Biallelic TUFT1 variants cause woolly hair, superficial skin fragility and desmosomal defects.

BACKGROUND: Desmosomes are complex cell junction structures that connect intermediate filaments providing strong cell-to-cell adhesion in tissues exposed to mechanical stress. OBJECTIVES: To identify causal variants in individuals with woolly hair and skin fragility of unknown genetic cause. METHODS: This research was conducted using whole-genome sequencing, whole-exome sequencing, clinical phenotyping, haplotype analysis, single-cell RNA sequencing data analysis, immunofluorescence microscopy and transmission electron microscopy. RESULTS: We identified homozygous predicted loss-of-function tuftelin-1 (TUFT1) variants in nine individuals, from three families, with woolly hair and skin fragility. One donor splice-site variant, c.60+1G>A, was present in two families, while a frameshift variant, p.Gln189Asnfs*49, was found in the third family. Haplotype analysis showed the c.60+1G>A substitution to be a founder variant in the Irish population that likely arose approximately 20 generations ago. Human and mouse single-cell RNA sequencing data showed TUFT1 expression to be enriched in the hair dermal sheath and keratinocytes. TUFT1 expression was highly correlated with genes encoding desmosomal components implicated in diseases with phenotypes that overlap with the cohort presented here. Immunofluorescence showed tuftelin-1 to be mainly localized to the peripheral cell membranes of keratinocytes in normal skin. Skin samples from individuals with TUFT1 variants showed markedly reduced immunoreactivity for tuftelin-1, with a loss of the keratinocyte cell membrane labelling. Light microscopy revealed keratinocyte adhesion, mild hyperkeratosis and areas of superficial peeling. Transmission electron microscopy showed panepidermal acantholysis with widening of intercellular spaces throughout the epidermis and desmosomal detachment through the inner plaques. CONCLUSIONS: Biallelic loss-of-function TUFT1 variants cause a new autosomal recessive skin/hair disorder characterized by woolly hair texture and early-onset skin fragility. Tuftelin-1 has a role in desmosomal integrity and function.

Mutations in the ribosome biogenesis factor gene LTV1 are linked to LIPHAK syndrome, a novel poikiloderma-like disorder.

In the framework of the UK 100 000 Genomes Project, we investigated the genetic origin of a previously undescribed recessive dermatological condition, which we named LIPHAK (LTV1-associated Inflammatory Poikiloderma with Hair abnormalities and Acral Keratoses), in four affected individuals from two UK families of Pakistani and Indian origins, respectively. Our analysis showed that only one gene, LTV1, carried rare biallelic variants that were shared in all affected individuals, and specifically they bore the NM_032860.5:c.503A > G, p.(Asn168Ser) change, found homozygously in all of them. In addition, high-resolution homozygosity mapping revealed the presence of a small 652-kb stretch on chromosome 6, encompassing LTV1, that was haploidentical and common to all affected individuals. The c.503A > G variant was predicted by in silico tools to affect the correct splicing of LTV1's exon 5. Minigene-driven splicing assays in HEK293T cells and in a skin sample from one of the patients confirmed that this variant was indeed responsible for the creation of a new donor splice site, resulting in aberrant splicing and in a premature termination codon in exon 6 of this gene. LTV1 encodes one of the ribosome biogenesis factors that promote the assembly of the small (40S) ribosomal subunit. In yeast, defects in LTV1 alter the export of nascent ribosomal subunits to the cytoplasm; however, the role of this gene in human pathology is unknown to date. Our data suggest that LIPHAK could be a previously unrecognized ribosomopathy.

Kindler epidermolysis bullosa-like skin phenotype and downregulated basement membrane zone gene expression in poikiloderma with neutropenia and a homozygous USB1 mutation.

Epidermolysis bullosa (EB) is a genotypically heterogeneous group of disorders characterized by cutaneous blistering and erosions with a tremendous spectrum of severity. One of the distinct forms of EB, Kindler EB (KEB), manifests with blistering and poikiloderma; this subtype of EB is caused by mutations in the FERMT1 gene encoding kindlin-1. In this study, we investigated a patient clinically diagnosed as KEB with reduced FERMT1 gene expression and intensity of immunostaining for kindlin-1. Transmission electron microscopy showed lamina densa reduplication, frequently observed in KEB. However, no mutations were identified in FERMT1 in this patient with consanguineous parents, and this gene resided outside of genomic regions of homozygosity (ROH). Instead, whole-exome sequencing and homozygosity mapping identified a homozygous sequence variant at the +4 position of intron 2 in the USB1 gene, encoding an exoribonuclease required for processing of U6 snRNA, a critical component of spliceosomes. Examination of the patient's RNA by RNA-Seq confirmed the pathogenicity of this variant, causing aberrant splicing predicted to result in loss of function of USB1. Mutations in this gene have been reported in patients with poikiloderma and neutropenia, with a few reported cases in association with skin fragility, a condition distinct from the KEB phenotype. Transcriptome analysis revealed that several genes, expressed in the cutaneous basement membrane zone and previously associated with different subtypes of EB, were differentially downregulated at the mRNA level. EB-associated mRNA downregulation was confirmed at protein levels by skin immunofluorescence. These observations provide a novel mechanism for blistering and erosions in the skin as a result reduced presence of adhesion complexes critical for stable association of epidermis and dermis at the level of cutaneous basement membrane zone.

