Fig. 2

Identification of fearless (Trpa1) mutant mice by forward genetics screening. a A flowchart of the recessive fear screen. b A graph of the fearless mutant pedigree consisting of seven reference (REF), six heterozygous (HET), and four phenovariant (VAR) individuals. A separate group of wild-type (WT) mice were included as controls every week. c A Manhattan plot showing a strong linkage (P = 2.13 × 10⁻¹¹) between the Trpa1 mutation and fearless phenotype. Horizontal yellow and blue lines represent the thresholds of P < 0.05 without or with Bonferroni correction, respectively. d The causative mutation was identified by sequencing the Trpa1 gene of REF, HET, and VAR mice. e A schematic of partial exon/intron structure of Trpa1 gene. The 5′ splice site (GT to GC) mutation results in the skipping of exon 15 and introduction of a premature stop codon. f, g Semi-quantitative (f) and quantitative (g) RT-PCR analysis showing that fearless mutant Trpa1 mRNA is diminished possibly owing to nonsense-mediated decay. Data are presented as mean ± SEM (n = 4, Student’s t-test, ***P < 0.001). h A schematic of the domain structure of Trpa1 protein, with the stop sign marking the premature stop codon and dash line representing the domains missing in mutant Trpa1 protein