Phenotypic spectrum of epidermolysis bullosa associated with α6β4 integrin mutations


  • Funding sources This work was supported by the Network Epidermolysis bullosa grant from the Federal Ministry for Education and Research (BMBF) to L.B.-T., the Excellence Initiative of the German Federal and State Governments and Freiburg Institute for Advanced Studies, School of Life Sciences to L.B.-T. and by the K. Kriezis scholarship from the National and Kapodistrian University of Athens to D.K.
  • Conflicts of interest None declared.


Leena Bruckner-Tuderman.




Integrin α6β4 is a transmembrane receptor and a key component of the hemidesmosome anchoring complex. It is involved in cell–matrix adhesion and signalling in various tissues. Mutations in the ITGA6 and ITGB4 genes coding for α6β4 integrin compromise dermal–epidermal adhesion and are associated with skin blistering and pyloric atresia (PA), a disorder known as epidermolysis bullosa with PA (EB-PA).


To elucidate the molecular pathology of skin fragility in eight cases, disclose the underlying ITGA6 and ITGB4 mutations and study genotype–phenotype correlations.


DNA was isolated from ethylenediaminetetraacetic acid–blood samples, and the coding exons and exon–intron boundaries of ITGA6 and ITGB4 were amplified by polymerase chain reaction (PCR), and directly sequenced. Skin samples were submitted to immunofluorescence mapping with antibodies to adhesion proteins of the dermal–epidermal junction. Primary keratinocytes were isolated, and used for RNA and protein extraction, reverse transcription PCR and immunoblotting. Ultrastructural analysis of the skin was performed in one patient.


We disclose 10 novel mutations, one in ITGA6 and nine in ITGB4. Skin cleavage was either intraepidermal or junctional. Lethal outcome and PA correlated with loss-of-function mutations in two cases. Solely mild skin involvement was associated with deletion of the C-terminus of β4 integrin. Combinations of missense, nonsense or frameshift mutations caused severe urinary tract involvement in addition to skin fragility in five cases.


The present study reveals novel ITGA6 and ITGB4 gene mutations and supports previous reports showing that the phenotype may lack PA and be limited to skin and nail involvement. In four out of six cases of EB-PA, life expectancy was not impaired. A high frequency of urinary tract involvement was found in this study, and represented the main cause of morbidity. Low levels of β4 integrin expression were compatible with hemidesmosomal integrity and a mild skin phenotype.

Inherited epidermolysis bullosa (EB) comprises a broad spectrum of bullous disorders affecting the skin and its appendices, nails and hair, mucous membranes (e.g. oral, urinary or gastrointestinal) or extracutaneous organs (e.g. muscle or kidney).[1] Typically, skin integrity is compromised by mechanical trauma. Apart from the ultrastructural level of skin cleavage, EB classification defines EB subtypes based on clinical features and inheritance pattern.[1] Four main EB types and several subtypes are distinguished: EB simplex (EBS) with basal and suprabasal subtypes; junctional EB (JEB) with the severe Herlitz and other milder subtypes; recessive and dominant dystrophic EB (DEB); and Kindler syndrome.[1] In basal EBS the level of split formation is within the basal keratinocytes, whereas in JEB, the tissue separation occurs within the lamina lucida of the cutaneous basement membrane.

