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Exonal Deletion of SLC24A4 Causes Hypomaturation Amelogenesis Imperfecta F. Seymen, K.-E. Lee, C.G. Tran Le, M. Yildirim, K. Gencay, Z.H. Lee and J.-W. Kim J DENT RES 2014 93: 366 originally published online 14 February 2014 DOI: 10.1177/0022034514523786 The online version of this article can be found at: http://jdr.sagepub.com/content/93/4/366
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On behalf of: International and American Associations for Dental Research
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research-article2014
JDR
93410.1177/0022034514523786
RESEARCH REPORTS Clinical
F. Seymen1, K.-E. Lee2, C.G. Tran Le2, M. Yildirim1, K. Gencay1, Z.H. Lee3, and J.-W. Kim2,4* 1
Department of Pedodontics, Faculty of Dentistry, Istanbul University, Istanbul, Turkey; 2Department of Pediatric Dentistry and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea; 3Department of Cell and Developmental Biology and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea; and 4Department of Molecular Genetics and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea; *corresponding author, [email protected]
Exonal Deletion of SLC24A4 Causes Hypomaturation Amelogenesis Imperfecta
J Dent Res 93(4):366-370, 2014
Abstract Amelogenesis imperfecta is a heterogeneous group of genetic conditions affecting enamel formation. Recently, mutations in solute carrier family 24 member 4 (SLC24A4) have been identified to cause autosomal recessive hypomaturation amelogenesis imperfecta. We recruited a consanguineous family with hypomaturation amelogenesis imperfecta with generalized brown discoloration. Sequencing of the candidate genes identified a 10-kb deletion, including exons 15, 16, and most of the last exon of the SLC24A4 gene. Interestingly, this deletion was caused by homologous recombination between two 354-bp-long homologous sequences located in intron 14 and the 3′ UTR. This is the first report of exonal deletion in SLC24A4 providing confirmatory evidence that the function of SLC24A4 in calcium transport has a crucial role in the maturation stage of amelogenesis.
KEY WORDS: enamel, tooth, hereditary, malformation, recombination, maturation.
DOI: 10.1177/0022034514523786 Received November 12, 2013; Last revision December 21, 2013; Accepted January 20, 2014 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research
Introduction
A
melogenesis imperfecta (AI) is a group of inherited defects in tooth enamel formation characterized by abnormalities in the thickness, structure, and/ or composition (Witkop, 1988). It is heterogeneous in clinical phenotype and genetic etiology. The prevalence of AI is various (between 1:4000 and 1:14,000), depending on study population and location, but is as high as 1:718 at certain locations (Backman and Holm, 1986). AI can be classified as hypoplastic, hypocalcified, and hypomaturation according to the clinical phenotypes. Hypoplastic enamel is thin but hard in most cases. Hypocalcified enamel is extremely soft, but the thickness is normal before the tooth eruption. Hypomaturation enamel is soft and discolored, but the thickness is normal (Witkop, 1988). However, it is difficult to determine exact type of AI in some cases. Mutations in amelogenin (AMELX; MIM *300391) cause X-linked hypoplastic or hypomaturation AI depending on the nature of the mutation (Lagerstrom et al., 1991; Kim et al., 2004). Enamelin (ENAM; MIM *606585) mutations cause hypoplastic AI with variable expressivity even in a single family (Rajpar et al., 2001; Kang et al., 2009). Family with sequence similarity 20 member A (FAM20A; MIM *611062) gene mutations cause hypoplastic AI with gingival hyperplasia and multiple eruption failures in autosomal recessive inheritance pattern (O’Sullivan et al., 2011; Cho et al., 2012). Family with sequence similarity 83 member H (FAM83H; MIM *611927) mutations cause autosomal dominant hypocalcified AI (Kim et al., 2008), while chromosome 4 open reading frame 26 (C4orf26; MIM *614829) mutations cause autosomal recessive hypocalcified AI (Parry et al., 2012). Mutations in enamelysin (MMP20; MIM *604629), kallikrein 4 (KLK4; MIM *603767), and WD repeat-containing protein 72 (WDR72; MIM *613214) genes cause autosomal recessive hypomaturation AI (Hart et al., 2004; Kim et al., 2005; El-Sayed et al., 2009). Recently, 2 homozygous mutations (NM_153646.3: c.1015C>T, p.Arg339* and c.1495A>T, p.Ser499Cys) in solute carrier family 24 member 4 (SLC24A4; MIM *609840) have been identified in 2 consanguineous families with hypomaturation AI (Parry et al., 2013). SLC24A4 encodes a member of the potassium-dependent sodium/calcium exchanger family, and those mutations are predicted to cause the loss or severe impairment of the SLC24A4 function. In this report, we recruited a Turkish consanguineous family with hypomaturation AI. We performed mutational screening based on the candidate gene approach.
