J Oral Pathol Med (2014) 43: 798–800 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
doi: 10.1111/jop.12198
wileyonlinelibrary.com/journal/jop
BRIEF REPORT
Novel complex disease allele mutations in cleidocranial dysplasia patients Robert P. Anthonappa1, Fan Yan-Hui2, Nigel M. King1, Abu Bakr M. Rabie3, Song You-Qiang2 1
School of Dentistry, The University of Western Australia, Perth, Australia; 2Department of Biochemistry and Centre for Reproduction, Development and Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; 3Orthodontist, Private Practice, Hong Kong SAR, China
This study reports a novel identical complex disease allele harboring two non-synonymous mutations that were identified in two southern Chinese individuals of the same family with cleidocranial dysplasia (CCD). Blood samples were obtained from the proband, his parents, plus 100 matched control subjects. Exons 0 to 7 of the RUNX2 gene were amplified using specific primers and sequenced. Multiple sequence alignment and protein structure modeling was performed using ClustalW2 and MODBASE software while PolyPhen-2 and MutationTaster applications were employed to predict the diseasecausing potential of the identified mutations. A complex disease allele in two affected individuals harboring two non-synonymous mutations in a cis-position on exons 4 (D273N) and 5 (P299L) were identified. The identified mutations were in the conserved region and changed the protein structure. J Oral Pathol Med (2014) 43: 798–800 Keywords: cleidocranial dysostosis; RUNX2; supernumerary teeth
cleidocranial
dysplasia;
Introduction Cleidocranial dysplasia (CCD, MIM#119600) is a rare genetic disorder characterized by skeletal dysplasia, which is caused by an impaired intra-membranous ossification commonly involving the cartilage ossification. It affects the entire skeletal system causing a range of abnormalities and a reported prevalence of 1 in 100,000 (1, 2). Cleidocranial dysplasia is inherited as an autosomal dominant trait with complete penetrance; nevertheless, sporadic cases have been Correspondence: Robert P. Anthonappa, BDS, MDS, PhD, AdvDipDS, FDS RCS (Edin), MPaed RCS (Edin), MRACDS (Paed Dent), Paediatric Dentistry, School of Dentistry, Faculty of Medicine, Dentistry and Health Sciences, University of Western Australia, 17 Monash Avenue, Nedlands, WA 6009, Australia. Tel: +61 8 9346 7868; Fax: +61 8 9346 7666, E-mail: [email protected] Accepted for publication March 27, 2014
reported (3). Furthermore, autosomal recessive form or germ line mosaicisms have been suggested as alternative causes for this condition. Mutations in the Runt-related transcription factor 2 (RUNX2, MIM # 600211) which is located on the chromosome 6p21 have been identified as the cause of CCD (4). To date, several mutations including deletions, insertions, translocations, missense, frame shift, and slice mutations in all the exons of RUNX2 gene, except exon 0 and exon 6, have been reported (2). Nevertheless, no mutations have been reported for the southern Chinese individuals with CCD. Therefore, the objectives of this study were to perform mutational analysis of the RUNX2 gene in individuals from a southern Chinese family with CCD.
Materials and methods Ethical approval was obtained from the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster, and the informed consent was obtained from all the participants in this study. Clinical and radiographic examinations were performed for the individuals with CCD [for review of diagnostic criteria, see Mundlos et al. (4)]. The stature was determined and compared with the norms for the southern Chinese population. Supernumerary teeth were identified using a panoramic radiograph; if a supernumerary tooth was not evident on the panoramic radiograph, then additional intra-oral radiographs were taken. The panoramic radiographs of the individuals affected with CCD in the present study are presented in Fig. 1a. The proband and his parents plus 100 matched control subjects (n = 100) were screened by standard DNA sequencing analysis of the RUNX2 gene. Blood samples were obtained and the DNA subsequently extracted. For all the samples, the complete RUNX2 coding regions (exons 0 to 7) were amplified using specific primers [see Xuan et al. (5)] and sequenced using the 3730 9 l DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA). Multiple sequence alignment of the RUNX2 gene was performed across five species using ClustalW2 software (6), while MODBASE was used for protein structure modeling.
