Dental Anomalies in Ciliopathies: Lessons from Patients with BBS2, BBS7, and EVC2 Mutations

Objective: To investigate dental anomalies and the molecular etiology of a patient with Ellis–van Creveld syndrome and two patients with Bardet–Biedl syndrome, two examples of ciliopathies. Patients and Methods: Clinical examination, radiographic evaluation, whole exome sequencing, and Sanger direct sequencing were performed. Results: Patient 1 had Ellis–van Creveld syndrome with delayed dental development or tooth agenesis, and multiple frenula, the feature found only in patients with mutations in ciliary genes. A novel homozygous mutation in EVC2 (c.703G>C; p.Ala235Pro) was identified. Patient 2 had Bardet–Biedl syndrome with a homozygous frameshift mutation (c.389_390delAC; p.Asn130ThrfsTer4) in BBS7. Patient 3 had Bardet–Biedl syndrome and carried a heterozygous mutation (c.389_390delAC; p.Asn130ThrfsTer4) in BBS7 and a homozygous mutation in BBS2 (c.209G>A; p.Ser70Asn). Her clinical findings included global developmental delay, disproportionate short stature, myopia, retinitis pigmentosa, obesity, pyometra with vaginal atresia, bilateral hydronephrosis with ureteropelvic junction obstruction, bilateral genu valgus, post-axial polydactyly feet, and small and thin fingernails and toenails, tooth agenesis, microdontia, taurodontism, and impaired dentin formation. Conclusions: EVC2, BBS2, and BBS7 mutations found in our patients were implicated in malformation syndromes with dental anomalies including tooth agenesis, microdontia, taurodontism, and impaired dentin formation.


Introduction
The primary cilium is an immotile single organelle found on the apical surface of the majority of cells in the human body [1,2]. During the development of vertebrates, primary cilia play a key role in sensory perception and coordinating various signaling Growth and development assessment showed delayed gross motor development. Chest radiography showed cardiomegaly with increased pulmonary blood flow. Echocardiography and cardiac computed tomography demonstrated a common atrium, cleft mitral valve, severe mitral regurgitation, small patent ductus arteriosus (PDA), bilateral superior vena cava (SVC), and left SVC draining into the left atrium and a patent ductus arteriosus ( Figure 1G,H). He underwent atrial septal defect closure, patent ductus arteriosus ligation, mitral valve repair, and connection of the left SVC to right atrium. He experienced a chylothorax during the post-operative period and was later discharged. In his last out-patient appointment, his clinical heart failure improved with normal weight gain but he still had developmental delay. Patient 2 A 1.5-month-old boy was admitted to Chiang Mai University Hospital with the chief complaints of dyspnea and central cyanosis for 2 weeks (Figure 2A). He was diagnosed with cyanotic heart disease and bacterial septicemia and treated with intravenous antibiotics and placed on respiratory support. He was born at home and his parents originated from a Northern Thai hill tribe and lived in the same village. His parents and elder brother were healthy with normal dentitions ( Figure 2B). His weight was 3.3 kg (<3 centile). He had central cyanosis with multiple anomalies including a macular hemangioma on the forehead, bulbous nose, cup-shaped ears, micrognathia, polydactyly of all extremities, and syndactyly of the left hand (Figure 2A-E). He had no murmur and a single S2 heart sound at the right side of his chest. Echocardiography and cardiac computed tomography revealed complex cyanotic heart disease and a single ventricle physiology with ductaldependent pulmonary circulation (dextrocardia, tricuspid atresia, atrial septal defect, ventricular septal defect, pulmonary atresia, non-confluent pulmonary artery, and patent Growth and development assessment showed delayed gross motor development. Chest radiography showed cardiomegaly with increased pulmonary blood flow. Echocardiography and cardiac computed tomography demonstrated a common atrium, cleft mitral valve, severe mitral regurgitation, small patent ductus arteriosus (PDA), bilateral superior vena cava (SVC), and left SVC draining into the left atrium and a patent ductus arteriosus ( Figure 1G,H). He underwent atrial septal defect closure, patent ductus arteriosus ligation, mitral valve repair, and connection of the left SVC to right atrium. He experienced a chylothorax during the post-operative period and was later discharged. In his last out-patient appointment, his clinical heart failure improved with normal weight gain but he still had developmental delay. Patient 2 A 1.5-month-old boy was admitted to Chiang Mai University Hospital with the chief complaints of dyspnea and central cyanosis for 2 weeks (Figure 2A). He was diagnosed with cyanotic heart disease and bacterial septicemia and treated with intravenous antibiotics and placed on respiratory support. He was born at home and his parents originated from a Northern Thai hill tribe and lived in the same village. His parents and elder brother were healthy with normal dentitions ( Figure 2B). His weight was 3.3 kg (<3 centile). He had central cyanosis with multiple anomalies including a macular hemangioma on the forehead, bulbous nose, cup-shaped ears, micrognathia, polydactyly of all extremities, and syndactyly of the left hand (Figure 2A-E). He had no murmur and a single S 2 heart sound at the right side of his chest. Echocardiography and cardiac computed tomography revealed complex cyanotic heart disease and a single ventricle physiology with ductal-dependent pulmonary circulation (dextrocardia, tricuspid atresia, atrial septal defect, ventricular septal defect, pulmonary atresia, non-confluent pulmonary artery, and patent ductus arteriosus with collateral vessels) ( Figure 2F). He underwent a central systemic to pulmonary shunt and pulmonary artery plasty. During the post-operative period, he had a prolonged ICU admission due to acute renal failure and systemic candidiasis. He was discharged to a provincial hospital 5 weeks after surgery. Unfortunately, he died 1 week after being discharged. The patient was too young for dental evaluation.
Genes 2023, 14, x FOR PEER REVIEW ductus arteriosus with collateral vessels) ( Figure 2F). He underwent a central syste pulmonary shunt and pulmonary artery plasty. During the post-operative period, a prolonged ICU admission due to acute renal failure and systemic candidiasis. H discharged to a provincial hospital 5 weeks after surgery. Unfortunately, he died 1 after being discharged. The patient was too young for dental evaluation.

