Hans Christian Hennies

Dupuytren’s disease (DD) is a highly heritable fibrotic disorder of the hand with incompletely understood etiology. A number of genetic loci, including Wnt signaling members, have been previously identified. Our overall aim was to identify novel genetic loci, to prioritize genes within the loci for functional studies, and to assess genetic correlation with associated disorders. We performed a meta-analysis of six DD genome-wide association studies from three European countries and extensive bioinformatic follow-up analyses. Leveraging 11,320 cases and 47,023 controls, we identified 85 genome-wide significant single nucleotide polymorphisms in 56 loci, of which 11 were novel, explaining 13.3–38.1% of disease variance. Gene prioritization implicated the Hedgehog and Notch signaling pathways. We also identified a significant genetic correlation with frozen shoulder. The pathways identified highlight the potential for new therapeutic targets and provide a basis for additional mechanistic studies for a common disorder that can severely impact hand function.

Hans C Hennies; Does the understanding of signalling pathways pave the way to therapies for keratinisation disorders?, British Journal of Dermatology, , ljad139

Keratosis follicularis spinulosa decalvans (KFSD) is a rare cornification disorder with an X-linked recessive inheritance in most cases. Pathogenic variants causing X-linked KFSD have been described in MBTPS2, the gene for a membrane-bound zinc metalloprotease that is involved in the cleavage of sterol regulatory element binding proteins important for the control of transcription. Few families have been identified with an autosomal dominant inheritance of KFSD. We present two members of an Austrian family with a phenotype of KFSD, a mother and her son. The disease was not observed in her parents, pointing to a dominant inheritance with a de novo mutation in the index patient. Using whole-exome sequencing, we identified a heterozygous missense variant in CST6 in DNA samples from the index patient and her affected son. In line with family history, the variant was not present in samples from her parents. CST6 codes for cystatin M/E, a cysteine protease inhibitor. Patient keratinocytes showed increased expression of cathepsin genes CTSL and CTSV and reduced expression of transglutaminase genes TGM1 and TGM3. A relative gain of active, cleaved transglutaminases was found in patient keratinocytes compared to control cells. The variant found in CST6 is expected to affect protein targeting and results in marked disruption of the balance between cystatin M/E activity and its target proteases and eventually transglutaminases 1 and 3. This disturbance leads to an impairment of terminal epidermal differentiation and proper hair shaft formation seen in KFSD.

Inherited ichthyoses represent a large heterogeneous group of skin disorders characterised by impaired epidermal barrier function and disturbed cornification. Current knowledge about disease mechanisms has been uncovered mainly through the use of mouse models or human skin organotypic models. However, most mouse lines suffer from severe epidermal barrier defects causing neonatal death and human keratinocytes have very limited proliferation ability in vitro. Therefore, the development of disease models based on patient derived human induced pluripotent stem cells (hiPSCs) is highly relevant. For this purpose, we have generated hiPSCs from patients with congenital ichthyosis, either non-syndromic autosomal recessive congenital ichthyosis (ARCI) or the ichthyosis syndrome trichothiodystrophy (TTD). hiPSCs were successfully differentiated into basal keratinocyte-like cells (hiPSC-bKs), with high expression of epidermal keratins. In the presence of higher calcium concentrations, terminal differentiation of hiPSC-bKs was induced and markers KRT1 and IVL expressed. TTD1 hiPSC-bKs showed reduced expression of FLG, SPRR2B and lipoxygenase genes. ARCI hiPSC-bKs showed more severe defects, with downregulation of several cornification genes. The application of hiPSC technology to TTD1 and ARCI demonstrates the successful generation of in vitro models mimicking the disease phenotypes, proving a valuable system both for further molecular investigations and drug development for ichthyosis patients.

