Whole-exome sequencing of a novel initiation codon mutation in RUNX2 in a Chinese family with cleidocranial dysplasia

Abstract Cleidocranial dysplasia (CCD) is mainly attributable to a variant of runt-related transcription factor 2 (RUNX2) on chromosome 6p21. CCD is an autosomal dominant skeletal disorder characterized by open/delayed closure of fontanels, clavicular hypoplasia, retention of deciduous teeth, and supernumerary permanent teeth. The aim of this study was to investigate potentially pathogenic mutations in 2 Chinese families. Genomic DNA was obtained from peripheral blood lymphocytes, and whole exome sequencing and Sanger sequencing were performed to detect gene variants. Real-time quantitative PCR was performed to determine the mRNA expression level of RUNX2 in the proband of family 1. Silico algorithms and conservation analyses were used to evaluate the functional impact. We identified a novel initiation codon mutation (c.2T>C) and a previously reported mutation (c.569G>A). Familial co-segregation verified an autosomal-dominant inheritance pattern. Our findings demonstrated that the novel mutation c.2T>C causes CCD. Quantitative real-time PCR suggested that downregulated RUNX2 levels and haploinsufficiency in RUNX2 lead to CCD. These results extend the spectrum of RUNX2 mutations in CCD patients and can be used for genetic consultation and prenatal diagnosis.

However, no apparent genotype-phenotype correlations have been investigated, and only a few RUNX2 mutations have been reported in Chinese CCD patients.
In the present study, we have reported a novel initiation codon variant in 1 RUNX2 allele (c.2T>C) as well as a previously reported missense mutation in 2 Chinese Han families with CCD.

Ethical approval and subjects
This work was approved by the Ethics Committee of the Affiliated of Stomatology Hospital of Hebei Medical University (No: [2016]004). All participants or their guardians signed written informed consent. The probands (Fig. 1, Family 1, II-1; Fig. 2, Family 2, II-1) were initially evaluated and diagnosed with CCD by Hebei Children's Hospital, and they came to the Hospital of Stomatology, Hebei Medical University, for dental treatment. All family members were checked by 2 experienced dentists, who performed oral and radiographic examinations.

DNA sample collection and extraction
The QIAmp DNA Blood Midi kit (Cat#51185; Qiagen, Hilden, Germany) was used to obtain genomic DNA from the peripheral blood lymphocytes of all participants, as per the manufacturer's protocols. DNA purity was analyzed with the NanoPhotometer spectrophotometer (Implen, Westlake Village, CA) and quantified with the Qubit DNA assay kit and Qubit 2.0 fluorimeter (Life Technologies, Carlsbad, CA).

Whole exome sequencing and Sanger sequencing
Whole exome sequencing was performed for the probands. Target enrichment and amplification were performed using the liquid-phase capture method with testing kits from iGeneTech

Clinical manifestations
The clinical features of the patients and phenotypic characteristics of the probands were as follows: short stature, broad forehead, frontal bossing, orbital hypertelorism, midface hypoplasia, and protruding mandible (Fig. 1B, Fig. 2B & F). Failure of eruption of permanent teeth, retention of deciduous teeth, and supernumerary teeth were confirmed by panoramic radiograph. Tooth deformity was observed because of the failure of eruption of several supernumerary teeth in the mandible or maxilla  Fig. 1D; Fig. 2C & G). Aplasia of the clavicle ( Fig. 1C; Fig. 2D & H) and delayed closure of cranial sutures in II:1 proband of family 2 were detected in the chest radiograph (Fig. 2E). On the basis of the clinical and radiological results, CCD was confirmed, and the clinical results of the other affected family members are listed in Table 1.

Analysis of RUNX2 variants
We identified a novel heterozygous RUNX2 missense variant c.2T>C (NM_001015051.4) that alters the initiation codon ATG to ACG in family 1 (Fig. 3A) and a previously reported mutation (c.569G>A, p.Arg190Pro) in family 2 (Fig. 3B). In family 1, the same mutation site was not detected in the patient's mother (Fig. 3A). We could not contact the patient's father because the parents were divorced, so the patient may have inherited the mutation from her father. Furthermore, the nucleotide alteration c.2T>C was not found in the healthy controls (n = 100) or NHLBI exome sequencing project Exome Variant Server (https://evs.gs.washington.edu/EVS/), indicating the substitution is a rare variant. Then, the candidate variant was confirmed for proband 1 and her mother by Sanger sequencing.
In family 2, we found a previously reported mutation (c.569G>A, p.Arg190Pro) [25] in the proband, his older sister, and their mother (Fig. 3B). Figure 3C shows the schematic representation of RUNX2 structure and annotated variants. The mother was heterozygotic for RUNX2. We could not get in touch with the father. On the basis of the autosomal dominant inheritance pattern of CCD, the siblings' mutation was inherited from their mother. The mutation (c. 569G>A) was shared by all affected members [25] but not detected in 100 unrelated healthy Chinese volunteers.

