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Familial adenomatous polyposis (FAP) is a genetically determined disorder that is inherited in an autosomal dominant manner. The occurrence of FAP is associated with mutations in the APC gene, which were described in 1991.1De novo mutations of the APC gene occur in one per 10 000 newborns. The APC gene is localised on chromosome 5q21 and consists of 21 exons. In most cases, mutations of the APC gene are small deletions or insertions: the AAAGA deletion at codon 1309, which occurs in 10% of families with FAP, and the ACAAA deletion at codon 1061, which occurs in 5% of families with FAP, are the most frequent mutations. Ninety-two percent of all mutations in the APC gene lead to truncations of the APC protein product. Dysfunction of the APC gene causes the accumulation of B-cathenin and the expression of genes that promote cell division.
The FAP syndrome contributes only a relatively low percentage of all colorectal carcinomas (1–2%) and is characterised by the presence of numerous (at least 100) polyps that line the mucosa of the large intestine and rectum. The occurrence of other gastrointestinal adenomas, cutaneous sebaceous cysts, osteomas (mostly in the jaw, scapula, and long bones), connective tissue neoplasms, desmoid tumours, and, in some cases, coexisting duodenal and thyroid carcinomas (which together are classified as Gardner syndrome) and less common structural deformations in the teeth may also be observed.2,3 The DNA bank of Polish families with FAP was established in 1997 at the Institute of Human Genetics, Polish Academy of Science in Poznań.
This study reports a spectrum of mutations of the APC gene in Polish patients with FAP.
MATERIAL AND METHODS
Clinical diagnoses of FAP in patients were established in genetic centres or gastroenterology clinics in Poznań, Szczecin, Kraków, Wrocław, Gdańsk, Warszawa, and Lódź, appropriate to the patients’ place of residence. Families with FAP came from the following regions of Poland: 79 from the central west, 17 from the northwest, seven from the south east, six from the southwest, two from the north, and nine from the central east. To date, samples from 315 people from 120 families with FAP have been collected in the Polish FAP DNA bank. In this group, 140 patients had typical FAP, while eight patients had atypical FAP. Mutations in the APC gene were analysed in 120 probands. If mutations were identified in a proband, the members of the proband’s families were also screened (if available).
We extracted DNA from peripheral blood cells with the classical phenol purification method. We screened fragments of the APC gene that encompassed exons 5–8, exons 10–14, and the fragment from A to L of exon 15 of the APC gene for mutations with heteroduplex analysis and single strand conformational polymorphism methods. DNA fragments that showed heteroduplex in heteroduplex analysis or additional patterns in single strand conformational polymorphism analysis were sequenced by direct polymerase chain reaction product sequencing and analysed with ALFExpress (Amersham-Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s specifications. New mutations were confirmed to be absent in 50 unrelated people in the control group, which consisted of 25 unrelated women and 25 unrelated men randomly chosen from the Polish population.
The aim of this study was to investigate mutations in the adenomatous polyposis coli (APC) gene in the Polish DNA bank for families with FAP.
120 Polish families with familial adenomatous polyposis (FAP) were screened for mutations in the part of the APC gene that encompasses exons 5-8, exons 10-14, and the 3′ part of exon 15. DNA was screened with heteroduplex analysis, single strand conformational polymorphism methods, and DNA sequencing.
Mutations in the APC gene were found in 42 (35%) Polish families with FAP, and 22 types of mutation in the APC gene were identified. Of these, 15 mutations were deletions of 1-11 base pairs, five were insertions of 1-8 base pairs, and two were substitutions. Overall, 14 of the identified mutations were not seen in other populations. De novo mutations occurred in three families; one of these mutations had not been described before.
Most of the detected mutations in 88% are localised at the 5′ end of exon 15 of the APC gene.
RESULTS AND DISCUSSION
We initially analysed DNA from 120 probands in the DNA bank for mutations in the APC gene. Mutations were found in 42 (35%) probands (table 1). We identified 22 types of mutations in the group studied; 14 of these were not seen in other populations. Five mutations recurred in the families that we examined (table 2). In three families, probands had de novo mutations.