EB subtypes associated with pyloric atresia (EB-PA) are caused by mutations in the genes coding for α6β4 integrin or plectin and are inherited in a recessive manner.[2, 3] More than 70 mutations in the gene encoding the β4 integrin subunit, ITGB4, have been reported and at least five mutations in the ITGA6 gene, coding for the α6 integrin subunit[3] (HGMD Professional 2012.3; A rare subset of EBS with PA is caused by mutations in the PLEC gene, coding for plectin, an intracellular hemidesmosomal protein, serving as a linker to the cytokeratin intermediate filament network.[4] In severe forms of EB-PA with lethal outcome, immunofluorescence staining shows negative or markedly reduced signals with α6β4 integrin or plectin antibodies due to mutations resulting either in premature termination codons, or amino acid substitutions affecting highly conserved regions.[3] In moderate forms of EB-PA the phenotype improves over time. Several cases with a mild phenotype and ITGB4 missense mutations in at least one allele[5, 6] have been reported and not all of them presented PA.[7-12] Urinary tract involvement is described in all four major EB subtypes, especially in JEB and in recessive DEB. However, Fine et al. suggested that the increased number of cases of urinary tract involvement in EB-PA might represent an over-reporting bias, as individuals severely affected with EB-PA are rapidly referred to specialist centres for evaluation and care.[13] Urethral meatal stenosis, urinary retention, development of bladder hypertrophy, hydronephrosis and pyelonephritis are complications described in several cases.[13, 14]

Integrin receptors are heterodimers consisting of an α and a β subunit. The α6β4 integrin consists of the 1130 amino acid α6 subunit linked to the 1822 amino acid β4 subunit.[15, 16] Apart from its crucial role as a structural protein in the hemidesmosome anchoring complex, it is important for signalling, activating pathways responsible for cell migration, invasion and survival.[17] It is expressed in various epithelial tissues, e.g. skin, mucous membranes, and urinary and gastrointestinal tract. In the skin, α6β4 integrin attaches basal keratinocytes to the basement membrane by linking the extracellular N-termini of the α and β subunits to laminin 332 and collagen XVII. The extracellular domain of β4 integrin contains repeated cysteine-rich regions, essential for building intra- and intermolecular disulfide bonds.[16] Mutations in cysteine are associated with lethal and nonlethal EB-PA forms.[7] The intracellular domain of β4 integrin is unusually large compared with other β integrin subunits and contains two pairs of fibronectin type-III domains (Fig. 1). The intracellular C-terminus is attached to the cytokeratin network via plectin.[17]

Figure 1.

Schematic representation of the α6 and β4 integrin polypeptides. Black area, extracellular domain; white area, intracellular (cytoplasmic) domain; light grey area, transmembrane domain; dark grey area, fibronectin type-III domains. The positions of the mutations of the present cases are marked with arrows. Mutations marked in bold are novel. VWFA, von Willebrand factor type A. Modified from Chung and Uitto.[3]

Here we report on eight patients with α6β4 integrin gene mutations and different phenotypes ranging from early lethality to mild skin fragility and nail dystrophy. PA was not an obligatory feature of the disease, whereas the urinary tract was frequently affected.

Materials and methods

Patients and tissue samples

Eight patients, clinically suspected to have EB, were investigated. In five patients, northern European heritage was documented; patient 5 was of Greek origin and patients 7 and 8 of Turkish origin. Following informed consent, ethylenediamine-tetraacetic acid (EDTA)–blood and skin samples were obtained from patients and, if available, from family members. The study was approved by the ethics committee of the University of Freiburg.

Immunofluorescence staining

Immunofluorescence staining of skin was performed using a panel of antibodies to components of the epidermal basement membrane zone as described previously.[18] The antibodies for α6β4 integrin were the monoclonal β4 integrin antibody 3E1 (Gibco, Karlsruhe, Germany) and monoclonal α6 integrin antibody NKI-GoH3 (Chemicon/Millipore, Temecula, CA, U.S.A.). The staining was observed with an Axiophot® fluorescence microscope (Carl Zeiss, Göttingen, Germany).

