Journal: Nature genetics

Article Title: POT1 loss-of-function variants predispose to familial melanoma

doi: 10.1038/ng.2947

Figure Lengend Snippet: POT1 variants identified in familial melanoma pedigrees

Article Snippet: Human POT1 in a T7 expression vector (OriGene) was mutated by site-directed mutagenesis to generate cDNAs carrying the POT1 Tyr89Cys, Gln94Glu and Arg273Leu variants.

Techniques: Variant Assay

Journal: Nature genetics

Article Title: POT1 loss-of-function variants predispose to familial melanoma

doi: 10.1038/ng.2947

Figure Lengend Snippet: a) Shown is the location of the POT1 residues Tyr89, Gln94 and Arg273 in the N-terminal two oligonucleotide-/oligosaccharide-binding (OB) domains, in green. A telomere-like polynucleotide sequence is shown in orange. Interacting nucleotides in the telomeric sequence are labeled in gray. All three substitutions are predicted to disrupt the association of POT1 with telomeres. b) Mutant Tyr89Cys, Gln94Glu and Arg273Leu POT1 proteins are unable to bind telomeric (TTAGGG) 3 sequences as revealed by an electromobility shift assay. The Tyr223Cys POT1 mutant was used as a positive control representing a known disruptive mutation . c) Calculation of telomere length from exome sequence data. The method used is analogous to the one described in Ref. . Adjusted relative telomere lengths for the three sequenced members of pedigree UF20 are shown alongside the mean telomere length of 38 (all) other melanoma cases who were sequenced alongside, but were wildtype for POT1 . The error bar indicates one standard deviation. A Wilcoxon rank sum test was performed comparing the telomere length of the 3 Tyr89Cys cases to the 38 non-carrier controls. d) PCR-based estimate of telomere length. Adjusted mean negative ΔCt values, which correlate positively with telomere length, for POT1 missense variant carriers and non-carrier family controls are shown against a distribution of values from 252 melanoma cases recruited from the Leeds Melanoma cohort that are wildtype at the abovementioned positions ( Online Methods ). All measurements have been adjusted for age at blood draw and gender. The black line represents a Gaussian kernel density estimate for this set using Silverman’s “rule-of-thumb” for bandwidth smoothing. Orange dots represent members of pedigree UF20; pink, UF31; blue, UF23; red, individual CT1663 from the Leeds Melanoma Case-control study carrying the Arg273Leu variant . The number of biological replicates for each case ranged from 1 to 4, each with two technical replicates for the POT1 missense variant carriers and non-carrier family controls. Two technical replicates were performed for the 252 POT1 non-carrier cases. Error bars indicate the standard error of the mean.

Article Snippet: Human POT1 in a T7 expression vector (OriGene) was mutated by site-directed mutagenesis to generate cDNAs carrying the POT1 Tyr89Cys, Gln94Glu and Arg273Leu variants.

Techniques: Binding Assay, Sequencing, Labeling, Mutagenesis, Electro Mobility Shift Assay, Positive Control, Standard Deviation, Variant Assay