Mutations in CCD patients Anthonappa et al.
A
799
B
C
Figure 1 (a) Panoramic radiographs of individuals with cleidocranial dysplasia; proband (II:1) exhibits multiple supernumerary teeth, while his father (I:1) presented with multiple missing teeth which were extracted previously; hence, the supernumerary teeth could not be accounted for. (b) RUNX2 gene sequencing results: Arrows point out mutations. (a) electropherogram of proband (II:1) illustrates the presence of two non-synonymous mutations: c.817G > A and c.896C > T on exons 4 and 5, respectively, (b) electropherogram of proband father (I:1) harboring identical mutations, and (c) electropherogram of proband mother (I:2) without any mutations. (c) Illustration of the change in the protein structure of the RUNX2 gene subsequent to the complex disease allele mutations.
Furthermore, PolyPhen-2 (7) and MutationTaster (8) applications were used to predict the disease-causing potential of identified mutations. Mutations in the present study are given in reference to the NCBI Reference Sequence: NM_004348.3 (http://www.ncbi.nlm.nih.gov/nuccore/NM_ 004348.3?report=genbank).
The identified missense mutations were in the proline/ serine/threonine (PST)-rich transactivation and protein interaction domain and changed the protein structure (Fig. 1c). Furthermore, both PolyPhen-2 and MutationTaster applications revealed that mutation P299L on exon 5 exhibited a possible damaging effect compared to D273N mutation on exon 4 (Table 1).
Results Mutational analysis of the RUNX2 gene in the proband revealed a complex disease allele harboring two nonsynonymous mutations, which has not been reported previously, in cis-position on exons 4 (D273N) and 5 (P299L), see Table 1 and Fig. 1b. The proband exhibited all the classic features of CCD with multiple supernumerary teeth. Furthermore, mutational analysis of his father, who also exhibited the clinical features of CCD, revealed identical mutations (Table 1 and Fig. 1b). However, the number of supernumerary teeth could not be accounted for, as he had several teeth missing that were extracted previously. Nevertheless, on the panoramic radiographs presented there was no evidence of supernumerary teeth.
Discussion We report a novel genetic abnormality that of an identical complex disease allele harboring two non-synonymous missense mutations in the RUNX2 gene of two affected individuals in the same family, which to our knowledge is the first report in patients with CCD. Furthermore, the identified mutations have not been reported previously as separate mutations in patients with CCD. The two novel missense mutations identified in this study were located in the PST domain which is considered to be involved in transactivation and protein interactions. To date, there are no experimentally determined 3D structures available for the PST domain. Therefore, we used homology modeling (MODBASE) to elucidate the J Oral Pathol Med
Mutations in CCD patients Anthonappa et al.
800
Table 1 Details of the novel complex disease allele mutations identified in the southern Chinese family with cleidocranial dysplasia Mutation Family 1
Exon
Forwarda
Reverseb
II:1 I:1 I:2 II:1 I:1 I:2
4
G->A G->A Normal C->T C->T Normal
C->T C->T Normal G->A G->A Normal
5
Position (genomic reference)
c.DNA
Protein
Gene feature
PolyPhen2 score
MutationTaster score
Disease causal probability
45567829
G817A
D273N
Missense
0.002
0.477774
No
– 45588039
– C896T
– P299L
– Missense
– 0.181
– 0.973369
– Yes
–
–
–
–
–
–
–
PolyPhen-2, polymorphism phenotyping version 2. a Forward sequencing. b Reverse sequencing. NCBI Reference Sequence: NM_004348.3.
