Genomic structure of the human MAD2 gene and mutation analysis in human lung and breast cancers.

Some of the many human cancers that exhibit chromosomal instability also carry mutations in mitotic checkpoint genes and/or reveal reduced expression of some of those genes, such as hMAD2. To facilitate investigation of alterations of hMAD2, we determined its genomic structure and intronic primers designed to amplify the entire coding region. Since general impairment of the mitotic checkpoint is frequently reported in lung cancers, and reduced expression of hMAD2 has been reported in breast cancers as well, we searched for mutations throughout the coding sequence of this gene in the genomic DNA of 30 primary lung tumors, 30 lung-cancer cell lines and 48 primary breast cancers. Our approach, which involved polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing, revealed nucleotide variants in only two of the 108 specimens. One was a cytosine-to-adenine substitution 3 bp upstream of exon 4 that occurred in one lung cancer cell line and one primary breast tumor, a change that did not alter transcriptional sequence. The other was an adenine-to-guanine substitution within exon 4, of the same lung cell line; this change already had been reported as a polymorphism. The results suggested that the hMAD2 gene is not commonly mutated in either lung nor breast cancers. Further studies should focus on other mechanisms that might account for reduced expression of the hMAD2 gene, and/or pursue analyses of other mitotic checkpoint genes for mutations in human cancer. Nevertheless, the genomic structure, the intronic primer sequences, and polymorphisms of the hMAD2 gene presented here will facilitate future studies to determine the full spectrum and frequency of the genetic events that can affect expression of the hMAD2 gene in human tumors.