Oro-dental phenotyping and report of three families with RELT-associated amelogenesis imperfecta

Amelogenesis imperfecta (AI) is a group of rare genetic conditions characterized by quantitative and/or qualitative tooth enamel alterations. AI can manifest as an isolated trait or as part of a syndrome. Recently, five biallelic disease-causing variants in the RELT gene were identified in 7 families with autosomal recessive amelogenesis imperfecta (ARAI). RELT encodes an orphan receptor in the tumor necrosis factor (TNFR) superfamily expressed during tooth development, with unknown function. Here, we report one Brazilian and two French families with ARAI and a distinctive hypomineralized phenotype with hypoplastic enamel, post-eruptive enamel loss, and occlusal attrition. Using Next Generation Sequencing (NGS), four novel RELT variants were identified (c.120+1G>A, p.(?); c.120+1G>T, p.(?); c.193T>C, p.(Cys65Arg) and c.1260_1263dup, p.(Arg422Glyfs*5)). Our findings extend the knowledge of ARAI dental phenotypes and expand the disease-causing variants spectrum of the RELT gene.


INTRODUCTION
Amelogenesis Imperfecta (AI) is a group of rare genetic disorders affecting tooth enamel in quantity and/or quality.The mode of inheritance may be X-linked, autosomal dominant, or autosomal recessive (AR).AI manifests as an isolated trait or as part of a syndrome [1,2].Presently, more than 100 genes with diseasecausing variants are recognized in both isolated and syndromic forms of AI [3,4].However, AI molecular basis is not yet completely understood.Candidate gene approaches have failed to identify the mutated gene in more than 50% of the studied cohorts [5,6].Recently, two studies reported 5 different RELT (receptor expressed in lymphoid tissues) disease-causing variants in 7 families with ARAI (OMIM; # 618386) [7,8].
RELT is an orphan Tumor Necrosis Receptor Factor (TNFR) superfamily member described in 2001 [9].RELT does not bind to the 19 currently known TNF ligands of the signaling TNFR pathways [10][11][12].Despite a major expression in lymphoid and hematopoietic tissues, RELT immunological function is unclear [13][14][15].Relt is expressed in the early developmental and secretion stages of mice amelogenesis, but its role in enamel formation is not yet elucidated [7].Here, we report three additional unrelated families of Brazilian and French origin with nonsyndromic ARAI and 4 novel RELT variants.Our findings expand the pathogenic variants spectrum and support that RELT-related cases have an isolated ARAI with a distinctive hypomineralized phenotype with additional hypoplastic enamel.

MATERIAL AND METHODS
See Supplementary Appendix.

Family 1
The proband was an 8-year-old Brazilian female (IV:2), born from first-degree consanguineous parents, referred to the Oral Care Center for Inherited Diseases, University Hospital of Brasilia, Brazil, with a complaint of tooth sensitivity and suspicion of AI.No systemic condition was reported in the family.Parents were not affected, but an elder sister (IV:1) also had a dental phenotype.Both sisters had pits on primary canines and molars.However, the enamel of the permanent dentition differed between siblings.In proband IV:2, it appeared severely affected presenting a yellowbrown discoloration and an association of hypoplastic and hypomineralized enamel defects.Whereas, the enamel of the elder sister (IV:1), showed generalized hypoplastic pits, yellowish-white discoloration, and diffuse opacities.All permanent molars of both siblings displayed a partial loss of occlusal enamel and a thin intact enamel ring covering the cervical third of the teeth.(Fig. 1A-C; Appendix S7).
Exome sequencing of the proband 1 (IV:2) detected a homozygous RELT variant in the splicing region of intron 3 c.120+1G>A, p.(?).Splice AI predicted a donor site splice loss with a score of 0.97 for this variant.Sanger sequencing confirmed the same homozygous variant in the affected sister (IV:1).The parents (III:1; III:2) were heterozygous for the variant (Table 1; Appendix S5A, S6).The unaffected sister was not tested.
Family 2 A 7-year-old French female (V:3), was referred to the Reference Center of Oral and Dental Rare Diseases at Hospital Rothschild in Paris, France, with suspicion of AI.She was the third child of consanguineous parents.Her brother and her sister were not affected by AI.Physical examination revealed facial dysmorphisms (mandibular prognathism, thin eyebrows, upslanted palpebral fissures), long fingers, intellectual disability, and tall stature.The oral examination revealed yellow-brown hypomineralized enamel on the primary molars and the third occlusal part of the primary canines.Permanent teeth presented variable alterations.Maxillary incisors showed a pitted hypoplastic enamel with brownish discoloration and loss of enamel in the occlusal part; although, mandibular incisors showed normal color with only small brown pits.Permanent molars and premolars presented a phenotype similar to primary molars.In addition, this patient presented an anterior open bite malocclusion (Fig. 1D, E; Appendix S8).
Exome and Sanger sequencing analysis revealed that the RELT variant c.120+1G>T was present at the homozygous state in the proband and the heterozygous state in both parents (Table 1; Appendix S6).Splice AI predicted a donor site splice loss with a score of 0.97 for this variant.

