PMC3860171_01
A. Family pedigree and genotypes. The proband is shaded. B. Flow Fish Telomere lengths of the family members in PBMCs isolated by Ficoll gradient centrifugation using fluorescein isothiocyanate (FITC)-conjugated (C3TA2)3 peptide nucleic acid (PNA) probe flow cytometry. Relative telomere length (RTL) results by comparing PBMCs with a control cell line, GM03671C, a tetraploid cell line with telomere length of 100%. The numbers refer to the individuals in the pedigree in panel A. C. TRAP (telomerase repeat amplification protocol) assay. Mutations identified in TERT genes were introduced into plasmids containing the wild-type (WT) gene using a QuickChange site-directed mutagenesis kit (Stratagene). Mutant or wild-type TERT and wild-type TERC plasmids (4 mug) were then transfected using Lipofectamine 2000 into WI-38 VA13 cells at 90% confluence. After 48 hours, diluted protein cell lysates (1:5) were assayed for telomerase activity using a PCR-based TRAP assay (Chemicon International). WT: wild-type TERT and wild-type TERC, HI: Heat Inactivated, LB: Lysis Buffer, IC: Internal Control (36-bp) for PCR amplification, ON: overnight.
nihms-536425-f0001.jpg
PMC3860171_02_nihms-536425-f0001.jpg
F1
Finally the synonymous DKC1 polymorphism, c.1461C>T, (rs1127051; minor allele frequency in 1000 genomes = 0.065), also segregating in this family but unlinked to the G486R mutation (Figure 1A), is a known polymorphism with an allele frequency of 5-13% in African populations.
A. Conservation of the TERT and DKC1 variants in different species. B. Sanger sequencing of DKC1 cDNA prepared from RNA from PBMCs of the proband's mother. The pedigree in Figure 1A establishes that the 1456G>A and 1461 C>T mutations are on different alleles.
nihms-536425-f0002.jpg
PMC3860171_02_nihms-536425-f0002.jpg
F2
We therefore extracted RNA from peripheral blood mononuclear cells (PBMCs) and examined the expression of DKC1 by reverse transcription and PCR (Figure 2C).
PMC3860171_02
A. Family pedigree and genotypes. The proband is shaded. B. Flow Fish Telomere lengths of the family members in PBMCs isolated by Ficoll gradient centrifugation using fluorescein isothiocyanate (FITC)-conjugated (C3TA2)3 peptide nucleic acid (PNA) probe flow cytometry. Relative telomere length (RTL) results by comparing PBMCs with a control cell line, GM03671C, a tetraploid cell line with telomere length of 100%. The numbers refer to the individuals in the pedigree in panel A. C. TRAP (telomerase repeat amplification protocol) assay. Mutations identified in TERT genes were introduced into plasmids containing the wild-type (WT) gene using a QuickChange site-directed mutagenesis kit (Stratagene). Mutant or wild-type TERT and wild-type TERC plasmids (4 mug) were then transfected using Lipofectamine 2000 into WI-38 VA13 cells at 90% confluence. After 48 hours, diluted protein cell lysates (1:5) were assayed for telomerase activity using a PCR-based TRAP assay (Chemicon International). WT: wild-type TERT and wild-type TERC, HI: Heat Inactivated, LB: Lysis Buffer, IC: Internal Control (36-bp) for PCR amplification, ON: overnight.
nihms-536425-f0001.jpg
PMC3860171_02_nihms-536425-f0001.jpg
F1
Finally the synonymous DKC1 polymorphism, c.1461C>T, (rs1127051; minor allele frequency in 1000 genomes = 0.065), also segregating in this family but unlinked to the G486R mutation (Figure 1A), is a known polymorphism with an allele frequency of 5-13% in African populations.
A. Conservation of the TERT and DKC1 variants in different species. B. Sanger sequencing of DKC1 cDNA prepared from RNA from PBMCs of the proband's mother. The pedigree in Figure 1A establishes that the 1456G>A and 1461 C>T mutations are on different alleles.
nihms-536425-f0002.jpg
PMC3860171_02_nihms-536425-f0002.jpg
F2
We therefore extracted RNA from peripheral blood mononuclear cells (PBMCs) and examined the expression of DKC1 by reverse transcription and PCR (Figure 2C).