Functional analysis of Ectodysplasin-A mutations causing selective tooth agenesis.

Mutations of the Ectodysplasin-A (EDA) gene are generally associated with the syndrome hypohidrotic ectodermal dysplasia (MIM 305100), but they can also manifest as selective, non-syndromic tooth agenesis (MIM300606). We have performed an in vitro functional analysis of six selective tooth agenesis-causing EDA mutations (one novel and five known) that are located in the C-terminal tumor necrosis factor homology domain of the protein. Our study reveals that expression, receptor binding or signaling capability of the mutant EDA1 proteins is only impaired in contrast to syndrome-causing mutations, which we have previously shown to abolish EDA1 expression, receptor binding or signaling. Our results support a model in which the development of the human dentition, especially of anterior teeth, requires the highest level of EDA-receptor signaling, whereas other ectodermal appendages, including posterior teeth, have less stringent requirements and form normally in response to EDA mutations with reduced activity

The WNT10A Gene in Ectodermal Dysplasias and Selective Tooth Agenesis

Mutations in the WNT10A gene were first detected in the rare syndrome odonto-onycho-dermal dysplasia (OODD, OMIM257980) but have now also been found to cause about 35-50% of selective tooth agenesis (STHAG4, OMIM150400), a common disorder that mostly affects the permanent dentition. In our random sample of tooth agenesis patients, 40% had at least one mutation in the WNT10A gene. The WNT10A Phe228Ile variant alone reached an allele frequency of 0.21 in the tooth agenesis cohort, about 10 times higher than the allele frequency reported in large SNP databases for Caucasian populations. Patients with bi-allelic WNT10A mutations have severe tooth agenesis while heterozygous individuals are either unaffected or have a mild phenotype. Mutations in the coding areas of the WNT10B gene, which is co-expressed with WNT10A during odontogenesis, and the WNT6 gene which is located at the same chromosomal locus as WNT10A in humans, do not contribute to the tooth agenesis phenotype. (C) 2014 Wiley Periodicals, Inc

Twist1- and Twist2-Haploinsufficiency Results in Reduced Bone Formation

<div><p>Background</p><p>Twist1 and Twist2 are highly homologous bHLH transcription factors that exhibit extensive highly overlapping expression profiles during development. While both proteins have been shown to inhibit osteogenesis, only Twist1 haploinsufficiency is associated with the premature synostosis of cranial sutures in mice and humans. On the other hand, biallelic Twist2 deficiency causes only a focal facial dermal dysplasia syndrome or additional cachexia and perinatal lethality in certain mouse strains. It is unclear how these proteins cooperate to synergistically regulate bone formation.</p><p>Methods</p><p>Twist1 floxed mice (<i>Twist1</i>^f/f) were bred with Twist2-Cre knock-in mice (<i>Twist2</i>^Cre/+) to generate Twist1 and Twist2 haploinsufficient mice (<i>Twist1</i>^f/+; <i>Twist2</i>^Cre/+). X-radiography, micro-CT scans, alcian blue/alizarin red staining, trap staining, BrdU labeling, immunohistochemistry, <i>in situ</i> hybridizations, real-time PCR and dual luciferase assay were employed to investigate the overall skeletal defects and the bone-associated molecular and cellular changes of <i>Twist1</i>^f/+;<i>Twist2</i>^Cre/+ mice.</p><p>Results</p><p>Twist1 and Twist2 haploinsufficient mice did not present with premature ossification and craniosynostosis; instead they displayed reduced bone formation, impaired proliferation and differentiation of osteoprogenitors. These mice exhibited decreased expressions of <i>Fgf2</i> and <i>Fgfr1–4</i> in bone, resulting in a down-regulation of FGF signaling. Furthermore, <i>in vitro</i> studies indicated that both Twist1 and Twist2 stimulated 4.9 kb <i>Fgfr2</i> promoter activity in the presence of E12, a Twist binding partner.</p><p>Conclusion</p><p>These data demonstrated that <i>Twist1</i>- and <i>Twist2</i>-haploinsufficiency caused reduced bone formation due to compromised FGF signaling.</p></div

Reduced bone formation in <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice.

<p>(A) Skeletons of 6-day-old control (left) and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> (right) mice stained with alcian blue (cartilage) and alizarin red (bone). The skeleton of the <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mouse is remarkably smaller. (B) Alcian blue- and alizarin red-stained skull from 6-day-old <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice (right) showed delayed fusion of interfrontal suture and open posterior fontanel (arrows), compared with the control mice (left). (C) Alcian blue- and alizarin red-stained hind foot of 6-day-old control (left) and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> (right) mice. Note the delayed ossification in metatarsals (mt) and phalanges (pl), and an additional toe (arrow) originating from the same (or duplicated) metatarsal as the hallux in <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice. (D) Plain X-radiography of the tibiae from 6-day-old control (left) and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice (right). The <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice had shorter tibiae and reduced radiopacity, compared to the control mice. (E) Representative three-dimensional μ-CT images of tibiae from 6-day-old control (left) and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> (right) mice. The <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice showed reduced trabecular (arrowheads) and cortical bones (arrows). (F–H) Quantitative μ-CT data showing that the 6-day-old <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice had a significant decrease in the ratio of bone volume (BV)/total volume (TV) (F) and in apparent bone density (G), compared to the control mice (n = 6, P<0.001). The <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice also presented reduced material density although no statistically significant difference was observed (H).</p

Primers used for real-time PCR.

<p>Primers used for real-time PCR.</p

Histological examination of <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice.

<p>(A–B) Femur sections of 6-day-old control and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice were stained with H&E. The <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice displayed reduced metaphyseal trabecular bone (A, red arrows) and a decreased thickness of the periosteum (B, blue arrows) and cortical bone (B, red arrows). (C) TRAP staining of femur sections of 6-day-old control and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice. Note that the osteoclasts (red arrows) appeared to be similar in size and distribution in the control and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice. The osteoclast densities were 0.55±0.06/0.01 mm² in the controls (<i>n</i> = 5) and 0.60±0.02/0.01 mm² in the <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice (<i>n</i> = 5, <i>P</i>>0.05). (D–F) <i>In situ</i> hybridization analyses (signal in blue) of the transcripts of <i>Alp</i> (D), <i>Ocn</i> (E) and <i>Dmp1</i> (F) in the femurs of one-week-old control and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice. (G, H) Immunohistochemical analyses (signal in brown) of the osterix (G) and biglycan (H) protein levels in the femurs of the 6-day-old control and <i>Twist1^flox/+</i>; <i>Twist2^Cre/+</i> mice. Scale bar = 100 µm.</p

Effects of Twist1, Twist2 and E12 on the activity of a 4.9<i>Fgfr2</i> promoter fragment.

<p>C3H10T1/2 (A) and MC3T3-E1 cells (B) were transiently co-transfected with a 4.9 kb <i>Fgfr2</i> promoter luciferase construct and the indicated expression constructs, along with a pRL-TK construct as an internal control. The luciferase activities were determined by a dual luciferase assay system, and the promoter activities were expressed as luciferase activities relative to that of the control. The values represented mean ± SD. n = 3 for each group. “a” indicates significant difference from the control (p<0.05); “b” denotes a significant difference from all other groups (p<0.05).</p