Clinical and molecular features in a cohort of Middle Eastern patients with epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) features skin and mucosal fragility due to pathogenic variants in genes encoding components of the cutaneous basement membrane. Based on the level of separation within the dermal-epidermal junction, EB is sub-classified into four major types including EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), and Kindler EB (KEB) with 16 EB-associated genes reported to date. METHODS: We ascertained a cohort of 151 EB patients of various Middle Eastern ethnic backgrounds. RESULTS: The cohort was comprised of EBS (64%, 97/151), DEB (21%, 31/151), JEB (12%, 18/151), and KEB (3%, 5/151). KRT14 and KRT5 variants were most common among EBS patients with 43% (42/97) and 46% (45/97) of EBS patients carrying mutations in either of these two genes, respectively. Truncal involvement was more common in KRT14-associated EBS as compared to EBS due to KRT5 mutations (p < .05). Mutations in COL17A1 and laminin 332-encoding genes were identified in 55% (10/18) and 45% (8/18) of JEB patients. Scarring alopecia, caries, and EB nevi were most common among JEB patients carrying COL17A1 mutations as compared to laminin 332-associated JEB (p < .05). Abnormal nails were evident in most DEB and JEB patients while poikiloderma was exclusively observed in KEB (p < .001). CONCLUSIONS: EB patients of Middle Eastern origin were found to feature specific phenotype-genotype correlations of relevance to the diagnosis and genetic counseling of patients in this region.

MicroRNA-200b-mediated reversion of a spectrum of epithelial-to-mesenchymal transition states in recessive dystrophic epidermolysis bullosa squamous cell carcinomas.

BACKGROUND: Cutaneous squamous cell carcinoma (SCC) is the leading cause of death in patients with recessive dystrophic epidermolysis bullosa (RDEB). However, the survival time from first diagnosis differs between patients; some tumours spread particularly fast, while others may remain localized for years. As treatment options are limited, there is an urgent need for further insights into the pathomechanisms of RDEB tumours, to foster therapy development and support clinical decision-making. OBJECTIVES: To investigate differences in RDEB tumours of diverging aggressiveness at the molecular and phenotypic level, with a particular focus on epithelial-to-mesenchymal (EMT) transition states and thus microRNA-200b (miR-200b) as a regulator. METHODS: Primary RDEB-SCC keratinocyte lines were characterized with respect to their EMT state. For this purpose, cell morphology was classified and the expression of EMT markers analysed using immunofluorescence, flow cytometry, semi-quantitative reverse transcriptase polymerase chain reaction and Western blotting. The motility of RDEB-SCC cells was determined and conditioned medium of RDEB-SCC cells was used to treat endothelial cells in an angiogenesis assay. In addition, we mined previously generated microRNA (miRNA) profiling data to identify a candidate with potential therapeutic relevance and performed transient miRNA transfection studies to investigate the candidate's ability to reverse EMT characteristics. RESULTS: We observed high variability in EMT state in the RDEB-SCC cell lines, which correlated with in situ analysis of two available patient biopsies and respective clinical disease course. Furthermore, we identified miR-200b-3p to be downregulated in RDEB-SCCs, and the extent of deregulation significantly correlated with the EMT features of the various tumour lines. miR-200b-3p was reintroduced into RDEB-SCC cell lines with pronounced EMT features, which resulted in a significant increase in epithelial characteristics, including cell morphology, EMT marker expression, migration and angiogenic potential. CONCLUSIONS: RDEB-SCCs exist in different EMT states and the level of miR-200b is indicative of how far an RDEB-SCC has gone down the EMT path. Moreover, the reintroduction of miR-200b significantly reduced mesenchymal features.

Dominant frontonasal dysplasia with ectodermal defects results from increased activity of ALX4.

