Jonkman, MF

Pasmooij, AM

Stellingsma, C

Yuen, WY

Acta Dermato-Venereologica

English

Yuen, Wing Yan

Pasmooij, Anna M. G.

Stellingsma, Cornelis

Jonkman, Marcel F.

University of Groningen

   University of GroningenEnamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis BullosaYuen, Wing Yan; Pasmooij, Anna M. G.; Stellingsma, Cornelis; Jonkman, Marcel F.Published in:Acta dermato-venereologicaDOI:10.2340/00015555-1341IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2012Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Yuen, W. Y., Pasmooij, A. M. G., Stellingsma, C., & Jonkman, M. F. (2012). Enamel Defects in Carriers of aNovel LAMA3 Mutation Underlying Epidermolysis Bullosa. Acta dermato-venereologica, 92(6), 695-696.https://doi.org/10.2340/00015555-1341CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019Acta Derm Venereol 92695Letters to the Editor© 2012 The Authors. doi: 10.2340/00015555-1341Journal Compilation © 2012 Acta Dermato-Venereologica. ISSN 0001-5555Junctional epidermolysis bullosa, non-Herlitz (JEB-nH) is an autosomal recessive EB subtype caused by mutations in the genes LAMA3, LAMB3, LAMC2 or COL17A1. The first three genes encode for laminin α3, laminin β3, and laminin γ2, respectively, which together form the heterotrimer laminin-332 (LM-332), whereas COL17A1 encodes for the homotrimer type XVII col-lagen (Col17) (1). LM-332 and Col17 are located in the lamina basale, and are important in epidermal–dermal adhesion. JEB-nH shows blistering after minor trauma, but also extracutaneous symptoms, such as dental ab-normalities (1). Both LM-332 and Col17 are crucial in ameloblast differentiation and enamel formation (2, 3), resulting in enamel defects of the entire dentition in all patients with JEB-nH, consisting of hypoplasia, pitting, roughness, thinning or furrowing of enamel (4). In 1996, McGrath et al. (5) reported the first heterozygous carriers of a COL17A1 mutation with enamel defects. The carriers of the glycine substitution p.G627V had no signs of skin fragility, but did have extensive enamel hypoplasia and pitting, thought to be caused by the dominant-negative effect of the mutant Col17. In 2007, Murrell et al. (6) reported enamel defects in carriers of a COL17A1 null mutation, which was attributed to haploinsufficiency. Dental abnormalities have not been reported previously in carriers of mutations in the genes encoding LM-332. We report here the first enamel defects in carriers of a LAMA3 mutation. CASE REPORTThe proband was a 23-year-old woman (EB050) diagnosed with JEB-nH. Starting at the age of 2 months she showed generalized blistering and erosions that healed with atrophic scarring. She had dystrophic nails, and, occasionally, oral erosions. There was no loss of primary or secondary hair. Her entire primary and secondary dentition was affected with enamel hypoplasia and enamel pits, which were treated with direct composite (Fig. 1a, b). Her non-consanguineous parents and her brother had no signs of skin fragility. However, her 53-year-old mother and her 25-year-old brother also had enamel defects in their dentition, consisting of roughness and pits, which had led to a higher susceptibility to caries in both (Fig. 1c, d). The mother was treated with maxillary dentures and numerous restorations (Fig. 1c). In contrast, the father’s teeth were not affected. Skin biopsies in the proband and her mother were taken, and immunofluorescence antigen mapping was performed on non-lesional skin. Staining of LM-332 with monoclonal antibody GB3 was slightly reduced in both mother and daughter, while staining for Col17 with monoclonal antibodies NCC-lu-226 and 233 was normal. Genomic DNA of all family members was extracted from peripheral blood. Previous molecular analysis of COL17A1, LAMB3, and LAMC2 yielded no abnormalities in the pro-band. Screening of LAMA3 (GenBank accession number NM_000227.3) showed a compound heterozygosity for the novel missense mutation c.4484C>T, and the novel nonsense mutation c.488delG in the proband (methods available on re-quest). The deletion c.488delG in exon 5 creates a frameshift that results in a premature termination codon (p.G163DfsX30), and the mutation c.4484C>T in exon 33 results in a substitution of alanine to valine at position 1495 (p.A1495V). Proving the pathogenicity of the mutation p.A1495V, are 2 unrelated patients with JEB-nH (EB169-01 and EB173-01) in our EB cohort, who both carry the LAMA3 mutation p.A1495V. Patient EB169-01 carries it together with the nonsense mutation p.R1126X, and patient 173-01 together with the missense mutation p.R262S (unpublished). Screening of LAMA3 in the family members of the proband showed that her father was heterozygous for c.4484C>T. Her mother and brother, showed heterozygosity for c.488delG. To investigate whether the enamel defects in the heterozygous carriers of c.488delG could be caused by a dominant-negative effect of an aberrant laminin α3 chain by alternatively spliced cDNA, nested PCRs surrounding both mutations were perfor-med with cDNA isolated from skin biopsies from the proband and her mother (methods available on request). There were no alternatively spliced cDNA products visible on agarose gel (Fig. S1a, b; available from http://www.medicaljournals.se/acta/content/?doi=10.2340/00015555-1341). The PCR products were sequenced and cDNA of both alleles of the proband were present (Fig. S1c, d). The allele bearing the mutation c.488delG in the mother was reduced compared with the normal wild-type (Fig. S1c). Together with the absence of alternative spliced products on agarose gel, we hypothesize that this indicates that haplo-insufficiency of LAMA3 is the cause of enamel defects in the mother and brother of the proband.Enamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis BullosaWing Yan Yuen1, Anna M.G. Pasmooij1, Cornelis Stellingsma2 and Marcel F. Jonkman1Departments of 1Dermatology and 2Oral and Maxillofacial Surgery and Maxillofacial Prosthetics, Center for Blistering Diseases, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, NL-9700 RB Groningen, The Netherlands. E-mail: w.y.yuen@umcg.nlAccepted December 13, 2011.Fig. 1. Features of enamel defects. (a, b) The proband (c) her mother, and (d) her brother had enamel defects in their dentition. (a) Rough pitted enamel is seen on the buccal side of the mandibular teeth, and (b) on the palatinal side of the maxillar teeth in the proband. The buccal side of the maxillar teeth are treated with direct composite. (c) The enamel defects on the labial side of the mandibular teeth are no longer visible due to multiple restorations in the mother. (d) In the brother there is typical pitted enamel in the insical area of the central incisors.696 Letters to the EditorDISCUSSIONEnamel is formed in 3 stages: (i) in the presecretory stage the ameloblasts differentiate and then the lamina basale, which supports the ameloblasts, disintegrates; (ii) in the secretory stage ameloblasts secrete enamel proteins that mineralize and form enamel crystals; (iii) in the maturation stage a lamina basale is deposited, adhering ameloblasts to the underlying enamel surface by hemidesmosomes. To harden the enamel, water and organic material are removed by ameloblasts (7). LM-332 is expressed by ameloblasts in all stages of enamel formation (3). In the presecretory and maturation stage LM-332 is a part of the lamina basale. In the secretory stage LM-332 most likely plays a role in the adhesion of ameloblasts to the enamel matrix (3). In patients with JEB-nH the reduction in LM-332 causes detachment from the underlying matrix and degeneration of ameloblasts. This causes enamel defects, such as pitting (4, 8). The enamel that does form has a different chemical composi-tion, as reduction in LM-332 can result in deficiencies in the junctions of ameloblasts that are important in main-taining channels for mineral ions and cell nutrients. This may lead to defective mineral transport or compromised ameloblast metabolism (8). Teeth of patients with JEB have reduced mineral content and presence of serum albumin, which is a known inhibitor of enamel crystal growth (8). It is possible that there is no coping mecha-nism that can manage the loss of one LAMA3 allele in this complex process of enamel formation, whereas these mechanisms are available in the skin (6). However, it remains peculiar that enamel defects have not been reported previously in carriers of LAMA3, LAMB3, or LAMC2 null mutations. Furthermore, none of the carriers of null mutations in LM-332 in the Dutch EB cohort have noted particular dental problems; however, they have not been examined systematically. It is possible that not all carriers are affected; a phenomenon also seen in carriers of COL17A1 null mutations (6, 9) or the enamel defects are so discrete that they are missed. Furthermore, it is possible that other factors contribute to the enamel defects seen in our carriers, such as the rare inherited enamel defects that occur in the absence of generalized syndromes designated as amelogenesis imperfecta (10), or due to environmental factors such as febrile diseases. However, this is less likely in our patients, considering the clinical dental symptoms (7). The enamel defects seen in the carriers of LAMA3 null mutations reported here are comparable to those seen in the carriers of missense COL17A1 mutations; although the carriers of COL17A1 null mutations described by Murrell et al. also showed horizontal ridging, a feature that was not present in the carriers we have described (5, 6).In conclusion, we have reported: (i) two novel LAMA3 mutations associated with JEB-nH; and (ii) the first car-riers of LAMA3 null mutations to have enamel defects probably caused by haploinsufficiency.ACkNOWLEDGEMENTSWe are grateful to the family for participation in this study. We thank krista van Dijk-Bos, Gonnie Meijer, Miranda Nijenhuis and Janny Zuiderveen for technical assistance. We are grateful to Geert Spijker and Svetislav Zaric for their contribution to the study.We wish to thank Vlinderkind (Dutch Butterfly Child Foun-dation) and the J. P. Nater Foundation for their financial support of the study.The authors declare no conflicts of interest.REFERENCES1. Fine JD, Eady RA, Bauer EA, Bauer JW, Bruckner-Tuder-man L, Heagerty A, et al. The classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB. J Am Acad Dermatol 2008; 58: 931–950.2. Asaka T, Akiyama M, Domon T, Nishie W, Natsuga k, Fujita Y, et al. Type XVII collagen is a key player in tooth enamel formation. Am J Pathol 2009; 174: 91–100.3. Yoshiba N, Yoshiba k, Aberdam D, Meneguzzi G, Perrin-Schmitt F, Stoetzel C, et al. Expression and localization of laminin-5 subunits in the mouse incisor. Cell Tissue Res 1998; 292: 143–149.4. Wright JT, Johnson LB, Fine JD. Development defects of enamel in humans with hereditary epidermolysis bullosa. Arch Oral Biol 1993; 38: 945–955.5. McGrath JA, Gatalica B, Li k, Dunnill MG, McMillan JR, Christiano AM, et al. Compound heterozygosity for a dominant glycine substitution and a recessive internal duplication mutation in the type XVII collagen gene results in junctional epidermolysis bullosa and abnormal dentition. Am J Pathol 1996; 148: 1787–1796.6. Murrell DF, Pasmooij AM, Pas HH, Marr P, klingberg S, Pfendner E, et al. Retrospective diagnosis of fatal BP180-deficient non-Herlitz junctional epidermolysis bullosa suggested by immunofluorescence (IF) antigen-mapping of parental carriers bearing enamel defects. J Invest Dermatol 2007; 127: 1772–1775.7. Nanci ARTCA. Ten Cate’s oral histology: development, structure, and function, 7th edn. Nanci A, editor. St Louis, MO: Mosby Elsevier, 2008.8. kirkham J, Robinson C, Strafford SM, Shore RC, Bonass WA, Brookes SJ, et al. The chemical composition of tooth enamel in junctional epidermolysis bullosa. Arch Oral Biol 2000; 45: 377–386.9. Pasmooij AM, Pas HH, Jansen GH, Lemmink HH, Jonkman MF. Localized and generalized forms of blistering in junc-tional epidermolysis bullosa due to COL17A1 mutations in the Netherlands. Br J Dermatol 2007; 156: 861–870.10. Hu JC, Chun YH, Al Hazzazzi T, Simmer JP. Enamel for-mation and amelogenesis imperfecta. Cells Tissues Organs 2007; 186: 78–85.Acta Derm Venereol 92

Enamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis Bullosa

W Yuen

A Pasmooij

C Stellingsma

M Jonkman

Crossref

Acta Dermato Venereologica

PEARL (Univ. of Plymouth)

University of PlymouthPEARL https://pearl.plymouth.ac.uk01 University of Plymouth Research Outputs University of Plymouth Research Outputs2012-11-01Enamel Defects in Carriers of a NovelLAMA3 Mutation UnderlyingEpidermolysis BullosaYuen, WYhttp://hdl.handle.net/10026.1/334710.2340/00015555-1341Acta Derm VenereolMedical Journals Sweden ABAll content in PEARL is protected by copyright law. Author manuscripts are made available in accordance withpublisher policies. Please cite only the published version using the details provided on the item record ordocument. In the absence of an open licence (e.g. Creative Commons), permissions for further reuse of contentshould be sought from the publisher or author.Version 1 08.05.12  PLYMOUTH UNIVERSITY  DEPOSIT LICENCE AGREEMENT FOR RESEARCH     PEARL supports the long-term preservation of research outputs, and open access to them. 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   University of GroningenEnamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis BullosaYuen, Wing Yan; Pasmooij, Anna M. G.; Stellingsma, Cornelis; Jonkman, Marcel F.Published in:Acta dermato-venereologicaDOI:10.2340/00015555-1341IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2012Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Yuen, W. Y., Pasmooij, A. M. G., Stellingsma, C., & Jonkman, M. F. (2012). Enamel Defects in Carriers of aNovel LAMA3 Mutation Underlying Epidermolysis Bullosa. Acta dermato-venereologica, 92(6), 695-696.https://doi.org/10.2340/00015555-1341CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 05-12-2023Acta Derm Venereol 92695Letters to the Editor© 2012 The Authors. doi: 10.2340/00015555-1341Journal Compilation © 2012 Acta Dermato-Venereologica. ISSN 0001-5555Junctional epidermolysis bullosa, non-Herlitz (JEB-nH) is an autosomal recessive EB subtype caused by mutations in the genes LAMA3, LAMB3, LAMC2 or COL17A1. The first three genes encode for laminin α3, laminin β3, and laminin γ2, respectively, which together form the heterotrimer laminin-332 (LM-332), whereas COL17A1 encodes for the homotrimer type XVII col-lagen (Col17) (1). LM-332 and Col17 are located in the lamina basale, and are important in epidermal–dermal adhesion. JEB-nH shows blistering after minor trauma, but also extracutaneous symptoms, such as dental ab-normalities (1). Both LM-332 and Col17 are crucial in ameloblast differentiation and enamel formation (2, 3), resulting in enamel defects of the entire dentition in all patients with JEB-nH, consisting of hypoplasia, pitting, roughness, thinning or furrowing of enamel (4). In 1996, McGrath et al. (5) reported the first heterozygous carriers of a COL17A1 mutation with enamel defects. The carriers of the glycine substitution p.G627V had no signs of skin fragility, but did have extensive enamel hypoplasia and pitting, thought to be caused by the dominant-negative effect of the mutant Col17. In 2007, Murrell et al. (6) reported enamel defects in carriers of a COL17A1 null mutation, which was attributed to haploinsufficiency. Dental abnormalities have not been reported previously in carriers of mutations in the genes encoding LM-332. We report here the first enamel defects in carriers of a LAMA3 mutation. CASE REPORTThe proband was a 23-year-old woman (EB050) diagnosed with JEB-nH. Starting at the age of 2 months she showed generalized blistering and erosions that healed with atrophic scarring. She had dystrophic nails, and, occasionally, oral erosions. There was no loss of primary or secondary hair. Her entire primary and secondary dentition was affected with enamel hypoplasia and enamel pits, which were treated with direct composite (Fig. 1a, b). Her non-consanguineous parents and her brother had no signs of skin fragility. However, her 53-year-old mother and her 25-year-old brother also had enamel defects in their dentition, consisting of roughness and pits, which had led to a higher susceptibility to caries in both (Fig. 1c, d). The mother was treated with maxillary dentures and numerous restorations (Fig. 1c). In contrast, the father’s teeth were not affected. Skin biopsies in the proband and her mother were taken, and immunofluorescence antigen mapping was performed on non-lesional skin. Staining of LM-332 with monoclonal antibody GB3 was slightly reduced in both mother and daughter, while staining for Col17 with monoclonal antibodies NCC-lu-226 and 233 was normal. Genomic DNA of all family members was extracted from peripheral blood. Previous molecular analysis of COL17A1, LAMB3, and LAMC2 yielded no abnormalities in the pro-band. Screening of LAMA3 (GenBank accession number NM_000227.3) showed a compound heterozygosity for the novel missense mutation c.4484C>T, and the novel nonsense mutation c.488delG in the proband (methods available on re-quest). The deletion c.488delG in exon 5 creates a frameshift that results in a premature termination codon (p.G163DfsX30), and the mutation c.4484C>T in exon 33 results in a substitution of alanine to valine at position 1495 (p.A1495V). Proving the pathogenicity of the mutation p.A1495V, are 2 unrelated patients with JEB-nH (EB169-01 and EB173-01) in our EB cohort, who both carry the LAMA3 mutation p.A1495V. Patient EB169-01 carries it together with the nonsense mutation p.R1126X, and patient 173-01 together with the missense mutation p.R262S (unpublished). Screening of LAMA3 in the family members of the proband showed that her father was heterozygous for c.4484C>T. Her mother and brother, showed heterozygosity for c.488delG. To investigate whether the enamel defects in the heterozygous carriers of c.488delG could be caused by a dominant-negative effect of an aberrant laminin α3 chain by alternatively spliced cDNA, nested PCRs surrounding both mutations were perfor-med with cDNA isolated from skin biopsies from the proband and her mother (methods available on request). There were no alternatively spliced cDNA products visible on agarose gel (Fig. S1a, b; available from http://www.medicaljournals.se/acta/content/?doi=10.2340/00015555-1341). The PCR products were sequenced and cDNA of both alleles of the proband were present (Fig. S1c, d). The allele bearing the mutation c.488delG in the mother was reduced compared with the normal wild-type (Fig. S1c). Together with the absence of alternative spliced products on agarose gel, we hypothesize that this indicates that haplo-insufficiency of LAMA3 is the cause of enamel defects in the mother and brother of the proband.Enamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis BullosaWing Yan Yuen1, Anna M.G. Pasmooij1, Cornelis Stellingsma2 and Marcel F. Jonkman1Departments of 1Dermatology and 2Oral and Maxillofacial Surgery and Maxillofacial Prosthetics, Center for Blistering Diseases, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, NL-9700 RB Groningen, The Netherlands. E-mail: w.y.yuen@umcg.nlAccepted December 13, 2011.Fig. 1. Features of enamel defects. (a, b) The proband (c) her mother, and (d) her brother had enamel defects in their dentition. (a) Rough pitted enamel is seen on the buccal side of the mandibular teeth, and (b) on the palatinal side of the maxillar teeth in the proband. The buccal side of the maxillar teeth are treated with direct composite. (c) The enamel defects on the labial side of the mandibular teeth are no longer visible due to multiple restorations in the mother. (d) In the brother there is typical pitted enamel in the insical area of the central incisors.696 Letters to the EditorDISCUSSIONEnamel is formed in 3 stages: (i) in the presecretory stage the ameloblasts differentiate and then the lamina basale, which supports the ameloblasts, disintegrates; (ii) in the secretory stage ameloblasts secrete enamel proteins that mineralize and form enamel crystals; (iii) in the maturation stage a lamina basale is deposited, adhering ameloblasts to the underlying enamel surface by hemidesmosomes. To harden the enamel, water and organic material are removed by ameloblasts (7). LM-332 is expressed by ameloblasts in all stages of enamel formation (3). In the presecretory and maturation stage LM-332 is a part of the lamina basale. In the secretory stage LM-332 most likely plays a role in the adhesion of ameloblasts to the enamel matrix (3). In patients with JEB-nH the reduction in LM-332 causes detachment from the underlying matrix and degeneration of ameloblasts. This causes enamel defects, such as pitting (4, 8). The enamel that does form has a different chemical composi-tion, as reduction in LM-332 can result in deficiencies in the junctions of ameloblasts that are important in main-taining channels for mineral ions and cell nutrients. This may lead to defective mineral transport or compromised ameloblast metabolism (8). Teeth of patients with JEB have reduced mineral content and presence of serum albumin, which is a known inhibitor of enamel crystal growth (8). It is possible that there is no coping mecha-nism that can manage the loss of one LAMA3 allele in this complex process of enamel formation, whereas these mechanisms are available in the skin (6). However, it remains peculiar that enamel defects have not been reported previously in carriers of LAMA3, LAMB3, or LAMC2 null mutations. Furthermore, none of the carriers of null mutations in LM-332 in the Dutch EB cohort have noted particular dental problems; however, they have not been examined systematically. It is possible that not all carriers are affected; a phenomenon also seen in carriers of COL17A1 null mutations (6, 9) or the enamel defects are so discrete that they are missed. Furthermore, it is possible that other factors contribute to the enamel defects seen in our carriers, such as the rare inherited enamel defects that occur in the absence of generalized syndromes designated as amelogenesis imperfecta (10), or due to environmental factors such as febrile diseases. However, this is less likely in our patients, considering the clinical dental symptoms (7). The enamel defects seen in the carriers of LAMA3 null mutations reported here are comparable to those seen in the carriers of missense COL17A1 mutations; although the carriers of COL17A1 null mutations described by Murrell et al. also showed horizontal ridging, a feature that was not present in the carriers we have described (5, 6).In conclusion, we have reported: (i) two novel LAMA3 mutations associated with JEB-nH; and (ii) the first car-riers of LAMA3 null mutations to have enamel defects probably caused by haploinsufficiency.ACkNOWLEDGEMENTSWe are grateful to the family for participation in this study. We thank krista van Dijk-Bos, Gonnie Meijer, Miranda Nijenhuis and Janny Zuiderveen for technical assistance. We are grateful to Geert Spijker and Svetislav Zaric for their contribution to the study.We wish to thank Vlinderkind (Dutch Butterfly Child Foun-dation) and the J. P. Nater Foundation for their financial support of the study.The authors declare no conflicts of interest.REFERENCES1. Fine JD, Eady RA, Bauer EA, Bauer JW, Bruckner-Tuder-man L, Heagerty A, et al. The classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB. J Am Acad Dermatol 2008; 58: 931–950.2. Asaka T, Akiyama M, Domon T, Nishie W, Natsuga k, Fujita Y, et al. Type XVII collagen is a key player in tooth enamel formation. Am J Pathol 2009; 174: 91–100.3. Yoshiba N, Yoshiba k, Aberdam D, Meneguzzi G, Perrin-Schmitt F, Stoetzel C, et al. Expression and localization of laminin-5 subunits in the mouse incisor. Cell Tissue Res 1998; 292: 143–149.4. Wright JT, Johnson LB, Fine JD. Development defects of enamel in humans with hereditary epidermolysis bullosa. Arch Oral Biol 1993; 38: 945–955.5. McGrath JA, Gatalica B, Li k, Dunnill MG, McMillan JR, Christiano AM, et al. Compound heterozygosity for a dominant glycine substitution and a recessive internal duplication mutation in the type XVII collagen gene results in junctional epidermolysis bullosa and abnormal dentition. Am J Pathol 1996; 148: 1787–1796.6. Murrell DF, Pasmooij AM, Pas HH, Marr P, klingberg S, Pfendner E, et al. Retrospective diagnosis of fatal BP180-deficient non-Herlitz junctional epidermolysis bullosa suggested by immunofluorescence (IF) antigen-mapping of parental carriers bearing enamel defects. J Invest Dermatol 2007; 127: 1772–1775.7. Nanci ARTCA. Ten Cate’s oral histology: development, structure, and function, 7th edn. Nanci A, editor. St Louis, MO: Mosby Elsevier, 2008.8. kirkham J, Robinson C, Strafford SM, Shore RC, Bonass WA, Brookes SJ, et al. The chemical composition of tooth enamel in junctional epidermolysis bullosa. Arch Oral Biol 2000; 45: 377–386.9. Pasmooij AM, Pas HH, Jansen GH, Lemmink HH, Jonkman MF. Localized and generalized forms of blistering in junc-tional epidermolysis bullosa due to COL17A1 mutations in the Netherlands. Br J Dermatol 2007; 156: 861–870.10. Hu JC, Chun YH, Al Hazzazzi T, Simmer JP. Enamel for-mation and amelogenesis imperfecta. Cells Tissues Organs 2007; 186: 78–85.Acta Derm Venereol 92