Investigative skin biology, analysis of human skin diseases, and numerous clinical and pharmaceutical applications rely on skin models characterized by reproducibility and predictability. Traditionally, such models include animal models, mainly rodents, and cellular models. While animal models are highly useful in many studies, they are being replaced by human cellular models in more and more approaches amid recent technological development due to ethical considerations. The culture of keratinocytes and fibroblasts has been used in cell biology for many years. However, only the development of co-culture and three-dimensional epidermis and full-skin models have fundamentally contributed to our understanding of cell–cell interaction and cell signalling in the skin, keratinocyte adhesion and differentiation, and mechanisms of skin barrier function. The modelling of skin diseases has highlighted properties of the skin important for its integrity and cutaneous development. Examples of monogenic as well as complex diseases including atopic dermatitis and psoriasis have demonstrated the role of skin models to identify pathomechanisms and drug targets. Recent investigations have indicated that 3D skin models are well suitable for drug testing and preclinical studies of topical therapies. The analysis of skin diseases has recognized the importance of inflammatory mechanisms and immune responses and thus other cell types such as dendritic cells and T cells in the skin. Current developments include the production of more complete skin models comprising a range of different cell types. Organ models and even multi-organ systems are being developed for the analysis of higher levels of cellular interaction and drug responses and are among the most recent innovations in skin modelling. They promise improved robustness and flexibility and aim at a body-on-a-chip solution for comprehensive pharmaceutical in vitro studies.

Background: Autosomal recessive congenital ichthyosis (ARCI) is a genetically and phenotypically heterogeneous skin disease, associated with defects in the skin permeability barrier. Several but not all genes with underlying mutations have been identified, but a clear correlation between genetic causes and clinical picture has not been described to date.Methods: Our study included 19 families from Saudi Arabia, Yemen, and Pakistan. All patients were born to consanguineous parents and diagnosed with ARCI. Mutations were analyzed by homozygosity mapping and direct sequencing.Results: We have detected mutations in all families in five different genes: TGM1, ABCA12, CYP4F22, NIPAL4, and ALOXE3. Five likely pathogenic variants were unknown so far, a splice site and a missense variant in TGM1, a splice site variant in NIPAL4, and missense variants in ABCA12 and CYP4F22. We attributed TGM1 and ABCA12 mutations to the most severe forms of lamellar and erythematous ichthyoses, respectively, regardless of treatment. Other mutations highlighted the presence of a phenotypic spectrum in ARCI.Conclusion: Our results contribute to expanding the mutational spectrum of ARCI and revealed new insights into genotype/phenotype correlations. The findings are instrumental for a faster and more precise diagnosis, a better understanding of the pathophysiology, and the definition of targets for more specific therapies for ARCI.

Objective: Dupuytren disease (DD) is a polygenic fibroproliferative disorder of the palmar fascia of the hand. Building on our previous work, we aimed to further characterize the genetic predisposition to DD by performing a genome-wide association study (GWAS) on a total of 7934 DD cases and 12°861 controls from the United Kingdom, Holland, and Germany. We then aimed to dissect the functional effects of the statistically associated variant within DD-derived myofibroblasts. Materials and Methods: We extracted DNA from saliva or blood of DD patients who had undergone fasciectomy or dermofasciectomy. The genotypes of each sample were investigated using Illumina HumanExomeCore microarray chips in the discovery phase, and other platforms for replication phase. For our functional analysis, we used single-gene TaqMan assays to measure messenger RNA (mRNA) expression of myofibroblast cells that were disaggregated from DD nodules. Primary myofibroblasts were stimulated with a combination of recombinant WNT3A, SFRP4, and DKK1 proteins for 48 hours, and mRNA expression was measured. Results: Our GWAS meta-analysis revealed a total of 24 DD-associated regions, 15 of which were novel ( P < 5 × 10⁻⁸). We observed a 4-fold difference in SFRP4 mRNA expression between the 2 homozygous genotypes of our most associated single-nucleotide polymorphism rs1687975. Interestingly, an inverse correlation was observed in extracellular SFRP4 protein secretion. Our WNT signaling pathway stimulation experiments demonstrated that SFRP4 attenuates WNT3A-induced connective tissue growth factor (CTGF) expression, showing a 20% decrease ( P < .05). SFRP4 alone has no effect on alpha-smooth muscle actin, COL1A1, and COL3A1 mRNA expression. Conclusions: We have performed a large 3-stage GWAS and have discovered 15 novel Dupuytren-associated regions. We have identified that our most significantly associated variant, rs16879765, has an effect on the mRNA expression of a WNT antagonist gene SFRP4. Further functional analysis on SFRP4 has revealed that it affects the expression of CTGF. This implies its anti-fibrotic effect is mediated by the non-canonical WNT signaling pathway, potentially via the YAP/TAZ pathway that is known to interact with transforming growth factor β (TGF BETA) pathway, and this plays a critical role in the development of DD. Our GWAS has also revealed multiple potential new therapeutic targets for DD. Genes within the associated regions include MMP14 and WNT4 and have effects on diverse functions such as inflammation, extracellular matrix remodeling, cellular differentiation, and proliferation, which are likely to be important processes in the development of DD and fibrotic diseases in general.

