Figure 5. (A) mRNA expression of TPE-OLD candidate genes in cells with normal/short telomeres and artificially elongated telomeres in the presence of hTERT. qPCR analysis was performed in a healthy human fibroblast cell line, a NBS fibroblast cell line, and in all 6 available lymphoblastoid cell lines (LCLs). Total mRNA was extracted from proliferating fibroblasts and from the same cell lines proliferating with experimentally elongated telomeres, after immortalization with hTERT. In LCLs, mRNA from the 6 different donors was compared. One of these cell lines (94P307) showed extremely elongated telomeres on p19. The genes were identified as TPE-OLD candidates by use of Affymetrix gene chip experiments in independent cell lines from healthy controls and HGP patients (as described in Supplementary Tables 7–8 and Supplementary Figures 5–8). All values were normalized to the level (=1-fold) of mRNA in unmodified and pre-senescent control fibroblasts. Each assay was performed in triplicate. Red arrows mark the genes with attenuated regulation in NBS (short telomeres in pre-senescent vs. long telomeres in hTERT infected cells). Green arrows mark the genes with reversed regulation in NBS (dark green: downregulated in pre-senescence in controls; light green upregulated). The blue arrow marks the LCL with extremely long telomeres on 19p. BSG: basigin; GAMT: guanidinoacetate methyl-transferase; SCAMP4: secretory carrier membrane protein 4; OLFM1: olfactomedin 1; UHRF1: ubiquitin like with PHD and ring finger domains 1; COL5A3: collagen type V alpha 3 chain; RNASEH2A: ribonuclease H2 subunit A; CACNA1A: calcium voltage-gated channel subunit alpha1 A; DDX39A: DExD-box helicase 39A; NOTCH3: Notch receptor 3. ND, non detectable (mRNA quantifications with Ct values above 35). Borderline Ct values were detected for NOTCH3 for the LCL 95P182 (mean Ct = 34.9) and 59P319 (mean Ct = 34.6), not shown in the figure. (B) TPE-OLD concept. Telomeres loop to specific loci to regulate gene expression, a process termed telomere position effect over long distance . The effect may likely extend to a distance of at least 10-15 Mbp from the telomere. The marked TPE-OLD gene candidates on 19p were investigated in experiments shown in Figure 5A: BSG, GAMT, SCAMP4, OLFM2, COL5A3, CACNA1A, NOTCH3 (upregulated in pre-senescent cells), and UHRF1, DDX39A and RNASEH2A (downregulated in senescent cells). TPE-OLD genes may form clusters with reverse regulation in NBS (green) further away from the telomere.