Ressor gene (TSG) loci [10?5]. However, few TSGs on chromosome 4 involved in CRC pathogenesis have been identified. We recently performed deletion mapping of chromosome 4 by loss of heterozygosity (LOH) study, and identified the D4S402 locus at 4q27 that exhibited the highest allelic loss frequency of 32.5 in 106 sporadic CRC (our unpublished data).Genetic Loss of NDST4 in Colorectal CancerIn the present study, we aimed to explore CRC-associated TSGs in the adjacent region of D4S402. Two approaches were conducted: (1) fine deletion mapping at chromosome 4q25-q28.2 to delineate the region harboring TSGs, and (2) analyses of alterations (gene expression and allelic deletion) of the candidate TSGs in primary CRC tumors. In BIBS39 web addition, the genetic loss of the candidate TSG was assessed for clinical relevance.Table 1. Association of genetic loss of NDST4 with clinicopathological characteristics of patients with colorectal cancer.Allelic loss of NDST4a Characteristic n 174 Positive 53 (30.5) Negative 121 (69.5) 0.964c 71.5 37?8 71 43?7 72 37?8 0.971 85 89 26 (30.6) 27 (30.3) 59 (69.4) 62 (69.7) 0.695 36 138 10 (27.8) 43 (31.2) 26 (72.2) 95 (68.8) 0.516 11 119 44 4 (36.4) 33 (27.7) 16 (36.4) 7 (63.6) 86 (72.3) 28 (63.6) 0.039 24 150 3 (12.5) 50 (33.3) 21 (87.5) 100 (66.7) 0.344 98 76 27 (27.6) 26 (34.2) 71 (72.4) 50 (65.8) 0.075 16985061 139 35 38 (27.3) 15 (42.9) 101 (72.7) 20 (57.1) 0.083d 21 65 53 35 3 (14.3) 21 (32.3) 14 (26.4) 15 (42.9) 18 (85.7) 44 (67.7) 39 (73.6) 20 (57.1) 0.584 31 87 8 (25.8) 27 (31.0) 23 (74.2) 60 (69.0)P valuebMaterials and Methods Patients and Tissue SpecimensA total of 174 patients with sporadic CRC, who underwent surgery at Cardinal Tien Hospital, Taiwan, were recruited between August 1997 and December 2008 (Table 1). Follow-ups were conducted until April 2010. All 174 patients were operated for histologically Asiaticoside A verified colorectal adenocarcinoma without preoperative chemotherapy and/or radiotherapy. Both paired tumor and adjacent normal mucosa samples were collected from each patient during surgery. In addition, adenomatous polyp tissues were collected from 57 patients who underwent colonoscopic polypectomy. All tissue specimens were immediately frozen after resection and stored in liquid nitrogen until nucleic acid extraction. All patients provided written informed consent, and the study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Cardinal Tien Hospital, Taiwan.Total patients Age at diagnosis (years) Median Range Gender Male Female Tumor location Proximal colon Distal colon Pathological differentiation Well Moderate Poor T stage T1 and T2 T3 and T4 N stage N0 N1 and N2 M stage M0 M1 Dukes’ stage A B C D Disease recurrencee Yes NoaLOH AnalysisDNA was extracted from frozen tissues by using the QIAamp DNA Mini Kit (Qiagen). For fine deletion mapping of chromosome 4q25-q28.2 (12.9 cM), LOH study with a panel of 11 microsatellites was conducted in 114 pairs of CRC tissue DNA (Figure 1A and Table 2). To further determine the allelic loss of NDST4 gene, LOH study with two microsatellite markers, MS5850 (UniSTS:536617) and D4S1580, was conducted in 174 CRC cases (Figure 1A and Table 2). In each 1676428 primer pair, the forward primer was synthesized with 6-FAM, VIC or NED fluorescent label depending on the amplicon size. PCR amplification was performed in a final volume of 6 mL by using 20 ng of DNA, 500 nM of each of respective primers, 200 mM of each dNTP, and 