Refined localization and cloning of a novel putative tumor suppressor gene associated with nasopharyngeal carcinoma on chromosome 9p21-22

Objectives: To further refine the extent of deletion on chromosome gp21-22 in nasopharyngeal carcinoma (NPC) and clone a new putative tumor suppressor gene associated with NPC. Methods: Loss of heterozygosity (LOH) on chromosome 9p21 -22 was analyzed in 25 paired blood and tumor samples by using 11 high-density microsatellite polymorphic markers; the expression of 3' end express sequence tags (ESTs) localized with in common deletion region was investigated tri NPC cell line HNE1, primary culture nasopharyngeal epithelial cells and BPC biopsies by using differential RT-PCR and Northern hybridization. Rapid amplification of cDNA ends (RACE) and bioinformatic data were used to clone putative EST, full-length cDNA. Results: Seventeen of 25 cases (68%) showed LOH at one or more loci. Higher frequencies of LOH were found at four loci: D9S161 (35. 0% ), D9S1678 (31.5% ), D9S263 (33. 3% ) and D9S1853 (33.3% ), where 6 cases had a contiguous stretch of allelic loss; 25 ESTs localized within D9S161 - D9S1853 were investigated and one EST (dbESI: 208825) was downexpressed in NPC cell line HNE1 and in 73% (11 / 15) NPC biopsies. Multiple tissue blot revealed that this putative EST hybridized to two different size transcripts (approximately 2.4 kb) and 3.5 kb) in fetal heart and brain tissues. A near full-length cDNA of 2.4 kb transcript of this EST was cloned(named NAG-6 gene) and there is no significant homology with any known genes. Conclusions: The minimal common region of deletion might he defined between D9S161 and D9S1853 (estimated about 2.7 cM in extent) at 9p21.1, the new gene NAG-6 located in this region may be a putative tumor suppressor gene associated with NPC.