ESPD-ESDR Joint Lecture: Harlequin Ichthyosis
According to the oral communication of Edel O'Toole
Edel O’Tool, Prof. of Molecular Dermatology - Lead of the Centre for Cell Biology and Cutaneous Research, Blizard Institute, Queen Mary University of London, gave a very interesting lecture on the etiology and pathogenesis of Harlequin ichthyosis, as well as on new therapeutic options for this disease.
Harlequin Ichthyosis (HI, OMIM 242500) is one of the most severe recessive congenital skin diseases. Affected infants develop large, armor-like skin plates separated by deep fissures. Although these skin plates are hard and thick, they are ineffective as a permeability barrier. The hard skin plates constrict body movements, and cause the malformation of ears, eyelids and lips during development. The barrier defects and the deep fissures lead to excessive water and heat loss, and render HI patients more susceptible to environmental insults. Other systemic consequences of harlequin infants, are increased caloric and nutrient requirements. This to a combination of overheating due to anhidrosis, increased epidermal turnover, and dermal inflammation, altogether resulting in a risk of growth failure. A further complication is increased susceptibility to bacterial infections, mainly due to skin fissures and erosions. Even with improvements during intensive perinatal care, many HI infants die soon after birth.
Among the many functions of the skin, the most important is to act as a barrier against unlimited transepidermal water loss (TEWL) and exposure to various environmental factors. The stratum corneum is the most essential component of the barrier, principally consisting of a cellular compartment (corneocytes) and an intercellular compartment (keratinocyte-derived lipids) bridged by numerous corneodesmosomes that undergo proteolytic degradation upon shedding of corneocytes. This proteolytic process is a complex function involving several enzymes and anti-proteases.
All cases of HI caused to date have been associated with Loss-of-function mutations in ABCA12 (ATP-binding cassette (ABC), sub-family A, member 12), a gene that belongs to the ABC family of transporters. Studies of human HI patients have revealed approximately 50 independent ABCA12 mutations that can lead to HI. These occur throughout the coding region and often result in protein truncation due to nonsense mutations, although there are also a number of splice site mutations that affect exon usage, as well as missense mutations.
The known roles of ABCA proteins in lipid transport have led to a focus on abnormal lipid metabolism in the epidermis as the primary cause of the HI pathology. The ABCA12 transporter is important in delivering glucosylceramides (GluCer) to the lipid lamellae through lamellar bodies (LBs). ABCA12 in a mouse model lacking ABCA12 expression, a profound reduction in the skin level of linoleic esters of ω-HACs has been reported, and by using this animal model, a defect in the lipid transfer to the intercellular space was found together with a lack of proteolytic enzymes (kallikreins) required for desquamation of corneocytes. KLK5, -7 and -14 are major serine proteases that mediate desquamation and are secreted into the extracellular spaces at the junction between the granular and cornified layers, where their enzymatic activities are initiated by self-cleavage of pro-KLK5. The activated KLKs then digest CD components that bind the corneocytes together, thus allowing these cellular structures to be shed.
In HI, it is now apparent that loss of ABCA12 function prevents the transfer of lipids and KLKs through the LBs, thus impairing both lipid barrier formation and desquamation. It was confirmed that there is p- STAT1 up-regulation in HI skin. JAC- STAT1 Pathway is also important for inflammation in HI. Therefore, HI is not simply a disease of deregulated lipid metabolism in the epidermis, but also a disease of profound desquamation defects.
Current treatments for HI and other diseases of the ARCI spectrum include treatment with retinoids and skin softeners, but these are limited in preventing disease progression.