Electron microscopy

The skin biopsy specimen was fixed in 3% glutaraldehyde solution in 0·1 mol L−1 cacodylate buffer, pH 7·4, at room temperature, cut into pieces of ~1 mm3, washed in buffer, postfixed for 1 h at 4 °C in aqueous 1% osmium tetroxide, rinsed in water, dehydrated through graded ethanol solutions, transferred into propylene oxide, and embedded in epoxy resin (glycid ether 100). Sections were cut with an ultramicrotome (Reichert Ultracut E; Reichert, Vienna, Austria) and examined with a Zeiss EM 900 electron microscope (Carl Zeiss).[19]

Keratinocyte cultures, RNA isolation and reverse transcription polymerase chain reaction

Primary epidermal keratinocytes were isolated from skin biopsies of patients 1 and 4 and of an unaffected control, and cultivated in keratinocyte growth medium (Invitrogen, Karlsruhe, Germany), as described previously.[20] Total RNA was isolated from keratinocytes using RNeasy® Plus Mini Kit (Qiagen, Hilden, Germany) and transcribed into cDNA (Fermentas, St Leon-Rot, Germany). Reverse transcription (RT) was performed with Advantage RT-for-PCR Kit (BD Biosciences, Heidelberg, Germany) with 0·5 μg of total RNA. The PCR products were separated on 1·5% agarose gels. The following primers were used: 2F-AGTGAAGAGCTGCACGGAGT and 4R-GGTGGTGTCAATCTGGGTCT (product spanning exons 2–4 of 158 b.p.), 8F-GTCTTCTCCACCGAGTCAGC and 9R-GATTGAAGGCCTCCTCCAG (spanning exons 8 and 9,316 b.p.) and 36F-ATATCGTCGGCTACCTGGTG and 38R-CTGCTGTACTCGCTTTGCAG (spanning exons 36–38, 277 b.p.), for ITGB4, and F-GCCTCCAAGGAGTAAGACC and R-AGGGGTCTACATGGCAACTG, for GAPDH.


For immunoblotting, keratinocytes were extracted with a buffer containing 0·1 mol L−1 NaCl, 20 mmol L−1 Tris–HCl, pH 7·4, 1% Nonidet P-40, Pefabloc and EDTA.[21, 22] Normalized amounts of the proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, immunoblotted and incubated with the monoclonal β4 integrin antibodies 450-11 A (BD Biosciences) targeting the endodomain and 3E1 (Gibco) targeting the ectodomain, as described previously. GAPDH antibodies (clone 6C5; Millipore) were used to control loading.

Mutation detection and RNA analysis

Genomic DNA was extracted from EDTA–blood using the QIAamp DNA Mini Kit (Qiagen). For amplification of all ITGA6 and ITGB4 exons and exon–intron boundaries (NM_000210.2, NM_001005731.1) primers were designed with Primer 3 (; they are listed in Table S1 (see Supporting Information). Sequencing was performed on an ABI 7330XL DNA analyser (Applied Biosystems, Foster City, CA, U.S.A.). Mutations were confirmed by resequencing.


Phenotypic variability in patients with integrin α6β4 mutations

Eight patients were included in this study. Their clinical and molecular characteristics are summarized in Table 1. The clinical parameters included skin fragility, mucosal involvement, nail abnormalities (Fig. 2), urinary tract and renal involvement, PA, dental anomalies and other features.

Table 1. Clinical features, mutations and immunofluorescence mapping findings in the patients with epidermolysis bullosa (EB)
PatientAgeSkin featuresNails and teethUrinary tract involvement (age at onset)Pyloric atresiaMutationsImmunofluorescence staining of the skin
132 yearsMild localized blistering predominantly at hands and feet, mild hyperkeratosis, hyperhidrosis on palms and solesOnychogryphosis, pachyonychia, enamel defects, all teeth cappedNoNo


c.4969_4990del, p.E1657fsX1, homozygous

No blister, α6 and β4 integrin slightly reduced, all other markers positive
226 yearsLocalized blistering on hands and feet, exacerbation in summer, numerous EB naeviPachyonychia or lost nails, enamel hypoplasia, discoloration, dystrophy or loss of teethNoYes


c.[1769G>A];[2251C>T], p.[C590Y];[R751X]