Arrhythmogenic right ventricular cardiomyopathy in patients with biallelic JUP-associated skin fragility.

Arrhythmogenic right ventricular cardiomyopathy (ARVC), with skin manifestations, has been associated with mutations in JUP encoding plakoglobin. Genotype-phenotype correlations regarding the penetrance of cardiac involvement, and age of onset have not been well established. We examined a cohort of 362 families with skin fragility to screen for genetic mutations with next-generation sequencing-based methods. In two unrelated families, a previously unreported biallelic mutation, JUP: c.201delC; p.Ser68Alafs*92, was disclosed. The consequences of this mutation were determined by expression profiling both at tissue and ultrastructural levels, and the patients were evaluated by cardiac and cutaneous work-up. Whole-transcriptome sequencing by RNA-Seq revealed JUP as the most down-regulated gene among 21 skin fragility-associated genes. Immunofluorescence showed the lack of plakoglobin in the epidermis. Two probands, 2.5 and 22-year-old, with the same homozygous mutation, allowed us to study the cross-sectional progression of cardiac involvements in relation to age. The older patient had anterior T wave inversions, prolonged terminal activation duration (TAD), and RV enlargement by echocardiogram, and together with JUP mutation met definite ARVC diagnosis. The younger patient had no evidence of cardiac disease, but met possible ARVC diagnosis with one major criterion (the JUP mutation). In conclusion, we identified the same biallelic homozygous JUP mutation in two unrelated families with skin fragility, but cardiac findings highlighted age-dependent penetrance of ARVC. Thus, young, phenotypically normal patients with biallelic JUP mutations should be monitored for development of ARVC.

Genomics-based treatment in a patient with two overlapping heritable skin disorders: Epidermolysis bullosa and acrodermatitis enteropathica.

Next-generation sequencing (NGS) is helpful in diagnosing complex genetic disorders and phenotypes, particularly when more than one overlapping condition is present. From a large cohort of 362 families with clinical manifestations of skin and mucosal fragility, referred by several major medical centers, one patient was found by NGS to have two overlapping heritable skin diseases, recessive dystrophic epidermolysis bullosa (RDEB; COL7A1 mutations) and acrodermatitis enteropathica (AE; SLC39A4 mutations). The pathogenicity of the variants was studied at gene expression as well as ultrastructural and tissue levels. Although there is no specific treatment for RDEB except avoiding trauma, supplementation with oral zinc (3 mg·kg-1 ·day-1 ) for the AE resulted in rapid amelioration of the skin findings. This case demonstrates the power of NGS in identifying two genetically unlinked diseases that led to effective treatment with major clinical benefits as an example of genomics-guided treatment.

Phase I/II open-label trial of intravenous allogeneic mesenchymal stromal cell therapy in adults with recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary blistering disorder due to a lack of type VII collagen. At present, treatment is mainly supportive. OBJECTIVES: To determine whether intravenous allogeneic bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs) are safe in RDEB adults and if the cells improve wound healing and quality of life. METHODS: We conducted a prospective, phase I/II, open-label study recruiting 10 RDEB adults to receive 2 intravenous infusions of BM-MSCs (on day 0 and day 14; each dose 2-4 × 106 cells/kg). RESULTS: BM-MSCs were well tolerated with no serious adverse events to 12 months. Regarding efficacy, there was a transient reduction in disease activity scores (8/10 subjects) and a significant reduction in itch. One individual showed a transient increase in type VII collagen. LIMITATIONS: Open-label trial with no placebo. CONCLUSIONS: MSC infusion is safe in RDEB adults and can have clinical benefits for at least 2 months.

Biallelic KRT5 mutations in autosomal recessive epidermolysis bullosa simplex, including a complete human keratin 5 "knock-out".

Epidermolysis bullosa simplex (EBS) is usually inherited as an autosomal dominant disease due to monoallelic gain-of-function mutations in KRT5 or KRT14. Although autosomal recessive forms of EBS have been associated with mutations in at least 10 genes, recessive EBS due to homozygous biallelic KRT5 mutations has not been reported previously; it has been hypothesized that it would result in prenatal lethality. We sought the genetic causes of EB in a cohort of 512 distinct EB families by performing whole exome sequencing (WES) and using an EB-targeting next-generation sequencing (NGS) panel of 21 genes. The pathogenicity and consequences of the mutations were determined by expression profiling and at tissue and ultrastructural levels. Two pathogenic, homozygous missense variants of KRT5 in two patients with generalized EBS and a homozygous null mutation in a patient who died as a neonate from complications of EB were found. The two missense mutations disrupted keratin 5 expression on immunofluorescence microscopy, and the human "knock-out" of KRT5 showed no RNA and protein expression. Collectively, these findings identify biallelic KRT5 mutations with a phenotypic spectrum varying from mild, localized and generalized to perinatal lethal, expanding the genotypic profile of autosomal recessive EBS.

