2. Methods
2.1. Patient Reports
Family I, patient I, II:
Two affected twin siblings were born to non-symptomatic consanguineous couple. A 32-yaer-old affected male selected genetic counseling to avoid his offspring inheriting the similar condition. He had complained about extensive blistering, skin fragility, digit fusion, pseudosyndactyly, fingers nail lost since childhood (fig1a). His twin sister had similar symptoms. Granulation tissue was observed on upper back of man but not her sister. Wounds were also observed on his elbows which biopsy revealed squamous cell carcinoma (fig1b). Mild mucosal involvement and oral blistering were clear in both cases and lead to malnutrition.
Family II, Patient III:
The third patient was a 2-year-old girl who referred with severe scars on the skin and oral cavity, recurrent blistering, skin erosion, nail dystrophy and partial digit fusion of hands from the third months of life (Fig 2a). She was a product of full term vaginal delivery with normal weight and length and no sign of skin lesions. At the end of the second month, red scars appeared on the skin especially the abdomen, lower legs and face and also showed nail dystrophy. Small progressive blisters were observed all over the body. Severe blisters and joint contractures were noted in 1 years old of age. A skin biopsy revealed reduced numbers of anchoring fibrils and dermis-epidermis cleavage. There was positive family history of skin disorder in her cousins. Genetic investigation was conducted to evaluate current pregnancy for identified mutation.
Family III, Patient IV
A 6-year-old girl, product of consanguineous couple, presented with skin erosion, mild milia especially around eyelid, loss of nails, digit fusion, joint contractures and scars of oral cavity. Laboratory evaluations showed low hemoglobin levels due to anemia. Electron microscopy of wounds revealed hypoplastic anchoring fibrils and cleavage at the level of the sublamina densa, which were consistent with DEB. The family was referred to genetic counseling to identify causative gene mutation.
Family IV, Patient V
The patient was 4 years 6 months old boy, he was referred with skin fragility, mucous membrane involvements and severe itching on the hands and feet. Physical examination showed blisters on the knees and elbows, joint contractures, ophthalmic abnormalities, oral ulcers and teeth deformities. Sparse hair and eyebrows were also observed.
Typical laboratory evaluations revealed low hemoglobin and vitamin D. His uncle was also affected by skin disorder and died at 4 years of age. The patient was the first child of healthy consanguineous couple and was a product of full term normal vaginal delivery. Parents did not give consent to skin biopsy because of pain and catastrophic condition of their child. However, the clinical symptoms were consistent with EB. Therefore, genetic study was recommended to clarify the disease-causing mutation and evaluate current pregnancy for identified mutation.
Family V, Patient VI:
The patient was a 27-year-old male, the only affected child of twelve siblings born to non- consanguineous parents. Scattered red spot, mild scarring was observed on the upper back, hands and lower leg at birth. Most of his blisters were particularly limited to trauma-exposed areas and bony prominences. He had no eye, nail, digit, oral involvements. He complained skin itching and recurrent blisters at knees and elbows at the time of experiment. Transmission electron microscopy of skin biopsy revealed a cleavage beneath the lamina densa and absence of a large number of anchoring fibrils. On the basis of clinical symptoms and no family history of skin disorder, dominant form of DEB was considered for the patient and was referred to genetic counseling to evaluate the mutation.
2.2. Genetic testing
WES was performed on the parents of dead patients or on the available patients to capture and enrich all exons of COL7A1 gene in addition to other essential flanking parts. Next generation sequencing was carried out using Illumina Hiseq 2000 machine to sequence about 100 million reads and standard Illumina protocol for pair-end 99 nucleotide sequencing. Aligning reads, identifying and annotating genetic variants were carried out using BWA aligner, GATK and ANNOVAR as bioinformatics tools, respectively (5-7).
Sanger sequencing was done on all individuals in order to confirm the WES results and also to find the genotype of other family members . Whole blood samples were collected from participants of all seven families in EDTA tube. DNA was extracted from blood and CVS samples using QIAamp DNA Minikit (Qiagen, Germany) according to the manufacturer’s instructions. DNA concentration was evaluated using NanoDrop C (Thermofisher, USA) and then kept at -20C until use. Table S1 shows primer pairs that were used to amplify the desired genome segment in each family.
Sanger sequencing was then carried out in both forward and reverse directions on the amplified DNA using ABI BigDye Terminator Cycle Sequencing kit (Applied Biosystems®, USA).
3. Results :
NGS data analysis of five families showed different variant in distinct part of COL7A1 gene.
In the first family a homozygous variant (COL7A1 , NM_000094, c.5018G>A, p.G1673E) of unknown significant inCOL7A1 gene was identified. The homozygous and heterozygous state of the mutation were confirmed by sanger sequencing in affected individuals (proband and his sister) and their parents, respectively. Therefore, this mutation is inherited as autosomal recessive pattern and mutation in this gene is associated with recessive dystrophic epidermolysis bullosa disorder (Fig 1c). According to mutation taster prediction (8), this glycine substitution variant is disease causing variant. Multiple sequence alignment using T-COFFE online software was performed across different animal kingdoms and confirmed the highly conservation of 1673 glycine amino acid (Fig 1d).
In family II, WES was performed on a 2-year-old girl who showed general DEB phenotypes. The results showed a homozygous mutation inCOL7A1 gene (COL7A1 , NM_000094, c.425A>G, p.K142R). The patients passed away due to accident at the time of confirmation and we didn’t have access to her DNA sample. Therefore, sanger sequencing was performed only on parents and the heterozygous mutation confirmed in her parents, which might show the autosomal recessive mode of inheritance in this family. Sanger sequencing analysis of CVS sample had showed that the first pregnancy had a homozygous mutant variant and the fetus was affected. The second pregnancy was heterozygous and healthy. This missense variant was reported previously as a disease-causing mutation (rs121912856) which could give rise to RDEB (Fig 2b). Multiple sequence alignment was performed across different animal kingdoms and confirmed the highly conservation of lysine 142 (Fig 2c).
In family III, the NGS results revealed a novel homozygous, non-frameshift deletion and a frameshift insertion in exon 2 ofCOL7A1 gene (COL7A1 , NM_000094: c.180_182del, p.60_61del & c.179-180 ins GAAA, p.F60fs). This mutation was confirmed as homozygous in patient and heterozygous variant in healthy sibling and parents. The autosomal recessive pattern was confirmed for mentioned mutation in this family (Fig 3a).
This variant was predicted as disease-causing mutation by online mutation taster software. The insertion of 4 nucleotides (GAAA) results in frameshift mutation after position 60 in amino acid sequence.
Analysis of WES data of patient in family IV showed a deleterious homozygous splice site mutation in exon 6 of COL7A1 gene (COL7A1 , NM_000094: c.682+1 G>A). The mutation previously was reported as a pathogenic variant. The mutation was confirmed in the parents by sanger sequencing. Heterozygous parents let us concluded the probable autosomal recessive mode of inheritance for mentioned mutation in the family (Fig3b).
In family V, WES test showed glycine substitution mutation in exon 73 ofCOL7A1 gene (COL7A1 , NM_000094: c.6101G>A, G2034E). Using sanger sequencing this heterozygous mutation was confirmed in proband. This mutation was not identified in parents, so this is considered as a de novo mutation with autosomal dominant mode of inheritance (Fig 4a).
Multiple sequence alignment using T-COFFE online software was done across distinct animal kingdoms and confirmed the highly conservation of 2034 glycine during evolution (Fig 4b).