The mechanism of action of retinoids in HI is mainly symptomatic, exerting rather unspecific ‘anti- keratinizing’ effects on hyperkeratotic skin. Retinoids bind to nuclear transcription factors, which assist in regulating numerous genes and hence modulate epidermal proliferation and differentiation, as well as inflammation. Ceramides (Cer) in the SC are mainly derived from GlcCer and are the major components of the lamellar layers between the corneocytes. GlcCer molecules are synthesized by differentiating keratinocytes, packaged into LBs, and secreted into the intercellular space between the granular layer and cornified layer, where they are enzymatically processed into Cer and included in the lipid lamellae. The cytokines IL-36α and IL-36γ were upregulated in HI skin, whereas the innate immune inhibitor IL-37 was strongly downregulated. There is also identified STAT1 and its downstream target inducible nitric oxide synthase (NOS2) as being upregulated in the in vitro HI 3D model and HI patient skin samples. Inhibition of NOS2 using the inhibitor 1400W or the JAK inhibitor tofacitinib dramatically improved the in vitro HI phenotype by restoring the lipid barrier in the HI 3D model.
Activation of STAT1 was also identified in other skin diseases, such as systemic lupus erythematosus and psoriasis. The JAK/STAT pathway is involved in many biological processes, including skin homeostasis.
Upregulation of the STAT1/NOS2 pathway has not been reported in HI before; however, it has been described in other inflammatory skin diseases, such as psoriasis and atopic dermatitis, sharing characteristics of skin barrier impairment and increased inflammation. It is interesting to note that tofacitinib, a potent inhibitor of the JAK family that has significantly improved the HI 3D phenotype, is already licensed for treating active psoriatic arthritis and rheumatoid arthritis, revealing the pertinence of targeting the JAK/STAT pathway in inflammatory skin diseases, such as HI. Additionally, the HI patient skin RNA-Seq data revealed upregulation of IL-17, a cytokine produced by Th17 immune cells, which is also an inducer of NOS2 expression, acting through STAT1, AP-1, and NF-κB transcription factors.
Interestingly the use of the anti–IL-17 antibody secukinumab, is currently under investigation in ichthyosis patients who express an elevated level of IL-17 and related cytokines (cohort not including HI patients). These findings strongly suggest that drugs targeting either NOS2 or the JAK/STAT1 pathway would be beneficial for treating HI patients and improving their quality of life.
"In memorial John I. Harper" MB BS MD FRCP FRCPCH (1950-2021)
Commemorative address by Peter Höger
Presentation of the ESPD Masters Award to his wife and family by Antonio Torrelo
Prof. Peter Hoger delivered a memorial speech in memory of Prof. John Harper, who passed away suddenly on April 5, 2021. Those who had the good fortune to know him will forever miss his sense of humour and humanity, the unmistakably gentle approach to patients and their families, and his stimulating art of linking clinical and scientific medicine. John was born on 26-th May 1950 and was brought up in Southend on Sea, Essex, a place he was proud of and always thought of as home. He studied at St. Mary ́s Hospital Medical School, University of London from where he graduated in 1973. He had his first taste of Paediatrics at St. Charles Hospital, London, where he was inspired by Professor David Harvey. He went on to work in Dermatology at Addenbrooke’s Hospital, Cambridge, under Drs Rook, Champion and Roberts and then in Dermatology at Wycombe General Hospital, under Dr Darrell Wilkinson. In 1980, John moved to Westminster Hospital, where he joined the Dermatology Department under Dr. Peter Copeman and Dr. Richard Staughton, two outstanding mentors who nurtured his passion for Dermatology. During his time at Westminster Hospital, John gained his accreditation in Dermatology in 1983 and in 1985 was awarded his MD on Graft-versus- Host Disease. In the same year, he was recipient of aBAD Research Travelling Scholarship to visit Professor Ramón Ruiz-Maldonado, Paediatric Dermatologist at the National Institute of Paediatrics, Mexico City, for several months in 1986. On his return to London in 1986, John was appointed Consultant Paediatric Dermatologist at Great Ormond Street Hospital joining Dr. David Atherton.
