Molecular epidemiology of junctional epidermolysis bullosa: discovery of novel and frequent LAMB3 mutations in Chilean patients with diagnostic significance

Dear Editor, Epidermolysis bullosa (EB) is an inherited disease encompassing a group of clinically and genetically heterogeneous disorders. It is characterized by mechanical fragility of epithelial tissue with blistering formation following minor trauma, most notably on the skin.1 The junctional form of EB (JEB, OMIM #226650 and #226700) is a monogenic autosomal recessive disorder characterized by blistering within the lamina lucida. JEB, among all EB types, can have the most severe clinical manifestations and the highest risk of infant mortality.2 To date, hundreds of mutations in six different genes have been described as causing JEB, provoking limitations for an assertive molecular diagnosis. However, although there is high allele diversity in patients with JEB, it has been shown that some populations have their own frequent and specific mutations.3 To date, more than 40 clinical trials have been conducted on different groups of patients with EB, including patients with JEB. However, because EB is such a heterogeneous disease, a successful therapy must be specifically designed for each patient, a concept also known as precision medicine. Thus, a genetic diagnosis is crucial for personalized therapy. In this study, we present the epidemiological and demographic data of 23 patients diagnosed with JEB in Chile over the last 19 years. Molecular data are also presented for 19 of the 23 patients, corresponding to 17 of the 19 distinct families of our cohort (Table 1). This study has been approved by three ethics committees following the Declaration of Helsinki protocols: (i) Facultad de Medicina, Clínica Alemana Universidad del Desarrollo; (ii) Facultad de Odontología, Universidad de Chile and (iii) Metropolitano Sur Oriente Health Service in Santiago. Overview of the 23 Chilean patients with junctional epidermolysis bullosa studied: demographics, protein expression and molecular characteristics GB3, antibody against laminin‐332; β3 chain, antibody against the laminin β3 chain; F, female; M, male; RM, Región Metropolitana; ND, not done. Only 19 of the 23 patients listed in this table were screened for mutations due to DNA availability. For patients 8, 13, 16 and 23 there are only epidemiological data. No mutations were found in patient 2. LAMB3 mutations were annotated using the MN_000228.2 reference sequence. aAge at death. Overview of the 23 Chilean patients with junctional epidermolysis bullosa studied: demographics, protein expression and molecular characteristics GB3, antibody against laminin‐332; β3 chain, antibody against the laminin β3 chain; F, female; M, male; RM, Región Metropolitana; ND, not done. Only 19 of the 23 patients listed in this table were screened for mutations due to DNA availability. For patients 8, 13, 16 and 23 there are only epidemiological data. No mutations were found in patient 2. LAMB3 mutations were annotated using the MN_000228.2 reference sequence. aAge at death. Based on the DEBRA Chile EB registry, between 1996 and March 2015 a total of 23 patients were diagnosed with JEB, and 19 of those were born in that period, resulting in a JEB incidence of 4 new cases per million live births. The Chilean JEB incidence is almost twice as high as that observed in the U.S.A.,4 and thirteen times higher than in Northern Ireland.5 This difference in JEB incidence between populations might be explained by genetic background and consanguineous unions, the latter being specially relevant for recessive disorders.6 The age of death in Chilean cases of JEB ranges from < 1 month to 28 years (Appendix S1; see Supporting Information). At March 2015, 11 of the 23 patients were alive, resulting in a JEB prevalence of 0·6 cases per million. Notably, all patients with JEB are concentrated in only five of the 16 geographic regions of the country, representing close to the 74% of the Chilean population. The molecular analysis of the 17 families with JEB failed to find a high prevalence of previously described frequent mutations, confirming that diagnostic strategies based on European and US molecular features are not applicable to all populations.3 7 Only one mutation, c.957ins77 in LAMB3, was present in a heterozygous state, in families 11 and 18. Using next‐generation sequencing technology, we found disease‐causing mutations in 16 of the 17 families analysed, all in the LAMB3 gene, and discovered two previously uncharacterized mutations, c.823−1G>A and c.3228+1G>A (no mutations were found in family number 2).8 These mutations disrupt splicing of LAMB3 transcripts at exons 9 and 21, respectively (Fig. S1 and Table S1; see Supporting Information). The three mutations c.3228+1G>A, c.823−1G>A and c.977ins77 were found in 15 (88%), 5 (29%) and 2 (12%) of the families and in 24 (71%), 5 (15%) and 2 (6%) of the alleles within those 17 families (34 alleles), respectively. Together, these three mutations were found in 91% of the LAMB3 alleles within our cohort of 17 families with JEB. Interestingly, the sequencing data strongly suggest that the most frequent mutation found in the Chilean JEB population, c.3228+1G>A, arose in a common ancestral allele, rather than being a mutational hotspot (Figs S2, S3; see Supporting Information). Our study is the first to investigate the molecular epidemiology of patients with JEB in South America, and is comparable only with a report on Chilean patients with dystrophic EB.9 These results are extremely relevant for patients with EB in Chile and can be immediately used in clinic to improve patient care and prognosis. This is because although different EB types can have a similar clinical appearance, especially in the neonatal period, they can have massive differences in patient prognosis, showing the great relevance of a proper diagnosis.10 We propose to start the diagnosis of every Chilean case suspicious for JEB by taking a small blood sample (minimum 0·2 mL) and performing the mutational analysis of three specific LAMB3 mutations: c.823−1G>A, c.957ins77 and c.3228+1G>A. According to our results, 91% of all LAMB3 alleles will carry at least one of these three mutations, thus allowing us to classify a large proportion of our patients in a few hours. Results like ours clearly show the current need to develop population‐specific diagnostic approaches for heritable diseases, especially for countries with low‐to‐zero governmental budget for molecular diagnosis. Funding sources: this work was supported by FONDECYT (grant number 11140440) and Universidad del Desarrollo (Fondo Concurso Interno de Investigación UDD 2014). Conflicts of interest: none declared. Appendix S1. Supplementary materials and methods. Fig S1. Reverse‐transcriptase polymerase chain reaction analysis of LAMB3 nMRA. Fig S2. Splicing isoforms generated by the recurrent LAMB3 mutations. Fig S3. Schematic representation of the LAMB3 locus, the two frequent mutations (in red) and the single‐nucleotide polymorphisms used to identify the origin of the c.3228+1G>A allele. Table S1. Oligonucleotide primer sequences.