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Case Report
Revised

Case Report: Whole exome sequencing reveals a novel frameshift deletion mutation p.G2254fs in COL7A1 associated with autosomal recessive dystrophic epidermolysis bullosa

[version 2; peer review: 3 approved]
PUBLISHED 05 Jul 2016
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This article is included in the Rare diseases collection.

Abstract

Dystrophic epidermolysis bullosa simplex (DEB) is a phenotypically diverse inherited skin fragility disorder. It is majorly manifested by appearance of epidermal bullae upon friction caused either by physical or environmental trauma. The phenotypic manifestations also include appearance of milia, scarring all over the body and nail dystrophy. DEB can be inherited in a recessive or dominant form and the recessive form of DEB (RDEB) is more severe. In the present study, we identify a novel p.G2254fs mutation in COL7A1 gene causing a sporadic case of RDEB by whole exome sequencing (WES). Apart from adding a novel frameshift Collagen VII mutation to the repertoire of known mutations reported in the disease, to the best of our knowledge, this is the first report of a genetically characterized case of DEB from India.

Keywords

Dystrophic epidermolysis bullosa, simplex whole exome sequencing, Collagen VII mutation

Revised Amendments from Version 1

  1. New references were added according to the reviewers comments
     
  2. Removed the confusing generalised statements from the introduction
No other changes were made to the methods, findings or conclusion about the study

See the authors' detailed response to the review by Robert Sidbury

Introduction

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 deformities13.

Case Report

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 patient46.

e423300d-1060-494a-a476-878c32a4f36d_figure1.gif

Figure 1.

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.

Discussion

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.

Consent

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.

Data availability

The raw exome sequencing data are available at the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra), accession number SRX1584466.

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Karuthedath Vellarikkal S, Jayarajan R, Verma A et al. Case Report: Whole exome sequencing reveals a novel frameshift deletion mutation p.G2254fs in COL7A1 associated with autosomal recessive dystrophic epidermolysis bullosa [version 2; peer review: 3 approved]. F1000Research 2016, 5:900 (https://doi.org/10.12688/f1000research.8380.2)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 2
VERSION 2
PUBLISHED 05 Jul 2016
Revised
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Reviewer Report 13 Jul 2016
Robert Sidbury, Dermatology Division, Seattle Children, Seattle, WA, USA 
Approved
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The authors ... Continue reading
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Sidbury R. Reviewer Report For: Case Report: Whole exome sequencing reveals a novel frameshift deletion mutation p.G2254fs in COL7A1 associated with autosomal recessive dystrophic epidermolysis bullosa [version 2; peer review: 3 approved]. F1000Research 2016, 5:900 (https://doi.org/10.5256/f1000research.9847.r14976)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Version 1
VERSION 1
PUBLISHED 17 May 2016
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Reviewer Report 27 Jun 2016
Mohamed Badawy Hassan Tawfik Abdel-Naser, Klinik für Dermatologie, Venerologie und Allergologie Städtisches Klinikum Dessau, Dessau, Germany 
Approved
VIEWS 26
  • Apart from few typo errors, the manuscript is well written and informative.
     
  • There are few too strong statements, e.g.,  "The disease also predisposes individuals to development of skin cancer and it is estimated
... Continue reading
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Abdel-Naser MBHT. Reviewer Report For: Case Report: Whole exome sequencing reveals a novel frameshift deletion mutation p.G2254fs in COL7A1 associated with autosomal recessive dystrophic epidermolysis bullosa [version 2; peer review: 3 approved]. F1000Research 2016, 5:900 (https://doi.org/10.5256/f1000research.9013.r14177)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 14 Jun 2016
Regina Fölster-Holst, Dermatologische Klinik, Universität Kiel, Kiel, Germany 
Approved
VIEWS 25
The authors describe a 5 year old boy with dystrophic epidermolysis bullosa (DEB), which is due to a novel mutation in the COL7A1 gene. The case is the first report of a genetically characterized case of DEB from India.
... Continue reading
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HOW TO CITE THIS REPORT
Fölster-Holst R. Reviewer Report For: Case Report: Whole exome sequencing reveals a novel frameshift deletion mutation p.G2254fs in COL7A1 associated with autosomal recessive dystrophic epidermolysis bullosa [version 2; peer review: 3 approved]. F1000Research 2016, 5:900 (https://doi.org/10.5256/f1000research.9013.r14364)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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33
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Reviewer Report 06 Jun 2016
Robert Sidbury, Dermatology Division, Seattle Children, Seattle, WA, USA 
Approved with Reservations
VIEWS 33
Though the authors distinguish dominant (DDEB) and recessive (RDEB) subtypes of dystrophic epidermolysis bullosa (DEB) at the outset in their abstract, they lapse into speaking of DEB in more monolithic terms later and conflating findings that are specific in some ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Sidbury R. Reviewer Report For: Case Report: Whole exome sequencing reveals a novel frameshift deletion mutation p.G2254fs in COL7A1 associated with autosomal recessive dystrophic epidermolysis bullosa [version 2; peer review: 3 approved]. F1000Research 2016, 5:900 (https://doi.org/10.5256/f1000research.9013.r14175)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 05 Jul 2016
    Vinod Scaria, GN Ramachandran Knowledge Center for Genome Informatics, CSIR Institute of Genomics and Integrative Biology, Delhi, India
    05 Jul 2016
    Author Response
    Dear Robert Sidbury,
    Thank you for reviewing our article and your valuable comments. We have incorporated your suggestions in the recent version.

    Thanks and Regards
    Authors 
    Competing Interests: No competing interests were disclosed.
COMMENTS ON THIS REPORT
  • Author Response 05 Jul 2016
    Vinod Scaria, GN Ramachandran Knowledge Center for Genome Informatics, CSIR Institute of Genomics and Integrative Biology, Delhi, India
    05 Jul 2016
    Author Response
    Dear Robert Sidbury,
    Thank you for reviewing our article and your valuable comments. We have incorporated your suggestions in the recent version.

    Thanks and Regards
    Authors 
    Competing Interests: No competing interests were disclosed.

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 17 May 2016
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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