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J Dent Res 93(4) 2014 367 Exonal Deletion of SLC24A4
Materials & Methods
Table. Primers Used to Amplify EDA Exons
Enrollment of Human Subjects
Primer
The study protocol and patient consents were independently reviewed and approved by the Institution Review Board at Seoul National University Dental Hospital and University of Istanbul. Blood samples were collected with the understanding and written consent of each participant according to the Declaration of Helsinki.
Polymerase Chain Reaction and Sequencing Genomic DNA was isolated from peripheral whole blood via the QuickGene DNA whole blood kit S with QuickGene-Mini80 equipment (Fujifilm, Tokyo, Japan). The purity and concentration of the DNA were quantitated by spectrophotometry, measured by the OD260/OD280 ratio. Mutational analyses including exons and nearby intron sequences were done for the genes involved in autosomal recessive hypomaturation AI. The primer pairs and polymerase chain reaction (PCR) conditions for amplification of MMP20, KLK4, and WDR72 genes were previously described (Hart et al., 2004; Kim et al., 2005; Lee et al., 2010). Primers for SLC24A4 gene were designed with Primer 3 on the Web (http://frodo.wi.mit.edu/primer3/) (Table). PCR amplifications were done with the HiPi DNA polymerase premix (Elpis Biotech, Taejeon, Korea). PCR amplification products were purified with a PCR Purification Kit and protocol (Elpis Biotech). DNA sequencing was performed at a DNA sequencing center (Macrogen, Seoul, Korea).
Array Comparative Genomic Hybridization Array comparative genomic hybridization via Affymetrix Genome-Wide Human SNP Array 6.0 was performed to detect pathologic copy number variation (CNV) and region of loss of heterozygosity. The data were analyzed with the CNV segmentation criteria, with the minimum markers in a segment being 1 and a minimum genomic segment size of 1 kb.
Confirmation of the Deleted Region and PCR across the Deletion Several sets of primers for the 3′ UTR (Ex17-2~5) were designed as described above (Table). PCR across the deletion was performed with a primer pair (Ex14F and Ex17-5R), and the amplicon was purified and sequenced as described above.
Results The proband was a 7-year-old girl in a consanguineous family (Fig. 1A), presenting generalized hypomaturation AI with brown discoloration (Fig. 1B-1D). She was born at full term, and her mother’s pregnancy and delivery were uneventful. She had no other medical problems. All teeth exhibited brown discoloration, and dental panoramic radiograph revealed the lack of contrast between the enamel and dentin because of the reduced mineral density of the affected enamel (Fig. 1E). Sanger sequencing of all exons and exon-intron boundaries of MMP20, KLK4, and WDR72 genes did not identify any
Ex1F Ex1R Ex2F Ex2R Ex3F Ex3R Ex4F Ex4R Ex5F Ex5R Ex6F Ex6R Ex7F Ex7R Ex8F Ex8R Ex9F Ex9R Ex10F Ex10R Ex11F Ex11R Ex12F Ex12R Ex13F Ex13R Ex14F Ex14R Ex15F Ex15R Ex16F Ex16R Ex17-1F Ex17-1R Ex17-2F Ex17-2R Ex17-3F Ex17-3R Ex17-4F Ex17-4R Ex17-5F Ex17-5R
Oligonucleotide Sequence 5′-ctcggccactgattgcac 5′-aaggaggggaaaacatctcg 5′-cagggctgttgctgacatag 5′-tggctgtaaccacccacata 5′-gaactctcagaagtcaagtgaggt 5′-agatctcagacacgccacg 5′-ctggtttggggtgtggtg 5′-ctcggtgtgacagtctttgc 5′-ttggctgtagagcgtccagt 5′-tgaggctcagagctgacaaa 5′-aaagggggacactgaggaag 5′-gctaccccaacctcttgtca 5′-gtggcctggagttaggaggt 5′-agtgccaggggcagagat 5′-gagcagctcagaaatggacc 5′-aacgattcagggaacccaac 5′-cacttctggacccctcattc 5′-ttctccctgctgtcacaaaa 5′-agggatggggtgtgatcc 5′-tctcttagggcacctgtggt 5′-tcacaaggtgaggggaagtc 5′-atcaatggcaccaggaagag 5′-tccccagggttgttgttcta 5′-acagattcgcctcctaagca 5′-ctagagtcacatcggtggca 5′-gtggttagccttgaacccag 5′-tgtttcctacgcttacagtgtctc 5′-aagtcaggcagggacgag 5′-ttccaaggaatggcactgat 5′-tagagcctctggctggaact 5′-gctgggatttctggatgga 5′-gtaagtgacgaggcggaatc 5′-tgaggatcagactgcagcac 5′-gcctatgcaggagagacctg 5′-CCTGAAGTTCCCTGTTGCAT 5′-GAAAATCACCCTCGTCCTCA 5′-TCTATCACCCAAGGGCAGTC 5′-AAGACGGGTATCCAGCAATG 5′-CATGTCTGCTGCTGGGATAC 5′-CAACAGTGGGAACAGGTGTG 5′-GAGCACAAGTTGTCTTTCCCTAA 5′-TGCGACCCTTATCCTTTTTG
Size (bp) 293 294 244 278 250 261 287 247 213 398 343 487 367 280 287 207 397 177 213 213 165
disease-causing mutation. During PCR amplifications of the SLC24A4 gene, we could not amplify the last 3 exons using the DNA sample of the proband. CNV analysis detected a homozygous deletion spanning 8 kb in the SLC24A4 gene (Fig. 2). The deleted region normally has exons 15 and 16 and most of exon 17 (NM_153646.3). The deletion was confirmed by PCR amplification through several sets of primers (Ex17-2~5). The PCR reaction of the proband DNA sample across the deletion via a primer pair that spanned the deletion (Ex14F and Ex17-5R) resulted in an amplicon of circa 5 kb. This reaction would usually result in an amplicon of around 15 kb for a DNA sample without a deletion. Sequencing of the amplified product confirmed a deletion region of 10,042 bp (Fig. 2).