Whilst at GOSH, John made landmark contributions to the establishment and recognition of Paediatric Dermatology as a sub-specialty. John ́s special clinical interests were atopic eczema and the ichthyoses as examples of skin barrier abnormalities, scleroderma, vascular and cutaneous developmental anomalies, and gene therapy for severe genetic skin disorders, in particular Netherton syndrome. One of John's many legacies is the establishment in 1994 of the Laser Unit at GOSH for the treatment of children with vascular birthmarks, and the foundation of a multi-disciplinary clinic for children with complex vascular malformations which he led from 2004-2014. John was instrumental in setting up and supporting a number of family support groups: The Ichthyosis Support Group; The Vitiligo Society; The Birthmark Support Group, the EB charity DEBRA, The Proteus Family Network UK and Sturge Weber UK.
Astrong ambassador for UK Paediatric Dermatology worldwide, John's international reputation attracted fellows and trainees from all over the world, many of whom have established new departments of Paediatric Dermatology in their own countries as well as many UK fellows, who are now actively practising Paediatric Dermatology. John was invited as guest speaker to give lectures all over the world, which were highly acclaimed; he maintained special links to colleagues in Mexico, Argentina, Uruguay and Peru, as well as India, China, Japan and many European countries. In 2001, John was awarded a personal chair and in recognition of his contribution to Paediatric Dermatology, became the first Professor of Paediatric Dermatology in the UK at GOSH and the Institute of Child Health. He applied the rigour of his training not only to clinical cases but also to his academic work.
He took a sabbatical in 2003 to pursue his interest in the genetics of eczema at the Wellcome Centre for Human Genetics in Oxford. His research contribution has been impressive with over 185 peer- reviewed publications. In 2012, with a generous donation from the Livingstone family, he established the Livingstone SkinResearch Centre at the Institute of Child Health, UCL, dedicated to understanding basic cell mechanisms responsible for skin diseases in children. In 2013, John was presented with the prestigious ILDS Certificate of Appreciation at the World Congress of Paediatric Dermatology in Madrid. John was the founder Secretary and Chairman of the British Society for Paediatric Dermatology from 1988 to 1991 and President of the European Society of Paediatric Dermatology from 1993 to1996. He held several administrative posts at GOSH (1999-2011), was Associate Editor of the Journal “Pediatric Dermatology” (1993-98) and Referee for major journals. John also ran a successful private practice at The Portland Hospital in London for 30 years. He was the first Paediatric Dermatologist to join the Hospital and helped expand its Paediatric Dermatology Service to include a further 6 consultants, as well as establishing the Laser Service and a Birthmark Service for newborn babies. Perhaps John’s greatest legacy is Harper’s Textbook of Pediatric Dermatology, first published in 2000 together with Arnold Oranje and Neil Prose, now in its 4th edition, published in 2020, which has become the gold standard for the specialty throughout the world. His shorter Handbook of Paediatric Dermatologyis a must for medical students and his textbook, Inherited Skin Disorders: The Genodermatoses, was published in 1996, before clinics for such dermatoses became established.
Recently, John learned that he was being honoured by the European Society for Paediatric Dermatology by being awarded the first ESPD Masters Award for Lifetime Achievement in Paediatric Dermatologyat the 20-th ESPD Conference in Vienna, which, sadly, will now be awarded posthumously. For those who knew John well, he remained humble and hungry for new knowledge. He was a very special person and a true gentleman. People appreciated him not only for his clinical expertise, but also for his kindness, professionalism, laughter and infectious enthusiasm. He was generous with his time, caring of patients and their families and an inspirational mentor to many generations of paediatric dermatologists. John was happily married for almost 46 years to Rowena. He was a devoted family man, immensely proud of his son, Peter, a solicitor and his daughter, Charlotte, a Paediatric Emergency Medicine Doctor. He will be dearly missed and his legacy in the field of Paediatric Dermatology will last forever.
