- Email: [email protected]
Enamel-renal syndrome in 2 patients with a mutation in FAM20 A and atypical hypertrichosis and hearing loss phenotypes
Accepted Manuscript Enamel-renal syndrome in two patients with a mutation in FAM20A and atypical hypertrichosis and hearing loss phenotypes Sabina Pena B. Pêgo, DDS, PhD, Ricardo D. Coletta, DDS, PhD, Simona Dumitriu, MD, PhD, Daniela Lancu, MD, PhD, Saleh Albanyan, MD, Robert Kleta, MD, PhD, Maria Teresa Auricchio, PhD, Luis Antônio Santos, DDS, PhD, Breno Rocha, DDS, Hercílio Martelli-Júnior, DDS, PhD PII:
S2212-4403(16)30614-9
DOI:
10.1016/j.oooo.2016.09.226
Reference:
OOOO 1620
To appear in:
Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology
Received Date: 14 July 2016 Revised Date:
14 September 2016
Accepted Date: 30 September 2016
Please cite this article as: Pêgo SPB, Coletta RD, Dumitriu S, Lancu D, Albanyan S, Kleta R, Auricchio MT, Santos LA, Rocha B, Martelli-Júnior H, Enamel-renal syndrome in two patients with a mutation in FAM20A and atypical hypertrichosis and hearing loss phenotypes, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology (2016), doi: 10.1016/j.oooo.2016.09.226. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Enamel-renal syndrome in two patients with a mutation in FAM20A and atypical hypertrichosis and hearing loss phenotypes
Sabina Pena B. Pêgo, DDS, PhD,a Ricardo D. Coletta, DDS, PhD,b Simona Dumitriu,
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MD, PhD,c Daniela Lancu, MD, PhD,c Saleh Albanyan, MD,c Robert Kleta, MD, PhD,c Maria Teresa Auricchio, PhD,d Luis Antônio Santos, DDS, PhD,a Breno Rocha,
SC
DDS,a Hercílio Martelli-Júnior, DDS, PhD,a
a
Claros, Minas Gerais, Brazil. b
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Stomatology Clinic, Dental School, State University of Montes Claros, Montes
Department of Oral Diagnosis, School of Dentistry, University of Campinas,
Piracicaba, São Paulo, Brazil. c
Center for Nephrology, University College London, London, United Kingdom.
d
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Center of Human Genome, Department of Genetics and Evolutionary Biology,
Biosciences Institute, University of São Paulo, São Paulo, Brazil.
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Short title: Enamel-renal syndrome with atypical features
Support: This work was supported by grants from the Fundação de Amparo a Pesquisa do Estado de Minas Gerais-FAPEMIG, Belo Horizonte, Brazil and the Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq, Brazil.
Word count: Abstract: 136; Text: 2118; References: 26; Figures: 4
1
ACCEPTED MANUSCRIPT ABSTRACT Enamel-renal syndrome (ERS) (OMIM #204690) is an uncommon disorder characterized by amelogenesis imperfecta and nephrocalcinosis, and is caused by mutations in FAM20A. We reported two additional patients with ERS which exhibited
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typical features of this syndrome and a homozygous nonsense mutation in FAM20A gene (c.406C>T), confirming genetically the diagnosis. They also exhibited two undescribed clinical features, such as hypertrichosis and hearing loss. Alterations in
SC
genes frequently associated with nonsyndromic hearing loss in the Brazilian population, including connexin 26 (GJB2), connexin 30 (GJB6) and mitochondrial
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12S rRNA (m.A1555G mutation), were not found. These results suggest a putative function of FAM20A in the development of the inner ear and in formation of hair. The presence of nephrocalcinosis is a risk factor for renal impairment, and it is important
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perform regular renal monitoring in order to avoid renal failure.
Key words: Enamel-renal syndrome; FAM20A; connexin, mitochondrial 12S rRNA,
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hypertrichosis; hearing loss.
2
ACCEPTED MANUSCRIPT INTRODUCTION Enamel-renal syndrome (ERS; OMIM #204690) is an rare autosomal recessive disorder characterized by amelogenesis imperfecta (AI) in association with nephrocalcinosis.1-6 This disorder is caused by mutations in FAM20A gene (family
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with sequence similarity 20, member A), located on chromosome 17q24.2.7-11 A number of reports in the literature has described patients with three main findings including hypoplastic AI, gingival fibromatosis and nephrocalcinosis.12
SC
In 2008, a new syndrome called AI and gingival fibromatosis syndrome (AIGFS, MIM #614253), which was characterized by gingival fibromatosis and dental
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abnormalities with thin generalized hypoplastic AI as the main dental feature, was described.13 This syndrome has many features in common with ERS, such as pulp calcifications, root dilacerations, hypodontia, failed or delayed eruption of the permanent dentition, particularly the posterior teeth, and pericoronal radiolucencies in
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unerupted teeth, although renal involvement was not observed in the original kindred.13 However, studies demonstrated that AIGFS is also caused by mutations in FAM20A,14 suggesting that both syndromes are not separate entities. Indeed, they
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probably represent the same condition with variable expressivity, particularly with kidney phenotypes not always present.9,12 Herein we report two additional patients
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affected with ERS showing FAM20A mutations and two undescribed clinical phenotypes such as hypertrichosis and hearing loss.
