For homozygous variants. It then searches for signatures of heterozygous insertions and deletions Title Loaded From File within the output of the basecaller phred run with the ?poly option [49]. After forming two vectors containing the bases with highest peak areas at each position of the read (or assigning the highest area peak to both vectors when the second largest peak has an area less than 10 the size of the largest peak), DIPDetector attempts to phase these vectors by inserting potential shifts of all possible sizes into all possible positions of the read, and scores these shifts according to how well the resulting shifted vectors match the observed bases within the trace. Human genome assembly hg18 (NCBI Build 36.1) was used as the reference sequence. Variant positions were cross-referenced to dbSNP (Build 129) entries to identify known polymorphisms. To determine whether novel variants were somatic mutations or germline polymorphisms, the appropriate tumor DNA and matched normal DNA were re-amplified in an independent PCR followed by sequence analysis of the variant position. The predicted impact of somatic mutations on protein function was evaluated in silico using Mutation Assessor release 2 (http://Cohesion Gene Mutations in Endometrial Cancerendometrial cancer cell lines (Figure S6); ESCO1 was variably expressed among these same cell lines. ESCO1, which encodes a lysine acetyltransferase that is essential for the establishment of sister chromatid cohesion in mammalian cells, was somatically mutated in 2.2 (1 of 45) of serous ECs, 10 (2 of 20) of clear cell ECs, and 2.4 (1 of 42) of endometrioid ECs. Two of the ESCO1 mutations are predicted to impact protein function. The ESCO1R786C missense mutant, within the acetyltransferase domain, is predicted to impact protein function by both the SIFT and Polyphen algorithms (Table 2). We speculate that the ESCO1E338X nonsense mutant, which we uncovered in a serous-EC, may be a loss-of function mutant since a protein produced by this allele would be Title Loaded From File prematurely truncated and fail to include the acetyltransferase domain. Alternatively, nonsensemediated decay of the ESCO1E338X transcript might lead to haploinsufficiency. CHTF18 was somatically mutated in 2.2 (1 of 45) of serous ECs and 2.4 (1 of 42) of endometrioid ECs. In human cells, the CHTF18-RFC complex regulates the acetylation of the SMC3 cohesion-subunit by ESCO1 and ESCO2 acetyltransferases [34], thereby contributing to the establishment of sister chromatid cohesion. The CHTF18-RFC complex has also been implicated in the stimulation of DNA polymerase g activity, and in the recruitment of DNA polymerase e to sites of gap-filling repair synthesis [55], [56]. Both of the CHTF18 mutants we uncovered in endometrial cancer localize to the carboxy-terminus of the protein (Figure 1), within a region (residues 576-876) that mediates binding to RFC2-5 [57]. The CHTF18R854W mutant is predicted to possibly affect protein function by the Mutation Assessor and SIFT algorithms (Table 2). Interestingly, the majority of CHTF18 mutations observed in other cancers also localize to the C-terminus of the encoded protein [58]. These observations raise the possibility that somatic missense mutations in the C-terminus of CHTF18, found here and in other cancers, might disrupt the CHTF18-RFC interaction. MRE11A was somatically mutated in 4.4 (2 of 45) of serous ECs. No MRE11A mutations were observed among clear cell or endometrioid tumors. MRE11A possesses both endonuc.For homozygous variants. It then searches for signatures of heterozygous insertions and deletions within the output of the basecaller phred run with the ?poly option [49]. After forming two vectors containing the bases with highest peak areas at each position of the read (or assigning the highest area peak to both vectors when the second largest peak has an area less than 10 the size of the largest peak), DIPDetector attempts to phase these vectors by inserting potential shifts of all possible sizes into all possible positions of the read, and scores these shifts according to how well the resulting shifted vectors match the observed bases within the trace. Human genome assembly hg18 (NCBI Build 36.1) was used as the reference sequence. Variant positions were cross-referenced to dbSNP (Build 129) entries to identify known polymorphisms. To determine whether novel variants were somatic mutations or germline polymorphisms, the appropriate tumor DNA and matched normal DNA were re-amplified in an independent PCR followed by sequence analysis of the variant position. The predicted impact of somatic mutations on protein function was evaluated in silico using Mutation Assessor release 2 (http://Cohesion Gene Mutations in Endometrial Cancerendometrial cancer cell lines (Figure S6); ESCO1 was variably expressed among these same cell lines. ESCO1, which encodes a lysine acetyltransferase that is essential for the establishment of sister chromatid cohesion in mammalian cells, was somatically mutated in 2.2 (1 of 45) of serous ECs, 10 (2 of 20) of clear cell ECs, and 2.4 (1 of 42) of endometrioid ECs. Two of the ESCO1 mutations are predicted to impact protein function. The ESCO1R786C missense mutant, within the acetyltransferase domain, is predicted to impact protein function by both the SIFT and Polyphen algorithms (Table 2). We speculate that the ESCO1E338X nonsense mutant, which we uncovered in a serous-EC, may be a loss-of function mutant since a protein produced by this allele would be prematurely truncated and fail to include the acetyltransferase domain. Alternatively, nonsensemediated decay of the ESCO1E338X transcript might lead to haploinsufficiency. CHTF18 was somatically mutated in 2.2 (1 of 45) of serous ECs and 2.4 (1 of 42) of endometrioid ECs. In human cells, the CHTF18-RFC complex regulates the acetylation of the SMC3 cohesion-subunit by ESCO1 and ESCO2 acetyltransferases [34], thereby contributing to the establishment of sister chromatid cohesion. The CHTF18-RFC complex has also been implicated in the stimulation of DNA polymerase g activity, and in the recruitment of DNA polymerase e to sites of gap-filling repair synthesis [55], [56]. Both of the CHTF18 mutants we uncovered in endometrial cancer localize to the carboxy-terminus of the protein (Figure 1), within a region (residues 576-876) that mediates binding to RFC2-5 [57]. The CHTF18R854W mutant is predicted to possibly affect protein function by the Mutation Assessor and SIFT algorithms (Table 2). Interestingly, the majority of CHTF18 mutations observed in other cancers also localize to the C-terminus of the encoded protein [58]. These observations raise the possibility that somatic missense mutations in the C-terminus of CHTF18, found here and in other cancers, might disrupt the CHTF18-RFC interaction. MRE11A was somatically mutated in 4.4 (2 of 45) of serous ECs. No MRE11A mutations were observed among clear cell or endometrioid tumors. MRE11A possesses both endonuc.
