Heterozygosity for a Novel Missense Mutation in the ITGB4 Gene Associated With Autosomal Dominant Epidermolysis Bullosa | Dermatology | JAMA Dermatology | JAMA Network
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Case Report/Case Series
May 2016

Heterozygosity for a Novel Missense Mutation in the ITGB4 Gene Associated With Autosomal Dominant Epidermolysis Bullosa

Author Affiliations
  • 1Department of Dermatology, Center for Blistering Diseases, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands,
  • 2Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
  • 3 Department of Otorhinolaryngology–Head and Neck Surgery, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
JAMA Dermatol. 2016;152(5):558-562. doi:10.1001/jamadermatol.2015.5236

Importance  Epidermolysis bullosa (EB) is a group of mechanobullous genodermatoses characterized by the fragility of skin and mucous membranes. Mutations in the ITGA6 and ITGB4 genes, encoding the hemidesmosomal protein α6β4-integrin, have been involved in the pathogenesis of EB. To date, the inheritance of these particular genes is known to be exclusively autosomal recessive. Herein, we report a novel heterozygous missense mutation in the ITGB4 gene exerting a dominant negative effect that cosegregates with the EB phenotype in an extended family.

Observations  The clinical phenotype of affected individuals is primarily characterized by nail dystrophy and late onset of mild skin fragility and acral blistering. Some patients developed granulation tissue in the larynx, urethra, lacrimal duct, and external auditory canal. Sequencing the complete set of genes associated with EB revealed a heterozygous missense mutation in exon 5 of ITGB4: c.433G>T, p.Asp145Tyr. The mutation was found in the affected relatives and was not present in unaffected relatives and control DNA samples.

Conclusions and Relevance  This study highlights, for the first time to our knowledge, the possibility of a dominant mode of inheritance for a missense ITGB4 mutation in EB, thus expanding the mutational database and genotype-phenotype correlation for this rare disease.


Epidermolysis bullosa (EB) comprises a group of heterogeneous inherited blistering diseases. Mutations in multiple genes encoding proteins responsible for the maintenance of dermoepidermal adhesion and skin integrity have been implicated in the disease pathophysiologic mechanisms. Based on the level of tissue cleavage, EB is further subdivided into epidermolysis bullosa simplex (EBS) with intraepidermal cleavage, junctional epidermolysis bullosa (JEB) with intralamina lucida cleavage, dystrophic epidermolysis bullosa (DEB) with sublamina densa cleavage, and Kindler syndrome (KS) with a mixed cleavage plane. Loss-of-function mutations in the ITGA6 and ITGB4 genes encoding the α6-integrin and β4-integrin subunits, respectively, have been implicated in EB.1 The possible subtypes include EBS with pyloric atresia (EBS-PA), JEB with pyloric atresia (JEB-PA), and junctional epidermolysis bullosa localized (JEB-loc). The latter subtypes are reported solely involving the integrin β4 subunit, whereas the subtypes with pyloric atresia involve either α6 or β4-integrin subunit.1 The clinical phenotype of the affected individuals presents as a spectrum ranging from neonatal death to mild skin fragility and nail dystrophy. Pyloric atresia and urethral strictures may occur; these symptoms are, however, not obligatory for diagnosis.2 α6β4-Integrin is a heterodimeric transmembrane polypeptide located at the core of the hemidesmosomes (HDs). This protein plays a major role in linking the intracellular hemidesmosomal plaque of the basal keratinocytes to the underlying basement membrane, thus providing mechanical resilience to the skin and mucous membranes. Also, integrins are known to execute agile responses to changes in the local environment and mediate transduction of signaling across plasma membranes for important cell functions such as migration, proliferation, and apoptosis.3 To date, EB caused by ITGB4 and ITGA6 gene mutations has been reported to be inherited exclusively in an autosomal recessive manner.1 Herein, we report a novel autosomal dominant missense mutation in the von Willebrand factor type A domain (VWFA domain) of β4-integrin associated with an EB phenotype.