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Discussion
Figure 1. Pedigree, clinical photos, and panoramic radiograph of the proband. (A) Pedigree of the family. (B-D) Frontal, maxillary, and mandibular clinical photos of the proband. Teeth have a brown discoloration and are abraded. (E) Panoramic radiograph of the proband revealed lack of contrast between the enamel and dentin due to the reduced mineral density of the affected enamel.
Figure 2. Mutational analysis. Copy number variation analysis identified a homozygous deletion in the SLC24A4 gene. Polymerase chain reaction amplifications using exon-specific primer pairs confirmed the deletion of exons 15, 16, and most of the last exon (17). Sanger sequencing of the polymerase chain reaction amplicon across the deletion revealed the specific deletion (chr14:92,957,680-92,967,722del). The locations of the primers (Ex14F and Ex17-5R) are indicated above the gene diagram. Red bars under the gene diagram indicate homologous sequences 354 bp long. Pink arrow under the gene diagram indicates the region of deletion.
SLC24A4 (NCKX4) was cloned from a human brain cDNA library through sequence blast searching with SLC24A3 (NCKX3). Based on the similarity in amino acid identity and exon boundaries with SLC24A3, it was proposed that SLC24A4 had arisen from a recent gene duplication (Li et al., 2002). SLC24A4 gene has multiple transcripts resulting from alternative splicing and use of alternative first exons. The longest isoform (encoded by NM_153646.3) has 17 exons and encodes 622 amino acid protein. SLC24A4, a member of the potassiumdependent sodium/calcium exchanger family, is a type II transmembrane protein with 11 predicted transmembrane domains and a glycosylated extracellular loop in the middle part (Parry et al., 2013). Two highly conserved and oppositely oriented regions known as alpha-1 and alpha-2 repeats form the ion-binding pockets (Visser et al., 2007). Like other potassium-dependent sodium/ calcium exchangers, SLC24A4 exchanges 4 sodium ion for 1 calcium and 1 potassium ion (Stephan et al., 2012). SLC24A4 was proposed as a novel candidate gene involved in dental enamel mineralization. By genomewide transcript profiling of the developing enamel organ, upregulation of SLC24A4 was identified and confirmed by Western blotting as well (Lacruz et al., 2012). Prominent expression of SLC24A4 was shown at the apical poles and at the lateral membrane proximal to the apical ends of ameloblasts during the mid- to late maturation stages of amelogenesis (Hu et al., 2012). The deletion identified in this study would result in a total loss of SLC24A4 because of nonsense-mediated decay. Even if the mutant transcript survived nonsensemediated decay, the mutant protein would lack the C-terminal 109 residues of the 622–amino acid wild-type protein, which would truncate the alpha-2 repeat, which forms the ion-binding pockets with the alpha-1 repeat (Parry et al., 2013). Interestingly, the sequence analysis of the deleted region identified homologous sequences in intron 14 and the 3′ UTR (Fig. 3). The homologous sequences, 354 bp long, were identical except for only 5 nucleotides (Appendix Fig.). The remaining sequences after deletion indicated that
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J Dent Res 93(4) 2014 369 Exonal Deletion of SLC24A4 the deletion occurred by homologous recombination between these 2 homologous sequences and that the intron 14 sequence survived after recombination. Blast search revealed that the homologous SLC24A4 sequences are unique and do not have any similarity with long interspersed nuclear elements (LINE) and Alu sequences. Most homologous recombinations are caused by Alumediated and rarely LINE-mediated events (Higashimoto et al., 2013). Therefore, the deletion caused by unusual homologous sequences in the same gene is quite rare, and these sequences may be remnants of gene duplication event. Our identification of the first exonal deletion in SLC24A4 gene, which would result in the loss or severe truncation of the SLC24A4 function, expands not only the list of the SLC24A4 mutations but also that of mutations caused by homologous recombination. It further provides confirmatory evidence that the function of SLC24A4 has a crucial role in the maturation stage of amelogenesis.
