Figure 1.
A: Pedigree of family 1 consistent with an autosomal dominant or X-linked pattern of inheritance. DNA was obtained from 8 persons, each indicated by a black dot. The arrow identifies the proband. A question mark indicates that the enamel phenotype was unknown. B: The proband's (X*Y; III:4) DNA sequencing chromatogram spanning the deleted segment of ARHGAP6 (g.302534_398773del96240) containing all of AMELX. C: Oral photographs of the proband, a 10 year old Caucasian male in the mixed dentition stage of dental development. Most of the occlusal enamel had abraded from the primary molars, revealing the thinness of retained enamel on the lateral and mesial/distal surfaces. D: Panoramic radiograph showing a thin layer of developing enamel on the unerupted bicuspids and second molars. The enamel is more radioopaque than dentin, but less so than normal enamel.
Figure 2.
Family 1 cousin (X*Y; III:1, Fig. 1A) of the proband.
A: 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: Panoramic radiograph reveals a thin layer of enamel on erupted and unerupted teeth that is more radioopaque than dentin, but less than normal enamel.
Figure 3.
Oral photographs of the proband's older (12 year old) brother (III:3, Fig. 1A).
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: 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.
Figure 4.
A: 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: DNA sequencing chromatogram spanning the deleted segment of ARHGAP6 (g.363924_416577del52654insA) containing all of AMELX and exon 2 of ARHGAP6. C: 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. D: Bitewing radiographs and E: a panoramic radiograph reveal a thin layer of enamel that is more radioopaque than dentin, but not as radiodense as normal enamel.
Figure 5.
Scanning Electron Micrographs (SEMs) of the primary left maxillary cuspid (tooth H) from the proband of Family 2.
A Left: Manually fractured surface of the cuspid showing the measured thickness of enamel at the cingulum and two positions moving up the cusp slope. A & B: The enamel surface appears to be smooth with extensive micro-pitting. C: Dentin with parallel dentinal tubules appears normal. Scale bar units are in µm.
Figure 6.
Mother (X*X; II:2, Fig. 4A) of family 2 proband.
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.
Figure 7.
Correlating genotypes and affection status.
A: Pedigrees for family 1 (left) and family 2 (right). B: PCR primers used to amplify across the deletion junctions. The numbers associated with the primers are the positions of the annealing sites in the ARHGAP6 genomic reference sequence (NG_012494.1). The same forward primer was paired first with a reverse primer within the deleted region to specifically detect wild-type, and then with a primer that annealed on the other side of the deletion to specifically detect the defective allele. (The wild-type wouldn't amplify because the product is too large.). C: PCR amplification for wild-type X-chromosome. D: PCR amplification for X-chromosome with deletion. Normal males (XY) and females (XX) only show a product in the first amplification. Affected females (X*X) show a product in both amplifications. Affected males (X*Y) show only a product in the second amplification.
Figure 8.
Summary of ARHGAP6 gene structure and locations of deletions.
A: Positions of the two ARHGAP6 deletions. B: ARHGAP6 gene structure inclusive of the four predicted promoters and AMELX.
Figure 9.
Alternative ARHGAP6 promoter usage in secretory and maturation ameloblasts.
A: Map of the 5′ end of ARHGAP6 inclusive of the four promoters (indicated by exons 1a through 1d). Arrows indicate primer annealing sites for RT-PCR. 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 Arhgap6 is expressed almost exclusively from promoter 1b in developing molars, and mostly during the maturation stage.