Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis

Abstract

Human Waardenburg syndrome 2A (WS2A) is a dominant hearing loss (HL) syndrome caused by mutations in the microphthalmia-associated transcription factor (MITF) gene. In mouse models with MITF mutations, WS2A is transmitted in a recessive pattern, which limits the study of hearing loss (HL) pathology. In the current study, we performed ENU (ethylnitrosourea) mutagenesis that resulted in substituting a conserved lysine with a serine (p. L247S) in the DNA-binding domain of the MITF gene to generate a novel miniature pig model of WS2A. The heterozygous mutant pig (MITF ) exhibits a dominant form of profound HL and hypopigmentation in skin, hair, and iris, accompanied by degeneration of stria vascularis (SV), fused hair cells, and the absence of endocochlear potential, which indicate the pathology of human WS2A. Besides hypopigmentation and bilateral HL, the homozygous mutant pig (MITF) and CRISPR/Cas9-mediated MITF bi-allelic knockout pigs both exhibited anophthalmia. Three WS2 patients carrying MITF mutations adjacent to the corresponding region were also identified. The pig models resemble the clinical symptom and molecular pathology of human WS2A patients perfectly, which will provide new clues for better understanding the etiology and development of novel treatment strategies for human HL.

Rescuing ocular development in an anophthalmic pig by blastocyst complementation

“…MITF L247S/L247S confers an eye developmental defect and can be observed as early as embryonic day 28 (E28; Hai et al , ). To further investigate the developmental competency of pig embryos derived from the blastocyst complementation, cloned embryos derived from MITF L247S/L247S male PEFs were used as the host and blastomeres of cloned embryos derived from female LW PEFs were used as donors for complementation.…”

“…The MITF L247S/L247S E44 fetus displayed an anophthalmic phenotype and showed a loss of RPE cells (Steingrimsson et al , ; Hai et al , 2017b; Fig A). The eyes of NW‐2 showed normal morphologically and were similar to those of the wild‐type (WT) fetus (Fig A).…”

“…The homozygote offspring MITF L247S/L247S were identified from a large‐scale ENU mutagenesis program (Hai et al , ) and developed defects in the eyeballs (Hai et al , ). This valuable anophthalmic pig model provided us a unique host with an empty developmental niche for exploring the possibility of producing an intact eye from xenogenic PSCs or an in vivo differentiation system.…”

Transcript Profiles of Stria Vascularis in Models of Waardenburg Syndrome

“…Waardenburg syndrome (WS) is a rare genetic condition characterized by at least some degree of congenital hearing loss and pigmentation deficiencies [ 1 ]. WS has more than 20 mutations in the Mitf allele [ 2 , 3 ], including Mitf mi-vga9 , Mitf mi-bw , and Mitf mi-ce , which have been identified to cause hearing loss and changes in pigmentation. Although the mouse model is widely used in disease phenotypes and pathogenic mechanisms of deafness-related research [ 4 – 8 ], many shortcomings have also been found in studying human genetic diseases.…”

Establishment of a Large Animal Model for Eustachian Tube Functional Study in Miniature Pigs

“…There has been a good deal of research regarding the histology, electrophysiology, and development of the miniature pig inner ear (Guo et al ., , ). In addition, gene expression (Wang et al ., ), gene regulation (Chen et al ., ; Hai et al ., , ), and gene transformation (Ma et al ., ; Shi et al ., ) studies of the auditory system have achieved remarkable results. The miniature pig has even been used to study cochlear implants (Chen et al ., ).…”