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APC Gene Mutations Causing Familial Adenomatous Polyposis in Polish Patients

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APC Gene Mutations Causing Familial Adenomatous Polyposis in Polish Patients J Appl Genet 49(4), 2008, pp. 407–414 Original article APC gene mutations causing familial adenomatous polyposis in Polish patients Andrzej Plawski, Ryszard Slomski Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland Abstract. Familial adenomatous polyposis (FAP) is a well-known hereditary condition characterised by alimen- tary system tumours. Tens to thousands of polyps occur in the colon and rectum of the patients. There is a high heterogeneity with regard to the number and time of the occurrence of polyps. The occurrence of FAP is associ- ated with mutations in the APC tumour suppressor gene, which was described in 1991. Since then, many studies have been done to analyse the distribution of mutations in individual populations and to determine the function of the gene and a diagnostic approach to FAP. Here the APC gene was studied with respect to the occurrence of small mutations and large rearrangements in 300 unrelated Polish FAP families. Ninety-seven mutations were identified in 164 families. Out of these mutations, 80 were small mutations, including 58 small mutations that were first identified in the Polish population (42 novel and 16 described previously). An increased frequency of mutation c.3927_3931delAAAGA was observed in 10% of the Polish group. Seventeen large rearrangements were found in 29 families. Out of those rearrangements, 8 repeat rearrangements occurred in 20 families. A prob- lem in fast molecular diagnostics of FAP is a high heterogeneity of mutations in the APC gene. It seems that a multiplex ligation-dependent probe amplification test and searching for small mutations by the use of screening methods at the 5’ end of exon 15 and exons 14, 9, 11, 13, 5, and 3, help to improve the molecular diagnostics of FAP in Polish patients. Keywords: FAP, APC gene, colorectal cancer, familial adenomatous polyposis, multiplex ligation-dependent probe amplification. Introduction 5-year-old child (Krush et al. 1988). The polyps have a high potential for development towards Familial adenomatous polyposis (FAP) is a disor- malignant tumours. This can occur from the late der that is inherited in an autosomal dominant childhood to the 70s. Attenuated adenomatous manner, with a predisposition to the occurrence of polyposis coli (AAPC) cases are characterised by a large number of polyps in the colon. First symp- a benign course of the disease, as opposed to the toms of FAP are diarrhoea and blood in stool. classic FAP. Symptoms of this disorder are ob- Weight loss and weakness are observed when tu- served at the age of 35–45. There are usually mour development is advanced. The incidence of 10–100 adenomatous polyps (adenomas) and flat FAP is one per 10 000 newborns (Cruz-Correa and adenomas in the colon. The occurrence of Giardiello 2003). The classic form of FAP is char- extraintestinal symptoms is not as common as in acterised by the occurrence of more than 100 pol- the classic FAP. Many papers have documented yps, which appear in the second decade of life. The symptoms like polyps of the fundus of the stomach average time of polyp occurrence is 15 years and adenomatous polyps or intra-ductal papillary (Cruz-Correa and Giardiello 2003). The earliest mucinous neoplasms of the pancreas (Chetty et al. symptoms of polyposis were observed in a 2005; Matsubara et al. 2000; Moisio et al. 2002; Received: June 11, 2008. Accepted: September 5, 2008. Correspondence: A. Plawski, Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60–479 Poznan, Po- land; email: [email protected] 408 A. Plawski, R. Slomski Soravia et al. 1998). In 1991, a relationship be- cerous disorders is very essential for the lowering tween the APC gene and FAP was discovered. of costs and improving the testing efficiency. To This was followed by a number of molecular stud- improve molecular diagnostics, it is important to ies, which generated a large number of data on the describe the nature and frequency of mutations in mutation spectrum in new countries or ethnic various ethnic groups. The aim of this study was to groups. Currently, the most up-to-date database of recognise the spectrum of small mutations and APC mutations of the APC gene can be found at the In- large rearrangements in the gene. This study, stitute of Medical Genetics in Cardiff, http://www. based on the investigation of 300 unrelated FAP hgmd.cf.ac.uk/ac/index.php (Stenson et al. 2003). patients from all over Poland, made it possible to APC It contains data on 858 types of mutations. Small determine the most effective way to detect deletions (356) are the majority of mutations in the gene mutations in Polish FAP patients. APC gene. They make a change in the open read- ing frame (ORF) and shorten the gene