Navigating the diagnostic journey of epidermolysis bullosa - a qualitative study on the lived experiences and needs of parents and patients.

BACKGROUND: Epidermolysis bullosa (EB) concerns a heterogeneous group of rare genetic skin fragility disorders that result in chronic blistering and wounding. EB significantly impacts the daily lives of patients with EB, as well as their families. While advances in diagnostics are improving the speed and accuracy of EB diagnosis, little is known about the experiences and needs of parents and patients throughout their diagnostic journey. OBJECTIVES: In this qualitative study, we explore the parent and patient perspective on the EB diagnostic trajectory to gain an in-depth understanding of their lived experiences and needs. METHODS: Participants were parents of paediatric patients (n=18), and adult patients (n=8) recruited from the Dutch EB Registry. After purposive sampling, they participated in semi-structured interviews via video-calls to discuss their personal diagnostic trajectory and the subsequent impact of EB diagnosis on their (family) life. Applying a constructivist approach, a reflexive thematic analysis was executed to facilitate a dynamic and iterative process, involving inductive open coding of transcripts and constant comparison of data. RESULTS: Ten major themes were developed, representing three distinct groups: (i) parents of children with JEB and RDEB, (ii) parents of children with EBS and DDEB, and (iii) adult patients with localized EB. The EB diagnostic process appeared to have a diversity of emotional consequences, varying from desperation and uncertainty about the future, to clarification and confirmation. The urgent need for a timely diagnosis and accurate prognosis was emphasized, particularly by parents of children with extensive presentation. Both parents and patients expressed shortcomings in clinical practice, with severity ratings in current EB disease terminology in particular seeming to have an adverse impact on illness perception, healthcare-seeking behaviour, research participation, and engagement in peer support. CONCLUSIONS: This study describes the lived experience and needs of parents of children and adult patients during the diagnostic process of EB. We show an urgent need to accelerate diagnostics and imply that the EB community should continue working towards ever faster diagnosis, public awareness, and education. While guiding patients along the diagnostic journey, clinicians should focus their support strategies on tailored medical communication, while refraining from value-connoted wording.

Epidermal growth factor receptor inhibition leads to cellular phenotype correction of DSP-mutated keratinocytes.

Desmoplakin (DSP) is a desmosomal component expressed in skin and heart, essential for desmosome stability and intermediate filament connection. Pathogenic variants in the DSP gene encoding DSP, lead to heterogeneous skin, adnexa and heart-related phenotypes, including skin fragility, woolly hair (WH), palmoplantar keratoderma (PPK) and arrhythmogenic/dilated cardiomyopathy (ACM/DCM). The ambiguity of computer-based prediction analysis of pathogenicity and effect of DSP variants, indicates a necessity for functional analysis. Here, we report a heterozygous DSP variant that was not previously described, NM_004415.4:c.3337C>T (NM_004415.4(NP_004406.2):p.(Arg1113*)) in a patient with PPK, WH and ACM. RNA and protein analysis revealed ~50% reduction of DSP mRNA and protein expression. Patient's keratinocytes showed fragile cell-cell connections and perinuclear retracted intermediate filaments. Epidermal growth factor receptor (EGFR) is a transmembrane protein expressed in the basal epidermal layer involved in proliferation and differentiation, processes that are disrupted in the development of PPK, and in the regulation of the desmosome. In skin of the abovementioned patient, evident EGFR upregulation was observed. EGFR inhibition in patient's keratinocytes strongly increased DSP expression at the plasma membrane, improved intermediate filament connection with the membrane edges and reduced the cell-cell fragility. This cell phenotypic recovery was due to a translocation of DSP to the plasma membrane together with an increased number of desmosomes. These results indicate a therapeutic potential of EGFR inhibitors for disorders caused by DSP haploinsufficiency.

Disruption of TUFT1, a Desmosome-Associated Protein, Causes Skin Fragility, Woolly Hair, and Palmoplantar Keratoderma.