Report of a Case

The index patient (denoted as IV:2 in Figure 1; EB 301-03) of this family had progressive nail dystrophy of the hands and feet since birth, and eventually total loss of nails on his feet. After puberty he developed mild acral blistering on palms, soles, and wrists (Figure 2A and B). In addition, this patient had chronic granulation tissue formation in the right external auditory canal, which led to a relapsing obstruction needing repeated surgery. The external auditory canal wound was finally closed by autologous split-skin graft transplantation (Figure 2C) from a donor site located on the upper left arm. Subsequently, a 3-year-long postoperative complication occurred, which consisted of delayed wound healing at the donor site with hypergranulation of the wound bed. Multiple attempts to facilitate healing with autologous cultured keratinocytes transplants were mostly unsuccessful. He also developed obstructed lacrimal ducts, leading to lacrimation. Another problem was the development of urethral strictures for which a urethroplasty was performed. At physical examination, his hair and teeth were unaffected, and he had no palmoplantar keratosis. His younger brother also had external auditory canal and urinary tract involvement. Interestingly, his youngest daughter developed laryngeal stenosis due to exuberant granulation tissue formation at age 5 years (Figure 2D), for which tracheostomy was performed. She underwent several endoscopic procedures, in which the granulation tissue was repeatedly removed by laser therapy, followed by local application of mitomycine. Finally, an endoscopic posterior cricoid split with rib cartilage interposition was performed, which resulted in successful decannulation. The child had 1 relapse for which she received local application of mitomycine once more. She was asymptomatic at the time of her 2-year follow-up.

Figure 1.  Clinical Pedigree of Family With Autosomal Dominant Epidermolysis Bullosa Due to ITGB4: p.Asp145Tyr Mutation
Clinical Pedigree of Family With Autosomal Dominant Epidermolysis Bullosa Due to ITGB4: p.Asp145Tyr Mutation

DNA was obtained from individuals III:2, IV:2, IV:4, V:1, V:2, V:3; m/wt indicates, mutation/wild type, or wt/wt, wild type/wild type underlines the genotype of the particular individual.

Figure 2.  Clinical Features of Affected Family Members
Clinical Features of Affected Family Members

A, Pachyonychia nail dystrophy consisting of nail thickening, transverse overcurvature, discoloration and brittleness in the index patient (IV-2 in Figure 1); B, Blistering on the sole of the index patient. C, Postoperative circular external auditory canal stenosis, due to granulation tissue formation in the index patient. D, Granulation tissue (arrowhead) in the larynx of his youngest daughter (V-2 in Figure 1).

The pedigree of this family clearly demonstrates an autosomal dominant mode of inheritance (Figure 1). The clinical phenotype predominantly manifests as pachyonychia dystrophic nails and late-onset, mild, acral blistering. Some family members developed extracutaneous complications, including granulation tissue in the larynx and involvement of urethra, lacrimal duct, and external auditory canal (Figure 2).

Immunofluorescence microscopy of nonlesional skin of the index patient and his daughters revealed normal staining for both extracellular and intracellular domains of integrin β4 with monoclonal antibodies 58XB4 and clone 7, respectively. Plectin, dystonin-e, type XVII collagen, laminin 332, and type VII collagen were also normally expressed (not shown). The plane of cleavage could not be determined because no lesional skin biopsy was available. Electron microscopy of uninvolved skin showed an adequate number of HDs; however, some were hypoplastic (Figure 3). The lamina densa was structurally abnormal with irregular thickness and some blind offshoots. The other components of the epidermal basement membrane zone were normal.

Figure 3.  Transmission Electron Microscopy of Nonlesional Skin in the Index Patient
Transmission Electron Microscopy of Nonlesional Skin in the Index Patient

Both normal (black arrowhead) and hypoplastic (red arrowhead) hemidesmosomes in adequate numbers along the epidermal basement membrane zone. The lamina densa displays irregular thickness and some blind off-shoots (asterisk). The intermediate tonofilaments are well inserted and the anchoring fibrils are present. Bar: 0.5 µm.

