Figure Legends
Figure 1. Severe piebaldism and profound hearing loss in mutant pigs. (a ) The pedigree of the G1-016105 mutant strain, including the ENU treated G0 male pig, the G1 male founder pig (G1-016105), and 63 G2 pigs produced by the wild-type sow. The black boxes and circles represented the pigs with pigmentation abnormalities. (b ) Statistical data of sex distribution and pigmentation pattern in the progenies of G1-016105 mating with ten wild-type females. (c ) The hair color of wild-type Bama pigs was “two-ends” black in head and hip, while the mutant pigs showed hypopigmentation in the head, ears, and hip. The retinal iris of the mutant pigs showed normal pigmentation and color. Scale bar: 5 cm. (d ) The results of ABR tests of wild-type and mutant pigs. The threshold of wild-type pigs was about 10–20 dB, while the mutant pigs showed profound bilateral deafness. (e ) Tone burst results of wild-type and mutant pigs. Wild-type pigs responded to sound stimuli from 1–32 kHz, while the mutant cochleae remained irresponsive at all frequencies.
Figure 2. Localization of the ENU-induced mutation site in exon 17 of the c-KIT gene. (a ) (b ) GWAS analysis results showed a highly significant linkage between piebaldism and the 32–48 cM region on chromosome 8. P < 1×10-5. LOD=7.8. (c ) 141 genes were annotated in the candidate linkage disequilibrium interval, including the candidate gene c-KIT , indicated as red. (d ) Sanger sequencing identified a site mutation (c.2430T>A) in exon 17 of the c-KIT gene, which altered aspartic acid (D) into glutamic acid (E) on the 810th site of the c-KIT protein. The p.D810E mutation was localized in the second tyrosine kinase domain. And the sequence of mutation site showed the transition (GAT > GAA). (e ) Alignment of the second kinase domain of c-KIT proteins showed that the Asp810 residue is highly conserved across different species and located in the DFG (Asp810, Phe811, and Gly812) motif at the N-terminus of the activation loop (A-loop).
Figure 3. The genotype of the c-KIT gene in Bama miniature pigs. (a ) Six different c-KIT alleles (i , iBe , iP , I1 , I2 , I3 ) exist in pigs. (b ) The picture of a wild type Bama pig with “two-ends” black hair color, which is similar to the belt phenotype in Hampshire. Scale bar: 5 cm. (c ) The primers and probes used in ddPCR for c-KITand ESR . (d ) One example of the ddPCR results ofc-KIT and ESR in the same DNA sample. The Y-axis shows the amplitude of fluorescence in every droplet, and the X-axis shows the number of droplets detected. The value of the purple line showed the threshold to decide positive or negative. (e ) The statistical data of the copy numbers of c-KIT and ESR in all five DNA samples. X: DNA sample; Y: 1000 copies/uL. (f ) The copy number of c-KIT in the Bama pig genome was decided by the ratio of c-KIT to ESRcopy number. The data implied the presence of a single copy ofc-KIT on one chromosome. (g ) The products of target fragment amplification with KIT17 primers and sequence analysis of the splice site mutation of exon 17.
Figure 4. D810E mutation disrupts the c-KIT-MITF signaling pathway. (a ) The crystal structure of the activated c-KIT protein. The DFG motif is localized in the core of the c-KIT protein active pocket and was manifested in the right side. ATP: blue; Mg2+: red; PTR568: orange. The DFG motif and the other important residues are shown. (b ) qPCR results of MITF-related genes (SOX10, PAX3, DCT, TYR, and MC1R ). (c ) The micro-CT scanning and 3-Dimensional reconstruction results of cochleae were shown at upper panel. ASC, anterior semicircular canal; PSC, posterior semicircular canal; LSC, lateral semicircular canal; OW, oval window; RW, round window. The results of celloidin embedding and hematoxylin-eosin staining (CE-HE) of wild-type and mutant cochleae were shown at lower panel. RM, the Reissner’s membrane; SG, spiral ganglion; SV, the stria vascularis.
Figure 5.Morphological changes of hair cells under scanning electron microscopy. The bundles of three rows of outer hair cells (OCHs) and one row of inner hair cells (IHCs) are regularly arranged in the apical, middle, and basal turn of the cochlea from a wild-type animal. Scale bar: 10 μm. High-resolution images show the bundles of OHCs from the apical and middle turn. Scale bar: 2 μm. An entire OHC can be observed in the basal turn. Scale bar: 10 μm. OHCs and IHCs ofc-KITc.2430A>T/+ animals appear to be completely degenerated in the apical, middle, and the basal turn. Scale bar: 10 μm. Stereociliary bundles are not present in the cuticular plates of the apical, middle, and basal turn. Scale bar:2 μm.
Figure 6. Representation of the structure of KIT, illustrating the observed missense mutations in patients with piebaldism.(a ) The complete list of KIT missense mutants retrieved from the literature for piebaldism. ECD, extracellular domain; TMD, transmembrane domain; JMD, juxtamembrane domain; KID, kinase insert domain; TK, tyrosine kinase domain. The red star represents the position 810, where the Asp is converted to Glu point mutation found in this article. (b ) The overall structure of activated c-KIT kinase labeled with the missense mutations located in the tyrosine kinase domain. The yellow labels Indicate that the sites belong to TK1, while the orange indicates TK2. The purple labels show the ATP binding pocket of KIT kinase. ATP: green; Mg2+: red; PTR568 from another KIT proteins: blue. Asp810 and Arg796 are indicated.