Dupuytren´s disease, a fibromatosis of the connective tissue in the palm, is a common complex disease with a strong genetic component. Up to date nine genetic loci have been found to be associated with the disease. Six of these loci contain genes that code for Wnt signalling proteins. In spite of this striking first insight into the genetic factors in Dupuytren´s disease, much of the inherited risk in Dupuytren´s disease still needs to be discovered. The already identified loci jointly explain ~1% of the heritability in this disease. To further elucidate the genetic basis of Dupuytren´s disease, we performed a genome-wide meta-analysis combining three genome-wide association study (GWAS) data sets, comprising 1,580 cases and 4,480 controls. We corroborated all nine previously identified loci, six of these with genome-wide significance (p-value < 5x10^-8). In addition, we identified 14 new suggestive loci (p-value < 10⁻⁵). Intriguingly, several of these new loci contain genes associated with Wnt signalling and therefore represent excellent candidates for replication. Next, we compared whole-transcriptome data between patient- and control-derived tissue samples and found the Wnt/β-catenin pathway to be the top deregulated pathway in patient samples. We then conducted network and pathway analyses in order to identify protein networks that are enriched for genes highlighted in the GWAS meta-analysis and expression data sets. We found further evidence that the Wnt signalling pathways in conjunction with other pathways may play a critical role in Dupuytren´s disease.

Autosomal recessive congenital ichthyosis (ARCI) caused by mutations in CYP4F22 is very rare. CyP4F22, a protein of the cytochrome‐P450 family 4, encodes an epidermal ω‐hydroxylase decisive in the formation of acylceramides, which is hypothesized to be crucial for skin‐barrier function. We report a girl with consanguineous parents presenting as collodion baby with contractures of the great joints and palmoplantar hyperlinearity. In the course of the disease she developed fine scaling of the skin with erythroderma, the latter disappearing until the age of 6 months. Her sister showed a generalized fine‐scaling phenotype, and, interestingly, was born without a collodion membrane. The analysis of all known candidate genes for ARCI in parallel with a next‐generation sequencing approach using a newly designed dermatogenetics gene panel revealed a previously unknown homozygous splice‐site mutation c.549+5G>C in CYP4F22 in both girls, confirming the diagnosis of ARCI. Ultrastructural analysis by transmission electron microscopy in both patients showed epidermal hyperplasia, orthohyperkeratosis with persistence of corneodesmosomes into the outer stratum corneum layers, fragmented and disorganized lamellar lipid bilayers, which could be ascribed to inhomogeneous lamellar body secretion, as well as lamellar body and lipid entombment in the corneocytes. These findings correlated with increased transepidermal water loss on the functional level. For the first time, we report a collodion baby phenotype and epidermal barrier impairment in CyP4F22‐deficient epidermis at both the ultrastructural and functional level, and corroborate the importance of CyP4F22 for epidermal maturation and barrier function.