Fig 1.
Overview of the Exome Sequencing workflow.
Workflow includes sample preparation, sequencing using Illumina NextSeq, and variant filtering. Key quality control steps include DNA purity, coverage, and population frequency thresholds.
Fig 2.
Pedigrees of the 12 families with epidermolysis bullosa (EB) patients included in this study.
The arrow indicates the index case (proband) in each family. Panels A to L represent Families 1 to 12, respectively. Enrolled family members are shown with their corresponding genotype results. A black symbol indicates affected individuals. A square denotes a male, and a circle denotes a female.
Table 1.
Demographic details, clinical types, and symptoms of index cases.
Fig 3.
A 7-year-old girl presenting dystrophic EB.
A. Blisters and erosions in the upper chest and lower part of the mouth causing difficulty with opening the mouth. B. crusts and atrophic scars. C. Thin, fragile, and translucent skin in the extremities. D. Repeated blistering and scarring of hands and feet cause fusion and webbing (pseudosyndacty) of the fingers and toes and absent nails.
Table 2.
Detailed annotations of the identified variants.
Table 3.
The molecular and clinical details of variants identified in EB patients.
Fig 4.
Sanger sequencing chromatograms of pathogenic variants identified in the COL7A1, COL17A1, and LAMB3 genes among patients diagnosed with epidermolysis bullosa (EB).
The highlighted regions indicate the position of each variant. Reverse complement sequences are displayed where applicable.
Table 4.
Details of the pathogenic prediction scores.
Fig 5.
Splice site variants in (a) LAMB3 c.1977-1G > A, (b) COL7A1 c.8305-1G > A, and (c) COL7A1 c.6751-1G > A are shown with exons and introns.
Each variant disrupts the respective splice acceptor site, leading to exon skipping, intron retention, or frameshift. These events likely result in premature stop codons and truncated proteins.
Fig 6.
RNA secondary structure and mountain plot for LAMB3 variants identified in EB patients (A) Wild-type RNA structure of the LAMB3 gene at the c.1977-1G > A splice acceptor site, showing a minimum free energy (MFE) of −132.75 kcal/mol.
(B) Mutant RNA structure of the LAMB3 gene after the c.1977-1G > A variant, with an MFE of −126.52 kcal/mol, indicates a stability decrease. The mountain plot comparison highlights structural changes between the wild-type and mutant RNA folds.
Fig 7.
RNA secondary structure analysis for two splice site variants in COL7A1.
(A) Wild-type RNA structure for the c.6751-1G > A variant with an MFE of −102.38 kcal/mol. (B) Mutant RNA structure for the c.6751-1G > A variant showing an MFE of −96.75 kcal/mol, suggesting destabilization. (C) Wild-type RNA structure for the c.8305-1G > A variant with an MFE of −116.85 kcal/mol. (D) Mutant RNA structure for the c.8305-1G > A variant showing an MFE of −111.45 kcal/mol, indicating reduced RNA stability. The mountain plots further illustrate the structural changes in both variants, reflecting the impact of the variants on RNA folding.
Fig 8.
Structural models of the COL7A1 protein (2,944 amino acids) generated using AlphaFold2, showing wild-type and mutant structures for the p.Gly1483Asp and p.Gly2749Arg variants.
The central image represents full-length protein, with close-up views highlighting the wild-type Gly1483 and Gly2749 residues (left) and their mutated counterparts, Asp1483 and Arg2749 (right).