Epidermolysis Bullosa: A Report of Three Cases with Novel Heterozygous Deletions in PLEC and Homozygous Non sense Mutations in COL7A1 Genes.

Epidermolysis bullosa (EB) is a group of rare inherited conditions that results in blistering of the skin and mucous membranes. Mutations in the PLEC gene cause epidermolysis bullosa simplex (EBS). Mutations in type VII collagen, encoded by COL7A1 lead to epidermolysis bullosa dystrophica (EBD). The report presents three autosomal recessive cases, one with epidermolysis bullosa simplex (EBS) with nail and muscular dystrophy showing heterozygous single base pair deletion in exon 31 (chr8:144998220delC; c. 6288del; p. Arg2097AlafsTer55) and a heterozygous two base pair deletion in exon 27 (chr8:145001693_145001694delCT; c. 4054_4055del; p. Ser1352CysfsTer68) of PLEC gene. Two cases of epidermolysis bullosa dystrophica (EBD), with a novel homozygous, nonsense mutations in exon 54 (c. 5047C > T) and exon 104 (c. 7762C > T) of COL7A1 gene. The findings of the case report, provide evidence for additional molecular heterogeneity, in epidermolysis bullosa and also emphasize the significance of PLEC and COL7A1 gene mutations in epidermolysis bullosa. Copyright:

Introduction

Epidermolysis bullosa (EB) is a group of rare genetic skin conditions characterized by fragile skin that blisters in response to friction, minor injury, or trauma. The incidence of epidermolysis bullosa is approximately 1 in 50,000 live births,[1] and the prevalence is 1:20,000–1:100,000 in the USA and Europe respectively. The subgroups of the condition include epidermolysis bullosa simplex (EBS), epidermolysis bullosa dystrophic (EBD), junctional epidermolysis bullosa (JEB), and Kindler syndrome.[2] The present study reports three autosomal recessive cases, one with epidermolysis bullosa simplex (EBS) with a nail and muscular dystrophy and two cases of epidermolysis bullosa dystrophica (EBD).

Case Presentations

Case 1

A 2-year-old male proband, born of a non-consanguineous marriage, presented with clinical indications of dystrophic nails, hypopigmented spots, blisters, and scars. The patient was referred to the institute with a history of jaundice and seizures. The patient was clinically diagnosed and suspected of epidermolysis bullosa simplex with muscular dystrophy. The patient DNA has been evaluated for pathogenic variants by targeted panel sequencing using Illumina next-generation sequencing (NGS) platform and the mean coverage was 80-100X. Which was further validated by Sanger sequencing. Genetic compound heterozygous single base pair deletion in exon 31 of the PLEC gene (chr8:144998220delC; c. 6288del; p. Arg2097AlafsTer55) and a heterozygous two base pair deletion in exon 27 of the PLEC gene (chr8:145001693_145001694delCT; c. 4054_4055del; p. Ser1352CysfsTer68) was detected. The deletions result in a frameshift and premature truncation of the protein 55 amino acids downstream to codon 2097 (p. Arg2097AlafsTer55) and 68 amino acids downstream to codon 1352 (p. Ser1352CysfsTer68) a novel genetic compound. Interestingly, both variants have not been reported in the 1000 genomes. As the proband was found to harbor heterozygous variations in the PLEC gene, his parents were also evaluated for the same variations [Pedigree 1]. Deletion in exon 31 (chr8:144998220delC; c. 6288del; p. Arg2097AlafsTer55) was detected in the heterozygous condition in the asymptomatic father of the patient, but not in the mother [Figure 1a and b]. Whereas, deletion in exon 27 (chr8:145001693_145001694delCT; c. 4054_4055del; p. Ser1352CysfsTer68) of the PLEC gene was detected in the heterozygous condition in the asymptomatic mother of the patient, but not in the father [Figure 2a and b]. The mother of the proband was referred on second pregnancy and advised for prenatal diagnosis. The amniotic fluid was collected by amniocentesis, and surprisingly, the same genetic compound variations reported in the proband were detected in the fetus. Genetic counseling was offered to the couple and informed the implications due to these mutations and suggested appropriate strategies.

