ß 2008 Wiley-Liss, Inc.

American Journal of Medical Genetics Part A 146A:343 – 349 (2008)

DLX3 c.561_562delCT Mutation Causes Attenuated

Phenotype of Tricho-Dento-Osseous Syndrome
J. Timothy Wright,1* Sung P. Hong,1 Darrin Simmons,1 Bill Daly,1
Daniel Uebelhart,2 and Hans U. Luder3
1
Department of Pediatric Dentistry, The University of North, Chapel Hill, North Carolina
2
Department of Rheumatology and Institute of Physical Medicine, Osteoporosis Center, University Hospital Zurich,
Zurich, Switzerland
3
Institute of Oral Biology, University of Zurich, Zurich, Switzerland
Received 9 May 2007; Accepted 18 September 2007

The distal-less homeobox gene DLX3 is expressed in a
variety of tissues including placenta, skin, hair, teeth, and
bone. Mutation of DLX3 (c.571_574delGGGG) causes
the tricho-dento-osseous syndrome (TDO), characterized
by abnormal hair, teeth, and bone. Evaluation of a kindred
segregating the DLX3 c.561_562delCT mutation revealed
distinct changes in the hair, teeth, and bones as has been
observed with the DLX3 c.571_574delGGGG mutation.
Previously, the DLX3 c.561_562delCT mutation was associ-
ated with autosomal dominant amelogenesis imperfecta with
taurodontism. The present study shows that the DLX3
c.560_561delCT mutation causes an attenuated TDO pheno-
type with less severe hair, tooth, and bone manifestations
compared with individuals having the DLX3 c.571_
574delGGGG mutation. Careful phenotyping of individuals
with allelic DLX3 mutations reveals marked differences in
phenotypic severity indicating that the carboxy-terminus of
the DLX3 protein is critical in determining its function during
development in these different tissues. ß 2008 Wiley-Liss, Inc.
Key words: distal-less; DLX3; bone; density; enamel;
hypoplasia; hair

How to cite this article: Wright JT, Hong SP, Simmons D, Daly B, Uebelhart D, Luder HU. 2008. DLX3
c.561_562delCT mutation causes attenuated phenotype of tricho-dento-osseous syndrome.
Am J Med Genet Part A 146A:343–349.

INTRODUCTION [Shapiro et al., 1983]. Identification of the molecular

etiology of TDO established that this phenotypic
The distal-less 3 homeobox gene, DLX3, is located
variance was due to variable expression that occur-
on chromosome 17q21 and has diverse temporal
red in people having identical DLX3 c.571_
expression in epithelial and mesenchymal tissues
574delGGGG mutations [Wright et al., 1997, 2000].
[Robinson and Mahon, 1994; Weiss et al., 1994;
A subsequent DLX3 gene mutation, c.561_
Berghorn et al., 2005]. Mutations in DLX3 (NM_
562delCT, was associated with a condition that
005220.2) cause autosomal dominant tricho-
affects formation of the enamel and morphology of
dento-osseous (TDO) syndrome (OMIM # 190320)
teeth, but lacked defects in any other tissues (i.e.,
comprising marked changes in the hair, teeth, and
hair and bone) [Dong et al., 2005]. This condition
bone [Lichenstein et al., 1972; Wright et al., 1997;
was classified as autosomal dominant hypoplastic,
Price et al., 1998a]. Individuals from several large
kindreds were found to have the same c.571_
574delGGGG DLX3 mutation, which predicts
p.Gly191ArgfsX66. This predicted truncated protein
retains the conserved homeobox region of the gene
[Price et al., 1998b]. Grant sponsor: General Clinical Research Centers Program of the
Marked phenotypic variability is seen in all of the Division of Research Resources; Grant number: RR00046; Grant sponsor:
affected tissues (i.e., hair, teeth, and bone), with National Institute of Dental and Craniofacial Research; Grant number:
alterations in fingernails, cranial morphology, and DE12202; Grant sponsor: National Institutes of Health.
mandibular prognathism also being variably present *Correspondence to: Dr. J. Timothy Wright, Department of Pediatric
Dentistry, Brauer Hall CB# 7450, School of Dentistry, The University of
[Jorgenson and Warson, 1973; Wright et al., 2000]. North Carolina, Chapel Hill, NC 27599.
This phenotypic variability in different tissues led to E-mail: Tim_Wright@Dentistry.unc.edu
the proposal that there were multiple TDO subtypes DOI 10.1002/ajmg.a.32132

