The present study was approved by the Research Ethics Committee of the Universidade Federal do Rio Grande do Sul (project 31608) and by Plataforma Brasil (CAAE 2,481,885).

The NGS gene panel was designed to be specific for EB, including the vast majority of cases of the disease at a viable cost for the Brazilian reality. Thus, the 11 main genes associated with the four types of EB were included in the panel (KRT5, KRT14, PLEC, TGM5, LAMA3, LAMB3, LAMC2, COL17A1, ITGB4, COL7A1, and FERMT1). The 62.72-kb and 574-amplicon panel was designed using the AmpliSeq Designer tool (Thermo Fisher Scientific – United States). Ion Torrent technology (Thermo Fisher Scientific – United States) was used for sequencing. Information about the running parameters, data analysis, investigation, classification, and details of the identified variants were described in a previous study.13

In order to obtain an estimate of the amount to be invested in the execution of IFM, TEM, and gene panel sequencing, a price survey was carried out in Brazilian hospitals/laboratories that perform each of the methods. For the TEM technique, a survey was carried out in laboratories in the United States, due to the lack of Brazilian laboratories that perform the method for the diagnosis of EB. Since the cost of gene panel sequencing is dependent on the number of samples to be analyzed, for calculation purposes, the price estimate took into account a number of 20 samples. The characteristics of each method, as well as their inherent advantages and disadvantages, have been reviewed in the specialized literature.

IFM and TEM are, historically, the most used methods for the diagnosis of EB and allow the determination of the level of cleavage in the skin and the consequent classification of the EB subtype.5 Although they have diagnostic accuracy for the main types of EB, these methods do not indicate the mode of genetic inheritance and depend on experience for analysis, being subject to frequent inaccurate or inconclusive results (Table 1).3,14 TEM, in particular, is a very laborious technique that requires notable technical knowledge and is therefore very little used worldwide. The identification of the associated pathogenic variant is the most definitive and precise criterion for the diagnosis of EB. The sequencing of candidate genes by the Sanger method is, therefore, performed in specialized centers, after IFM and/or TEM.5,14 Although informative in most cases, the combination of IFM, TEM, and Sanger sequencing methods fails in approximately 15% of cases, either due to uncertainty in the clinical examination or due to intrinsic limitations of the techniques.8 Moreover, this combined approach can be costly and requires a great deal of work, since several of the genes associated with EB contain multiple exons, such as COL7A1 (118 exons) and COL17A1 (56 exons).8

Unlike Sanger sequencing, the NGS gene panel allows the simultaneous sequencing of a large number of genes with a known association with the disease,1 waiving the need of previous determination of candidate genes. Genetic analysis by gene panel is fast, highly accurate, and conclusive for the diagnosis of EB, without requiring previous tests such as IFM and TEM (Table 1).

The IFM and TEM methods require skin biopsy, usually performed on a recent intact blister. This examination is in itself highly invasive, but in patients with EB, who have frail skin, it becomes even more aggressive. Furthermore, the need for repeated biopsy due to inconclusive results is not uncommon. Since NGS analyses do not require, can be performed on a saliva sample, and provide diagnosis with speed and high precision, some authors have advocated rethinking current methods for assessing patients with EB.15 The identification of causal variants associated with EB allows the indication of the prognosis of the disease, prenatal and pre-implantation genetic diagnosis, and precise genetic counseling.3

Although NGS sequencing is a powerful tool for genetic diagnosis, some limitations must be considered. The main challenge concerns bioinformatics analyses, which are essential for the identification of genetic variants and, even more important, for indicating their causality.8 In this sense, the interpretation of variants of uncertain meaning can be quite difficult, causing dilemmas for clinicians and uncertainties on how to inform patients and provide appropriate genetic counseling. In addition, large rearrangements and repetitive sequences are complex to analyze by NGS and, therefore, are often not correctly detected. Thus, it is important to note that, although highly effective, NGS technology cannot solve all cases of genetic diseases.

The gene panel developed by the present group showed high efficiency (94.3%) in the studied sample, when compared with the results obtained by other researchers who used panels with a larger number of genes.10–12 The choice of the 11 main genes associated with EB was made in order to establish an economically viable methodology, although other genes were also reported in the literature.2,3 Although the panel developed fails to diagnose rare cases associated with other genes (such as STD, EXPH5, KLHL24, CD151, ITGA3, and ITGA6), the efficiency obtained here demonstrates that even a smaller gene panel can be of remarkable relevance for the diagnosis of EB. It should be noted that the NGS methodology does not allow for an adequate identification of large genetic rearrangements, which presupposes that even a panel including all the genes already associated with EB does not guarantee 100% efficiency.

Aside from the immediate benefits, genetic analysis can be an essential tool for the development and employment of EB treatment. Although there is currently no treatment, recent preclinical studies have suggested potential therapies for EB, many of them dependent on the associated pathogenic variant.16–20 The advancement in genetic diagnosis through NGS technology, together with the development of new molecular therapies, promises to bring significant benefits to this group of patients.21 Although it is not a current reality, it is possible that, in the not far away future, patients with specific pathogenic variants may be treated.