protein structure information; which provided a theoretical 3D structure model of the PST domain. However, we did not perform an in vitro functional analysis of the identified mutations on DNA binding and transactivation, due to technical constraints and financial limitations, which could be considered a limitation of the present study. Nevertheless, the identified sequence variations were not evident in the control samples which confirm that they do not represent common polymorphisms in the southern Chinese population. Polyphen-2, an automatic tool used to predict the possible impact of an amino acid substitution on the structure and function of a human protein, revealed a possible damaging effect of the mutation P299L on exon 5. Furthermore, MutationTaster application, a tool used for rapid evaluation of the disease-causing potential of DNA sequence alterations and considered to be more accurate than PolyPhen-2, also revealed a possible damaging effect of the mutation P299L on exon 5 compared to D273N mutation on exon 4. In the present case, both the father and son exhibited concordance in their genotype and phenotype, except for the unaccounted supernumerary teeth in the father. Therefore, it can be considered that mutation P299L on exon 5 was responsible to affect the gene function, thus resulting in the CCD phenotypes. Identification of a complex disease allele harboring two non-synonymous mutations in two CCD individuals in the same family makes this report novel. The existence of more than one common mutation can also be an important factor influencing manifestation and clinical variability of disorders such as CCD which would help to elucidate further the role of RUNX2 gene in the etiology of CCD.
J Oral Pathol Med
References 1. Chitayat D, Hodgkinson KA, Azouz EM. Intra-familial variability in cleidocranial dysplasia: a three-generation family. Am J Hum Genet 1993; 42: 298–303. 2. D’Alessandro G, Tagariello T, Piana G. Cleidocranial dysplasia: etiology and stomatognathic and craniofacial abnormalities. Minerva Stomatol 2010; 59: 117–27. 3. Ramesar RS, Greenberg J, Martin R, et al. Mapping of the gene for cleidocranial dysplasia in the historical Cape Town (Arnold) Kindred and evidence for locus homogeneity. J Med Genet 1996; 33: 511–14. 4. Mundlos S, Otto F, Mundlos C, et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 1997; 89: 773–9. 5. Xuan D, Sun X, Yan Y, Xie B, Xu P, Zhang J. Effect of cleidocranial dysplasia-related novel mutation of RUNX2 on characteristics of dental pulp cells and tooth development. J Cell Biochem 2008; 111: 1473–81. 6. Larkin MA, Blackshields G, Brown NP, et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007; 23: 2947–8. 7. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods 2010; 7: 248–9. 8. Schwarz JM, R€ odelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 2010; 7: 575–6.
Acknowledgments We thank the family members for their cooperation. This work was supported by the department fund (Paediatric Dentistry and Orthodontics, The University of Hong Kong).
doi: 10.1111/jop.12198
wileyonlinelibrary.com/journal/jop
BRIEF REPORT
Novel complex disease allele mutations in cleidocranial dysplasia patients Robert P. Anthonappa1, Fan Yan-Hui2, Nigel M. King1, Abu Bakr M. Rabie3, Song You-Qiang2 1
School of Dentistry, The University of Western Australia, Perth, Australia; 2Department of Biochemistry and Centre for Reproduction, Development and Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; 3Orthodontist, Private Practice, Hong Kong SAR, China
This study reports a novel identical complex disease allele harboring two non-synonymous mutations that were identified in two southern Chinese individuals of the same family with cleidocranial dysplasia (CCD). Blood samples were obtained from the proband, his parents, plus 100 matched control subjects. Exons 0 to 7 of the RUNX2 gene were amplified using specific primers and sequenced. Multiple sequence alignment and protein structure modeling was performed using ClustalW2 and MODBASE software while PolyPhen-2 and MutationTaster applications were employed to predict the diseasecausing potential of the identified mutations. A complex disease allele in two affected individuals harboring two non-synonymous mutations in a cis-position on exons 4 (D273N) and 5 (P299L) were identified. The identified mutations were in the conserved region and changed the protein structure. J Oral Pathol Med (2014) 43: 798–800 Keywords: cleidocranial dysostosis; RUNX2; supernumerary teeth
cleidocranial
dysplasia;
Introduction Cleidocranial dysplasia (CCD, MIM#119600) is a rare genetic disorder characterized by skeletal dysplasia, which is caused by an impaired intra-membranous ossification commonly involving the cartilage ossification. It affects the entire skeletal system causing a range of abnormalities and a reported prevalence of 1 in 100,000 (1, 2). Cleidocranial dysplasia is inherited as an autosomal dominant trait with complete penetrance; nevertheless, sporadic cases have been Correspondence: Robert P. Anthonappa, BDS, MDS, PhD, AdvDipDS, FDS RCS (Edin), MPaed RCS (Edin), MRACDS (Paed Dent), Paediatric Dentistry, School of Dentistry, Faculty of Medicine, Dentistry and Health Sciences, University of Western Australia, 17 Monash Avenue, Nedlands, WA 6009, Australia. Tel: +61 8 9346 7868; Fax: +61 8 9346 7666, E-mail: [email protected] Accepted for publication March 27, 2014
reported (3). Furthermore, autosomal recessive form or germ line mosaicisms have been suggested as alternative causes for this condition. Mutations in the Runt-related transcription factor 2 (RUNX2, MIM # 600211) which is located on the chromosome 6p21 have been identified as the cause of CCD (4). To date, several mutations including deletions, insertions, translocations, missense, frame shift, and slice mutations in all the exons of RUNX2 gene, except exon 0 and exon 6, have been reported (2). Nevertheless, no mutations have been reported for the southern Chinese individuals with CCD. Therefore, the objectives of this study were to perform mutational analysis of the RUNX2 gene in individuals from a southern Chinese family with CCD.