Junctional blister, α6 and β4 integrin reduced, all other markers positive
317 yearsIntermittent mild blistering after mechanical traumaMild nail dystrophy, enamel defectsNumerous episodes of colics followed by severe dysuria, recurrent vesicoureteral occlusion, development of nephroliths, no further symptoms in the urinary tract in 2-year follow-up (12 years)Duodenal atresiaITGB4 c.[599C>T];[914C>T], p.[P200L];[P305L]Intraepidermal microblister, slightly reduced α6β4 integrin at the blister floor, all other markers positive
411 yearsMild blistering at birth, in mechanically stressed areasNail dystrophy, enamel defectsChronic unspecific cystitis resulting in bladder wall changes, ulcers and erosions in the urothelium, dysuria, haematuria, no further symptoms in the urinary tract in 2-year follow-up (8 years)YesITGB4 c.[914C>T];[917C>T], p.[P305L];[S306L]No blister, all markers positive
53 yearsMild blistering, predominantly on hands and feet from the age of 2 months, no mucosal involvementDystrophic toe nails at birthHaematuria, dysuria, hydronephrosis, ulcers in the thickened bladder wall (3 years)No, but recurrent vomiting in first 4 months


c.[3842G>C];[4295_4298dup], p.[R1281P];[T1434LfsX69]

Junctional and basal intraepidermal blisters, β4 integrin slightly reduced, all other markers positive
65 yearsAt birth single blister on the head and mucous membranes, haemorrhagic blisters on the feet and palms since infancy at places of mechanical irritationNail dystrophyAcute pyelonephritis and haematuria at 4 months, urethra dilatation performed at 5 months. At the age of 16 months dilatation of kidney pelvis, hydronephrosis, thickening of the bladder wall was found, vesicostomy was placed at 5 months (5 months)Yes


c.[3809C>T];[?], p.[Q1237X];[?]

Intraepidermal microblister, β4 integrin reduced, integrin α6 slightly reduced
7Died at age 5 weeksSevere blistering predominantly on the legs, mucous membrane involvement at birthNo nail anomaliesNoYes, death by septicaemia


c.566 + 3_+6delAAGT, homozygous

Junctional blister, β4 integrin negative, α6 integrin, collagen XVII and plectin reduced
8Died at age 3 monthsGeneralized blistering, development of widespread erosionsNo nail anomaliesDilated renal pelvis, thickened bladder wall, haematuria (first weeks of life)Yes, wheezing, artificial ventilation, cholestasis


c.388-5T>G, homozygous

Junctional blister, α6 and β4 integrin negative
Figure 2.

Clinical manifestations of epidermolysis bullosa caused by β4 integrin mutations. (a) Onychogryphosis in 32-year-old patient 1; (b) discoloration of teeth with enamel defects and (c) patchy hyperpigmentation on both elbows in 26-year-old patient 2; (d) severe skin fragility, mechanical ventilation and feeding by gastrostomy in patient 8; (e) very mild skin involvement with a single pretibial scab on the right leg of patient 3.

Patient 1 (age 32 years, female)

The patient presented infrequent mechanically induced blistering, predominantly on the hands and feet, and mild diffuse keratosis on the soles. Palmoplantar hyperhidrosis was prominent during summertime. The teeth were carious due to enamel hypoplasia since childhood, and were completely capped with crowns in early adulthood. Onychogryphosis, pachyonychia and nail dystrophy were present (Fig. 2a). Oral blisters were rare and did not cause dysphagia.

Patient 2 (age 26 years, female)

Blistering of hands and feet was present at birth, and continued predominantly on mechanically stressed regions. PA was corrected directly after birth. Blistering improved over time, and deteriorated mostly in summer. Numerous patchy hyperpigmented macules and naevi were present on the elbows (Fig. 2c), fingers and feet. Oral and nasal mucosae were involved occasionally. The teeth were carious, partially lost and revealed discoloration but no pitting (Fig. 2b). Apart from three lost toe nails, all other nails were either dystrophic or thickened (pachyonychia). Over the last few years, the patient developed recurrent duodenal reflux and gastritis.

