorectal tumors and correlates with the loss of gene expression. In our study, we used bisulfite treatment and direct sequencing of 2 regulatory regions of APC containing a total of 25 CpG dinucleotides, to investigate the possible role of germline hypermethylation of the APC promoter in FAP and AFAP families that were negative for APC and MUTYH mutations. Analysis of 21 FAP and 39 AFAP families did not identify signs of abnormal promoter methylation, indicating that this form of epigenetic silencing is not a common cause of FAP/AFAP. These results substantially contribute to clarify the potential role of germline epimutations as a cause of inherited predisposition to cancer. © 2007 Wiley-Liss, Inc.

Familial Adenomatous Polyposis (FAP) is the second most common syndrome of hereditary predisposition to colorectal cancer, and in the majority of cases it is inherited as a highly penetrant autosomal dominant trait. It is characterized by the presence of hundreds to thousands of adenomatous polyps in the colon and rectum of affected individuals, and would lead to colorectal cancer in virtually all the patients if left untreated. An attenuated form of FAP (AFAP) is characterized by a reduced number of polyps compared to classical FAP and a later age of onset. Together, FAP and AFAP affect up to 1 in 5,000 individuals. Inherited mutation or deletion of 1 allele of the adenomatous polyposis coli (APC) gene is responsible for 80% and 30% of FAP or AFAP cases, respectively.1–3 A small proportion of the FAP/AFAP individuals that do not have germline mutations in APC carry homozygous or compound-heterozygous mutations in the gene MutY homolog (MUTYH).4 However, the underlying genetic cause of FAP/AFAP is not known for a large proportion of the affected families.1–4 Precise identification of the genetic cause of this condition has a profound impact on the management of FAP/AFAP family members, and there is therefore an acute need to identify new genetic abnormalities that could be responsible for a significant number of these adenomatous polyposis syndromes in APC/MUTYH mutation negative families.

Methylation of cytosines in CpG dinucleotides located in the promoter region of tumor suppressor genes can result in transcriptional silencing. These epigenetic changes are conserved when cells divide, and in many cases are functionally equivalent to genetic changes such as mutations or deletions. Importantly, these epigenetic events can be inherited and cause cancer predisposition in affected individuals. This is the case of hereditary nonpolyposis colorectal cancer (HNPCC), another syndrome causing predisposition to colorectal cancer. HNPCC is the most common form of colorectal cancer predisposition and in most cases is caused by inheritance of a mutant allele of a gene involved in DNA mismatch repair (such as MLH1 or MSH2). It has recently been shown that germline hypermethylation of the MLH1 or MSH2 promoter is functionally equivalent to inactivating mutations and results in a clinical phenotype that resembles HNPCC.5–8 Importantly, although the APC promoter is hypermethylated in ∼20% of sporadic human colorectal tumors, and has been shown to be functionally equivalent to APC inactivating mutations, it is currently not known whether inherited germline hypermethylation of the APC promoter can be responsible for the FAP/AFAP phenotype observed in a subset of APC/MUTYH mutation negative families.

Material and methods

Clinical samples

Blood samples were obtained from 21 classical FAP and 39 AFAP unrelated individuals from Spain and Germany (see Table I). Screening for APC mutations was performed on DNA extracted from peripheral blood leukocytes using the protein truncation test for mutations in exon 15, denaturing high performance liquid chromatography for mutations in exons 1–14 and the first 400 base pairs of exon 15, and multiplex ligation-dependent probe amplification (MLPA) for the presence of large genomic deletions (German series) or direct sequencing of the full coding region and MLPA (Spanish series), as previously described.9, 10 MUTYH was genotyped by means of direct sequencing of the complete coding region (German series) or fluorescent hybridization probe melting curves using the Light Cycler instrument (Spanish series), as described.11, 12 All included patients were negative for APC or MUTYH mutations. Informed consent for genetic analysis was obtained from each patient, according to the Human Investigations and Ethical Committee approved research proposal of the Institution.

