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 associated 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 mutations 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. 12, and 15M occurred in 2 families. Amplifica- tions were identified in 6 families, including the amplification of exon 11 in 3 families. The largest Results deletions concerned the whole APC gene, exon 14 and whole exon 15, or exons from 10 to 14 and Small sequence alterations from 11 to 13. Large rearrangements occurred in 9.6% of the studied families. We screened 300 probands for small mutations in the APC gene. Eighty types of small mutations in 135 families were identified (Table 1). The most Discussion frequent mutation in the Polish FAP patients was the deletion c.3927_3931delAAAGA. This muta- The analysis was performed for 300 Polish fami- tion occurred in 10.3% of families. The second de- lies with FAP, and 97 mutations were found in 164 letion, c.3183_3187delACAAA, was observed in families. Most of the detected mutations were 4% of families. Mutation c.3202_3205delTCAA small. Six mutations occurring in introns close to was found in 5 probands (1.7%). Ten repeat muta- the exon–intron border were identified. Two of tions occurred in 65 families, which constituted them were described previously, and the remain- 47.4% of all the diagnosed families with small ing 4 did not occur in the studied control group. mutations, or 21% of all studied patients. Out of The IVS3-14T>G substitution, which is 14 nu- the 80 small mutations found in the Polish FAP pa- cleotides away from the exon–intron border, is tients, 42 were novel mutations, 16 mutations novel. No studies were performed on the mRNA were first described by our research team in the level but the absence of the substitution Polish population previously (Plawski et al. 2004; IVS3-14T>G in the control group and its Plawski et al. 2007a; Plawski et al. 2007b), and 22 cosegregation with the phenotype of this disorder mutations were earlier described in other popula- in the family indicates the pathogenic character of tions. In total, 58 mutations were first observed in IVS3-14T>G. The most common mutation in our the Polish population. population studied was c.3927_3931delAAAGA. Fifty-four mutations that occurred in 102 fami- The frequency of c.3927_3931delAAAGA varied lies were ORF mutations, from which 40 deletions depending on the population and it was the most ranged from 1 to 11 bp. One mutation (deletion of common mutation in the Polish FAP patients. Its 3 adenines from codons 793 and 794) caused a frequency was more than 10% and was one of the change of glutamine and serine to arginine. Other highest in the studied groups (Gavert et al. 2002; mutations led to a change in the ORF and to the de- Plawski et al. 2007b; Ruiz-Ponte et al. 2001; velopment of the Stop codon in close proximity to the site of mutation. The remaining 14 mutations Schnitzler et al. 1998; Varesco et al. 1993b). were insertions or duplications ranging from 1 to The frequency of the second most common muta- 8 bp. All insertions and duplications led to ORF al- tion (c.3183_3187delACAAA) in the Polish pa- terations and premature termination of the transla- tients reached 4% . The high heterogeneity of the APC tion of APC protein. In 33 families, 26 gene mutation is noteworthy. Out of the 80 substitutions were observed, of which 12 led to small mutations that were found in the Polish FAP 410

