N was decreased substantially, the adenomas showed a similar level of expression as normal mucosae (Figure 3B). In Rp loss in lipid-free apoA-I exposed to 0 (black bars) or 15 mM addition, all 10 CRC cell lines studied expressed extremely low or undetectable levels of NDST4 mRNA (Figure 3C). A dramatic reduction of NDST4 expression in CRC sustains that NDST4 is a novel candidate TSG, and that the loss of its function might play a role in colorectal tumorigenesis.Allelic Loss of NDST4 Gene is Significantly Associated with Advanced Pathological Stage and Poor Survival in CRCTo precisely determine the genetic deletion of NDST4 in CRC, the WebSat, web software for microsatellite marker development, was used to identify short tandem repeats within NDST4 gene [19]. A new marker, MS5850, which was designed in this study, and D4S1580 were used for LOH analysis in 174 pairs of primary CRC tissues (Figure 1A). The correlations between the genetic alteration and clinicopathological characteristics are listed in Table 1. In total, 53 (30.5 ) of the 174 tumors were positive for allelic loss of NDST4 gene. The genetic aberration was increased considerably in tumors with higher pathological stages (T3 and T4) (P = 0.039). In addition, although not statistically significant, an increasing trend was observed in the progression of distantGenetic Loss of NDST4 in Colorectal CancerFigure 4. Comparison of patients’ overall survival in correlation with genetic loss of NDST4. Kaplan-Meier analysis was performed to compare patients with allelic loss of NDST4 gene to the others (no allelic loss). Genetic loss of NDST4 shows significant association with poorer survival (log-rank test). doi:10.1371/journal.pone.0067040.gmetastasis and Dukes’ stage (P = 0.075 and 0.083, respectively). Nevertheless, the genetic loss was not associated with other clinicopathological features. The correlation of allelic loss of NDST4 gene with patient survival was further evaluated. OS analysis was performed on 174 patients, of whom the OS rate was 67.8 (n = 118) with a mean survival of 94 months. By applying Kaplan-Meier survival curve analysis, the allelic loss of NDST4 gene predicted a worse OS (P = 0.036) (Figure 4). Fifty-three patients with this genetic aberration had an OS of 60.4 and a mean survival of 74 months, whereas the other 121 patients had an OS of 71.1 and a mean 23148522 survival of 100 months. In addition, DFS analysis was performed on 118 patients with Dukes’ stages B and C, of whom the DFS rate was 73.7 (n = 87) with a mean DFS of 104 months. Nevertheless, the genetic feature was not identified as a significant predictor of DFS for the patient group with Dukes’ stages B and C.DiscussionIn the present study, we identified NDST4 gene as a novel candidate TSG at chromosome 4q26, which is a common deletion region in CRC. In Title Loaded From File contrast to normal colonic mucosa with NDST4 expression, a majority of CRC tumors and cell lines showed a dramatic reduction in gene expression. In addition, we developed an LOH assay with two microsatellite markers, and revealed that the genetic loss of NDST4 was significantly associated with advanced pathological stage and poor survival, supporting the tumor suppressor function of NDST4 in CRC. Despite certain molecular pathways underlying the elucidated colorectal tumorigenesis, different genetic alterations can result in a specific phenotype that is correlated with various tumor behaviors and patient outcomes [20]. Therefore, the identification of novel molecular markers is necessary to improve strategies for t.N was decreased substantially, the adenomas showed a similar level of expression as normal mucosae (Figure 3B). In addition, all 10 CRC cell lines studied expressed extremely low or undetectable levels of NDST4 mRNA (Figure 3C). A dramatic reduction of NDST4 expression in CRC sustains that NDST4 is a novel candidate TSG, and that the loss of its function might play a role in colorectal tumorigenesis.Allelic Loss of NDST4 Gene is Significantly Associated with Advanced Pathological Stage and Poor Survival in CRCTo precisely determine the genetic deletion of NDST4 in CRC, the WebSat, web software for microsatellite marker development, was used to identify short tandem repeats within NDST4 gene [19]. A new marker, MS5850, which was designed in this study, and D4S1580 were used for LOH analysis in 174 pairs of primary CRC tissues (Figure 1A). The correlations between the genetic alteration and clinicopathological characteristics are listed in Table 1. In total, 53 (30.5 ) of the 174 tumors were positive for allelic loss of NDST4 gene. The genetic aberration was increased considerably in tumors with higher pathological stages (T3 and T4) (P = 0.039). In addition, although not statistically significant, an increasing trend was observed in the progression of distantGenetic Loss of NDST4 in Colorectal CancerFigure 4. Comparison of patients’ overall survival in correlation with genetic loss of NDST4. Kaplan-Meier analysis was performed to compare patients with allelic loss of NDST4 gene to the others (no allelic loss). Genetic loss of NDST4 shows significant association with poorer survival (log-rank test). doi:10.1371/journal.pone.0067040.gmetastasis and Dukes’ stage (P = 0.075 and 0.083, respectively). Nevertheless, the genetic loss was not associated with other clinicopathological features. The correlation of allelic loss of NDST4 gene with patient survival was further evaluated. OS analysis was performed on 174 patients, of whom the OS rate was 67.8 (n = 118) with a mean survival of 94 months. By applying Kaplan-Meier survival curve analysis, the allelic loss of NDST4 gene predicted a worse OS (P = 0.036) (Figure 4). Fifty-three patients with this genetic aberration had an OS of 60.4 and a mean survival of 74 months, whereas the other 121 patients had an OS of 71.1 and a mean 23148522 survival of 100 months. In addition, DFS analysis was performed on 118 patients with Dukes’ stages B and C, of whom the DFS rate was 73.7 (n = 87) with a mean DFS of 104 months. Nevertheless, the genetic feature was not identified as a significant predictor of DFS for the patient group with Dukes’ stages B and C.DiscussionIn the present study, we identified NDST4 gene as a novel candidate TSG at chromosome 4q26, which is a common deletion region in CRC. In contrast to normal colonic mucosa with NDST4 expression, a majority of CRC tumors and cell lines showed a dramatic reduction in gene expression. In addition, we developed an LOH assay with two microsatellite markers, and revealed that the genetic loss of NDST4 was significantly associated with advanced pathological stage and poor survival, supporting the tumor suppressor function of NDST4 in CRC. Despite certain molecular pathways underlying the elucidated colorectal tumorigenesis, different genetic alterations can result in a specific phenotype that is correlated with various tumor behaviors and patient outcomes [20]. Therefore, the identification of novel molecular markers is necessary to improve strategies for t.