interestingly, the fumarate hydratase gene along with the neighboring kynurenine 3 - monooxygenase ( kmo ) gene, had a log2 - fold copy ratio of < - 1 in 293e, 293 - f and freestyle 293 - f cell lines compared to the parental hek293 ( fig. 2b and supplementary fig. s4 ), suggesting that these cells have half the number of copies compared to the parental cell line. moreover, the 293t and 293 - h cell lines have a gain of the genomic loci surrounding the fh gene, while maintaining the copy number of the fh gene compared to hek293. interestingly, the resulting fh expression levels of the cell lines only partly reflected the gene copy number changes ( fig. 2b and supplementary fig. s4 ). even though the gene copy number of the parental hek293 strain is the same as for 293t and 293 - h lineages, the fh mrna levels of hek293 was as low as the expression levels of the lineages with only half the number of fh gene copies. moreover, the expression levels of kmo was comparably low in all cell lines but did not correlate with gene copy number. besides the changes in gene copy number of the fh locus, a locus around the transducin - like enhancer protein 4 ( tle4 ) gene, encoding a transcriptional co - repressor of wnt signaling pathway members was found to have a log2 - fold copy number gain of > 1. 5 in all progeny cell lines except for 293e ( fig. 2b ). this gain in the tle4 locus was accordingly reflected in the transcription level of the gene with a higher level of expression in 293t, 293 - h, 293 - f and freestyle 293 - f compared to 293e and hek293 ( supplementary fig. s4 ). in addition, a major loss of copy number of the adam3a pseudogene was observed for all cell lines except 293 - h with a maintained low or no expression of the pseudogene observed in the cell lines ( fig. 2a, b and supplementary fig. s4 ). figure 2copy number variation analysis of hek293 progeny cells compared to the parental hek293 revealed conserved patterns of copy number gain and loss. ( genomic copy number gain ( red ) or loss ( blue ) of chromosomes 1, 8, 9, 13 and 18 of progeny hek293 cell lines compared to parental hek293 cells. the black line indicates the centromere position of each chromosome. ( genomic copy number gain or loss ( log2 fold change ) compared to hek293 for each cell line of the fh, kmo, tle4 and adam3a genes. copy number variation analysis of hek293 progeny cells compared to the parental hek293 revealed conserved patterns of copy number gain and loss. ( genomic copy number gain ( red ) or loss ( blue ) of chromosomes 1, 8, 9, 13 and 18 of progeny hek293 cell lines compared to parental hek293 cells. the black line indicates the centromere position of each chromosome. ( genomic copy number gain or loss ( log2 fold change ) compared to hek293 for each cell line of the fh, kmo, tle4 and adam3a genes. due to the observed pattern of common genomic changes to progeny cell lines compared to the parental hek293, an evaluation of common snps amongst all progeny cell lines but not hek293 was performed. go enrichment analysis of common genes with high or moderate impact snps different in all progeny cell lines compared to the original hek293 ( supplementary table s4 ), showed significant ( adjusted p - value < 0. 05 ) enrichment of homophilic cell adhesion via plasma membrane adhesion molecules ( go: 0007156; adjusted p - value 0. 025; fold enrichment 10. 26; data not shown ) and cell – cell adhesion via plasma - membrane adhesion molecules ( go: 0098742; adjusted p - value 0. 032; fold enrichment 7. 53; data not shown ). all genes with moderate or high impact snps in progeny cell lines compared to hek293 found amongst both these go - terms were protocadherins ( pcdh12, pcdhb10, pcdhb13, pcdhb15, pcdhb16 pcdhga2, pcdhga3 and pcdhgb2 ). in addition, the teneurin - 2 gene ( tenm2 ) ( within go: 0098742 ) had an altered snp allele in all progeny cell lines compared to hek293. these snps all result in missense mutations with unknown biological impact on the gene products. however, the enrichment of common snps within this group of genes in all hek293 progeny cell lines may suggest an impact on the protein function and a selective advantage of such phenotypic changes during continuous cell line cultivation. based on the overall genomic and transcriptomic profiles of the different hek293 cell lines, the parental hek293 strain stood out as different compared to all other cell lines. in order to evaluate common changes between all progeny cell lines and the parental hek293, differential expression analysis was performed. results showed a significant consensus of down - regulation of genes involved in extracellular matrix organization, locomotion and cell adhesion in progeny cells compared to the parental hek293 strain ( fig. 3a ). moreover, amino acid metabolism and metabolic process of small molecules were found up - regulated in all progeny cell - lines. along with changes in extracellular matrix genes, there is also a consensus amongst progeny cell lines compared to hek293 of differential expression of genes involved in other types of cellular component organization such as cell morphogenesis, cytoskeleton -, membrane - and cell junction organization. a comparison between gene expression fold changes and copy number variation of the differentially expressed genes ( log2 - fold change > ± 1 ) for each progeny cell line compared to hek293 showed a trend of gained gene copies amongst the majority of genes with up - regulated mrna levels ( supplementary fig. s4 ). however, there was not a clear trend of loss in gene copy number amongst transcriptionally down-regulate d genes for any cell line. figure 3differential expression analysis emphasized processes and genes with common changes in all progeny cell lines compared to the parental hek293. ( consensus heatmap of go biological processes with a different expression pattern between progeny cell lines compared to the parental hek293. low consensus scores, represented by a dark blue color, indicate more significant differences. ( common differentially expressed ( de ) genes in pairwise comparisons of all hek293 cells. blue bars show number of de genes in each pairwise comparison. green bar shows 329 common de genes in pairwise comparisons of progeny with hek293 parental cells. red bar shows common 38 de genes in the comparison of suspension cells against adherent cells. ( top ten significant go cellular components of the 329 common de genes in pairwise comparisons between progeny cells and hek293. differential expression analysis emphasized processes and genes with common changes in all progeny cell lines compared to the parental hek293. ( consensus heatmap of go biological processes with a different expression pattern between progeny cell lines compared to the parental hek293. low consensus scores, represented by a dark blue color, indicate more significant differences. ( common differentially expressed ( de ) genes in pairwise comparisons of all hek293 cells. blue bars show number of de genes in each pairwise comparison. green bar shows 329 common de genes in pairwise comparisons of progeny with hek293 parental cells. red bar shows common 38 de genes in the comparison of suspension cells against adherent cells. ( top ten significant go cellular components of the 329 common de genes in pairwise comparisons between progeny cells and hek293. for further evaluation of the transcriptomic similarities and changes between hek293 cell lines, pairwise differential expression comparisons between all cell lines were performed. as expected, the parental cell line had the highest number of differentially expressed genes when compared to all other cell lines ( fig. 3b, supplementary fig. s5 and supplementary table s5 ).