Most mutations were found in the 5′ region of exon 15; in addition, two mutations were found in exon 5, one in exon 8, and one in exon 11. No mutations outside exon 15 had been described previously. In exon 11, we saw insertion T in position 1491 in two families. In both families, differences were seen in the age of onset of polyps. In family 9129, symptoms were noted when patients were 10 and 12 years old, while in family 9001, symptoms were noted later (table 1). Different genetic backgrounds in these families must have influenced the time of onset of the disease. In both families, probands had brain cancer, and proband 9001 also had a desmoid tumour, which is linked with mutations located between codons 1403 and 1578p; in both cases in our study, the affected proband had a mutation in exon 11.2–4
The mutation 636delA in exon 5 is a new mutation that occurred de novo in family 9017. We detected two other de novo mutations in the Polish population: 2413C>T (Arg>Stop) and 4393–4394Ins GA both were described in 1996 by Dobbie and colleagues.5 The youngest patient with FAP who we studied was a girl who at the age of four years already had numerous polyps in the colon and sparse duodenal polyps and who was a carrier for the known mutations 3921–3924delAAAA.1
The most frequent mutations in Polish families with FAP were 3927–3931del AAAGA (del 5 bp at 1309), which occurred in 15 (12.5%) families, and 3183–3187delACAAA (del 5 bp at 1061), which was seen in six (5%) families. The frequency of these mutations varies depending on populations. The frequency of the most common deletion, 5 bp at 1309, in other populations varies from 0% in northwest Spain through 2.4% in Australian populations, 5% in Dutch populations, and 7% in Israeli populations up to 16% in the group reported by Varsco and colleagues.6–9 The deletion 5 bp at 1061 also occurs with a range of frequencies: for example, 0% in northwest Spain, 1.5% in Israeli populations, and 8.4% in Australia.6–8 A study of more than 100 Dutch families showed equal frequency of these most frequent mutations. In another study of 680 families from Germany, the two most frequent mutations had frequencies of 4.9% for deletion 5 bp at 1061 and 7% for deletion 5 bp at 1309.10,11 In our group, the deletion 5 bp at 1309 occurred more than twice as frequently as the deletion 5 bp at 1061. In worldwide populations, differences in the frequency of these two mutations are seen. Polish populations of patients with FAP belong to the group in which both mutations occur with high frequency.12
In exon 15, we saw another two recurrent mutations, each in two families. One was a 2626C>T substitution and the other a 3202–3205delTCAA.13,14 Mutations in the region between codons 1445 and 1578 are associated with the occurrence of numerous features outside the colon (desmoid tumours, osteomas, epidermoid cysts, and upper gastrointestinal polyps), which are classified as Gardner syndrome.2,3 In this region of the gene, we identified two mutations: 4386–4387insGA in the 9123 family (de novo) and 4667insA in the 9027 family. One of those families (9027) had features of Gardner syndrome, while the patients from family 9123 did not have any cancerous features outside the colon. The second case of Gardner syndrome had a mutation outside the expected region: a TCTTCCAGATA deletion that started at codon 1422 and lead to the stop codon at codon 1433(fig 1). This may indicate that the region responsible for Gardner syndrome cannot be determined exactly. In Polish patients with FAP, most of the detected mutations were localised at the 5′ end of exon 15 of the APC gene. Of the detected mutations, 50% were in the fragment that encompassed nucleotides 1040 to 1309. In our group of patients, we did not see mutations in exons 6, 7, 10, 12, 13, and 14, in which mutations were expected with the frequency of 1–2% on the basis of their occurrence in other populations.15
In our study, we examined 120 Polish families with FAP for the occurrence of mutations in the APC gene. For cost-efficiency reasons, we screened only the part of the APC gene in which mutations were most expected. Exon 9 of the APC gene was omitted, because of the absence of late onset of the disease and of variations in phenotypic manifestations in the identified families, which are characteristic for mutations in exon 9.16–18 The study region was chosen on the basis of previous studies in other populations, especially the report published by Wallis and colleagues, which considered >200 families with FAP from the United Kingdom.1,15,19,20
We found mutations in 35% of the families we studied. A study in the biggest referred group from Germany reported mutations in 48% of 680 studied families.11 The percentage of detected mutations in our study was lower, which could have been caused by the occurrence of mutations outside the studied region or the lower efficiency of the methods we used to detect mutations. In all of the abovementioned studies in other populations, in vitro translation was used for mutation screening, and differences in rates of mutation detection ranged from 30% to 85%.11,15,21,22 In our study, known polymorphic single base substitutions were visible during the analysis (data not shown), but the detected rate of substitutions in the studied group was low compared with the large German study (3/120 v 87/680).11 This can be attributed to the efficiency of the applied methods or the occurrence of mutations specific for Polish families with FAP.
To rule out the occurrence of large deletions, structural rearrangements of APC gene, or reduced expression of one allele of the APC gene in Polish patients with FAP as a frequent cause of FAP in Poland is impossible.23,24 In addition, the occurrence of recessive mutations in other genes (for example, the MYH gene), especially in de novo cases (>25% in our group), may contribute to the occurrence of the disease.25,26 Further studies to look at the remaining regions of the APC gene and to search for large deletions in Polish families with FAP will be necessary. A study of recessive mutations of the MYH gene will have to be performed in families who lack mutations in the APC gene as the next step towards explaining causes of FAP in Poland.
The studies were financed by State Committee of Scientific Research Project KBV No6PO5A13021.