Although the estimated cost per sample for the gene panel (R$ 800.00) is higher when compared with that of IFM (R$ 500.00), it is considerably lower than that of TEM (US$ 980.00; R$ 3,920.00). However, it is important to note that the cost of TEM was based on prices charged in laboratories in the United States, since, to the best of the authors’ knowledge, the method is not offered commercially in Brazil. Although some Brazilian universities have an electron microscope, the method used to diagnose the disease is used in these academic centers only, for cases of specific scientific interest. Sanger sequencing has a mean price of R$ 35.00 per analyzed gene fragment (usually an exon), per sample. Thus, the final value of this methodology depends on the number of exons of the analyzed genes. Hypothetically, if an individual with dystrophic EB (associated with the COL7A1 gene) was analyzed by Sanger sequencing, a value of approximately R$ 2,520.00 would be invested with this methodology alone (taking into account a number of 72 pairs of primers that include the 118 exons of the gene, as described by Christiano et al.22).

Thus, although the gene panel is not a low-cost method, the result obtained from it is more informative and conclusive than that obtained by IFM and TEM. In comparison to Sanger sequencing, the cost is lower, as well as the processing time, showing the cost-effectiveness of the gene panel for the diagnosis of EB. In addition, the price of this methodology has been decreasing over time, which should make the method more accessible in the coming years.10

It is worth mentioning, however, that the estimated cost in this study for diagnosis through the gene panel did not include the panel purchase price (approximately R$ 20,000.00) and the investment to validate it. The present proposal is that genetic diagnosis is provided in the context of public health, aiming, in the long term, to include the largest number of patients with EB in Brazil. Thus, as more patients are analyzed, the cost invested in the panel, per patient, decreases to the point of being negligible. In addition, the cost to be invested in an analyst with expertise to assess the results was not added to the calculation. In the authors’ experience, in an academic context, this question doesn’t apply; however, it should be taken into account before providing the method in the scope of public assistance.

The diagnosis of EB in Brazil is made, in most cases, through the evaluation of the clinical signs presented. There are few specialized centers that offer EB diagnostic methods, which results in difficulties in diagnosing and subclassifying patients and, consequently, providing correct genetic monitoring and counseling.

Even when available, IFM often results in inconclusive results, due to the lack of experienced analysts, especially in poorer and more remote Brazilian regions. Taking into account the damage caused by skin biopsy exams and the fact that most patients do not have the appropriate care after the exam (including appropriate dressings and healthcare), some physicians choose not to indicate IFM to subclassify EB. Consequently, the precise diagnosis and definition of the type of EB rely on a complete clinical examination. However, even when performed by dermatologists specialized in EB, clinical examination is not sufficient to solve all cases, in view of the extreme phenotypic variability observed. Table 2 presents an example of a clinical case whose genetic result did not coincide with the previous clinical hypothesis, surprising dermatologists. Similar cases have been described in previous studies and indicated that the effect of genetic variants may result in atypical clinical phenotypes.23,24

In the authors’ experience in Brazil, in most cases an accurate diagnosis of the type of associated EB was only possible after the genetic results, which in turn allowed for correct genetic counseling. In addition, some peculiarities of the country are also important. The rates of endogamy and consanguinity are higher in some Brazilian populations, especially in the Northeast, suggesting a high prevalence of recessive genetic inheritance.25,26 In fact, in a previous study, the authors found that 82.7% of Brazilian patients with DEB subtypes had recessive inheritance.13 Determining the pattern of inheritance is especially important for the diagnosis of localized DEB subtypes, since the dominant and recessive forms are clinically indistinguishable, hindering correct counseling in the absence of genetic testing.

Considering the difficulty of making an accurate clinical diagnosis and, particularly, the complexity of classifying EB, genetic results proved to be essential for the correct determination of the type of associated EB.

The survey carried out in this study demonstrated that gene panel is an effective tool for the conclusive diagnosis of EB, with high efficiency, diagnostic precision, and reduced analysis time (approximately ten days), being economically viable and waiving the need for invasive tests (Fig. 1). Some factors, however, are paramount for the effectiveness of the method. Processing the results of the gene panel requires support from bioinformatics, and the time taken to analyze the variants depends on the researcher's experience. However, when compared with the methods of IFM and TEM, the interpretation of the results of the gene panel is simpler, not requiring a team with extensive expertise.3

In light of the greater diagnostic efficiency, the robustness of the results obtained, the price comparable to the techniques currently adopted, and the possibility of genetic counseling, prognosis, and prenatal diagnosis, the present study supports the use of the gene panel as the first choice for the diagnosis of EB in Brazil. Taking into account the continental dimensions of the Brazilian territory and the difficulty of introducing a detailed technique such as the gene panel, the establishment of a reference center for the diagnosis of EB in the country would bring many benefits. The centralization of analyses in a single place eliminates the need to create multiple specialized teams and reduces the cost of processing per sample.

The difficulty of diagnosing patients with EB in Brazil can be mitigated with the methodological approach proposed in the present study. For this plan to become a reality, financial resources and government support are needed. It is essential that the authorities pay attention to this group of patients who have a serious genetic disease and who, although rare, represent a group of more than 900 Brazilians.