Collagen (<i>COL1A1</i> and <i>COL1A2</i>) sequence variations identified in the proband.

<p>Collagen (<i>COL1A1</i> and <i>COL1A2</i>) sequence variations identified in the proband.</p

Family pedigree, missing teeth and disease causing mutations. <i>A:</i>

<p>The family pedigree follows the tooth agenesis trait for 3 generations and is consistent with an autosomal-dominant pattern of inheritance. <b>Key</b>: A filled icon indicates tooth agenesis. A dot indicates individual who donated samples. <b><i>B:</i></b> Chart of missing teeth in mother (II:5) and the proband (III:1). <b><i>C:</i></b> DNA sequencing chromatograms show that the affected mother (II:5) and proband (III:1) had a T or A (W) (arrowhead) at position g.5368 (NCBI Ref. Seq. NC_000014.8). This <i>PAX9</i> mutation (g.5368T>A; c.43T>A; p.Phe15Ile) caused the tooth agenesis. <b><i>D:</i></b> DNA sequencing chromatograms show both parents (II:5; II:6) had the wild-type G, while the proband had a G or A (R) (arrowhead) at position g.15941 (NCBI Ref. Seq. NG_007405.1). This spontaneous <i>COL1A2</i> mutation (g.15941G>A; c.1171G>A; p.Gly391Ser) caused the dentin defects in the proband. <b><i>E:</i></b> Radiographs of the mother (II:5) and proband (III:1) document the missing teeth (arrowheads) and the peg lateral (*) in the proband. Oral photos show the proband’s primary anterior teeth show the brownish discoloration and attrition. His maxillary incisors were removed because of severe attrition and a pediatric partial denture was placed. The proband’s radiographs show the bulbous crowns with cervical constrictions and thin, narrow roots.</p

Images of <i>FAM20A</i>^−/− tooth #18.

<p><i>A:</i> Photographs of #18 after cutting it sagitally. <i>B:</i> Photographs of a wild-type molar after cutting it sagitally. <i>C:</i> Photograph of #18 before sectioning. <i>D:</i> Occlusal view of #18 by photograph (top) and 3-D μ-CT image. <i>E:</i> 3-D μ-CT image of inside #18. Note the hollow area in the crown and the calcified pulp chamber. <i>F:</i> 3-D μ-CT image of #18. Note the shortness of the crown, which as apparently greatly diminished by resorption.</p

Scanning Electron Micrographs (SEMs) of molar (#18) occlusal surface.

<p><i>A:</i> Low magnification view of occlusal surface after partially cutting and then splitting the tooth sagitally (mesial-distal direction) for SEM analyses (bar: 1 mm). The boxes, from top to bottom, are locations of higher magnification views shown in B–E, respectively. <i>B:</i> Region showing knob-like calcifications (bar: 100 µm). <i>C:</i> Region where dentinal tubules reach the surface (bar: 10 µm); <i>D:</i> Region showing a relatively smooth surface (bar: 10 µm). <i>E:</i> Region from edge of crown (bar: 100 µm); <i>F:</i> Higher magnification of box in panel E showing no true enamel and apparent resorption lacunae (bar: 10 µm).</p

Family 1 from the Caribbean with <i>FAM20A</i> mutation c.992G>A; g.63853G>A; p.G331D.

<p><i>A:</i> Pedigree. A dot marks person who donated samples for DNA sequencing. <i>B: FAM20A</i> exon 7 DNA sequencing chromatograms. The proband's parents (II:1 and II:2) were both heterozygous (R = A or G) at cDNA position 992 (arrowheads). The proband (III-1) had the c.992G>A transition mutation in both alleles of <i>FAM20A</i>. This mutation changed a conserved glycine with an aspartic acid (p.G331D). The proband's affected younger sister (III-4) and her infant niece (IV:1) were also homozygous for this mutation (not shown). II:1 and III:8 were heterozygous for a recognized polymorphism (rs2302234) in exon 7 (K = A or C) unrelated to the phenotype. <i>C:</i> Proband's panoramic radiograph. Note the many unerupted teeth. The mandibular and maxillary unerupted second molars show concave occlusal surfaces without enamel (arrowheads). <i>D:</i> Proband's oral photos. The maxillary central incisors are restored. The clinical crowns were short with hypoplastic enamel. There was a deep anterior overbite, a posterior cross-bite, and retained mandibular primary molars (letters K, L, S, T).</p

Backscatter Scanning Electron Micrographs (bSEMs) of molar (#18) roots.

