Cleidocranial dysplasia and novel RUNX2 variants: dental, craniofacial, and osseous manifestations

Abstract Cleidocranial dysplasia (CCD) is a skeletal disorder affecting cranial sutures, teeth, and clavicles, and is associated with the RUNX2 mutations. Although numerous patients have been described, a direct genotype–phenotype correlation for RUNX2 has been difficult to establish. Further cases must be studied to understand the clinical and genetic spectra of CCD. Objectives To characterize detailed phenotypes and identify variants causing CCD in five unrelated patients and their family members. Methodology Clinical and radiographic examinations were performed. Genetic variants were identified by exome and Sanger sequencing, data were analyzed by bioinformatics tools. Results Three cases were sporadic and two were familial. Exome sequencing successfully detected the heterozygous pathogenic RUNX2 variants in all affected individuals. Three were novel, comprising a frameshift c.739delA (p.(Ser247Valfs*)) in exon 6 (Patient-1), a nonsense c.901C>T (p.(Gln301*)) in exon 7 (Patient-2 and affected mother), and a nonsense c.1081C>T (p.(Gln361*)) in exon 8 (Patient-3). Two previously reported variants were missense: the c.673C>T (p.(Arg225Trp)) (Patient-4) and c.674G>A (p.(Arg225Gln)) (Patient-5) in exon 5 within the Runt homology domain. Patient-1, Patient-2, and Patient-4 with permanent dentition had thirty, nineteen, and twenty unerupted teeth, respectively; whereas Patient-3 and Patient-5, with deciduous dentition, had normally developed teeth. All patients exhibited typical CCD features, but the following uncommon/unreported phenotypes were observed: left fourth ray brachymetatarsia (Patient-1), normal clavicles (Patient-2 and affected mother), phalangeal malformations (Patient-3), and normal primary dentition (Patient-3, Patient-5). Conclusions The study shows that exome sequencing is effective to detect mutation across ethnics. The two p.Arg225 variants confirm that the Runt homology domain is vital for RUNX2 function. Here, we report a new CCD feature, unilateral brachymetatarsia, and three novel truncating variants, expanding the phenotypic and genotypic spectra of RUNX2 , as well as show that the CCD patients can have normal deciduous teeth, but must be monitored for permanent teeth anomalies.


Introduction
Cleidocranial dysplasia (CCD, MIM #119600) is a congenital disorder that affects bone growth and teeth formation. 1 The incidence of CCD is about one in a million. 2,3 CCD is also known as Scheuthauer-Marie-Sainton syndrome, cleidocranial dysostosis, osteodental dysplasia, generalized dysostosis, mutational dysostosis, and cleidocranial-pubic dysostosis. 1,4 The main clinical features of CCD are hypoplastic or aplastic clavicles, defective ossification of anterior fontanelle, and dental anomalies (retention teeth, supernumerary teeth, impacted teeth, and cysts). 1,3 The unique facial appearances of CCD are prominent forehead, midfacial hypoplasia, and hypertelorism. Other bone deformities include wide pelvic joint, coxa vara (less than 120 degrees angle between the femoral head and shaft), coxa valga (increased angle between the femoral neck and shaft), genu valgum (knock-knee deformity), delayed growth of the pubic bone, merging failure of the lower jaw, scoliosis, and brachydactyly. 3,5 Treatment for CCD depends on the symptoms. The pentapeptide sequence (amino acids 517-521). 10,11 Both the QA and PST regions act as the transactivation regions. The RHD is highly conserved and plays a role in DNA binding. 10 The VWRPY pentapeptide sequence functions as a transcriptional repression region. 10 RUNX2 is a transcription factor that either activates or inhibits the gene expression by regulating transcription through binding specific DNA sequences or interacting with transcriptional co-inducers and co-depressors to regulate osteogenesis. 12 In humans, the heterozygous loss-of-function variants in RUNX2 are the major cause of CCD (60-70% of patients).  The variants were identified as novel if they were not published in the ClinVar-NCBI (https://www.ncbi.nlm. nih.gov/clinvar) and the gnomAD (https://gnomad. broadinstitute.org/).

