Status of the DPC4 tumor suppressor gene in sporadic colon adenocarcinoma of Croatian patients: identification of a novel somatic mutation.
Loss of heterozygosity (LOH) of loci on chromosome 18q occurs in a majority of colorectal cancers. The DPC4 (Smad4) tumor suppressor gene, located at 18q21.1, may be a predisposing gene for Juvenile Polyposis Syndrome. To investigate alterations of the DPC4 gene in sporadic colon adenocarcinoma, a panel of 60 tumor specimens from Croatian patients was surveyed for evidence of LOH and also for mutations within the entire DPC4 coding region (exons 1-11). Using three pairs of specific primers for the three DPC4 microsatellite repetitive sequences, we investigated the frequency of LOH. The presence of single nucleotide change at restriction sites of specific codons in exons 2, 8, 10, and 11 (which belong to the conserved region of the gene) was examined by RFLP analysis. The investigation was extended to search for any other mutation within the entire coding region of the DPC4 gene by single strand conformation polymorphism (SSCP) analysis. Our results show a high frequency of heterozygosity in 58 of 60 (97%) colon adenocarcinoma samples. LOH at any one of the three flanking markers was observed in 26 (45%) of the 58 informative cases. The loss of one allele of the DPC4 gene was negatively correlated with tumor size; more frequent in smaller tumors (<5 cm) than in larger ones. A mutation was found in exon 11 in only one tumor sample (T18), and the mutation was verified by sequencing. Sequencing demonstrated a novel mutation-a deletion in exon 11 (134-153 del TAGACGAAGTACTTCATACC) of the DPC4 gene in the MH2 domain. These data suggest that inactivation of the DPC4 gene contributes to the genesis of colorectal carcinoma through allelic loss whereas mutation in the coding region of the DPC4 gene is infrequently detected in Croatian patients with A, B or C stages of colorectal cancers.
Popović Hadzija
Marijana
M
Division of Molecular Medicine, Rudjer Bosković Institute, Bijenicka c.54, 10000 Zagreb, Croatia. mhadzija@rudjer,irb.hr
Radosevic
Senka
S
Kovacević
Duje
D
Lukac
Josip
J
Hadzija
Mirko
M
Spaventi
Radan
R
Pavelić
Kresimir
K
Kapitanović
Sanja
S
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D002887:Chromosomes, Human, Pair 18 / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics*; D017523:Croatia / Q000453:epidemiology; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D006801:Humans; D019656:Loss of Heterozygosity; D008297:Male; D018895:Microsatellite Repeats; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics*
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D015534:Trans-Activators