Institute for Nuclear Sciences “vinča”, Laboratory for Radiobiology and Molecular Genetics, Belgrade, Serbia

The structure and function of numerous genes involved in process of carcinogenesis have been established through explosive development of molecular oncogenetics during last 15 years. Depending on the function in the cell, these genes are divided into proto-oncogenes and tumor suppressor genes. Proto-oncogenes are normal cellular genes that perform and control essential functions in the cell: growth, proliferation and differentiation. Tumor suppressor genes are normal cellular genes which carry out the control of the cellular cycle and apoptosis. Protein products of proto-oncogenes and tumor suppressor genes are found in all cellular structures / compartments. Changes in nucleotide sequence and consequently alteration of their function may generate diverse class of genes (oncogenes) that lead to the neoplastic transformation of the cell. Mutations in tumor suppressor genes lead to the complete loss of their function, which also causes the process of oncogenesis. Several translocations involving different proto-oncogenes have been reported to be associated with a chi-like signal sequence similar to the signal sequence for the prokaryotic activator of recombination (14-16). The activity of chi-sites is reported to be influenced by their locations and by the number of chi-octamers at each site. A single chi-site stimulates recombination, but combinations of chi-sites on the two homologous are synergistic (17). In a case of chronic myelogenous leukemia (CML) in blastic crisis with t(3;21) near the breakpoint on chromosome 21, four chi-like sequences, potential consensus signals for activating recombination were found (18). In some NF1 patients, (MIM# 162200) recombination events occurred in a discrete 2-kb recombination hotspot. Recombination events were accompanied by apparent gene conversion involving the locus associated with NF1 tumor suppressor. A search for recombination-prone motifs revealed a chi-like sequence (19). Two closely spaced chi-like sequences were identified within 70-bp of the chromosome 1 breakpoint site associated with deregulation by chromosomal translocation in malignant lymphoma (20). At the beginning of the third millennium, the stage was set for the practical implementation of the results of molecular oncogenetics in diagnostics, prognosis, therapy, and the main objective in oncology, in preventive medicine. The implementation of standard gene tests in clinical practice of hereditary forms of cancer syndromes should primarily be emphasized (2). The basic prerequisite for the establishment of the standard gene test is that there is complete information about the structure of the carrier gene (structure of promoters, exons, introns). The second prerequisite is that there is a sufficient amount epidemiologic data that reliably correlate disease presentation with the appearance of certain mutations – adequate genotype / phenotype correlation. So far, 5 standard gene tests have been integrated into routine clinical practice – detection of hereditary mutations in the RET, VHL, AP, MEN 1 and RB genes. The RET gene (Rearranged during Transfection) belongs to the group of proto-oncogenes and its protein product has been proven in the membrane of almost all cell types. Nevertheless, the highest level of expression of the RET protein has been confirmed in cells which originate from bronchial arches (parathyroid), the neural crest (CNS, C-cells, and thyroid), and in tissue of the urogenital system (3,4). Heredity mutations in the RET gene have been detected in persons with type 2 multiple endocrine neoplasia (MEN 2). The main characteristic of this disease is hereditary medullar thyroid carcinoma (MTC) and pheochromocytoma, each in 50% of cases, presented as 2A and 2B forms (Table 1). So far, mutation in the RET gene has been detected in 98% examined patients with a developed MEN 2A and 2B disease (4). Familial medullar thyroid carcinoma (FMTC) develops due to the hereditary mutation in the RET gene in 85% of examined families (3,4). It is interesting that the RET gene consists of 20 exons, and, in all examined cases so far, mutations have been found in only 6 exons (10, 11 and 13-16). Namely, hereditary mutations in persons with MEN 2 disease are found in two main functional domains of the RET protein: extracellular domain which binds the ligand (in persons with the MEN 2A and FMTC form) and intracellular catalytic domain (in persons with the MEN 2B and FMTC form). In over 85% of cases in persons with the MEN 2A and FMTC form, mutations appear in exon 10 in one of the 4 cysteine domains (codons 609, 611, 618, 620) and in the exon 11 (codon 634). The most frequent mutation appears in codon 634 in 80% of cases of MEN 2A and in 30% cases of FMTC (2-4). In MEN 2B, almost all cases have mutations in exon 16 and in codon 918 (4-6). If the clinical picture suggests the possibility of hereditary nature of an established tumor one should not delay confirmation by gene tests of the MEN2 disease. A standard gene test consists of 2 main steps: the detection of the mutation and its characterization. When the screening for a hereditary mutation is involved, DNA is isolated from mononuclear blood cells, the exons of interest are amplified using the PCR technique and the detection of mutations follows. The detection of mutation is performed using different techniques of molecular genetics, and in gene tests, the so-called rapid screening techniques are used as they shorten the time necessary for analysis and they cut down the financial costs. In the analysis of RET, the technique of single strand fragments or Single Strand Conformation Polymorphism (SSCP) technique is most frequently used and direct sequencing as well. The SSCP technique, as its very name indicates, is based on the difference in conformation and consequently also on the mobility in the electrophoretic field, of single-strand DNA molecules, which can occur even as a consequence of one single basic substitution (7). The sensitivity of this technique depends on the amplicon being analyzed, and amounts to 80% to >90%, actually, there is a rare possibility for the production of false negative results, and false positive results are impossible. Contrary to the SSCP, the technique of direct gene sequencing enables absolute precision in the detection of the present mutations, so called “gold standard”. Conference paper UDC: 616-006.441:614.44:577.21