CASE REPORTS The proband, a 10-year-old girl, was referred to the University of Montes Claros Stomatology Clinic for gingival evaluation. She is the second child of a consanguineous couple (first degree cousins) and was born normally at term after an
3
ACCEPTED MANUSCRIPT uneventful pregnancy and no perinatal complications. Intraoral examination revealed mixed dentition and permanent teeth showed yellow to yellowish-brown discoloration, rough surfaces, irregular defects and a lack of contact points (Figure 1A). She exhibited anterior and posterior cross-bite. Her gingiva was enlarged, which was first
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noted after eruption of the primary dentition. The panoramic radiograph revealed delayed eruption of several teeth, no density differences between enamel and dentin, and intrapulpal calcifications. Pericoronal radiolucencies delimited by sclerotic margin
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were also observed around unerupted teeth (Figure 1B). The patient’s mother informed us hearing difficulties perceived at age of 7, which was confirmed as
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conductive hearing loss by tonal audiometry. The hearing loss was mild and affected mainly the frequency between 500-4000 Hz. After 1 year, a second audiometric test was performed and showed normal results. Both patient and her mother denied exposition to environmental factors frequently associated with hearing loss, including
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exposition to high intensity sounds, acoustic trauma, infections such as measles and meningitis, and ototoxic drugs. Computed tomography did not reveal temporal bone fractures or cochlear otoesclerosis, as well as cerebral calcifications. Physical
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examination also revealed hypertrichosis, characterized by thick eyebrows and eyelashes and excessive amount of hair in the forehead, lateral portions of the face,
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arms and back close to sacral region (Figure 1C). Renal ultrasound showed bilateral nephrocalcinosis without evidence of impaired renal function (Figure 1D). Computed tomography scan of abdomen and pelvis, and laboratory findings, including serum electrolytes, calcium, phosphate, urea, creatinine, alkaline phosphatase and parathyroid hormone level, were unremarkable. A diagnosis of ERS was made and 20 family members spanning the 3 latest generations, were examined showing an autosomal recessive pattern (Figure 2). The initial treatment consisted of oral
4
ACCEPTED MANUSCRIPT hygiene
orientation
and
plaque
control.
Gingivectomy
in
association
with
gingivoplasty was performed in the anterior upper region and the patient is under orthodontic treatment for correction of dental positioning. She remained under regular medical follow-up. Genetic orientation was given to the family emphasizing the
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pattern of gene transmission, consequences of phenotypes, and the need to regular follow up with a nephrologist.
During clinical examination of the patient’s family members, a 31 year old man
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(III-14, Figure 2), mother’s first cousin of patient 1, reported kidney failure at age of 21, hemodialysis for 4 years and kidney transplant at age of 25 years. The kidney
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was donated by his sister. After 7 years, transplanted organ showed signs of rejection and the patient returned to dialysis. He is waiting to make new kidney transplant. Family history did not confirm consanguineous parents. Although the patient was edentulous and wears total prosthesis, he stated that was submitted to
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extraction of all teeth, fifteen years ago, due to gingival enlargement and small and yellowish teeth. He reported no gingival overgrowth after extractions. The patient has arterial hypertension related to renal impairment and takes medications to control
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blood pressure. He has no mental or behavioral changes. As observed in the proband, this family member showed sensorineural hearing loss confirmed by tonal
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audiometry (4 KHz) and hypertrichosis. He denied exposition to environmental factors frequently associated with hearing loss, and computed tomography revealed normal inner ear and brain structures. A diagnosis of ERS was proposed. Genetic orientation was given to him emphasizing the pattern of gene transmission and consequences of phenotypes.
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ACCEPTED MANUSCRIPT METHODS AND RESULTS Since the clinical and radiological findings in these two patients were compatible with the diagnosis of ERS, FAM20A sequencing analysis was performed. Genomic DNA was extracted from oral mucosa cells, and exons and flanking splice junctions of
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FAM20A were PCR amplified with specific primers,7 followed by bidirectional sequencing in an ABI Prism 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Both patients were homozygous for a c.406C>T nonsense mutation in
genetically the diagnosis of ERS (Figure 3).
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exon 2, that caused a premature termination at p.Arg136*, thus confirming
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We also verified whether causative mutations frequently associated with nonsyndromic deafness in the Brazilian population were presented in these 2 patients.15 Among the investigated mutations are those located in connexin 26 (GJB2) and connexin 30 (GJB6) which accounts for a large proportion of cases of
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congenital nonsyndromic recessive deafness, and the m.A1555G mutation in 12S rRNA mitochondrial gene, the most frequent mitochondrial mutation related to hearing loss which is associated with aminoglycoside-induced deafness and non-
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syndromic deafness in families of various population backgrounds including those of Brazil. Analysis of GJB2 and GJB6 genes and m.A155G mutation were performed
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after the methods of Abreu-Silva et al.15, using the same DNA isolated from oral mucosa cells. None of those mutations was detected. The gingival tissues removed during the surgical procedures were fixed in
formalin, embedded in paraffin, and the sections were subjected to regular hematoxylin and eosin stain. The histologic features included a well-structured epithelium with elongated and thin papillae inserted in fibrous connective tissue, which showed an increased amount of collagen fiber bundles running in all directions
6
ACCEPTED MANUSCRIPT and areas of small calcified particles close to nests of odontogenic epithelium (Figure 4). The informed consent was obtained from patients and patient's parents before the procedures, which were performed according to the guidelines of the ethics of
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research involving human subjects and were approved by the Institutional Ethics Committee.