To identify the underlying genetic mutation for this disease, we applied in the index patient and his affected daughters our diagnostic next-generation sequencing gene panel test, consisting of a comprehensive set of 33 genes associated with EB, which is based on targeted SureSelect enrichment (Agilent Technologies Inc), and subsequent sequencing on a MiSeq sequencer (Illumina Inc). We identified a heterozygous c.433G>T substitution GenBank NM_ 000213.3 in exon 5 of the ITGB4 gene resulting in the p.Asp145Tyr missense mutation (Figure 4A). Sanger sequencing of genomic DNA confirmed the presence of this mutation in the patient and other affected family members. This mutation was not found in the Genome of the Netherlands,4 1000 genomes, or the ExAc Browser databases and, to our knowledge, was not earlier described in the literature. The pathogenicity prediction software tool Alamut (version 2.0, Interactive Biosoftware), classifies the missense mutation as probably pathogenic. The p.Asp145Tyr missense mutation changes the acidic side-chained aspartate to hydroxyl side-chained tyrosine at codon 145 in the extracellular VWFA domain of β4-integrin. Alignment of β4-integrin orthologs illustrated that residue p.Asp145 is highly conserved among species, which advocates its functional significance (Figure 4B). To exclude that this mutation induced alternative splicing, nested polymerase chain reactions surrounding the mutation were performed with complimentary DNA (cDNA) isolated from skin biopsy specimens from the index patient and his youngest daughter. No alternatively spliced cDNA products were observed (Figure 4D); this result was in congruence with the assessment of in silico splice-site prediction software.

Figure 4.  Schematic Representation of the β4-Integrin Subunit, Conservation of Residues and RT-PCR Analysis
Schematic Representation of the β4-Integrin Subunit, Conservation of Residues and RT-PCR Analysis

A, All known ITGB4 missense mutations involved in epidermolysis bullosa (EB) are indicated above the schematic polypetide with evident clustering within the Von Willebrand factor type A (VWFA) domain. The heterozygous p.Asp145Tyr substitution reported herin is shown in red below the schematic structure. B, Conservation of β4-integrin residue Asp145 (D letter code) between species. C, Alignment of β-integrin subunits sequences shows evident conservation (except β8-integrin) of DDL peptide sequence (boxed area). D, Electrophoresis gel analysis of messenger RNA amplified by nested PCRs in the index patient and his youngest daughter identified no alternatively spliced products in the index patient (IV:2, lane 3) and his youngest daughter (V:2, lane 2), compared with control (lane 1). Bp indicates base pair; COOH, carboxyl; NH2, amino terminus; RT-PCR, real time polymerase chain reaction.


Our findings emphasize the importance of considering heterozygous ITGB4 gene mutations as a possible cause for EB, in which the pachyonychia dystrophic nails are typically the presenting symptom in affected individuals. Also, this study provides information on the natural history of this particular mutation in later life, with prognostic repercussions for younger patients, such as acral blistering after puberty.

Considering the dominant mode of inheritance of this disorder and the predominant feature of pachyonychia nail dystrophy, we considered pachyonychia congenita (PC) as a differential diagnosis. Other typical PC symptoms, such as oral leukokeratosis, cysts, and follicular keratosis, were not part of the phenotype. In addition, involvement of the external auditory canal, lacrimal duct, and urinary tract, as well as delayed wound healing, and granulation tissue formation in the larynx, have been reported in JEB but not in PC.5,6 Pachyonychia congenita was excluded ultimately as a diagnosis through our extended EB gene panel test, as no mutations in the genes KRT6A, KRT6B, KRT6C, KRT16, and KRT17, known to underlie PC,6 were identified.

Notably, granulation tissue formation in the larynx of individual V:2 is a clinical feature reminiscent of junctional epidermolysis bullosa- laryngo-onycho-cutaneous syndrome (JEB-LOC). This diagnosis was, however, excluded because no mutations have been found in LAMA3 and its isoform LAMA3A.

Results for the immunofluorescence studies with monoclonal antibodies against β4-integrin in the index patient and his daughters were normal, which may not be surprising considering that the heterozygous missense mutation is not likely to result in diminished protein production. Notably, unaltered immunofluorescence staining for α6β4-integrin has also been reported in a patient with homozygous missense mutations in ITGB4.2 The electron microscopic findings in the skin sample of the index patient revealed a combination of normal and hypoplastic HDs. α6β4-Integrin is essential for the assembly of HDs.7 In fact, this protein is one of the first to emerge at the basement membrane zone and is the nucleating factor for HD formation. The occurrence of hypoplastic HDs in the presence of this mutation confirms the important role for β4-integrin in the maturation of these structures. In nonlethal cases of JEB caused by mutations in the ITGB4 gene, HDs are present, although they are often incomplete.8 Abnormal assembly of mutated β4-integrin and normal α6-integrin polypeptides could explain the occurrence of hypoplastic HDs. Electron microscopy also revealed abnormal architecture of the basement membrane, which may be caused by the central role which β4-integrin plays in its formation,9 or due to constant restoration after microscopic dermoepidermal cleavage. Such findings have been previously noted in patients with EB with underlying ITGB4 mutations.2