Acknowledgment This work was supported by grants from the Bio and Medical Technology Development Program (2013037491) and the Science Research Center grant to Bone Metabolism Research Center (2008-0062614) by the Korea Research Foundation. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
References
Figure 3. Sequences of a part of SLC24A4 gene. Homologous sequences are denoted by a pink color, and deleted sequences are underlined. Exon sequences are denoted by bold characters and the numbers of the exons are shown on the left margin. Stop codon (TAG) is shown by red color in exon 17.
Backman B, Holm AK (1986). Amelogenesis imperfecta: prevalence and incidence in a northern Swedish county. Community Dent Oral Epidemiol 14:43-47. Cho SH, Seymen F, Lee KE, Lee SK, Kweon YS, Kim KJ, et al. (2012). Novel FAM20A mutations in hypoplastic amelogenesis imperfecta. Hum Mutat 33:91-94. El-Sayed W, Parry DA, Shore RC, Ahmed M, Jafri H, Rashid Y, et al. (2009). Mutations in the beta propeller WDR72 cause autosomalrecessive hypomaturation amelogenesis imperfecta. Am J Hum Genet 85:699-705. Hart PS, Hart TC, Michalec MD, Ryu OH, Simmons D, Hong S, et al. (2004). Mutation in kallikrein 4 causes autosomal recessive hypomaturation amelogenesis imperfecta. J Med Genet 41:545-549. Higashimoto K, Maeda T, Okada J, Ohtsuka Y, Sasaki K, Hirose A, et al. (2013). Homozygous deletion of DIS3L2 exon 9 due to non-allelic homologous recombination between LINE-1s in a Japanese patient with Perlman syndrome. Eur J Hum Genet 21:1316-1319. Hu P, Lacruz RS, Smith CE, Smith SM, Kurtz I, Paine ML (2012). Expression of the sodium/calcium/potassium exchanger, NCKX4, in ameloblasts. Cells Tissues Organs 196:501-509.
Kang HY, Seymen F, Lee SK, Yildirim M, Tuna EB, Patir A, et al. (2009). Candidate gene strategy reveals ENAM mutations. J Dent Res 88:266269. Kim JW, Simmer JP, Hu YY, Lin BP, Boyd C, Wright JT, et al. (2004). Amelogenin p.M1T and p.W4S mutations underlying hypoplastic X-linked amelogenesis imperfecta. J Dent Res 83:378-383. Kim JW, Simmer JP, Hart TC, Hart PS, Ramaswami MD, Bartlett JD, et al. (2005). MMP-20 mutation in autosomal recessive pigmented hypomaturation amelogenesis imperfecta. J Med Genet 42:271275. Kim JW, Lee SK, Lee ZH, Park JC, Lee KE, Lee MH, et al. (2008). FAM83H mutations in families with autosomal-dominant hypocalcified amelogenesis imperfecta. Am J Hum Genet 82:489-494. Lacruz RS, Smith CE, Bringas P Jr, Chen YB, Smith SM, Snead ML, et al. (2012). Identification of novel candidate genes involved in mineralization of dental enamel by genome-wide transcript profiling. J Cell Physiol 227:2264-2275. Lagerstrom M, Dahl N, Nakahori Y, Nakagome Y, Backman B, Landegren U, et al. (1991). A deletion in the amelogenin gene (AMG) causes X-linked amelogenesis imperfecta (AIH1). Genomics 10:971-975.
