archives of oral biology 60 (2015) 919–922

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Novel FAM20A mutation causes autosomal recessive amelogenesis imperfecta Michael Volodarsky a,1, Uri Zilberman b,1, Ohad S. Birk a,c,* a

The Morris Kahn Laboratory of Human Genetics at the National Institute for Biotechnology in the Negev (NIBN) and Faculty of Health Sciences, Ben Gurion University, Beer-Sheva, Israel b Pediatric Dental Unit, Barzilai Medical Center, Ashkelon, Israel c The Genetics Institute, Soroka Medical Center, Beer-Sheva, Israel

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Article history:

Objective: To relate the peculiar phenotype of amelogenesis imperfecta in a large Bedouin

Accepted 20 February 2015

family to the genotype determined by whole genome linkage analysis. Design: Amelogenesis imperfecta (AI) is a broad group of inherited pathologies affecting


enamel formation, characterized by variability in phenotypes, causing mutations and


modes of inheritance. Autosomal recessive or compound heterozygous mutations in


FAM20A, encoding sequence similarity 20, member A, have been shown to cause several

Linkage analysis

AI phenotypes. Five members from a large consanguineous Bedouin family presented with


hypoplastic amelogenesis imperfecta with unerupted and resorbed permanent molars.


Following Soroka Medical Center IRB approval and informed consent, blood samples were

Stop codon

obtained from six affected offspring, five obligatory carriers and two unaffected siblings. Whole genome linkage analysis was performed followed by Sanger sequencing of FAM20A. Results: The sequencing unravelled a novel homozygous deletion mutation in exon 11 (c.1523delC), predicted to insert a premature stop codon (p.Thr508Lysfs*6). Conclusions: We provide an interesting case of novel mutation in this rare disorder, in which the affected kindred is unique in the large number of family members sharing a similar phenotype. # 2015 Elsevier Ltd. All rights reserved.



Amelogenesis imperfecta (AI) represents a broad spectrum of genetic diseases affecting enamel formation in both primary and permanent dentition. AI is the oldest hereditary disorder affecting enamel, observed in early hominids. It has been described in a Homo erectus child from Melka Kunture Ethiopia (Garba IV) dated to circa 1.5 MY.1 AI has been classified into 14 different subtypes according to the clinical appearance of the

enamel and the Mendelian mode of inheritance2; however, the molecular genetic basis for only some of the phenotypes has been defined. The prevalence of AI has been reported to be 1:14,000 in the USA,2 1:8000 in Israel,3 1:4000 in Sweden4 and as high as 1:700 in the Vasterbotten country of Sweden.5 The enamel abnormalities have been categorized into three major groups (hypocalcified, hypomaturation and hypoplastic), and the inheritance patterns reported include autosomal dominant or recessive as well as X-linked dominant or recessive heredity.2 Distinctive clinical features may be observed in each

* Corresponding author at: Genetics Institute, Soroka University Medical Center, P.O.B. 151, Beer-Sheva 84101, Israel. Tel.: +972 8 6400111. E-mail addresses: [email protected] (M. Volodarsky), [email protected] (U. Zilberman), [email protected] (O.S. Birk). 1 These authors contributed equally to this work. 0003–9969/# 2015 Elsevier Ltd. All rights reserved.


archives of oral biology 60 (2015) 919–922

Fig. 1 – Clinical and molecular studies. (A) Affected individual – frontal view. Note the very thin enamel on the gingival half of the teeth crown and the exposed dentine on the occlusal half of the crowns. (B) Affected individual – panoramic view. Note the missing enamel on all teeth, the un-erupted permanent molars and upper left canine teeth and the absorption of the first molars crowns. (C) The end of stage 1 repair procedure. Note the composite restorations on anterior permanent teeth,

archives of oral biology 60 (2015) 919–922

variant.6 However, regardless of the mode of heredity, all AI patients are afflicted with clinical problems of poor aesthetics, teeth sensitivity and loss of occlusal vertical dimensions. The mildest problems were found in the pitted hypoplastic type, whereas the most severe problems were encountered in the hypocalcified type of AI.7 The mean enamel mineral content in hypomaturation and hypocalcified AI is reduced, while in the hypoplastic variant, enamel mineral content varies from normal to reduced compared to normal enamel. The decreased enamel content is associated with increased protein content in AI teeth.8 In the hypomaturation type, the enamel shows increased proline content compared with normal enamel or other AI types, while the enamel in hypocalcified AI is characterized by increased tyrosine content.8 The occurrence of hypocalcified AI may be in part due to malfunction of matrix protein degradation during the maturation phase.9 Autosomal recessive (homozygous and compound heterozygous), autosomal dominant, or X-linked forms of isolated AI have been reported to be caused by mutations in AMELX (MIM #301200), ENAM (MIM #104500), MMP20 (MIM #612529), KLK4 (MIM #204700), FAM83H (MIM #130900), WDR72 (MIM #613211), C4orf26 (MIM # 614832) and FAM20A (MIM #204690) and a yet undefined gene at the AIH3 (MIM #301201) locus. The FAM20 family contains three separate subfamilies which are referred to as FAM20A (NM_017565.3), FAM20B (NM_014864.3) and FAM20C (NM_020223.3) in humans.10 FAM20A is expressed in secretory ameloblasts, maturation stage ameloblasts, suprabasal cells of the gingivae, odontoblasts, and dental pulp cells, indicating that FAM20A plays a fundamental role in enamel development and gingival homeostasis.11 All FAM20A mutations causing AI described to date are found in autosomal recessive forms of the disease (homozygous or compound heterozygous). Most mutations lead to transcript degradation as a result of premature stop codons12 and alteration of splice sites.13 Missense mutations are rare but have also been reported.14 Here we report a genome-wide linkage analysis in a large Bedouin consanguineous kindred of the Negev area of Israel affected with a novel FAM20A mutation leading to autosomal recessive hypoplastic AI with absorbed un-erupted permanent molars and no renal impairment.


