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American Journal of Medical Genetics Part A 143A:390 – 394 (2007)
Research Letter
A Novel Gln358Glu Mutation in Ectodysplasin A Associated With X-Linked Dominant Incisor Hypodontia Patrick Tarpey,1 Trevor J. Pemberton,2 David W. Stockton,4,5,6,10 Parimal Das,7 Vasiliki Ninis,7 Sarah Edkins,1 P. Andrew Futreal,1 Richard Wooster,1 Sushanth Kamath,8 Rabindra Nayak,9 Michael R. Stratton,1 and Pragna I. Patel2,3* 1
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK 2 Institute for Genetic Medicine, University of Southern California, Los Angeles, California 3 Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, California 4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 5 Department of Internal Medicine, Baylor College of Medicine, Houston, Texas 6 Department of Ophthalmology, Baylor College of Medicine, Houston, Texas 7 Department of Neurology, Baylor College of Medicine, Houston, Texas 8 Kasturba Medical College, Mangalore, India 9 Ambedkar Medical College, Kadugondanahalli, Bangalore, India 10 Departments of Pediatrics and Internal Medicine, Wayne State University School of Medicine, Detroit, Michigan Received 15 May 2006; Accepted 26 September 2006
How to cite this article: Tarpey P, Pemberton TJ, Stockton DW, Das P, Ninis V, Edkins S, Futreal PA, Wooster R, Kamath S, Nayak R, Stratton MR, Patel PI. 2007. A novel Gln358Glu mutation in ectodysplasin A associated with X-linked dominant incisor hypodontia. Am J Med Genet Part A 143A:390–394.
To the Editor:
The congenital lack of one or more permanent teeth (hypodontia), is a common anomaly [Pemberton et al., 2005]. Eighty to eighty-five percent of reported cases of hypodontia involve the agenesis of just one or two teeth [Muller et al., 1970], indicating that most affected individuals have a mild form of the disorder. The tooth most commonly missing is the third molar (or wisdom tooth), which is absent in as much as 20% of the population. Tooth agenesis involving the mandibular central and lateral permanent incisors has the lowest incidence [Pemberton et al., 2005]. Hypodontia can occur in association with other developmental anomalies (syndromic) or as an isolated condition (non-syndromic). Genes associated with several syndromic conditions that present hypodontia have been identified, including the EDA gene underlying ectodermal dysplasia [Kere et al., 1996]. Three genes underlying nonsyndromic hypodontia have been identified to date: PAX9 [Stockton et al., 2000], MSX1 [Vastardis et al., 1996], and AXIN2 [Mostowska et al., 2006]. An Indian family (DEN11) segregating an apparent X-linked dominant form of hypodontia (Fig. 1A) affecting predominantly incisor teeth (Fig. 1B,C) was ascertained (Baylor College of Medicine IRB Protocol
H8007). Thirty-seven members of the family were subjected to an intraoral examination by an orthodontist (R.N.) (Fig. 1A). The most striking feature in almost all affected males was the lack of all mandibular incisors and maxillary lateral incisors in both the primary and permanent dentition, with maxillary central incisors also missing in some cases (Fig. 1B). The absence of at least one incisor, typically of the maxillary lateral incisors, was also observed in affected females, with at least one female patient presenting as severe a dental phenotype as the affected males (Fig. 1B; V:32). Phenotypic characteristics of scalp and body hair, skin, and nails, tolerance to heat and ability to sweat were examined in eight individuals (IV:4, 7, 9, 13 and V:12, 15, 17, 18; Fig. 1A) by questionnaire. All of the latter individuals reported normal sweating and had no complaints about intolerance to heat, and their facial features, skin, and nails all appeared normal. An objective test
Patrick Tarpey and Trevor Pemberton contributed equally to this work. Grant sponsor: NIH; Grant number: DE014102; Grant sponsor: Wellcome Trust. *Correspondence to: Pragna I. Patel, Ph.D., Institute for Genetic Medicine, University of Southern California, 2250 Alcazar Street, IGM240, Los Angeles, CA 90033. E-mail:
[email protected] DOI 10.1002/ajmg.a.31567