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Lee SK, Seymen F, Kang HY, Lee KE, Gencay K, Tuna B, et al. (2010). MMP20 hemopexin domain mutation in amelogenesis imperfecta. J Dent Res 89:46-50. Li XF, Kraev AS, Lytton J (2002). Molecular cloning of a fourth member of the potassium-dependent sodium-calcium exchanger gene family, NCKX4. J Biol Chem 277:48410-48417. O’Sullivan J, Bitu CC, Daly SB, Urquhart JE, Barron MJ, Bhaskar SS, et al. (2011). Whole-exome sequencing identifies FAM20A mutations as a cause of amelogenesis imperfecta and gingival hyperplasia syndrome. Am J Hum Genet 88:616-620. Parry DA, Brookes SJ, Logan CV, Poulter JA, El-Sayed W, Al-Bahlani S, et al. (2012). Mutations in C4orf26, encoding a peptide with in vitro hydroxyapatite crystal nucleation and growth activity, cause amelogenesis imperfecta. Am J Hum Genet 91:565-571. Parry DA, Poulter JA, Logan CV, Brookes SJ, Jafri H, Ferguson CH, et al. (2013). Identification of mutations in SLC24A4, encoding a
potassium-dependent sodium/calcium exchanger, as a cause of amelogenesis imperfecta. Am J Hum Genet 92:307-312. Rajpar MH, Harley K, Laing C, Davies RM, Dixon MJ (2001). Mutation of the gene encoding the enamel-specific protein, enamelin, causes autosomal-dominant amelogenesis imperfecta. Hum Mol Genet 10:16731677. Stephan AB, Tobochnik S, Dibattista M, Wall CM, Reisert J, Zhao H (2012). The Na(+)/Ca(2+) exchanger NCKX4 governs termination and adaptation of the mammalian olfactory response. Nat Neurosci 15:131-137. Visser F, Valsecchi V, Annunziato L, Lytton J (2007). Exchangers NCKX2, NCKX3, and NCKX4: identification of Thr-551 as a key residue in defining the apparent K(+) affinity of NCKX2. J Biol Chem 282:44534462. Witkop CJ Jr (1988). Amelogenesis imperfecta, dentinogenesis imperfecta and dentin dysplasia revisited: problems in classification. J Oral Pathol 17:547-553.
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Exonal Deletion of SLC24A4 Causes Hypomaturation Amelogenesis Imperfecta F. Seymen, K.-E. Lee, C.G. Tran Le, M. Yildirim, K. Gencay, Z.H. Lee and J.-W. Kim J DENT RES 2014 93: 366 originally published online 14 February 2014 DOI: 10.1177/0022034514523786 The online version of this article can be found at: http://jdr.sagepub.com/content/93/4/366
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - Mar 17, 2014 OnlineFirst Version of Record - Feb 14, 2014 What is This?
Downloaded from jdr.sagepub.com at Universitaetsbibliothek Bern on October 1, 2014 For personal use only. No other uses without permission. © International & American Associations for Dental Research
research-article2014
JDR
93410.1177/0022034514523786
RESEARCH REPORTS Clinical
F. Seymen1, K.-E. Lee2, C.G. Tran Le2, M. Yildirim1, K. Gencay1, Z.H. Lee3, and J.-W. Kim2,4* 1
Department of Pedodontics, Faculty of Dentistry, Istanbul University, Istanbul, Turkey; 2Department of Pediatric Dentistry and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea; 3Department of Cell and Developmental Biology and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea; and 4Department of Molecular Genetics and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea; *corresponding author, [email protected]
Exonal Deletion of SLC24A4 Causes Hypomaturation Amelogenesis Imperfecta
J Dent Res 93(4):366-370, 2014
Abstract Amelogenesis imperfecta is a heterogeneous group of genetic conditions affecting enamel formation. Recently, mutations in solute carrier family 24 member 4 (SLC24A4) have been identified to cause autosomal recessive hypomaturation amelogenesis imperfecta. We recruited a consanguineous family with hypomaturation amelogenesis imperfecta with generalized brown discoloration. Sequencing of the candidate genes identified a 10-kb deletion, including exons 15, 16, and most of the last exon of the SLC24A4 gene. Interestingly, this deletion was caused by homologous recombination between two 354-bp-long homologous sequences located in intron 14 and the 3′ UTR. This is the first report of exonal deletion in SLC24A4 providing confirmatory evidence that the function of SLC24A4 in calcium transport has a crucial role in the maturation stage of amelogenesis.