University of GroningenEnamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis BullosaYuen, Wing Yan; Pasmooij, Anna M. G.; Stellingsma, Cornelis; Jonkman, Marcel F.Published in:Acta dermato-venereologicaDOI:10.2340/00015555-1341IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2012Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Yuen, W. Y., Pasmooij, A. M. G., Stellingsma, C., & Jonkman, M. F. (2012). Enamel Defects in Carriers of aNovel LAMA3 Mutation Underlying Epidermolysis Bullosa. Acta dermato-venereologica, 92(6), 695-696.https://doi.org/10.2340/00015555-1341CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 30-12-2025Acta Derm Venereol 92695Letters to the Editor© 2012 The Authors. doi: 10.2340/00015555-1341Journal Compilation © 2012 Acta Dermato-Venereologica. ISSN 0001-5555Junctional epidermolysis bullosa, non-Herlitz (JEB-nH) is an autosomal recessive EB subtype caused by mutations in the genes LAMA3, LAMB3, LAMC2 or COL17A1. The first three genes encode for laminin α3, laminin β3, and laminin γ2, respectively, which together form the heterotrimer laminin-332 (LM-332), whereas COL17A1 encodes for the homotrimer type XVII col-lagen (Col17) (1). LM-332 and Col17 are located in the lamina basale, and are important in epidermal–dermal adhesion. JEB-nH shows blistering after minor trauma, but also extracutaneous symptoms, such as dental ab-normalities (1). Both LM-332 and Col17 are crucial in ameloblast differentiation and enamel formation (2, 3), resulting in enamel defects of the entire dentition in all patients with JEB-nH, consisting of hypoplasia, pitting, roughness, thinning or furrowing of enamel (4). In 1996, McGrath et al. (5) reported the first heterozygous carriers of a COL17A1 mutation with enamel defects. The carriers of the glycine substitution p.G627V had no signs of skin fragility, but did have extensive enamel hypoplasia and pitting, thought to be caused by the dominant-negative effect of the mutant Col17. In 2007, Murrell et al. (6) reported enamel defects in carriers of a COL17A1 null mutation, which was attributed to haploinsufficiency. Dental abnormalities have not been reported previously in carriers of mutations in the genes encoding LM-332. We report here the first enamel defects in carriers of a LAMA3 mutation. CASE REPORTThe proband was a 23-year-old woman (EB050) diagnosed with JEB-nH. Starting at the age of 2 months she showed generalized blistering and erosions that healed with atrophic scarring. She had dystrophic nails, and, occasionally, oral erosions. There was no loss of primary or secondary hair. Her entire primary and secondary dentition was affected with enamel hypoplasia and enamel pits, which were treated with direct composite (Fig. 1a, b). Her non-consanguineous parents and her brother had no signs of skin fragility. However, her 53-year-old mother and her 25-year-old brother also had enamel defects in their dentition, consisting of roughness and pits, which had led to a higher susceptibility to caries in both (Fig. 1c, d). The mother was treated with maxillary dentures and numerous restorations (Fig. 1c). In contrast, the father’s teeth were not affected. Skin biopsies in the proband and her mother were taken, and immunofluorescence antigen mapping was performed on non-lesional skin. Staining of LM-332 with monoclonal antibody GB3 was slightly reduced in both mother and daughter, while staining for Col17 with monoclonal antibodies NCC-lu-226 and 233 was normal. Genomic DNA of all family members was extracted from peripheral blood. Previous molecular analysis of COL17A1, LAMB3, and LAMC2 yielded no abnormalities in the pro-band. Screening of LAMA3 (GenBank accession number NM_000227.3) showed a compound heterozygosity for the novel missense mutation c.4484C>T, and the novel nonsense mutation c.488delG in the proband (methods available on re-quest). The deletion c.488delG in exon 5 creates a frameshift that results in a premature termination codon (p.G163DfsX30), and the mutation c.4484C>T in exon 33 results in a substitution of alanine to valine at position 1495 (p.A1495V). Proving the pathogenicity of the mutation p.A1495V, are 2 unrelated patients with JEB-nH (EB169-01 and EB173-01) in our EB cohort, who both carry the LAMA3 mutation p.A1495V. Patient EB169-01 carries it together with the nonsense mutation p.R1126X, and patient 173-01 together with the missense mutation p.R262S (unpublished). Screening of LAMA3 in the family members of the proband showed that her father was heterozygous for c.4484C>T. Her mother and brother, showed heterozygosity for c.488delG. To investigate whether the enamel defects in the heterozygous carriers of c.488delG could be caused by a dominant-negative effect of an aberrant laminin α3 chain by alternatively spliced cDNA, nested PCRs surrounding both mutations were perfor-med with cDNA isolated from skin biopsies from the proband and her mother (methods available on request). There were no alternatively spliced cDNA products visible on agarose gel (Fig. S1a, b; available from http://www.medicaljournals.se/acta/content/?doi=10.2340/00015555-1341). The PCR products were sequenced and cDNA of both alleles of the proband were present (Fig. S1c, d). The allele bearing the mutation c.488delG in the mother was reduced compared with the normal wild-type (Fig. S1c). Together with the absence of alternative spliced products on agarose gel, we hypothesize that this indicates that haplo-insufficiency of LAMA3 is the cause of enamel defects in the mother and brother of the proband.Enamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis BullosaWing Yan Yuen1, Anna M.G. Pasmooij1, Cornelis Stellingsma2 and Marcel F. Jonkman1Departments of 1Dermatology and 2Oral and Maxillofacial Surgery and Maxillofacial Prosthetics, Center for Blistering Diseases, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, NL-9700 RB Groningen, The Netherlands. E-mail: w.y.yuen@umcg.nlAccepted December 13, 2011.Fig. 1. Features of enamel defects. (a, b) The proband (c) her mother, and (d) her brother had enamel defects in their dentition. (a) Rough pitted enamel is seen on the buccal side of the mandibular teeth, and (b) on the palatinal side of the maxillar teeth in the proband. The buccal side of the maxillar teeth are treated with direct composite. (c) The enamel defects on the labial side of the mandibular teeth are no longer visible due to multiple restorations in the mother. (d) In the brother there is typical pitted enamel in the insical area of the central incisors.696 Letters to the EditorDISCUSSIONEnamel is formed in 3 stages: (i) in the presecretory stage the ameloblasts differentiate and then the lamina basale, which supports the ameloblasts, disintegrates; (ii) in the secretory stage ameloblasts secrete enamel proteins that mineralize and form enamel crystals; (iii) in the maturation stage a lamina basale is deposited, adhering ameloblasts to the underlying enamel surface by hemidesmosomes. To harden the enamel, water and organic material are removed by ameloblasts (7). LM-332 is expressed by ameloblasts in all stages of enamel formation (3). In the presecretory and maturation stage LM-332 is a part of the lamina basale. In the secretory stage LM-332 most likely plays a role in the adhesion of ameloblasts to the enamel matrix (3). In patients with JEB-nH the reduction in LM-332 causes detachment from the underlying matrix and degeneration of ameloblasts. This causes enamel defects, such as pitting (4, 8). The enamel that does form has a different chemical composi-tion, as reduction in LM-332 can result in deficiencies in the junctions of ameloblasts that are important in main-taining channels for mineral ions and cell nutrients. This may lead to defective mineral transport or compromised ameloblast metabolism (8). Teeth of patients with JEB have reduced mineral content and presence of serum albumin, which is a known inhibitor of enamel crystal growth (8). It is possible that there is no coping mecha-nism that can manage the loss of one LAMA3 allele in this complex process of enamel formation, whereas these mechanisms are available in the skin (6). However, it remains peculiar that enamel defects have not been reported previously in carriers of LAMA3, LAMB3, or LAMC2 null mutations. Furthermore, none of the carriers of null mutations in LM-332 in the Dutch EB cohort have noted particular dental problems; however, they have not been examined systematically. It is possible that not all carriers are affected; a phenomenon also seen in carriers of COL17A1 null mutations (6, 9) or the enamel defects are so discrete that they are missed. Furthermore, it is possible that other factors contribute to the enamel defects seen in our carriers, such as the rare inherited enamel defects that occur in the absence of generalized syndromes designated as amelogenesis imperfecta (10), or due to environmental factors such as febrile diseases. However, this is less likely in our patients, considering the clinical dental symptoms (7). The enamel defects seen in the carriers of LAMA3 null mutations reported here are comparable to those seen in the carriers of missense COL17A1 mutations; although the carriers of COL17A1 null mutations described by Murrell et al. also showed horizontal ridging, a feature that was not present in the carriers we have described (5, 6).In conclusion, we have reported: (i) two novel LAMA3 mutations associated with JEB-nH; and (ii) the first car-riers of LAMA3 null mutations to have enamel defects probably caused by haploinsufficiency.ACkNOWLEDGEMENTSWe are grateful to the family for participation in this study. We thank krista van Dijk-Bos, Gonnie Meijer, Miranda Nijenhuis and Janny Zuiderveen for technical assistance. We are grateful to Geert Spijker and Svetislav Zaric for their contribution to the study.We wish to thank Vlinderkind (Dutch Butterfly Child Foun-dation) and the J. P. Nater Foundation for their financial support of the study.The authors declare no conflicts of interest.REFERENCES1. Fine JD, Eady RA, Bauer EA, Bauer JW, Bruckner-Tuder-man L, Heagerty A, et al. The classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB. J Am Acad Dermatol 2008; 58: 931–950.2. Asaka T, Akiyama M, Domon T, Nishie W, Natsuga k, Fujita Y, et al. Type XVII collagen is a key player in tooth enamel formation. Am J Pathol 2009; 174: 91–100.3. Yoshiba N, Yoshiba k, Aberdam D, Meneguzzi G, Perrin-Schmitt F, Stoetzel C, et al. Expression and localization of laminin-5 subunits in the mouse incisor. Cell Tissue Res 1998; 292: 143–149.4. Wright JT, Johnson LB, Fine JD. Development defects of enamel in humans with hereditary epidermolysis bullosa. Arch Oral Biol 1993; 38: 945–955.5. McGrath JA, Gatalica B, Li k, Dunnill MG, McMillan JR, Christiano AM, et al. Compound heterozygosity for a dominant glycine substitution and a recessive internal duplication mutation in the type XVII collagen gene results in junctional epidermolysis bullosa and abnormal dentition. Am J Pathol 1996; 148: 1787–1796.6. Murrell DF, Pasmooij AM, Pas HH, Marr P, klingberg S, Pfendner E, et al. Retrospective diagnosis of fatal BP180-deficient non-Herlitz junctional epidermolysis bullosa suggested by immunofluorescence (IF) antigen-mapping of parental carriers bearing enamel defects. J Invest Dermatol 2007; 127: 1772–1775.7. Nanci ARTCA. Ten Cate’s oral histology: development, structure, and function, 7th edn. Nanci A, editor. St Louis, MO: Mosby Elsevier, 2008.8. kirkham J, Robinson C, Strafford SM, Shore RC, Bonass WA, Brookes SJ, et al. The chemical composition of tooth enamel in junctional epidermolysis bullosa. Arch Oral Biol 2000; 45: 377–386.9. Pasmooij AM, Pas HH, Jansen GH, Lemmink HH, Jonkman MF. Localized and generalized forms of blistering in junc-tional epidermolysis bullosa due to COL17A1 mutations in the Netherlands. Br J Dermatol 2007; 156: 861–870.10. Hu JC, Chun YH, Al Hazzazzi T, Simmer JP. Enamel for-mation and amelogenesis imperfecta. Cells Tissues Organs 2007; 186: 78–85.Acta Derm Venereol 92