Patient 3 (age 17 years, male)

After birth, PA was treated surgically. Skin blistering was noticed for the first time when the boy started walking. It occurred predominantly on the feet. Nail dystrophy developed in early childhood. The patient suffered from repetitive renal colics, caused by vesicoureteral occlusion, and the presence of a vesicoureteral valve was suspected. Episodes of painful loss of soft tissue fragments through the urethra were reported and discharge of nephroliths was suspected. Continuous pain treatment resulted in abuse of pain medication at the age of 12 years. A stepwise reduction of the pain medication resulted in a pain-free interval over the last 2 years, and no further urinary tract involvement was observed. Teeth demonstrated enamel hypoplasia. At the last visit no blistering had been perceived for several months (Fig. 2e). Oral and subungual blistering occurred only rarely.

Patient 4 (age 11 years, male)

At birth, skin blistering was reported in the medical records, but the parents recalled first trauma-induced blistering at the age of 5 years on the hands and feet. PA was diagnosed after birth and treated surgically. Nail dystrophy first occurred from the age of 1·5 years and enamel hypoplasia resulted in early caries. Chronic cystitis was diagnosed at the age of 8 years, and erosions and ulcers were found in tissue samples of the bladder. A magnetic resonance imaging scan showed a thickened bladder wall. As a bladder tumour was suspected, a biopsy was taken, revealing a bladder wall of 2-cm diameter, with polypus exophytic areas. Histopathological examination showed a hypertrophic muscular layer and interstitial fibrosis with sparse eosinophilic infiltrates, without any indication for malignancies.

Patient 5 (age 3 years, male)

At birth, only dysplastic toe nails were present, whereas mild blistering developed from the age of 2 months. During the first 4 months of life, the patient suffered from recurrent vomiting, but ultrasound excluded PA. The mucous membranes were never involved and the primary teeth appeared normal. In the third year of life an episode of haematuria, dysuria and renal colics occurred, and moderate hydronephrosis of the left kidney and thickened bladder wall were found. Cystoscopy revealed ulcers in the urinary bladder, whereas the histopathological examination showed eosinophilic infiltrate and adenomatous hyperplastic lesions. This was considered to be severe chronic cystitis and nephrogenic adenoma as a result of chronic irritation. Another episode of severe haematuria and dysuria followed soon after, during which the renal function deteriorated with a glomerular filtration rate 38 mL min−1 1·73 m−2 (normally > 90) and microalbuminuria. Ultrasound demonstrated bilateral hydronephrosis, dilated ureters and a thick bladder wall. Treatment was initiated with oxybutynin (Ditropan®; Sanofi-Aventis, Paris, France) at a dose of 0·6 mg kg−1 daily, which led to gradual improvement of the renal function and no recurrence for a 2-month follow-up.

Interestingly, the father of patient 5, a carrier of the ITGB4 mutation c.4295_4298dup (p.T1434LfsX69), had dystrophic great toenails. Onychomycosis was excluded by repeated diagnostic tests.

Patient 6 (age 5 years, male)

At birth a single blister on the scalp was present and PA was treated surgically. Within the first weeks of life serous blisters developed, but disappeared in short order. Over the next years haemorrhagic blisters appeared on the limbs and palms. Nails became dystrophic in the first 2 years of life. Blistering is now seen only in mechanically stressed areas. Acute pyelonephritis with haematuria and proteinuria was diagnosed at the age of 4 months. Due to a urethral stenosis, surgical dilatation was performed at the age of 5 months and a vesicostomy was performed. At 16 months, dilatation of the renal pelvis, thickening of the bladder wall and progressive proliferation of urinary epithelium resulted in recurrent obliterations. A reduced creatinine clearance developed and a closure of the vesicostomy developed by the age of 18 months, resulting in accumulation of urine in the upper urinary tract and secondary infections. At the age of 3 years a cystoscopy was performed and Pseudomonas aeruginosa was identified. After intravenous colistin (colistimethate sodium) treatment, vesicostomy function improved. No further infections of the urinary system have occurred for over 3 years.