KEY WORDS: enamel, tooth, hereditary, malformation, recombination, maturation.
DOI: 10.1177/0022034514523786 Received November 12, 2013; Last revision December 21, 2013; Accepted January 20, 2014 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research
Introduction
A
melogenesis imperfecta (AI) is a group of inherited defects in tooth enamel formation characterized by abnormalities in the thickness, structure, and/ or composition (Witkop, 1988). It is heterogeneous in clinical phenotype and genetic etiology. The prevalence of AI is various (between 1:4000 and 1:14,000), depending on study population and location, but is as high as 1:718 at certain locations (Backman and Holm, 1986). AI can be classified as hypoplastic, hypocalcified, and hypomaturation according to the clinical phenotypes. Hypoplastic enamel is thin but hard in most cases. Hypocalcified enamel is extremely soft, but the thickness is normal before the tooth eruption. Hypomaturation enamel is soft and discolored, but the thickness is normal (Witkop, 1988). However, it is difficult to determine exact type of AI in some cases. Mutations in amelogenin (AMELX; MIM *300391) cause X-linked hypoplastic or hypomaturation AI depending on the nature of the mutation (Lagerstrom et al., 1991; Kim et al., 2004). Enamelin (ENAM; MIM *606585) mutations cause hypoplastic AI with variable expressivity even in a single family (Rajpar et al., 2001; Kang et al., 2009). Family with sequence similarity 20 member A (FAM20A; MIM *611062) gene mutations cause hypoplastic AI with gingival hyperplasia and multiple eruption failures in autosomal recessive inheritance pattern (O’Sullivan et al., 2011; Cho et al., 2012). Family with sequence similarity 83 member H (FAM83H; MIM *611927) mutations cause autosomal dominant hypocalcified AI (Kim et al., 2008), while chromosome 4 open reading frame 26 (C4orf26; MIM *614829) mutations cause autosomal recessive hypocalcified AI (Parry et al., 2012). Mutations in enamelysin (MMP20; MIM *604629), kallikrein 4 (KLK4; MIM *603767), and WD repeat-containing protein 72 (WDR72; MIM *613214) genes cause autosomal recessive hypomaturation AI (Hart et al., 2004; Kim et al., 2005; El-Sayed et al., 2009). Recently, 2 homozygous mutations (NM_153646.3: c.1015C>T, p.Arg339* and c.1495A>T, p.Ser499Cys) in solute carrier family 24 member 4 (SLC24A4; MIM *609840) have been identified in 2 consanguineous families with hypomaturation AI (Parry et al., 2013). SLC24A4 encodes a member of the potassium-dependent sodium/calcium exchanger family, and those mutations are predicted to cause the loss or severe impairment of the SLC24A4 function. In this report, we recruited a Turkish consanguineous family with hypomaturation AI. We performed mutational screening based on the candidate gene approach.
366 Downloaded from jdr.sagepub.com at Universitaetsbibliothek Bern on October 1, 2014 For personal use only. No other uses without permission. © International & American Associations for Dental Research
J Dent Res 93(4) 2014 367 Exonal Deletion of SLC24A4
Materials & Methods
Table. Primers Used to Amplify EDA Exons
Enrollment of Human Subjects
Primer
The study protocol and patient consents were independently reviewed and approved by the Institution Review Board at Seoul National University Dental Hospital and University of Istanbul. Blood samples were collected with the understanding and written consent of each participant according to the Declaration of Helsinki.
Polymerase Chain Reaction and Sequencing Genomic DNA was isolated from peripheral whole blood via the QuickGene DNA whole blood kit S with QuickGene-Mini80 equipment (Fujifilm, Tokyo, Japan). The purity and concentration of the DNA were quantitated by spectrophotometry, measured by the OD260/OD280 ratio. Mutational analyses including exons and nearby intron sequences were done for the genes involved in autosomal recessive hypomaturation AI. The primer pairs and polymerase chain reaction (PCR) conditions for amplification of MMP20, KLK4, and WDR72 genes were previously described (Hart et al., 2004; Kim et al., 2005; Lee et al., 2010). Primers for SLC24A4 gene were designed with Primer 3 on the Web (http://frodo.wi.mit.edu/primer3/) (Table). PCR amplifications were done with the HiPi DNA polymerase premix (Elpis Biotech, Taejeon, Korea). PCR amplification products were purified with a PCR Purification Kit and protocol (Elpis Biotech). DNA sequencing was performed at a DNA sequencing center (Macrogen, Seoul, Korea).