RUNX2 mRNA expression
qPCR was performed to evaluate the variant effects and determine mRNA expression levels of RUNX2 of the proband in family 1. GAPDH was used as the internal reference (control group was set to 1), and the relative quantity of RUNX2 mRNA in the proband was 0.616. The results indicated that the mRNA levels of RUNX2 were downregulated in the patient.

Bioinformatics analyses
SIFT, Polyphen2, and MutationTaster predicted that the mutation (c.2T>C) was "deleterious" (0.00), "possibly damaging" (0.838), and "disease-causing" (1.00), respectively, suggesting the variant is highly pathogenic. No other candidate variants were identified in the evaluation of the exome file of family 1 to rule out the possibility of the contribution of any variation in other known causative genes for CCD. A cross-species amino acid sequence alignment of the reported mutation (c.569G>A, [p.Arg190Pro]) showed that Arg190 was highly conserved among humans, rhesus macaques, chickens, mice, rats, and chimpanzees (Fig. 3D).

Discussion
CCD is a skeletal dysplasia that ranges from classical CCD (dental abnormalities, hypoplastic/aplastic clavicles, and delayed closure of cranial sutures) to mild CCD to isolated dental anomalies without the skeletal features. [10] Among the 202 registered RUNX2 variants (HGMD Professional 2020.4 http:// www.hgmd.cf.ac.uk/), 99% (201/202) cause classical CCD. In this study, the 2 probands and family 2 members all showed classic features such as short stature, midface hypoplasia, frontal bossing, retention of deciduous teeth, failure of eruption of permanent teeth, aplasia of the clavicle, and supernumerary teeth, and the proband II:1 of family 2 exhibited delayed closure of cranial sutures. These phenotypes are consistent with previous findings in which most cases exhibited classical features.
In this study, we found a previously reported missense variant (p.Arg190Pro) in RUNX2 that has been previously found to be responsible for CCD, [11] but the mutation detected in the initiation codon (c.2T>C) has not yet been reported. The computational programs all predicted that the c.2T>C missense change is damaging to the resultant protein function and structure. Further, according to the criteria for classifying pathogenic variants proposed by ACMG, [12] the initiation codon variant should be considered as very strong evidence of pathogenicity (PVS1).
On the basis of Kozak principles, [13] we hypothesized that the translation is mostly initiated 304 nucleotides downstream for type 2 RUNX2 (NM_001015051.3; Fig. 4). Consequently, this initiation codon mutation results in a frameshift mutation and truncated proteins (lack of the Q/A domain and part of the runt domain), which is consistent with the previous consensus that most mutations that cause premature termination in the runt domain produce a classic CCD phenotype. [14] It is also possible that the mRNAs are degraded via the nonsense-mediated mRNA decay quality-control mechanism. [15] The etiology of CCD is heterozygous variants in RUNX2, which encodes a transcription factor essential for osteoblast differentiation. Previous studies have shown that bones are malformed in case of a homozygous deletion in this gene in animals. Thus, the knockout mice with Runx2 À/À lack osteoblasts and bones, [8] whereas the heterozygous mice  indicates CCD; in contrast, >79% levels produce a normal skeleton. [14,16] Our result is consistent with those of previous studies [6,16] and confirms the mechanism underlying this case is haploinsufficiency.
Of the previously reported 202 RUNX2 variants, 63% (51/80) of the missense/nonsense mutations occurred in the runt domain, which is the most important variation hotspot of RUNX2. [2,6,7,[17][18][19][20][21][22][23][24] In this study, we located the variant (p. Arg190Pro) in the runt domain, confirming the above-mentioned point. Sequence alignment results showed that ARG190 is highly conserved. A functional study showed that the R190Q variant exhibited no DNA binding and markedly reduced transactivation activities. [25] This genotype is correlated with the classic CCD phenotype [25,26] and similar dental abnormalities. [11,27] Interestingly, heterozygotic Runx2 mice have no dental phenotypes, in contrast to the hyperdontia phenotype in humans; this may be attributable to composition differences in the mouse monophydont dentition, as it parallels the more simply patterned human primary dentition. A previous study demonstrated that RUNX2 suppresses the expression of Wnt inhibitors in the dental mesenchyme; increased mesenchymal Wnt signaling inhibits the sequential formation of teeth and is attributable to supernumerary teeth caused by RUNX2 variants in humans. [6,16] However, the detailed mechanism underlying this process needs to be studied further.

Conclusion
This study demonstrated that a novel heterozygous initiation codon variant (c.2T>C) in RUNX2 causes CCD. This study expands the pathogenic variant spectrum of RUNX2 and could help in genetic counseling and prenatal screening and contribute to disease status prediction for CCD families.