Childhood Epidermolysis Bullosa Acquisita: Confirmation of Diagnosis by Skin Deficient in Type VII Collagen, Enzyme-linked Immunosorbent Assay, and Immunoblotting

Abstract

Epidermolysis bullosa acquisita (EBA) is an acquired subepidermal bullous disorder characterized by autoantibodies against Type VII collagen. It usually affects adults; childhood EBA is rare. We describe a 10-year-old girl presenting with recurrent tense blisters predominantly on legs, dorsa of hands and feet accompanied by oral erosions since the age of 5 years. Direct immunofluorescence (IF) microscopy showed linear deposition of IgG and C3 along the basement membrane zone (BMZ); indirect IF microscopy on salt-split skin revealed staining of IgG to the dermal side of the split. The patient's serum did not show BMZ staining in recessive dystrophic epidermolysis bullosa skin deficient for Type VII collagen, thus confirming autoantibody reactivity against Type VII collagen. Circulating antibodies against the immunodominant noncollagenous 1 domain of Type VII collagen were detected by ELISA and immunoblotting studies. The patient was treated with oral corticosteroids and dapsone with good improvement.

What was known?

• Epidermolysis bullosa acquisita (EBA) is an acquired, subepidermal, and immunobullous disease characterized by autoantibodies against Type VII collagen

• It is typically seen in adults. Childhood EBA is rare

• Autoantibodies in childhood EBA usually target triple-helical domain of Type VII collagen.

Introduction

Epidermolysis bullosa acquisita (EBA) is an acquired, chronic subepidermal bullous disease affecting the skin and mucosa.[1] It is characterized by autoantibodies to Type VII collagen, the major component of anchoring fibrils that attaches the epidermis to the dermis.[2] The two main phenotypic presentations of EBA are the classic mechanobullous and the inflammatory vesiculobullous type. The mechanobullous phenotype is characterized by noninflammatory blisters on trauma-prone areas such as elbows, knees as well as the dorsum of hands and feet that often heals with scarring and milia formation. On the other hand, the inflammatory form of EBA manifests as widespread blisters on an erythematous base that mimics other bullous diseases, including bullous pemphigoid (BP). Some patients may have a mixed phenotype.[3]

Histopathology of lesional skin shows a subepidermal blister with varying degree of dermal inflammatory infiltrate, depending on the clinical phenotype.[3] By direct immunofluorescence (IF) microscopy of perilesional skin, linear IgG and/or C3 deposits are detected along the basement membrane zone (BMZ). Indirect IF microscopy on 1 mol/L NaCl-split skin demonstrates circulating autoantibodies that localize to the dermal side of the artificially split skin.[4]

EBA mainly affects adults. It was initially thought to be rare in children;[5] however, an increasing number of cases of childhood EBA have been reported over the last few years.[6,7,8] We here report a child with the mechanobullous phenotype of EBA with autoantibodies targeting the noncollagenous 1 domain (NC1) of Type VII collagen.

Case Report

A 10-year-old girl, born out of nonconsanguineous marriage, presented with recurrent blisters all over the body since the age of 5 years. Blisters developed initially on the legs and gradually progressed to involve the trunk and the upper limbs. Birth and development history were unremarkable, and none of her family members had a similar skin problem. Examination revealed multiple tense blisters (clear as well as hemorrhagic), crusted ulcers and erosions as well as milia, predominantly over legs, and dorsa of hands and feet [Figure 1]. In the oral cavity, erosions on the hard palate and buccal mucosa were found.

Figure 1.

Tense blisters, crusts, and milia (indicated by arrows) on the (a) dorsum of hands and (b) on lower legs

Direct IF microscopy of a perilesional skin biopsy revealed linear staining of IgG and C3 at the BMZ. Indirect IF microscopy on 1 M NaCl-split skin showed binding of IgG along the dermal side of the split at a titer of 1:100 [Figure 2]. When the serum was treated with a panel of hereditary epidermolysis bullosa (EB) skin samples, no BMZ reactivity was seen with recessive dystrophic EB (RDEB) skin deficient for Type VII collagen [Figure 3]. This indicated that autoantibodies in our patient indeed target Type VII collagen. Enzyme-linked immunosorbent assay (ELISA) based on the recombinant immunodominant NC1 domain of Type VII collagen was positive (59 U/ml). By immunoblotting with dermal extracts and the recombinant NC1 domain of Type VII collagen, a 290 kDa band and 145 kDa band were recognized by our patient's serum, respectively [Figure 4]. She was treated with prednisolone 1 mg/kg body weight and dapsone 50 mg/day with good improvement of skin and mucous membrane lesions.

Figure 2.

Direct immunofluorescence microscopy showing linear deposits of IgG at the basement membrane zone (a); indirect immunofluorescence microscopy on salt-split skin revealing deposits of IgG on the dermal side of the split (b) (fluorescein isothiocyanate, ×200)

Figure 3.

