Investigation of polymorphic variation within intron 7 of the human cathepsin B gene

Abstract

Cathepsin B is a lysosomal cysteine protease whose activity has been implicated in a variety of physiological and pathological states including the development of lung disease and the progression of cancers. Cathepsin B has been implicated in the pathogenesis such lung diseases as emphysema and chronic bronchitis. Although no direct associations between cathepsin B and cystic fibrosis are evident, cathepsin B may be implicated to the disease progression through the diseases associated elevation in neutrophil elastase activity. The expression, post translational processing and targeting of cathepsin B is frequently altered in transformed and malignant cancer cells. Associations between malignancy and cathepsin B have been reported for human colon, breast, prostrate and bladder tumours (Keppler and Sloane, 1996). Cathepsin B could have a role in the detachment of cancerous cells from the tumour, and lead hence into metastasis. MacKenzie (1995) identified a 19 nucleotide insertion within intron 7 of the human cathepsin B gene. Sequencing of a B allele also revealed a nucleotide substitution of G to C at position 319 in intron 7. This study reports that this substitution occurs in all B allele homozygotes tested and that polymorphic linkage is present between the absence of the 19 nucleotide insertion and the nucleotide substitution within the B allele. This study also reports the screening of 19 normal DNA samples, 30 DNA samples from cystic fibrosis patients, and 22 colon carcinoma tissue DNA samples (all samples unrelated). A allele frequencies were determined to be 0.62, 0.65, & 0.64 respectively. Chi-square analysis revealed no significant (p=0.99) difference between normal, cystic fibrosis and colon cancer sample populations. The two alleles have been shown to conform to Hardy-Weinberg population distribution within each sample population so are thought to be stably inherited according to Mendalian segregation. Studies have indicated that cathepsin B has multiple forms generated by different cell processing of RNA and glycosylation of the precursor protein. These polymorphisms have been shown to introduce a potential alternative splicing site within intron 7 which, if functional would produce larger sized mRNA. These polymorphisms also have the potential to interfere with the existing intron 7/exon 8 splicing site. This study reports the development of a RNA extraction technique from human blood, and the design and partial optimisation of reverse transcriptase-PCR CRT-PCR). These techniques will be used in the future to study mRNA derived from homozygote individuals blood, and observe any differences in the length or amount of mRNA between the 2 alleles.