Epidermolysis Bullosa (EB) contemplates a group of genetic diseases that are characterized by severe skin fragility and recurrent blistering.1,2 Most cases begin soon after birth or in early childhood, although the age of onset of the skin lesions is variable. Skin lesions usually occur due to minimal trauma. Since in EB, the basement membrane is fragile and easily damaged, there is constant flux and subsequent accumulation of extracellular fluid in the cleavage generated through the epidermis.

Overall, EB comprises a group of rare congenital disorders caused by pathogenic variants in nearly 20 genes related to the expression of proteins related to skin adherence, especially those located in the basement membrane zone. The prevalence is estimated to be approximately 8.22:1,000,000 and the incidence in the USA is considered to be 19.6:1,000,000 newborns, with similar numbers in other countries and apparently no major distinction between ethnicities. Over 30 different types of EB have been described, with a recent attempt to divide them into four large groups (2020): EB Simplex (EBS), Dystrophic EB (DEB), Junctional EB (JEB) and hybrid form, also known as Kindler Syndrome (KEB). Such classification is based mainly on inheritance pattern (autosomal dominant or recessive), severity of phenotype, and histological/immunomapping analysis.

In earlier times, electronic microscopic analysis, and afterward immunomapping through a skin biopsy, were the former gold standard for EB diagnosis. Even though either can identify the cutaneous level in which the cleavage of the blisters resides (intradermal or subepidermal) most of the time, both are usually unable to indicate with certainty the affected gene and, thus, the inheritance pattern of each patient. Besides that, skin biopsy is an invasive procedure (with the need for local anesthesia and subsequent suture) and when its results are inconclusive, there is often a delay and need for a second sample, which can be particularly problematic in EB patients.3

In this scenario, molecular analysis has become an important tool for a more precise diagnosis of EB, especially given the recent consolidation and easier access to NGS techniques.4–8 Here we compare the results of immunomapping and WES of a Brazilian cohort of 67 patients with EB.

There was a clear predominance of DEB (associated with a mutation in COL7A1 gene), with 51 of the 67 patients (76%) presenting this type of disease, which usually represents a more severe phenotype, especially in the autosomal recessive form. Considering that the patients in this study were attending the group of Pain and Palliative Care, an elevated frequency of severeness was already expected; still, the total count of deaths under 20 years stands apart from the reported Brazilian and global death rates, perhaps indicating the lack of proper intense care support for these patients in some local hospitals. Other than that, phenotypes were similar to the ones described in the literature for each respective gene.

Regarding familiar history, it is important to point out that it was somewhat biased, given that 11 of the 60 families presented some level of consanguinity; notoriously, 8 of these cases presented homozygous mutations in the COL7A1 gene. Still, autosomal recessive forms of the disease were the majority even in the families without consanguinity, with 41 families presenting variants in compound heterozygosis (meaning the patient received a different deleterious variant in the same gene from each health parent). As expected for autosomal dominant forms, there was a majority of 6 cases of de novo mutations in heterozygosis, not inherited from either parent; vertical transmission was observed in only 2 families, meaning that the child and its father or mother were both affected.

In earlier times, electronic microscopic analysis had been the former gold standard for EB diagnosis, but its expressive costs and difficulty of access (few centers have this equipment at disposition and perhaps even fewer people have experience using it) gradually determined its decline.

As mentioned before, until recently the gold standard for diagnosis has been immunomapping through skin biopsy. Even though it can usually identify the cutaneous level in which the cleavage of the blisters resides (intradermal or subepidermal), it is, however, unable to indicate with certainty the affected gene and, thus, the inheritance pattern of each patient. Besides that, it is an invasive procedure (with the need for local anesthesia and subsequent sutures) and its results are rarely inconclusive, with often a need for a second skin biopsy, which can be particularly problematic in EB patients.

Nowadays, molecular analysis has become an important tool for a more precise diagnosis of EB. Unsurprisingly, its use has been strongly recommended not only due to its precision and potential lesser cost but also because it would avoid the harm of damaging the skin in such vulnerable patients since it works fine with just one drop of blood or even a spit of saliva from the patient.