Materials and methods Ethical approval was obtained from the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster, and the informed consent was obtained from all the participants in this study. Clinical and radiographic examinations were performed for the individuals with CCD [for review of diagnostic criteria, see Mundlos et al. (4)]. The stature was determined and compared with the norms for the southern Chinese population. Supernumerary teeth were identified using a panoramic radiograph; if a supernumerary tooth was not evident on the panoramic radiograph, then additional intra-oral radiographs were taken. The panoramic radiographs of the individuals affected with CCD in the present study are presented in Fig. 1a. The proband and his parents plus 100 matched control subjects (n = 100) were screened by standard DNA sequencing analysis of the RUNX2 gene. Blood samples were obtained and the DNA subsequently extracted. For all the samples, the complete RUNX2 coding regions (exons 0 to 7) were amplified using specific primers [see Xuan et al. (5)] and sequenced using the 3730 9 l DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA). Multiple sequence alignment of the RUNX2 gene was performed across five species using ClustalW2 software (6), while MODBASE was used for protein structure modeling.
Mutations in CCD patients Anthonappa et al.
A
799
B
C
Figure 1 (a) Panoramic radiographs of individuals with cleidocranial dysplasia; proband (II:1) exhibits multiple supernumerary teeth, while his father (I:1) presented with multiple missing teeth which were extracted previously; hence, the supernumerary teeth could not be accounted for. (b) RUNX2 gene sequencing results: Arrows point out mutations. (a) electropherogram of proband (II:1) illustrates the presence of two non-synonymous mutations: c.817G > A and c.896C > T on exons 4 and 5, respectively, (b) electropherogram of proband father (I:1) harboring identical mutations, and (c) electropherogram of proband mother (I:2) without any mutations. (c) Illustration of the change in the protein structure of the RUNX2 gene subsequent to the complex disease allele mutations.
Furthermore, PolyPhen-2 (7) and MutationTaster (8) applications were used to predict the disease-causing potential of identified mutations. Mutations in the present study are given in reference to the NCBI Reference Sequence: NM_004348.3 (http://www.ncbi.nlm.nih.gov/nuccore/NM_ 004348.3?report=genbank).
The identified missense mutations were in the proline/ serine/threonine (PST)-rich transactivation and protein interaction domain and changed the protein structure (Fig. 1c). Furthermore, both PolyPhen-2 and MutationTaster applications revealed that mutation P299L on exon 5 exhibited a possible damaging effect compared to D273N mutation on exon 4 (Table 1).
Results Mutational analysis of the RUNX2 gene in the proband revealed a complex disease allele harboring two nonsynonymous mutations, which has not been reported previously, in cis-position on exons 4 (D273N) and 5 (P299L), see Table 1 and Fig. 1b. The proband exhibited all the classic features of CCD with multiple supernumerary teeth. Furthermore, mutational analysis of his father, who also exhibited the clinical features of CCD, revealed identical mutations (Table 1 and Fig. 1b). However, the number of supernumerary teeth could not be accounted for, as he had several teeth missing that were extracted previously. Nevertheless, on the panoramic radiographs presented there was no evidence of supernumerary teeth.
