Kyung-Eun Lee

FAM83H Mutations in Families with Autosomal-Dominant Hypocalcified Amelogenesis Imperfecta

Amelogenesis imperfecta (AI) is a collection of diverse inherited disorders featuring dental-enamel defects in the absence of significant nondental symptoms. AI phenotypes vary and are categorized as hypoplastic, hypocalcified, and hypomaturation types. Phenotypic specificity to enamel has focused research on genes encoding enamel-matrix proteins. We studied two families with autosomal-dominant hypocalcified AI and have identified nonsense mutations (R325X and Q398X) in the FAM83H gene on chromosome 8q24.3. The mutations perfectly cosegregate with the disease phenotype and demonstrate that FAM83H is required for proper dental-enamel calcification.

Mutational Spectrum of FAM83H: The C-Terminal Portion is Required for Tooth Enamel Calcification

Dental enamel forms through the concerted activities of specialized extracellular matrix proteins, including amelogenin, enamelin, MMP20, and KLK4. Defects in the genes encoding these proteins cause non‐syndromic inherited enamel malformations collectively designated as amelogenesis imperfecta (AI). These genes, however, account for only about a quarter of all AI cases. Recently we identified mutations in FAM83H that caused autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI). Unlike other genes that cause AI, FAM83 H does not encode an extracellular matrix protein. Its location inside the cell is completely unknown, as is its function. We here report novel FAM83H mutations in four kindreds with ADHCAI. All are nonsense mutations in the last exon (c.1243G>T, p.E415X; c.891T>A, p.Y297X; c.1380G>A, p.W460X; and c.2029C>T, p.Q677X). These mutations delete between 503 and 883 amino acids from the C‐terminus of a protein normally comprised of 1179 residues. The reason these mutations cause such extreme defects in the enamel layer without affecting other parts of the body is not known yet. However it seems evident that the large C‐terminal part of the protein is essential for proper enamel calcification.

A Novel Mutation in the DSPP Gene Associated with Dentinogenesis Imperfecta Type II

Hereditary dentin defects are divided into dentinogenesis imperfecta and dentin dysplasia. We identified a family segregating severe dentinogenesis imperfecta. The kindred spanned four generations and showed an autosomal-dominant pattern of inheritance. The proband was a child presenting with a severely affected primary dentition, with wide-open pulp chambers and multiple pulp exposures, resembling a DGI type III (DGI-III) pattern. We hypothesized that a mutation in the DSPP gene is responsible for this severe phenotype. Mutational analyses revealed a novel mutation (c.53T>A, p.V18D) near the intron-exon boundary in the third exon of the DSPP gene. We analyzed the effect of the mutation by means of an in vitro splicing assay, which revealed that the mutation did not affect pre-mRNA splicing. Further studies are needed for a better understanding of the nature of the disease and the development of an appropriate treatment strategy.

Novel FAM20A mutations in hypoplastic amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a genetically and clinically heterogeneous group of inherited dental enamel defects without any other nonoral symptoms. Recently, a disease‐causing nonsense mutation (c.406C>T) in a novel gene, FAM20A, was identified in a large consanguineous family affected by AI with gingival hyperplasia. We performed mutational analyses on nine AI families with similar phenotypes and identified three homozygous mutations (c.34_35delCT, c.813‐2A>G, c.1175_1179delGGCTC) in three families and a compound heterozygous mutation (c.[590‐2A>G] + [c.826C>T]) in one family. An in vitro splicing assay with a minigene confirmed the mutations located in the splicing acceptor site caused the deletion of exons 3 and 6, respectively. Taking into consideration the locations of the nonsense and frameshift mutations, the mutant transcripts are most likely degraded by nonsense‐mediated mRNA degradation and it results in a loss of the FAM20A protein. This study confirms the importance of the FAM20A protein in enamel biomineralization as well as tooth eruption. Hum Mutat 33:91–94, 2012.

LAMB3 Mutations Causing Autosomal-dominant Amelogenesis Imperfecta

Amelogenesis imperfecta (AI) can be either isolated or part of a larger syndrome. Junctional epidermolysis bullosa (JEB) is a collection of autosomal-recessive disorders featuring AI associated with skin fragility and other symptoms. JEB is a recessive syndrome usually caused by mutations in both alleles of COL17A1, LAMA3, LAMB3, or LAMC2. In rare cases, heterozygous carriers in JEB kindreds display enamel malformations in the absence of skin fragility (isolated AI). We recruited two kindreds with autosomal-dominant amelogenesis imperfecta (ADAI) characterized by generalized severe enamel hypoplasia with deep linear grooves and pits. Whole-exome sequencing of both probands identified novel heterozygous mutations in the last exon of LAMB3 that likely truncated the protein. The mutations perfectly segregated with the enamel defects in both families. In Family 1, an 8-bp deletion (c.3446_3453del GACTGGAG) shifted the reading frame (p.Gly 1149Glufs*8). In Family 2, a single nucleotide substitution (c.C3431A) generated an in-frame translation termination codon (p.Ser1144*). We conclude that enamel formation is particularly sensitive to defects in hemidesmosome/basement-membrane complexes and that syndromic and non-syndromic forms of AI can be etiologically related.