Desmosomes are dynamic complex protein structures involved in cellular adhesion. Disruption of these structures by loss-of-function variants in desmosomal genes leads to a variety of skin- and heart-related phenotypes. In this study, we report TUFT1 as a desmosome-associated protein, implicated in epidermal integrity. In two siblings with mild skin fragility, woolly hair, and mild palmoplantar keratoderma but without a cardiac phenotype, we identified a homozygous splice-site variant in the TUFT1 gene, leading to aberrant mRNA splicing and loss of TUFT1 protein. Patients' skin and keratinocytes showed acantholysis, perinuclear retraction of intermediate filaments, and reduced mechanical stress resistance. Immunolabeling and transfection studies showed that TUFT1 is positioned within the desmosome and that its location is dependent on the presence of the desmoplakin carboxy-terminal tail. A Tuft1-knockout mouse model mimicked the patients' phenotypes. Altogether, this study reveals TUFT1 as a desmosome-associated protein, whose absence causes skin fragility, woolly hair, and palmoplantar keratoderma.

Expanding the phenotype of anauxetic dysplasia caused by biallelic NEPRO mutations: A case report.

The cartilage hair hypoplasia and anauxetic dysplasia (CHH-AD) spectrum encompasses a group of rare skeletal disorders, with anauxetic dysplasia (ANXD) at the most severe end of the spectrum. Biallelic variants in RMRP, POP1, and NEPRO (C3orf17) have previously been associated with the three currently recognized ANXD types. Generally, all types are characterized by severe short stature, brachydactyly, skin laxity, joint hypermobility and dislocations, and extensive skeletal abnormalities visible on radiological evaluation. Thus far, only five patients with type 3 anauxetic dysplasia (ANXD3) have been reported. Here, we describe one additional ANXD3 patient. We provide a detailed physical and radiological evaluation of this patient, in whom we identified a homozygous variant, c.280C > T, p.(Arg94Cys), in NEPRO. Our patient presented with clinically relevant features not previously described in ANXD3: atlantoaxial subluxation, extensive dental anomalies, and a sagittal suture craniosynostosis resulting in scaphocephaly. We provide an overview of the literature on ANXD3 and discuss our patient's characteristics in the context of previously described patients. This study expands the phenotypic spectrum of ANXD, particularly ANXD3. Greater awareness of the possibility of atlantoaxial subluxation, dental anomalies, and craniosynostosis may lead to more timely diagnosis and treatment.

Heterogeneity of reported outcomes in epidermolysis bullosa clinical research: a scoping review as a first step towards outcome harmonization.

BACKGROUND: Epidermolysis bullosa (EB) is a rare, genetically and clinically heterogeneous group of skin fragility disorders. No cure is currently available, but many novel and repurposed treatments are upcoming. For adequate evaluation and comparison of clinical studies in EB, well-defined and consistent consensus-endorsed outcomes and outcome measurement instruments are necessary. OBJECTIVES: To identify previously reported outcomes in EB clinical research, group these outcomes by outcome domains and areas and summarize respective outcome measurement instruments. METHODS: A systematic literature search was performed in the databases MEDLINE, Embase, Scopus, Cochrane CENTRAL, CINAHL, PsycINFO and trial registries covering the period between January 1991 and September 2021. Studies were included if they evaluated a treatment in a minimum of three patients with EB. Two reviewers independently performed the study selection and data extraction. All identified outcomes and their respective instruments were mapped onto overarching outcome domains. The outcome domains were stratified according to subgroups of EB type, age group, intervention, decade and phase of clinical trial. RESULTS: The included studies (n = 207) covered a range of study designs and geographical settings. A total of 1280 outcomes were extracted verbatim and inductively mapped onto 80 outcome domains and 14 outcome areas. We found a steady increase in the number of published clinical trials and outcomes reported over the past 30 years. The included studies mainly focused on recessive dystrophic EB (43%). Wound healing was reported most frequently across all studies and referred to as a primary outcome in 31% of trials. Great heterogeneity of reported outcomes was observed within all stratified subgroups. Moreover, a diverse range of outcome measurement instruments (n = 200) was identified. CONCLUSIONS: We show substantial heterogeneity in reported outcomes and outcome measurement instruments in EB clinical research over the past 30 years. This review is the first step towards harmonization of outcomes in EB, which is necessary to expedite the clinical translation of novel treatments for patients with EB.

Uncombable hair syndrome due to maternal uniparental disomy of chromosome 1.

Uncombable hair syndrome is a hair shaft condition in which the hair is frizzy, light in color (silver to light brown), and cannot be combed flat. Autosomal dominant (with complete or incomplete penetrance), autosomal recessive, and sporadic cases have been reported. In 2016 causative mutations in three genes were identified for uncombable hair syndrome, all with an autosomal recessive inheritance pattern: PADI3, TGM3, and TCHH. In many cases, however, there is still no molecular diagnosis. Here, we describe a case of autosomal recessive uncombable hair syndrome resulting from maternal uniparental disomy of chromosome 1.