Frontonasal dysplasia (FND) refers to a group of rare developmental disorders characterized by abnormal morphology of the craniofacial region. We studied a family manifesting with clinical features typical for FND2 including neurobehavioral abnormalities, hypotrichosis, hypodontia, and facial dysmorphism. Whole-exome sequencing analysis identified a novel heterozygous frameshift insertion in ALX4 (c.985_986insGTGC, p.Pro329Argfs*115), encoding aristaless homeobox 4. This and a previously reported dominant FND2-causing variant are predicted to result in the formation of a similar abnormally elongated protein tail domain. Using a reporter assay, we showed that the elongated ALX4 displays increased activity. ALX4 negatively regulates the Wnt/ß-catenin pathway and accordingly, patient keratinocytes showed altered expression of genes associated with the WNT/ß-catenin pathway, which in turn may underlie ectodermal manifestations in FND2. In conclusion, dominant FND2 with ectodermal dysplasia results from frameshift variants in ALX4 exerting a gain-of-function effect.

Defective cathepsin Z affects EGFR expression and causes autosomal dominant palmoplantar keratoderma.

BACKGROUND: The abnormal function of epidermal growth factor receptor (EGFR) has recently been shown to underlie various disorders of cornification. OBJECTIVES: To delineate the genetic basis of a novel dominant form of palmoplantar keratoderma (PPK). METHODS: Whole-exome (WES) and direct sequencing, quantitative real-time polymerase chain reaction, protein modelling, confocal immunofluorescence microscopy, immunoblotting, three-dimensional skin equivalents and an enzyme activity assay were used to delineate the genetic basis of a novel dominant form of PPK. RESULTS: WES revealed heterozygous variants (c.274T > C and c.305C > T) in CTSZ (encoding cathepsin Z) in four individuals (belonging to three unrelated families) with focal PPK. Bioinformatics and protein modelling predicted the variants to be pathogenic. Previous studies have suggested that EGFR expression may be subject to cathepsin regulation. Immunofluorescence revealed reduced cathepsin Z expression in the upper epidermal layers and concomitant increased epidermal EGFR expression in patients harbouring CTSZ variants. Accordingly, human keratinocytes transfected with constructs expressing PPK-causing variants in CTSZ displayed reduced cathepsin Z enzymatic activity, as well as increased EGFR expression. In line with the role played by EGFR in the regulation of keratinocyte proliferation, human keratinocytes transfected with the PPK-causing variants showed significantly increased proliferation that was abolished upon exposure to erlotinib, an EGFR inhibitor. Similarly, downregulation of CTSZ resulted in increased EGFR expression and increased proliferation in human keratinocytes, suggestive of a loss-of-function effect of the pathogenic variants. Finally, three-dimensional organotypic skin equivalents grown from CTSZ-downregulated cells showed increased epidermal thickness and EGFR expression as seen in patient skin; here, too, erlotinib was found to rescue the abnormal phenotype. CONCLUSIONS: Taken collectively, these observations attribute to cathepsin Z a hitherto unrecognized function in epidermal differentiation.

Woolly hair in tricho-dento-osseous syndrome.

Tricho-dento-osseous syndrome (TDOS) is a rare ectodermal dysplasia caused by mutations in the DLX3 gene and it is not usually included as a cause of syndromic woolly hair. We present a new case of TDOS with a novel DLX3 variant and woolly hair.

Ustekinumab therapy for Netherton syndrome.

Netherton syndrome (NS) is a rare disorder of cornification associated with high morbidity. It is caused by bi-allelic mutations in SPINK5 encoding the serine protease inhibitor LEKTI. Previous studies have shown Th17 skewing with IL-23 upregulation in NS, raising the possibility that targeting these inflammatory pathways may alleviate disease manifestations. We ascertained the therapeutic efficacy of six doses of ustekinumab administered to three patients with NS over a period of 13 months using the Ichthyosis Area and Severity Index (IASI), the Dermatology Life Quality Index (DLQI), a visual analogue scale (VAS) for itch and the peak-pruritus numeric rating scale (PP-NRS). Histopathology analysis including CD3, CD4, CD8 and interleukin 17 (IL-17) immunostaining, was performed at baseline and 4 weeks following the last ustekinumab dose. Total IASI scores were reduced by 28% in two patients at week 16 with sustained response by week 56. No consistent improvement in DLQI, VAS for itch and PP-NRS scores was observed. The inflammatory infiltrate and the degree of acanthosis were slightly reduced at week 56 as compared to baseline. No significant change in immunostaining of the various inflammatory markers was observed at week 56. In conclusion, this case series did not demonstrate a significant therapeutic effect of ustekinumab in NS.