Table 1. Characteristics of the sequence alterations in the APC gene detected in Polish FAP patients Mutation Exon/ Consequence Affected Source intron of mutation families 1234 5 IVS3-14T>G 3 splice site 1 novel IVS3-1G>C 3 splice site 1 (Brozek et al. 2008; Hutter et al. 2001) c.517_521delCCTT 4 p.Pro173fs 1 (Plawski et al. 2007a; Stekrova et al. 2007) c.601delG 5 p.Glu201fs 1 (Plawski et al. 2004) c.636delA 5 p.Lys212fs 1 (Plawski et al. 2004) c.646C>T 6 p.Arg216X 1 (Lamlum et al. 1999) c.892_893delCA 8 p.Ser294fs 1 (Plawski et al. 2004) IVS9-1G>C 9 splice site 1 novel IVS9+5G>C 9 splice site 1 (Mihalatos et al. 2005) c.1111_1112delGG 9 p.Gly371fs 1 novel c.1288_1291delGGCA 9 p.Gly430fs 1 novel c.1297C>T 9 p.Pro433X 1 (Friedl et al. 2001) c.1429G>T 11 p.Glu477X 2 novel c.1490_1491insT 11 p.Leu497fs 2 (Plawski et al. 2004) c.1500T>A 11 p.Tyr500X 4 novel IVS13-3T>G 13 splice site 1 novel IVS13 +1 G>A 13 splice site 1 novel c.1744G>T 14 p.Glu582X 1 novel c.1817delG 14 p.Ile606fs 1 novel c.1816_1817InsA 14 p.Ile606fs 1 novel c.1829delA 14 p.Asp610fs 2 novel c.1873delC 14 p.Gln625fs 1 novel c.1875_1878delGACA 14 p.Gln625fs 1 novel c.2292_2293insA 15 p.Phe764fs 1 novel c.2365_2366insC 15 p.Gln789fs 1 novel c.2377_2379delAAA 15 p.Gln793fs 1 novel c.2413C>T 15 p.Arg805X 1 (Dobbie et al. 1996) c.2482A>G 15 p.Thr828Ala 1 novel c.2527_2530delAGTT 15 p.Ser843fs 1 (Chen et al. 2006) c.2609delC 15 p.Pro870fs 1 (Plawski et al. 2004) C.2626C>T 15 P.Arg876X 2 (Ficari et al. 2000; Furuuchi et al. 2000; Nordling et al. 1997; Prosser et al. 1994; Valanzano et al. 1996) c.2645_2649delCCACT 15 p.Thr882fs 1 novel c.2797_2800dAACA 15 p.Asn933fs 1 novel c.2802_2805delTTAC 15 p.Thr934fs 1 (Truta et al. 2005) c.2805C>A 15 p.Tyr935X 1 (Groden et al. 1993) c.2815_2816delAA 15 p.Lys939fs 1 novel c.2853T>G 15 p.Tyr951X 1 novel c.2922_2923insG 15 p.Gly974fs 1 (Plawski et al. 2004) c.2932C>T 15 p.Gly978X 3 (Kim et al. 2001) c.3090_3091insA 15 p.Asn1031fs 1 novel c.3093T>G 15 p.Asn1031X 1 (Homfray et al. 1998) c.3109_3110delAA 15 p.Asn1037fs 1 novel c.3118_3119dupCTCTGGAA 15 p.Arg1040fs 1 (Plawski et al. 2004) c.3164_3168delTAATA 15 p.Ile1055fs 1 (Mandl et al. 1994b; Miyoshi et al. 1992) c.3183_3184insA 15 p.Gln1062fs 1 novel C.3183_3187delACAAA 15 P.Lys1061fs 12 (Groden et al. 1991; Miyoshi et al. 1992; Wakatsuki et al. 1998) c.3183delA 15 p.Lys1061fs 1 novel c.3184_3187delCAAA 15 p.Gln1062fs 1 (Mandl et al. 1994a) c.3202_3205delTCAA 15 p.Ser1068fs 5 (Paul et al. 1993) c.3225T>A 15 p.Tyr1075X 1 (Palmirotta et al. 1995) c.3371delA 15 p.Asn1124fs 1 (Plawski et al. 2004) c.3473_3474delGA 15 p.Arg1158fs 1 (Plawski et al. 2004) APC mutations in Polish FAP patients 411

Table 1 continue. 1234 5 c.3515delA 15 p.Ser1171fs 1 (Plawski et al. 2004) c.3538delA 15 p.Val1173fs 1 novel c.3577_3580delCAGT 15 p.Gln1193fs 1 novel c.3580delT 15 p.Ser1194fs 1 (Plawski et al. 2004) c.3613delA 15 p.Ser1205fs 1 (Plawski et al. 2004) c.3645T>G 15 p.Ser1215Arg 1 novel c.3685T>C 15 p.Leu1129Ser 1 novel c.3807_3808delAT 15 p.Ile1269fs 1 novel c.3919T>A 15 p.Ile1307Leu 1 (Laken et al. 1997) c.3921_3924DelAAAA 15 p.Ile1307fs 1 (Walon et al. 1997) c.3923A>G 15 p.Glu1308Arg 1 novel c.3927_3931delAAAGA 15 p.Glu1309fs 31 (Groden et al. 1991) c.3929delA 15 p.Glu1309fs 1 novel c.3930_3931insG 15 p.Lys1310fs 1 novel c.3933delT 15 p.Ile1311fs 1 novel c.3949G>C 15 p.Glu1317Gln 1 (Frayling et al. 1998; van Puijenbroek et al. 2008) c.3977_3978insC 15 p.Val1326fs 1 novel c.3977_3978insT 15 p.Val1326fs 1 novel c.4129_4130delGT 15 p.Val1377fs 2 (Plawski et al. 2007b) c.4144C>T 15 p.Ser1382Phe 1 novel