Family 3
A 12-year-old male (II-2) was referred to the Reference Center of Oral and Dental Rare Diseases at the Hospital Rothschild in Paris, France, with suspicion of AI without tooth sensitivity.Neither family consanguinity, nor systemic involvement was reported, and the dental examination revealed a pitted hypoplastic enamel in the half-occlusal part of all permanent dentition.On the halfcervical part of the crown, the enamel was thinner, yellow-brown, and smooth (Fig. 1F, G    was also identified in the unaffected father (I:1) (Table 1, Appendix S4-S6).
Panoramic radiographs of all index cases showed a thin enamel with normal radiodensity (Fig. 1).

DISCUSSION
This study characterized the enamel phenotype of three families of Brazilian and French origin with nonsyndromic ARAI and reported 4 novel RELT disease-causing variants.
RELT presents 11 exons and codes for a 430 amino acids protein whose function during amelogenesis remains unclear.To date, all reported disease-causing variants appear to be clustered in two regions between intron 3 and exon 4, and between exons 10 and 11.Variants altering Arginine 422 have been reported in three families (Table 1; Appendix S6).These variants affect a CpG residue in the last exon of RELT and despite the small number of RELT variants reported, it could be a mutational hotspot playing an important role in amelogenesis.In addition, three different splicesite variants in intron 3 were reported in three families suggesting another mutational hotspot.The description of splice-site variants, small deletions, duplication, and frameshift variants supports that RELT-associated AI is caused by biallelic loss-of-function variants as previously suggested [8].Compound heterozygosity was only described in family 3 (proband II:2) (Table 1; Appendix S6).Also, haplotype analysis in three Pakistani cases showed a single origin for the same homozygous variant [8].Those results demonstrate the rarity of this disease [16].A larger number of disease-causing variants are necessary to better understand RELT-related AI's pathogenesis.
The three families' medical history and physical examination do not support a syndromic phenotype.The facial dysmorphisms, intellectual disability, and tall stature of family 2 (proband V:3) appear to co-segregate with the AI phenotype and further genetic investigation is being undertaken.Also, a milder AI phenotype was previously suggested in heterozygous parents of RELT probands [7,8].In this study, no enamel defects were observed in the heterozygous carriers.
All the RELT reported cases including ours share variable degrees of a distinctive hypomineralized AI phenotype with variable expression of hypoplastic enamel.The common features include a hypomineralized enamel ranging from white to brown, generalized pits most often localized on the middle of the crown, and post-eruptive enamel loss by occlusal attrition of posterior teeth.We suggest that brownish enamel could be considered hypomineralized enamel, explaining the susceptibility to posteruptive loss of enamel.The dental phenotype reported here is in accordance with previously reported families [7,8] showing enamel loss and variable enamel defects between patients and within the same patient's teeth.It is noted that the AI phenotype observed in our patients differs from the OMIM (618386) description that classifies RELT-related cases as hypocalcified AI.Further reports with detailed dental phenotypes are needed to appreciate the full clinical spectrum of the disease.
Patients carrying RELT mutations and Relt −/− mice showed similarities in the enamel phenotype such as rough surface and rapid attrition.In mice, highly mineralized initial enamel near the dentin-enamel junction (DEJ) was observed, which could explain the reduction in DEJ mechanical resistance [7].Furthermore, previous micro-computed tomography and Scanning Electron Microscopy analyses of permanent and primary teeth of two RELTrelated AI cases showed prismatic disorganization in the inner enamel and a mild decrease in mineral content, suggesting that the prismatic disorganization could affect the enamel mechanical resistance to masticatory forces [8].Mild taurodontism involving permanent molar teeth was also reported by Kim et al. [7] No alteration in the crown/root proportion was observed in the four affected cases.Taurodontism does not appear to be a consistent feature among RELT-related cases.Mice Relt expression is restricted to pre-secretory and secretory ameloblasts and odontoblasts, however, its function in odontogenesis has not yet been elucidated [7].More recently, it has been suggested that RELT may play a role in ameloblasts p38 kinase activation, to provide the signaling necessary for proper enamel formation.ADAM10-mediated shedding of RELT from the ameloblasts cell surface may modulate this signaling [17].Further studies are necessary to better understand the role of RELT in tooth development.
In conclusion, we described three additional families with disease-causing RELT variants.We suggest that RELT-associated phenotype is an isolated hypomineralized AI with variable hypoplastic enamel in addition, characterized by variability of expression between teeth in the same patient.This phenotype could be considered pathognomonic for RELT-associated cases, aiding diagnosis, future molecular analyses, and better follow-up of ARAI patients.Thus, RELT-associated AI classification must be revisited.