Discussion We report a novel genetic abnormality that of an identical complex disease allele harboring two non-synonymous missense mutations in the RUNX2 gene of two affected individuals in the same family, which to our knowledge is the first report in patients with CCD. Furthermore, the identified mutations have not been reported previously as separate mutations in patients with CCD. The two novel missense mutations identified in this study were located in the PST domain which is considered to be involved in transactivation and protein interactions. To date, there are no experimentally determined 3D structures available for the PST domain. Therefore, we used homology modeling (MODBASE) to elucidate the J Oral Pathol Med
Mutations in CCD patients Anthonappa et al.
800
Table 1 Details of the novel complex disease allele mutations identified in the southern Chinese family with cleidocranial dysplasia Mutation Family 1
Exon
Forwarda
Reverseb
II:1 I:1 I:2 II:1 I:1 I:2
4
G->A G->A Normal C->T C->T Normal
C->T C->T Normal G->A G->A Normal
5
Position (genomic reference)
c.DNA
Protein
Gene feature
PolyPhen2 score
MutationTaster score
Disease causal probability
45567829
G817A
D273N
Missense
0.002
0.477774
No
– 45588039
– C896T
– P299L
– Missense
– 0.181
– 0.973369
– Yes
–
–
–
–
–
–
–
PolyPhen-2, polymorphism phenotyping version 2. a Forward sequencing. b Reverse sequencing. NCBI Reference Sequence: NM_004348.3.
protein structure information; which provided a theoretical 3D structure model of the PST domain. However, we did not perform an in vitro functional analysis of the identified mutations on DNA binding and transactivation, due to technical constraints and financial limitations, which could be considered a limitation of the present study. Nevertheless, the identified sequence variations were not evident in the control samples which confirm that they do not represent common polymorphisms in the southern Chinese population. Polyphen-2, an automatic tool used to predict the possible impact of an amino acid substitution on the structure and function of a human protein, revealed a possible damaging effect of the mutation P299L on exon 5. Furthermore, MutationTaster application, a tool used for rapid evaluation of the disease-causing potential of DNA sequence alterations and considered to be more accurate than PolyPhen-2, also revealed a possible damaging effect of the mutation P299L on exon 5 compared to D273N mutation on exon 4. In the present case, both the father and son exhibited concordance in their genotype and phenotype, except for the unaccounted supernumerary teeth in the father. Therefore, it can be considered that mutation P299L on exon 5 was responsible to affect the gene function, thus resulting in the CCD phenotypes. Identification of a complex disease allele harboring two non-synonymous mutations in two CCD individuals in the same family makes this report novel. The existence of more than one common mutation can also be an important factor influencing manifestation and clinical variability of disorders such as CCD which would help to elucidate further the role of RUNX2 gene in the etiology of CCD.
J Oral Pathol Med
References 1. Chitayat D, Hodgkinson KA, Azouz EM. Intra-familial variability in cleidocranial dysplasia: a three-generation family. Am J Hum Genet 1993; 42: 298–303. 2. D’Alessandro G, Tagariello T, Piana G. Cleidocranial dysplasia: etiology and stomatognathic and craniofacial abnormalities. Minerva Stomatol 2010; 59: 117–27. 3. Ramesar RS, Greenberg J, Martin R, et al. Mapping of the gene for cleidocranial dysplasia in the historical Cape Town (Arnold) Kindred and evidence for locus homogeneity. J Med Genet 1996; 33: 511–14. 4. Mundlos S, Otto F, Mundlos C, et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 1997; 89: 773–9. 5. Xuan D, Sun X, Yan Y, Xie B, Xu P, Zhang J. Effect of cleidocranial dysplasia-related novel mutation of RUNX2 on characteristics of dental pulp cells and tooth development. J Cell Biochem 2008; 111: 1473–81. 6. Larkin MA, Blackshields G, Brown NP, et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007; 23: 2947–8. 7. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods 2010; 7: 248–9. 8. Schwarz JM, R€ odelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 2010; 7: 575–6.
Acknowledgments We thank the family members for their cooperation. This work was supported by the department fund (Paediatric Dentistry and Orthodontics, The University of Hong Kong).