product. Materials and methods The 235 mutations are missense or nonsense mu- tations that proceed to the Stop codon at the site of The studied group consisted of 300 unrelated FAP mutation. So far, only 131 small insertions have patients, derived from all over Poland, whose been described. Large deletions (54), mutations in DNA samples were collected in the DNA bank of splicing areas (49), small deletions connected with Polish FAP families, established in 1997 at the In- insertions (17), large insertions (7), and rearrange- stitute of Human Genetics in Poznan. FAP patients ments (6) were rare, and in regulatory sequences were diagnosed in the health centres appropriate to only (3) mutations were found. Most mutations in patients’ place of residence, cooperating with this the APC gene contribute to the change in the ORF Institute. Patients who agreed to the genetic tests and shortening of the gene product. Deletions or were directed to molecular tests by gastro- insertions of a few base pairs (68% of all muta- enterology clinics or genetic centres. The control tions) lead to the frame shift. As a result, the Stop group comprised 100 individuals, who consisted codon develops close to the mutation site. Substi- of 50 unrelated men and 50 unrelated women ran- domly chosen from the Polish population. tutions constitute 30% of all cases, and where the Our investigations were approved by the Ethi- Stop codon appears, it is the site of change. Most cal Committee of the Medical University in of germline mutations are at the 5’ end of the gene. Poznan. The mutation cluster region (MCR) with a higher frequency of germline mutations was identified in DNA extraction the APC gene. The MCR is between codons 1250 and 1464 (Cetta and Dhamo 2007). Mutations are The collected blood samples on anticoagulant, to- heterogeneous in the APC gene. Two of them ex- gether with the patients’ permissions to carry out hibit an increased frequency: deletion of 5 bp at genetic tests, were sent to the Institute of Human codon 1309 (c.3927_3931delAAAGA) and 5 bp at Genetics in Poznan. DNA was extracted from pe- ripheral blood cells with the phenol purification codon 1061 (c.3183_3187delACAAA). The mu- method (Lahiri and Schnabel 1993). tation c.3927_3931delAAAGA is the most fre- APC quent mutation in the gene. Its frequency Molecular analysis of small mutations varies from 0% in southwest Spain through 2.4% in the Australian population, 5% in the Dutch pop- The earlier described primers were used to am- ulation, 7% in the Israeli population, and up to plify the fragments that included individual 16% in Italian FAP patients (Gavert et al. 2002; exon-splicing sites. The amplified fragments of APC Ruiz-Ponte et al. 2001; Schnitzler et al. 1998; the gene were screened for mutations involv- ing the heteroduplex analysis (HD) and detection Varesco et al. 1993a). The frequency of the second of single-stranded conformational polymorphism mutation, c.3183_3187delACAAA, varies from (SSCP) (Plawski et al. 2001). The heteroduplex 0% in north Spain through 1.5% in the Israeli pop- DNA fragments detected in the HD analysis or ulation, and up to 8.4% in FAP patients from Aus- various patterns in the SSCP analysis were further tralia (Gavert et al. 2002; Ruiz-Ponte et al. 2001; analysed by direct PCR product sequencing, using Schnitzler et al. 1998; Varesco et al. 1993a). MEGABace 500 sequencer (General Electric The optimization of molecular diagnostics of Health Care USA) according to the manufac- hereditary predispositions to the incidence of can- turer’s specifications. APC mutations in Polish FAP patients 409 Detection of large rearrangements in the APC Stop codons. One of them was an Ashkenazi gene variant. In 8 cases, a change in amino acids was observed, which did not appear in the control Multiplex ligation-dependent probe amplification group . Six substitutions occurred at splice sites. (MLPA) analysis was conducted on extracted DNA from 165 probands, in which previously no APC gene rearrangements small sequence alterations were identified. The MLPA kit was used for the analysis of locus A total of 165 unrelated patients were studied, in APC; SALSA MLPA KIT P043 APC (MCR Hol- which no small sequence mutations were previ- land Amsterdam, The Netherlands). The analysis ously found but 17 large changes in 29 FAP fami- was conducted according to the manufacturer’s rec- lies were observed (Table 2). Eight large ommendations (http://www.mrc-holland.com).The rearrangements occurred more frequently than in analysis of fragments was conducted with the use of one family. The most common deletion occurred the MegaBACE 500 kit (General Electric Health within promoter sequence 2. It was identified in 4 Care USA). The ET-550 marker (General Electric cases. The deletion of the whole APC gene was de- Health Care USA) was employed as a molecular tected in 3 families. Each deletion of exons 3, 4, weight marker.
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