Identification of New Splice Variants and Differential Expression of the Human Kallikrein 10 Gene, a Candidate Cancer Biomarker

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Abstract
The human kallikrein gene 10 (KLK10) is a member of the kallikrein gene family on chromosome 19q13.4. This gene was identified by its downregulation in breast cancer, and preliminary evidence suggests that it may act as a tumor suppressor. A computer-based analysis was performed on EST and SAGE clones from the Cancer Genome Anatomy Project and other databases. Experimental verification of differential expression of KLK10 in cancer was performed by PCR using gene-specific primers. The mRNA and EST analysis allowed the construction of the longest transcript of the gene and characterization of a 5' extension of the reported mRNA. In addition, seven new splice variants of KLK10 were identified. One of these variants, named KLK10 splice variant 3 (KLK10-SV3) which starts with a novel first exon, was experimentally verified. This variant is predicted to encode for the same protein as the 'classical' KLK10 mRNA, since the first exon is untranslated. One variant mRNA partially matches with the sequence of KLK10, while the rest of the mRNA matches with a portion of the polycystic kidney disease gene, found on chromosome 15. This variant could not be experimentally verified in either normal or cancerous tissues. There are 39 reported single nucleotide polymorphisms (SNPs) for the gene, in which three result in amino acid substitutions. SAGE analysis shows a clear upregulation of KLK10 in ovarian, pancreatic, colon, and gastric cancers. The gene is, however, downregulated in breast and prostate cancers. A three-fold decrease in expression levels was noted in actinic keratosis, compared to normal skin from the same patient. The differential regulation of KLK10 in ovarian and prostate cancers was experimentally verified by RT-PCR analysis. In addition, a significant number of clones were isolated from carcinomas of the head and neck. Fewer clones were found in carcinomas of the skin, brain and prostate. Orthologues were identified in three other species, with the highest degree of homology observed with the mouse and rat orthologues (42% in each). In conclusion new splice variants of the KLK10 gene were identified. These in silico analyses show a differential expression of the gene in various malignancies and provide the basis for directing experimental efforts to investigate the possible role of the gene as a cancer biomarker.