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. Author manuscript; available in PMC: 2018 Apr 12.
Published in final edited form as: Nat Neurosci. 2018 Jan 8;21(2):207–217. doi: 10.1038/s41593-017-0053-5

Fig. 7. An L1224R mutation in VPS15 is associated with neurodevelopmental disease.

Fig. 7

a, Structure of the family harboring a missense mutation in VPS15. The father and mother are heterozygous for the variant, whereas the proband (filled box) is homozygous for the L1224R mutation. Unaffected siblings were either heterozygous or wild-type. b,c, MRI images of the affected patient (at age 16) who presents with bilateral cortical and optic nerve atrophy, regions of cortical dysplasia (arrowheads), ataxia, intellectual impairment and epilepsy. d, Structural model of the VPS15-VPS34-BECLIN1 complex, with VPS15 shown in pink. The L1224R mutation lies in the WD40 domain in the fourth beta strand and is part of a hydrophobic core (red dot). e, Western blot analysis of VPS15, VPS34, BECLIN1 and P62 in protein lysates from dermal fibroblasts obtained from the father (F), mother (M) and proband (L1224R). f–i, Normalization with GAPDH reveals a highly significant reductions in VPS15, VPS34 and BECLIN1 in the affected proband and mild reductions in VPS15 and VPS34 in the father (n = 3 separate flasks per individual, one-way ANOVA with Tukey’s multiple comparison; VPS15: father vs. mother, P = 0.036; father vs. proband, P = 0.0007; mother vs. proband, P < 0.0001; VPS34: father vs. mother, P = 0.01; father vs. proband, P = 0.009; mother vs. proband, P = 0.0002; BECLIN1: father vs. proband, P = 0.004; mother vs. proband, P= 0.0052; P62: father vs. proband, P = 0.02; mother vs. proband, P=0.006). These experiments were performed twice with similar results. j, Western blot analysis of lentiviral rescue experiment. L1224R fibroblasts were infected with a lentivirus expressing either mCherry (control), or mCherry + wild-type VPS15 (rescue). k–m, This resulted in significant increases in VPS15, VPS34 and BECLIN1 in the rescue cells (n = 3 separate flasks per individual, one-tailed unpaired t test; VPS15: P = 0.002; VPS34: P= 0.03; BECLIN1: P= 0.001). These experiments were performed twice with similar results. n, Ectopic expression of VPS15 in L1224R cells resulted in a significant reduction in P62 (n = 3 separate flasks per individual, one-tailed unpaired t test, P = 0.015). All immunoblots shown are cropped; see Supplementary Fig. 11 for full-length blots). Error bars show mean ± s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.