Patient 3
A 12-year-old Thai girl was born to non-consanguineous parents. Both parent healthy with unremarkable dentitions. Her father passed away a few years ago. Sh sented with short stature, night-blindness, and bilateral knee pain ( Figure 3A,B). S born following an uneventful pregnancy at full term with a birthweight of 2.95 k neonatal and infancy period was complicated by pyometra with vaginal atresia, required colpostomy with percutaneous drainage and vaginoplasty. She also exper recurrent urinary tract infections. Investigations revealed bilateral hydronephros ureteropelvic junction obstruction.

Patient 3
A 12-year-old Thai girl was born to non-consanguineous parents. Both parents were healthy with unremarkable dentitions. Her father passed away a few years ago. She presented with short stature, night-blindness, and bilateral knee pain ( Figure 3A,B). She was born following an uneventful pregnancy at full term with a birthweight of 2.95 kg. Her neonatal and infancy period was complicated by pyometra with vaginal atresia, which required colpostomy with percutaneous drainage and vaginoplasty. She also experienced recurrent urinary tract infections. Investigations revealed bilateral hydronephrosis with ureteropelvic junction obstruction.
Her developmental milestones were globally delayed. Physical examination revealed a weight of 38.3 kg (P50), height 121 cm (<P3), and upper/lower segment ratio 1.2:1 (normal 1:1), indicating disproportionate short stature, body mass index (BMI) of 26.2 kg/m 2 , obesity, bilateral genu valgus, and post-axial polydactyly of the feet. Fingernails and toes nails were small ( Figure 3C,D). Eye examination revealed myopia and retinal dystrophy, which was consistent with early retinitis pigmentosa. Oral manifestations consisted of agenesis of the mandibular left permanent first molar and microdontia of the mandibular premolars ( Figure 3E). Panoramic radiograph showed taurodontism of all permanent molars, absence of the mandibular left permanent first molar, and generalized large root canal spaces, indicating impaired dentin formation ( Figure 3F). Her developmental milestones were globally delayed. Physical examination rev a weight of 38.3 kg (P50), height 121 cm (<P3), and upper/lower segment ratio 1.2:1 mal 1:1), indicating disproportionate short stature, body mass index (BMI) of 26.2 k obesity, bilateral genu valgus, and post-axial polydactyly of the feet. Fingernails and nails were small ( Figure 3C,D). Eye examination revealed myopia and retinal dystro which was consistent with early retinitis pigmentosa. Oral manifestations consist agenesis of the mandibular left permanent first molar and microdontia of the mandi premolars ( Figure 3E). Panoramic radiograph showed taurodontism of all permanen lars, absence of the mandibular left permanent first molar, and generalized large ro nal spaces, indicating impaired dentin formation ( Figure 3F).

