A technique used to decipher the DNA sequence, called next-generation sequencing (NGS), was successfully used to identify two new mutations in a patient diagnosed with both simplex and junctional epidermolysis bullosa (EB).
The findings were reported in the study “Next generation sequencing identifies double homozygous mutations in two distinct genes (EXPH5 and COL17A1) in a patient with concomitant simplex and junctional epidermolysis bullosa” and published in the journal Human Mutation.
DNA sequencing is the process of determining the precise order of the DNA building blocks, the nucleotides, within the DNA molecule. Specifically, NGS represents a cutting-edge DNA sequencing technology, which provides results quickly and efficiently.
NGS is now being used to diagnose several diseases, representing an important tool for the implementation of precision medicine. NGS allows, for example, the identification of two concurrent diseases with overlapping symptoms. It also raises hope for the identification of genes in conditions with genetic heterogeneity, such as EB.
In the study, a team of researchers used NGS to assess the expression of 21 genes associated with different skin fragility syndromes, 19 of which are known to harbor EB-causing mutations.
NGS analysis involved the collection of DNA from blood samples of 91 EB patients, with clinical diagnosis based on skin blistering and erosions, the patients’ parents and other family members.
While EXPH5 gene codes for the exophilin-5, a protein involved in vesicle trafficking inside the cell, the COL17A1 gene gives rise to a collagen protein, which plays a role in strengthening and supporting connective tissue like the skin.
The EXPH5 and COL17A1 mutations were found on chromosomes 10 and 11, respectively. Also, they were found to be homozygous mutations, meaning that both copies of the gene (one inherited from the mother and one from the father) had the mutation.
The results were confirmed using RNAseq, a technique that involves sequencing of the RNA, the molecule that originates from the DNA and that will give rise to proteins. The analysis predicted the absence (in the case of EXPH5) or very low levels (for COL17A1) of the corresponding protein products given the mutations found.
Histopathology analysis, transmission electron microscopy (TEM) and immunofluorescence staining of skin biopsy samples further confirmed the findings. These techniques also revealed skin cell disruption and death, as well as blister formation, in two areas of the skin consistent with the presence of two subtypes of EB, the simplex and junctional forms.
The existence of small droplets, or vesicles, inside skin cells and the reduced number of hemidesmosomes (structures connecting skin cells to the matrix surrounding them) also confirmed the diagnosis of both forms of EB.
The patient was the only child of consanguineous, clinically unaffected parents. This means that, although neither parent was affected, each carried a mutated and a functional copy of the gene, making them healthy individuals, but they both transmitted the mutated copies to the child.
The researchers concluded that, in this case, NGS was successfully used to identify two new mutations in a patient with concomitant forms of EB, with implications at both genetic (genotype) and physical (phenotype; the traits of an individual) levels.
“This case illustrates the power of next-generation sequencing in identifying mutations in patients with complex EB phenotype, with implications for genotype-phenotype correlations, prenatal testing, and genetic counseling of families,” the researchers said.