Generation and characterization of a murine amelogenesis imperfecta model

Amelogenesis imperfecta (AI) refers to a group of rare yet complex genetic disorders that affect the quantity and/or quality of tooth enamel. Recently, in AI patients, we identified mutations that disrupt a conserved alternative splicing pattern of the AMELX gene, which encodes amelogenin, the most abundant enamel matrix protein. These mutations led to the retention of exon 4, which is normally skipped during the pre-mRNA splicing process, resulting in the characteristic pitted, hypoplastic, and hypomineralized enamel defects. To observe the impact of retention of exon 4 within AMELX, a gene edited knock-in mouse model was generated.

A single-nucleotide knock-in mouse model was generated using CRISPR/Cas9 technology to introduce a silent mutation (NM_001415990.1: c.120 T>C, p.(Ala40=)) that abrogated alternative splicing of exon 4. Following genomic sequence validation, the successfully-targeted mice were propagated, and their offspring genotyped for characterization. Micro-computed tomography analysis and immunohistochemistry analysis were performed on the hemi-mandibles of the wild-type and the knock-in mice.

The enamel of the knock-in mice was chalky white and lacked translucency, due to faulty mineralization. This defective enamel broke down soon after tooth eruption. During the maturation stage, the ameloblast layer lost its cellular polarity and homogeneity, and intermingled with adjacent cell types to form disorganized clusters.

The validated and characterized Amelx c.120 T>C mouse model provides a useful platform for investigating the molecular pathophysiology associated with retention of the exon 4 sequence. Following systemic characterization, this mouse model will serve as an important tool for assessing therapeutic strategies aimed at ameliorating the disease phenotype.