Patient 7 (died 5 weeks after birth, female)

The child was born in the 28th week of gestation and showed PA and severe blistering predominantly on the legs. Oral mucosa was involved with blistering and erosions. PA was treated surgically after birth. The child died due to septicaemia 5 weeks after birth. At that time point, no urinary tract involvement or nail abnormalities were documented.

Patient 8 (died age 3 months, male)

From birth on, generalized blistering and severe skin fragility were noted. PA was surgically treated, but feeding was maintained via gastrostomy. Stridor developed within the first weeks and because of respiratory distress, a tracheostomy was placed and the child was mechanically ventilated until death (Fig. 2d). Bronchoscopy revealed tracheal ulceration in the area of the tracheostomy. Urinary stasis, with dilated pelvis and ureter, and haematuria developed. Ultrasound examination demonstrated thickened bladder walls, cholestasis and irregular inner surface pattern and a strongly reduced inner lumen. The child died at the age of 3 months due to disease complications.

Molecular diagnostics: novel ITGA6 and ITGB4 mutations and their consequences

Immunofluorescence staining of skin biopsies showed a junctional split within the lamina lucida of the basement membrane in patients 2, 5, 7 and 8. A reduced immunofluorescence staining with antibodies to β4 integrin was found in patients 1 (Fig. 3c,d), 2 (Fig. 3e,f), 3, 5 and 6, and no signal was obtained in patients 7 and 8. All other markers of the dermal–epidermal junction zone stained positive. In patients 1, 3 and 4 with mild skin fragility, the precise level of skin splitting was difficult to resolve, as they did not show blistering for weeks or months. In patient 1, intensive rubbing of the skin resulted in an artificial subcorneal split (Fig. 3c,d) and in patient 3, only microblisters were found, with signals of both integrins α6 and β4 at the blister floor (Fig. 3g,h), indicating a deep basal intraepidermal split.

Figure 3.

Different levels of split formation in β4 integrin-associated epidermolysis bullosa forms. (a, b) Control staining of normal human skin. The skin of patient 1 exhibited no split at the dermal–epidermal junction, but β4 integrin (c) and α6 integrin (d) staining was reduced. (e, f) Patient 2 showed a junctional split and reduced β4 integrin (e) and α6 integrin (f) staining at the blister roof. Patient 3 presented a basal intraepidermal split and slightly reduced β4 (g) and α6 integrin (h) staining at the blister floor. Yellow asterisks mark blister cavities. Scale bar = 50 μm.

In the skin of patient 1, electron microscopy showed occasional junctional split formation and minute detachments of the basal lamina from parts of basal keratinocytes (Fig. 4a). Although hemidesmosomes appeared normal in different areas of the sample, systematic analysis revealed a slightly reduced quality and reduced number and size of hemidesmosomes, which sometimes lacked the sub-basal dense plates and the cytoplasmic plaques. Doubled basement membrane was a quite regular finding and is probably due to the continuous reconstruction process after split formation (Fig. 4b).

Figure 4.

(a) Junctional split formation detected as detachment of basal lamina from basal keratinocytes in some areas and an overall reduced quality of the hemidesmosomes with reduced size, rarefication, lack of sub-basal dense plates and cytoplasmic plaques in some places; (b) doubled basement membrane (indicated by arrows) was a quite common finding and is probably due to the continuous reconstruction process after split formation.