Pedigree 1

Represents case 1, where the proband harbours heterozygotic mutations with single base pair deletion in exon 31 (chr8:144998220delC; c. 6288del; p. Arg2097AlafsTer55) and two base pair deletion in exon 27 (chr8:145001693_145001694delCT; c. 4054_4055del; p. Ser1352CysfsTer68) of PLEC gene. The father is detected with heterozygous variant in exon 31(chr8:144998220delC; c. 6288del; p. Arg2097AlafsTer55) and mother with heterozygote variant in exon 27 (chr8:145001693_145001694delCT; c. 4054_4055del; p. Ser1352CysfsTer68) of PLEC gene

Figure 1

Sequence chromatogram and alignment to the reference sequence showing the variation in exon 31 of the PLEC gene (chr8:144998220delC; c. 6288del; p. Arg2097AlafsTer55) detected in heterozygous condition in the father of the proband (a) and not detected in the mother of the proband (b)

Figure 2

Sequence chromatogram and alignment to the reference sequence showing the variation in exon 27 of the PLEC gene (chr8:145001693_145001694delCT; c. 4054_4055del; p. Ser1352CysfsTer68) detected in heterozygous condition in the mother of the proband (b) and not detected in the father of the proband (a)

Case 2

A female proband aged 5 years, the first child born to a third-generation consanguineous couple, presented multiple blistering scars all over the body and skin fusion of digits on hands and toes. The proband mother had three spontaneous miscarriages at 2nd month of pregnancy, after the birth of proband, and presently came for genetic counseling on being three months pregnant [Pedigree 2]. The patient is clinically evaluated and suspected for epidermolysis bullosa dystrophica (EBD) and evaluated for the gene variants related to the phenotype by targeted panel sequencing using Illumina next-generation sequencing (NGS) platform and the mean coverage was 80-100X. A homozygous nonsense variation in exon 54 of the COL7A1 gene (c. 5047C >T) that results in a stop codon and premature truncation of the protein at codon 1683 (p. Arg1683Ter) was identified. The observed variant has a minor allele frequency of 0.0008% in the genome AD database and is not reported in the 1000 GnomAD database.

Pedigree 2

Represents the family of case 2, where the third-generation consanguineous marriage is observed. A homozygous nonsense variation in exon 54 (c. 5047C > T) (p. Arg1683Ter) of the COL7A1 gene was identified in the proband

Case 3

A 7-year-old, female patient, born to a consanguineous parent, presented with scaly and dried skin, a fusion of fingers and toes, absent nails, and abnormal teeth. The patient was clinically evaluated and suspected to be affected with epidermolysis bullosa dystropica (EBD). The proband mother reported two spontaneous abortions and one neonatal death due to sepsis and epidermolysis bullosa, before the birth of the proband [Pedigree 3]. Respective genes were screened for the patient by targeted panel sequencing using Illumina next-generation sequencing (NGS) platform, and the mean coverage was 80-100X. A homozygous nonsense variation (c. 7762C > T) in exon 104 of the COL7A1 gene that results in a stop codon and premature truncation of the protein at codon 2588 (p. Gln2588Ter) was detected. The validation of the variant is done by Sanger sequencing [Figure 3]. The observed variant is not present in both 1000 Genomes and GnomAD databases.

