D. The WNT7A gene is located between markers D3S2385, D3S2403 and D3S1252. A loss of heterozygosity at least with one marker was found in 85 (23/27) of informative cases. Moreover, for the first time we have shown a loss of the microsatellite markers D3S2385 and D3S2403 in cancer. It should be recalled that the D3S1252 marker was lost in 14 of informative cases of head and neck carcinomas [54]. To verify the correlation between hypermethylation/LOH presence and gene expression, the level of the WNT7A gene expression was investigated. Hypermethylation of 59-CpG island of the WNT7A gene/LOH presence coincided with decreased expression of the gene in 88 (15/17) of CASIN site selected clear cell RCC samples. The methylation/LOH status and expression of WNT7A gene have been studied on the same set of samples. Also, we observed that WNT7A expression was restored after 5-aza-29deoxycytidine treatment of the RCC cell line. In addition, it was reported that expression of 22948146 the WNT7A gene is frequently reduced in 
lung cancer [55], and that restoration of WNT7A gene0.89 (16/18) 78 (7/9)*p-value is referred to correlation between clinical-pathological characteristics and hypermethylation status. doi:10.1371/journal.pone.0047012.t(14/15), 82 (18/22) and 29 (2/7) of informative cases, respectively. Among the analyzed samples, 27 cases were informative for at least one LOH marker. Overall, we detected 23 (85 ) samples that contained at least one LOH and 4 samples without LOH. In 12 samples one LOH was detected, and in 11 samples – two LOH simultaneously. Data from LOH assays are presented in Figure 2 and Table S1.Expression of the WNT7A Gene in Clear Cell RCCWe examined whether the expression of the WNT7A gene was correlated with DNA methylation/presence of LOH in clear cell RCCs. For this purpose, the expression of the WNT7A gene in 17 clear cell renal cell carcinomas was determined by qRT-PCR. A decrease of gene expression was detected in 88 of clear cell RCCs (15/17 samples) (Figure 3 and Table S1). A correlation was detected between the decrease of WNT7A gene expression and hypermethylation of the WNT7A gene or the presence of LOH (rs = 0.917, p,0.05). The mean AVP web values of expression in the form of the logarithmic ratio of tumor/normal tissue for samples with stages I I (20.53+/21.0) and stages III V (21.54+/20.8) tend to be different (p = 0.09).Colony Formation and Proliferation TestsThe effect of WNT7A re-expression on colony formation of the A498 and KRC/Y cell lines was investigated. A high level of the WNT7A mRNA was detected in the A498 and KRC/Y cells after transfection with the WNT7A-pcDNA3.1 vector in comparison with the empty vector (Figure 4A). The ectopic expression of the WNT7A gene in the A498 and KRC/Y cell lines led to a significant reduction in colony number (p,0.05). The number ofFigure 2. The LOH assays: status 
of the informative cases of clear cell RCC for the 3q25 region surrounding the WNT7A gene. D3S2403, D3S2385, D3S1252?microsatellite markers, white circles – homozygotes (non informative cases), grey circles ?absence of the LOH, black circles ?presence of the LOH, IC – informative cases, “+” – LOH positive sample, “2” – LOH negative sample. doi:10.1371/journal.pone.0047012.gWNT7A Inactivated in Clear Cell RCCFigure 3. Panel of WNT7A gene expression in clear cell RCC samples. R ?values of expression in the form of the logarithmic ratio of tumor/ normal tissue of the WNT7A gene relatively to the TBP gene. doi:10.1371/journal.po.D. The WNT7A gene is located between markers D3S2385, D3S2403 and D3S1252. A loss of heterozygosity at least with one marker was found in 85 (23/27) of informative cases. Moreover, for the first time we have shown a loss of the microsatellite markers D3S2385 and D3S2403 in cancer. It should be recalled that the D3S1252 marker was lost in 14 of informative cases of head and neck carcinomas [54]. To verify the correlation between hypermethylation/LOH presence and gene expression, the level of the WNT7A gene expression was investigated. Hypermethylation of 59-CpG island of the WNT7A gene/LOH presence coincided with decreased expression of the gene in 88 (15/17) of selected clear cell RCC samples. The methylation/LOH status and expression of WNT7A gene have been studied on the same set of samples. Also, we observed that WNT7A expression was restored after 5-aza-29deoxycytidine treatment of the RCC cell line. In addition, it was reported that expression of 22948146 the WNT7A gene is frequently reduced in lung cancer [55], and that restoration of WNT7A gene0.89 (16/18) 78 (7/9)*p-value is referred to correlation between clinical-pathological characteristics and hypermethylation status. doi:10.1371/journal.pone.0047012.t(14/15), 82 (18/22) and 29 (2/7) of informative cases, respectively. Among the analyzed samples, 27 cases were informative for at least one LOH marker. Overall, we detected 23 (85 ) samples that contained at least one LOH and 4 samples without LOH. In 12 samples one LOH was detected, and in 11 samples – two LOH simultaneously. Data from LOH assays are presented in Figure 2 and Table S1.Expression of the WNT7A Gene in Clear Cell RCCWe examined whether the expression of the WNT7A gene was correlated with DNA methylation/presence of LOH in clear cell RCCs. For this purpose, the expression of the WNT7A gene in 17 clear cell renal cell carcinomas was determined by qRT-PCR. A decrease of gene expression was detected in 88 of clear cell RCCs (15/17 samples) (Figure 3 and Table S1). A correlation was detected between the decrease of WNT7A gene expression and hypermethylation of the WNT7A gene or the presence of LOH (rs = 0.917, p,0.05). The mean values of expression in the form of the logarithmic ratio of tumor/normal tissue for samples with stages I I (20.53+/21.0) and stages III V (21.54+/20.8) tend to be different (p = 0.09).Colony Formation and Proliferation TestsThe effect of WNT7A re-expression on colony formation of the A498 and KRC/Y cell lines was investigated. A high level of the WNT7A mRNA was detected in the A498 and KRC/Y cells after transfection with the WNT7A-pcDNA3.1 vector in comparison with the empty vector (Figure 4A). The ectopic expression of the WNT7A gene in the A498 and KRC/Y cell lines led to a significant reduction in colony number (p,0.05). The number ofFigure 2. The LOH assays: status of the informative cases of clear cell RCC for the 3q25 region surrounding the WNT7A gene. D3S2403, D3S2385, D3S1252?microsatellite markers, white circles – homozygotes (non informative cases), grey circles ?absence of the LOH, black circles ?presence of the LOH, IC – informative cases, “+” – LOH positive sample, “2” – LOH negative sample. doi:10.1371/journal.pone.0047012.gWNT7A Inactivated in Clear Cell RCCFigure 3. Panel of WNT7A gene expression in clear cell RCC samples. R ?values of expression in the form of the logarithmic ratio of tumor/ normal tissue of the WNT7A gene relatively to the TBP gene. doi:10.1371/journal.po.