Whole Exome Sequencing and Mutation Analysis
Genomic DNA was extracted from blood according to the standard procedure DNA samples of the patient and unaffected parents or siblings were exome-seque with the SureSelect V6 + UTR-Post Target Capture Kit. Genomics Analysis To (GATK) germline mutation workflow version 3.8.1 [12] was utilized to identify var The sequencing reads were aligned to hg19 using BWA-MEM version 0.7.17 [13] to erate BAM files. These BAM files were processed by GATK HaplotypeCaller to ide SNVs and small indels, resulting in individual GVCF files. These GVCF files were co idated into a single joint-genotyped VCF file format, listing all genotypes in separat umns. Ensembl Variant Effect Predictor Tool (version 95) [14] was used to predi pathogenic effects of those variants.

Whole Exome Sequencing and Mutation Analysis
Genomic DNA was extracted from blood according to the standard procedure. The DNA samples of the patient and unaffected parents or siblings were exome-sequenced with the SureSelect V6 + UTR-Post Target Capture Kit. Genomics Analysis Toolkit (GATK) germline mutation workflow version 3.8.1 [12] was utilized to identify variants. The sequencing reads were aligned to hg19 using BWA-MEM version 0.7.17 [13] to generate BAM files. These BAM files were processed by GATK HaplotypeCaller to identify SNVs and small indels, resulting in individual GVCF files. These GVCF files were consolidated into a single joint-genotyped VCF file format, listing all genotypes in separate columns. Ensembl Variant Effect Predictor Tool (version 95) [14] was used to predict the pathogenic effects of those variants.

Patient 2
Whole exome sequencing showed a homozygous two-base deletion mutation (chr4:g.122780285G>T; NM_018190.3:c.389_390del; NP_789794.1:p.Asn130ThrfsTer4; rs863224530) in BBS7. This base deletion is predicted to result in an amino acid residue change from Asn to Thr at amino acid position 130 and subsequent truncation of the protein four amino acids later (p.Asn130ThrfsTer4) (Figure 4).

Patient 2
Whole exome sequencing showed a homozygous two-base deletion mutation (chr4:g. 122780285G>T; NM_018190.3:c.389_390del; NP_789794.1:p.Asn130ThrfsTer4; rs863224530) in BBS7. This base deletion is predicted to result in an amino acid residue change from Asn to Thr at amino acid position 130 and subsequent truncation of the protein four amino acids later (p.Asn130ThrfsTer4) (Figure 4).

Discussion
We report three patients, one with EVC and two with BBS. The EVC patient (patient 1) was a boy who had a novel homozygous mutation in EVC2 (c.703G>C; p.Ala235Pro). This mutation was predicted to be possibly damaging, polymorphism, and tolerated by PolyPhen-2, MutationTaster, and SIFT. His oral features were tooth agenesis or delayed dental development, and multiple frenula. His sister, who died a few weeks after birth, also had EVC with a common atrium and postaxial polydactyly. Patient 2, who was affected with BBS, carried a homozygous mutation (c.389_390delAC; p.Asn130ThrfsTer4) in BBS7. This mutation was predicted to be disease causing by MutationTaster. He was too young to have a dental evaluation. Patient 3 was affected with BBS with tooth agenesis, microdontia, taurodontism, and impaired dentin formation. She was heterozygous for the BBS7 variant (c.389_390delAC; p.Asn130ThrfsTer4), the variant found in patient 2. Interestingly, patients 2 and 3 shared the rare p.Asn130ThrfsTer4 variant. Although parents of both parents were not aware that they were related, it is important to note that these parents and their families were Hmong living in Thailand. This suggests a possible founder effect. In addition to the heterozygous BBS7 frameshift mutation, which has been previously described in BBS patients, patient 3 also carried a homozygous variant in BBS2 (c.209G>A; p.Ser70Asn; rs4784677). According to gnomAD, this BBS2 variant is common with an allele frequency of 0.9944. It is considered benign by Varsome and ACMG Classification. Interestingly, this variant has been reported in previous cases with a digenic triallelic inheritance of BBS. BBS is a complex genetic disease, which in some patients require two mutations in one BBS gene, accompanied by a third BBS gene variant, which is considered as a contributing factor to manifest the BBS phenotype [15]. However, this is the first time that that two purportedly benign variants (BBS2) have been seen in combination with a heterozygous pathogenic mutation (BBS7). We therefore hypothesize that the phenotype in patients 2 and 3 are likely to be the consequences of stoichiometric disruption of the BBSome complex in the cilium as both BBS2 and BBS7 proteins are important components [16,17].