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DISCUSSION
The patients reported here exhibited typical features of ERS, such as hypoplastic AI,
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nephrocalcinosis, gingival overgrowth, and other dental abnormalities. Furthermore, novel characteristics as hearing loss, conductive in the proband and sensorineural in the proband’s cousin, and hypertrichosis were observed. Although those features could represent a coincidence of phenotypes in a same family with deleterious
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recessive mutations, other sporadic findings increasing ERS clinical spectrum were also describe, including heterotopic calcification in the lungs and early cessation of limited menstruation.9 Regarding hearing loss, it can be caused by a variety of
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environmental and genetic factors, and genetic origin contributes to at least 60% of the cases.16 Among the most frequent hearing loss-associated environmental factors
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reported in the Brazilian population are prenatal infections by measles and citomegalovirus
(CMV),
contact
with
teratogenic
agents during pregnancy,
prematurity, perinatal anoxia, and postnatal factors including meningitis, exposure to ototoxic medications and head trauma.17 Cochlear ossification, frequently due to a sequel to meningitis, is a major cause of hearing loss,16 accounting to approximately 6% of the cases in the Brazilian population.17 Mutations in GJB2 gene, which encodes connexin 26, are found in up to 50% of patients with autosomal recessive
7
ACCEPTED MANUSCRIPT nonsyndromic hearing loss.18 Another mutations associated with hearing loss are located in GJB6 and in the mitochondrial 12S rRNA gene.19 The affected patients described in this study did not describe exposition to environmental factors frequently related to hearing loss and neither showed any of the classical mutations frequently
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found in nonsyndromic cases of hearing loss, supporting a causative effect for FAM20A mutation.
Mutations in FAM20C, another member of the FAM20 family, cause Raine
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syndrome, an autosomal recessive osteosclerotic bone dysplasia characterized by craniofacial dimorphism, including midface hypoplasia, hypoplastic nose with choanal
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atresia, proptosis, microcephaly, triangular mouth, gingival hyperplasia, cleft palate and low-set ears, intracranial calcification and generalized osteosclerosis.20 In most of the cases the disorder is lethal in the perinatal period, with few cases surviving due to milder phenotypes.21 Although there are some phenotypic overlapping between
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Raine syndrome and ERS, in the few non-lethal cases of Raine syndrome, hearing loss associated with ossification of the inner ear structures was never reported. To date different mutations including missense, nonsense, splice site and
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insertion/deletion mutations were described in FAM20A.12 In our patients, the mutation c.406C>T in exon 2 was identified, and the presences of hearing loss and
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hypertrichosis suggest a putative function of FAM20A in cells of cochlea and epithelial cells of hair follicles, supporting future studies of FAM20A in the physiological and pathological conditions involving inner ear and hair development. Furthermore, deafness mechanisms underlying connexin mutation-induced hearing loss and the detailed cellular mechanisms underlying these pathological changes remain unclear.22
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ACCEPTED MANUSCRIPT FAM20A-/- knockout mice exhibited very specific features, including enamel alterations, widespread and severe ectopic calcifications of muscular arteries, predominantly in the kidneys, and calcifications in lungs, suggesting an involvement in the biomineralization processes.23 However, FAM20A expression was detect in a
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wide variety of tissues, and demonstrated potential functions in regulating differentiation and function of hematopoietic and other tissues.24 Thus, deafness and hypertrichosis identified in those patients may be related to FAM20A function.
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Furthermore, gingival fibromatosis has been associated with a wide spectrum of syndromes, with several of them showing hypertrichosis and/or hearing loss.25
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The calcified structures found in the gingival tissues in close proximity to the nests of odontogenic epithelium observed in our patients have been previously described by us13 and others.8,10,12 We have hypothesized that the odontogenic epithelial cells might have roles in the formation of these calcified bodies,13 but a
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significant proportion of myofibroblasts was found in the gingival connective tissue and a recent study revealed that the differentiation of valvular interstitial cells to myofibroblasts is a key mechanistic step in the mineralization process of calcific
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aortic valve disease.26 Similar calcifications occur in the dental pulp, dental follicles, lungs and in the kidneys, causing nephrocalcinosis. As suggested by Wang and
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collaborators,8 this pattern of ectopic mineralization might be explained by failure to catalyze
appropriate
post-translational
modifications
on
extracellular
matrix
molecules that inhibit mineralization when FAM20A is absent.
CONCLUSIONS In closing, we described two additional cases of ERS and suggested an expansion of the phenotypic spectrum of the disease by describe patients showing two
9
ACCEPTED MANUSCRIPT undescribed clinical features such as hypertrichosis and hearing loss. This allows us to think a putative function of FAM20A in cells of cochlea and epithelial cells of hair follicles and propose future studies of FAM20A in conditions involving inner ear and hair development. Although most patients are asymptomatic and had normal renal
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function until adulthood, the presence of nephrocalcinosis is a risk factor for renal impairment. Thus, it is important perform regular renal monitoring in order to avoid
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renal failure.
We thank Dr. Regina Célia Mingroni Netto, Center of Human Genome, Department
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of Genetics and Evolutionary Biology, Biosciences Institute, University of São Paulo, São Paulo, Brazil, for her assistance in the genetic analysis.