Rudolf Happle Lecture: Mosaicism in human skin: from bedside to bench and back Lecturer: Prof. Cristina Has – Laboratory of Molecular Dermatology Freiburg, Germany
Mosaicism is a powerful biologic concept, originally developed from studying plants and animals. All cutaneous neoplasms, both benign and malignant, reflect mosaicism, which is the necessary basis to explain numerous human skin disorders. For example, various mosaic patterns visualize the embryonic development of human skin and X-linked skin disorders explain why women live longer than men, and so on. The concept of cutaneous mosaicism has today been proven at the cellular level in at least fifteen different skin disorders. Mosaicism denotes an individual who has at least two populations of cells with distinct genotypes that are derived from a single fertilized egg. Genetic variation among the cell lines can involve whole chromosomes, structural or copy-number variants, small or single-nucleotide variants, or epigenetic variants. The mutational events that underlie mosaic variants occur during mitotic cell divisions after fertilization and zygote formation. The initiating mutational event can occur in any types of cell at any time in development, leading to enormous variation in the distribution and phenotypic effect of mosaicism. A number of classification proposals have been put forward to classify genetic mosaicism into categories based on the location, pattern, and mechanisms of the disease. Prof. Happle and al. have proposed a new classification of genetic mosaicism in 2020 that considers the affected tissue, the pattern and distribution of the mosaicism, the pathogenicity of the variant, the direction of the change (benign to pathogenic vs. pathogenic to benign), and the postzygotic mutational mechanism. The accurate and comprehensive categorization and subtyping of mosaicisms is important and has potential clinical utility to define the natural history of these disorders, tailor follow-up frequency and interventions, estimate recurrence risks, and guide therapeutic decisions. The authors distinguished six different patterns of mosaicism, including the phylloid pattern and the lateralization pattern. Etiologically, cutaneous mosaics can be divided into two large categories, epigenetic mosaicism and genomic mosaicism. All forms of epigenetic mosaicism known so far, including the various patterns of X-inactivation, appear to be caused by the action of retrotransposons. A new concept is functional autosomal mosaicism transmittable through the action of retrotransposons, which has been described in mice and dogs and may explain, for example, the familial occurrence of pigmentary mosaicism along the Blaschko lines in human skin. Among the examples of mosaicism of autosomal lethal mutations, phylloid hypomelanosis is a recently recognized neurocutaneous entity caused by mosaic trisomy 13. Possible examples of a type 2 segmental manifestation now include at least fifteen different autosomally dominant skin disorders.
This phenomenon is most frequently found in glomangiomatosis, cutaneous leiomyomatosis, and disseminated superficial actinic porokeratosis. Recently proposed examples of didymosis (twin spotting) include cutis tricolor, paired patches of excessive or absent involvement in Darier disease, and didymosis aplasticosebacea characterized by coexistent aplasia cutis congenita and nevus sebaceus. To the list of possible examples of paradominant inheritance, cutis marmorata telangiectatica congenita and speckled lentiginous nevus syndrome have now been added. Revertant mosaicism giving rise to unaffected skin areas in autosomally recessive cutaneous traits will certainly likewise be recognized more often when clinicians are bearing this concept in mind. Such cases can be taken as examples of “natural gene therapy”.
X-chromosome inactivation: role in skin disease expression.
The occurrence of X inactivation in mammals has the consequence that all women are functional mosaics. In X-linked skin disorders, Lyonization usually gives rise to a mosaic pattern, as manifest by the appearance of the lines of Blaschko. This arrangement of lesions is observed in male-lethal X- linked traits, such as incontinentia pigmenti, focal dermal hypoplasia, Conradi-Hünermann-Happle syndrome, oral-facial-digital syndrome type 1 and MIDAS (microphthalmia, dermal aplasia and sclerocornea) syndrome, as well as in various X-linked non-lethal phenotypes, such as hypohidrotic ectodermal dysplasia of Christ-Siemens-Touraine, IFAP (ichthyosis follicularis-alopecia-photophobia) syndrome and X-linked dyskeratosis congenita. Analogous X-inactivation patterns have been documented in human bones, teeth, eyes and, possibly, the brain. Patterns that are distinct from the lines of Blaschko are also seen, such as the lateralization observed in CHILD (congenital hemidysplasia with ichthyosiform nevus and limb defects) syndrome, and the chequerboard pattern seen in women heterozygous for X-linked congenital hypertrichosis. Exceptional cases of either severe or absent involvement in a woman heterozygous for an X-linked trait can be explained by skewing of X inactivation. Some X-linked skin disorders are caused by genes that escape inactivation, which is why heterozygous female 'carriers' of these disorders do not show mosaicism. A well-known example is X-linked recessive ichthyosis due to steroid sulphatase deficiency, the locus for which is situated at the tip of the short arm of the X chromosome and does not undergo Lyonization. On the other hand, in the case of Fabry disease, the gene encoding alpha-galactosidase A is subject to inactivation. Remarkably, however, the skin lesions of women do not show a mosaic pattern.