Table I. Clinical Phenotype in 60 FAP/AFAP Patients with APC/MUTYH Mutation-Negative familial Adenomatous Polyposis
Family IDAge at presentationNumber of polypsExtra-intestinal manifestationsFamily historyClassification
F-0010-9838>100YesYesFAP
F-0162-9931>100NoYesFAP
F-0163-9933>100NoNoFAP
F-0220-9932>100NoNoFAP
F-0323-0024>100NoYesFAP
F-0519-0039>100NoNoFAP
F-0651-0049>100NoYesFAP
F-1641-0451>100YesNoFAP
F-1714-0462>100NoNoFAP
F-2105-0548>100NoNoFAP
G105019 NoYesFAP
G105720 NoYesFAP
G118120MultipleNoYesFAP
G125920MultipleNoYesFAP
G131414MultipleNoYesFAP
G133340>200NoYesFAP
G140238 NoYesFAP
G62930>100NoYesFAP
G67824MultipleNoYesFAP
G80725MultipleYesYesFAP
G91818 NoYesFAP
F-0373-003031–100YesNoAFAP
F-0016-985015–50NoNoAFAP
F-0058-995415–50NoNoAFAP
F-0070-994315–50NoNoAFAP
F-0119-994515–50NoNoAFAP
F-0184-997215–50NoNoAFAP
F-0426-003315–50NoNoAFAP
F-0443-005415–50NoNoAFAP
F-0485-005615–50NoNoAFAP
F-0528-004315–50NoNoAFAP
F-0545-007115–50NoNoAFAP
F-0615-006515–50NoNoAFAP
F-0722-017215–50NoNoAFAP
F-1198-026015–50NoNoAFAP
F-1378-033515–50NoNoAFAP
F-1793-045215–50NoNoAFAP
F-1903-047815–50NoNoAFAP
F-1968-047315–50NoNoAFAP
F-2000-044915–50NoNoAFAP
F-2002-044750–100NoNoAFAP
F-2125-053015–50NoNoAFAP
F-2489-064815–50NoNoAFAP
G115540>20NoYesAFAP
G115649MultipleNoNoAFAP
G117760MultipleYesYesAFAP
G119351FewNoYesAFAP
G127251<100NoYesAFAP
G1362 MultipleNoYesAFAP
G14136040NoYesAFAP
G142556MultipleNoYesAFAP
G1427392NoYesAFAP
G143247 NoYesAFAP
G143362 NoYesAFAP
G70369 NoYesAFAP
G71548MultipleNoYesAFAP
G72140<100YesYesAFAP
G79462MultipleNoYesAFAP
G86166MultipleYesYesAFAP
G90676MultipleNoNoAFAP

Bisulfite treatment and sequencing

We used bisulfite sequencing of germline genomic DNA from a total of 60 FAP/AFAP unrelated individuals that were negative for APC and MUTYH mutations to investigate the levels of APC promoter methylation in a region spanning 229 bp (from −209 to +20) and containing 16 CpG dinucleotides (APC promoter 1A; Accession number U02509). In addition, we also studied a second regulatory region containing a CpG island (APC promoter 1B; Accession number D13981). The amplified 155-bp fragment contains 9 CpG dinucleotides.13, 14

Unmethylated cytosine residues in DNA samples were converted to uracil after sodium bisulfite treatment. Methylated cytosine residues are protected from this treatment and are observed as cytosine after PCR amplification with specific primers and sequencing, whereas unmethylated cytosines will be detected as thymidine residues. The primers for the bisulfite-modified APC promoter 1A were: 5′-GGG TTA GGG TTA GGT AGG TTG T-3′ (sense) and 5′-ACA CCT CCA TTC TAT CTC CAA TAA C-3′ (antisense). APC promoter 1B: 5′-AGG TTA GTA AGT GTT GTA ATT GAG ATT-3′ (sense) and 5′-AAA TAT TAC TAA CTT CCC ACA ACC C-3′ (antisense). The PCR conditions used were: hot-started at 94°C for 10 min, 35 cycles of 94°C for 30 sec, annealing at 65.5 or 61°C (Promoter 1A and 1B, respectively) for 30 sec and 72°C for 45 sec and 1 cycle of 72°C for 10 min. The PCR fragments obtained were sequenced using BigDye Terminator and an ABI PRISM 3100 sequencer (Applied Biosystems, Foster City, CA) as previously reported.15