344
hypomaturation amelogenesis imperfecta with
taurodontism (ADHHTAI) (OMIM# 104510) due to
its pattern of inheritance, thin and poorly minera-
lized enamel, and enlarged pulp chamber that
occurs due to apical displacement of the root
furcation. This second DLX3 mutation predicts
p.Tyr188GlnfsX13, which truncates slightly earlier
than the c.571_574delGGGG mutation [Dong et al.,
2005].
Many cases of TDO have been erroneously
reported as being ADHHTAI due to the subtle clinical
features of the hair and bone or lack of adequate
radiographic evaluation to identify osseous changes
[Crawford and Aldred, 1990; Seow, 1993a]. Changes
in bone density must be identified by radiographic
or bone density studies [Haldeman et al., 2004]. The
hair, which is kinky or curly at birth in about 85% of
infants with TDO will straighten and can appear
normal in nearly half of individuals having the DLX3
c.571_574delGGGG mutation [Wright et al., 1997].
Here we report the hair, tooth, and bone mani-
festations in a newly identified kindred having a
DLX3 c.561_562delCT mutation. Detailed phenotyp-
ing of this kindred suggests that this allelic DLX3
mutation causes TDO with an attenuated phenotype
and not ADHHTAI, as previously reported.
METHODS
This study was approved by the Institutional
Ethical Committee. Family members were identified
by their clinical and radiographic dental manifes-
tations that included thin enamel and taurodontism.
DNA was extracted from peripheral blood samples
using standard techniques (Qiagen, Valencia, CA).
The DLX3 gene was sequenced using primers for all
three exons and splice sites (primers available upon
request). Amplicons were sequenced using an ABI
770 and verified as previously described [Price et al.,
1998a].
Study participants provided a medical and dental
history with attention given to questions regarding
changes in hair, fingernails, or bone manifestation
FIG. 1. The family pedigree shows inheritance of the trait in an autosomal dominant fashion with male to male transmission and a near 50/50 differential between
affected and unaffected individuals.
American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a
WRIGHT ET AL.
associated with TDO. Family members were clini-
cally evaluated by one investigator. Three random
hair samples per individual were mounted on
scanning electron microscopy stubs and sputter-
coated with 10–15 nm of platinum using a MED010
apparatus (BAL-TEC, Balzers, Lichtenstein). Using a
Tescan VEGA scanning electron microscope (SEM;
Tescan, Brno, Czech Republic), three sites per
specimen were evaluated with respect to hair
shaft size and morphology. The average hair shaft
diameter at each sampling site was determined by
measuring the projected shaft area and dividing it by
the length of the longitudinal shaft axis.
Panographic and cephalometric radiographs were
obtained to assess the dental and cranial phenotypes.
Molar teeth with completed root formation were
measured from the panographs and the crown/root
(C/R) ratio determined to assess the severity of
taurodontism [Seow and Lai, 1989]. Bone mineral
density (BMD) was assessed using Dual Energy
X-Ray Absorptometry (DXA) as described previously
[Haldeman et al., 2004] using a Hologic QDR 4500 A
Scanner (Hologic, Inc., Bedford, MA) and norma-
lized for sex and age using Z-scores and compared
with young controls and T-scores. Hair shaft
diameters and BMD measures were evaluated by
Analysis of Covariance with repeated measure-
ments using SPSS (SAS Institute, Cary, North
Carolina) accepting P < 0.05 as significant.
RESULTS
Twelve family members from a three-generation
family demonstrated an autosomal dominant mode
of inheritance (Fig. 1). Mutation analysis revealed
that all affecteds were heterozygous for the DLX3
c.561_562delCT mutation and unaffecteds were
homozygous normal.
The dental phenotype included teeth that
appeared small and varied from having areas of
opaque white enamel with yellow-brown discolora-
tions to teeth that were very yellow brown in
coloration (Fig. 2). The small tooth size gave an
 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

ATTENUATED TRICHO-DENTO-OSSEOUS PHENOTYPE 345

FIG. 2. The dental phenotype in these two affected individuals varied markedly ranging from a slight yellow brown discoloration (A: individual III:4, age 16 years
6 months) to a severe and generalized yellow brown discoloration (B: individual III:3, age 14 years 2 months). [Color figure can be viewed in the online issue, which is
available at www.interscience.wiley.com.]