Cutaneous and Developmental Effects of CARD14 Overexpression in Zebrafish.

BACKGROUND: Gain-of-function mutations in CARD14 have recently been shown to be involved in the pathogenesis of psoriasis and pityriasis rubra pilaris (PRP). Those mutations were found to activate the NF-kB signaling pathway. OBJECTIVE: Zebrafish is often used to model human diseases in general, and in skin disorders more particularly. In the present study, we aimed to examine the effect of CARD14 overexpression in zebrafish with the aim to validate this model for future translational applications. METHODS: We used light microscopy, scanning electron microscopy, histological analysis and whole mount in situ hybridization as well as real-time PCR to ascertain the effect of CARD14 overexpression in the developing zebrafish. RESULTS: Overexpression of human CARD14 had a marked morphological and developmental effect on the embryos. Light microscopy demonstrated a characteristic cutaneous pattern including a granular surface and a spiky pigment pattern. In situ hybridization revealed keratinocytes of uneven size and shape. Scanning electron microscopy showed aberrant production of actin microridges and a rugged keratinocyte cell surface, reminiscent of the human hyperkeratotic phenotype. Developmentally, overexpression of CARD14 had a variable effect on anterior-posterior axis symmetry. Similar to what has been observed in humans with psoriasis or PRP, NF-kB expression was higher in CARD14-overexpressing embryos compared to controls. CONCLUSIONS: Overexpression of CARD14 results in a distinct cutaneous pattern accompanied by hyperactivation of the NF-kB pathway, suggesting that the zebrafish represents a useful system to model CARD14-associated papulosquamous diseases.

Autosomal recessive congenital ichthyosis caused by a pathogenic missense variant in CLDN1.

Autosomal recessive congenital ichthyosis (ARCI) refers to a large and genetically heterogenous group of non-syndromic disorders of cornification featuring diffuse scaling. Ichthyosis, leukocyte vacuoles, alopecia, and sclerosing cholangitis (ILVASC) syndrome is a rare autosomal recessive syndromic form of ichthyosis. The disease usually results from premature termination codon-causing pathogenic variants in CLDN1 encoding CLAUDIN-1 (CLDN1). We used whole exome sequencing (WES), Sanger sequencing, 3D protein modeling, Western blotting, and immunofluorescence confocal microscopy to delineate the genetic basis of ichthyosis in two siblings with ichthyosis but no other ectodermal abnormalities. One of the two siblings underwent liver transplantation in early childhood due to biliary atresia. Both patients were found to carry a homozygous missense pathogenic variant, c.242G>A (p.Arg81His), in CLDN1. The variant resulted in decreased CLDN1 expression in patient skin. 3D protein modeling predicted that p.Arg81His induces deleterious conformational changes. Accordingly, HaCaT cells transfected with a construct expressing the mutant CLDN1 cDNA featured decreased levels and mislocation of CLDN1 as compared with cells expressing the wildtype cDNA. In conclusion, we describe the first pathogenic missense variant in CLDN1 shown to result in ARCI.

A unique skin phenotype resulting from a large heterozygous deletion spanning six keratin genes.