Conclusion: In the various X-linked skin disorders, affected women show quite dissimilar degrees of involvement and forms of manifestation because X inactivation may give rise to different patterns of functional mosaicism. Paradoxically, no such pattern is observed in women with Fabry disease. Like many X-linked diseases, Fabry disease should neither be called recessive nor dominant, because these dichotomous terms are obscured by the mechanism of X inactivation.
Mosaicism in monogenic skin disorders.
Mosaic skin diseases may show different patterns of clinical involvement such as lines of Blaschko, a checkerboard pattern, a phylloid pattern, a patchy pattern without midline separation, and a lateralization pattern. Mosaicism has been demonstrated at the cellular level in more than 15 monogenic skin disorders to date. Recently, the specific molecular mechanisms governing three particular forms of cutaneous mosaicism have been unraveled and reported in the JCI. In 2004, has shown at the cellular and molecular level that the type 2 segmental manifestation of Hailey-Hailey disease, being superimposed on the ordinary nonsegmental lesions of this autosomal dominant trait, results from postzygotic loss of heterozygosity (LOH). These data provided the first molecular evidence supporting this genetic concept, which was postulated ten years ago. If LOH results from postzygotic crossing-over, this would give rise to two different daughter cells, one of them being homozygous for the underlying mutation. Conversely, the other cell would be homozygous for the wild-type allele, resulting in a band or patch of completely healthy tissue. This would reflect “natural gene therapy” in an autosomal dominant skin disease in a way analogous to that proposed by Pasmooij et al. in this issue of the JCI for autosomal recessive cutaneous traits. After ex vivo expansion of cells from such healthy skin areas, cell sheets could be generated and used for autologous transplantation.
Over the last 20 years, the phenotypic reversion of a clinically severe autosomal recessively inherited disease by one or several correcting somatic mutations has been described in various human disorders. This particular category of human mosaicism was first described in Lesch-Nyhan syndrome in 1988. Various molecular mechanisms governing this kind of naturally occurring phenotypic rescue have been proposed and demonstrated, including true reverse point mutations, deletions, nondisjunction, crossing-over events, and gene conversion. Moreover, revertant mosaicism can be induced by transposable elements known as retrotransposons as documented in Duchenne muscular dystrophy. In 1997, Jonkman et al. were the first to demonstrate at the molecular level the occurrence of revertant mosaicism in human skin in a patient suffering from generalized atrophic benign epidermolysis bullosa (EB), who showed some healthy patches of skin in which blistering could not be evoked. The authors proved that within these areas, phenotypic reversion was caused by mitotic gene conversion in one of the two mutated collagen type XVII α1 (COL17A1) alleles. Based on these data, they suggested that revertant mosaicism in autosomal recessive skin diseases is as an example of natuRevertant mosaicism in EB: guiding the path toward gene therapy. The two patients had autosomal recessive non-Herlitz junctional EB caused by compound heterozygous or homozygous germline mutations in the laminin β3 (LAMB3) gene, respectively. Molecular analysis of biopsy specimens derived from areas of healthy-appearing skin led to the identification of five different correcting somatic mutations that predominantly affected mRNA splicing. Interestingly, one of the patients studied by these authors revealed expansion of the healthy skin regions harboring a spontaneously occurring correcting second-site mutation. It is tempting to speculate that some of the keratinocytes in which revertant mutational events occurred must have been epidermal stem cells.