BBS2 and BBS7 Mutations and Impaired BBSome Complex
BBS2 and BBS7 are two of the eight highly conserved BBS proteins that form the BBSome, the heterooctameric protein complex required for ciliary membrane biogenesis. BBS7 is a unique subunit that stabilizes BBS2 in the BBSome and has a direct physical interaction with the BBS chaperonin complex (made up of BBS6, BBS10, and BBS12). The presence of a mutant subunit in a multi-subunit complex like the BBSome results in the malfunction of the entire complex [18,19]. BBS7 has an important role in the localization of the ciliary membrane proteins and specific ciliary membrane trafficking. Mice lacking in Bbs7 have primary cilia and IFT complexes [17], but there is an abnormal accumulation of the dopamine D1 receptor to the ciliary membrane as a result of abnormal IFT. Therefore in the case of patient 3, although the variant in BBS2 (p.Ser70Asn) is predicted to generally be benign, it is hypothesized that the accompanying BBS7 frameshift mutation (albeit heterozygous) is enough to upset the stoichiometric balance of the BBSome by "pushing" the BBS2 p.Ser70Asn beyond a threshold of pathogenicity. Absence of BBS7 also results in the accumulation of Smo within the cilia [20,21], which results in aberrant Hh signaling and subsequent Hh signaling phenotypes including dental anomalies ( Figure 5).

Oral Manifestations in EVC and BBS
Both EVC and BBS share a number of clinical findings including dental anomalies, the malformations related to cilia-associated impaired Hh and Wnt signaling. Patient 3, who was heterozygous for a BBS7 variant and homozygous for a BBS2 variant, had tooth agenesis, microdontia, taurodontism, and large dental pulp spaces, indicating impaired dentin formation. Oral manifestations of patient 1 consisted of tooth agenesis or delayed tooth eruption, and multiple frenula. Regarding multiple frenula found in patient 1, who had EVC, to the best of our knowledge, multiple frenula are found only in patients affected with ciliopathies including EVC or EVC2-associated EVC, OFD1-associated orofaciodigital syndrome, GLI3-associated Pallister-Hall syndrome, and CEP120-associated short rib thoracic dysplasia 13 with or without polydactyly, CEP120-associated Joubert syndrome, and WDR35-associated short rib thoracic dysplasia 7 with or without polydactyly [22]. The current knowledge on the embryology of the frenulum is not sufficient to explain why only mutations in ciliary genes cause multiple frenula. Interestingly, the syndromes that have multiple frenula as a feature also have natal teeth as a feature.

EVC, BBS, and Abnormal Hh and Wnt Signaling
Hh and Wnt signaling are crucial for tooth development. Mutations in both signaling pathways are known to cause various kinds of human malformations including dental anomalies. Cilia promotes Hh signaling, but restrains canonical Wnt signaling, indicating their functional antagonism is mediated by cilia [23][24][25]. Loss of BBS protein function results in abnormalities in the suppression of non-canonical Wnt signaling, with an increase in targets of canonical Wnt signaling [26]. Therefore, mutations in BBS genes would result in malformations similar to those with the upregulation of canonical Wnt signaling [27] (Figure 5).
Regarding tooth development, EVC2, BBS2, and BBS7 mutations found in our patients are predicted to result in abnormal BBSome function, disrupted intraflagellar transport, abnormal cilia biogenesis and function, impaired Hh and Wnt signaling, and subsequent dental anomalies including tooth agenesis, microdontia, taurodontism, and impaired dentin formation, among other phenotypes ( Figure 5). In vertebrates, Hh signaling is initiated at the primary cilia by the ligand-triggered accumulation of Hh signaling effector protein Smoothened (Smo) in the ciliary membrane, which is absolutely required for Hh signaling in all tissues [28]. EVC and EVC2, positive tissue-specific regulators of Hh signaling pathways, form the EVC-EVC2 complex and acts on Smo in Hh signal transduction [29]. The accumulation of Smo in cilia in response to Hh signaling results in a physical association of Smo and EVC2 in the EVC-EVC2 complex in the EVC zone [28]. The EVC-EVC2 complex localizes in the basal body of the primary cilia by anchoring itself with ciliary protein complex EFCAB7-IQCE in a signaling microdomain. EFCAB7 functions as an adaptor protein linking IQCE to the EVC-EVC2 complex at the C-terminal disordered region in EVC2. The role of EVC2 in Hh signaling is to translate Smo activation to the inhibition of SuFu and PKA ( Figure 5). Therefore, localization of the EFCAB7-IQCE protein complex in the basal body of cilia is a requirement for Hh signaling [29,30] and mutations in EVC2 are predicted to cause impaired linking of the EFCAB7-IQCE complex to the EVC-EVC2 complex, which results in the dysregulation of Hh signaling because the Smo-EVC2 signaling complex at the EVC zone is required for Hh signal transduction ( Figure 5).
EVC and EVC2 are required for Hh signaling, therefore, Evc-like genes are absent in organisms that do not have Hh signaling (worms) or do not possess cilia for Hh signaling (flies). Patients with EVC2 mutations have clinical findings of abnormal Hh signaling including dental anomalies because in patients with EVC2 mutations, EVC2 proteins are not functional because they fail to localize in the cilia, and function as dominant inhibitors of Hh signaling [28]. Mice lacking Evc or Evc2 have defective biomineralization [31]. In humans, patients with mutations in EVC and EVC2 have impaired Hh signaling phenotypes including congenital heart defects, limb anomalies, and dental anomalies [28]. Evc-/mice have microdontia and the fusion of roots [32][33][34]. The phenotypes of the patients with EVC2 mutations are associated with the failure of signaling complex EVC2-Smo to assemble in the EVC zone [28] (Figure 5).

