Correlations of Clinical, Histomorphologic and Molecular Findings. in the Diagnostics and Management of Patients. with Selected Genetic Skin Disorders

Transcription

1 Aus der Klinik und Poliklinik für Dermatologie und Venerologie der Universität zu Köln Direktor: Universitätsprofessor Dr. med. Dr. h.c. Th. Krieg Correlations of Clinical, Histomorphologic and Molecular Findings in the Diagnostics and Management of Patients with Selected Genetic Skin Disorders Korrelationen klinischer, histomorphologischer und molekularer Befunde in der Diagnostik und im Management von Patienten mit ausgewählten Genodermatosen Habilitationsschrift zur Erlangung der venia legendi für das Fach Dermatologie und Venerologie an der Hohen Medizinischen Fakultät der Universität zu Köln vorgelegt von Dr. med. Iliana Tantcheva-Poór aus Sofia, Bulgarien Köln 2017

2 Table of contents Table of contents Abbreviations.. 5 I. Introduction Classification Principles of diagnostics Clinical evaluation and history Clinical evaluation Family history Laboratory tests Morphologic studies Biochemical and enzymatic tests Molecular genetic diagnostics Ethical issues Correlations of clinical and laboratory data Clinico-pathologic correlation Genotype-phenotype correlation Principles of management German networks for genetic skin disorders.24 II. Objectives..26 III. Selected clinical cases and case studies Inherited epidermolysis bullosa (EB) EB simplex Ogna (MIM #131950)

3 Table of contents p.r1303q-related junctional EB/ junctional EB late-onset (MIM #226650) A mild phenotype of recessive dystrophic EB related to two compound heterozygous COL7A1 mutations (MIM #226600) Mendelian disorders of cornification Ichthyoses Further evidence for a disordered ceramide composition in the epidermis of ALOX-related ARCI patients (MIM PS242300) Heritable connective tissue disorders Cutis laxa congenital Autosomal recessive cutis laxa, type 2A (MIM #219200) Cancer-associated genodermatoses Brooke-Spiegler syndrome (MIM #605041) Hereditary leiomyomatosis and renal cell cancer (MIM #150800) Nevi as manifestations of somatic (postzygotic) mosaicism Fatty tissue nevi Nevus psiloliparus: a phenotypic link between oculoectodermal syndrome and encephalocraniocutaneous lipomatosis (MIM #613001) Epidermal nevi Nevus trichilemmocysticus...73 IV. Discussion Gaining new insights in the pathogenesis of genodermatoses

4 Table of contents 4.2. Development of pathogenesis-based disease classifications Implementation of New Diagnostic Markers and Methods Further phenotypic and genotypic disease characterization Better characterization of some very rare subtypes Identification of new entities Expanding genotype-phenotype correlations and identification of new mutations Improved management of patients with genetic skin disorders More accurate genetic counselling with improved opportunities for prenatal and preimplantation diagnostics Early detection, interdisciplinary treatment and, if possible prevention of disease-related complications Development of causal therapeutic approaches and perspectives Gene therapy Protein Replacement Therapy Cell-based therapies Targeting the biochemical or signaling pathway Better understanding the pathogenesis of common multifactorial disorders 102 V. Summary 104 VI. VII. Zusammenfassung..106 References.108 Acknowledgment..121 Appendix (list of tables and figures)

5 Abbreviations Abbreviations (most commonly used) AD AR ARCL2A ARCI BCAC-HG BM-MSC BSS CIE CL CRISPR/ Cas9 DDEB DEB ECCL EB EBS-MD EBS-Ogna EBS-PA EM FH HLRCC IFM ipsc JEB JEB-lo autosomal dominant autosomal recessive autososomal recessive cutis laxa type 2A autosomal recessive congenital ichthyosis basal cell adenocarcinoma-like pattern, high-grade bone marrow-derived mesenchymal stromal cells Brooke-Spiegler syndrome congenital ichthyosiform erythroderma cutis laxa clustered regularly interspaced palindromic repeats dominant dystrophic EB dystrophic EB encephalocraniocutaneous lipomatosis epidermolysis bullosa EBS with muscular dystrophy epidermolysis bullosa Ogna EBS with pyloric atresia electron microscopy fumarate hydratase hereditary leiomyomatosis and renal cell cancer syndrome immunofluorescence mapping inducible pluripotent stem cells junctional EB junctional EB late-onset 5

