Functional evaluation of disease-modifying and somatic reversion SAMD9L variants. (A-C) 293FT cells were transiently transfected (Tx) with TFP-SAMD9L wild-type (WT) or potentially disease-modifying p.Thr233Asn variant, as indicated. (A) Western blots of recombinant SAMD9L variant expression, as indicated. (B) Cell proliferation in 293FT cells assessed by dye dilution assays of TFP-SAMD9L–transfected 293FT cells. Dye levels were monitored in TFP− cells (filled gray histograms) and compared with cells expressing uniformly intermediate levels of TFP-SAMD9L variants, as indicated. A single representative experiment is shown. (C) Cumulative data from independent experiments on growth inhibition associated with specific TFP-SAMD9L variants, as indicated. According to the index, 0 denotes growth of vector-transfected cells, whereas −1 approximates TFP-SAMD9L wild-type transfected cells. Significance was determined by 1-way analysis of variance (*P < .05; **P < .005; ***P < .0005. (D) Overview of SAMD9L structure, including SAM domain (blue). Positions of identified disease-associated germ line SAMD9L gain-of-function mutations (red), germ line loss-of-function variants (blue), and somatic reversion mutations (green) are indicated. Disease-associated germ line SAMD9L mutations reported by Chen and colleagues38 are included (red).
