Keywords
Dystrophic epidermolysis bullosa, simplex whole exome sequencing, Collagen VII mutation
This article is included in the Rare diseases collection.
Dystrophic epidermolysis bullosa, simplex whole exome sequencing, Collagen VII mutation
See the authors' detailed response to the review by Robert Sidbury
Dystrophic epidermolysis bullosa (DEB) is an extremely rare subtype of epidermolysis bullosa with an estimated incidence of approximately 6.5 per million newborns. The disease is caused by mutations in collagen VII (COL7A1)1. Collagen VII is a major structural macromolecule of the skin and plays an important component of the anchoring fibrils, which connect the epidermis and dermis of the skin. The disease affects the skin, the mucosa (including that of the oral cavity) and gastrointestinal tract. The blisters are further followed by scarring and development of deformities1–3.
A 4.5-year-old South Indian female child presented to the outpatient clinic with a history of multiple vesicular and bullous lesions induced by trauma since perinatal period. The child was born out of a third degree consanguineous marriage with no known history of similar illness. The child had severe blistering and scarring all over the body, nail dystrophy and milia. The oral mucosa was involved along with tongue blistering, dental calculus, and chipping of teeth with difficulty in opening the mouth. The child also had flexural deformities resulting in contractures and pseudo-syndactyly of the fingers. The clinical picture (Figure 1a,b) corroborated the diagnosis of dystrophic epidermolysis bullosa (DEB). There is no center in India offering genetic diagnosis for the disease using targeted gene sequencing. Given that targeted gene sequencing can be quite expensive, tedious and time-consuming to standardise, we attempted whole-exome sequencing (WES). Moreover no background genetic map of mutations in the disease from India was available. Previous reports, including from our laboratory suggest WES as an alternative to traditional approaches; WES is fast, less tedious, and cost-effective and also provides a holistic view of the mutation spectrum in the patient4–6.
a) Hands and thoracic region showing generalized bullae, scarring and milia b) Lower legs showing scarring, bullae, milia and characteristic dystrophic nails c) Pedigree of the family d) The chromatogram depicting capillary sequencing results of c.6759_6760del in the trio. The mutation loci (ΔCT) is highlighted with asterisks e) Domain structure of COL7A1 protein showing Von Willebrand factor type A domain (VWA), Fibronectin type III domain (fn3), collagen triple helix domain (blue) and Kunitz domain (yellow). Each needle represents disease causing variation site and the red needle represent p.G2254fs (c.6759_6760del) variation. Panel at the bottom represents COL7A1 p.G2254fs induced PTC compared to the normal protein.
Approximately 5 ml of blood was collected from the affected individual and the parents after obtaining signed informed consent and approval from the institutional ethical committee (BSC0212 IHECC proposal No.08). Genomic DNA was isolated by using salting out method7. 50ng of high quality DNA was used for whole exome sample preparation using a Nextera (Illumina Inc, USA) expanded exome kit according to manufacturer supplied instruction. The exome was sequenced using Illumina Hiseq2500 according to the manufacturer’s protocols (Illumina Inc, USA). Paired-end reads of 150 bases were generated, which was quality and adapter trimmed at a Phred quality score of 20. Alignment was performed on the human reference genome (hg19) using Burrows-Wheeler Alignment (version 0.5.10-evan.9)8. The mean mapped coverage on target region was 12.2x. Variants were called using Platypus pipelines (version 0.7.9.1)9. Analysis revealed a novel homozygous frameshift deletion (chr3:g.48610366CT>-) c.6759_6760del (p.G2254fs) in COL7A1 gene. The c.6759_6760del was predicted to be deleterious (confidence score 0.858) and introduce a premature termination codon (PTC) at 2273th amino acid position according to SIFT10. Homozygous PTCs in COL7A1 is previously reported to reduce overall stability of anchoring filaments and cause mild to very severe generalised RDEB1. Secondary structure analysis shows that p.G2254fs resultant PTC leads to loss of function of several collagen triple helix repeats and kunitz domain (Figure 1e). We also found a homozygous nonsynonymous variation c.5716C>T (p.P1906S) in COL7A1, which was predicted to be ‘tolerated’ by SIFT (0.5)11.
The variant was verified independently using capillary sequencing in the child and parents. The variant was not found in ExAC or our internal cohort of 122 exomes, confirming its rarity and novelty. Parents were provided detailed genetic counselling by the consulting clinical geneticist.
Dystrophic EB could be inherited in both recessive and dominant form1. Several cases of DEB have been reported from India. A recent paper reported a cohort of 17 DEB patients using immunofluorescence mapping12, though the patients were not genetically characterized. Our earlier report characterized a novel mutation in KRT5 associated with epidermolysis bullosa (EB) simplex in West India6. Taken together, we suggest a large and potentially uncharacterized repertoire of genetic variations causing EB in India, which might benefit from genetic screening approaches.
In this study, we show the application of next-generation sequencing to identify the mutation in a sporadic case of autosomal recessive EB in clinical settings. Apart from adding a novel frameshift collagen VII deletion mutation to the repertoire of known mutations in the disease, to the best of our knowledge, this is the first report of a genetically characterized patient of DEB from India. We suggest that next-generation sequencing approach would significantly benefit the understanding and genetic characterization of this rare disease in India.
Written informed consent was obtained from the parent of the patients for publication of this case report and any accompanying images and/or other details that could potentially reveal the patient’s identity.
The raw exome sequencing data are available at the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra), accession number SRX1584466.
SN performed the clinical evaluation and sent the blood for DNA analyses and provided genetic counselling. SKV, RJ, RR, AV and VSV isolated the DNA, conducted the quality checks, prepared the exome capture and sequencing library, performed the exome sequencing, performed data quality checks on the reads, reference alignments, variant call and computational prioritization of the variants, designed and performed the validation experiments and contributed to writing the manuscript. SS and VS conceptualized and oversaw the all the experiments and analysis and contributed to writing the manuscript.
Authors acknowledge funding from the Council of Scientific and Industrial Research (CSIR), India through Grant No. BSC0212 (Wellness Genomics Project) granted to SS and VS. Authors acknowledge support from the GUaRDIAN consortium.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
References
1. McGrath JA: Recently Identified Forms of Epidermolysis Bullosa.Ann Dermatol. 2015; 27 (6): 658-66 PubMed Abstract | Publisher Full TextCompeting Interests: No competing interests were disclosed.
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