Epidermolysis bullosa (EB), the prototype of skin fragility disorders, manifests with blistering and erosions of the skin and mucous membranes (Has et al., 2020Has C. Bauer J.W. Bodemer C. Bolling M.C. Bruckner-Tuderman L. Diem A. et al.Consensus reclassification of inherited epidermolysis bullosa and other disorders with skin fragility.Br J Dermatol. 2020; 183: 614-627Crossref PubMed Scopus (253) Google Scholar; Vahidnezhad et al., 2019aVahidnezhad H. Youssefian L. Saeidian A.H. Uitto J. Phenotypic spectrum of epidermolysis bullosa: the paradigm of syndromic versus non-syndromic skin fragility disorders.J Invest Dermatol. 2019; 139: 522-527Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The classic forms of EB are associated with 16 distinct genes expressed in the cutaneous basement membrane zone (Has et al., 2020Has C. Bauer J.W. Bodemer C. Bolling M.C. Bruckner-Tuderman L. Diem A. et al.Consensus reclassification of inherited epidermolysis bullosa and other disorders with skin fragility.Br J Dermatol. 2020; 183: 614-627Crossref PubMed Scopus (253) Google Scholar). One such gene is ITGA3 encoding α3 integrin subunit, which combines with β1 subunit to form α3β1 integrin. Mutations in the ITGA3 gene have been reported in 10 cases with EB, and the characteristic feature in the majority of these patients is severe respiratory and renal involvement causing early postnatal demise (Has et al., 2012Has C. Spartà G. Kiritsi D. Weibel L. Moeller A. Vega-Warner V. et al.Integrin α3 mutations with kidney, lung, and skin disease.N Engl J Med. 2012; 366: 1508-1514Crossref PubMed Scopus (168) Google Scholar). This subtype of EB is known as EB with renal and respiratory involvement or interstitial lung disease, nephrotic syndrome, and EB (Online Mendelian Inheritance in Man #614748). The pathogenic sequence variants are characteristically loss-of-function mutations, and EB with renal and respiratory involvement has been considered to be a severe lethal condition. In this study, we report two adult males aged 45 and 30 years with EB associated with a homozygous missense mutation in ITGA3. A written informed consent was obtained from both patients to participate in this study, and they gave their permission to the publication of their images. The proband (Figure 1a, II-1 ) was a male aged 45 years with blisters and erosions that were reported from age 22 years on and limited to lower extremities (Figure 1c). He had dysmorphic facial features, distal onycholysis with nail dystrophy since birth, and loss of scalp hair and eyebrows since his teenage years, whereas eyelashes were present (Figure 1b and e). He had renal involvement with proteinuria (1.4–1.5 g/day) since he turned 26 years. The proband’s younger brother (II-5), a male aged 30 years, had similar cutaneous lesions (Figure 1d), with bullae and erosions on the lower legs, which were reported from age 8 years on. The lesions healed with atrophic scars. He also has sparse hair, a loss of eyebrows since his teenage years, and distal onycholysis since birth. At age 7 years, he was diagnosed with hydronephrosis and pyeloureteral stenosis requiring surgery. He also had surgery for congenital nasolacrimal duct obstruction. Proteinuria (2.8–3.2 g/day) was documented at age 13 years leading to end-stage renal disease, and he was placed on hemodialysis at the age of 26 years. Renal biopsy documented focal segmental glomerulosclerosis, perihilar form (Figure 1i). Electron microscopy demonstrated that glomerular structures were partially retained and were hypertrophic, with villous podocytes. Effacement of the visceral epithelial cell foot processes and microvillous transformation as well as thickened glomerular basement membrane and swollen endothelial cells, which contained myelin-like structures, were also noted (Figure 1j). Skin biopsy of the younger brother at age 17 years revealed evidence of separation of the epidermis from the dermis at the level of dermal‒epidermal junction (Figure 1f). Transmission electron microscopy revealed thin and fragmented tonofilaments with electron-dense patches. Hemidesmosomes were numerous, mostly of normal size (Figure 1g). There was focal widening within the lamina lucida, cleavage within the lower pole of keratinocytes, and widening of the spaces between the basal cells. The parents (deceased) or the children of the proband had no clinical findings suggestive of EB or renal disease. Consequently, the diagnosis of a syndromic form of EB with autosomal recessive inheritance was made. Because the clinical features of our patients were very similar to those encountered in patients with mutations in the CD151 gene (Vahidnezhad et al., 2018Vahidnezhad H. Youssefian L. Saeidian A.H. Mahmoudi H. Touati A. Abiri M. et al.Recessive mutation in tetraspanin CD151 causes Kindler syndrome-like epidermolysis bullosa with multi-systemic manifestations including nephropathy.Matrix Biol. 2018; 66: 22-33Crossref PubMed Scopus (41) Google Scholar), immunostaining of a skin biopsy with an antibody recognizing CD151 was performed, and it revealed altered staining of this protein with a linear pattern along the basement membrane zone, compared with the peripheral immunoreactivity of the basal keratinocytes in control skin (Figure 1h). Omission of the primary antibody revealed no staining (not shown). However, RT-PCR amplification of exonic and flanking intronic sequences of CD151 followed by Sanger sequencing failed to identify pathogenic mutations in this gene. Subsequently, DNA isolated from the peripheral blood of the proband was subjected to whole-exome sequencing, which identified 76,648 sequence variants (Figure 2b ). No pathogenic variants were identified in CD151. Filtering of the variants by bioinformatics steps indicated in Figure 2b reduced the total number of candidate variants to 216. Considering the possibility that our patients have homozygous mutations owing to restricted ethnic background (Roma), homozygosity mapping of the proband’s DNA was performed on the basis of the nucleotide sequence data derived from whole-exome sequencing (Vahidnezhad et al., 2019bVahidnezhad H. Youssefian L. Saeidian A.H. Zeinali S. Touati A. Abiri M. et al.Genome-wide single nucleotide polymorphism-based autozygosity mapping facilitates identification of mutations in consanguineous families with epidermolysis bullosa.Exp Dermatol. 2019; 28: 1118-1121Crossref PubMed Scopus (24) Google Scholar). Four runs of homozygosity >4 megabases were found (Figure 2a). Superimposing the 216 candidate variants with the homozygosity map revealed that only one of them overlapped with a run of homozygosity, one of 13.7 megabases within chromosome 17 (Figure 2a). Examination of the sequence data revealed a homozygous ITGA3 mutation NM_002204: exon 25:c.3056T>C:p.Phe1019Ser. This variant was confirmed to be homozygous in the two patients and heterozygous in the parents by Sanger sequencing (Figure 2c). This missense mutation was predicted by bioinformatics programs, including Sorting Intolerant From Tolerant, to be damaging with a Combined Annotation-Dependent Depletion score of 20.3. Its count in homozygous and heterozygous states in 172,424 healthy individuals (gnomAD r2.1) was zero and one, respectively, yielding an allele frequency of 0.0000058. Immunostaining of the proband’s skin revealed an altered pattern of CD151, which could be explained by perturbed protein–protein interactions between CD151 and α3β1 integrin, both being transmembrane proteins as part of focal adhesion complexes (Berditchevski et al., 2001Berditchevski F. Gilbert E. Griffiths M.R. Fitter S. Ashman L. Jenner S.J. Analysis of the CD151-alpha3beta1 integrin and CD151-tetraspanin interactions by mutagenesis.J Biol Chem. 2001; 276: 41165-41174Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) (Figure 2d). Loss of these interactions may render CD151 unstable and susceptible to degradation, supported by the observation