The search of clinical records from the relevant institutions in Macedonia resulted in a selection of six patients with a histopathologically confirmed diagnosis of multiple polyposis, suspected for FAP. On the basis of population size and number of colorectal cancer patients, we calculated the incidence and prevalence of FAP in Republic of Macedonia to be 0.25 x 10^–6 and 2 x 10^–-6, respectively, and FAP to be responsible for approximately 0.06% of all colorectal cancers in our country.

      Detailed molecular analyses of the APC gene did not reveal any pathogenic point mutation or small deletions/insertions in the 15 exons and their flanking intronic sequences. Four common polymorphisms in exons 11, 13 and 15, and in the 3’UTR were detected (Table 2). Patients #1, #2, #3 and #5 were heterozygous for (most of) the intragenic polymorphisms, thus excluding the possibility of germ line hemizygosity at the APC locus. However, patients #4a and #6 were homozygous for all intragenic markers, leaving the possibility that a deletion that removed the entire APC gene could be responsible for the clinical phenotype. Linkage analysis of the APC gene region was possible only for the family of patient #4a, whose brother had FAP and was homozygous for different alleles at all four intragenic polymorphisms (Table 2). This strongly suggests the deletion of the entire APC gene in this family.  To confirm and delineate this suggested deletion, six microsatellite loci [(D5S299, D5S122 and D5S82 (all 5’) and D5S346, MCC and D5S2055 (all 3’)] to the APC gene were used for linkage analysis of these two brothers. Their APC gene region haplotypes are shown in Figure 2. The results indicated that the 5’ breakpoint of the deletion is between D5S82 and APC exon 11, while the 3’ breakpoint is within the region flanked by the MCC and D5S2055 markers (Fig. 3).

Figure 1. Scheme of the methods used for mutation detection analysis of various regions in the APC gene.

In three of the other patients aberrant transcripts in the peripheral blood were detected after reverse transcription (RT)-PCR analysis of PTT segment 1 (codons 1-804): an ~600 bp fragment in patient #1, a 1.7 kb fragment in patient #2 and ~1.9 kb fragment in patient #3. These fragments were excised from agarose gels and analyzed by DNA sequencing. This revealed that the ~600 bp fragment lacks exons 2-14 of the APC gene (Fig. 4); the 1.7 kb fragment lackes exons 8-13, while the 1.9 kb fragment arose from aberrant splicing of exon 9 and absence of exon 14. Gene mapping analysis of peripheral blood DNA in patients #1 and #2 did not reveal the presence of a deletion, that explains the occurrence of their abnormal fragments. This analysis was not done in patient #3 due to lack of sufficient DNA material.

      Tumours of all patients were microsatellite stable, except for patient #1, whose adenomas and carcinoma exhibited MSI in all of the five markers used. A summary of the results obtained by APC gene analysis is presented in Table 3. The Y165C and G382 mutations in the MYH gene were not detected in any of the patients analyzed.