Nevertheless, it is important to point out a few disadvantages regarding the genetic diagnosis of EB. First of all, more generalized methods and insufficient resources dedicated to molecular testing, particularly in distant regions. Other than that, there is the fact that there are at least 18 genes associated with the various types of EB, with over 1000 different mutations described; previous approaches with Sanger sequencing of one gene or even specific known mutations could not warrant confirmation of etiology, often representing a long and more expensive path to diagnosis.

Fortunately, the advances in NGS technique can now provide simultaneous analysis of one or hundreds of genes not only with the same cost but also in a much faster and more automatic way; besides, the possibility of collecting blood samples in primary health centers or even the use saliva kits at home, both which can be rather simply sent through the mail to reference facilities, represents an obvious expansion of the range of testing.

Here it is important to point out that Whole-Exome Sequencing (WES) analyzes not only genes related with EB, but actually all human genes. This of course represents a higher cost for the test when compared to a panel, but still, in the authors’ experience, it can often be more difficult for laboratories to establish a cheaper focused NGS panel for one single condition rather than offering WES for several patients with various genetic hypothesis (from intellectual disability to short stature). Considering all that, even though the present study used WES for genetic analysis, the authors believe these results can be applied to all NGS techniques since germline mutations in genes related to EB would be detected by either WES or a focused NGS panel.

Despite being considered more expensive than immunomapping at first, it is important to express that NGS costs have kept constantly fallen over the last years. The potential cost-effectiveness of the NGS panel focused in genes related with EB for diagnosis in a country with continental dimensions such as Brazil has already been suggested by another recent study.7

At last, the potential application of gene therapy in a rare and, so far, incurable disable such as EB cannot be undermined. Despite the numerous challenges on this path, more than one research has shown promising results in the use of these new therapeutic approaches9–11 in patients with different types of EB. As the knowledge of the genotype is absolutely necessary for such selected cases, once again the authors highlight that immunomapping alone would provide insufficient information in order to decide if each patient is eligible or not for specific treatment. On a final note, considering the growing availability and demand of IVF (in vitro fertilization),12 genetic diagnosis of EB also becomes all the more necessary.

EB contemplates a group of genetic skin diseases that are marked by severe fragility and recurrent blistering. As a rare congenital disorder caused by mutations in multiple genes, proper diagnosis can be challenging for a number of reasons.

Although immunomapping has been useful in services where molecular studies are not available, genetic analysis performed through NGS has shown a higher tax of diagnosis. Besides, all positive results will lead to the confirmation of the inheritance pattern of each family, which often immunomapping cannot predict. Additionally, in the not-so-distant future, knowledge of the genotype will be necessary in order to decide if each patient is eligible or not for specific forms of gene therapy, and also assisted reproduction techniques.

Finally, we conclude that the molecular study (either with NGS focused panel or WES) is cost-effective for the diagnosis of EB, not only due to its high efficiency and accuracy but also because it eliminates the need for skin biopsy. Thus, we consider it will provide the patients overall better prognosis, prenatal genetic diagnosis, and genetic counseling.

Kim CA CNPQ304897/2020-5; FAPESP2019/21644-0.

Matsumoto N - Supported by the Japan Agency for Medical Research and Development (AMED) [grant numbers JP22ek0109486, JP22ek0109549, JP22ek0109493]; the Takeda Science Foundation.

Samantha Vernaschi Kelmann: Contributed largely to the conception and design of the article, as well as the dermatological evaluation and interpretation of data.

Bruno de Oliveira Stephan: Contributed largely to the conception and design of the article, as well as the genetic analysis and interpretation of data.

Silvia Maria de Macedo Barbosa: Contributed to the clinical follow-up and management of patients.

Rita Tiziana Verardo Polastrini: Contributed to the clinical follow-up and management of patients.

Zilda Najjar Prado de Oliveira: Contributed to the execution and interpretation of immunomapping.

Maria Cecília Rivitti-Machado: Contributed to the execution and interpretation of immunomapping.

Gustavo Marquezani Spolador: Contributed to the genetic counseling and management of patients.

Rachel Sayuri Honjo: Contributed to the draft and critical revision of the article.

Ken Saida: Contributed to the execution and interpretation of the molecular testing.

Naomichi Matsumoto: Contributed to the execution and interpretation of the molecular testing.

Chong Ae Kim: Contributed to the draft and critical revision of the article.

None declared.