American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a NOVEL EDA MUTATION UNDERLYING X-LINKED HYPODONTIA
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FIG. 1. A: Pedigree of family DEN11 segregating X-linked dominant incisor hypodontia. Affected males and females are indicated by filled squares and circles, respectively, and carrier females are identified by circles with a dot at their center. Affection status is depicted as used in linkage analysis, except for females who were identified as carriers by molecular analysis of the c.1072C > G mutation. The proband is identified by the arrow. Individuals for whom DNA samples were obtained have their pedigree number highlighted in gray, and those who underwent an oral examination for this study are identified by an ‘X’ below their pedigree number. Affection status in the remaining individuals was obtained from history. B: Schematic representation of congenitally missing teeth in available affected males (highlighted in gray) and carrier females of the family showing the predominantly incisor hypodontia phenotype. C: Panoramic X-ray of an adult affected male (A; V:15) showing congenital absence of all mandibular and lateral maxillary incisors, maxillary first premolars, and mandibular canines in both left and right quadrants. Arrows indicate positions of missing teeth with assignations as in (B). D: Electropherograms from an unaffected individual (not from family DEN11) (upper) and an affected individual from family DEN11 (lower; IV:9) showing the C (wild-type) to G (mutant) transversion that is predicted to result in a p.Q358E substitution in EDA. Electropherograms for the reverse sequence were reversed to align with the forward sequence using an in-house software package at the Sanger Center. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
of sweating was not feasible. Mild hypotrichosis was present in some individuals (Fig. 1A; IV:4, IV:13, V:12, and V:15). Genotypes were determined for 50 members of family DEN11 using 32 fluorescent dye-labeled oligonucleotide primers flanking short tandem repeat polymorphisms on the X chromosome selected from the ABI PRISM linkage mapping set (Applied Biosystems, Foster City, CA) as described by Stockton et al. [Stockton et al., 1998]. Linkage analysis was conducted using FASTLINK [Schaffer, 1996] and yielded a peak two-point LOD score of 3.28 (y ¼ 0) with the marker DXS1039 in Xq21.3 (Table I). An additional 13 markers were similarly genotyped and haplotypes constructed to identify four key
recombinants that allowed delineation of the critical region to a 22.7 Mb interval in Xp11.22–Xq13.2 between markers DXS8023 and DXS8079 containing 175 known and predicted genes. In order to identify the causative mutation, DNA from two affected males (IV:9 and V:15 in Fig. 1A) was included in a high-throughput automated mutation screen of genes on the X chromosome using Applied Biosystems 3730 DNA analyzers and the BigDye Terminator 3.1 fluorescent sequencing technology (Applied Biosystems). Automated comparative sequence analysis of 136 genes within the candidate interval identified 68 single nucleotide sequence variations within 48 genes. All but three of these were ruled out as a causative mutation because
American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a
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TARPEY ET AL. TABLE I. Two-Point LOD Scores Obtained With 13 X-Linked Microsatellite Markers From the Pericentromeric Region y
Marker
0.001
0.01
0.05
0.1
0.2
0.3
0.4
DXS1214 DXS1068 DXS993 DXS8080 DXS8083 DXS1065 DXS1039 DXS991 DXS1216 DXS986 DXS1196 DXS1217 DXS990
2.206 4.136 4.597 3.474 6.109 1.155 3.281 0.106 0.360 1.851 0.744 0.424 2.416
1.199 2.447 2.634 1.776 4.221 1.130 3.237 0.101 0.351 1.826 0.706 0.447 2.395
0.475 1.106 1.253 0.515 2.621 1.010 3.036 0.080 0.311 1.700 0.577 0.521 2.287
0.167 0.581 0.653 0.072 1.657 0.846 2.766 0.058 0.261 1.509 0.482 0.567 2.122
0.087 0.157 0.103 0.204 0.675 0.485 2.161 0.028 0.164 1.038 0.437 0.540 1.698
0.149 0.008 0.095 0.223 0.237 0.146 1.466 0.011 0.078 0.496 0.494 0.390 1.159
0.107 0.028 0.107 0.138 0.046 0.018 0.689 0.002 0.019 0.094 0.404 0.158 0.528
The peak two-point LOD score is shown in bold.