PRDM10 directs FLCN expression in a novel disorder overlapping with Birt-Hogg-Dubé syndrome and familial lipomatosis.

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder characterized by fibrofolliculomas, pulmonary cysts, pneumothoraces and renal cell carcinomas. Here, we reveal a novel hereditary disorder in a family with skin and mucosal lesions, extensive lipomatosis and renal cell carcinomas. The proband was initially diagnosed with BHD based on the presence of fibrofolliculomas, but no pathogenic germline variant was detected in FLCN, the gene associated with BHD. By whole exome sequencing we identified a heterozygous missense variant (p.(Cys677Tyr)) in a zinc-finger encoding domain of the PRDM10 gene which co-segregated with the phenotype in the family. We show that PRDM10Cys677Tyr loses affinity for a regulatory binding motif in the FLCN promoter, abrogating cellular FLCN mRNA and protein levels. Overexpressing inducible PRDM10Cys677Tyr in renal epithelial cells altered the transcription of multiple genes, showing overlap but also differences with the effects of knocking out FLCN. We propose that PRDM10 controls an extensive gene program and acts as a critical regulator of FLCN gene transcription in human cells. The germline variant PRDM10Cys677Tyr curtails cellular folliculin expression and underlies a distinguishable syndrome characterized by extensive lipomatosis, fibrofolliculomas and renal cell carcinomas.

Revertant Mosaicism in Genodermatoses: Natural Gene Therapy Right before Your Eyes.

Revertant mosaicism (RM) is the intriguing phenomenon in which nature itself has successfully done what medical science is so eagerly trying to achieve: correcting the effect of disease-causing germline variants and thereby reversing the disease phenotype back to normal. RM was molecularly confirmed for the first time in a genodermatosis in 1997, the genetic skin condition junctional epidermolysis bullosa (EB). At that time, RM was considered an extraordinary phenomenon. However, several important discoveries have changed this conception in the past few decades. First, RM has now been identified in all major subtypes of EB. Second, RM has also been identified in many other genodermatoses. Third, a theoretical mathematical exercise concluded that reverse mutations should be expected in all patients with a recessive subtype of EB or any other genodermatosis. This has shifted the paradigm from RM being an extraordinary phenomenon to it being something that every physician working in the field of genodermatoses should be looking for in every patient. It has also raised hope for new treatment options in patients with genodermatoses. In this review, we summarize the current knowledge on RM and discuss the perspectives of RM for the future treatment of patients with genodermatoses.

A translation re-initiation variant in KLHL24 also causes epidermolysis bullosa simplex and dilated cardiomyopathy via intermediate filament degradation.

This study shows that gain-of-function variants in KLHL24 causing EBS and DCM, do not only originate in the start-codon and suggest that any nonsense-inducing variant affecting nucleotides c.4_84 will likely cause the same effect on protein level and a similar potential lethal phenotype.

In Vitro Models for the Evaluation of Antisense Oligonucleotides in Skin.

The genodermatosis dystrophic epidermolysis bullosa (DEB) is caused by mutations in the COL7A1 gene which encodes type VII collagen (C7). In the cutaneous basement membrane zone, C7 secures attachment of the epidermal basal keratinocyte to the papillary dermis by means of anchoring fibril formation. The complete absence of these anchoring fibrils leads to severe blistering of skin and mucosa upon the slightest friction and early mortality. To date, although preclinical advances toward therapy are promising, treatment for the disease is merely symptomatic. Therefore, research into novel therapeutics is warranted.Antisense oligonucleotide (ASO)-mediated exon skipping is such a therapy . Clinical examination of naturally occurring exon skipping suggested that this mechanism could most likely benefit the most severely affected patients. The severe form of DEB is caused by biallelic null mutations. Exon skipping aims to bind an ASO to the mutated exon of the pre-mRNA in the cell nucleus. Thereby, the ASO inhibits the recognition of the mutated exon by the splicing machinery, and as a result, the mutated exon is spliced out from the mRNA with its surrounding introns, i.e., it is skipped. Here, we describe in vitro methods to evaluate ASO-mediated exon skipping in a preclinical setting.

In Vivo Models for the Evaluation of Antisense Oligonucleotides in Skin.

