P112 Addressing the problem of variants of uncertain significance in genetic diagnosis of vascular pulmonary disease: a role for transcript expression in blood monocytes?

Functional filter for whole-genome sequencing data identifies HHT and stress-associated non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA

“…This study was approved by the East of Scotland Research Ethics Service (EoSRES: 16/ES/0095), and the 6 participants provided written informed consent. Using methods we have developed to perform experimental treatments on human cells while resuspended in endogenous plasma, 47 , 48 , 49 peripheral blood mononuclear cells (PBMCs) were prepared using BD Vacutainer CPT tubes (Bunzl Healthcare) according to manufacturer’s instructions with minor modifications. As detailed further in the supplemental methods , these were to provide comparative resources of cells in stressed and unstressed states, where alternate transcripts/exon region use might be impacted by modified efficiency of final AAUAAA cleavage and polyadenylation due to the 3′ UTR variants.…”

“…After PBMC resuspension in plasma, for each donor, equal volumes were distributed to separate experimental treatment tubes, prewarmed at 37°C for 10 min, then subjected to 4 different treatment conditions for 1 h including control at 37°C and low temperature in a 32°C waterbath for 1 h to mimic the stress incurred at the threshold between mild and moderate hypothermia. 50 , 51 Additional stresses previously optimized in our laboratory 47 , 48 , 49 , 52 , 53 were inhibition of translation by cycloheximide 100 μg/mL (cycloheximide inhibits eukaryotic translation elongation by mechanisms including binding to the 60S ribosomal subunit E-site 54 , 55 ) and a clinically relevant mild reactive oxygen species (ROS) stress using ferric citrate 10 μmol/L. 53 , 56 After 1 h, all tubes were centrifuged at 520 RCF at room temperature for 15 min.…”