Array Comparative Genomic Hybridization Array comparative genomic hybridization via Affymetrix Genome-Wide Human SNP Array 6.0 was performed to detect pathologic copy number variation (CNV) and region of loss of heterozygosity. The data were analyzed with the CNV segmentation criteria, with the minimum markers in a segment being 1 and a minimum genomic segment size of 1 kb.
Confirmation of the Deleted Region and PCR across the Deletion Several sets of primers for the 3′ UTR (Ex17-2~5) were designed as described above (Table). PCR across the deletion was performed with a primer pair (Ex14F and Ex17-5R), and the amplicon was purified and sequenced as described above.
Results The proband was a 7-year-old girl in a consanguineous family (Fig. 1A), presenting generalized hypomaturation AI with brown discoloration (Fig. 1B-1D). She was born at full term, and her mother’s pregnancy and delivery were uneventful. She had no other medical problems. All teeth exhibited brown discoloration, and dental panoramic radiograph revealed the lack of contrast between the enamel and dentin because of the reduced mineral density of the affected enamel (Fig. 1E). Sanger sequencing of all exons and exon-intron boundaries of MMP20, KLK4, and WDR72 genes did not identify any
Ex1F Ex1R Ex2F Ex2R Ex3F Ex3R Ex4F Ex4R Ex5F Ex5R Ex6F Ex6R Ex7F Ex7R Ex8F Ex8R Ex9F Ex9R Ex10F Ex10R Ex11F Ex11R Ex12F Ex12R Ex13F Ex13R Ex14F Ex14R Ex15F Ex15R Ex16F Ex16R Ex17-1F Ex17-1R Ex17-2F Ex17-2R Ex17-3F Ex17-3R Ex17-4F Ex17-4R Ex17-5F Ex17-5R
Oligonucleotide Sequence 5′-ctcggccactgattgcac 5′-aaggaggggaaaacatctcg 5′-cagggctgttgctgacatag 5′-tggctgtaaccacccacata 5′-gaactctcagaagtcaagtgaggt 5′-agatctcagacacgccacg 5′-ctggtttggggtgtggtg 5′-ctcggtgtgacagtctttgc 5′-ttggctgtagagcgtccagt 5′-tgaggctcagagctgacaaa 5′-aaagggggacactgaggaag 5′-gctaccccaacctcttgtca 5′-gtggcctggagttaggaggt 5′-agtgccaggggcagagat 5′-gagcagctcagaaatggacc 5′-aacgattcagggaacccaac 5′-cacttctggacccctcattc 5′-ttctccctgctgtcacaaaa 5′-agggatggggtgtgatcc 5′-tctcttagggcacctgtggt 5′-tcacaaggtgaggggaagtc 5′-atcaatggcaccaggaagag 5′-tccccagggttgttgttcta 5′-acagattcgcctcctaagca 5′-ctagagtcacatcggtggca 5′-gtggttagccttgaacccag 5′-tgtttcctacgcttacagtgtctc 5′-aagtcaggcagggacgag 5′-ttccaaggaatggcactgat 5′-tagagcctctggctggaact 5′-gctgggatttctggatgga 5′-gtaagtgacgaggcggaatc 5′-tgaggatcagactgcagcac 5′-gcctatgcaggagagacctg 5′-CCTGAAGTTCCCTGTTGCAT 5′-GAAAATCACCCTCGTCCTCA 5′-TCTATCACCCAAGGGCAGTC 5′-AAGACGGGTATCCAGCAATG 5′-CATGTCTGCTGCTGGGATAC 5′-CAACAGTGGGAACAGGTGTG 5′-GAGCACAAGTTGTCTTTCCCTAA 5′-TGCGACCCTTATCCTTTTTG
Size (bp) 293 294 244 278 250 261 287 247 213 398 343 487 367 280 287 207 397 177 213 213 165
disease-causing mutation. During PCR amplifications of the SLC24A4 gene, we could not amplify the last 3 exons using the DNA sample of the proband. CNV analysis detected a homozygous deletion spanning 8 kb in the SLC24A4 gene (Fig. 2). The deleted region normally has exons 15 and 16 and most of exon 17 (NM_153646.3). The deletion was confirmed by PCR amplification through several sets of primers (Ex17-2~5). The PCR reaction of the proband DNA sample across the deletion via a primer pair that spanned the deletion (Ex14F and Ex17-5R) resulted in an amplicon of circa 5 kb. This reaction would usually result in an amplicon of around 15 kb for a DNA sample without a deletion. Sequencing of the amplified product confirmed a deletion region of 10,042 bp (Fig. 2).