Over time, clonal expansion of these stem cells led to ral gene therapy. Amelioration of the phenotype within a defined area of skin due to a selection advantage of revertant stem cells compared with their deficient counterparts.
These observations have far-reaching consequences for possible future strategies of gene therapy for autosomal recessive cutaneous disorders. In mosaic individuals, autologous skin grafts derived from areas with normal-appearing skin could be transplanted to affected de-epidermized skin regions on the same patient, thereby following the example of natural gene therapy and avoiding immunologic reactions that might cause graft rejection. Pasmooij et al. conclude that in LAMB3 revertant mosaicism, one might expand in vitro the patient’s own revertant cells and use such naturally corrected cells for grafting.
Gene therapy in EB: where are we now? When designing gene therapy approaches as a future method for causal treatment of severe inherited cutaneous diseases some 10–15 years ago, investigators convinced of the benefits of this therapeutic regimen were often confronted with skepticism and concerns. Today, gene therapy for EB is no longer wishful thinking but is at the verge of being introduced into clinical practice. Mavilio et al. reported the first successful ex vivo gene therapy approach in a patient suffering from LAM5-β3–deficient junctional EB, a disorder that is severe and often lethal due to dysfunctional skin adhesion, manifesting with extensive blistering at birth and serious complications such as recurrent infections. In a phase I/II clinical trial, epidermal stem cells from this patient were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-β3) and used to create genetically corrected epidermal grafts. After surgical preparation, these grafts were transplanted onto the patient’s legs resulting in development of fully functional and adherent epidermis. One year later, the grafts were stable and did not show any blistering, indicating that this therapeutic approach was curative. This encouraging report is just the beginning of a new era in which laboratory researchers and clinicians will intensify their efforts to develop and improve strategies of gene therapy for potentially fatal skin diseases. The concept of revertant mosaicism as presented in this issue by Pasmooij et al. will be an important contribution toward this goal. In particular, the discovery that postzygotic mutations giving rise to revertant mosaicism occur far more frequently than so far assumed may serve as a starting point to develop new strategies of gene therapy that we are sorely missing in our clinical practice today.
In her lecture Prof. Cristina Has focuses on Epidermal nevi - from clinical-histological classification to genetic and molecular pathway. More recently, the molecular basis of linear epidermal nevi of the common, nonorganoid and nonepidermolytic type was also reported in the JCI. These benign keratinocytic skin lesions present at birth but become more conspicuous during early childhood. Using a SNaPshot Multiplex assay, Hafner et al. detected activating somatic mutations in the FGF receptor 3 (FGFR3) gene in about one-third of the nevi studied. These findings could provide a basis for the future development of noninvasive therapeutic strategies for skin tumors, such as, for example, siRNA specific to the mutant allele. A significant therapeutic benefit of RNA interference in vivo has recently been demonstrated for amyotrophic lateral sclerosis. Another example Prof. Has gave was linear and disseminated porokeratosis. Deffect of Mevalonate pathway (MVP) decrease production of cholesterol, steroid, vitamin D and lipoproteins. Prof. Has investigated 15 cases of porokeratosis – 6 cases with linear porokeratosis (LP) and 9 cases with disseminated porokeratosis (DP). In all these cases, Prof. Has finds that Mevalonate pathway controls calcium-induced differentiation of keratinocytes. Consequences from MVP mutations in DP are unstable enzymes, reduced enzymatic activity, reduction of cholesterol biosynthesis and increased apoptosis of keratinocytes. Pathogenesis- based therapy for treatment of porokeratosis include use of topical statin/cholesterol. Another way to treat the patients with LP is targeting inflammation. After 10 weeks of anti-inflammatory treatment distinct reduction of erythema, scaling and infiltration it was found.
Prof. Has outlined the following perspectives: clinical and genetic characterization of patients is essential to understand abnormality of the skin, genetic testing is important for identification of potential inheritance and complications, genetic defect and affected biological process/pathway are rationale for pathogenesis-based therapy. Finally, Prof. Cristina Has thanks to Prof. Rudolph Happle.