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