Patients. A computer search was conducted of the database at the Institute for Oncology and Radiotherapy, Faculty of Medicine, Skopje, Republic of Macedonia, in order to find records of patients with multiple polyposis of the colon that were histopathologically confirmed in the last 10 years. Records of patients with multiple polyposis from the Clinic for Abdominal Surgery and Clinic for Gastroenterology, Facualty of Medicine, Skopje, were also evaluated. The term “multiple polyposis” was defined as the presence of more than 10 adenomatous polyps in the large intestine. Using these criteria seven patients from six unrelated families (six males and one female), were selected for detailed molecular investigation (Table 1). The number of polyps varied from 12 to more than a 1000. Adenocarcinoma was present at diagnosis in four patients. A positive family history of polyposis was obtained in two patients: patient #4a, whose brother (patient #4b) had a similar phenotype, and in patient #6, whose father died of disseminated malignant disease. Extracolonic features, namely desmoid-like tumours, were present in patient #1, that together with the low polyp count, warranted the APC gene analysis in this patient. Mean age at diagnosis of the patients was 33.5 years.

      Patients were contacted by their attending physicians and peripheral blood was collected with EDTA as anticoagulant. Informed consent was obtained from all patients. Paraffin-embedded tissue samples of available adenomas and carcinomas available from these patients, were also included in the molecular analysis.

      Mutation Detection Analyses. DNA was isolated from peripheral white blood cells using standard phenol-chloroform extraction/ethanol precipitation method [12]. Peripheral blood mononuclear cells were isolated by Ficoll gradient centrifugation and total cellular RNA was isolated using the acid guanidinium thiocyanate/phenol-chloroform method [13]. The initial mutation screening for the two most common mutations (deletions in codons 1309 and 1061) was done by polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE). Extensive screening of exons 1 through 14 and the 5’ end of exon 15 (codons 1-804) was done by protein truncation test (PTT) [14]. Denaturing gradient gel electrophoresis (DGGE) [15] was used for analysis of exons 1-14 and heteroduplex analysis (HD) [16] for the screening of exon 15. The mutation cluster region (MCR) (codons 1286-1513) of the APC gene was analysed by DNA sequencing. All analyses were performed on DNA only, while PTT was done on both DNA and RNA. Detailed mapping of part of the APC gene was done by Southern blot analysis [17]. Figure 1 lists the methods used for analysis of various regions of the APC gene. The familial relationship among available members from families was confirmed by DNA fingerprinting using a commercial kit (AmpFSTR Identifiler, Applied Biosystems Corporation, PaloAlto, CA, USA).

      Haplotype Analysis of the APC Gene Region. Four intragenic markers [codon 486 (TAC®TAT), codon 545 ( GCA®GCG ), codon 1493 ( ACA®ACG ) and T®C in 3’UTR ( 3’ untranslated region )] and six markers flanking the APC gene ( D5S2055, D5S299, D5S122, D5S346, MCC and D5S2027 ) were used to construct the haplotypes in the region of this gene. The analysis was done by DGGE, PCR-RFLP ( restriction fragment length polymorphism ) and/or by fluorescent PCR/capillary electrophoresis procedures.