Fig. 5
Prioritizing candidate disease-associated mutations through shared disruption profiles. a Schematic of interaction disruption profiles for disease-associated mutation D32N and rare variants T152I and T149M. Stable expression of FLAG-tagged wild-type and mutant PSPH proteins was validated by Western blot using α-FLAG. α-γ-Tubulin was used as a loading control. A brief diagram of PSPH phosphatase activity is shown. * indicates 37 kDa marker b Enzymatic activity of purified recombinant wild-type and mutant PSPH proteins using phosphoserine substrate was measured in vitro using a malachite green assay performed in triplicate. Enzymatic activities for PSPH mutants are shown in proportion to wild-type activity. Error bars indicate +SE of mean. P value by one-tailed t-test. c Schematic of interaction disruption profiles for SEPT12 rare variant G169E and disease-associated mutation D197N. d Homology model of SEPT12–SEPT1 interaction. PDB ID 5CYO chains A and B used as template. Disruptive mutations on interaction interface are labeled. e Disruption of SEPT12 interaction with SEPT1 by G169E and D197N was validated by co-IP. SEPT12 bait proteins were detected using α-FLAG. SEPT1 prey was detected using α-HA. α-GAPDH was used as a loading control. f Fertility tests of 2–6-month-old WT (n = 2 males, avg = 8.9 ± 0.51; n = 2 females, avg = 8.6 ± 0.61) and Sept12G169E/G169E (n = 3 males, avg = 4.0 ± 1.3; n = 2 females, avg = 9.2 ± 0.57) mice bred to age-matched controls. Litter sizes were recorded. Green = males. Blue = females. All comparisons are not significant except for male WT vs. male Sept12G169E/G169E (P = 0.00052; by two-tailed t-test). g Assessment of sperm motility of WT (n = 2, sperm = 166), Sept12G169E/+ (n = 4, sperm = 484), and Sept12G169E/G169E (n = 3, sperm = 416) mice. See also Supplementary Figs. 7b, 8, and 9