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Discussion
Figure 1. Pedigree, clinical photos, and panoramic radiograph of the proband. (A) Pedigree of the family. (B-D) Frontal, maxillary, and mandibular clinical photos of the proband. Teeth have a brown discoloration and are abraded. (E) Panoramic radiograph of the proband revealed lack of contrast between the enamel and dentin due to the reduced mineral density of the affected enamel.
Figure 2. Mutational analysis. Copy number variation analysis identified a homozygous deletion in the SLC24A4 gene. Polymerase chain reaction amplifications using exon-specific primer pairs confirmed the deletion of exons 15, 16, and most of the last exon (17). Sanger sequencing of the polymerase chain reaction amplicon across the deletion revealed the specific deletion (chr14:92,957,680-92,967,722del). The locations of the primers (Ex14F and Ex17-5R) are indicated above the gene diagram. Red bars under the gene diagram indicate homologous sequences 354 bp long. Pink arrow under the gene diagram indicates the region of deletion.
SLC24A4 (NCKX4) was cloned from a human brain cDNA library through sequence blast searching with SLC24A3 (NCKX3). Based on the similarity in amino acid identity and exon boundaries with SLC24A3, it was proposed that SLC24A4 had arisen from a recent gene duplication (Li et al., 2002). SLC24A4 gene has multiple transcripts resulting from alternative splicing and use of alternative first exons. The longest isoform (encoded by NM_153646.3) has 17 exons and encodes 622 amino acid protein. SLC24A4, a member of the potassiumdependent sodium/calcium exchanger family, is a type II transmembrane protein with 11 predicted transmembrane domains and a glycosylated extracellular loop in the middle part (Parry et al., 2013). Two highly conserved and oppositely oriented regions known as alpha-1 and alpha-2 repeats form the ion-binding pockets (Visser et al., 2007). Like other potassium-dependent sodium/ calcium exchangers, SLC24A4 exchanges 4 sodium ion for 1 calcium and 1 potassium ion (Stephan et al., 2012). SLC24A4 was proposed as a novel candidate gene involved in dental enamel mineralization. By genomewide transcript profiling of the developing enamel organ, upregulation of SLC24A4 was identified and confirmed by Western blotting as well (Lacruz et al., 2012). Prominent expression of SLC24A4 was shown at the apical poles and at the lateral membrane proximal to the apical ends of ameloblasts during the mid- to late maturation stages of amelogenesis (Hu et al., 2012). The deletion identified in this study would result in a total loss of SLC24A4 because of nonsense-mediated decay. Even if the mutant transcript survived nonsensemediated decay, the mutant protein would lack the C-terminal 109 residues of the 622–amino acid wild-type protein, which would truncate the alpha-2 repeat, which forms the ion-binding pockets with the alpha-1 repeat (Parry et al., 2013). Interestingly, the sequence analysis of the deleted region identified homologous sequences in intron 14 and the 3′ UTR (Fig. 3). The homologous sequences, 354 bp long, were identical except for only 5 nucleotides (Appendix Fig.). The remaining sequences after deletion indicated that
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J Dent Res 93(4) 2014 369 Exonal Deletion of SLC24A4 the deletion occurred by homologous recombination between these 2 homologous sequences and that the intron 14 sequence survived after recombination. Blast search revealed that the homologous SLC24A4 sequences are unique and do not have any similarity with long interspersed nuclear elements (LINE) and Alu sequences. Most homologous recombinations are caused by Alumediated and rarely LINE-mediated events (Higashimoto et al., 2013). Therefore, the deletion caused by unusual homologous sequences in the same gene is quite rare, and these sequences may be remnants of gene duplication event. Our identification of the first exonal deletion in SLC24A4 gene, which would result in the loss or severe truncation of the SLC24A4 function, expands not only the list of the SLC24A4 mutations but also that of mutations caused by homologous recombination. It further provides confirmatory evidence that the function of SLC24A4 has a crucial role in the maturation stage of amelogenesis.
Acknowledgment This work was supported by grants from the Bio and Medical Technology Development Program (2013037491) and the Science Research Center grant to Bone Metabolism Research Center (2008-0062614) by the Korea Research Foundation. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
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Figure 3. Sequences of a part of SLC24A4 gene. Homologous sequences are denoted by a pink color, and deleted sequences are underlined. Exon sequences are denoted by bold characters and the numbers of the exons are shown on the left margin. Stop codon (TAG) is shown by red color in exon 17.
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