RUNX2 mutations in Taiwanese patients with cleidocranial dysplasia
Wei-De Lin1,2, Shuan-Pei Lin3, Chung-Hsing Wang4, Yushin Tsai5, Chih-Ping Chen6and Fuu-Jen Tsai1,4,5,7
1Department of Medical Research, China Medical University Hospital, Taichung, Taiwan.
2School of Post Baccalaureate Chinese Medicine, China Medical University, Taichung, Taiwan.
3Department of Pediatrics, MacKay Memorial Hospital, Taipei, Taiwan.
4Department of Pediatrics, China Medical University Hospital, Taichung, Taiwan.
5Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan.
6Department of Obstetrics and Gynecology, MacKay Memorial Hospital, Taipei, Taiwan.
7Department of Biotechnology and Bioinformatics, Asia University, Taichung, Taiwan.
Cleidocranial dysplasia (CCD) is an autosomal dominant human skeletal disorder comprising hypoplastic clavicles,
wide cranial sutures, supernumerary teeth, short stature, and other skeletal abnormalities. It is known that mutations
in the human RUNX2 gene mapped at 6p21 are responsible for CCD. We analyzed the mutation patterns of the
RUNX2 gene by direct sequencing in six Taiwanese index cases with typical CCD. One of the patients was a familial
case and the others were sporadic cases. Sequencing identified four mutations. Three were caused by single nucle-
otide substitutions, which created a nonsense (p.R391X), two were missense mutations (p.R190W, p.R225Q), and
the forth was a novel mutation (c.1119delC), a one-base deletion. Real time quantitative PCR adapted to determine
copy numbers of the promoter, all exons and the 3’UTR region of the RUNX2 gene detected the deletion of a single
allele in a sporadic case. The results extend the spectrum of RUNX2 mutations in CCD patients and indicate that
complete deletions of the RUNX2 gene should be considered in those CCD patients lacking a point mutation de-
tected by direct sequencing.
Key words: cleidocranial dysplasia, CCD, RUNX2, RUNX2 deletion mutation.
Received: June 23, 2010; Accepted: December 12, 2010.
Cleidocranial dysplasia, (CCD, MIM#119600), is an
autosomal dominant skeletal dysplasia comprising defec-
tive skull ossification with frontal bossing, open fontanels,
clavicular hypoplasia, delayed ossification of the pelvis,
late eruption of permanent teeth, malformed dental roots,
supernumerary teeth, and normal intelligence (Mundlos,
Mutations in the core-binding factor A1 gene
(CBFA1) located on chromosome 6p21 were identified as
the cause of CCD (Gelb et al., 1995; Mundlos et al., 1997).
This gene is composed of eight exons and spans a region of
more than 220 kb in 6p21. The CBFA1 protein contains a
highly conserved runt domain and therefore, the gene is
also referred to as RUNX2 (GenBank accession number
AF001450) (Yoshida et al., 2002). The runt domain inter-
acts with the core binding factor consensus sequence
TGT/CGGT located in the promoter region of target genes.
RUNX2 regulates the expression of multiple genes ex-
pressed in osteoblasts and it is a cis-acting element in the
erozygous RUNX2 mutated mice display some of the hall-
marks of human CCD, including open fontanelles and hy-
The RUNX2 protein contains two regions divided by
the runt domain. On the N-terminal side of the runt domain
are glutamine-alanine repeats (Q/A domain) and on the
C-terminal side is a region rich in proline-serine-threonine
(PST domain). The PST domain is thought to be the tran-
sactivation domain. The last five amino acids of RUNX2
(VWRPY) compose a conserved motif in all runt proteins
(Quack et al., 1999; Zhang et al., 2000; Otto et al., 2002).
Several mutations of RUNX2 were identified in pa-
tients with CCD (Otto et al., 2002). In this study, we
screened six patients with typical CCD for mutations in the
RUNX2 gene. Mutations were detected in five cases, these
including an entire deletion of the RUNX2 coding region.