Materials and methods


Genome-wide linkage analysis

Following Soroka Medical Center IRB approval and informed consent, blood samples were obtained from 6 affected offspring (Fig. 1E: V:2, V:3, V:5, V:6, V:9 and V:10), five obligatory


carriers (Fig. 1E: IV:1, IV:4, IV:5, IV:6 and IV:7) and two unaffected siblings (Fig. 1E: V:4 and V:8). DNA was extracted from all blood samples using FlexiGene DNA kit (Qiagen). Genome-wide linkage analysis was performed on individuals V:2, V:5, V:6, V:9 and V:10 (Fig. 1E) using Affymetrix GeneChip Human Mapping 250K Set Nsp (Affymetrix, Santa Clara, CA) according to the Affymetrix GeneChip Mapping Assay protocol. Homozygosity mapping was performed using HomozygosityMapper web-based tool.15


PCR and sequencing

Primers used for amplification of all FAM20A exons and their flanking exon–intron boundaries were designed using the online Primer3 software.16 REDTaq1 ReadyMixTM PCR Reaction Mix (Sigma, Rehovot, Israel) was used for all PCR reactions. PCR amplification products were purified using AccuPrep1 PCR Purification Kit (Bioneer, Seoul, South Korea). Sequencing was performed in the DNA microarray and sequencing unit at Ben-Gurion University of the Negev, Beer Sheva, Israel.




Clinical diagnosis

A sixteen year old Bedouin Israeli girl was examined at Barzilai paediatric dental clinic with a major complaint of ‘‘ugly teeth’’. Apart from the dentition pathology, her medical history and physical exam were normal. Per medical records, her primary dentition had also been affected and discoloured. Clinical examination demonstrated brown upper anterior teeth with hypoplastic enamel and missing permanent molars (Fig. 1A). The panoramic view showed impacted and crown absorbed permanent molars and un-erupted canines and premolars (Fig. 1B). Very thin enamel was observed. Based on the clinical and radiographic examination, a diagnosis of hypoplastic amelogenesis imperfecta with un-erupted and absorbed permanent teeth was made. Five other family members had similar dentition. Due to the complexity and extent of the treatment, it was performed under general anaesthesia. The stage one treatment included extraction of deciduous teeth, coverage of all erupted teeth with either composite material on anterior teeth and stainless steel crowns (SSC) on the premolars. The upper left first molar was exposed and covered with SSC. Root canal treatment was performed when necessary (Fig. 1C and D). Follow-up every three months was performed and the final stage will include ceramic bridges. One affected individual was available for ultrasound of kidneys (Fig. 1: EV:2). A single minor hyperechogenic finding

exposure of the upper left first permanent molar and coverage of all posterior teeth with SSC. The treatment was performed under general anaesthesia in one visit. (D) Panoramic view at the end of stage 1 repair procedure. Note the root canal treatment of right laterals and canines and the SSC cemented on all exposed posterior teeth. (E) Pedigree of affected kindred. (F) Homozygosity mapping. Arrow pointing to the candidate region on chromosome 17. (G) Sanger sequencing of wild-type (WT), obligate carrier (HET) and affected individual (HOM). Rectangle highlights the A/T substitution followed by deletion of cytosine.


archives of oral biology 60 (2015) 919–922

was evident in one of the kidneys. The medical history of all other individuals was insignificant.


Mutational analysis

Autosomal recessive inheritance was inferred from the family tree (Fig. 1E). Genome-wide homozygosity mapping identified a single substantial homozygous 0.66Mb locus on chr.17: 66152910-66817866 (rs41322951-rs9897551) shared by all five affected individuals (Fig. 1F). Among the six genes within this locus, only FAM20A had known AI-related association. Sanger sequencing of all exons and exon–intron boundaries of FAM20A in an affected individual demonstrated a single homozygous mutation: NM_017565.3 (transcript variant 1): n.1811del; c.1523delC; p.Thr508Glyfs*6. Namely, an A > T substitution followed by a single base deletion in exon 11 (hg19 chr17:g.[66533721delG]) (Fig. 1G). The mutation segregated as expected within the family (based on sequencing of all 13 participants) and was not previously reported in any database.