Fig. 1
Fig.1Dental phenotype of three families with AI caused by homozygous or compound heterozygous RELT variants.Clinical analysis shows variable severity of enamel defect from mild phenotype showing mainly hypoplastic enamel (family 3) to severe enamel defect showing an association with hypoplastic grooves and hypomineralized enamel (family 1, proband IV:2) with enamel detachment.A Pedigree family 1.B Proband IV:2 of family 1 was an 8-year-old female.The primary canines showed normal color but hypoplastic pitted enamel on the occlusal part.Permanent incisors presented a brownish vitreous enamel with hypoplastic grooves, dentin exposition, generalized hypoplastic pits, and a rough surface.Posterior teeth were brownish hypomineralized with loss of enamel of occlusal surfaces.Panoramic radiograph revealed enamel thickness reduction with normal radiodensity.C Sister IV:1 of family 1: Primary canine presents a mild enamel alteration.Permanent incisors showed a pitted enamel hypoplastic with a white to yellow discoloration.Posterior teeth exhibited occlusal loss of enamel in both primary and permanent teeth.Panoramic radiograph revealed thin enamel with normal radiodensity.D Pedigree family 2. E Family 2 (proband V:3) shows a severe hypoplastic and hypomineralized brownish enamel of primary and permanent teeth.The lower permanent incisor presented a very mild phenotype with only a brown demarcated opacity.Posterior teeth presented flat cuspids and severe occlusal attrition with a ring of thin remaining enamel covering the cervical third of the crown.Panoramic radiograph revealed enamel thickness reduction.F Pedigree family 3. G Family 3 (proband II:2) shows a pitted hypoplastic and hypomineralized yellowish enamel.Permanent upper incisors were restored and the remaining permanent teeth showed a pitted enamel in the middle half of the crown.Also, the pits showed a brown discoloration.Posterior teeth were brown with enamel loss.Panoramic radiograph revealed enamel thickness reduction.

Table 1 .
Description of the reportedRELT variants according to geographic origin, sex, variant classification, systemic features and enamel phenotype.

Table 1 .
continued Common haplotype IVA= # separate genetic investigation is being undertaken; ## IVA= isovaleric acidemia, a causative genetic variant in the IVD gene was previously identified in the family. *