The six unrelated patients included in this study were
first evaluated because of short stature. They received the
see Mundlos, 1999). Patient 4 was a familial case, whereas
Genetics and Molecular Biology, 34, 2, 201-204 (2011)
Copyright © 2011, Sociedade Brasileira de Genética. Printed in Brazil
Send correspondence to Fuu-Jen Tsai. Department of Medical Ge-
netics, China Medical University Hospital, number 2, Yuh-Der
Road, 404 Taichung, Taiwan. E-mail: email@example.com.
the others were sporadic. Except for Patients 5 and 6, their
height was below the 5thcentile for the Taiwanese popula-
tion. All patients showed brachycephaly, mild frontal boss-
merary teeth, as well as clavicular hypoplasia. The upper
part of the thorax was small and a funnel chest was noted.
Scoliosis of the thoracic spine with convexity to the right
and mild lordosis were also found. Chest radiographs
showed hypoplasia of the clavicles with absence of the
outer half. Three-dimensional head CT revealed enlarged
anterior fontanelles beyond two years of age. Physical data
for the six patients are summarized in Table 1.
Informed consent was obtained from the patients or
the guardians of these patients. Genomic DNA was ex-
tracted from peripheral blood leukocytes using a MagNA
cording to Quack et al. (1999) and Napierala et al. (2005).
PCR products were purified from the agarose gel using a
QIAEX II kit (Qiagen, Hilden, Germany) and subjected to
direct sequencing using a BigDye 3.1 Terminator cycle se-
quencing kit (Applied Biosystems, Forst City, CA, U.S.A.)
with ABI Prism 377 DNA Sequencer or ABI 3100 Genetic
Analyzer (Applied Biosystems, Forst City, CA, U.S.A.).
To determine carrier rates for the novel mutations in the
Taiwanese population, the RUNX2 gene profile of
100 healthy individuals was analyzed using the same pro-
cedures. Reference sequence and base-pair numbers refer
to GenBank accession numbers AF001443-AF001450 and
RUNX2 mutations were detected in four patients. Pa-
tient 1 was heterozygous for a C-to-T transition at cDNA
nucleotide position 1171 in exon 7 of RUNX2, this creating
a stop codon at amino acid position 391 (p.R391X). The
nonsense mutation, which caused early termination and
produced a truncated protein lacking the PST-domain and
the VWRPY motif (Thirunavukkarasu et al., 1998), has
been previously reported (Tsai et al., 2000; Zhang et al.,
2000). The mutation seen in Patient 4 was in heterozygosis
and represented a C-to-T transition at nucleotide 568 in
exon 1 of RUNX2, this causing the substituation of arginine
by tryptophan at amino acid position 190 (p.R190W) in the
runt domain (Otto et al., 2002). An arginine stretch located
in the runt domain plays an important role in DNA binding.
Therefore, disruption of this region may lead to the loss of
DNA binding ability (Yoshida et al., 2002). As the side
chain of arginine is the major part that interacts with DNA,
replacing it by another amino acids of different hydropho-
bicity and charge, such as tryptophan, disrupts the structure
of the runt domain and decreases DNA binding affinity.
Patient 6 was heterozygous for a G-to-A transition at
cDNA nucleotide position 674 in exon 3 of RUNX2, this
causing arginine to be substituted by glutamine at amino
acid position 225 (p.R225Q) at the C-terminal end of the
runt domain (Otto et al., 2002). This amino acid substitu-
tion and might affect the structure stability and interaction
with other proteins.
amino acid 373, creating a stop codon at position 483 (Fig-
ure 1). This deletion has not been reported previously and
was not present in the patient’s parents. It disrupts both the
repression subdomain and the VWRPY motif and is pre-
dicted to abolish RUNX2 function. The C-terminal region
of the PST-domain, which contains a region that interacts
with Smad proteins, is considered necessary for RUNX2-
mediated transcriptional regulation (Zhang et al., 2000).
The VWRPY motif is an interaction partner for the binding
of transcriptional corepressors of the TLE/Grg family
(Aronson et al., 1997; Levanon et al., 1998). Thus, even in
caseswhere the runt domain is complete, the mutated
tution of G for A. This polymorphism site was described
previously in another population (Machuca-Tzili et al.,
202Lin et al.