that the Phe1019 residue is evolutionarily conserved in α3 integrin between human and rat (Figure 2e). Interestingly, all previously published ITGA3 mutations reside within the large extracellular segment of the α3 integrin polypeptide, which interacts not only with CD151 but also with the G domain of the α3 polypeptide subunit of laminin 332 (Figure 2d). Thus, mutations in either CD151 or ITGA3 can result in a similar blistering phenotype in the spectrum of EB. Although most patients with interstitial lung disease, nephrotic syndrome, and EB die in early childhood, ITGA3 mutations were previously reported in two siblings aged 13 and 9 years with skin manifestations and pulmonary involvement but without nephrotic impairment (Colombo et al., 2016Colombo E.A. Spaccini L. Volpi L. Negri G. Cittaro D. Lazarevic D. et al.Viable phenotype of ILNEB syndrome without nephrotic impairment in siblings heterozygous for unreported integrin alpha3 mutations.Orphanet J Rare Dis. 2016; 11: 136Crossref PubMed Scopus (19) Google Scholar). These patients were compound heterozygous for missense variants, p.Gly125Arg and p.Arg274Gln. In addition, delayed presentation of respiratory and renal symptoms and prolonged survival were reported in a patient who died at age 9 years (Tarur et al., 2020Tarur S.U. Srinivasan S. Seeralar A. Delayed presentation of respiratory symptoms and prolonged survival in homozygous a3 integrin deficiency.Indian Pediatr. 2020; 57: 268-269Crossref PubMed Scopus (4) Google Scholar). This patient had a homozygous splice site mutation ITGA3: c.1825-1G>A; p.Val609Serfs∗31. Finally, a male patient in his late teens with cutaneous findings similar to those of our patients but without renal and respiratory involvement was recently reported (Cohen-Barak et al., 2019Cohen-Barak E. Danial-Farran N. Khayat M. Chervinsky E. Nevet J.M. Ziv M. et al.A nonjunctional, nonsyndromic case of junctional epidermolysis bullosa with renal and respiratory involvement.JAMA Dermatol. 2019; 155: 498-500Crossref PubMed Scopus (7) Google Scholar). Whole-exome sequencing identified a homozygous missense mutation p.Arg274Gln in ITGA3. In the case of our patients, at ages 45 and 30 years, no evidence of lung disease was noted by routine X-rays, and there was no clinical evidence of pulmonary involvement. Thus, the absence of pulmonary involvement may explain the survival of our patients beyond the early postnatal period and subsequent development into adulthood. Datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/sra/, hosted at National Library of Medicine Sequence Read Archive with submission number SUB9249514. Ágnes Kinyó: https://orcid.org/0000-0002-9827-2690 András László Kovács: https://orcid.org/0000-0002-2620-1172 Péter Degrell: https://orcid.org/0000-0002-0198-0333 Endre Kálmán: https://orcid.org/0000-0002-3995-9732 Nikoletta Nagy: https://orcid.org/0000-0001-8576-7953 Sarolta Kárpáti: https://orcid.org/0000-0002-8472-0712 Rolland Gyulai: https://orcid.org/0000-0002-3286-8846 Amir Hossein Saeidian: https://orcid.org/0000-0003-3512-0654 Leila Youssefian: https://orcid.org/0000-0002-4253-6503 Hassan Vahidnezhad: https://orcid.org/0000-0003-4298-9147 Jouni Uitto: https://orcid.org/0000-0003-4639-807X The authors state no conflict of interest. HV should be considered a co-corresponding author to whom questions of the technical aspects of next-generation sequencing should be addressed. The authors would like to acknowledge the contributions of Katalin Farkas, Márta Medvecz, and Irwin McLean. Carol Kelly assisted in manuscript preparation. This study was approved by the Institutional Review Boards of Thomas Jefferson University (Philadelphia, PA) and the University of Pécs (Hungary). This study was supported by Debra International. Conceptualization: AK, HV, JU; Data Curation: PD, EK, RG; Formal Analysis: AHS, LY; Funding Acquisition: AK, JU; Investigation: AK, ALK, NN, SK, AHS, LY
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