Results and discussion

Members of FAP/AFAP families greatly benefit from genetic counseling, but the identification of the genetic cause underlying this syndrome in each family is essential for optimal management. Since 1991 it is known that the most common cause of FAP/AFAP is the inheritance of an inactivated allele of APC.2, 3 Point mutations in APC or deletion of large genomic segments involving 1 allele of APC are responsible for ∼80% of the FAP cases and up to 30% of AFAP patients.1–3 APC is an important regulator of Wnt signaling in the normal intestinal epithelium. In the absence of a functional APC protein, β-catenin accumulates in the cytoplasm and translocates to the nucleus, where it binds to TCF/LEF transcription factors and regulates the expression of important regulators of the cell cycle such as c-Myc and Cyclin-D1.16–18 Mutations in APC are the hallmark of sporadic colorectal cancer and are present in more than 80% of these somatic tumors.19 Genetic analyses aimed at the identification of the cause of FAP/AFAP normally involve direct sequencing of the full coding region of APC and gene deletion analysis. These analyses, however, fail to identify APC genetic abnormalities in more than 20% of the FAP/AFAP families. Although MUTYH mutations are responsible for the phenotype observed in a small number of these families, no genetic defects can be identified in the vast majority of them. Therefore, although the phenotype observed in these families is compatible with the presence of an inactive allele of APC, cancer risk assessment for individual family members can only be achieved through individual genetic testing, and this requires the identification of the underlying mechanism of APC inactivation in the family.

Epigenetic silencing of the promoter of tumor suppressor genes through hypermethylation of regulatory regions rich in CpG dinucleotides known as CpG islands, is frequently observed in colorectal tumors and has been shown to significantly contribute to tumor initiation and/or progression. Hypermethylation of CpG islands in the APC promoter is a common event in sporadic colorectal tumors, and this epigenetic event has been shown to be functionally equivalent to genetic inactivation of APC. Like genetic alterations, epigenetic changes or “epimutations” can be inherited. Moreover, it has recently been reported that germline epimutation of MLH1 and MSH2 two genes involved in the repair of DNA mismatches—can cause an HNPCC phenotype.5–8

In our study, we used bisulfite sequencing of germline genomic DNA from 21 FAP and 39 AFAP families that are negative for mutations in APC and MUTYH (Table I) to investigate the levels of APC promoter methylation in a region spanning 229 bp (from −209 to +20), and containing 16 CpG dinucleotides (APC promoter 1A). This region has previously been reported to be hypermethylated in sporadic colorectal tumors, and the methylated state has been shown to result in a loss of APC expression.14, 20 In addition, we also studied a second regulatory region containing a CpG island (APC promoter 1B) containing 9 additional CpG dinucleotides.13, 14

As a control, DNA samples methylated in vitro with SssI CpG methylase (CpG Genome Universal Methylated DNA; Chemicon International, Temecula, CA) were bisulfite treated and the fragments corresponding to the APC promoter 1A and 1B were PCR-amplified. All cytosine residues in CpG dinucleotides were methylated and thus detected as cytosines after sequencing, whereas the remaining unmethylated cytosines were observed as thymidine residues (Fig. 1d). Moreover, sequencing of bisulfite-treated DNA samples from LS1034, a colon cancer cell line reported to carry a methylated and an unmethylated copy of APC promoter 1A,21 readily detected the level of methylation of this sample (Figs. 1c and 1d). Because DNA samples from FAP/AFAP patients carrying bona fide germline epimutations would be expected to contain approximately equimolar amounts of methylated and unmethylated alleles, these results indicate that this methodology can be used to investigate the presence of germline hypermethylation of the APC promoter. However, we did not detect germline CpG methylation in any of the 60 FAP/AFAP families investigated (Fig. 1), suggesting that germline promoter hypermethylation in the APC promoter is not a common mechanism of APC inactivation causing the FAP/AFAP syndrome observed in APC/MUTYH mutation negative families.