Table 1 - Summary of clinical manifestations and RUNX2 gene mutation sites in five Taiwanese cases with cleidocranial dysplasia.
CaseType Age, years SexStature, cm
Mutation site on RUNX2
1Sporadic4M 91 (< 3rd)YYY
c.1171 C ? T, R391X
2Sporadic11M 127 (< 5th)YYY c.1119delC, frameshift
3Sporadic2F 78 (< 3rd)YYYexon 0~7, 3’UTR deletion
4Familial 10M 122 (< 5th)YYY
c.568 C ? T, R190W
40F 144 (< 5th)YYY
c.568 C ? T, R190W
5Sporadic 12M 141 (10~25 th)YYYN.D.4
6Sporadic8F 127 (75 th)YYY
c.674 G ? A, R225Q
Sex: M, male; F, female. Y: present; N: not present. N.D.: not detected.
moter and the coding regions of RUNX2, similar to previ-
ous results showing that only 33~50% of patients with an
unambiguous clinical and radiographic diagnosis of CCD
carry mutations in the RUNX2 gene (Quack et al., 1999).
Large deletions or duplication of the RUNX2 gene are
rarely seen in CCD patients (Lee et al., 2008). Thus, to in-
vestigate the possibility of a gross RUNX2 deletion, the
genomic DNA of Patients 3 and 5 were subjected to real
conditions described by Lee et al. (2008). Briefly, three in-
dependent experiments were performed to determine copy
number variation in CCD patients and normal individuals.
The RT-qPCR primers were designed to detect the pro-
moter, exons, and 3’UTR regions of RUNX2. The glyceral-
dehyde-3-phosphate dehydrogenase (GAPDH) gene was
used as control. The formula for calculating copy number
was: copy number = 2 * 2-(?Ctp - ?Ctn) where Ct is the thresh-
old cycle defined as the mean cycle at which the fluores-
cence curve reached an arbitrary threshold; ?Ct is calcu-
lated as Ct of RUNX2 - Ct of GAPDH, ?Ctp is the ?Ct for
patients, and ?Ctn that for normal individuals. While two
copies of the tested RUNX2 regions were present in Patient
5, Patient 3 had only one copy of theses regions (Figure 2).
Thus, we concluded that Patient 3 carried a deletion of one
of the RUNX2 alleles.
Although no mutation was found in Patient 5, follow-
ing sequencing and RT-qPCR analysis, these result do not
exclude other mutation types in the RUNX2 gene, such as
the other hand, a report showed that cytogenetic anomalies
at chromosomal band 8q22 have been associated with a
CCD-like phenotype case (Brueton et al., 1992). Thus,
more detailed analyses are required to identify the genetic
abnormality seen in Patient 5.
The patients with any of the four point mutations in
the RUNX2 gene were similar in their clinical symptoms,
independent of whether their RUNX2 protein retained the
normal runt domain or not. The patients presented here
lacking the PST domain and VWRPY motif exhibited typi-
cal CCD. This suggests that the C-terminal region plays an
important role in RUNX2 function, with different geno-
types seemingly generating a similar CCD phenotype.
From the findings of Patient 3, who carried the deletion of
ilar to those of point mutations. We furthermore inferred
that CCD patients not showing a RUNX2 mutation in di-
or duplication, and RT-qPCR appears as a sensitive ap-
proach to this end.
The work was supported by grants from the National
Science Council (NSC-93-2314-B-039-011) and the China
Medical University Hospital (CMU98-S-02).
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RUNX2 mutations in Taiwanese CCD patients 203
Figure 1 - Sequence analyses of genomic DNA of the RUNX2 from Pa-
tient 2. A heterozygous cytosine deletion at cDNA position 1119 was de-
tected (c.1119delC). The normal sequence is shown above the elec-
Figure 2 - Analysis of RUNX2 copy number using RT-qPCR. Copy num-
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the 3’-UTR. Patient 3 had only one copy of these regions. ?: normal;
¡ : Patient 3; o : Patient 5. Each bar indicates mean ? SD of three inde-
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Associate Editor: John M. Opitz
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204Lin et al.