0.3 units.Ressor gene (TSG) loci [10?5]. However, few TSGs on chromosome 4 involved in CRC pathogenesis have been identified. We recently performed deletion mapping of chromosome 4 by loss of heterozygosity (LOH) study, and identified the D4S402 locus at 4q27 that exhibited the highest allelic loss frequency of 32.5 in 106 sporadic CRC (our unpublished data).Genetic Loss of NDST4 in Colorectal CancerIn the present study, we aimed to explore CRC-associated TSGs in the adjacent region of D4S402. Two approaches were conducted: (1) fine deletion mapping at chromosome 4q25-q28.2 to delineate the region harboring TSGs, and (2) analyses of alterations (gene expression and allelic deletion) of the candidate TSGs in primary CRC tumors. In addition, the genetic loss of the candidate TSG was assessed for clinical relevance.Table 1. Association of genetic loss of NDST4 with clinicopathological characteristics of patients with colorectal cancer.Allelic loss of NDST4a Characteristic n 174 Positive 53 (30.5) Negative 121 (69.5) 0.964c 71.5 37?8 71 43?7 72 37?8 0.971 85 89 26 (30.6) 27 (30.3) 59 (69.4) 62 (69.7) 0.695 36 138 10 (27.8) 43 (31.2) 26 (72.2) 95 (68.8) 0.516 11 119 44 4 (36.4) 33 (27.7) 16 (36.4) 7 (63.6) 86 (72.3) 28 (63.6) 0.039 24 150 3 (12.5) 50 (33.3) 21 (87.5) 100 (66.7) 0.344 98 76 27 (27.6) 26 (34.2) 71 (72.4) 50 (65.8) 0.075 16985061 139 35 38 (27.3) 15 (42.9) 101 (72.7) 20 (57.1) 0.083d 21 65 53 35 3 (14.3) 21 (32.3) 14 (26.4) 15 (42.9) 18 (85.7) 44 (67.7) 39 (73.6) 20 (57.1) 0.584 31 87 8 (25.8) 27 (31.0) 23 (74.2) 60 (69.0)P valuebMaterials and Methods Patients and Tissue SpecimensA total of 174 patients with sporadic CRC, who underwent surgery at Cardinal Tien Hospital, Taiwan, were recruited between August 1997 and December 2008 (Table 1). Follow-ups were conducted until April 2010. All 174 patients were operated for histologically verified colorectal adenocarcinoma without preoperative chemotherapy and/or radiotherapy. Both paired tumor and adjacent normal mucosa samples were collected from each patient during surgery. In addition, adenomatous polyp tissues were collected from 57 patients who underwent colonoscopic polypectomy. All tissue specimens were immediately frozen after resection and stored in liquid nitrogen until nucleic acid extraction. All patients provided written informed consent, and the study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Cardinal Tien Hospital, Taiwan.Total patients Age at diagnosis (years) Median Range Gender Male Female Tumor location Proximal colon Distal colon Pathological differentiation Well Moderate Poor T stage T1 and T2 T3 and T4 N stage N0 N1 and N2 M stage M0 M1 Dukes’ stage A B C D Disease recurrencee Yes NoaLOH AnalysisDNA was extracted from frozen tissues by using the QIAamp DNA Mini Kit (Qiagen). For fine deletion mapping of chromosome 4q25-q28.2 (12.9 cM), LOH study with a panel of 11 microsatellites was conducted in 114 pairs of CRC tissue DNA (Figure 1A and Table 2). To further determine the allelic loss of NDST4 gene, LOH study with two microsatellite markers, MS5850 (UniSTS:536617) and D4S1580, was conducted in 174 CRC cases (Figure 1A and Table 2). In each 1676428 primer pair, the forward primer was synthesized with 6-FAM, VIC or NED fluorescent label depending on the amplicon size. PCR amplification was performed in a final volume of 6 mL by using 20 ng of DNA, 500 nM of each of respective primers, 200 mM of each dNTP, and 0.3 units.
