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The adenomatous polyposis coli (APC) gene, localized on chromosome 5, consists of 15 exons. The largest is the exon 15, which comprises more than 75% of the coding sequence of the gene and is the most common target for both germline and somatic mutations (Groden et al, 1993). The germline mutations in the APC gene are in charge of the familial adenomatous polyposis (FAP), a rare autosomal inherited disease (Hegde and Roa, 2006). These mutations predispose to develop colonic polyps and other extraintestinal neoplasms, including desmoid tumour in subjects between 15 and 60 years. The estimated risk of developing a desmoid tumour in patients with FAP is between 4 and 20 percent. This risk increases about 2 years after colectomy (Groen et al, 2008).
We studied a woman (56 years old) affected with FAP and desmoid tumour and her three children (two daughters and one son). This study was carried out in accordance with the World Medical Association Helsinki Declaration, adopted in 1964 and amended in 1975, 1983, 1989, 1996 and 2000 (World Medical Association, 1998). Informed consents were obtained from all subjects, and the study was approved and conducted according to the ethical guidelines of the Second School of Naples. The pedigree was generated by the genetic counsellor and the case-histories of the subjects were collected.
The pedigree and the main clinical data are summarised in Figure 1. The family originated from the Campania, a region of southern Italy. The proband affected with FAP had surgical treatment with restorative proctocolectomy and ileal J-pouch at the age of 54 years. Two years after the pouch procedure the proband developed desmoid tumour, consistent with previous reports (for example, Groen et al, 2008).
Out of three children of proband, a daughter and a son were affected with FAP, diagnosed at the ages of 28 year and 18 year, respectively. Both siblings received ileal J-pouch procedure. The eldest daughter (33 years old) did not display the disease phenotype.
Peripheral blood from subjects was collected using standard procedures and all 15 exons of the APC gene and the flanking splice site regions were amplified using a set of 28 primer pairs (Table 1). One set of primers was used to amplify each exon and the adjacent intronic region except for exon 15, which was amplified as 14 separate PCR fragments due to it large size.
PCR products were sequenced using the ABI PRISM di-Deoxy Terminator Cycle sequencing kit in an ABI 9700 Thermal Cyclers and ABI PRISM 310 Genetic Analyzer. Deviations from the wild-type sequence were identified using Mutation Surveyor, sequence analysis and assembly software.
The analysis of the APC gene in the DNA sample from proband revealed the presence of the novel deletion, 1605_1606delTG. This frameshift mutation is located in exon 12 of APC gene and is characterized by a deletion of two nucleotides, generating a premature stop codon between codons 537 and 538 (Figure 2). The presence of the mutation was confirmed by resequencing a second DNA sample from the patient.
The genetic analysis conducted in family members showed the same mutation in the daughter and the son affected with FAP, while the healthy daughter did not have this mutation (Figure 1).
In this study, we identified in the proband, affected with FAP and desmoid tumour the novel mutation 1605_1606delTG. The proband passed on the mutation to one of her daughters and the son resulting in FAP phenotype. The different onset age of FAP in proband, daughter and son (54, 28 and 18 years, respectively) indicates intrafamilial variability. In the FAP, the sites of the mutation determine the presence or the absence of extracolonic manifestation of the disease, as desmoid tumours. The identified frameshift mutation is located in exon 12 at codon 535 in the 5′end of APC gene, a region of APC gene not usually associated with desmoid tumor. Indeed, the previous studies show that in patients affected with FAP and desmoid tumour the mutations of APC gene are localized predominantly in exon 15 (Lips et al, 2009). It is possible that the site of the mutation may also influence the severity of desmoid phenotype (Couture et al, 2000). It has been previously reported that the most severe desmoid phenotype (penetrance=100%) presents in families which carry mutations in the 3′ end of the APC gene (Eccles et al, 2001).
In the proband, after three months of medical treatment with Toremifene, a selective oestrogen receptor modulator, the abdominal ultrasound showed no detectable hydronephrosis and the magnetic resonance imaging documented a downsizing of the mass. These data suggest that the mutations that are not in the 3′ end of the APC gene may be associated with a less severe phenotype.
Both siblings affected with FAP and positive for APC gene mutation received colectomy nearly an year ago and at present are undergoing a clinical follow-up program. We propose that a longer follow-up could clarify the association between this novel mutation and the development of desmoid tumour. The identification of this novel mutation in APC gene and the outcome of the clinical follow-up of the subjects of this study are likely to be helpful in the FAP patient treatment and care strategies in the future.
This study was supported by the Regione Campania Research Grant Program 2005.