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Version 2. F1000Res. 2016; 5: 900.
Published online 2016 July 5. doi:  10.12688/f1000research.8380.2
PMCID: PMC4926754
Other versions

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

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

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 deformities 13.

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 patient 46.

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 ...

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 method 7. 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 2273 th amino acid position according to SIFT 10. Homozygous PTCs in COL7A1 is previously reported to reduce overall stability of anchoring filaments and cause mild to very severe generalised RDEB 1. 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 form 1. Several cases of DEB have been reported from India. A recent paper reported a cohort of 17 DEB patients using immunofluorescence mapping 12, though the patients were not genetically characterized. Our earlier report characterized a novel mutation in KRT5 associated with epidermolysis bullosa (EB) simplex in West India 6. 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 data referenced by this article are under copyright with the following copyright statement: Copyright: © 2016 Karuthedath Vellarikkal S et al.

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

Acknowledgements

Authors acknowledge help from Dr. Vamsi Krishna for preparation of the manuscript.

Notes

[version 2; referees: 3 approved]

Funding Statement

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.

Notes

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

References

1. Chung HJ, Uitto J.: Type VII collagen: the anchoring fibril protein at fault in dystrophic epidermolysis bullosa. Dermatol Clin. 2010;28(1):93–105. 10.1016/j.det.2009.10.011 [PMC free article] [PubMed] [Cross Ref]
2. McGrath JA.: Recently Identified Forms of Epidermolysis Bullosa. Ann Dermatol. 2015;27(6):658–66. 10.5021/ad.2015.27.6.658 [PMC free article] [PubMed] [Cross Ref]
3. Vanden Oever MJ, Tolar J.: Advances in understanding and treating dystrophic epidermolysis bullosa. F1000Prime Rep. 2014;6:35. 10.12703/P6-35 [PMC free article] [PubMed] [Cross Ref]
4. Sawyer SL, Hartley T, Dyment DA, et al. : Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care. Clin Genet. 2016;89(3):275–84. 10.1111/cge.12654 [PMC free article] [PubMed] [Cross Ref]
5. Gupta A, Sharma YK, Vellarikkal SK, et al. : Whole-exome sequencing solves diagnostic dilemma in a rare case of sporadic acrokeratosis verruciformis. J Eur Acad Dermatol Venereol. 2016;30(4):695–7. 10.1111/jdv.12983 [PubMed] [Cross Ref]
6. Vellarikkal SK, Patowary A, Singh M, et al. : Exome sequencing reveals a novel mutation, p.L325H, in the KRT5 gene associated with autosomal dominant Epidermolysis Bullosa Simplex Koebner type in a large family from western India. Hum Genome Var. 2014;1: 14007. 10.1038/hgv.2014.7 [PMC free article] [PubMed] [Cross Ref]
7. Miller SA, Dykes DD, Polesky HF.: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16(3):1215. 10.1093/nar/16.3.1215 [PMC free article] [PubMed] [Cross Ref]
8. Li H, Durbin R.: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754–60. 10.1093/bioinformatics/btp324 [PMC free article] [PubMed] [Cross Ref]
9. Rimmer A, Phan H, Mathieson I, et al. : Integrating mapping-, assembly- and haplotype-based approaches for calling variants in clinical sequencing applications. Nat Genet. 2014;46(8):912–8. 10.1038/ng.3036 [PMC free article] [PubMed] [Cross Ref]
10. Hu J, Ng PC.: SIFT Indel: predictions for the functional effects of amino acid insertions/deletions in proteins. PLoS One. 2013;8(10):e77940. 10.1371/journal.pone.0077940 [PMC free article] [PubMed] [Cross Ref]
11. Ng PC, Henikoff S.: SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res. 2003;31(13):3812–4. 10.1093/nar/gkg509 [PMC free article] [PubMed] [Cross Ref]
12. Hiremagalore R, Kubba A, Bansel S, et al. : Immunofluorescence mapping in inherited epidermolysis bullosa: a study of 86 cases from India. Br J Dermatol. 2015;172(2):384–91. 10.1111/bjd.13305 [PubMed] [Cross Ref]

Review Summary Section

Review dateReviewer name(s)Version reviewedReview status
2016 July 13Robert SidburyVersion 2Approved
2016 June 27Mohamed Badawy Abdel-NaserVersion 1Approved
2016 June 14Regina Fölster-HolstVersion 1Approved
2016 June 6Robert SidburyVersion 1Approved with Reservations

Approved

Robert Sidbury, Referee1
1Dermatology Division, Seattle Children, Seattle, WA, USA
Competing interests: No competing interests were disclosed.
Review date: 2016 July 13. Status: Approved

The authors have responded satisfactorily.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Approved

1Klinik für Dermatologie, Venerologie und Allergologie Städtisches Klinikum Dessau, Dessau, Germany
Competing interests: No competing interests were disclosed.
Review date: 2016 June 27. Status: Approved

  • 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 that almost all affected members develop cancers in the third or fourth decade of life". Perhaps authors can insert a reference (s) that supports this statement.
  • Fig. 1. needs some corrections. Please insert (a) to refer to the upper limb, thorax and abdomen (not hands and thoracic region). b. should refer to legs and feet.
  • Is the mentioned "our internal cohort of 122 exomes" published or available online? Furthermore, they may mention some of the weaknesses of the Nextera platform, such as coverage bias.
  • In the discussion sections, authors may mention the clinical relevance of their finding.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Approved

1Dermatologische Klinik, Universität Kiel, Kiel, Germany
Competing interests: No competing interests were disclosed.
Review date: 2016 June 14. Status: Approved

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.

It is a well written manuscript which is worthy to be indexed with F1000Research. To understand the new mutation as the cause of severe DEB in the boy, it would be interesting to know what are the differences to other mutations of the COL7A1 gene. In other words, what does this mean on the protein level?

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Approved with Reservations

Robert Sidbury, Referee1
1Dermatology Division, Seattle Children, Seattle, WA, USA
Competing interests: No competing interests were disclosed.
Review date: 2016 June 6. Status: Approved with Reservations

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 cases only to one  subtype.  There is a reason for this as there is considerable overlap but there are differences particularly with certain features like the propensity for developing skin cancer.  The authors state  "the development of  skin cancer....in almost all affected members in the third or fourth decade of life."  This is true for RDEB phenotype but not DDEB in whom the development of squamous cell carcinoma as well as the distinctive psuedosyndactyly type of scarring much less common.  This may not be the forum for parsing such details but this struck me.

Similarly, while I realize this is not an EB review article a brief internal reference to the Vander Oever article might allow interested readers easy access to a therapeutic update.

Finally,  I would want someone other than I with expertise in the genetic methods used to weigh in on their suitability. The methods and results to my untrained --relative to a geneticist--- eye appear sound.

Otherwise I approve indexing this article.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

References

1. McGrath JA.: Recently Identified Forms of Epidermolysis Bullosa. Ann Dermatol.2015;27(6) : 10.5021/ad.2015.27.6.658 658-66 10.5021/ad.2015.27.6.658 [PMC free article] [PubMed] [Cross Ref]
2. Vanden Oever MJ, Tolar J.: Advances in understanding and treating dystrophic epidermolysis bullosa. F1000Prime Rep.2014;6: 10.12703/P6-35 35 10.12703/P6-35 [PMC free article] [PubMed] [Cross Ref]

Articles from F1000Research are provided here courtesy of F1000 Research Ltd