REFERENCES
J. 1972;17:61-63.
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1. MacGibbon D. Generalized enamel hypoplasia and renal dysfunction. Aust Dent
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2. Lubinsky M, Angle C, Marsh PW, Witkop CJ Jr. Syndrome of amelogenesis imperfecta, nephrocalcinosis, impaired renal concentration, and possible
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abnormality of calcium metabolism. Am J Med Genet. 1985;20:233-243. 3. Dellow EL, Harley KE, Unwin RJ, Wrong O, Winter GB, Parkins BJ. Amelogenesis imperfecta, nephrocalcinosis, and hypocalciuria syndrome in two siblings from a large family with consanguineous parents. Nephrol Dial Transplant. 1998;13:3193-3196. 4. Elizabeth J, Lakshmi Priya E, Umadevi KMR, Ranganathan K. Amelogenesis imperfecta with renal disease: a report of two cases. J Oral Pathol Med. 2007;36:625-628.
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ACCEPTED MANUSCRIPT 5. Martelli-Junior H, Santos Neto PE, Aquino SN, et al. Amelogenesis imperfecta and nephrocalcinosis syndrome: a case report and review of the literature. Nephron Physiol. 2011;118:62-65. 6. Jouad IC, Alloussi ME, El Alaoui C, Laarabi FZ, Lyahyai J, Sefiani A. Further
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evidence for causal FAM20A mutations and first case of amelogenesis imperfecta and gingival hyperplasia syndrome in Marocco: a case report. BMC Oral Health. 2015;15:14.
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7. Jaureguiberry G, De la Dure-Molla M, Parry D, et al. Nephrocalcinosis (enamel
Physiol. 2012;122:1-6.
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renal syndrome) caused by autosomal recessive FAM20A mutations. Nephron
8. Wang SK, Aref P, Hu Y, et al. FAM20A mutations can cause enamel-renal syndrome (ERS). PLoS Genet. 2013;9:1-18.
9. Kantaputra PN, Bongkochwilawan C, Kaewgahya M, et al. Enamel-renal-gingival
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syndrome, hypodontia, and a novel FAM20A mutation. Am J Med Genet A 2014;164A:2124-2128.
10. Kantaputra PN, Kaewgahya M, Khemaleelakul U, et al. Enamel-renal-gingival
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syndrome and FAM20A mutations. Am J Med Genet A 2014;164A:1-9. 11. Wang SK, Reid BM, Dugan SL, et al. FAM20A mutations associated with enamel
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renal syndrome. J Dent Res, 2014;93:42-48. 12. De la Dure-Molla M, Quentric M, Yamaguti PM, et al. Pathognomonic oral profile of enamel renal syndrome (ERS) caused by recessive FAM20A mutations. Orphanet J Rare Dis. 2014;9:1-13. 13. Martelli-Junior H, Bonan PR, Dos Santos LA, Santos SM, Cavalcanti MG, Coletta RD. Case reports of a new syndrome associating gingival fibromatosis and
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ACCEPTED MANUSCRIPT dental abnormalities in a consanguineous family. J Periodontol. 2008;79:12871296. 14. O’Sullivan J, Bitu CC, Daly SB, et al. Whole-exome sequencing identifies FAM20A mutations as a cause of amelogenesis imperfecta and gingival
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hyperplasia syndrome. Am J Hum Genet. 2011;88:616-620.
15. Abreu-Silva RS, Rincon D, Horimoto AR, et al. The search of a genetic basis for noise-induced hearing loss (NIHL). Ann Hum Biol. 2011;38:210-218.
causes,
diagnosis,
2013;113:1527-1538.
rehabilitation.
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population:
and
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16. Deltenre P, Van Maldergem L. Hearing loss and deafness in the pediatric
severe
Clin
Neurol.
17. Ramos PZ, de Moraes VC, Svidnicki MC, Soki MN, Castilho AM, Sartorato EL. Etiologic
and
diagnostic
evaluation:
algorithm
for
to
profound
sensorineural hearing loss in Brazil. Int J Audiol. 2013;52:746-752.
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18. Snoeckx RL, Huygen PLM, Feldmann D, et al. GJB2 mutations and degree of hearing loss: A multicenter study. Am J Hum Genet. 2005;77:945-957. 19. Moreira D, Silva D, Priscila Lopez P, Mantovani JC. Screening of connexin 26 in
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nonsyndromic hearing loss. Int Arch Otorhinolaryngol. 2015;19:30-33. 20. Seidahmed MZ, Alazami AM, Abdelbasit OB, et al. Report of a case of Raine
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syndrome and literature review. Am J Med Genet A. 2015;167A:2394-2398. 21. Fradin M, Stoetzel C, Muller J, et al. Osteosclerotic bone dysplasia in siblings with a Fam20C mutation. Clin Genet. 2011;80:177-183. 22. Wingard JC, Zhao H-B. Cellular and deafness mechanisms underlying connexin mutation-induced hearing loss – a common hereditary deafness. Front Cell Neurosci. 2015;9:202.
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ACCEPTED MANUSCRIPT 23. Vogel P, Hansen GM, Read RW, et al. Amelogenesis imperfecta and other biomineralization defects in Fam20a and Fam20c null mice. Vet Pathol. 2012;49:998-1017. 24. Nalbant D, Youn H, Nalbant SI, et al. FAM20: an evolutionarily conserved family
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of secreted proteins expressed in hematopoietic cells. BMC Genomics 2005;6:11.
25. Coletta RD, Graner E. Hereditary gingival fibromatosis: A systematic review. J
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Periodontol. 2006;77:753-764.
26. Hjortnaes J, Goettsch C, Hutcheson JD, et al. Simulation of early calcific aortic
Cardiol. 2016;94:13-20.