The phenotypic spectrum of genodermatoses is continuously expanding. Three siblings were referred because of a highly unusual phenotype comprising alopecia, dystrophic nails, palmoplantar keratoderma and trauma-induced skin blistering. Whole-exome sequencing analysis identified a heterozygous large genomic alteration of around 116 0000 bp resulting in the deletion of the KRT9, KRT14, KRT15, KRT16 and KRT19 genes, as well as part of KRT17. This genomic change leads to the generation of a truncated keratin 17 (KRT17) protein encoded by the first three exons of the gene and part of intron 3. The three patients were found to carry the heterozygous genomic deletion while their healthy parents did not, indicative of germline mosaicism. The genomic alteration was found to result in reduced KRT17 expression in patient skin. More importantly, the abnormal truncated KRT17 was found to exert a deleterious effect on keratinocyte cytoskeleton formation, leading to keratin aggregation. Coexpression of wildtype and truncated KRT17 proteins also caused keratin aggregation, demonstrating that the deletion exerts a dominant negative effect. In conclusion, we are reporting on a novel clinical phenotype that was found to result from germline mosaicism for a large genomic deletion spanning six keratin genes, thus expanding the spectrum of clinical manifestations associated with keratin disorders. What is already known about this topic? Various conditions known as keratinopathies have been shown over recent years to be associated with dominant or recessive variants in several individual keratin genes. What does this study add? We report three patients presenting with a unique clinical phenotype that was found to result from germline mosaicism for a large genomic deletion spanning six keratin genes. The genomic variant is predicted to result in a truncated form of keratin 17, which was found in an in vitro assay to disrupt keratinocyte cell cytoskeleton formation.

Coexistence of pachyonychia congenita and hidradenitis suppurativa: more than a coincidence.

BACKGROUND: The coexistence of pachyonychia congenita (PC) and hidradenitis suppurativa (HS) has been described in case reports. However, the pathomechanism underlying this association and its true prevalence are unknown. OBJECTIVES: To determine the genetic defect underlying the coexistence of PC and HS in a large kindred, to delineate a pathophysiological signalling defect jointly leading to both phenotypes, and to estimate the prevalence of HS in PC. METHODS: We used direct sequencing and a NOTCH luciferase reporter assay to characterize the pathophysiological basis of the familial coexistence of HS and PC. A questionnaire was distributed to patients with PC registered with the International Pachyonychia Congenita Research Registry (IPCRR) to assess the prevalence of HS among patients with PC. RESULTS: Direct sequencing of DNA samples obtained from family members displaying both PC and HS demonstrated a missense variant (c.275A>G) in KRT17, encoding keratin 17. Abnormal NOTCH signalling has been suggested to contribute to HS pathogenesis. Accordingly, the KRT17 c.275A>G variant resulted in a significant decrease in NOTCH activity. To ascertain the clinical importance of the association of HS with PC, we distributed a questionnaire to all patients with PC registered with the IPCRR. Seventy-two of 278 responders reported HS-associated clinical features (25·9%). Disease-causing mutations in KRT17 were most prevalent among patients with a dual phenotype of PC and HS (43%). CONCLUSIONS: The coexistence of HS and KRT17-associated PC is more common than previously thought. Impaired NOTCH signalling as a result of KRT17 mutations may predispose patients with PC to HS. What is already known about this topic? The coexistence of pachyonychia congenita (PC) and hidradenitis suppurativa (HS) has been described in case reports. However, the pathomechanism underlying this association and its true prevalence are unknown. What does this study add? A dual phenotype consisting of PC and HS was found to be associated with a pathogenic variant in KRT17. This variant was found to affect NOTCH signalling, which has been previously implicated in HS pathogenesis. HS was found to be associated with PC in a large cohort of patients with PC, especially in patients carrying KRT17 variants, suggesting that KRT17 variants causing PC may also predispose to HS. What is the translational message? These findings suggest that patients with PC have a higher prevalence of HS than previously thought, and hence physicians should have a higher level of suspicion of HS diagnosis in patients with PC.

Up-regulation of ST18 in pemphigus vulgaris drives a self-amplifying p53-dependent pathomechanism resulting in decreased desmoglein 3 expression.