Novel WDR72 Mutation and Cytoplasmic Localization

The proven candidate genes for amelogenesis imperfecta (AI) are AMELX, ENAM, MMP20, KLK4, FAM83H, and WDR72. We performed mutation analyses on seven families with hypomaturation AI. A novel WDR72 dinucleotide deletion mutation (g.57,426_57,427delAT; c.1467_ 1468delAT; p.V491fsX497) was identified in both alleles of probands from Mexico and Turkey. Haplotype analyses showed that the mutations arose independently in the two families. The disease perfectly segregated with the genotype. Only persons with both copies of the mutant allele were affected. Their hypomineralized enamel suffered attrition and orange-brown staining following eruption. Expression of WDR72 fused to green fluorescent protein showed a cytoplasmic localization exclusively and was absent from the nucleus. We conclude that WDR72 is a cytoplasmic protein that is critical for dental enamel formation.

A dentin sialophosphoprotein mutation that partially disrupts a splice acceptor site causes type II dentin dysplasia.

The dentin sialophosphoprotein (DSPP) gene on chromosome 4q21.3 encodes the major noncollagenous protein in tooth dentin. DSPP mutations are the principal cause of dentin dysplasia type II, dentinogenesis imperfecta type II, and dentinogenesis imperfecta type III. We have identified a DSPP splice junction mutation (IVS2-6T>G) in a family with dentin dysplasia type II. The primary dentition is discolored brown with severe attrition. The mildly discolored permanent dentition has thistle-shaped pulp chambers, pulp stones, and eventual pulp obliteration. The mutation is in the sixth nucleotide from the end of intron 2, perfectly segregates with the disease phenotype, and is absent in 200 normal control chromosomes. An in vitro splicing assay shows that pre-mRNA splicing of the mutant allele generates wild-type mRNA and mRNA lacking exon 3 in approximately equal amounts. Skipping exon 3 might interfere with signal peptide cleavage, causing endoplasmic reticulum stress, and also reduce DSPP secretion, leading to haploinsufficiency.

Expression patterns of the Fam83h gene during murine tooth development

AIM Recently a novel gene, FAM83H, was identified by a genetic linkage study in the hypocalcified form of the amelogenesis imperfecta family with an autosomal dominant hereditary pattern. Little is known about this novel gene, and so we investigated the expression pattern of Fam83h in murine tooth development using serial sectional in situ hybridisation. METHODS AND MATERIALS Using mandibles of ICR mouse at specific developmental stages, in situ hybridisation was performed by DIG-labeled RNA probe. RESULTS Faint expression was detected in limited cells at embryonic day 14 (E14) in the molar. At the bell stage, E16, Fam83h was localised in the outer and inner enamel epithelium, as well as dental papilla. Fam83h expression begins on E15 in the developing incisor. At E18, Fam83h was expressed in the inner enamel epithelium of the apical bud, ameloblasts and odontoblasts. The expression was stronger in the presecretory stages than the secretory stages. CONCLUSION Fam83h was detected in the ameloblasts from the presecretory to the secretory stage, and also the odontoblasts layer and surrounding alveolar bone.

Effects of Fam83h overexpression on enamel and dentine formation

OBJECTIVE The aim of this study was to determine if FAM83H over-expression causes dentine or enamel malformations. MATERIALS AND METHODS The full-length mouse Fam83h cDNA was inserted into the pCAGIG vector between a β-actin promoter and β-globin enhancer for ubiquitous expression in transgenic mice. Recombinant mouse FAM83H was expressed and used to generate polyclonal antibodies. Western blots showed enhanced expression of the Fam83h transgene. The effects of transgene expression on tooth development were assessed by microhardness measurements of enamel and dentine. Total thickness of incisor enamel at the level of the alveolar crest was measured and decussating rod patterns were visualized by scanning electron microscopy (SEM). RESULTS Three transgenic mouse lines were selected based upon their transgene expression levels. There was no statistically significant difference in the Vickers microhardness values of enamel or dentine between the transgenic lines or between the transgenic lines and wild type mice. No statistically significant differences in enamel thickness were observed between the transgenic lines and the wild type mice. SEM analysis revealed no apparent differences in the enamel crystal and rod morphologies. CONCLUSION Our findings demonstrate that over-expression of FAM83H in mice does not produce a phenotype in dentine or enamel.

A novel mutation in the AMELX gene and multiple crown resorptions

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders with regard to genetic aetiology and clinical phenotype and affects tooth enamel with no other non-oral syndromic conditions. X-linked AI is caused by mutations in the amelogenin (AMELX) gene, the only AI candidate gene located on the X chromosome. To date, 15 mutations in the AMELX gene have been found to cause AI. We identified a proband with generalized hypoplastic enamel and unusual multiple crown resorption in premolars and molars. Pedigree analysis suggested an X-linked hereditary pattern. We performed mutational analysis for the AMELX gene based on the candidate gene approach. Sequencing analysis revealed a novel mutation in exon 6 (g.4090delC, c.517delC, p.Pro173LeufsX16). This frameshift mutation produces a premature stop codon within exon 6 and is predicted to replace 33 amino acids at the C-terminus with 15 novel amino acids if the mutant mRNA escapes the nonsense-mediated decay system. Although crown resorptions occur frequently in patients with the hypoplastic type of A1, an association with the AMELX mutation has not been previously reported. We believe that these findings will broaden our understanding of the clinical phenotype and pathogenesis of X-linked AI.