Figure 1.

Bisulfite sequencing of APC promoter sequences. (a) The genomic structure of APC (5q21-22) is shown. APC has 16 exons represented by black boxes numbered 1–16. The 2 regulatory regions studied are indicated by arrows (Promoter 1A and B; accession numbers U02509 and D13981, respectively). (b) Representative results of sequencing chromatograms are shown. (c) The location of the CpG dinucleotides studied is represented by circles in a subset of representative cases studied. Open circles represent unmethylated CpG dinucleotides, filled circles methylated CpGs and partially methylated CpGs are indicated by open/filled circles. The published CpG dinucleotide located at position 89–90 of the APC promoter 1B (D13981), but not found in our samples, is marked by an asterisk. IVD: in vitro methylated DNA. (d) Methylation status of the 2 CpG dinucleotides shown within the box in panel (c). The sequence of an in vitro methylated bisulfite treated sample is shown on the top; DNA sequence of LS1034 cells with 1 methylated and 1 unmethylated copy of APC promoter 1A is shown in the middle; an unmethylated sample from a FAP patient is shown in the bottom. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

A detailed inspection of the remaining of the bisulfite transformed sequence corresponding to the 2 regulatory regions investigated did not reveal any consistent changes from the published sequences (U02509 and D13981), with the exception of one of the CpG dinucleotides located at position 89–90 of the APC promoter 1B (D13981; marked with an asterisk in Fig. 1c). The CG dinucleotide in this position should be observed as a TG or CG in bisulfite-treated samples depending on the unmethylated or methylated state, respectively. Instead, a homozygous GT was observed at this position, which was shown to be derived from a GC in the genomic untransformed sequence. In addition, we found a homozygous insertion of 1 G in position 108 of the APC promoter 1B (D13981) in all the cases studied. However, because these changes were found also in homozygosis in the 7 healthy blood donor samples studied, it is unlikely to be linked to FAP/AFAP and they most likely represent errors in the published sequence (D13981).

In summary, we report here that germline hypermethylation of the CpG islands in the APC promoter regions investigated is not a common mechanism responsible for the FAP/AFAP syndrome observed in APC/MUTYH mutation negative families. These results are in good agreement with an earlier study where the presence of methylation of 3 CpG dinucleotides within the APC promoter 1A was investigated in FAP and AFAP cases.22 Here we extended the analysis to a total of 16 CpGs in APC promoter 1A and further investigated the possible contribution of a second CpG island in APC promoter 1B. It remains possible, however, that CpG hypermethylation of other regulatory regions may account for this phenotype in a subset of these families. Although no gross abnormalities were observed in the bisulfite-transformed sequence of the regulatory sequences studied (384 bp in total), it is also possible that mutation of other APC promoter regions and/or intronic sequences that are not routinely investigated could be the cause of a FAP/AFAP phenotype. However, these results further contribute to the idea that mutations in genes other than APC and MUTYH could account for a significant subset of FAP/AFAP cases.

Acknowledgements

The authors thank the Vall d'Hebron Blood and Tissue Bank for supplying the control samples used in these investigations. This study was partly funded by Fondo de Investigaciones Sanitarias (FIS 05/1394 and CP05/00256) and Fundación de Investigación Médica Mutua Madrileña to Diego Arango and Spanish Networks RCESP (C03/09) and RTICCC (C03/10) from Instituto de Salud Carlos III to Gabriel Capellá.

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