Mutation analysis and gene mapping in keratinization disorders
Autosomal recessive congenital ichthyosis (ARCI), including lamellar ichthyosis (LI) and non-bullous congenital ichthyosiform erythroderma (NBCIE), forms a clinically and genetically heterogeneous group of severe keratinization disorders with a prevalence of 1 in 100,000 to 200,000 persons in the European and Northern American populations. It is characterized by generalized scaling of the skin and variable erythema and includes extensive clinical and genetic heterogeneity. Affected newborns often present with a collodion membrane. This encasement is parchment-like and disappears within the first days of life. After loosing the membrane patients show a generalized scaling that varies from patient to patient in scale size, colour, and adherence. An underlying erythema is often seen, it is mostly mild and sometimes invisible. Secondary effects may include alopecia, hypohydrosis, and marked hyperkeratosis and hyperlinearity on palms and soles.
Although up to date five chromosomal regions with the genes TGM1, ALOX12B, ALOXE3, ABCA12, Ichthyin, and CYP4F22 have been mapped, further loci must exist. At least 30% of all ARCI patients do not have mutations in one of the known genes. We have been performing comprehensive mutation analyses in all known genes for ARCI and gene mapping studies to characterize the mutation spectrum and search for unknown ARCI genes.
This study is funded by the BMBF in the German “Network for ichthyoses and related keratinization disorders” (NIRK). Major collaborators also include Eli Sprecher, Reuven Bergman (Haifa), Moien Kanaan, and Adnan Shqueir (Bethlehem). We have a close interaction with the German support group Selbsthilfe Ichthyose. Further information on ARCI in English can also be found at the website of F.I.R.S.T.
We are looking for blood samples from ARCI patients and their siblings and parents and for skin samples from ARCI patients. Moreover, we are looking for families with several affected family members, especially such with a consanguineous background.
Details on the diagnostic opportunities provided within our research activities can be found here. Technical instructions about how to obtain and send blood or DNA samples and skin biopsies can be downloaded here (all documents in German; further information can be obtained upon request).
Identifying novel genes for congenital ichthyosis
Autosomal recessive congenital ichthyosis (ARCI) encompasses a large, heterogeneous group of disorders of cornification. Isolated forms and ichthyosis associated with other signs of disease can be differentiated. The association of congenital diffuse ichthyosis with follicular atrophoderma and hypotrichosis is a rare disorder that represents a unique autosomal recessive syndrome. We have recruited two families with a similar phenotype from the United Arab Emirates and from Turkey in collaboration with Gilles Lestringant and Ümit Türsen. Five sibs of the Emirati family showed normal stature, dispersed congenital ichthyosis, patchy follicular atrophoderma, generalized and diffuse non-scarring hypotrichosis, and hypohidrosis. The parents are first cousins and free of any skin and hair lesions as are three other unaffected sibs. The affected girl of the Turkish family showed diffuse congenital ichthyosis, follicular atrophoderma, hypotrichosis, and additionally woolly hair, the parents and her sister were free of skin disease. Histopathologically, the epidermis was of regular thickness, with the stratum granulosum thinned and the stratum corneum ortho-hyperkeratotic. Hair follicle epithelium was thinned, hair infundibulum showed hyperkeratosis and a very thin stratum granulosum. EM analysis showed deposits of lamellar bodies in the lower lamellae of the stratum corneum.
In order to identify the gene for this disorder, we have conducted a genome-wide linkage scan in the Emirati family. Two different regions were identified by linkage analysis with significant scores of 3.4 and 3.5, respectively. Analysis for homozygosity in both families mapped the underlying gene and reduced the critical interval to 4.1 Mb in length. Identification of the underlying mutations will give further insight into the biochemical pathways and molecular mechanisms behind the process of epidermal differentiation and the epidermal barrier dysfunction seen in congenital ichthyosis.
3D organotypic skin models mimicking congenital ichthyosis
Upon characterizing autosomal recessive congenital ichthyosis (ARCI) we have mainly focused on the investigation of the epidermal lipoxygenase genes ALOX12B and ALOXE3 on chromosome 17p13 and more recently also on Ichthyin on chromosome 5q33. In order to investigate the roles of ichthyin, 12R-LOX, and eLOX-3 in the pathogenesis of ARCI, we have established 3D organotypic skin models (skin equivalents/epidermis equivalents) with primary keratinocytes and fibroblasts from healthy voluntary donors. We have analysed the structure of the 3D epidermis model histopathologically and immunohistochemically with particular regard to the suprabasal layers including the stratum corneum. To stratify the effects of inactivation of different genes involved in ARCI, single genes were knocked down using RNA interference in primary keratinocytes from healthy volunteers, which were then used for our skin models. We found the typical histopathological features seen in patient samples.