A question that remains to be answered is, why do we consider this specific substitution to be pathogenic? Review of the literature reveals that most missense ITGB4 mutations reside in the extracellular domain of β4-integrin with evident clustering within the VWFA domain (Figure 4A). The amino acid substitution p.Asp145Tyr is also located within the VWFA domain. This domain, since its discovery, has drawn great scientific interest owing to its wide variety of important cellular functions. These include, among others, basement membrane formation, cell migration, ligand binding, and signaling.10,11 Specific-site mutations in this domain might, accordingly, have a detrimental effect on each of these functions, although interpretation of the role of specific residues is a challenge. Mutagenesis studies at the Asp145 residue within the β4-integrin subunit have not been reported. Interestingly, Pasqualini et al12 have investigated the analog protein region in β3-integrin. According to their data, the Asp-Asp-Leu (DDL) portion (Figure 4C), of which Asp145 is the middle residue, represents the contact domain for the Arg-Gly-Asp (RGD)-containing integrin ligands.12 Such observations emphasize the importance this sequence plays in ligand binding. The high conservation of this sequence through several β-integrin subunits (except β8) supports the idea of functional consequences in case of mutations (Figure 4C).

In eukaryotic cells, phosphorylation usually occurs on serine, threonine, and tyrosine residues.13 The latest advances in extracellular signaling research have provided mounting evidence of extracellular phosphorylation for a large number of extracellular matrix proteins and extracellular domains of transmembrane proteins.14 In fact, Yalak et al15 reported 6 experimentally verified phosphorylation sites within the extracellular domain of β4-integrin, including one on a tyrosine residue. In regard to our study, the p.Asp145Tyr substitution is intriguing because such events may create a novel phosphorylation site in the extracellular domain of β4-integrin, and modify its function.

The lack of literature reports regarding dominant β4-integrin subtypes of EB may be better comprehended if only particular mutations, such as p.Asp145Tyr, in very specific regions, lead to clinical manifestations. For instance, in the case of EBS-Ogna, a plectinopathy, the dominant p.Arg2000Trp substitution results in a diseased phenotype. Another example is EBS with mottled pigmentation, a rare entity owing to the p.Pro24Leu substitution in keratin 5 or the p.Met119Thr in keratin 14.1 Evidently, more investigation is necessary to address the exact mechanism by which the p.Asp145Tyr mutation changes dynamics of ligand interaction and signal transduction in this particular mutated protein domain of β4-integrin. Nevertheless, the extensive segregation in a dominant manner, exclusion of mutations in other EB-related genes, and the typical clinical features suggest that this mutation is responsible for the EB phenotype in the affected individuals.

Given that not all patients developed exuberant granulation tissue in the mucous membranes, it is possible that other unidentified modifying genetic or epigenetic factors determine whether the patient will develop this particular clinical feature.


In contrast with previous findings, our data indicate that heterozygous ITGB4 missense mutations can cause an autosomal dominant subtype of EB. This expands our current knowledge on the genotype-phenotype correlation of EB. The phenotype of the affected individuals primarily includes pachyonychia nail dystrophy and mild acral skin fragility developing after puberty. Some patients developed extracutaneous complications in the external auditory canal, lacrimal duct, larynx, and urethra. The molecular mechanism by which this particular mutation modifies β4-integrin function and leads to EB is yet to be established.

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Article Information

Corresponding Author: Marcel F. Jonkman, MD, PhD, Department of Dermatology, University Medical Centre Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands (m.f.jonkman@umcg.nl).

Published Online: January 27, 2016. doi:10.1001/jamadermatol.2015.5236.

Author Contributions: Drs Turcan and Jonkman had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Turcan, Pasmooij, Van den Akker, Sinke and Jonkman.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Turcan, Jonkman.

Critical revision of the manuscript for important intellectual content: All authors.

Administrative, technical, or material support: Turcan, Lemmink, Sinke, Jonkman.

Study supervision: Halmos, Sinke, Jonkman.

Conflict of Interest Disclosures: None reported.

Funding/Support: None reported.