Indirect immunofluorescence on salt split skin with patient's sera showing floor binding pattern (a); modified indirect immunofluorescence using patient's sera and recessive dystrophic epidermolysis bullosa skin as a substrate showing absence of basement membrane zone staining (b) (fluorescein isothiocyanate, ×200)

Figure 4.

Western blotting of the patient's serum with dermal extract showing binding, like in a control epidermolysis bullosa acquisita patient, to a 290 kDa band representing full-length Type VII collagen (upper arrow). Another control serum from a patient with anti-p200 pemphigoid reacts with a 200 kDa protein (lower arrow) (a). Western bot analysis with the recombinant NC1 domain of Type VII collagen shows reactivity with this 145 kDa protein in both an epidermolysis bullosa acquisita control and the patient's serum (b)

Discussion

EBA is a rare disease with a prevalence of approximately 0.2 per million people.[3] It typically starts in the fourth or fifth decade of life; childhood EBA (16 years old or younger) is rare and <50 cases have been reported in the literature.[3] The inflammatory vesiculobullous phenotype is the most common mode of presentation of EBA both in adult as well as in childhood.[5,6,8,9,10,11]

There are some differences between EBA in adults and children. Mucosal involvement is more common in children as compared to adults, and the prognosis in childhood EBA seems to be much better than in adult EBA. The adult form of the disease is known to be difficult to treat in contrast to childhood EBA, which usually responds well to dapsone and low-dose prednisolone.[5,6,8,9,10,12,13] Our patient presented with the mechanobullous phenotype with tense blisters over trauma-prone areas which healed with scarring, atrophy, and milia formation.

The routine screening test for diagnosing EBA is direct and indirect IF microscopy; particularly, indirect IF microscopy on salt-split skin can distinguish EBA from BP. Patients with EBA have immune deposits on the dermal side of salt-split skin (“floor pattern”), whereas in BP, the deposits are seen on the epidermal side (“roof pattern”). This technique, however, does not distinguish EBA from other “floor” binding diseases such as anti-p200 pemphigoid and anti-laminin 332 pemphigoid.[14,15] In thin sections and with higher magnification (≤600-fold), labeling of the BMZ appears as a u-serrated pattern with arches open at the top in EBA skin.[16,17] If this method, as in our patient, cannot be applied, autoantigen identification using a panel of EB skin as the substrate may help to confirm the diagnosis. The advantage of this test is that it can be carried out in any laboratory with a facility for IF microscopy.[18] Our patient's serum did not demonstrate BMZ staining in RDEB skin deficient in type VII collagen, thus confirming the diagnosis of EBA.

Type VII collagen consists of a triple-helical domain flanked by the large 145 kDa NC1 amino-terminal and a small 34 kDa noncollagenous carboxyl-terminal (NC2) domain.[9] Mayuzumi et al. reviewed 6 cases of childhood EBA in whom immunoblotting was performed; 2 children showed immunoreactivity against all 3 domains of Type VII collagen while 2 other demonstrated reactivity against the triple-helical and NC2 domains only. One patient had antibodies targeting triple-helical domain only while the other child had antibodies directed against NC1 and NC2 domain of Type VII collagen.[9] This is in contrast to adult cases with classical EBA where antibodies are directed against NC1.[19]

Recently, several ELISA systems based on the immunodominant NC1 domain of Type VII collagen (alone or in combination with NC2) have been established with high sensitivity (66–100%) and specificity (98.2–100%).[20,21] These ELISA systems are useful both for the diagnosis of EBA and evaluation of disease severity.[14,22] In our case, ELISA with the recombinant NC1 domain of Type VII collagen confirmed the diagnosis of EBA.

In this report, we describe a case of childhood EBA that presented with the classical mechanobullous phenotype. The diagnosis was confirmed by a combination of IF microscopy, antigen identification using EB skin, ELISA, and immunoblotting. Although IF tests on organ sections are useful for the diagnosis of EBA, target antigens can be detected by indirect IF microscopy on EB skin, ELISA, and immunoblotting. Among them, antigen identification using EB skin is a simple procedure which can be carried out in any laboratory with a facility for IF microscopy.[18] However, availability of EB skin may be a limiting factor. On the other hand, though immunoblotting is technically more demanding and expensive, it is useful to identify target epitopes within Type VII collagen.[14] Commercially available ELISA and indirect IF tests based on the recombinant NC1 domain of Type VII collagen have recently produced promising results.[21]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

What is new?

• We report a case of childhood EBA with mechanobullous phenotype

• Final diagnosis was confirmed by a combination of tests which included indirect immunofluorescence microscopy, antigen identification using skin deficient in Type VII collagen, ELISA, and immunoblotting

• Immunoblotting studies in our case revealed antibodies targeting only NC1 domain of Type VII collagen in contrast to previous reports of childhood EBA.