appearance of increased tooth spacing in most cases.
Two affecteds had abnormal second mandibular and
maxillary molars that were heart-shaped with only
three major cusps. Radiographically, the teeth had a
decreased enamel thickness and an increased pulp
chamber dimension. The molar teeth had varying
degrees of apical displacement of the root furcation
resulting in taurodontism. Panographic radiographs
and C/R ratio measurements revealed that the
molar taurodontism (Fig. 3) ranged from normal
(cynodont, C/R ratio <1.10) to mesotaurodont
(moderate apical displacement of the molar root
furcation C/R ratio
2.0) morphology [Shaw, 1928].
None of the molars had the hypertaurodont mor-
phology (severe apical displacement of the furca-
tion. C\R ratio >2.0) that is often seen in individuals
with the DLX3 c.571_574delGGGG mutation (Fig. 3).
One affected individual had essentially normal pulp
morphology but had severely abnormal enamel and
markedly yellow-brown discoloration of the teeth.
Cephalometric radiographs showed the affected
individuals did not exhibit markedly increased bone
density, obliteration of the diplöe or mastoid air cells,
or frontal sinus (Fig. 4). DXA bone scans were

FIG. 3. The enamel was thin in both of these affected individuals and the pulp morphology is essentially normal in one (B: individual III:4) and showed a moderate
degree of taurodontism in the second (A: individual III:3).

346
FIG. 4. This cephalographs of an affected adult (individual II:1, age 43 years
6 months) showed the frontal sinus and diplöe (arrows) was visible and the
bone density was normal.
evaluated for the family as a group (N ¼ 12) and then
for the adults (N ¼ 7), separately. No significant
difference of affected and unaffected individuals was
observed when the entire group was evaluated.
However, the affected adults did show a statistically
significant increase in spine BMD (Table I) compared
with the unaffecteds. The increase in hip BMD was
approaching a significant level (P ¼ 0.08), whereas
the total femur, femoral neck, wrist, and full body
BMD did not show significant differences.
Affecteds had coarse, unruly hair compared
with the unaffecteds (Fig. 5). Electron microscopy
showed smaller diameter hair shafts compared with
unaffecteds (Fig. 6). Hair shafts in the affecteds
frequently displayed abnormal shaft morphology
with horizontal grooves (Fig. 7). Hair shaft diameters
were not as reduced, as seen in individuals with
the DLX3 c.571_574delGGGG mutation [Wright
et al., 1994].
DISCUSSION
Detailed evaluation of the hair, tooth, and bone
phenotype in a family with a DLX3 c.560_561delCT
mutation showed marked differences in severity
TABLE I. DEXA Bone Density of Adult Family Members
Individuals with DLX3 CT del mutation
Z-scores N Mean SD
Spine 4 0.7 0.5
Femoral neck 4 0.4 0.5
Hip 4 0.1 0.5
Radius þ ulna 4 0.8 0.8
American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a
WRIGHT ET AL.
compared with individuals previously reported with
the DLX3 c.571_574delGGGG mutation. Variation in
the TDO phenotype has lead to the proposal
of different TDO subtypes [Shapiro et al., 1983],
erroneous classification of many cases [Crawford
and Aldred, 1990], and controversy as to whether
ADHHAI is a distinct entity [Crawford and Aldred,
1990; Seow, 1993b]. The present study indicates
that the DLX3 c.561_562delCT mutation causes an
attenuated TDO phenotype involving hair, teeth,
and bone. We conclude that this mutation causes
TDO and not ADHHAI.
The kinky-curly TDO hair phenotype is known to
change in about half of the children, becoming
normal in appearance or in some cases becoming
coarse and unmanageable [Wright et al., 1997]. All
individuals in the kindred reported here had coarse
unmanageable hair with a diminished shaft diameter
and morphology similar to that seen in people with
the DLX3 c.571_574delGGGG mutation [Wright
et al., 1994].
The dental phenotype was present in all individ-
uals with the DLX3 c.561_562delCT mutation, but
was highly variable. The severity of the enamel
defect varied considerably, ranging from a near
normal color to a severe generalized yellow brown
color. All of the affecteds showed decreased enamel
thickness based on radiographic and histologic (data
not shown) evaluations. Alterations in tooth size and
the severity of taurodontism were both less severe
in people with the DLX3 c.561_562delCT mutation
compared with individuals having the DLX3 c.571_
574delGGGG mutation [Wright et al., 1997]. Pulp
morphology ranged from normal to mesotaurodont
with none of the teeth displaying hypertaurodont-
ism, as is often seen in individuals with the DLX3
c.571_574delGGGG mutation. Pulp morphology in
the case illustrated by Dong et al. [2005] did not
exhibit taurodontism of the first permanent molar,
which is similar to most of the affecteds evaluated in
the present study. Quantitative evaluation of the pulp
morphology showed that the permanent molars in
people with the DLX3 c.561_562delCT mutation can
have mild taurodontism or pyramidal shaped root
morphology.
The third major feature of TDO, the bone
phenotype, is highly variable in individuals having
the DLX3 c.571_574delGGGG mutation [Wright
et al., 2000; Haldeman et al., 2004]. The bone density
Unaffected family members
Min Max N Mean SD Min Max P-value
1.2 0.0 3 1.2 1.5 2.9 0.0 P ¼ 0.01
1.0 0.2 2 0.8 0.9 1.4 0.1 P > 0.1
0.5 0.7 2 0.0 1.1 0.8 0.8 P ¼ 0.08
1.8 0.0 3 0.9 0.9 1.5 0.2 P > 0.1
 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