Reprint requests:
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valve disease in a 3D platform: A role for myofibroblast differentiation. J Mol Cell
Assistant Professor
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Sabina Pena Borges Pêgo, DDS, PhD
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Rua Novo Horizonte, 131
39508-000 Jaíba, Minas Gerais, Brazil
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[email protected]
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ACCEPTED MANUSCRIPT Figure Legends Fig 1. Clinical features of proband (A) Intra-oral photographs of the proband. Teeth exhibited severe enamel hypoplasia, and the gingiva showed generalized but mild hyperplasia, which was of normal color and fibrous consistency. (B) Panoramic
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radiography showed mix dentition with generalized enamel hypoplasia, intrapulpal calcifications and unerupted teeth with pericoronal radiolucent areas. (C) Hypertrichosis was observe in the forehead, lateral portions of the face, arms and
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back close to sacral region, and as thick eyebrows. (D) Ultrasound revealed
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nephrocalcinosis involving both kidneys.
Fig. 2. Pedigree of family affected with ERS, showing an autosomal recessive trait. Affected individuals are indicated by blackened symbols. Circles denote females and squares denote males, a slash through a symbol denotes a deceased individual, and
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arrow indicates the proband.
Fig. 3. Sequencing chromatograms of FAM20A exon 2. This homozygous nonsense
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mutation, charcaterized by the c.406C>T transition in both alleles of FAM20A, caused a premature termination at p.Arg136*. Electropherogram in A represent an
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unaffected family member (control) and in B of index case.
Fig. 4. Histologic features of the gingival overgrowth. Numerous concentric calcified particles in a psammomatoid pattern in close contact with the odontogenic epithelial rests were distributed in the dense fibrous connective tissue. (A: original magnification x100 and B: original magnification x200)
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S2212-4403(16)30614-9
DOI:
10.1016/j.oooo.2016.09.226
Reference:
OOOO 1620
To appear in:
Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology
Received Date: 14 July 2016 Revised Date:
14 September 2016
Accepted Date: 30 September 2016
Please cite this article as: Pêgo SPB, Coletta RD, Dumitriu S, Lancu D, Albanyan S, Kleta R, Auricchio MT, Santos LA, Rocha B, Martelli-Júnior H, Enamel-renal syndrome in two patients with a mutation in FAM20A and atypical hypertrichosis and hearing loss phenotypes, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology (2016), doi: 10.1016/j.oooo.2016.09.226. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Enamel-renal syndrome in two patients with a mutation in FAM20A and atypical hypertrichosis and hearing loss phenotypes
Sabina Pena B. Pêgo, DDS, PhD,a Ricardo D. Coletta, DDS, PhD,b Simona Dumitriu,
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MD, PhD,c Daniela Lancu, MD, PhD,c Saleh Albanyan, MD,c Robert Kleta, MD, PhD,c Maria Teresa Auricchio, PhD,d Luis Antônio Santos, DDS, PhD,a Breno Rocha,
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DDS,a Hercílio Martelli-Júnior, DDS, PhD,a
a
Claros, Minas Gerais, Brazil. b
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Stomatology Clinic, Dental School, State University of Montes Claros, Montes
Department of Oral Diagnosis, School of Dentistry, University of Campinas,
Piracicaba, São Paulo, Brazil. c
Center for Nephrology, University College London, London, United Kingdom.
d
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Center of Human Genome, Department of Genetics and Evolutionary Biology,
Biosciences Institute, University of São Paulo, São Paulo, Brazil.
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Short title: Enamel-renal syndrome with atypical features
Support: This work was supported by grants from the Fundação de Amparo a Pesquisa do Estado de Minas Gerais-FAPEMIG, Belo Horizonte, Brazil and the Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq, Brazil.
Word count: Abstract: 136; Text: 2118; References: 26; Figures: 4
1
ACCEPTED MANUSCRIPT ABSTRACT Enamel-renal syndrome (ERS) (OMIM #204690) is an uncommon disorder characterized by amelogenesis imperfecta and nephrocalcinosis, and is caused by mutations in FAM20A. We reported two additional patients with ERS which exhibited
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typical features of this syndrome and a homozygous nonsense mutation in FAM20A gene (c.406C>T), confirming genetically the diagnosis. They also exhibited two undescribed clinical features, such as hypertrichosis and hearing loss. Alterations in
SC
genes frequently associated with nonsyndromic hearing loss in the Brazilian population, including connexin 26 (GJB2), connexin 30 (GJB6) and mitochondrial
M AN U
12S rRNA (m.A1555G mutation), were not found. These results suggest a putative function of FAM20A in the development of the inner ear and in formation of hair. The presence of nephrocalcinosis is a risk factor for renal impairment, and it is important
TE D
perform regular renal monitoring in order to avoid renal failure.
Key words: Enamel-renal syndrome; FAM20A; connexin, mitochondrial 12S rRNA,
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EP
hypertrichosis; hearing loss.