Here, we describe an in vivo model in which antisense oligonucleotides were preclinically evaluated in reconstituted patient and healthy control skin. The aim was to investigate the effect of antisense oligonucleotides upon local or systemic administration. This allows for clinically relevant evaluation of antisense oligonucleotides in an in vivo setting. In this model, primary human keratinocytes and fibroblasts were placed into silicone grafting chambers, implanted onto the back of athymic nude mice. After sufficient cells were expanded, within a few weeks, human skin grafts were generated with a high success rate. These mice bearing grafts were subsequently treated with antisense oligonucleotides targeting exon 105 of the COL7A1 gene which encodes type VII collagen. Patients completely lacking expression of type VII collagen develop severe blistering of skin and mucosa, i.e., recessive dystrophic epidermolysis bullosa. In this chapter, we describe the in vivo model used for the preclinical evaluation of antisense oligonucleotides as therapeutic approach for recessive dystrophic epidermolysis bullosa.

Therapeutic Prospects of Exon Skipping for Epidermolysis Bullosa.

Epidermolysis bullosa is a group of genetic skin conditions characterized by abnormal skin (and mucosal) fragility caused by pathogenic variants in various genes. The disease severity ranges from early childhood mortality in the most severe types to occasional acral blistering in the mildest types. The subtype and severity of EB is linked to the gene involved and the specific variants in that gene, which also determine its mode of inheritance. Current treatment is mainly focused on symptomatic relief such as wound care and blister prevention, because truly curative treatment options are still at the preclinical stage. Given the current level of understanding, the broad spectrum of genes and variants underlying EB makes it impossible to develop a single treatment strategy for all patients. It is likely that many different variant-specific treatment strategies will be needed to ultimately treat all patients. Antisense-oligonucleotide (ASO)-mediated exon skipping aims to counteract pathogenic sequence variants by restoring the open reading frame through the removal of the mutant exon from the pre-messenger RNA. This should lead to the restored production of the protein absent in the affected skin and, consequently, improvement of the phenotype. Several preclinical studies have demonstrated that exon skipping can restore protein production in vitro, in skin equivalents, and in skin grafts derived from EB-patient skin cells, indicating that ASO-mediated exon skipping could be a viable strategy as a topical or systemic treatment. The potential value of exon skipping for EB is supported by a study showing reduced phenotypic severity in patients who carry variants that result in natural exon skipping. In this article, we review the substantial progress made on exon skipping for EB in the past 15 years and highlight the opportunities and current challenges of this RNA-based therapy approach. In addition, we present a prioritization strategy for the development of exon skipping based on genomic information of all EB-involved genes.

Genotype-phenotype correlations for pancreatic cancer risk in Dutch melanoma families with pathogenic CDKN2A variants.

BACKGROUND: Pathogenic variants in the CDKN2A gene are generally associated with the development of melanoma and pancreatic ductal adenocarcinoma (PDAC), but specific genotype-phenotype correlations might exist and the extent of PDAC risk is not well established for many variants. METHODS: Using the Dutch national familial melanoma database, we identified all families with a pathogenic CDKN2A variant and investigated the occurrence of PDAC within these families. We also estimated the standardised incidence ratio and lifetime PDAC risk for carriers of a highly prevalent variant in these families. RESULTS: We identified 172 families in which 649 individuals carried 15 different pathogenic variants. The most prevalent variant was the founder mutation c.225_243del (p16-Leiden, 484 proven carriers). Second most prevalent was c.67G>C (55 proven carriers). PDAC developed in 95 of 163 families (58%, including 373 of 629 proven carriers) harbouring a variant with an effect on the p16INK4a protein, whereas PDAC did not occur in the 9 families (20 proven carriers) with a variant affecting only p14ARF. In the c.67G>C families, PDAC occurred in 12 of the 251 (5%) persons at risk. The standardised incidence ratio was 19.1 (95% CI 8.3 to 33.6) and the cumulative PDAC incidence at age 75 years (lifetime risk) was 19% (95% CI 7.5% to 30.1%). CONCLUSIONS: Our results support the notion that pathogenic CDKN2A variants affecting the p16INK4a protein, including c.67G>C, are associated with increased PDAC risk and carriers of such variants should be offered pancreatic cancer surveillance. There is no clinical evidence that impairment of only the p14ARF protein leads to an increased PDAC risk.