IADR Abstract Archives
Autosomal Recessive Amelogenesis Imperfecta Phenotype in Acp4 Defective Mice
Objectives: Recently, Testicular Acid Phosphatase (ACP4) gene defects were identified in six families with autosomal recessive, non-syndromic amelogenesis imperfecta (AI). ACP4 had not previously been suspected of functioning during enamel formation. The genetic findings are currently the only evidence that ACP4 is critical for amelogenesis. The objectives is to generate Acp4 mutant mice to characterize the enamel they form, and to evaluate these mice for non-dental phenotypes.
Methods: One of the reported human AI-causing ACP4 mutations was c.331C>T/p.Arg111Cys. Arg111 is highly conserved and its mutation to Cys111 has a PolyPhen-2 score of 1.0. The homologous residue in mouse Acp4 is Arg110. We worked with the University of Michigan Transgenic Animal Model Core to generate genomic Acp4 exon 4 edited mice (p.Arg110Cys) using CRISPR-Cas9. The entire Acp4 coding region was characterized by DNA sequencing in multiple founders and only mice with the p.Arg110Cys alteration and silent variations introduced as part of the procedure (but no other changes) were bred for subsequent characterization. The mice teeth were characterized by microscopic photography. Day 14 and 7-week first molars were characterized by backscatter Scanning Electron Microscopy (bSEM). The continuously growing mandibular incisors at 7-weeks were cross-sectioned at 1-mm increments and characterized by bSEM.
Results: Acp4 heterozygous (Acp4+/R110C) mice showed no phenotype. Acp4 homozygous (Acp4R110C/R110C) mice exhibited enamel hypoplasia, with chaulky-white maxillary and mandibular incisors, which was apparent by simple, unaided inspection. The molar surfaces were rough and incisor cross-sections showed the forming enamel was thinner than the wild-type. No phenotype outside of the dentition was observed. Mouse and human EST databases show only trace expression of ACP4 in non-dental tissues.
Conclusions: Characterizaton of Acp4R110C/R110C mutant mice confirm that ACP4 is essential for proper dental enamel formation and contributes to the genetic etiology of autosomal recessive, non-syndromic amelogenesis imperfecta.
Methods: One of the reported human AI-causing ACP4 mutations was c.331C>T/p.Arg111Cys. Arg111 is highly conserved and its mutation to Cys111 has a PolyPhen-2 score of 1.0. The homologous residue in mouse Acp4 is Arg110. We worked with the University of Michigan Transgenic Animal Model Core to generate genomic Acp4 exon 4 edited mice (p.Arg110Cys) using CRISPR-Cas9. The entire Acp4 coding region was characterized by DNA sequencing in multiple founders and only mice with the p.Arg110Cys alteration and silent variations introduced as part of the procedure (but no other changes) were bred for subsequent characterization. The mice teeth were characterized by microscopic photography. Day 14 and 7-week first molars were characterized by backscatter Scanning Electron Microscopy (bSEM). The continuously growing mandibular incisors at 7-weeks were cross-sectioned at 1-mm increments and characterized by bSEM.
Results: Acp4 heterozygous (Acp4+/R110C) mice showed no phenotype. Acp4 homozygous (Acp4R110C/R110C) mice exhibited enamel hypoplasia, with chaulky-white maxillary and mandibular incisors, which was apparent by simple, unaided inspection. The molar surfaces were rough and incisor cross-sections showed the forming enamel was thinner than the wild-type. No phenotype outside of the dentition was observed. Mouse and human EST databases show only trace expression of ACP4 in non-dental tissues.
Conclusions: Characterizaton of Acp4R110C/R110C mutant mice confirm that ACP4 is essential for proper dental enamel formation and contributes to the genetic etiology of autosomal recessive, non-syndromic amelogenesis imperfecta.