Pedigree 3

Represents the family of case 3, with parental consanguineous marriage, and the neonatal death is reported in the proband generation due to sepsis and epidermolysis bullosa. A homozygous nonsense variation (c. 7762C > T) (p. Gln2588Ter) in exon 104 of the COL7A1 gene was identified in the proband

Figure 3

Sanger sequencing data (electropherogram) from case 3. showing the homozygous nucleotide change C >T at position c. 7762 in the COL7A1 gene. The variation was confirmed by sequencing with both forward and reverse primers

The GATK best practices framework was followed for the identification of variants in all the three probands using Sentieon (v201808.01).[3] The sequences obtained are aligned to the human reference genome (GRCh37/hg19) using Sentieon aligner[34] and analyzed using Sentieon for removing duplicates, recalibration, and re-alignment of indels.[3] Sentieon haplotypeCaller has been used to identify variants that are relevant to the clinical indication. Gene annotation of the variants is performed using the VEP program[5] against the Ensemble release 91 human gene model.[6] In addition to SNVs and small Indels, copy number variants (CNVs) were detected from targeted sequence data using the Exome Depth (v1.1.10) method.[7] This algorithm detects rare CNVs based on a comparison of the read-depths of the test data with the matched aggregate reference dataset.

Clinically relevant mutations were annotated using published variants in literature and a set of diseases databases-ClinVar, OMIM, GWAS, HGMD (v2018.3) and SwissVar.[89101112] Common variants were filtered based on allele frequency in 1000Genome Phase 3, ExAC (v1.0), gnomAD (v2.1), EVS, dbSNP (v151), 1000 Japanese Genome and Indian population database.[1314151617] Non-synonymous variants effect was calculated using multiple algorithms such as PolyPhen-2, SIFT, MutationTaster2, and LRT. Only non-synonymous and splice site variants found in the epidermolysis bullosa panel genes were used for clinical interpretation. Silent variations that do not result in any change in amino acid in the coding region are not reported.

Discussion

Epidermolysis bullosa simplex with muscular dystrophy is caused due to mutations in the PLEC gene, which encodes for plectin-1, a multifunctional cytolinker expressed in tissues exposed to mechanical stress, and plays a crucial role in cytoskeleton stability, cell and tissue integrity, and regulating signal complexes.[18] In the present report, case 1 showed autosomal recessive, epidermolysis bullosa simplex with a nail and muscular dystrophy with compound heterozygous variants in exon 27 and 31 of the PLEC gene respectively. A heterozygous single base pair deletion in exon 31 (proband, father, and fetus) and a heterozygous two base pair deletion in exon 27 (proband, mother, and fetus) of the PLEC gene were detected, resulting in a frameshift and premature truncation of the protein. The parents of the patient are asymptomatic heterozygous carriers harboring each genetic variation identified in the proband and thus found compound heterozygous pathogenic variations in the onset of epidermolysis bullosa simplex (EBS).

The COL7A1 gene encodes for type VII collagen, a major stabilizing molecule of dermo-epidermal junction, and consists of 118 coding exons.[19] More than 841 mutations have been identified in the COL7A1 gene, which is family specific and leads to variable structural and functional impairment of anchoring fibrils.[202122] The present study reports case 2 and case 3 with autosomal recessive, epidermolysis bullosa dystrophica, wherein a homozygous nonsense variation is detected in exon 54 (c. 5047C >T) and (c. 7762C >T) in exon 104 of the COL7A1 gene. The creation of cryptic stop codon results in premature truncation of the protein leading to the disease condition in a dominant-negative effect, as collagen 7A is a multimer protein.

To the best of our knowledge, the mutations detected in three of these cases were not reported earlier in the literature. This is the first case report to identify novel mutations in PLEC and COL7A1 genes in epidermolysis bullosa highlighting the ethnicity and genetic diversity of South Indian origin. Thus, the present study provides a better understanding of the possible role of genetic mutations in patients and families with epidermolysis bullosa which will help in early diagnosis of the condition, carrier screening, prenatal diagnosis and to offer genetic counseling to the families affected with the condition to combat the situation.

Financial support and sponsorship

Financial support from Department of Biotechnology (File No.: BT/ HRD/01/02/2017), Ministry of Science and Technology, Govt. of India, New Delhi is kindly acknowledged.

Conflicts of interest

There are no conflicts of interest.