Our 3D skin equivalent model, mimicking congenital ichthyosis, will be further useful to apply the recombinant proteins and/or missing metabolites and analyse their effects on the epidermis. The approach has clear advantages over "classical" gene therapy for skin diseases. These experiments are complementary to the analysis of animal models and will further contribute to developing alternatives to animal experiments.
Characterization of epidermal lipoxygenases in the barrier function of the skin
At least four different gene products involved in the pathogenesis of congenital ichthyosis take part in pathways of the lipid metabolism of the skin. Key enzymes of the epidermal 12-lipoxygenase pathway include 12R-LOX and eLOX-3. We and others have shown that mutations in ALOX12B and ALOXE3, encoding these two epidermal lipoxygenases, can cause ARCI. To assess the impairment of enzyme activity, we have established a functional assay to express the wild type and mutated ALOX12B and ALOXE3 in vitro and determined the activity of the recombinant proteins towards their genuine substrates. We have shown that almost all missense mutations result in a complete ablation of enzyme activity. Until today we have functionally analysed 24 missense mutations and in-frame deletions that result in complete loss of lipoxygenase activity.
In a collaboration with Peter Krieg, German Cancer Research Center Heidelberg, we are further characterizing the underlying molecular mechanisms using comparative analyses in samples from patients, human skin equivalents, and the Alox12b knockout mouse model. We are performing expression profiling studies to elucidate the molecular network that underlies the severe disturbance of keratinization and epidermal barrier defect in these mice.
Database at roughskin.de
A consistent genotype/phenotype correlation for autosomal recessive congenital ichthyosis (ARCI) has not been described so far. In order to generate criteria for analyzing a genotype/phenotype correlation and further characterize the phenotypic outcome of specific mutations, we have developed a web-based database service that collects, stores, and provides data from patients with ARCI and other related skin disorders. For each case family members, family history (of the disease), and pedigree are stored. The family history is automatically transferred into the LINKAGE data format and a graphical output is included using the software HaploPainter. Biochemical, histological, and clinical data, even pictures can be further included. Results of direct and indirect genetic analyses, as performed in our department, and results from enzyme assays are recorded to the database. The database is closely linked with the database of the German “Network for ichthyoses” (NIRK).
The service runs on an Apache 2 web server and is powered by a MySQL database management system and accessible through the web using an HTTP interface. For data protection reasons, sample names are stored on a separate server connected with the database by anonymous 32 bit sample IDs. Data requests are handled semi-automatically through email using temporary IDs, each valid for immediate access and one dataset, following the recommendations of the commissioner for data protection of North Rhine-Westphalia. The service is open to external academic users on a collaborative basis and includes different user levels. Medical doctors and scientists who have provided samples or work within the NIRK or similar networks can get access by personal passwords. A standardized questionnaire for clinical examination can be downloaded either from roughskin.de or NIRK.
Rare genetic skin diseases
Advancing diagnosis, management and public awareness through a European network (GeneSkin) the number of genetic skin diseases considered as distinct entities has largely increased in recent years because of the identification of mutations in several novel genes. Such studies, however, can only be achieved through networks of researchers since most of the phenotypes are rare. The European network on genetic skin disorders aims at advancing diagnosis, management, and public awareness of such disorders in Europe. This is not only a prerequisite for further genetic studies but also for the future translation of molecular genetic knowledge into other clinical approaches and novel therapies.
Palmoplantar keratodermas (PPK) form an exceedingly heterogeneous group of keratinization disorders characterized by marked thickening of the epidermis of palms and soles. The disease is often associated with severe complications including recurrent infections, mutilation, unremitting pain and contractures resulting in significant functional disability. PPK are classified based on the pattern of skin hyperkeratosis (thickening), mode of inheritance, and the existence of associated symptoms. With the elucidation of the molecular basis of many of these disorders, it has become clear that clinical manifestations poorly correlate with underlying genetic defects, significantly complicating the diagnosis of PPK and indicating the need for the development and implementation of a systematic approach to the clinical and molecular diagnosis of these disorders.
Autosomal dominant punctate PPK, also referred to as PPK type Buschke-Fischer-Brauer, is a rare hereditary skin disease clinically characterized by hyperkeratotic papules irregularly distributed on palms and soles. Lesions usually start to develop in late childhood to adolescence but may also start to appear as late as in the fifth decade in life. Walking may be painful because of larger hyperkeratoses over pressure points. Two loci for punctate PPK were described recently, located on chromosomes 8q24 and 15q22-q24. We have recruited two large families with punctate PPK and several core families. We have analysed the larger families for linkage and confirmed the locus on chromosome 15 with a combined maximum lod score of 5.2 at D15S983. We are analysing positional candidates with mutation screening in patients and families.
