J.T. Wright

Mutation in kallikrein 4 causes autosomal recessive hypomaturation amelogenesis imperfecta

Serine protease functionality is based on nucleophilic attack of a targeted peptidic bond by a serine. The serine protease superfamily is extremely diverse and includes proteases such as plasminogen, prostatin, hepsin, the kallikrein family ( KLK genes clustered on chromosome 19.13), and a recently discovered cluster of tryptic-like serine proteases located on human chromosome 16p13.1,2 Serine protease mutations have been reported as causative in only a few autosomal recessive human hereditary conditions, which produce diverse pathological conditions.3,4 We report the first human kallikrein mutation and describe its association with a rare autosomal recessive form of amelogenesis imperfecta. The amelogenesis imperfectas are a clinically and genetically heterogeneous group of disorders characterised by faulty development of the tooth enamel due to hypoplasia or hypomineralisation.5 The amelogenesis imperfecta phenotypes vary widely depending on the specific gene involved, the location and type of mutation, and the corresponding putative change at the protein level.6,7 The amelogenesis imperfecta enamel defects can be broadly divided into hypoplastic (enamel crystallites do not grow to the correct length) and hypomineralised (crystallites fail to grow in thickness or width) phenotypes. The prevalence of amelogenesis imperfecta varies in different countries (ranging from 1 in 700 in Sweden to 1 in 14 000 in the United States) suggesting allele frequency differences between populations.8–11 Amelogenesis imperfecta can be inherited as an autosomal dominant, autosomal recessive, or X-linked Mendelian trait. While autosomal dominant amelogenesis imperfecta types are most common in the United States and Europe, autosomal recessive amelogenesis imperfecta types are more common in the Middle East.8,10,11 Dental enamel is the most highly mineralised tissue in the human body with 85% of its volume occupied by highly organised carbonate substituted hydroxyapatite crystals.12 These crystallites are packed into a highly ordered decussating prism …

Relationship of phenotype and genotype in X-linked amelogenesis imperfecta

X-linked amelogenesis imperfectas (AI) resulting from mutations in the amelogenin gene (AMELX) are phenotypically and genetically diverse. Amelogenin is the predominant matrix protein in developing enamel and is essential for normal enamel formation. To date, 12 allelic AMELX mutations have been described that purportedly result in markedly different expressed amelogenin protein products. We hypothesize that these AMELX gene mutations result in unique and functionally altered amelogenin proteins that are associated with distinct amelogenesis imperfecta phenotypes. The AMELX mutations and associated phenotypes fall generally into three categories. (1) Mutations (e.g., signal peptide mutations) causing a total of loss of amelogenin protein are associated with a primarily hypoplastic phenotype (though mineralization defects also can occur). (2) Missense mutations affecting the N-terminal region, especially those causing changes in the putative lectin-binding domain and TRAP (tyrosine rich amelogenin protein) region of the amelogenin molecule, result in a predominantly hypomineralization/hypomaturation AI phenotype with enamel that is discolored and has retained amelogenin. (3) Mutations causing loss of the amelogenin C terminus result in a phenotype characterized by hypoplasia. The consistent association of similar hypoplastic or hypomineralization/hypomaturation AI phenotypes with specific AMELX mutations may help identify distinct functional domains of the amelogenin molecule. The phenotype-genotype correlations in this study suggest there are important functional domains of the amelogenin molecule that are critical for the development of normal enamel structure, composition, and thickness.

Amelogenin p.M1T and p.W4S Mutations Underlying Hypoplastic X-linked Amelogenesis Imperfecta

Mutations in the human amelogenin gene (AMELX, Xp22.3) cause a phenotypically diverse set of inherited enamel malformations. We hypothesize that the effects of specific mutations on amelogenin protein structure and expression will correlate with the enamel phenotype, clarify amelogenin structure/function relationships, and improve the clinical diagnosis of X-linked amelogenesis imperfecta (AI). We have identified two kindreds with X-linked AI and characterized the AMELX mutations underlying their AI phenotypes. The two missense mutations are both in exon 2 and affect the translation initiation codon and/or the secretion of amelogenin (p.M1T and p.W4S), resulting in hypoplastic enamel. Primary anterior teeth from affected females with the p.M1T mutation were characterized by light and scanning electron microscopy. The thin enamel had defective prism organization, and the surface was rough and pitted. Dentin was normal. The severity of the enamel phenotype correlated with the predicted effects of the mutations on amelogenin expression and secretion.

A common DLX3 gene mutation is responsible for tricho-dento-osseous syndrome in Virginia and North Carolina families.