Pemphigus vulgaris (PV) is a life-threatening autoimmune mucocutaneous blistering disease which is to a large extent genetically determined, and results, at least in part, from the deleterious activity of autoantibodies directed against desmoglein (DSG)3, a prominent intra-epidermal adhesion molecule. Those autoantibodies lead to decreased membranal DSG3 expression in keratinocytes (KCs), thereby destabilizing cell-cell adhesion within the epidermis and leading to blister formation. We previously showed that rs17315309, a strong risk variant for PV within the promoter of the ST18 transcription factor gene, promotes epidermal ST18 up-regulation in a p53/p63-dependent manner. Accordingly, ST18 was found to be overexpressed in the skin of PV patients. Increased ST18 expression was then shown to markedly augment PV autoantibodies-mediated loss of KCs cohesion. Here, we demonstrate that ST18 overexpression significantly increases autoantibody-mediated DSG3 down-regulation in keratinocytes. In addition, DSG3 decreased expression boosts p53 function through p38 mitogen-activated protein kinase (p38MAPK) activation and dramatically augments p53-dependent ST18 promoter activity. Finally, the PV risk variant rs17315309 is associated with increased p53 expression in PV skin. Taken collectively, these observations reveal a novel self-amplifying pathomechanism involving ST18, DSG3, p38 and p53, capable of perpetuating disease activity, and therefore indicative of novel actionable molecular targets in PV.

Loss-of-function variants in KLF4 underlie autosomal dominant palmoplantar keratoderma.

PURPOSE: Palmoplantar keratodermas (PPKs) form a group of disorders characterized by thickening of palm and sole skin. Over the past 2 decades, many types of inherited PPKs have been found to result from abnormal expression, processing, or function of adhesion proteins. METHODS: We used exome and direct sequencing to detect causative pathogenic variants. Functional analysis of these variants was conducted using reverse transcription quantitative polymerase chain reaction, immunofluorescence confocal microscopy, immunoblotting, a promoter reporter assay, and chromatin immunoprecipitation. RESULTS: We identified 2 heterozygous variants (c.1226A>G and c.633_634dupGT) in KLF4 in 3 individuals from 2 different unrelated families affected by a dominant form of PPK. Immunofluorescence staining for a number of functional markers revealed reduced epidermal DSG1 expression in patients harboring heterozygous KLF4 variants. Accordingly, human keratinocytes either transfected with constructs expressing these variants or downregulated for KLF4 displayed reduced DSG1 expression, which in turn has previously been found to be associated with PPK. A chromatin immunoprecipitation assay confirmed direct binding of KLF4 to the DSG1 promoter region. The ability of mutant KLF4 to transactivate the DSG1 promoter was significantly decreased when compared with wild-type KLF4. CONCLUSION: Loss-of-function variants in KLF4 cause a novel form of dominant PPK and show its importance in the regulation of epidermal differentiation.

Neonatal inflammatory skin and bowel disease type 1 caused by a complex genetic defect and responsive to combined anti-tumour necrosis factor-α and interleukin-12/23 blockade.

ADAM17, encoding ADAM metallopeptidase domain 17, is a membrane-bound shedding protease, which plays an essential role during normal development and in the regulation of inflammation. Biallelic variants in ADAM17, resulting in complete loss of ADAM17 expression, have been reported in individuals affected by rare neonatal inflammatory skin and bowel disease 1 (NISBD1). Here, we report on a young female individual with NISBD1 featuring erythroderma, atrichia, nail dystrophy, oesophageal strictures, intractable diarrhoea, profound failure to thrive and recurrent cutaneous and systemic infections. In this case, NISBD1 was found to result from a complex compound heterozygous defect consisting of a large genomic deletion spanning exons 6 and 7 in addition to a splice site variant causing exon 17 skipping. Skin manifestations dramatically improved in response to combined anti-tumour necrosis factor-α and interleukin-12/23 blockade, while gastrointestinal symptoms were controlled with budesonide. Our study further expands the phenotypic and genetic spectrum of NISBD1 and suggests that combined immunosuppressive treatments may be indicated in this complex condition. What is already known about this topic? Biallelic loss-of-function variants in ADAM17, encoding ADAM metallopeptidase domain 17, have to date been reported in only four families with neonatal inflammatory skin and bowel disease 1 (NISBD1). NISBD1 features variable disease phenotypes associated with different degrees of severity. What does this study add? We report a case of a patient with NISBD1 caused by a unique genetic defect consisting of a combination of a splice site variant leading to exon 17 skipping and a large deletion spanning exons 6-7 of the ADAM17 gene. The severe cutaneous phenotype in our patient responded to a combination of ustekinumab and certolizumab.