2
ACCEPTED MANUSCRIPT INTRODUCTION Enamel-renal syndrome (ERS; OMIM #204690) is an rare autosomal recessive disorder characterized by amelogenesis imperfecta (AI) in association with nephrocalcinosis.1-6 This disorder is caused by mutations in FAM20A gene (family
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with sequence similarity 20, member A), located on chromosome 17q24.2.7-11 A number of reports in the literature has described patients with three main findings including hypoplastic AI, gingival fibromatosis and nephrocalcinosis.12
SC
In 2008, a new syndrome called AI and gingival fibromatosis syndrome (AIGFS, MIM #614253), which was characterized by gingival fibromatosis and dental
M AN U
abnormalities with thin generalized hypoplastic AI as the main dental feature, was described.13 This syndrome has many features in common with ERS, such as pulp calcifications, root dilacerations, hypodontia, failed or delayed eruption of the permanent dentition, particularly the posterior teeth, and pericoronal radiolucencies in
TE D
unerupted teeth, although renal involvement was not observed in the original kindred.13 However, studies demonstrated that AIGFS is also caused by mutations in FAM20A,14 suggesting that both syndromes are not separate entities. Indeed, they
EP
probably represent the same condition with variable expressivity, particularly with kidney phenotypes not always present.9,12 Herein we report two additional patients
AC C
affected with ERS showing FAM20A mutations and two undescribed clinical phenotypes such as hypertrichosis and hearing loss.
CASE REPORTS The proband, a 10-year-old girl, was referred to the University of Montes Claros Stomatology Clinic for gingival evaluation. She is the second child of a consanguineous couple (first degree cousins) and was born normally at term after an
3
ACCEPTED MANUSCRIPT uneventful pregnancy and no perinatal complications. Intraoral examination revealed mixed dentition and permanent teeth showed yellow to yellowish-brown discoloration, rough surfaces, irregular defects and a lack of contact points (Figure 1A). She exhibited anterior and posterior cross-bite. Her gingiva was enlarged, which was first
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noted after eruption of the primary dentition. The panoramic radiograph revealed delayed eruption of several teeth, no density differences between enamel and dentin, and intrapulpal calcifications. Pericoronal radiolucencies delimited by sclerotic margin
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were also observed around unerupted teeth (Figure 1B). The patient’s mother informed us hearing difficulties perceived at age of 7, which was confirmed as
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conductive hearing loss by tonal audiometry. The hearing loss was mild and affected mainly the frequency between 500-4000 Hz. After 1 year, a second audiometric test was performed and showed normal results. Both patient and her mother denied exposition to environmental factors frequently associated with hearing loss, including
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exposition to high intensity sounds, acoustic trauma, infections such as measles and meningitis, and ototoxic drugs. Computed tomography did not reveal temporal bone fractures or cochlear otoesclerosis, as well as cerebral calcifications. Physical
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examination also revealed hypertrichosis, characterized by thick eyebrows and eyelashes and excessive amount of hair in the forehead, lateral portions of the face,
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arms and back close to sacral region (Figure 1C). Renal ultrasound showed bilateral nephrocalcinosis without evidence of impaired renal function (Figure 1D). Computed tomography scan of abdomen and pelvis, and laboratory findings, including serum electrolytes, calcium, phosphate, urea, creatinine, alkaline phosphatase and parathyroid hormone level, were unremarkable. A diagnosis of ERS was made and 20 family members spanning the 3 latest generations, were examined showing an autosomal recessive pattern (Figure 2). The initial treatment consisted of oral
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orientation
and
plaque
control.
Gingivectomy
in
association
with
gingivoplasty was performed in the anterior upper region and the patient is under orthodontic treatment for correction of dental positioning. She remained under regular medical follow-up. Genetic orientation was given to the family emphasizing the
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pattern of gene transmission, consequences of phenotypes, and the need to regular follow up with a nephrologist.
During clinical examination of the patient’s family members, a 31 year old man
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(III-14, Figure 2), mother’s first cousin of patient 1, reported kidney failure at age of 21, hemodialysis for 4 years and kidney transplant at age of 25 years. The kidney
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was donated by his sister. After 7 years, transplanted organ showed signs of rejection and the patient returned to dialysis. He is waiting to make new kidney transplant. Family history did not confirm consanguineous parents. Although the patient was edentulous and wears total prosthesis, he stated that was submitted to
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extraction of all teeth, fifteen years ago, due to gingival enlargement and small and yellowish teeth. He reported no gingival overgrowth after extractions. The patient has arterial hypertension related to renal impairment and takes medications to control
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blood pressure. He has no mental or behavioral changes. As observed in the proband, this family member showed sensorineural hearing loss confirmed by tonal
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audiometry (4 KHz) and hypertrichosis. He denied exposition to environmental factors frequently associated with hearing loss, and computed tomography revealed normal inner ear and brain structures. A diagnosis of ERS was proposed. Genetic orientation was given to him emphasizing the pattern of gene transmission and consequences of phenotypes.
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ACCEPTED MANUSCRIPT METHODS AND RESULTS Since the clinical and radiological findings in these two patients were compatible with the diagnosis of ERS, FAM20A sequencing analysis was performed. Genomic DNA was extracted from oral mucosa cells, and exons and flanking splice junctions of
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FAM20A were PCR amplified with specific primers,7 followed by bidirectional sequencing in an ABI Prism 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Both patients were homozygous for a c.406C>T nonsense mutation in
genetically the diagnosis of ERS (Figure 3).