Safety and early efficacy outcomes for lentiviral fibroblast gene therapy in recessive dystrophic epidermolysis bullosa.

BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is a severe form of skin fragility disorder due to mutations in COL7A1 encoding basement membrane type VII collagen (C7), the main constituent of anchoring fibrils (AFs) in skin. We developed a self-inactivating lentiviral platform encoding a codon-optimized COL7A1 cDNA under the control of a human phosphoglycerate kinase promoter for phase I evaluation.METHODSIn this single-center, open-label phase I trial, 4 adults with RDEB each received 3 intradermal injections (~1 × 106 cells/cm2 of intact skin) of COL7A1-modified autologous fibroblasts and were followed up for 12 months. The primary outcome was safety, including autoimmune reactions against recombinant C7. Secondary outcomes included C7 expression, AF morphology, and presence of transgene in the injected skin.RESULTSGene-modified fibroblasts were well tolerated, without serious adverse reactions or autoimmune reactions against recombinant C7. Regarding efficacy, there was a significant (P < 0.05) 1.26-fold to 26.10-fold increase in C7 mean fluorescence intensity in the injected skin compared with noninjected skin in 3 of 4 subjects, with a sustained increase up to 12 months in 2 of 4 subjects. The presence of transgene (codon-optimized COL7A1 cDNA) was demonstrated in the injected skin at month 12 in 1 subject, but no new mature AFs were detected.CONCLUSIONTo our knowledge, this is the first human study demonstrating safety and potential efficacy of lentiviral fibroblast gene therapy with the presence of COL7A1 transgene and subsequent C7 restoration in vivo in treated skin at 1 year after gene therapy. These data provide a rationale for phase II studies for further clinical evaluation.TRIAL REGISTRATIONClincalTrials.gov NCT02493816.FUNDINGCure EB, Dystrophic Epidermolysis Bullosa Research Association (UK), UK NIHR Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London, and Fondation René Touraine Short-Exchange Award.

Mutations in PLOD3, encoding lysyl hydroxylase 3, cause a complex connective tissue disorder including recessive dystrophic epidermolysis bullosa-like blistering phenotype with abnormal anchoring fibrils and type VII collagen deficiency.

Epidermolysis bullosa (EB), the paradigm of heritable skin fragility disorders, is associated with mutations in as many as 20 distinct genes. One of the clinical variants, recessive dystrophic EB (RDEB), demonstrates sub-lamina densa blistering accompanied by alterations in anchoring fibrils due to mutations in COL7A1. In this study, we characterized a patient with widespread connective tissue abnormalities, including skin blistering similar to that in RDEB. Whole exome sequencing, combined with genome-wide homozygosity mapping, identified a homozygous missense mutation in PLOD3 encoding lysyl hydroxylase 3 (LH3). No mutations in COL7A1, the gene previously associated with RDEB, were detected. The level of LH3 was dramatically reduced in the skin and fibroblast cultures from the patient. The blistering in the skin occurred below the lamina densa and was associated with variable density and morphology of anchoring fibrils. The level of type VII collagen expression in the skin was markedly reduced. Analysis of hydroxylysine and its glycosylated derivatives (galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine) revealed marked reduction in glycosylated hydroxylysine. Collectively, these findings indicate that PLOD3 mutations can result in a dystrophic EB-like phenotype in the spectrum of connective tissue disorders and add it to the list of candidate genes associated with skin fragility.

Next generation sequencing identifies double homozygous mutations in two distinct genes (EXPH5 and COL17A1) in a patient with concomitant simplex and junctional epidermolysis bullosa.

Epidermolysis bullosa (EB) is a heterogeneous group of heritable blistering diseases. We developed a next generation sequencing (NGS) panel covering 21 genes associated with skin fragility disorders, and it was applied to DNA from 91 probands with the diagnosis of EB. In one patient, novel homozygous mutations were disclosed in two different, unlinked EB-associated genes: EXPH5, chr11 g.108510085G > A; p.Arg1808Ter and COL17A1, chr10 g.104077423delT; p.Thr68LeufsTer106. Consequences of the COL17A1 mutation were examined by RNAseq which revealed a complex splicing pattern predicting synthesis of a truncated polypeptide (85%) or in-frame deletion of exon 4 (15% of transcripts). Transmission electron microscopy (TEM) and immunostaining revealed findings consistent with EB simplex (EBS) and junctional EB (JEB), and clinical examination revealed a complex phenotype with features of both subtypes. This case illustrates the power of next generation sequencing in identifying mutations in patients with complex EB phenotype, with implications for genotype-phenotype correlations, prenatal testing, and genetic counseling of families at risk for recurrence.