NARCIS 

University of Groningen Research Database

  
 University of Groningen
Enamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis Bullosa
Yuen, Wing Yan; Pasmooij, Anna; Stellingsma, Cornelis; Jonkman, Marcel F.
Published in:
Acta Dermato-Venereologica
DOI:
10.2340/00015555-1341
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2012
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Yuen, W. Y., Pasmooij, A. M. G., Stellingsma, C., & Jonkman, M. F. (2012). Enamel Defects in Carriers of a
Novel LAMA3 Mutation Underlying Epidermolysis Bullosa. Acta Dermato-Venereologica, 92(6), 695-696.
DOI: 10.2340/00015555-1341
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
Acta Derm Venereol 92
695Letters to the Editor
© 2012 The Authors. doi: 10.2340/00015555-1341
Journal Compilation © 2012 Acta Dermato-Venereologica. ISSN 0001-5555
Junctional epidermolysis bullosa, non-Herlitz (JEB-
nH) is an autosomal recessive EB subtype caused by 
mutations in the genes LAMA3, LAMB3, LAMC2 or 
COL17A1. The first three genes encode for laminin α3, 
laminin β3, and laminin γ2, respectively, which together 
form the heterotrimer laminin-332 (LM-332), whereas 
COL17A1 encodes for the homotrimer type XVII col-
lagen (Col17) (1). LM-332 and Col17 are located in the 
lamina basale, and are important in epidermal–dermal 
adhesion. JEB-nH shows blistering after minor trauma, 
but also extracutaneous symptoms, such as dental ab-
normalities (1). Both LM-332 and Col17 are crucial in 
ameloblast differentiation and enamel formation (2, 3), 
resulting in enamel defects of the entire dentition in all 
patients with JEB-nH, consisting of hypoplasia, pitting, 
roughness, thinning or furrowing of enamel (4). In 1996, 
McGrath et al. (5) reported the first heterozygous carriers 
of a COL17A1 mutation with enamel defects. The carriers 
of the glycine substitution p.G627V had no signs of skin 
fragility, but did have extensive enamel hypoplasia and 
pitting, thought to be caused by the dominant-negative 
effect of the mutant Col17. In 2007, Murrell et al. (6) 
reported enamel defects in carriers of a COL17A1 null 
mutation, which was attributed to haploinsufficiency. 
Dental abnormalities have not been reported previously 
in carriers of mutations in the genes encoding LM-332. 
We report here the first enamel defects in carriers of a 
LAMA3 mutation. 
CASE REPORT
The proband was a 23-year-old woman (EB050) diagnosed with 
JEB-nH. Starting at the age of 2 months she showed generalized 
blistering and erosions that healed with atrophic scarring. She 
had dystrophic nails, and, occasionally, oral erosions. There 
was no loss of primary or secondary hair. Her entire primary 
and secondary dentition was affected with enamel hypoplasia 
and enamel pits, which were treated with direct composite 
(Fig. 1a, b). Her non-consanguineous parents and her brother 
had no signs of skin fragility. However, her 53-year-old mother 
and her 25-year-old brother also had enamel defects in their 
dentition, consisting of roughness and pits, which had led to a 
higher susceptibility to caries in both (Fig. 1c, d). The mother 
was treated with maxillary dentures and numerous restorations 
(Fig. 1c). In contrast, the father’s teeth were not affected. 
Skin biopsies in the proband and her mother were taken, and 
immunofluorescence antigen mapping was performed on non-
lesional skin. Staining of LM-332 with monoclonal antibody 
GB3 was slightly reduced in both mother and daughter, while 
staining for Col17 with monoclonal antibodies NCC-lu-226 
and 233 was normal. 
Genomic DNA of all family members was extracted from 
peripheral blood. Previous molecular analysis of COL17A1, 
LAMB3, and LAMC2 yielded no abnormalities in the pro-
band. Screening of LAMA3 (GenBank accession number 
NM_000227.3) showed a compound heterozygosity for the 
novel missense mutation c.4484C>T, and the novel nonsense 
mutation c.488delG in the proband (methods available on re-
quest). The deletion c.488delG in exon 5 creates a frameshift 
that results in a premature termination codon (p.G163DfsX30), 
and the mutation c.4484C>T in exon 33 results in a substitution 
of alanine to valine at position 1495 (p.A1495V). Proving the 
pathogenicity of the mutation p.A1495V, are 2 unrelated patients 
with JEB-nH (EB169-01 and EB173-01) in our EB cohort, who 
both carry the LAMA3 mutation p.A1495V. Patient EB169-01 
carries it together with the nonsense mutation p.R1126X, and 
patient 173-01 together with the missense mutation p.R262S 
(unpublished). 
Screening of LAMA3 in the family members of the proband 
showed that her father was heterozygous for c.4484C>T. Her 
mother and brother, showed heterozygosity for c.488delG. To 
investigate whether the enamel defects in the heterozygous 
carriers of c.488delG could be caused by a dominant-negative 
effect of an aberrant laminin α3 chain by alternatively spliced 
cDNA, nested PCRs surrounding both mutations were perfor-
med with cDNA isolated from skin biopsies from the proband 
and her mother (methods available on request). There were no 
alternatively spliced cDNA products visible on agarose gel (Fig. 
S1a, b; available from http://www.medicaljournals.se/acta/co
ntent/?doi=10.2340/00015555-1341). The PCR products were 
sequenced and cDNA of both alleles of the proband were present 
(Fig. S1c, d). The allele bearing the mutation c.488delG in the 
mother was reduced compared with the normal wild-type (Fig. 
S1c). Together with the absence of alternative spliced products 
on agarose gel, we hypothesize that this indicates that haplo-
insufficiency of LAMA3 is the cause of enamel defects in the 
mother and brother of the proband.
Enamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis Bullosa
Wing Yan Yuen1, Anna M.G. Pasmooij1, Cornelis Stellingsma2 and Marcel F. Jonkman1
Departments of 1Dermatology and 2Oral and Maxillofacial Surgery and Maxillofacial Prosthetics, Center for Blistering Diseases, University of Groningen, 
University Medical Center Groningen, Hanzeplein 1, PO Box 30001, NL-9700 RB Groningen, The Netherlands. E-mail: w.y.yuen@umcg.nl
Accepted December 13, 2011.
Fig. 1. Features of enamel defects. (a, b) The proband (c) her mother, and (d) 
