## 3 Results
### Clinical Phenotypes
Screening of Pathogenic Sequence Variants  
Analysis of the WES data results in identification of two, homozygous novel mutations (c.7034G>A, p. Gly2345 Asp and c.385 del (p. His129 MetfsTer18) and one known homozygous variant (c.1573 C>T (p. R525*) in the _COL7A1_ gene in diseased individuals of three families (A, B, and C), causing RDEB. The obligate carriers were heterozygous for the detected pathogenic variations, whereas the affected individuals were homozygous. The 100 ethnically matched healthy controls were evaluated to rule out the chance of polymorphism among the identified mutations., removing the possibility of nonpathogenic polymorphisms for those unique mutations. In \"Family A\", the identified a novel missense variant c.7034G>A, p. Gly2345 Asp lies in exon 91 of _COL7A1_. Sanger sequencing confirmed identified variant (c.7034G>A) in the proband (V-2). Afterward, the variant was screened in parents (IV-1 and IV-2) and found to cosegregate within the family with the phenotype of disease. Patient (V-2) was carrying the mutation in homozygous state (Figure 3(Ca)), whereas his parents (IV-1 and IV-2) were heterozygous for the mutant allele (Figure 3(Pb)). The homozygous mutation c.7034G>A in _COL7A1_ changes an amino acid Gly to Asp at 2345 codon in 91 number exon. SIFT, [PERSON] and Fathmm predicted the variant c.7034G>A to be damaging, likely pathogenic by Varsome and MutPred, disease causing by Mutation Taster and deleterious by PROVEAN. It was also shown to have decreased stability by MUPRO (Table 2). In Family B, WES revealed a frameshift mutation c.385 del (p. His129 MetfsTer18) in homozygous form in the 3 rd exon of _COL7A1_. The selected c.385 del mutation of _COL7A1_ was confirmed in homozygous state in patient IV-4 through Sanger sequencing (Figure 3(Da)). The variant was co-segregated within the family with the disease phenotype. Consistent with the autosomal recessive mode of inheritance, the parents (II:1 and II:2) and the phenotypically normal individual (III:2) were in a heterozygous state for the c.385 del mutation (Figure 3(Db,c)). The homozygous variant c.385 del on _COL7A1_ deletes nucleotides 'C' from exon 3, which creates frameshift change in the protein coding sequence. The deletion is expected to cause protein truncation. This is likely to result in normal protein function loss, due to nonsense-mediated mRNA decaying. These two variants in _COL7A1_ are predicted to be novel, as they have never been shown in gnomAD [8] HGMD [9], Clinvar [10] or the Pakistan Genetic Mutation Database (PGMD) [11]. Figure 3: _Cont._  
A nonsense variation (c.1573 C\(>\)T; p. Arg525 Ter) in the _COL7A1_ 12 th exon was discovered in Family C, and its segregation was verified by Sanger sequencing in other participants (Figure 3E(a-c)). In the _COL7A1_ gene, this genetic alteration causes a premature translational stop signal (p. Arg525\"). It is likely to result in a protein product that is missing or disrupted. In population databases, this variant can be found (rs368007918, ExAC 0.006%). Individuals with autosomal recessive epidermolysis bullosa have been found to carry this variation (PMID: 10504458, 21448560). This variation has a record in ClinVar (Variation ID: 279785). This variant may produce or brace a splicing site, according to algorithms built to anticipate the effect of sequence alterations on RNA splicing, although not verified yet. The _COL7A1_ function loss mutation is considered to be disease causing (PMID: 16971478) (Table 2). This variation has been labeled as pathogenic as a result of these factors. Figure 4A-C display multiple sequence alignment of Human Gly2345, His129, and Arg525 with their orthologs in different species. \begin{table}
\begin{tabular}{c c c c c c c c c c c c c} \hline \hline
**Gene** & **Variant** & **Chr. ** & **Chr. ** & **Variant** & **Cyto** & **GenRank** & **Ref. ** & **Alt. ** & **RS** & **UniProt** & **MAF** & **gramAD** \\  & & & **Position** & **Type** & **Band** & **Seq.