The finding of this specific novel FAM20A mutation in a large affected kindred is unique in that it allowed for close genotype–phenotype correlation in many affected individuals sharing a single mutation. We unravelled a novel FAM20A mutation (c.1522delC) leading to AI (MIM #204690) in a large consanguineous kindred. The FAM20A protein (NP_060035.1) consists of 541 amino acid (aa) and contains a conserved C-terminal domain (CCD) which overlaps with the unknown function DUF1193 (aa 305–524; Pfam: PF06702) domain.10 Within the CCD of each of the FAM20 family members, there is a set of eight cysteine residues that are perfectly conserved, that may participate in inter or intra-molecular disulphide bond formation.10 Though nonsense mediated decay (NMD) of FAM20A transcripts was not established in these patients, the mutation we describe here is predicted to truncate the protein after 507aa, abrogating the last 34aa of the CCD region, including the 17aa long C-terminal end of the DUF1193 domain and the eighth cysteine residue. It is thus likely that the protein encoded by the aberrant transcript (in case that NMD is not occurring) has dramatically compromised function as predicted by PROVEAN software (Deleterious; PROVEAN score of 32.399), leading to the phenotype we describe.

Funding None.

Competing interests None declared.

Ethical approval Not required.

Acknowledgments This research was supported by the Israel Science Foundation (grant No. 1689/12). We deeply thank the Morris Kahn family foundation for the generous support of this study, and the patients and their families for kind participation.


1. Zilberman U, Smith P, Piperno M, Condemi S. Evidence of amelogenesis imperfecta in an early African Homo erectus. J Hum Evol 2004;46(6):647–53. 2. Witkop CJ, Sauk JJ. Heritable defects of enamel. In: Stewart RE, Prescott GH, editors. Oral facial genetics. St. Louis: Mosby; 1976. p. 151–226. 3. Chosak A, Eidelman E, Wisotski I, Cohen T. Amelogenesis imperfecta among Israeli Jews and the description of a new type of local hypoplastic autosomal recessive amelogenesis imperfecta. Oral Surg Oral Med Oral Pathol 1979;47(2):148–56. 4. Ba¨ckman B, Holmgren G. Amelogenesis imperfecta: a genetic study. Hum Hered 1988;38(4):189–206. 5. Ba¨ckman B, Holm AK. Amelogenesis imperfecta: prevalence and incidence in a northern Swedish country. Community Dent Oral Epidemiol 1986;14(1):43–7. 6. Witkop CJ, Stewart RE. Amelogenesis imperfecta. In: Stewart RE, Barber TK, Troutman KC, Wei SHY, editors. Pediatric dentistry. St. Louis: Mosby; 1982. p. 110–7. 7. Seow WK. Clinical diagnosis and management strategies of amelogenesis imperfecta variants. Pediatr Dent 1993;15(16):384–93. 8. Wright JT, Hall KI, Yamauche M. The enamel proteins in human amelogenesis imperfecta. Arch Oral Biol 1997;42(2):149–59. 9. Takagi Y, Fujita H, Katano H, Shimokawa H, Kuroda T. Immunochemical and biochemical characteristics of enamel proteins in hypocalcified amelogenesis imperfecta. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85(4):424–30. 10. Nalbant D, Youn H, Nalbant SI, Sharma S, Cobos E, Beale EG, et al. FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells. BMC Genom 2005;6:11. 11. O’Sullivan J, Bitu CC, Daly SB, Urquhart JE, Barron MJ, Bhaskar SS, et al. Whole-exome sequencing identifies FAM20A mutations as a cause of amelogenesis imperfecta and gingival hyperplasia syndrome. Am J Hum Genet 2011;88(5):616–20. 12. Kantaputra PN, Bongkochwilawan C, Kaewgahya M, Ohazama A, Kayserili H, Erdem AP, et al. Enamel-renal gingival syndrome, hypodontia, and a novel FAM20A mutation. Am J Med Genet A 2014;9999:1–5. 13. Cho SH, Seymen F, Lee KE, Lee SK, Kweon YS, Kim KJ, et al. Novel FAM20A mutations in hypoplastic amelogenesis imperfecta. Hum Mutat 2012;33(1):91–4. 14. Wang SK, Reid BM, Dugan SL, Roggenbuck JA, Read L, Aref P, et al. FAM20A mutations associated with enamel renal syndrome. J Dent Res 2014;93(1):42–8. 15. Seelow D, Schuelke M, Hildebrandt F, Nu¨rnberg P. Homozygosity Mapper – an interactive approach to homozygosity mapping. Nucl Acids Res 2009;37(Web Server Issue):W593–9. 16. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al. Primer3 – new capabilities and interfaces. Nucl Acids Res 2012;40(15):e115.

Novel FAM20A mutation causes autosomal recessive amelogenesis imperfecta.

To relate the peculiar phenotype of amelogenesis imperfecta in a large Bedouin family to the genotype determined by whole genome linkage analysis...
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