## 4 Discussion
The mutation c.385 del was predicted to be disease producing by MutationTaster and pathogenic by VarSome (Table 2). In Family C, a 13-year-old girl (IV-2) presented with typical features of DEB, including abnormal blistering of the skin, erosions and atypical scarring with erythematous hyperkeratotic papules and keloids of skin affecting the trunk and extremities, showing no improvement with advancing age. Other symptoms included mitten deformities of phalanges, joint contractures, severe blistering of oral mucosa and esophageal strictures, feeding difficulties, severe anemia, growth delay, frequent corneal and conjunctival erosions, symblepharon, anterior blebaritis, exposure keratitis and conjunctival complications, as reported in other studies [37; 38; 39]. WES detected a nonsense sequence variant (c.1573 C>T; p. Arg525 Ter) in _COL7A1_ at the 12 th exon, cosegregating with the diseased members of the family (Table 1). This genetic mutation in the _COL7A1_ gene causes a premature translation stop signal (p. Arg525*) and is classed as pathogenic (Table 2). Commonly practicing consanguineal marriages has turned out to be a distressing matter in an ethnically rich, religious country like Pakistan, further increasing the risk of many monogenic disorders (autosomal recessive). Under such circumstances, Next-Generation Sequencing technologies (WES and Whole-Genome Sequencing, WGS) are a preferred state-of-the-art molecular diagnostic tool for quick and efficient diagnosis of single-gene disorders. These novel mutations identified in the _COL7A1_ gene causing RDEB in three different families will further elaborate the phenotypic and mutational spectrum of RDEB, and will also aid in the development of clinical and gene-based therapeutic strategies and targeted genome-editing techniques which, along with the procedures for inducing pluripotent stem cells, are predicted to permit and expand the usage of patient-specific autologous stem cell(renewable) therapies [40; 41], especially in patients affected by _COL7A1_ mutations, especially in Pakistan, where _COL7A1_ mutations prevail in increased numbers, as shown in this article. In this sense, detecting and analyzing individual genetic mutation is a critical first step in devising suitable personalized treatment strategies, as well as the identification of individuals who could gain an advantage from particular innovative genomic therapies. The present study adds to the knowledge of clinical heterogeneity associated with _COL17A1_ variants, and it will also help in genetic counseling and accurate prenatal diagnosis for parents of at-risk individuals in order to facilitate decision making regarding the continuation of upcoming pregnancies, especially in a region like Pakistan, where the custom of consanguineous marriage prevails at a much higher rate. The following are available online at [[https://www.mdpi.com/article/10.3390/diagnostics12071525/s1](https://www.mdpi.com/article/10.3390/diagnostics12071525/s1)]([https://www.mdpi.com/article/10.3390/diagnostics12071525/s1](https://www.mdpi.com/article/10.3390/diagnostics12071525/s1)), Table S1: Primers used for the amplification of the regions of interest. Conceptualization, F.F. and R.N. ; methodology, F.F. and R.N. ; software, F.F., S.A.K. ; validation, F.F., R.N., M.M.A., M.A.M.A.-S. and I.A. ; formal analysis, F.F., I.A., M.M.A. ; investigation, F.F. ; resources, F.F., I.A. ; visualization: M.M.A., H.K.G. ; data curation, R.N., I.A. ; writing--original draft preparation, F.F. and S.A.K. ; writing--review and editing, R.N., Z.U.H., M.J. and F.F., M.A.M.A.-S., M.B. and H.K.G. ; supervision, R.N. ; project administration, R.N., I.A. ; funding acquisition, F.F. and M.M.A. All authors have read and agreed to the published version of the manuscript. **Funding:** The authors extend their appreciation to the Deanship of Scientific Research, King Saud University for funding through Vice Deanship of Scientific Research Chairs; Research Chair of Medical and Molecular Genetics.