Tricho-dento-osseous syndrome (TDO) is characterised by a variable clinical phenotype primarily affecting the hair, teeth, and bone. Different clinical features are observed between and within TDO families. It is not known whether the variable clinical features are the result of genetic heterogeneity or clinical variability. A gene for TDO was localised recently to chromosome 17q21 in four North Carolina families, and a 4 bp deletion in the human distal-less 3 gene (DLX3) was identified in all affected members. A previous genetic linkage study in a large Virginia kindred with TDO indicated possible linkage to the ABO, Gc, and Kell blood group loci. To examine whether TDO exhibits genetic heterogeneity, we have performed molecular genetic analysis to determine whether affected members of this Virginia kindred have the DLX3 gene deletion identified in North Carolina families. Results show that affected subjects (n=3) from the Virginia family have the same four nucleotide deletion previously identified in the North Carolina families. A common haplotype for three genetic markers surrounding the DLX3 gene was identified in all affected subjects in the North Carolina and Virginia families. These findings suggest that all people with TDO who have been evaluated have inherited the same DLX3 gene deletion mutation from a common ancestor. The variable clinical phenotype observed in these North Carolina and Virginia families, which share a common gene mutation, suggests that clinical variability is not the result of genetic heterogeneity at the major locus, but may reflect genetic heterogeneity at other epigenetic loci or contributing environmental factors or both.

Identification of cathepsin C mutations in ethnically diverse Papillon-Lefèvre syndrome patients

INTRODUCTION Papillon-Lefèvre syndrome (PLS) is an autosomal recessive disorder characterised by palmoplantar keratoderma and severe, early onset periodontitis, which results from deficiency of cathepsin C activity secondary to mutations in the cathepsin C gene. To date, 13 different cathepsin C mutations have been reported in PLS patients, all of which are homozygous for a given mutation, reflecting consanguinity. AIM To evaluate the generality of cathepsin C mutations in PLS, we studied an ethnically diverse group of 20 unrelated families. METHODS Mutations were identified by direct automated sequencing of genomic DNA amplified for exonic regions and associated splice site junctions of the cathepsin C gene. Long range PCR was performed to determine the genomic structure of the cathepsin C gene. RESULTS The cathepsin C gene spans over 46 kb, with six introns ranging in size from 1.6 to 22.4 kb. Eleven novel mutations and four previously reported mutations were identified in affected subjects from 14 families. Missense mutations were most common (9/15), followed by nonsense mutations (3/15), insertions (2/15), and deletions (1/15). Among these 14 probands, two were compound heterozygotes. Affected subjects with transgressions of the dermal lesions onto the knees or elbows or both had mutations in both the pro- and mature regions of the enzyme, although most were in the mature region. CONCLUSION Mutations in the mature region of cathepsin C were more likely to be associated with the transgressions of the dermatological lesions, although the results were not statistically significant. A comprehensive list of all cathepsin C mutations described to date, representing 25 mutations from 32 families with PLS and related conditions, is also presented.

Identification of the enamelin (g.8344delG) mutation in a new kindred and presentation of a standardized ENAM nomenclature

The amelogenesis imperfectas (AI) are a genetically heterogeneous group of diseases that result in defective development of tooth enamel. Although X-linked, autosomal dominant and autosomal recessive forms of AI have been clinically characterized, only two genes (AMELX and ENAM) have been associated with AI. To date, three enamelin (ENAM) mutations have been identified. These mutations cause phenotypically diverse forms of autosomal dominant AI. Detailed phenotype-genotype correlations have not been performed for autosomal dominant AI due to ENAM mutations. We identified a previously unreported kindred segregating for the ENAM mutation, g.8344delG. Light and electron microscopy analyses of unerupted permanent teeth show the enamel is markedly reduced in thickness, lacks a prismatic structure and has a laminated appearance. Taken together these histological features support the enamelin protein as being critical for the development of a normal enamel thickness and that it likely has a role in regulating c-axis crystallite growth. Because there is growing molecular and phenotypic diversity in the enamelin defects, it is critical to have a nomenclature and numbering system for characterizing these conditions. We present a standardized nomenclature for ENAM mutations that will allow consistent reporting and communication.