Ciliopathy, WNT Signaling, and Dental Anomalies
In addition to Bardet-Biedl syndrome and Ellis-van Creveld syndrome, dental anomalies have been reported in other ciliopathy-associated syndromes including Weyers acrofacial dysostosis (MIM 193530 When the Wnt ligand binds to its receptor complex, it inhibits the function of the β-catenin destruction complex, allowing for the accumulation of β-catenin in the cy-toplasm and its nuclear translocation as well as the transcription activation via T-cell factor/lymphoid enhancer-binding factor transcription factors. Loss of Hh signaling generally leads to the overactivation of Wnt signaling, and impaired ciliary function in ciliary mutants [24]. Non-canonical and canonical Wnt signaling are associated with the cilia and basal body. BBS proteins regulate proteasome function and transport Inversin from the basal body to the cytoplasm, where it reduces the cytoplasmic levels of disheveled protein (DVL) via phosphorylation. Mutations in BBS disrupt retrograde IFT and disrupt the transport of Inversin to the cytoplasm, preventing its interaction with DVL [27,37] (Figure 5).
Deletion of IFT140, a subunit of IFT complex A, which is important for retrograde transportation of cilia, leads to poor odontogenic differentiation, abnormal primary cilia, decreased Shh signaling, and poor dentin formation [38]. The thinning of dentin in patient 3, who had mutations in BBS7 and BBS2, suggests that its pathogenetic mechanism is similar to the deletion of IFT140 or the abnormal retrograde transportation of cilia [39]. Primary cilia regulate Shh activity in the control of tooth number. Mice mutant of IFT88/Polaris show an upregulation of Shh activity, and subsequent ectopic tooth formation [40].
In addition to Bardet-Biedl syndrome and Ellis-van Creveld syndrome, dental anomalies have been reported in other ciliopathy-associated syndromes including Weyers acrofacial dysostosis (MIM 193530 When the Wnt ligand binds to its receptor complex, it inhibits the function of the βcatenin destruction complex, allowing for the accumulation of β-catenin in the cytoplasm and its nuclear translocation as well as the transcription activation via T-cell factor/lymphoid enhancer-binding factor transcription factors. Loss of Hh signaling generally leads to the overactivation of Wnt signaling, and impaired ciliary function in ciliary mutants [24]. Non-canonical and canonical Wnt signaling are associated with the cilia and basal body. BBS proteins regulate proteasome function and transport Inversin from the basal body to the cytoplasm, where it reduces the cytoplasmic levels of disheveled protein (DVL) via phosphorylation. Mutations in BBS disrupt retrograde IFT and disrupt the transport of Inversin to the cytoplasm, preventing its interaction with DVL [27,37] (Figure 5).

Conclusions
The EVC2, BBS2, and BBS7 mutations found in our patients are predicted to result in an abnormal BBSome complex, disrupted intraflagellar transport, abnormal cilia biogenesis and function, impaired Hh and Wnt signaling, and subsequent dental anomalies including tooth agenesis, microdontia, taurodontism, and impaired dentin formation. The phenotypes of the patients with EVC2 mutations are associated with the failure of signaling complex EVC2-Smo to assemble in the EVC zone. This is the first time that two benign BBS2 variants have been seen in combination with a heterozygous pathogenic BBS7 mutation. It is hypothesized that the BBS phenotypes in our patients are the likely consequences of the stoichiometric disruption of the BBSome complex in the cilium as both BBS2 and BBS7 proteins are important components.