ITGB4 and ITGA6 mutation analysis revealed 10 previously unpublished sequence variants (HGMD Professional 2012.3) (Fig. 1). In seven cases (patients 1–7), ITGB4 mutations were found, whereas ITGA6 was involved only in patient 8, who had severe disease manifestations (Table 1).

Interestingly, the mutation c.4969_4990del, p.E1657fsX1, found in patient 1 with the mildest phenotype in this cohort, is located in the cytoplasmic domain, close to the C-terminus of integrin β4. RT-PCR showed no evidence for mRNA decay, but revealed a shortened transcript (Fig. 5a,b). Immunoblot analysis with domain-specific antibodies targeting the extracellular domain of β4 integrin showed a strongly decreased signal (Fig. 5c), probably through degradation of unstable polypeptides. The staining with antibodies targeting the intracellular domain also showed a reduced signal in immunoblot (not shown).

Figure 5.

Consequences of ITGB4 mutations in patients 1 and 4. (a) Reverse transcription polymerase chain reaction with cDNA from keratinocytes of a control (Co), patient 1 (P1) and patient 4 (P4) and primers to ITGB4 exons 2–4, 8 and 9, and 36–38, and GAPDH. Note similar levels of ITGB4 mRNA in the control and patients and a shorter amplicon spanning exons 36–38 in patient 1 (red arrow). N, negative control without DNA; M, DNA ladder. (b) Partial sequence of the amplicon spanning exons 36–38 in the control and patient 1. The deletion (Del) is marked with a red rectangle on the normal sequence, and the premature termination codon resulting from the frameshift is indicated (red line, X) on the patient's sequence. (c) Immunoblot analysis with antibodies targeting the extracellular domain of β4 integrin showed a strongly decreased amount of a truncated polypeptide in patient 1, but not in patient 4 compared with the GAPDH loading control.

The missense mutations p.C590Y, p.P200L, p.P305L, p.S306L and p.R1281P were associated with mild skin, nail and dental involvement, but extensive fragility of the urinary tract mucosa in patients 3–5. All these sequence variants affect conserved amino acids and are predicted to be ‘disease causing’ (PolyPhen-2; The amino acids P200, P305, S306 and C590 are located in the extracellular domain of integrin β4, within the von Willebrand factor type A domain, or the fourth cysteine-rich tandem repeat, whereas R1281 is located in the cytoplasmic, fibronectin type-III 2 domain (Fig. 1). Keratinocytes of patient 4, compound heterozygous for p.P305L and p.S306L, were available for analysis. RT-PCR demonstrated that the mutation affected neither splicing nor the mRNA levels (Fig. 5a), and immunoblot analysis revealed that the amount of β4 integrin in the cells of patient 4 was comparable with the control (Fig. 5c).

The mutations found in patients 2, 5, 6, 7 and 8 in ITGB4 c.2251C>T (p.R751X), c.4295_4298dup (p.T1434LfsX69), c.3809C>T (p.Q1237X), c.566 + 3_+6delAAGT and ITGA6 c.388-5T>G, respectively, presumably lead to mRNA decay and absence of protein expression.


In this study, we present a broad spectrum of clinical and morphological EB manifestations associated with 10 novel α6β4 integrin mutations. The clinical spectrum ranges from nonlethal forms with very mild skin features, with or without PA and/or genitourinary involvement, to severe lethal phenotypes. Our study adds two patients (patients 1 and 5) to cases with EB caused by ITGB4 mutations, but no gastrointestinal atresia,[9-12, 23] raising the question as to whether the designation EB-PA is appropriate. This suggestion is supported by individual case reports in the literature. Inoue et al.[10] investigated a 68-year-old patient with the homozygous ITGB4 mutation p.G931D, associated with JEB and a life-long history of trauma-induced blistering, recurrent urethral stenosis and early loss of teeth, but neither PA nor other gastrointestinal manifestations.[10] A 49-year-old patient with intraepidermal blistering on the hands and feet, pachyonychia, onychogryphosis, plantar hyperkeratosis and enamel disruption, but neither PA nor urinary tract affection, had the ITGB4 mutation c.4733delCT, which led to skipping of exon 36 and an in-frame deletion of 50 amino acids Δ1450_1499.[9] Dang et al.[11] described two siblings with JEB and compound heterozygous ITGB4 mutations c.264G>A and 3111-1G>A; PA was found in the brother, but not in the sister. Finally, Yuen et al. reported on two patients with the ITGB4 mutations c.3040C>T, p.R1014W and c.4975G>T, p.E1659X, who had onycholysis and enamel defects, but no gastrointestinal atresia or stenosis.[23]