Vorinostat, a histone deacetylase inhibitor, as a potential novel treatment for psoriasis.

BACKGROUND: Psoriasis is characterized by aberrant activation of several pro-inflammatory circuits as well as abnormal hyperproliferation and dysregulated apoptosis of keratinocytes (KCs). Most currently available therapeutic options primarily target psoriasis-associated immunological defects rather than epidermal abnormalities. OBJECTIVE: To investigate the efficacy of the histone deacetylase (HDAC) inhibitor, Vorinostat, in targeting hyperproliferation and impaired apoptosis in psoriatic skin. METHODS: Vorinostat effect was investigated in primary KCs cell cultures using cell cycle analysis by flow cytometry, apoptosis assays (Annexin V-FICH and caspase-3/7) and antibody arrays, qRT-PCR and immunohistochemistry. Vorinostat impact on clinical manifestations of psoriasis was investigated in a chimeric mouse model. RESULTS: Vorinostat was found to inhibit KCs proliferation and to induce their differentiation and apoptosis. Using a chimeric mouse model, vorinostat was found to result in marked attenuation of a psoriasiform phenotype with a significant decrease in epidermal thickness and inhibition of epidermal proliferation. CONCLUSIONS: Our results support the notion that vorinostat, a prototypic HDAC inhibitor, may be of potential use in the treatment of psoriasis and other hyperproliferative skin disorders.

Molecular epidemiology of non-syndromic autosomal recessive congenital ichthyosis in a Middle-Eastern population.

Autosomal recessive congenital ichthyosis (ARCI) is a rare and heterogeneous skin cornification disorder presenting with generalized scaling and varying degrees of erythema. Clinical manifestations range from lamellar ichthyosis (LI), congenital ichthyosiform erythroderma (CIE) through the most severe form of ARCI, Harlequin ichthyosis (HI). We used homozygosity mapping, whole-exome and direct sequencing to delineate the relative distribution of pathogenic variants as well as identify genotype-phenotype correlations in a cohort of 62 Middle Eastern families with ARCI of various ethnic backgrounds. Pathogenic variants were identified in most ARCI-associated genes including TGM1 (21%), CYP4F22 (18%), ALOX12B (14%), ABCA12 (10%), ALOXE3 (6%), NIPAL4 (5%), PNPLA1 (3%), LIPN (2%) and SDR9C7 (2%). In 19% of cases, no mutation was identified. Our cohort revealed a higher prevalence of CYP4F22 and ABCA12 pathogenic variants and a lower prevalence of TGM1 and NIPAL4 variants, as compared to data obtained in other regions of the world. Most variants (89%) in ALOX12B were associated with CIE and were the most common cause of ARCI among patients of Muslim origin (26%). Palmoplantar keratoderma associated with fissures was exclusively a result of pathogenic variants in TGM1. To our knowledge, this is the largest cohort study of ARCI in the Middle-Eastern population reported to date. Our data demonstrate the importance of population-tailored mutation screening strategies and shed light upon specific genotype-phenotype correlations.

Epidermolytic epidermal nevus caused by a somatic mutation in KRT2.

Superficial epidermolytic ichthyosis (formerly Ichthyosis bullosa of Siemens) is an uncommon condition caused by dominant mutations in KRT2 encoding keratin 2. Epidermolytic epidermal nevus due to somatic mutations in KRT2 is even rarer. Here, we report the third case of KRT2-associated epidermal nevus and review the literature.

Epidermolysis bullosa simplex due to bi-allelic DST mutations: Case series and review of the literature.