In some types of PPK, hyperkeratosis spreads from the palms and soles to other parts of the body, such as dorsal aspects of the hands and feet, elbows, knees, and lower legs. Besides Naxos disease and Papillon-Lefèvre syndrome, Mal de Meleda (MDM) is a severe PPK with autosomal recessive inheritance. It is characterized by diffuse transgressive hyperkeratosis that progresses with age and extends from the palms and soles onto the dorsal surface of the hands and feet, keratotic lesions over the joints, elbows and knees, perioral erythema, brachydactyly, nail abnormalities, hyperhydrosis, and inflammation without involvement of other organs. We and others have identified mutations in SLURP1 on chromosome 8q24-qter in patients with MDM from several different countries. We have been performing mutation analyses for SLURP1 in patients with a diagnosis of transgressive PPK. Expression profiling for MDM samples is performed using primary keratinocytes obtained from punch biopsies of MDM patients, and SLURP1 is analysed by Western blotting and immunohistochemistry.
Molecular genetics of alopecia areata
Alopecia areata is a common and often chronic disease characterized by patchy hair loss on the scalp with a lifetime risk of approx. 2% in the general population. The disease first manifests through one or several circular hairless areas, mostly without signs of inflammation. The subsequent fast and extended hair loss often leads to social isolation and thoughts of suicide, particularly in juveniles. It involves strong genetic susceptibility towards autoimmune disease. The genetic basis for the disease is completely unknown and genetic factors involved have not been identified in a systematic approach so far. In order to characterize the molecular pathogenesis of alopecia areata, we are using a combined strategy characterizing the basis for hair loss in an established animal model, the Dundee epidermal bald (DEB) rat, using genetic, gene expression, and immunohistologic analyses, and identifying genetic factors underlying susceptibility to alopecia areata in humans by linkage analysis and association studies, primarily using an affected sib pair study design. The strategy of analysing affected sib pairs and an animal model promises synergistic effects and provides us with a system to further characterize genes and proteins involved in the pathophysiology. These are important steps for the development of pathogenesis-based therapeutic approaches for alopecia areata.
For the human study, we are using an affected sib pair approach for the analysis of the human genome, i.e., the analysis of samples from clinically well characterized affected siblings and their unaffected parents. In addition to the affected sib pair study design, we are collecting samples from ‘trio’ families, i.e., affected individuals and their parents, and also from single cases for further studies including whole-genome association studies to confirm mapping results and as an additional, independent approach into gene mapping in multifactorial diseases.
We are recruiting families with alopecia areata in an international study group (AASG). Our request for samples can be downloaded here. Further information about how to take part in the study can be obtained from Hella Blech, University of Cologne und Privatpraxis für Dermatologie, or from Hans Christian Hennies.
This project includes close collaborations with Peter Nürnberg, CCG, and Rolf Hoffmann, University of Freiburg and Privatpraxis Dermaticum. It is also supported by the German support group for alopecia areata, Alopecia Areata Deutschland e. V., and has been approved as a funded project for the Center for Molecular Medicine Cologne (ZMMK, research project B 11) in the new funding period 2008 - 2010.
Isolation and characterization of the gene for Cohen syndrome, VPS13B (COH1)
The Cohen syndrome is a rare autosomal recessive disorder with a rather variable clinical picture, mainly characterized by mental retardation, microcephaly, facial dysmorphism, retinopathy, and myopia. We and others have found mutations in the gene VPS13B (COH1), located on human chromosome 8, after genome-wide linkage studies. VPS13B is one of the largest human genes, covering 846 kb on chromosome 8q22 and comprising 62 exons. It was described to encode various splice forms, however, we have found only one transcript coding for 3997 amino acids, which is conserved through evolution and ubiquitously expressed.
In a collaboration with Denise Horn, Charité Berlin, we have collected samples from more than 100 patients with Cohen syndrome, probably the largest collection in the world, and have been analysing the mutational spectrum. A detailed clinical characterization has resulted in a refined definition of the phenotypic spectrum and deals with the question of a genotype/phenotype correlation. The similarity of COH1 with Vps13p from S. cerevisiae suggested a role in protein sorting and transport between trans-Golgi network and endosomes. We have generated polyclonal antibodies against COH1 and cloned the full-length human cDNA. We are conducting experiments in humans and mice including analysis of overexpression, co-localization, interaction, and studies of truncated proteins.