Importantly, in our cohort of eight patients, five developed urinary tract complications including unspecific cystitis, dysuria, recurrent vesicourethral occlusion, bladder wall changes, hydronephrosis, pyelonephritis and nephroliths (Table 1). In a review of 33 cases with α6β4 integrin mutations reported in the literature, only eight patients had urological complications.[11] The frequency of urinary tract complications correlated with the severity of the skin involvement.[13] In contrast, in our study, urinary tract involvement was present in 60% of patients with only mild skin fragility (patients 3, 4 and 5), implying that urinary tract symptoms can be the major or only clinical sign. All combinations of organ involvement were observed. Urinary tract involvement persisted after the skin was no longer or only mildly affected (patients 3 and 4; Fig. 2e), or also occurred in individuals without PA (patient 5). Our findings are consistent with a recent report by Diociaiuti et al. presenting urinary tract involvement in combination with very mild skin effects.[24] Interestingly, in two cases, severe urinary tract symptoms were transient and resolved within 2 years (patients 3 and 4). Therefore, paediatricians and especially paediatric urologists should be aware of the possibility that urinary tract problems in association with minor skin blistering may represent a genetic disorder caused by α6β4 integrin mutations.

The above facts render molecular diagnostics of this group of patients challenging. In our patients immunofluorescence staining was not always conclusive, as either no cleavage (patients 1 and 4), or intraepidermal (patients 3 and 6) or junctional split (patients 2, 5, 7 and 8) was observed. The results must be evaluated with care and may even be misleading. For example, patient 1 with a truncating mutation in the cytoplasmic domain of β4 integrin exhibited ultrastructural separation along the lamina lucida. This is in contrast to a previous report on a similar mutation leading to EBS and cleavage within the basal layer.[9] In our cases, reduced integrin β4 staining was the only distinctive feature for the diagnosis (Table 1). However, in discrete cases the mildly reduced signals can easily be overlooked.

Patient history, including urinary tract symptoms and inspection of skin, teeth and nails may give a clue to underlying α6β4 integrin mutations (Fig. 2). Clinical hallmarks can help to distinguish the EB subtypes; however, PA is not a suitable criterion for prognostication – both lethal and mild courses of the disease can follow. Mutation analysis is recommended to unravel the molecular background in these challenging cases.

In conclusion, α6β4 integrin-associated EB forms can present with involvement of the skin, and the gastrointestinal and the urinary tract, but different oligosymptomatic forms also exist.[11, 25] Although mutations allowing residual protein expression seem to be associated with milder phenotypes than null mutations, clear genotype–phenotype correlations and prognostic predictions still need to be established. Our findings imply that mutations in the cytoplasmic tail of β4 integrin (p.E1657fsX1 and p.R1281P) are less disruptive for the gastrointestinal tract. Secondary modifiers may account for the phenotypic variability of this EB subtype.[11, 26] According to the current EB classification, EB-PA is associated with death in many cases (+3 and +4 subsequently on a 0 to +4 scale[1]); however, this study and others[6, 23-25] report an increasing number of patients with nonlethal forms.


We thank all patients who participated in this study. The excellent technical support by Gabriele Grüninger, Vera Morand, Margit Schubert, Nadja Chmel and Kaethe Thoma is gratefully acknowledged.