her brother had enamel defects in their dentition. (a) Rough pitted enamel is 
seen on the buccal side of the mandibular teeth, and (b) on the palatinal side 
of the maxillar teeth in the proband. The buccal side of the maxillar teeth 
are treated with direct composite. (c) The enamel defects on the labial side 
of the mandibular teeth are no longer visible due to multiple restorations in 
the mother. (d) In the brother there is typical pitted enamel in the insical area 
of the central incisors.
696 Letters to the Editor
DISCUSSION
Enamel is formed in 3 stages: (i) in the presecretory 
stage the ameloblasts differentiate and then the lamina 
basale, which supports the ameloblasts, disintegrates; 
(ii) in the secretory stage ameloblasts secrete enamel 
proteins that mineralize and form enamel crystals; (iii) 
in the maturation stage a lamina basale is deposited, 
adhering ameloblasts to the underlying enamel surface 
by hemidesmosomes. To harden the enamel, water and 
organic material are removed by ameloblasts (7). LM-
332 is expressed by ameloblasts in all stages of enamel 
formation (3). In the presecretory and maturation stage 
LM-332 is a part of the lamina basale. In the secretory 
stage LM-332 most likely plays a role in the adhesion 
of ameloblasts to the enamel matrix (3). In patients with 
JEB-nH the reduction in LM-332 causes detachment from 
the underlying matrix and degeneration of ameloblasts. 
This causes enamel defects, such as pitting (4, 8). The 
enamel that does form has a different chemical composi-
tion, as reduction in LM-332 can result in deficiencies in 
the junctions of ameloblasts that are important in main-
taining channels for mineral ions and cell nutrients. This 
may lead to defective mineral transport or compromised 
ameloblast metabolism (8). Teeth of patients with JEB 
have reduced mineral content and presence of serum 
albumin, which is a known inhibitor of enamel crystal 
growth (8). It is possible that there is no coping mecha-
nism that can manage the loss of one LAMA3 allele 
in this complex process of enamel formation, whereas 
these mechanisms are available in the skin (6). However, 
it remains peculiar that enamel defects have not been 
reported previously in carriers of LAMA3, LAMB3, or 
LAMC2 null mutations. Furthermore, none of the carriers 
of null mutations in LM-332 in the Dutch EB cohort 
have noted particular dental problems; however, they 
have not been examined systematically. It is possible that 
not all carriers are affected; a phenomenon also seen in 
carriers of COL17A1 null mutations (6, 9) or the enamel 
defects are so discrete that they are missed. Furthermore, 
it is possible that other factors contribute to the enamel 
defects seen in our carriers, such as the rare inherited 
enamel defects that occur in the absence of generalized 
syndromes designated as amelogenesis imperfecta (10), 
or due to environmental factors such as febrile diseases. 
However, this is less likely in our patients, considering 
the clinical dental symptoms (7). 
The enamel defects seen in the carriers of LAMA3 null 
mutations reported here are comparable to those seen in 
the carriers of missense COL17A1 mutations; although the 
carriers of COL17A1 null mutations described by Murrell 
et al. also showed horizontal ridging, a feature that was 
not present in the carriers we have described (5, 6).
In conclusion, we have reported: (i) two novel LAMA3 
mutations associated with JEB-nH; and (ii) the first car-
riers of LAMA3 null mutations to have enamel defects 
probably caused by haploinsufficiency.
ACkNOWLEDGEMENTS
We are grateful to the family for participation in this study. We 
thank krista van Dijk-Bos, Gonnie Meijer, Miranda Nijenhuis 
and Janny Zuiderveen for technical assistance. We are grateful 
to Geert Spijker and Svetislav Zaric for their contribution to 
the study.
We wish to thank Vlinderkind (Dutch Butterfly Child Foun-
dation) and the J. P. Nater Foundation for their financial support 
of the study.
The authors declare no conflicts of interest.
REFERENCES
1. Fine JD, Eady RA, Bauer EA, Bauer JW, Bruckner-Tuder-
man L, Heagerty A, et al. The classification of inherited 
epidermolysis bullosa (EB): report of the Third International 
Consensus Meeting on Diagnosis and Classification of EB. 
J Am Acad Dermatol 2008; 58: 931–950.
2. Asaka T, Akiyama M, Domon T, Nishie W, Natsuga k, 
Fujita Y, et al. Type XVII collagen is a key player in tooth 
enamel formation. Am J Pathol 2009; 174: 91–100.
3. Yoshiba N, Yoshiba k, Aberdam D, Meneguzzi G, Perrin-
Schmitt F, Stoetzel C, et al. Expression and localization of 
laminin-5 subunits in the mouse incisor. Cell Tissue Res 
1998; 292: 143–149.
4. Wright JT, Johnson LB, Fine JD. Development defects of 
enamel in humans with hereditary epidermolysis bullosa. 
Arch Oral Biol 1993; 38: 945–955.
5. McGrath JA, Gatalica B, Li k, Dunnill MG, McMillan 
JR, Christiano AM, et al. Compound heterozygosity for 
a dominant glycine substitution and a recessive internal 
duplication mutation in the type XVII collagen gene results 
in junctional epidermolysis bullosa and abnormal dentition. 
Am J Pathol 1996; 148: 1787–1796.
6. Murrell DF, Pasmooij AM, Pas HH, Marr P, klingberg S, 
Pfendner E, et al. Retrospective diagnosis of fatal BP180-
deficient non-Herlitz junctional epidermolysis bullosa 
suggested by immunofluorescence (IF) antigen-mapping of 
parental carriers bearing enamel defects. J Invest Dermatol 
2007; 127: 1772–1775.
7. Nanci ARTCA. Ten Cate’s oral histology: development, 
structure, and function, 7th edn. Nanci A, editor. St Louis, 
MO: Mosby Elsevier, 2008.
8. kirkham J, Robinson C, Strafford SM, Shore RC, Bonass 
WA, Brookes SJ, et al. The chemical composition of tooth 
enamel in junctional epidermolysis bullosa. Arch Oral Biol 
2000; 45: 377–386.
9. Pasmooij AM, Pas HH, Jansen GH, Lemmink HH, Jonkman 
MF. Localized and generalized forms of blistering in junc-
tional epidermolysis bullosa due to COL17A1 mutations in 
the Netherlands. Br J Dermatol 2007; 156: 861–870.
10. Hu JC, Chun YH, Al Hazzazzi T, Simmer JP. Enamel for-
mation and amelogenesis imperfecta. Cells Tissues Organs 
2007; 186: 78–85.
Acta Derm Venereol 92