c.4266_4276delTCTTCCAGATA 15 p.Asp1422fs 1 (Plawski et al. 2004) c.4394_4395dupGA 15 p.Ser1465fs 1 (Plawski et al. 2004) c.4394_4395insAG 15 p.Ser1465fs 1 novel c.4588delG 15 p.Thr1530fs 1 novel c.4609delA 15 p.Thr1537fs 1 novel c.4650_4651delGA 15 p.Glu1550fs 1 novel c.4666_4667insA 15 p.Thr1556fs 1 (Bjork et al. 2001; Shitoh et al. 2000) c.5465t>a 15 p.Asp1822Val 1 (Wallis et al. 1999) Novel: mutations absent in The Human Mutation Database at the Institute of Medical Genetics in Cardiff http://www.hgmd.cf.ac.uk/ac/index.php and in reviewed literature. Mutations described in the Polish population for the first time are marked as “novel” (42), or the remaining ones (16) were described earlier by P³awski The cDNA numbering is based on reference sequence (GenBank NM_000038); +1 corresponds to the A of the ATG translation initiation codon.

Table 2. Characteristics of the large rearrangements of patients, 58 were identified for the first time and, out APC the gene detected in Polish FAP patients of them, only one mutation (c.517_521delCCT) No. Mutation Exons Affected was later found in the Czech population (Plawski families et al. 2007a; Stekrova et al. 2007). Most of the de- 1 deletion whole APC gene 3 2 deletion promoter 1 2 tected mutations (72.5%) are characteristic for our 3 deletion promoter 2 4 Polish populations. These mutations were found in 4 deletion 3 2 58 families diagnosed with a small mutation (43% 5 deletion 4 2 of all diagnosed families). Mutations characteris- 6 deletion 10 1 tic for Polish patients do not occur with increased 7 deletion 10–14 1 frequencies. One of the newly identified mutations 8 deletion 11–13 1 was c.1500T>A, which was detected in 4 families. 9 deletion 12 2 Three other mutations were found in 2 families. 10 deletion 14 1 The contribution of these mutations to the process 11 deletion 14-15M 1 of determining the risk of the disorder was lower 12 deletion 14-15 1 13 deletion 15M 2 in the diagnosed families in spite of the fact that 14 amplification 3 1 those mutations constituted more than 70% of all 15 amplification 7 1 detected mutations. We observed 10 small muta- 16 amplification 11 3 tions that occurred in more than one family. These 17 amplification 15M 1 mutations augmented the risk of the disorder in 65 families. No small mutations at exons 1, 2, 3, 7, 10, 12, and 13 were identified in our studies. Most 412 A. Plawski, R. Slomski of the diagnosed small mutations were detected at the most common mutations, the remaining repeat exon 15 of the APC gene. These mutations condi- mutations determined the occurrence of the disor- tioned the risk of the disorder in 106 diagnosed der only in 21 families. Thus, for the optimization families. Mostly, the mutations occurred at the 5’ of molecular diagnostics of the APC gene in the end of exon 15. A considerable rearrangement in Polish population, the most useful way will be to the APC gene was found in 10% of the studied apply the MLPA test and next study the 5’ end part families. Seventeen mutations occurred in 29 fam- at exon 15 of the APC gene (codons 784–1556) ilies, of which 8 were repeated mutations deter- and then successively exons 14, 9, 11, 13, 5, and 3. mining the occurrence of the disorder in 20 families. Frequent changes were found within Acknowledgements. This study was supported by the promoter, although the deletion of the entire the Ministry of Education and Science Poland (grant APC gene was by no means infrequent. 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