Additional Contributions: We would like to thank Janny Zuiderveen and Gonnie Meijer, laboratory technicians at the Department of Dermatology, Center for Blistering Diseases, University Medical Center Groningen, for their excellent technical assistance performing immunofluorescence staining, Miranda Nijenhuis, Department of Dermatology, Center for Blistering Diseases, University Medical Center Groningen, for performing agarose gel analysis of cDNA products, and Dr Frederic G. Dikkers, Department of Otorhinolaryngology–Head and Neck Surgery, University Medical Center Groningen, for performing laryngeal endoscopy. They were not compensated for their contributions. We also thank the patient for granting permission to publish this information.

Fine  JD, Bruckner-Tuderman  L, Eady  RA,  et al.  Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification.  J Am Acad Dermatol. 2014;70(6):1103-1126.PubMedGoogle ScholarCrossref
Schumann  H, Kiritsi  D, Pigors  M,  et al.  Phenotypic spectrum of epidermolysis bullosa associated with α6β4 integrin mutations.  Br J Dermatol. 2013;169(1):115-124.PubMedGoogle ScholarCrossref
de Pereda  JM, Lillo  MP, Sonnenberg  A.  Structural basis of the interaction between integrin alpha6beta4 and plectin at the hemidesmosomes.  EMBO J. 2009;28(8):1180-1190.PubMedGoogle ScholarCrossref
Genome of the Netherlands Consortium.  Whole-genome sequence variation, population structure and demographic history of the Dutch population.  Nat Genet. 2014;46(8):818-825.PubMedGoogle ScholarCrossref
Fine  JD, Mellerio  JE.  Extracutaneous manifestations and complications of inherited epidermolysis bullosa, part I: Epithelial associated tissues.  J Am Acad Dermatol. 2009;61(3):367-384.PubMedGoogle ScholarCrossref
Shah  S, Boen  M, Kenner-Bell  B, Schwartz  M, Rademaker  A, Paller  AS.  Pachyonychia congenita in pediatric patients: natural history, features, and impact.  JAMA Dermatol. 2014;150(2):146-153.PubMedGoogle ScholarCrossref
Dowling  J, Yu  QC, Fuchs  E.  Beta4 integrin is required for hemidesmosome formation, cell adhesion and cell survival.  J Cell Biol. 1996;134(2):559-572.PubMedGoogle ScholarCrossref
Eady  RA, McGrath  JA, McMillan  JR.  Ultrastructural clues to genetic disorders of skin: the dermal-epidermal junction.  J Invest Dermatol. 1994;103(5)(suppl):13S-18S.PubMedGoogle ScholarCrossref
de Pereda  JM, Ortega  E, Alonso-García  N, Gómez-Hernández  M, Sonnenberg  A.  Advances and perspectives of the architecture of hemidesmosomes: lessons from structural biology.  Cell Adh Migr. 2009;3(4):361-364.PubMedGoogle ScholarCrossref
Tuckwell  DS, Humphries  MJ.  A structure prediction for the ligand-binding region of the integrin beta subunit: evidence for the presence of a von Willebrand factor A domain.  FEBS Lett. 1997;400(3):297-303.PubMedGoogle ScholarCrossref
Colombatti  A, Bonaldo  P.  The superfamily of proteins with von Willebrand factor type A-like domains: one theme common to components of extracellular matrix, hemostasis, cellular adhesion, and defense mechanisms.  Blood. 1991;77(11):2305-2315.PubMedGoogle Scholar
Pasqualini  R, Koivunen  E, Ruoslahti  E.  A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins.  J Cell Biol. 1995;130(5):1189-1196.PubMedGoogle ScholarCrossref
Frijns  E, Kuikman  I, Litjens  S,  et al.  Phosphorylation of threonine 1736 in the C-terminal tail of integrin β4 contributes to hemidesmosome disassembly.  Mol Biol Cell. 2012;23(8):1475-1485.PubMedGoogle ScholarCrossref
Yalak  G, Olsen  BR.  Proteomic database mining opens up avenues utilizing extracellular protein phosphorylation for novel therapeutic applications.  J Transl Med. 2015;13(125). doi:10.1186/s12967-015-0482-4.Google Scholar
Yalak  G, Vogel  V.  Extracellular phosphorylation and phosphorylated proteins: not just curiosities but physiologically important.  Sci Signal. 2012;5(255):1-13.PubMedGoogle ScholarCrossref