they were either reported in dbSNP or because they were also found in some of the 192 patients with mental retardation that were part of a parallel screen. Two of these three remaining sequence variants, c.437T > C in the inter-alpha (globulin) inhibitor H5-like (ITIH5L; UniGene Hs.454272) gene and c.40C > T in the pyrimidinergic receptor P2Y, G-protein coupled, 4 (P2RY4; UniGene Hs.11042) gene, were ruled out because they failed to segregate with the disease in family DEN11 (data not shown). The third sequence variant, a c.1072C > G nucleotide substitution in exon 8 of the EDA gene (UniGene Hs.105407) encoding ectodysplasin A, was present in individuals IV:9 and V:15 but was not present in any of the 192 individuals with mental retardation (Fig. 1D). This substitution resulted in the non-synonymous substitution of glutamic acid for glutamine at amino acid residue position 358(p.Q358E) in the EDA protein. The c.1072C > G substitution created a SmaI restriction endonuclease site which allowed examination of all 50 available members by a restrictionfragment length polymorphism assay using an amplicon containing exons 8 and 9 [Schneider et al., 2001]. The mutant G allele was present in all affected males, and in affected and obligate carrier females, but none of the unaffected individuals (data not shown). The c.1072C > G nucleotide substitution was not found in any of 67 control individuals of the same ethnic background strongly suggesting that this is the causative mutation in this family. Mutations in ectodysplasin A have been previously identified as causing X-linked hypohidrotic ectodermal dysplasia (ED1, XLHED) (OMIM 305100), a rare condition characterized by sparse hair, oligodontia or anodontia, and an inability to sweat due to the lack of eccrine and sebaceous sweat glands [Kere et al., 1996]. Facial deformities, such as frontal bossing, saddle nose, a pointed chin, a prominent supraorbital ridge with periorbital hyperpigmentation, are also observed in affected individuals. Heterozygous carriers of XLHED generally show no obvious clinical
manifestations and typically exhibit minor to moderate degrees of the phenotype. XLHED manifests principally in young children and exhibits substantial morbidity and a high rate of childhood mortality. Severe fever associated with the hypohidrosis can cause mental retardation, convulsions, and seizures. An increased risk of chest infection and atopic disease despite a normal immune system has also been reported [Clarke et al., 1987]. Recently a family with a missense mutation (p.R65G) in the juxtamembrane region of EDA where, similar to the family we report, affected members do not show other XLHED characteristics, except hypodontia, has been reported [Tao et al., 2006]. The hypodontia in the latter family involves all classes of teeth and is more severe than that observed in family DEN11. The EDA gene product is a type-II transmembrane protein with a small N-terminal intracellular domain followed by a larger C-terminal extracellular domain that contains a collagen-like repeat domain and a tumor necrosis factor (TNF) homology domain [Ezer et al., 1999]. The C-terminal extracellular TNF domain has been shown to homotrimerize [Ezer et al., 1999] and this is believed to be required for receptor interactions as other TNF proteins bind to their respective receptors at the monomer–monomer interface [Hymowitz et al., 2000]. The p.Q358E substitution observed in this family is located at the beginning of the penultimate b-strand in the TNF domain of the EDA protein [Hymowitz et al., 2003]. The glutamine is found to be completely conserved at this position in the other known EDA proteins (Fig. 2A), suggesting that it has an important function in the protein. It is located on the outer surface of the protein with its side chain occupying a space between two b-strands (Fig. 2B), and neighboring the side chains of leucine at position 354 in the preceding b-strand, and the valine and aspartic acid at positions 324 and 325, respectively, in an adjacent b-strand. Both glutamine and aspartic acid are polar amino acids (basic and acidic, respectively), and
American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a NOVEL EDA MUTATION UNDERLYING X-LINKED HYPODONTIA
FIG. 2. A: Multiple-species sequence comparison of EDA showing complete conservation of glutamine at position 358 (marked by arrows). B: Location of glutamine 358 within TNF domain of ectodysplasin A and in the quaternary structure of the EDA homotrimers (inset). (Multiple-species sequence comparison prepared in ClustalX version 1.83 [Thompson et al., 1997]. Images produced in RasMol version 2.7.3 [Sayle and Milner-White, 1995] from PDB file 1RJ7 [Hymowitz et al., 2003].) [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