   University of GroningenEnamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis BullosaYuen, Wing Yan; Pasmooij, Anna M. G.; Stellingsma, Cornelis; Jonkman, Marcel F.Published in:Acta dermato-venereologicaDOI:10.2340/00015555-1341IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2012Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Yuen, W. Y., Pasmooij, A. M. G., Stellingsma, C., & Jonkman, M. F. (2012). Enamel Defects in Carriers of aNovel LAMA3 Mutation Underlying Epidermolysis Bullosa. Acta dermato-venereologica, 92(6), 695-696.https://doi.org/10.2340/00015555-1341CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 26-04-2023Acta Derm Venereol 92695Letters to the Editor© 2012 The Authors. doi: 10.2340/00015555-1341Journal Compilation © 2012 Acta Dermato-Venereologica. ISSN 0001-5555Junctional epidermolysis bullosa, non-Herlitz (JEB-nH) is an autosomal recessive EB subtype caused by mutations in the genes LAMA3, LAMB3, LAMC2 or COL17A1. The first three genes encode for laminin α3, laminin β3, and laminin γ2, respectively, which together form the heterotrimer laminin-332 (LM-332), whereas COL17A1 encodes for the homotrimer type XVII col-lagen (Col17) (1). LM-332 and Col17 are located in the lamina basale, and are important in epidermal–dermal adhesion. JEB-nH shows blistering after minor trauma, but also extracutaneous symptoms, such as dental ab-normalities (1). Both LM-332 and Col17 are crucial in ameloblast differentiation and enamel formation (2, 3), resulting in enamel defects of the entire dentition in all patients with JEB-nH, consisting of hypoplasia, pitting, roughness, thinning or furrowing of enamel (4). In 1996, McGrath et al. (5) reported the first heterozygous carriers of a COL17A1 mutation with enamel defects. The carriers of the glycine substitution p.G627V had no signs of skin fragility, but did have extensive enamel hypoplasia and pitting, thought to be caused by the dominant-negative effect of the mutant Col17. In 2007, Murrell et al. (6) reported enamel defects in carriers of a COL17A1 null mutation, which was attributed to haploinsufficiency. Dental abnormalities have not been reported previously in carriers of mutations in the genes encoding LM-332. We report here the first enamel defects in carriers of a LAMA3 mutation. CASE REPORTThe proband was a 23-year-old woman (EB050) diagnosed with JEB-nH. Starting at the age of 2 months she showed generalized blistering and erosions that healed with atrophic scarring. She had dystrophic nails, and, occasionally, oral erosions. There was no loss of primary or secondary hair. Her entire primary and secondary dentition was affected with enamel hypoplasia and enamel pits, which were treated with direct composite (Fig. 1a, b). Her non-consanguineous parents and her brother had no signs of skin fragility. However, her 53-year-old mother and her 25-year-old brother also had enamel defects in their dentition, consisting of roughness and pits, which had led to a higher susceptibility to caries in both (Fig. 1c, d). The mother was treated with maxillary dentures and numerous restorations (Fig. 1c). In contrast, the father’s teeth were not affected. Skin biopsies in the proband and her mother were taken, and immunofluorescence antigen mapping was performed on non-lesional skin. Staining of LM-332 with monoclonal antibody GB3 was slightly reduced in both mother and daughter, while staining for Col17 with monoclonal antibodies NCC-lu-226 and 233 was normal. Genomic DNA of all family members was extracted from peripheral blood. Previous molecular analysis of COL17A1, LAMB3, and LAMC2 yielded no abnormalities in the pro-band. Screening of LAMA3 (GenBank accession number NM_000227.3) showed a compound heterozygosity for the novel missense mutation c.4484C>T, and the novel nonsense mutation c.488delG in the proband (methods available on re-quest). The deletion c.488delG in exon 5 creates a frameshift that results in a premature termination codon (p.G163DfsX30), and the mutation c.4484C>T in exon 33 results in a substitution of alanine to valine at position 1495 (p.A1495V). Proving the pathogenicity of the mutation p.A1495V, are 2 unrelated patients with JEB-nH (EB169-01 and EB173-01) in our EB cohort, who both carry the LAMA3 mutation p.A1495V. Patient EB169-01 carries it together with the nonsense mutation p.R1126X, and patient 173-01 together with the missense mutation p.R262S (unpublished). Screening of LAMA3 in the family members of the proband showed that her father was heterozygous for c.4484C>T. Her mother and brother, showed heterozygosity for c.488delG. To investigate whether the enamel defects in the heterozygous carriers of c.488delG could be caused by a dominant-negative effect of an aberrant laminin α3 chain by alternatively spliced cDNA, nested PCRs surrounding both mutations were perfor-med with cDNA isolated from skin biopsies from the proband and her mother (methods available on request). There were no alternatively spliced cDNA products visible on agarose gel (Fig. S1a, b; available from http://www.medicaljournals.se/acta/content/?doi=10.2340/00015555-1341). The PCR products were sequenced and cDNA of both alleles of the proband were present (Fig. S1c, d). The allele bearing the mutation c.488delG in the mother was reduced compared with the normal wild-type (Fig. S1c). Together with the absence of alternative spliced products on agarose gel, we hypothesize that this indicates that haplo-insufficiency of LAMA3 is the cause of enamel defects in the mother and brother of the proband.Enamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis BullosaWing Yan Yuen1, Anna M.G. Pasmooij1, Cornelis Stellingsma2 and Marcel F. Jonkman1Departments of 1Dermatology and 2Oral and Maxillofacial Surgery and Maxillofacial Prosthetics, Center for Blistering Diseases, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, NL-9700 RB Groningen, The Netherlands. E-mail: w.y.yuen@umcg.nlAccepted December 13, 2011.Fig. 1. Features of enamel defects. (a, b) The proband (c) her mother, and (d) her brother had enamel defects in their dentition. (a) Rough pitted enamel is seen on the buccal side of the mandibular teeth, and (b) on the palatinal side of the maxillar teeth in the proband. The buccal side of the maxillar teeth are treated with direct composite. (c) The enamel defects on the labial side of the mandibular teeth are no longer visible due to multiple restorations in the mother. (d) In the brother there is typical pitted enamel in the insical area of the central incisors.696 Letters to the EditorDISCUSSIONEnamel is formed in 3 stages: (i) in the presecretory stage the ameloblasts differentiate and then the lamina basale, which supports the ameloblasts, disintegrates; (ii) in the secretory stage ameloblasts secrete enamel proteins that mineralize and form enamel crystals; (iii) in the maturation stage a lamina basale is deposited, adhering ameloblasts to the underlying enamel surface by hemidesmosomes. To harden the enamel, water and organic material are removed by ameloblasts (7). LM-332 is expressed by ameloblasts in all stages of enamel formation (3). In the presecretory and maturation stage LM-332 is a part of the lamina basale. In the secretory stage LM-332 most likely plays a role in the adhesion of ameloblasts to the enamel matrix (3). In patients with JEB-nH the reduction in LM-332 causes detachment from the underlying matrix and degeneration of ameloblasts. This causes enamel defects, such as pitting (4, 8). The enamel that does form has a different chemical composi-tion, as reduction in LM-332 can result in deficiencies in the junctions of ameloblasts that are important in main-taining channels for mineral ions and cell nutrients. This may lead to defective mineral transport or compromised ameloblast metabolism (8). Teeth of patients with JEB have reduced mineral content and presence of serum albumin, which is a known inhibitor of enamel crystal growth (8). It is possible that there is no coping mecha-nism that can manage the loss of one LAMA3 allele in this complex process of enamel formation, whereas these mechanisms are available in the skin (6). However, it remains peculiar that enamel defects have not been reported previously in carriers of LAMA3, LAMB3, or LAMC2 null mutations. Furthermore, none of the carriers of null mutations in LM-332 in the Dutch EB cohort have noted particular dental problems; however, they have not been examined systematically. It is possible that not all carriers are affected; a phenomenon also seen in carriers of COL17A1 null mutations (6, 9) or the enamel defects are so discrete that they are missed. Furthermore, it is possible that other factors contribute to the enamel defects seen in our carriers, such as the rare inherited enamel defects that occur in the absence of generalized syndromes designated as amelogenesis imperfecta (10), or due to environmental factors such as febrile diseases. However, this is less likely in our patients, considering the clinical dental symptoms (7). The enamel defects seen in the carriers of LAMA3 null mutations reported here are comparable to those seen in the carriers of missense COL17A1 mutations; although the carriers of COL17A1 null mutations described by Murrell et al. also showed horizontal ridging, a feature that was not present in the carriers we have described (5, 6).In conclusion, we have reported: (i) two novel LAMA3 mutations associated with JEB-nH; and (ii) the first car-riers of LAMA3 null mutations to have enamel defects probably caused by haploinsufficiency.ACkNOWLEDGEMENTSWe are grateful to the family for participation in this study. We thank krista van Dijk-Bos, Gonnie Meijer, Miranda Nijenhuis and Janny Zuiderveen for technical assistance. We are grateful to Geert Spijker and Svetislav Zaric for their contribution to the study.We wish to thank Vlinderkind (Dutch Butterfly Child Foun-dation) and the J. P. Nater Foundation for their financial support of the study.The authors declare no conflicts of interest.REFERENCES1. Fine JD, Eady RA, Bauer EA, Bauer JW, Bruckner-Tuder-man L, Heagerty A, et al. The classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB. J Am Acad Dermatol 2008; 58: 931–950.2. Asaka T, Akiyama M, Domon T, Nishie W, Natsuga k, Fujita Y, et al. Type XVII collagen is a key player in tooth enamel formation. Am J Pathol 2009; 174: 91–100.3. Yoshiba N, Yoshiba k, Aberdam D, Meneguzzi G, Perrin-Schmitt F, Stoetzel C, et al. Expression and localization of laminin-5 subunits in the mouse incisor. Cell Tissue Res 1998; 292: 143–149.4. Wright JT, Johnson LB, Fine JD. Development defects of enamel in humans with hereditary epidermolysis bullosa. Arch Oral Biol 1993; 38: 945–955.5. McGrath JA, Gatalica B, Li k, Dunnill MG, McMillan JR, Christiano AM, et al. Compound heterozygosity for a dominant glycine substitution and a recessive internal duplication mutation in the type XVII collagen gene results in junctional epidermolysis bullosa and abnormal dentition. Am J Pathol 1996; 148: 1787–1796.6. Murrell DF, Pasmooij AM, Pas HH, Marr P, klingberg S, Pfendner E, et al. Retrospective diagnosis of fatal BP180-deficient non-Herlitz junctional epidermolysis bullosa suggested by immunofluorescence (IF) antigen-mapping of parental carriers bearing enamel defects. J Invest Dermatol 2007; 127: 1772–1775.7. Nanci ARTCA. Ten Cate’s oral histology: development, structure, and function, 7th edn. Nanci A, editor. St Louis, MO: Mosby Elsevier, 2008.8. kirkham J, Robinson C, Strafford SM, Shore RC, Bonass WA, Brookes SJ, et al. The chemical composition of tooth enamel in junctional epidermolysis bullosa. Arch Oral Biol 2000; 45: 377–386.9. Pasmooij AM, Pas HH, Jansen GH, Lemmink HH, Jonkman MF. Localized and generalized forms of blistering in junc-tional epidermolysis bullosa due to COL17A1 mutations in the Netherlands. Br J Dermatol 2007; 156: 861–870.10. Hu JC, Chun YH, Al Hazzazzi T, Simmer JP. Enamel for-mation and amelogenesis imperfecta. Cells Tissues Organs 2007; 186: 78–85.Acta Derm Venereol 92