ATTENUATED TRICHO-DENTO-OSSEOUS PHENOTYPE 347

FIG. 7. Scanning electron micrographs show the differences in hair shaft

diameters from a normal individual and those with the DLX3 mutations.

compared with the unaffecteds. This slight increase

FIG. 5. All the affected individuals had coarse, unmanageable hair as seen in
this affected person (III:5 age 11 years).
ranged from decreased in some individuals but on
average is markedly increased. Based on the limited
sample evaluated in the present study, it appears that
the affecteds had only a slight increase in BMD
did not manifest at all sites evaluated. Indeed, a
significant increase in BMD was only observable at
the spine location. Interestingly, BMD evaluation in
individuals with the DLX3 c.571_574delGGGG
mutation showed the spine to be the most affected
having mean Z-scores over 3 [Haldeman et al., 2004].
Other sites such as the wrist and hip had a lesser
increase in density and did not increase with age as
was observed at the spine. None of the affecteds
evaluated in this study had the magnitude of
increased BMD as seen in individuals having the
DLX3 c.571_574delGGGG mutation
Taken together, the hair, tooth, and bone pheno-
types in individuals with the DLX3 c.561_562delCT
mutation indicate a diagnosis of TDO and not AI with
taurodontism as has been proposed. All three tissues

FIG. 6. Means and standard deviations of hair shaft diameters as a function of age. Hair shaft diameter was significantly reduced in the affecteds compared with the
unaffected family members and as seen in the older individuals, this difference diminishes. [Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]
 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

348 WRIGHT ET AL.

FIG. 8. A: Comparison of DLX3 mutations leading to differing DLX3 proteins and TDO phenotypes. B: Kyte-Doolittle mean hydrophobicity plot (window size ¼ 7)
demonstrating divergence of the protein characters of wild type and c.564_567delGGGG mutation, which is near the second activation domain (aa 200). The novel C-
terminal peptide may lead to stearic hindrance of additional components of the transcription complex necessary for DLX3 mediated transcription, which is not an issue
in the protein predicted to result from the c.560_561delCT mutation.

are indeed affected but present an attenuated
phenotype. It is interesting that these two DLX3
mutations, that are only four base pairs apart,
produce such different phenotypic severities. There
are several fundamental differences in the proteins
that result from these two mutations that could
account for the resulting phenotypic differences
(Fig. 8). One might predict that the DLX3 c.561_
562delCT mutation which is predicted to alter the
homeobox sequence and truncate C-terminal of the
homeobox would be a more severe mutation
compared to the DLX3 c.571_574delGGGG mutation
which occurs outside the homeobox. The putative
proteins produced from these two mutations differ in
the amount of novel peptide at the C-terminus
(Fig. 8). The DLX3 c.561_562delCT mutation predicts
a protein that is 56 amino acids shorter than the DLX3
c.571_574delGGGG mutation. The c.561_562delCT
mutation predicts a 12 aa novel peptide sequence
while the c.571_574delGGGG mutation predicts a
67 aa novel peptide. Hydrophobicity evaluation of
these peptides differs most significantly in the 190–
210 aa region encompassing most of the C-terminal
transactivation domain. We hypothesize that this
novel peptide alters the protein functionality and
explains the differences in the phenotypes caused by
these two mutations.
ACKNOWLEDGMENTS
This work was supported in part by a grant
(RR00046) from the General Clinical Research Centers
Program of the Division of Research Resources and
a grant (DE12202) from the National Institute of
Dental and Craniofacial Research, National Institutes
of Health.
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