valine and leucine are both neutral non-polar amino acids. We would expect the replacement of glutamine by glutamic acid to be a favored change, as the only difference is the change in charge of the terminal side chain group, aminyl to carboxyl. This is unlikely to affect the interaction of the side chain with valine-324 and leucine-354 as these are both neutral and non-polar, and therefore, such a change should not alter their reactive properties to this side chain. The only possible conflict induced by this substitution would be a change in the interaction between this side chain and that of aspartic acid-325, which now both have negatively charged side chains. This could induce a small repulsive force between the two side chains, but given their location, such a small effect would not be expected to disrupt the TNF domain. In the trimeric quaternary structure of the complexed EDA protein, the p.Q358E substitution lies on the outside of the trimer and away from the points of interaction between the individual monomers (Fig. 2B). It is, however, in a cluster of other known EDA mutations associated with XLHED (p.D298H [Bayes et al., 1998], p.G299S [Bayes et al., 1998; Monreal et al., 1998], p.A356D [Monreal et al., 1998], and p.R357P [Monreal et al., 1998]). Given their locations, it is unlikely that these mutations would
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interfere with the interactions of the monomers. The p.A356D mutation was found to cause abnormal protein folding [Schneider et al., 2001], but none of the remaining three mutations have had their associated biochemical effect reported. Other EDA mutations within the TNF domain have been reported to reduce or eliminate the interaction EDA with its target receptors [Schneider et al., 2001]. The cluster of mutations that includes the p.Q358E substitution reported here is located close to the cleft that is created by the interaction of the different monomers when in their homotrimeric state, and these mutations are all present in components of the cleft’s secondary structures. They may therefore, disrupt such an interaction in this region of the homotrimer resulting in the inability of EDA to interact with its target receptors. This leads us to hypothesize that the relatively neutral p.Q358E substitution identified here only partially disrupts the interaction of the EDA homotrimers and their target receptors, affecting their function significantly only in the dental tissues, resulting in the observed unique hypodontia phenotype rather than the full XLHED phenotype associated with other mutations in the EDA gene. ACKNOWLEDGMENTS
This work was supported by NIH grant DE014102 (P.I.P.), and by the Wellcome Trust (M.S.). Support for genotyping was provided by the Kleberg Foundation. This investigation was partly conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 (RR10600-01, CA62528-01, RR14514-01) from the NCRR, NIH. REFERENCES Bayes M, Hartung AJ, Ezer S, Pispa J, Thesleff I, Srivastava AK, Kere J. 1998. The anhidrotic ectodermal dysplasia gene (EDA) undergoes alternative splicing and encodes ectodysplasin-A with deletion mutations in collagenous repeats. Hum Mol Genet 7:1661–1669. Clarke A, Phillips DIM, Brown R, Harper PS. 1987. Clinical aspects of X-linked hypohidrotic ectodermal dysplasia. Arch Dis Child 62:989–996. Ezer S, Bayes M, Elomaa O, Schlessinger D, Kere J. 1999. Ectodysplasin is a collagenous trimeric type II membrane protein with a tumor necrosis factor-like domain and colocalizes with cytoskeletal structures at lateral and apical surfaces of cells. Hum Mol Genet 8:2079–2086. Hymowitz SG, O’Connell MP, Ultsch MH, Hurst A, Totpal K, Ashkenazi A, de Vos AM, Kelley RF. 2000. A unique zincbinding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL. Biochemistry 39:633–640. Hymowitz SG, Compaan DM, Yan M, Wallweber HJA, Dixit VM, Starovasnik MA, de Vos AM. 2003. The crystal structures of EDA-A1 and EDA-A2: Splice variants with distinct receptor specificity. Structure 11:1513–1520. Kere J, Srivastava AK, Montonen O, Zonana J, Thomas N, Ferguson B, Munoz F, Morgan D, Clarke A, Baybayan P, et al. 1996. X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein. Nat Genet 13:409–416.
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