University of GroningenEnamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis BullosaYuen, Wing Yan; Pasmooij, Anna M. G.; Stellingsma, Cornelis; Jonkman, Marcel F.Published in:Acta dermato-venereologicaDOI:10.2340/00015555-1341IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2012Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Yuen, W. Y., Pasmooij, A. M. G., Stellingsma, C., & Jonkman, M. F. (2012). Enamel Defects in Carriers of aNovel LAMA3 Mutation Underlying Epidermolysis Bullosa. Acta dermato-venereologica, 92(6), 695-696.https://doi.org/10.2340/00015555-1341CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 24-12-2025Acta Derm Venereol 92695Letters to the Editor© 2012 The Authors. doi: 10.2340/00015555-1341Journal Compilation © 2012 Acta Dermato-Venereologica. ISSN 0001-5555Junctional epidermolysis bullosa, non-Herlitz (JEB-nH) is an autosomal recessive EB subtype caused by mutations in the genes LAMA3, LAMB3, LAMC2 or COL17A1. The first three genes encode for laminin α3, laminin β3, and laminin γ2, respectively, which together form the heterotrimer laminin-332 (LM-332), whereas COL17A1 encodes for the homotrimer type XVII col-lagen (Col17) (1). LM-332 and Col17 are located in the lamina basale, and are important in epidermal–dermal adhesion. JEB-nH shows blistering after minor trauma, but also extracutaneous symptoms, such as dental ab-normalities (1). Both LM-332 and Col17 are crucial in ameloblast differentiation and enamel formation (2, 3), resulting in enamel defects of the entire dentition in all patients with JEB-nH, consisting of hypoplasia, pitting, roughness, thinning or furrowing of enamel (4). In 1996, McGrath et al. (5) reported the first heterozygous carriers of a COL17A1 mutation with enamel defects. The carriers of the glycine substitution p.G627V had no signs of skin fragility, but did have extensive enamel hypoplasia and pitting, thought to be caused by the dominant-negative effect of the mutant Col17. In 2007, Murrell et al. (6) reported enamel defects in carriers of a COL17A1 null mutation, which was attributed to haploinsufficiency. Dental abnormalities have not been reported previously in carriers of mutations in the genes encoding LM-332. We report here the first enamel defects in carriers of a LAMA3 mutation. CASE REPORTThe proband was a 23-year-old woman (EB050) diagnosed with JEB-nH. Starting at the age of 2 months she showed generalized blistering and erosions that healed with atrophic scarring. She had dystrophic nails, and, occasionally, oral erosions. There was no loss of primary or secondary hair. Her entire primary and secondary dentition was affected with enamel hypoplasia and enamel pits, which were treated with direct composite (Fig. 1a, b). Her non-consanguineous parents and her brother had no signs of skin fragility. However, her 53-year-old mother and her 25-year-old brother also had enamel defects in their dentition, consisting of roughness and pits, which had led to a higher susceptibility to caries in both (Fig. 1c, d). The mother was treated with maxillary dentures and numerous restorations (Fig. 1c). In contrast, the father’s teeth were not affected. Skin biopsies in the proband and her mother were taken, and immunofluorescence antigen mapping was performed on non-lesional skin. Staining of LM-332 with monoclonal antibody GB3 was slightly reduced in both mother and daughter, while staining for Col17 with monoclonal antibodies NCC-lu-226 and 233 was normal. Genomic DNA of all family members was extracted from peripheral blood. Previous molecular analysis of COL17A1, LAMB3, and LAMC2 yielded no abnormalities in the pro-band. Screening of LAMA3 (GenBank accession number NM_000227.3) showed a compound heterozygosity for the novel missense mutation c.4484C>T, and the novel nonsense mutation c.488delG in the proband (methods available on re-quest). The deletion c.488delG in exon 5 creates a frameshift that results in a premature termination codon (p.G163DfsX30), and the mutation c.4484C>T in exon 33 results in a substitution of alanine to valine at position 1495 (p.A1495V). Proving the pathogenicity of the mutation p.A1495V, are 2 unrelated patients with JEB-nH (EB169-01 and EB173-01) in our EB cohort, who both carry the LAMA3 mutation p.A1495V. Patient EB169-01 carries it together with the nonsense mutation p.R1126X, and patient 173-01 together with the missense mutation p.R262S (unpublished). Screening of LAMA3 in the family members of the proband showed that her father was heterozygous for c.4484C>T. Her mother and brother, showed heterozygosity for c.488delG. To investigate whether the enamel defects in the heterozygous carriers of c.488delG could be caused by a dominant-negative effect of an aberrant laminin α3 chain by alternatively spliced cDNA, nested PCRs surrounding both mutations were perfor-med with cDNA isolated from skin biopsies from the proband and her mother (methods available on request). There were no alternatively spliced cDNA products visible on agarose gel (Fig. S1a, b; available from http://www.medicaljournals.se/acta/content/?doi=10.2340/00015555-1341). The PCR products were sequenced and cDNA of both alleles of the proband were present (Fig. S1c, d). The allele bearing the mutation c.488delG in the mother was reduced compared with the normal wild-type (Fig. S1c). Together with the absence of alternative spliced products on agarose gel, we hypothesize that this indicates that haplo-insufficiency of LAMA3 is the cause of enamel defects in the mother and brother of the proband.Enamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis BullosaWing Yan Yuen1, Anna M.G. Pasmooij1, Cornelis Stellingsma2 and Marcel F. Jonkman1Departments of 1Dermatology and 2Oral and Maxillofacial Surgery and Maxillofacial Prosthetics, Center for Blistering Diseases, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, NL-9700 RB Groningen, The Netherlands. E-mail: w.y.yuen@umcg.nlAccepted December 13, 2011.Fig. 1. Features of enamel defects. (a, b) The proband (c) her mother, and (d) her brother had enamel defects in their dentition. (a) Rough pitted enamel is seen on the buccal side of the mandibular teeth, and (b) on the palatinal side of the maxillar teeth in the proband. The buccal side of the maxillar teeth are treated with direct composite. (c) The enamel defects on the labial side of the mandibular teeth are no longer visible due to multiple restorations in the mother. (d) In the brother there is typical pitted enamel in the insical area of the central incisors.696 Letters to the EditorDISCUSSIONEnamel is formed in 3 stages: (i) in the presecretory stage the ameloblasts