Craniofacial features
Open/delayed closure of fontanelle  (Figure 2A -D). Chest radiograph disclosed absence of both clavicles ( Figure 2E). Skull radiographs showed unclosed fontanelle, frontal and parietal bossing, persistent metopic suture, multiple Wormian bones along the bilateral lambdoid and posterior aspect of squamosal sutures, and prominent adenoid tissue and bilateral palatine tonsils. Maxillary hypoplasia was not evident (Figure 2F -G). Anteroposterior hip radiograph showed a lack of ossification of pubic bones with the resultant apparent widening of the pubic symphysis and a characteristic "chef's hat" appearance of the femoral head ( Figure 2H). Hand radiograph exhibited partial pseudoepiphysis of 2 nd and 5 th proximal metacarpal bones, shortening of 2 nd and 5 th middle phalanges, and fusion of 1 st distal phalanx ( Figure 2I). Oral examination revealed deciduous   16 Her parents were unaffected and did not carry the missense. The variant was previously reported in the familial and sporadic cases with CCD. 13, 16,20 Patient-5 (5-II:1), a 3-year-old boy, was referred for genetic analysis due to congenital malformations of skull and face bones, deformed clavicles, and a history of bilateral clavicular fractures. At 4 months old, his whole-body bone mineral density (BMD) was 0.267 g/cm 2 within normal range (0.25 + 0.04 g/cm 2 ); total body less head BMD was 0.267 g/cm 2 ; lumbar spine BMD was 0.142 g/cm 2 ; and whole-body mineral content was 163.14 g. At age 3, his height was 86.2 cm (<3 rd percentile). Examinations showed that 5-II:1 had macrocephaly (OCF 52 cm, >97 th percentile), metopic depression, narrow sloping shoulders opposing at the midline, and lordosis ( Figure 2S -10; ‡2, 5, 6, 8, 11-20 (References are provided in the supplementary figure 1) With variants in the PST region and normal clavicles, previous CCD cases were reported with multiple supernumerary teeth. 25,26 Similarly, supernumerary teeth were found in patients 2-II:1 and 2-I:2.
Further studies are needed to confirm this genotypephenotype correlation.
During tooth development, RUNX2 is expressed from early stage to the formation of roots and periodontium. 27 It plays a role in osteoblast and odontoblast differentiation, alveolar bone remodeling essential for tooth eruption, and the maintenance of the periodontal ligament. 28 In RUNX2 knockout mice, molar tooth development arrested at the late bud stage. 29 Interestingly, both Runx2 -/and Runx2 +/mice developed lingual buds in front of the upper molars more than in wild-type mice, representing the extension of dental lamina for successional teeth. 30 It was suggested that Runx2 may have different effects at different stages of tooth development.
Accordingly, the development of permanent dentition in CCD patients is severely disturbed while the primary dentition is rarely affected. 1,28,31 Patients 1-I:2, 2-II:1, 2-I:2, and 4-II:2 with permanent dentition, showed several dental anomalies, such as malocclusion, clinically missing of permanent teeth, unerupted teeth, and supernumerary teeth; whereas patients 3-II:2 and 5-II:1, with primary dentition, showed no remarkable tooth abnormalities. It may also be assumed that the effects of RUNX2 haploinsufficiency on the development of the primary dentition may be too subtle to be detected clinically. 28 Apart from typical CCD features, 1-I:2 also presented with brachydactyly and fourth-ray brachymetatarsia of the left foot and 3-II:1 had partial pseudoepiphysis of the 2 nd and 5 th proximal metacarpal bone, shortening of the 2 nd and 5 th middle phalanges, and fusion of the 1 st distal phalanx. Hand abnormalities, including brachydactyly, tapering fingers, and short and broad thumbs, are frequently observed in CCD patients. 17,[32][33][34] A decrease or loss-of-function of RUNX2 is associated with CCD; while gain-of-function due to intragenic duplications is related to metaphyseal dysplasia with maxillary hypoplasia and brachydactyly (MDMHB, MIM #156510). 35,36 These suggest that fine-tuning the expression level of RUNX2 is important for normal development of the phalanges or metacarpals. There have been only few clinical case reports describing brachymetatarsia in CCD cases, but without genetic variant identification. 37-39 Our findings could lead to genotype-phenotype correlation in the future.