Family 1 cousin (X*Y; III:1, Fig. 1A) of the proband.

<p><b><i>A:</i></b> Oral photographs of the proband's affected cousin with an anterior open bite at age 12. The enamel is whitest and thickest along the incisal edges and cusp tips. Enamel on the lateral surfaces is thin and rough and retains plaque. <b><i>B:</i></b> Panoramic radiograph reveals a thin layer of enamel on erupted and unerupted teeth that is more radioopaque than dentin, but less than normal enamel.</p

Summary of <i>ARHGAP6</i> gene structure and locations of deletions.

<p><b><i>A:</i></b> Positions of the two <i>ARHGAP6</i> deletions. <b><i>B:</i></b><i> ARHGAP6</i> gene structure inclusive of the four predicted promoters and <i>AMELX</i>.</p

Amelogenesis Imperfecta in Two Families with Defined <em>AMELX</em> Deletions in <em>ARHGAP6</em>

<div><p><em>Amelogenesis imperfecta</em> (AI) is a group of inherited conditions featuring isolated enamel malformations. About 5% of AI cases show an X-linked pattern of inheritance, which are caused by mutations in <em>AMELX</em>. In humans there are two, non-allelic amelogenin genes: <em>AMELX</em> (Xp22.3) and <em>AMELY</em> (Yp11.2). About 90% of amelogenin expression is from <em>AMELX</em>, which is nested within intron 1 of the gene encoding Rho GTPase activating protein 6 (<em>ARHGAP6</em>). We recruited two AI families and determined that their disease-causing mutations were partial deletions in <em>ARHGAP6</em> that completely deleted <em>AMELX</em>. Affected males in both families had a distinctive enamel phenotype resembling “snow-capped” teeth. The 96,240 bp deletion in family 1 was confined to intron 1 of <em>ARHGAP6</em> (g.302534_398773del96240), but removed alternative <em>ARHGAP6</em> promoters 1c and 1d. Analyses of developing teeth in mice showed that <em>ARHGAP6</em> is not expressed from these promoters in ameloblasts. The 52,654 bp deletion in family 2 (g.363924_416577del52654insA) removed <em>ARHGAP6</em> promoter 1d and exon 2, precluding normal expression of ARHGAP6. The male proband of family 2 had slightly thinner enamel with greater surface roughness, but exhibited the same pattern of enamel malformations characteristic of males in family 1, which themselves showed minor variations in their enamel phenotypes. We conclude that the enamel defects in both families were caused by amelogenin insufficiency, that deletion of <em>AMELX</em> results in males with a characteristic snow-capped enamel phenotype, and failed <em>ARHGAP6</em> expression did not appreciably alter the severity of enamel defects when <em>AMELX</em> was absent.</p> </div

Oral photographs of the proband's older (12 year old) brother (III:3, Fig. 1A).

<p>All teeth are permanent (the secondary dentition). The enamel of the posterior teeth was thickest along the cusp tips and thinner on the lateral surfaces of the crowns, which were stained and showed the underlying dentin. Attrition is evident, particularly on the first molars and the enamel is chipped in many locations. <b><i>B:</i></b> Oral photographs of the proband's younger (4 year old) brother (III:5, Fig. 1A). All teeth are from the primary dentition. Attrition of the molar occlusal surfaces shows the thinness of the enamel. The incisal edges of the anterior teeth are worn and chipped. Some teeth are secondarily affected by dental caries.</p

Scanning Electron Micrographs (SEMs) of the primary left maxillary cuspid (tooth H) from the proband of Family 2.

<p><b><i>A Left:</i></b> Manually fractured surface of the cuspid showing the measured thickness of enamel at the cingulum and two positions moving up the cusp slope. <b><i>A & B:</i></b> The enamel surface appears to be smooth with extensive micro-pitting. <b><i>C:</i></b> Dentin with parallel dentinal tubules appears normal. Scale bar units are in µm.</p

Alternative <i>ARHGAP6</i> promoter usage in secretory and maturation ameloblasts.

<p><b><i>A:</i></b> Map of the 5′ end of <i>ARHGAP6</i> inclusive of the four promoters (indicated by exons 1a through 1d). Arrows indicate primer annealing sites for RT-PCR. <b><i>B:</i></b> PCR amplifcation products Key to RNA sources: Lanes 1: laser captured day 5 ameloblasts; Lanes 2: laser captured day 12 ameloblasts; Lanes 3: day 5 enamel organ epithelia; Lanes 4: day 12 enamel organ epithelia; Lanes 5: Positive PCR controls, spleen for 1a, lung for 1b, 1c, and 1d). These results indicate that <i>Arhgap6</i> is expressed almost exclusively from promoter 1b in developing molars, and mostly during the maturation stage.</p

Family 2.

<p><b><i>A:</i></b> Pedigree of family 2 consistent with an autosomal dominant or X-linked pattern of inheritance. DNA was obtained from 5 persons, each indicated by a black dot. The arrow identifies the proband. A diagonal line indicates the family member is deceased. <b><i>B:</i></b> DNA sequencing chromatogram spanning the deleted segment of <i>ARHGAP6</i> (g.363924_416577del52654insA) containing all of <i>AMELX</i> and exon 2 of <i>ARHGAP6</i>. <b><i>C:</i></b> Oral photographs of the proband, a 12 year old Caucasian male. The color of dentin showed through the thin enamel surfaces of the permanent central incisors, which had pitted, rough surfaces and whiter incisal edges. The enamel of the permanent first molars was thickest along the cusp tips and marginal ridges, and thinner on the occlusal and lateral surfaces. There were spaces between most teeth. <b><i>D:</i></b> Bitewing radiographs and <b><i>E:</i></b> a panoramic radiograph reveal a thin layer of enamel that is more radioopaque than dentin, but not as radiodense as normal enamel.</p

Mother (X*X; II:2, Fig. 4A) of family 2 proband.

<p>Oral photographs depicting multiple vertical cracks in incisor enamel and bitewing radiographs showing thin enamel that contrasts with dentin but is not as radioopaque as normal enamel.</p

Definitions of caries experience based on age and DMFT (Decayed, Missing due to caries, Filled Teeth) scores used in the Filipino families.

a<p>DMFT cut-offs were modified from the World Health Organization (World Health Organization, 2003),</p>b<p>Standard deviation.</p

Correlations of enamel microhardness values in the several experimental points.

<p>Statistically significant correlations are presented in bold font.</p