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Whole Exome Sequencing Confirms Molecular Diagnostics of Three Pakhtun Families With Autosomal Recessive Epidermolysis Bullosa.

ABSTRACT: Epidermolysis bullosa (EB) is a genetic skin disorder that shows heterogeneous clinical fragility. The patients develop skin blisters congenitally or in the early years of life at the dermo-epithelial junctions, including erosions, hyperkeratosis over the palms and soles. The other associated features are hypotrichosis on the scalp, absent or dystrophic nails, and dental anomalies. Molecular diagnosis through whole-exome sequencing (WES) has become one of the successful tool in clinical setups. In this study, three Pakhtun families from the Khyber Pakhtunkhwa province of Pakistan were ascertained. WES analysis of a proband in each family revealed two novel variants (COL17A1: NM_000494.4: c.4041T>G: p.Y1347^* and PLEC: NM_201380.3: c.1283_1285delGCT: p.L426del) and one previously known COL17A1: NM_000494.4:c.3067C>T: p.Q1023^*) variant in homozygous forms. Sanger sequencing of the identified variants confirmed that the heterozygous genotypes of the obligate carriers. The identified variants have not only increased the mutation spectrum of the COL17A1 and PLEC but also confirms their vital role in the morphogenesis of skin and its associated appendages. WES can be used as a first-line diagnostic tool in genetic testing and counselling families from Khyber Pakhtunkhwa, Pakistan.

SUBMITTER: Fozia F

PROVIDER: S-EPMC8369263 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundRecessive dystrophic epidermolysis bullosa is an incurable, often fatal mucocutaneous blistering disease caused by mutations in COL7A1, the gene encoding type VII collagen (C7). On the basis of preclinical data showing biochemical correction and prolonged survival in col7 −/− mice, we hypothesized that allogeneic marrow contains stem cells capable of ameliorating the manifestations of recessive dystrophic epidermolysis bullosa in humans.MethodsBetween October 2007 and August 2009, we treated seven children who had recessive dystrophic epidermolysis bullosa with immunomyeloablative chemotherapy and allogeneic stem-cell transplantation. We assessed C7 expression by means of immunofluorescence staining and used transmission electron microscopy to visualize anchoring fibrils. We measured chimerism by means of competitive polymerase-chain-reaction assay, and documented blister formation and wound healing with the use of digital photography.ResultsOne patient died of cardiomyopathy before transplantation. Of the remaining six patients, one had severe regimen-related cutaneous toxicity, with all having improved wound healing and a reduction in blister formation between 30 and 130 days after transplantation. We observed increased C7 deposition at the dermal-epidermal junction in five of the six recipients, albeit without normalization of anchoring fibrils. Five recipients were alive 130 to 799 days after transplantation; one died at 183 days as a consequence of graft rejection and infection. The six recipients had substantial proportions of donor cells in the skin, and none had detectable anti-C7 antibodies.ConclusionsIncreased C7 deposition and a sustained presence of donor cells were found in the skin of children with recessive dystrophic epidermolysis bullosa after allogeneic bone marrow transplantation. Further studies are needed to assess the long-term risks and benefits of such therapy in patients with this disorder. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00478244.)

Project description:BackgroundRevertant mosaicism has been described previously in recessive dystrophic epidermolysis bullosa (RDEB), manifesting as regions of skin with normal mechanical and biological characteristics. Here we report the discovery of revertant dermal fibroblasts, unique in that all other documented cases of revertant mosaicism occur in epidermal keratinocytes.ObjectivesTo determine the cause of revertant mosaicism found in a patient with RDEB from isolated epidermal keratinocytes and dermal fibroblasts in blister and mosaic skin regions.MethodsSkin biopsies were taken from blister and mosaic skin regions of a patient with RDEB. Allele identification was confirmed and the type VII collagen (C7) content and COL7A1 expression profile of isolated keratinocytes and fibroblasts was determined.ResultsKeratinocytes isolated from the mosaic area had a slight increase in C7, although overall expression of COL7A1 was unchanged between blister and mosaic fibroblasts. Differential allele expression was identified in blister and mosaic fibroblasts using targeted RNA sequencing (TREx), where the allele harbouring a point mutation was preferentially expressed over that containing a frameshift mutation. A crossing over event was identified in mosaic fibroblasts that was not present in blister fibroblasts, yielding a functional COL7A1 allele in a subset of cells.ConclusionsIn documenting a novel case of revertant mosaicism in RDEB, we have identified dermal fibroblasts as having the capacity to correct blistering functionally. We have also pioneered the use of TREx in quantifying allele-specific expression. Using fibroblasts instead of keratinocytes for RDEB therapies offers advantages in the local and systemic therapy of RDEB. What's already known about this topic? Revertant mosaicism has been previously documented in patients with recessive dystrophic epidermolysis bullosa (RDEB), however, it has only been found in epidermal keratinocytes. What does this study add? We have demonstrated that COL7A1 gene reversion in dermal fibroblasts occurs and is able to form functional skin in a patient with RDEB. Additionally, we have pioneered a new application for targeted RNA sequencing in quantifying allele-specific expression in fibroblasts and keratinocytes. What is the translational message? This opens up possibilities for using fibroblasts as local and systemic therapy for patients with RDEB.

Project description:Small fibres in the skin are vulnerable to damage in metabolic or toxic conditions such as diabetes mellitus or chemotherapy resulting in small fibre neuropathy and associated neuropathic pain. Whether injury to the most distal portion of sensory small fibres due to a primary dermatological disorder can cause neuropathic pain is still unclear. Recessive dystrophic epidermolysis bullosa (RDEB) is a rare condition in which mutations of proteins of the dermo-epidermal junction lead to cycles of blistering followed by regeneration of the skin. Damage is exclusive to the skin and mucous membranes, with no known direct compromise of the nervous system. It is increasingly recognized that most RDEB patients experience daily pain, the aetiology of which is unclear but may include inflammation (in the wounds), musculoskeletal (due to atrophy and retraction scars limiting movement) or neuropathic pain. In this study we investigated the incidence of neuropathic pain and examined the presence of nerve dysfunction in RDEB patients. Around three quarters of patients presented with pain of neuropathic characteristics, which had a length-dependent distribution. Quantitative sensory testing of the foot revealed striking impairments in thermal detection thresholds combined with an increased mechanical pain sensitivity and wind up ratio (temporal summation of noxious mechanical stimuli). Nerve conduction studies showed normal large fibre sensory and motor nerve conduction; however, skin biopsy showed a significant decrease in intraepidermal nerve fibre density. Autonomic nervous system testing revealed no abnormalities in heart rate and blood pressure variability however the sympathetic skin response of the foot was impaired and sweat gland innervation was reduced. We conclude that chronic cutaneous injury can lead to injury and dysfunction of the most distal part of small sensory fibres in a length-dependent distribution resulting in disabling neuropathic pain. These findings also support the use of neuropathic pain screening tools in these patients and treatment algorithms designed to target neuropathic pain.

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Whole Exome Sequencing Confirms Molecular Diagnostics of Three Pakhtun Families With Autosomal Recessive Epidermolysis Bullosa.

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