BACKGROUND: Epidermolysis bullosa simplex (EBS) is a heterogeneous group of inherited disorders characterized by skin fragility due to intraepidermal separation. Most cases result from heterozygous mutations in KRT5 or KRT14; however, a minority of affected individuals carry mutations in non-keratin genes including DST encoding an epithelial isoform of dystonin. DST-associated EBS is transmitted as an autosomal recessive trait. Here, we report a series of EBS patients carrying bi-allelic DST mutations and review previously reported cases aiming to delineate phenotype-genotype correlations. METHODS: Whole-exome and direct sequencing were used for variant analysis. Review of previously reported cases was performed. RESULTS: Mutation analysis revealed DST mutations in five patients belonging to three families. Two variants have not been previously reported: c.7097dupA (p.Tyr2366X) and c.7429delC (p.Leu2477Serfs*13). We identified an additional six cases in the literature, bringing the total number of individuals affected with EBS due to DST variants to 11. Patients displayed distinctive phenotypes regardless of the causative variant. CONCLUSIONS: The current study expands the clinical and genetic spectrum of DST-associated EBS subtype.

Phenotypic suppression of acral peeling skin syndrome in a patient with autosomal recessive congenital ichthyosis.

Autosomal recessive congenital ichthyosis (ARCI) manifests with generalized scaling often associated with generalized erythema. Mutations in at least 13 different genes have been reported to cause ARCI. Acral peeling skin syndrome (APSS) is a rare autosomal recessive disorder manifesting with peeling over the distal limbs and dorsal surfaces of hands and feet. APSS is mostly due to mutations in TGM5, encoding transglutaminase 5. Both ARCI and APSS are fully penetrant genetic traits. Here, we describe a consanguineous family in which one patient with mild ARCI was found to carry a homozygous mutation in ALOXE3 (c.1238G > A; p.Gly413Asp). The patient was also found to carry a known pathogenic homozygous mutation in TGM5 (c.1335G > C; p.Lys445Asn) but did not display acral peeling skin. Her uncle carried the same homozygous mutation in TGM5 but carried the ALOXE3 mutation in a heterozygous state and showed clinical features typical of APSS. Taken collectively, these observations suggested that the ALOXE3 mutation suppresses the clinical expression of the TGM5 variant. We hypothesized that ALOXE3 deficiency may affect the expression of a protein capable of compensating for the lack of TGM5 expression. Downregulation of ALOXE3 in primary human keratinocytes resulted in increased levels of corneodesmosin, which plays a critical role in the maintenance of cell-cell adhesion in the upper epidermal layers. Accordingly, ectopic corneodesmosin expression rescued the cell-cell adhesion defect caused by TGM5 deficiency in keratinocytes as ascertained by the dispase dissociation assay. The present data thus provide evidence for phenotypic suppression in a human hereditary skin disorder.

Loss-of-function variants in C3ORF52 result in localized autosomal recessive hypotrichosis.

PURPOSE: Localized autosomal recessive hypotrichosis (LAH) has been associated with pathogenic variants in DSG4, encoding a desmosomal protein as well as in LIPH and LPAR6, encoding respectively lipase H, which catalyzes the formation of 2-acyl-lysophosphatidic acid (LPA), and lysophosphatidic acid receptor 6, a receptor for LPA. LPA promotes hair growth and differentiation. In this study we aimed at delineating the genetic basis of LAH in patients without pathogenic variants in these three genes. METHODS: Variant analysis was conducted using exome and direct sequencing. We then performed quantitative reverse transcription polymerase chain reaction (RT-qPCR), immunofluorescence staining, immunoblotting, enzymatic, and coimmunoprecipitation assays to evaluate the consequences of potential etiologic variants. RESULTS: We identified homozygous variants in C3ORF52 in four individuals with LAH. C3ORF52 was found to be coexpressed with lipase H in the inner root sheath of the hair follicle and the two proteins were found to directly interact. The LAH-causing variants were associated with decreased C3ORF52 expression and resulted in markedly reduced lipase H-mediated LPA biosynthesis. CONCLUSION: LAH can be caused by abnormal function of at least three proteins which are necessary for proper LPA biosynthesis.