Developmental defects of enamel in humans with hereditary epidermolysis bullosa

Developmental defects of enamel are often reported as a feature of the more severe forms of epidermolysis bullosa (EB). The purpose of this investigation was to determine the prevalence and character of enamel defects in each of the major hereditary EB types. Clinical evaluations were made on 237 individuals representing all of the major EB types and 58 unaffected individuals. All EB cases were categorized by phenotype, mode of inheritance and skin biopsy. The frequency of individuals having developmental enamel defects ranged from 8.6% in recessive dystrophic EB to 100% in junctional EB; 27.5% of the control population had these defects. Generalized hypoplasia characterized by either severe pitting and/or thin enamel was seen in all junctional EB cases but not in any other EB type. There was a tendency for the severe Herlitz form of junctional EB to have thin enamel while non-Herlitz junctional EB cases had less severe pitting and generally no reduction in enamel thickness. The prevalence of individuals with hypoplastic enamel bands was greater in the EB population (9.7%) than controls (1.9%). Thus individuals with simplex and dominant dystrophic EB typically have enamel defects that are similar in frequency and distribution to those of unaffected individuals. Developmental defects of enamel are a consistent feature of junctional EB, although the clinical expression is highly variable.

Amelogenesis imperfecta phenotype-genotype correlations with two amelogenin gene mutations.

Amelogenin, the predominant matrix protein in developing dental enamel, is considered essential for normal enamel formation, but its exact functions are undefined. Mutations in the AMELX gene that encodes for amelogenin protein cause X-linked amelogenesis imperfecta (AI), with phenotypes characterized by hypoplastic and/or poorly mineralized enamel. Eight different AMELX deletion and substitution mutations have been reported to date. The purpose here was to evaluate the genotype and phenotype of two large kindreds segregating for X-linked AI. Phenotypically affected males in family 1 had yellowish-brown, poorly mineralized enamel; those in family 2 had thin, smooth, hypoplastic enamel. Heterozygous females in both kindreds had vertical hypoplastic grooves in their enamel. DNA was obtained from family members; exons 1-7 of AMELX were amplified and sequenced. Mutational analysis of family 1 revealed a single-base-pair change of A-->T at nucleotide 256, resulting in a His-->Leu change. Analysis of family 2 revealed deletion of a C-nucleotide in codon 119 causing a frameshift alteration of the next six codons, and a premature stop codon resulting in truncation of the protein 18 amino acids shorter than the wild-type. To date, all mutations that alter the C-terminus of amelogenin after the 157th amino acid have resulted in a hypoplastic phenotype. In contrast, other AMELX mutations appear to cause predominantly mineralization defects (e.g. the mutation seen in family 1). This difference suggests that the C-terminus of the normal amelogenin protein is important for controlling enamel thickness.

Unique Enamel Phenotype Associated with Amelogenin Gene (AMELX) Codon 41 Point Mutation

Different mutations in the amelogenin gene (AMELX) result in the markedly different enamel phenotypes that are collectively known as amelogenesis imperfecta (AI). We hypothesize that unique phenotypes result from specific genetic mutations. The purpose of this study was to characterize the enamel compositional and structural features associated with a specific AMELX mutation in three families with X-linked AI. We performed mutational analysis by amplifying AMELX exons and sequencing the products. Permanent and primary affected (N = 6) and normal (N = 3) teeth were collected and examined by light, scanning, and transmission electron microscopy. Enamel proteins were evaluated by immunolocalization of amelogenin and amino acid analysis. AI-affected individuals all shared a common AMELX point mutation (C to A change at codon 41). The dental phenotypic findings were remarkably consistent in all affected individuals. The AI enamel was opaque, with numerous prism defects or holes encompassing the entire prism width. Affected crystallites appeared more radiolucent and morphologically less uniform, compared with that of normal enamel. Immunogold labeling with anti-amelogenin antibodies localized amelogenin to the crystallites but not to the inter-crystalline spaces. No immunogold labeling was seen in normal enamel. There was an increased and amelogenin-like protein content in AI enamel (0.95%) compared with normal enamel (0.13%). We conclude that this codon 41 C to A missense point mutation, in a highly conserved region of the AMELX gene, results in a remarkably consistent phenotype.

A nomenclature for X-linked amelogenesis imperfecta.

Mutations of the X-chromosome amelogenin gene (AMELX) are associated with amelogenesis imperfecta (AI) phenotypes (OMIM no. 301200). Currently, 12 different AMELX mutations have been identified in individuals with abnormal enamel characteristic of AI. A notable feature of AI is the variable clinical phenotype, spurring interest in genotype-phenotype correlations. It is important that researchers and clinicians have an informative and reliable means of reporting and communicating these molecular defects. Therefore, the purpose here was to present a systematic nosology for reporting the genomic, cDNA and protein consequences of AMELX mutations associated with AI. The proposed nomenclature adheres to conventions proposed for other conditions and can be adopted for the autosomal forms of AI as the molecular basis of these conditions becomes known.