Amelogenesis

A MUTATION IN THE MOUSE AMELX TRI-TYROSYL DOMAIN RESULTS IN IMPAIRED SECRETION OF AMELOGENIN AND PHENOCOPIES HUMAN X-LINKED
AMELOGENESIS IMPERFECTA

Martin J. Barron1,{, Steven J. Brookes2,{, Jennifer Kirkham2,_, Roger C. Shore2, Charlotte Hunt1,
Aleksandr Mironov1, Nicola J. Kingswell2, Joanne Maycock2, C. Adrian Shuttleworth1
and Michael J. Dixon1
1Faculty of Life Sciences and School ofDentistry, Manchester Academic Health Sciences Centre, University of
Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK and 2Department of Oral Biology, Leeds
Dental Institute, University of Leeds, Clarendon Way, Leeds LS2 9LU, UK
Received November 13, 2009; Revised and Accepted January 3, 2010
Amelogenesis imperfecta (AI) describes a broad group of clinically andgenetically heterogeneous inherited
defects of dental enamel bio-mineralization. Despite identification of a number of genetic mutations underlying
AI, the precise causal mechanisms have yet to be determined. Using a multi-disciplinary approach,
we describe here a mis-sense mutation in the mouse Amelx gene resulting in a Y!H substitution in the
tri-tyrosyl domain of the enamel extracellular matrixprotein amelogenin. The enamel in affected animals phenocopies
human X-linked AI where similar mutations have been reported. Animals affected by the mutation
have severe defects of enamel bio-mineralization associated with absence of full-length amelogenin protein
in the developing enamel matrix, loss of ameloblast phenotype, increased ameloblast apoptosis and formation
of multi-cellular masses.We present evidence to demonstrate that affected ameloblasts express
but fail to secrete full-length amelogenin leading to engorgement of the endoplasmic reticulum/Golgi apparatus.
Immunohistochemical analysis revealed accumulations of both amelogenin and ameloblastin in
affected cells. Co-transfection of Ambn and mutant Amelx in a eukaryotic cell line also revealed intracellular
abnormalitiesand increased cytotoxicity compared with cells singly transfected with wild-type Amelx, mutant
Amelx or Ambn or co-transfected with both wild-type Amelx and Ambn. We hypothesize that intracellular
protein–protein interactions mediated via the amelogenin tri-tyrosyl motif are a key mechanistic factor underpinning
the molecular pathogenesis in this example of AI. This study therefore successfullylinks phenotype
with underlying genetic lesion in a relevant murine model for human AI.

INTRODUCTION
Amelogenesis imperfecta (AI) is a common group of inherited defects of dental enamel formation that exhibit marked genetic and clinical heterogeneity with at least 14 different sub-typesbeing recognized on the basis of their clinical appearance and mode of inheritance (1–3). Affectedindividuals show eitherhypoplastic (thin but seemingly correctly mineralized) or hypomineralized enamel but an overlap of these characteristics is seen in many cases (2). Autosomal dominant, autosomal recessive and X-linked forms of AI are recognized. To date, mutations in the genes encoding amelogenin (AMELX), enamelin (ENAM), enamelysin (MMP20), kallikrein 4 (KLK4) and the FAM83H gene have been shown tounderlie nonsyndromic forms of AI (4–6). Despite these advances, the underlying molecular pathogenesis of the various forms of AI remains poorly characterized.
Dental enamel is a highlymineralized tissue with _85%of its volume occupied by unusually large hydroxyapatite crystals that are organized into prisms (7). Enamel is unique among the mineralized tissues as it is produced by ectodermallyderived ameloblasts which pass through a series of discrete differentiation states that correlate with the various stages of enamel formation
(5). During the pre-secretory stage, ameloblasts are separated from the adjacent neural crest cell-derived odontoblasts by a
basement membrane which is subsequently removed as the ameloblasts enter their secretory phase. During this latter stage, the...