<p><i>A:</i> The bSEM of molar after it was cut sagitally (mesial-distally). <i>B:</i> Higher magnification of smaller box in A showing the layered build-up resembling cellular cementum. Arrowheads mark the dentin-cementum border. <i>C–D:</i> Higher magnifications of the larger box in A showing the thick layers of “cellular cementum” covering the roots. In panel D a dark line is placed at the dentin surface. <i>E:</i> Higher magnification of the larger box in panel C showing the thick layers of “cellular cementum” covering the roots and how the lamellar pattern suggests that deposition of these layers was punctuated by periods of resorption that sometimes penetrated into the dentin. <i>F–G:</i> Higher magnification of the smaller box in panel C also showing how deposition of the layers of acellular cementum was punctuated by resorption that sometimes penetrated into the dentin.</p

Scanning Electron Micrographs (SEMs) of mineral covering coronal dentin in a molar (#18) split for SEM examination.

<p><i>Left:</i> Enamel layer in normal molar <i>Right:</i> Mineral covering dentin in <i>FAM20</i>^−/− molar. No long thin crystals with rod/interrod organization are observed in the <i>FAM20</i>^−/− molar.</p

Backscatter Scanning Electron Micrographs (bSEMs) of molar (#18) crown.

<p><i>A:</i> The bSEM of molar after it was cut sagitally (mesial-distally). <i>B:</i> Higher magnification of region boxed in A showing regions magnified in C–F. The bowtie-shaped structure in the lower right is the calcified pulp chamber. Most of the coronal dentin has been resorbed, with some of it replaced by well-formed lamellar bone (b). <i>C–E:</i> Region showing dense, rough, crusty mineral in place of enamel (e) covering sclerotic dentin (d) that is fused to lamellar bone (b). There appears to be sites of active resorption of the dentin and bone (arrowheads). <i>F:</i> The pulp calcification (pc) is comprised of coalesced spheres that resemble the crusty “enamel” in mineral density embedded in a second, less mineralized material like dentin or acellular cementum that lacks osteocyte lacunae.</p

Localization of FAM20A to the Golgi.

<p>HEK293 cells were co-transfected with two plasmids. Cells that expressed the Golgi-GFP exhibited green fluorescence in the Golgi (A–F). FAM20A-flag was immunodetected and exhibited red fluorescence (H–J). Cells expressing both plasmids exhibited yellow fluorescence indicating superimposition of the two signals (K–M).</p

Backscatter Scanning Electron Micrographs (bSEMs) of molar (#32).

<p><i>A:</i> The bSEM of molar after it was cut sagitally (mesial-distally). <i>B:</i> Rough “enamel” (e) covering sclerotic dentin. <i>C:</i> Acellular cementum covering sclerotic root dentin. <i>D–E:</i> Highly mineralized pulp or radicular calcifications (pc) comprised of coalesced spheres above the root furcation and associated with a less mineralized material that contacts dentin (d). <i>F:</i> The radicular area appears to be comprised entirely of acellular cementum (ac) or lamellar bone from the furcation to the highly mineralized coalesced spheres. <i>G:</i> Root dentin covered with a thick layer of acellular cementum (ac) or bone. A thin line of more highly mineralized material, possibly cementum (c), separates these layers. <i>H:</i> The material covering root dentin is deposited in layers and sometimes fills in areas of localized root resorption.</p