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exon 2, that caused a premature termination at p.Arg136*, thus confirming
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We also verified whether causative mutations frequently associated with nonsyndromic deafness in the Brazilian population were presented in these 2 patients.15 Among the investigated mutations are those located in connexin 26 (GJB2) and connexin 30 (GJB6) which accounts for a large proportion of cases of
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congenital nonsyndromic recessive deafness, and the m.A1555G mutation in 12S rRNA mitochondrial gene, the most frequent mitochondrial mutation related to hearing loss which is associated with aminoglycoside-induced deafness and non-
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syndromic deafness in families of various population backgrounds including those of Brazil. Analysis of GJB2 and GJB6 genes and m.A155G mutation were performed
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after the methods of Abreu-Silva et al.15, using the same DNA isolated from oral mucosa cells. None of those mutations was detected. The gingival tissues removed during the surgical procedures were fixed in
formalin, embedded in paraffin, and the sections were subjected to regular hematoxylin and eosin stain. The histologic features included a well-structured epithelium with elongated and thin papillae inserted in fibrous connective tissue, which showed an increased amount of collagen fiber bundles running in all directions
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ACCEPTED MANUSCRIPT and areas of small calcified particles close to nests of odontogenic epithelium (Figure 4). The informed consent was obtained from patients and patient's parents before the procedures, which were performed according to the guidelines of the ethics of
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research involving human subjects and were approved by the Institutional Ethics Committee.
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DISCUSSION
The patients reported here exhibited typical features of ERS, such as hypoplastic AI,
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nephrocalcinosis, gingival overgrowth, and other dental abnormalities. Furthermore, novel characteristics as hearing loss, conductive in the proband and sensorineural in the proband’s cousin, and hypertrichosis were observed. Although those features could represent a coincidence of phenotypes in a same family with deleterious
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recessive mutations, other sporadic findings increasing ERS clinical spectrum were also describe, including heterotopic calcification in the lungs and early cessation of limited menstruation.9 Regarding hearing loss, it can be caused by a variety of
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environmental and genetic factors, and genetic origin contributes to at least 60% of the cases.16 Among the most frequent hearing loss-associated environmental factors
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reported in the Brazilian population are prenatal infections by measles and citomegalovirus
(CMV),
contact
with
teratogenic
agents during pregnancy,
prematurity, perinatal anoxia, and postnatal factors including meningitis, exposure to ototoxic medications and head trauma.17 Cochlear ossification, frequently due to a sequel to meningitis, is a major cause of hearing loss,16 accounting to approximately 6% of the cases in the Brazilian population.17 Mutations in GJB2 gene, which encodes connexin 26, are found in up to 50% of patients with autosomal recessive
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ACCEPTED MANUSCRIPT nonsyndromic hearing loss.18 Another mutations associated with hearing loss are located in GJB6 and in the mitochondrial 12S rRNA gene.19 The affected patients described in this study did not describe exposition to environmental factors frequently related to hearing loss and neither showed any of the classical mutations frequently
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found in nonsyndromic cases of hearing loss, supporting a causative effect for FAM20A mutation.
Mutations in FAM20C, another member of the FAM20 family, cause Raine
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syndrome, an autosomal recessive osteosclerotic bone dysplasia characterized by craniofacial dimorphism, including midface hypoplasia, hypoplastic nose with choanal
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atresia, proptosis, microcephaly, triangular mouth, gingival hyperplasia, cleft palate and low-set ears, intracranial calcification and generalized osteosclerosis.20 In most of the cases the disorder is lethal in the perinatal period, with few cases surviving due to milder phenotypes.21 Although there are some phenotypic overlapping between
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Raine syndrome and ERS, in the few non-lethal cases of Raine syndrome, hearing loss associated with ossification of the inner ear structures was never reported. To date different mutations including missense, nonsense, splice site and
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insertion/deletion mutations were described in FAM20A.12 In our patients, the mutation c.406C>T in exon 2 was identified, and the presences of hearing loss and
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hypertrichosis suggest a putative function of FAM20A in cells of cochlea and epithelial cells of hair follicles, supporting future studies of FAM20A in the physiological and pathological conditions involving inner ear and hair development. Furthermore, deafness mechanisms underlying connexin mutation-induced hearing loss and the detailed cellular mechanisms underlying these pathological changes remain unclear.22
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ACCEPTED MANUSCRIPT FAM20A-/- knockout mice exhibited very specific features, including enamel alterations, widespread and severe ectopic calcifications of muscular arteries, predominantly in the kidneys, and calcifications in lungs, suggesting an involvement in the biomineralization processes.23 However, FAM20A expression was detect in a
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wide variety of tissues, and demonstrated potential functions in regulating differentiation and function of hematopoietic and other tissues.24 Thus, deafness and hypertrichosis identified in those patients may be related to FAM20A function.
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Furthermore, gingival fibromatosis has been associated with a wide spectrum of syndromes, with several of them showing hypertrichosis and/or hearing loss.25
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The calcified structures found in the gingival tissues in close proximity to the nests of odontogenic epithelium observed in our patients have been previously described by us13 and others.8,10,12 We have hypothesized that the odontogenic epithelial cells might have roles in the formation of these calcified bodies,13 but a
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significant proportion of myofibroblasts was found in the gingival connective tissue and a recent study revealed that the differentiation of valvular interstitial cells to myofibroblasts is a key mechanistic step in the mineralization process of calcific
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aortic valve disease.26 Similar calcifications occur in the dental pulp, dental follicles, lungs and in the kidneys, causing nephrocalcinosis. As suggested by Wang and
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collaborators,8 this pattern of ectopic mineralization might be explained by failure to catalyze
appropriate
post-translational
modifications
on
extracellular
matrix
molecules that inhibit mineralization when FAM20A is absent.
