Identification of a Novel NLRP12 Frameshift Mutation (Val730Glyfs41) by Whole-Exome Sequencing in Patients with Crohn’s Disease

Figure 4

Functional consequences of the identified NLRP12 mutation. (a) The WB results showed that NLRP12 protein levels in the peripheral blood of the proband and his father and sister were significantly lower than those of the heathy control (his mother). (b) The mRNA level of NLRP12 transcribed from cells transfected with the mutant V730Gfs41 plasmid was significantly lower than that from cells transfected with the WT plasmid. NMDI14 significantly increased NLRP12 mRNA transcribed from the V730Gfs41 plasmid but made no difference in mRNA levels transcribed from the WT plasmid. (c) The WB results showed that the expression of the mutant FLAG-NLRP12-V730Gfs41 protein (~97 kDa) was significantly lower than that of the NLRP12-WT protein (~127 kDa). Mutant protein levels significantly increased after treatment with NMDI14, but the treatment caused no difference in WT protein levels. (d) Effects of NLRP12-WT and NLRP12-mutant proteins on NF-κB activity. The NF-κB transcriptional activity induced by p65 was significantly suppressed by overexpression of NLRP12-WT protein but was almost rescued by NLRP12 V730Gfs41 protein. Similarly, the NLRP12-WT protein also inhibited TNF-α-induced NF-κB activation, and this inhibition was dramatically abrogated by the NLRP12-mutant protein. WT: wild type; EV: empty vector; V730Gfs41: FLAG-NLRP12-V730Gfs41; WB: Western blotting; mRNA: messenger RNA. represents a -value less than 0.01.

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