Discussion
In this study, we identified the novel mutation of c-KIT (p. Asp810Glu) which has not been reported previously and induced severe piebaldism with profound hearing loss in pigs. The heterozygousc-KITc.2430T>A/+ pig is the first big mammalian animal with a loss-of-function mutation in thec-KIT gene, thereby providing an important experimental model for exploring the pathological effect of c-KIT mutation and the candidate therapies for c-KIT mutation related diseases.
As a type III receptor tyrosine kinase, the binding of SCF to the first three Ig-like domains causes dimerization of two c-KIT proteins and promotes the kinase domains to act in trans as a substrate and enzyme for each other(Lemmon, Pinchasi, Zhou, Lax, & Schlessinger, 1997; Liu, Chen, Focia, & He, 2007; Yuzawa et al., 2007). To date, a total of 35 missense mutations, 17 deletions, four insertions, three nonsense mutations, seven nucleotide splice-site mutations, and one pericentric chromosomal inversion in c-KIT have been described (Table S3). Of the 35 missense mutations, three are located in the extracellular domain, 19 in the first tyrosine kinase domain (TK1), and 13 in the TK2 domain (Fig. 6a). Moreover, only R796G induced congenital and profound hearing loss in offspring(Spritz & Beighton, 1998), while A608D induced unilateral deafness in only one patient(Hamadah et al., 2019).
To further explore the mechanism under c-KIT mutations inducing hearing loss, we labeled and reanalyzed the missense mutations in the crystal structure of the active c-KIT protein (Fig. 6b). Two types of active sites of type III receptor tyrosine kinase are essential for the normal kinase activity of KIT: ATP binding sites and polypeptide substrate binding sites. A total of 4/19 sites (L595P, G601R, A621D, and A621T) in TK1 are ATP binding sites, while 2/13 (D792Y and P832L) in TK2 are polypeptide substrate binding sites (Fig. 6b). Only the R796G mutation is located in the core of the active pocket, and Arg796 functions as both types of the active sites. As for the residue Asp810, it is directed towards the positively charged guanidinium group of Arg815 in the c-KIT autoinhibited conformation and is rotated to ligate the Mg2+ ion and nucleotide phosphates during the activation process(Mol et al., 2004; Mol et al., 2003). The carboxylate side chain of Asp810 maintains approximately the same position in both enzyme conformations. Herein, in the mutated D810E, glutamic acid is similar to aspartic acid in polarity and acidity but with additional carbon. Therefore, there would be no space in the active pocket to accommodate the elongated side chain of Glu810. Thus, we deduced that the core location and function of Arg796 and Asp810 make them indispensable for the c-KIT function, and the induced mutation expanded the hearing loss with piebaldism.
Previous studies have reported approximately 163 c-KIT mutations in mice, although only KITW-V and KITWads homozygotes exhibit a recessive hearing loss trait(Cable, Jackson, & Steel, 1995; J. CABLE, 1994; Ruan et al., 2005). However, the phenotypes induced by the D810E mutation in Bama pig were transmitted in an autosomal dominant pattern, and the homozygotes faced embryonic lethality. This phenomenon indicates that thec-KIT dosage may exert a critical effect on pigs than mice and that the genetic, physiological, and structural similarities between pigs and humans make the pigs superior to mice for studying inherited deafness. Reportedly, the homozygous mutation (F809L) of Phe811 in mice induces hair pigmentation, macrocytic anemia, hepatic steatosis, and postnatal lethality (Magnol et al., 2007); however, no hearing phenotype was described. A missense mutation (G812V) of Gly812 has been identified in a patient with severe piebaldism, showing white forelocks and extensive leukoderma of the forehead without hearing loss (Kuster, 1987; Spritz, Holmes, Itin, & Kuster, 1993). These different phenotypes derived from the three sites of the DFG motif may be related to the function and characteristic of specific and substituted amino acids, respectively. The specific association among c-KIT mutation, the residual activity of the KIT protein, and hearing loss still need further study.
Materials and Methods