CONCLUSIONS In closing, we described two additional cases of ERS and suggested an expansion of the phenotypic spectrum of the disease by describe patients showing two
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ACCEPTED MANUSCRIPT undescribed clinical features such as hypertrichosis and hearing loss. This allows us to think a putative function of FAM20A in cells of cochlea and epithelial cells of hair follicles and propose future studies of FAM20A in conditions involving inner ear and hair development. Although most patients are asymptomatic and had normal renal
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function until adulthood, the presence of nephrocalcinosis is a risk factor for renal impairment. Thus, it is important perform regular renal monitoring in order to avoid
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renal failure.
We thank Dr. Regina Célia Mingroni Netto, Center of Human Genome, Department
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of Genetics and Evolutionary Biology, Biosciences Institute, University of São Paulo, São Paulo, Brazil, for her assistance in the genetic analysis.
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2. Lubinsky M, Angle C, Marsh PW, Witkop CJ Jr. Syndrome of amelogenesis imperfecta, nephrocalcinosis, impaired renal concentration, and possible
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abnormality of calcium metabolism. Am J Med Genet. 1985;20:233-243. 3. Dellow EL, Harley KE, Unwin RJ, Wrong O, Winter GB, Parkins BJ. Amelogenesis imperfecta, nephrocalcinosis, and hypocalciuria syndrome in two siblings from a large family with consanguineous parents. Nephrol Dial Transplant. 1998;13:3193-3196. 4. Elizabeth J, Lakshmi Priya E, Umadevi KMR, Ranganathan K. Amelogenesis imperfecta with renal disease: a report of two cases. J Oral Pathol Med. 2007;36:625-628.
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ACCEPTED MANUSCRIPT 5. Martelli-Junior H, Santos Neto PE, Aquino SN, et al. Amelogenesis imperfecta and nephrocalcinosis syndrome: a case report and review of the literature. Nephron Physiol. 2011;118:62-65. 6. Jouad IC, Alloussi ME, El Alaoui C, Laarabi FZ, Lyahyai J, Sefiani A. Further
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evidence for causal FAM20A mutations and first case of amelogenesis imperfecta and gingival hyperplasia syndrome in Marocco: a case report. BMC Oral Health. 2015;15:14.
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8. Wang SK, Aref P, Hu Y, et al. FAM20A mutations can cause enamel-renal syndrome (ERS). PLoS Genet. 2013;9:1-18.
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ACCEPTED MANUSCRIPT dental abnormalities in a consanguineous family. J Periodontol. 2008;79:12871296. 14. O’Sullivan J, Bitu CC, Daly SB, et al. Whole-exome sequencing identifies FAM20A mutations as a cause of amelogenesis imperfecta and gingival
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hyperplasia syndrome. Am J Hum Genet. 2011;88:616-620.
15. Abreu-Silva RS, Rincon D, Horimoto AR, et al. The search of a genetic basis for noise-induced hearing loss (NIHL). Ann Hum Biol. 2011;38:210-218.
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16. Deltenre P, Van Maldergem L. Hearing loss and deafness in the pediatric
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18. Snoeckx RL, Huygen PLM, Feldmann D, et al. GJB2 mutations and degree of hearing loss: A multicenter study. Am J Hum Genet. 2005;77:945-957. 19. Moreira D, Silva D, Priscila Lopez P, Mantovani JC. Screening of connexin 26 in
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nonsyndromic hearing loss. Int Arch Otorhinolaryngol. 2015;19:30-33. 20. Seidahmed MZ, Alazami AM, Abdelbasit OB, et al. Report of a case of Raine
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ACCEPTED MANUSCRIPT 23. Vogel P, Hansen GM, Read RW, et al. Amelogenesis imperfecta and other biomineralization defects in Fam20a and Fam20c null mice. Vet Pathol. 2012;49:998-1017. 24. Nalbant D, Youn H, Nalbant SI, et al. FAM20: an evolutionarily conserved family
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Reprint requests:
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valve disease in a 3D platform: A role for myofibroblast differentiation. J Mol Cell
Assistant Professor
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Sabina Pena Borges Pêgo, DDS, PhD
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Rua Novo Horizonte, 131
39508-000 Jaíba, Minas Gerais, Brazil
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[email protected]
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ACCEPTED MANUSCRIPT Figure Legends Fig 1. Clinical features of proband (A) Intra-oral photographs of the proband. Teeth exhibited severe enamel hypoplasia, and the gingiva showed generalized but mild hyperplasia, which was of normal color and fibrous consistency. (B) Panoramic
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radiography showed mix dentition with generalized enamel hypoplasia, intrapulpal calcifications and unerupted teeth with pericoronal radiolucent areas. (C) Hypertrichosis was observe in the forehead, lateral portions of the face, arms and
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back close to sacral region, and as thick eyebrows. (D) Ultrasound revealed
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nephrocalcinosis involving both kidneys.
Fig. 2. Pedigree of family affected with ERS, showing an autosomal recessive trait. Affected individuals are indicated by blackened symbols. Circles denote females and squares denote males, a slash through a symbol denotes a deceased individual, and
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arrow indicates the proband.
Fig. 3. Sequencing chromatograms of FAM20A exon 2. This homozygous nonsense
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mutation, charcaterized by the c.406C>T transition in both alleles of FAM20A, caused a premature termination at p.Arg136*. Electropherogram in A represent an
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unaffected family member (control) and in B of index case.
Fig. 4. Histologic features of the gingival overgrowth. Numerous concentric calcified particles in a psammomatoid pattern in close contact with the odontogenic epithelial rests were distributed in the dense fibrous connective tissue. (A: original magnification x100 and B: original magnification x200)
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