differentiate and then the lamina basale, which supports the ameloblasts, disintegrates; (ii) in the secretory stage ameloblasts secrete enamel proteins that mineralize and form enamel crystals; (iii) in the maturation stage a lamina basale is deposited, adhering ameloblasts to the underlying enamel surface by hemidesmosomes. To harden the enamel, water and organic material are removed by ameloblasts (7). LM-332 is expressed by ameloblasts in all stages of enamel formation (3). In the presecretory and maturation stage LM-332 is a part of the lamina basale. In the secretory stage LM-332 most likely plays a role in the adhesion of ameloblasts to the enamel matrix (3). In patients with JEB-nH the reduction in LM-332 causes detachment from the underlying matrix and degeneration of ameloblasts. This causes enamel defects, such as pitting (4, 8). The enamel that does form has a different chemical composi-tion, as reduction in LM-332 can result in deficiencies in the junctions of ameloblasts that are important in main-taining channels for mineral ions and cell nutrients. This may lead to defective mineral transport or compromised ameloblast metabolism (8). Teeth of patients with JEB have reduced mineral content and presence of serum albumin, which is a known inhibitor of enamel crystal growth (8). It is possible that there is no coping mecha-nism that can manage the loss of one LAMA3 allele in this complex process of enamel formation, whereas these mechanisms are available in the skin (6). However, it remains peculiar that enamel defects have not been reported previously in carriers of LAMA3, LAMB3, or LAMC2 null mutations. Furthermore, none of the carriers of null mutations in LM-332 in the Dutch EB cohort have noted particular dental problems; however, they have not been examined systematically. It is possible that not all carriers are affected; a phenomenon also seen in carriers of COL17A1 null mutations (6, 9) or the enamel defects are so discrete that they are missed. Furthermore, it is possible that other factors contribute to the enamel defects seen in our carriers, such as the rare inherited enamel defects that occur in the absence of generalized syndromes designated as amelogenesis imperfecta (10), or due to environmental factors such as febrile diseases. However, this is less likely in our patients, considering the clinical dental symptoms (7). The enamel defects seen in the carriers of LAMA3 null mutations reported here are comparable to those seen in the carriers of missense COL17A1 mutations; although the carriers of COL17A1 null mutations described by Murrell et al. also showed horizontal ridging, a feature that was not present in the carriers we have described (5, 6).In conclusion, we have reported: (i) two novel LAMA3 mutations associated with JEB-nH; and (ii) the first car-riers of LAMA3 null mutations to have enamel defects probably caused by haploinsufficiency.ACkNOWLEDGEMENTSWe are grateful to the family for participation in this study. We thank krista van Dijk-Bos, Gonnie Meijer, Miranda Nijenhuis and Janny Zuiderveen for technical assistance. We are grateful to Geert Spijker and Svetislav Zaric for their contribution to the study.We wish to thank Vlinderkind (Dutch Butterfly Child Foun-dation) and the J. P. Nater Foundation for their financial support of the study.The authors declare no conflicts of interest.REFERENCES1. Fine JD, Eady RA, Bauer EA, Bauer JW, Bruckner-Tuder-man L, Heagerty A, et al. The classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB. J Am Acad Dermatol 2008; 58: 931–950.2. Asaka T, Akiyama M, Domon T, Nishie W, Natsuga k, Fujita Y, et al. Type XVII collagen is a key player in tooth enamel formation. Am J Pathol 2009; 174: 91–100.3. Yoshiba N, Yoshiba k, Aberdam D, Meneguzzi G, Perrin-Schmitt F, Stoetzel C, et al. Expression and localization of laminin-5 subunits in the mouse incisor. Cell Tissue Res 1998; 292: 143–149.4. Wright JT, Johnson LB, Fine JD. Development defects of enamel in humans with hereditary epidermolysis bullosa. Arch Oral Biol 1993; 38: 945–955.5. McGrath JA, Gatalica B, Li k, Dunnill MG, McMillan JR, Christiano AM, et al. Compound heterozygosity for a dominant glycine substitution and a recessive internal duplication mutation in the type XVII collagen gene results in junctional epidermolysis bullosa and abnormal dentition. Am J Pathol 1996; 148: 1787–1796.6. Murrell DF, Pasmooij AM, Pas HH, Marr P, klingberg S, Pfendner E, et al. Retrospective diagnosis of fatal BP180-deficient non-Herlitz junctional epidermolysis bullosa suggested by immunofluorescence (IF) antigen-mapping of parental carriers bearing enamel defects. J Invest Dermatol 2007; 127: 1772–1775.7. Nanci ARTCA. Ten Cate’s oral histology: development, structure, and function, 7th edn. Nanci A, editor. St Louis, MO: Mosby Elsevier, 2008.8. kirkham J, Robinson C, Strafford SM, Shore RC, Bonass WA, Brookes SJ, et al. The chemical composition of tooth enamel in junctional epidermolysis bullosa. Arch Oral Biol 2000; 45: 377–386.9. Pasmooij AM, Pas HH, Jansen GH, Lemmink HH, Jonkman MF. Localized and generalized forms of blistering in junc-tional epidermolysis bullosa due to COL17A1 mutations in the Netherlands. Br J Dermatol 2007; 156: 861–870.10. Hu JC, Chun YH, Al Hazzazzi T, Simmer JP. Enamel for-mation and amelogenesis imperfecta. Cells Tissues Organs 2007; 186: 78–85.Acta Derm Venereol 92

Compound heterozygosity for a dominant glycine substitution and a recessive internal duplication mutation in the type XVII collagen gene results in junctional epidermolysis bullosa and abnormal dentition.

Development defects of enamel in humans with hereditary epidermolysis bullosa. Arch Oral Biol

Enamel formation and amelogenesis imperfecta. Cells Tissues Organs

Expression and localization of laminin-5 subunits in the mouse incisor. Cell Tissue Res

Localized and generalized forms of blistering in junctional epidermolysis bullosa due to COL17A1 mutations in the Netherlands. Br J Dermatol

Retrospective diagnosis of fatal BP180-deficient non-Herlitz junctional epidermolysis bullosa suggested by immunofluorescence (IF) antigen-mapping of parental carriers bearing enamel defects.

Ten Cate’s oral histology: development, structure, and function, 7th

The chemical composition of tooth enamel in junctional epidermolysis bullosa. Arch Oral Biol

The classification of inherited epidermolysis bullosa

Type XVII collagen is a key player in tooth enamel formation.

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Enamel Defects in Carriers of a Novel LAMA3 Mutation Underlying Epidermolysis Bullosa

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