6 Abbreviations LI MEDOC MFT MSC NBS NGS NIRK OES PID RCC RDEB RDEB-O RDEBsg SCC SMA TALENs TGF TSC WES WGS XP ZFNs lamellar ichthyosis Mendelian Disorders of Cornification multiple familial trichoepitheliomas mesenchymal stromal cells newborn screening next generation sequencing German Network for Ichthyoses oculo-ectodermal syndrome preimplantation diagnostics renal cell carcinoma recessive dystrophic EB RDEB generalized other severe generalized RDEB squamous cell carcinoma smooth muscle actin transcription activator-like effector nucleases transforming growth factor tuberous sclerosis complex whole-exome sequencing whole-genome sequencing xeroderma pigmentosum zinc-finger nucleases 6

7 Introduction I. Introduction In Europe, there are at least 30 Million people living with rare diseases that are defined as disorders having a prevalence of less than 1 in % of these orphan diseases are hereditary and approximately one-third of them show characteristic skin alterations [1, 2]. Classic genodermatoses represent cutaneous single-gene disorders which include ca. 560 distinct diseases [3]. They mostly manifest in early childhood, may affect more than one family members, have a chronic course and are associated with significant morbidity, and even mortality. The estimated number of patients with genodermatoses in Germany is at least [4, 5]. Given the rarity and diversity of genodermatoses, their overlapping and heterogeneous phenotypes, the vast amount of new information available, as well as complicated nomenclature including eponyms or old designations such as Bourneville-Pringle syndrome for tuberous sclerosis syndrome (TSC), the diagnosis of hereditary skin disorders remains a challenging task even for experienced dermatologists [6]. In many cases, patients experience an odyssey before getting the correct diagnosis. According to a recent survey conducted in the US and UK, it takes 6-8 years and up to 8 medical visits (4 primary care physicians and 4 specialists) for patients with rare diseases to receive a proper diagnosis [ impact.com]. With this thesis, we are going to explore the importance of having an interdisciplinary network and, in particular, a flexible multistep approach when evaluating patients with rare genetic skin disorders. Based on examples from our own experience, we are going to focus on the necessity to closely correlate clinical and laboratory data in the diagnostics of genodermatoses patients. Referral to specialized centers with setting- 7

8 Introduction up national and international registries would (i) allow the accurate stratification of patients with similar, atypical and mild phenotypes; (ii) enable better understanding of disease mechanisms and (iii) consequently assist in developing pathogenesisoriented therapeutic approaches Classification Thanks to the immense advances in genetic technology, most of the classical genodermatoses have been recently mapped to specific loci [5]. For most genodermatoses, however, no unified classifications integrating both clinical and molecular findings exist so far. First advances in this direction have been achieved with the recently revised classification of inherited epidermolysis bullosa (EB) [7] and the first classification of inherited ichthyoses [8]. A broad classification of the major genetic skin disorders based mainly on clinical features and morphologic criteria is presented in Table 1. 8

9 Introduction Table 1: Classification of genetic skin disorders (modified after Irvine and McLean [2]) Group of genodermatoses Type / Disorder EB simplex Inherited bullous dermatoses EB junctional EB dystrophica Kindler syndrome Ichthyoses (syndromic and non-syndromic) Palmoplantar keratodermas (diffuse, focal and Disorders of keratinization punctate) Follicular keratoses e.g. Darier disease Erythrokeratodermas Albinism Disorders with abnormal pigmentation Piebaldism Waardenburg syndrome DNA repair disorders e.g. Xeroderma pigmentosum (XP), Cockayne syndrome Disorders associated with malignancy Familial tumor syndromes e.g. Muir-Torre syndrome, Gardner syndrome Epidermodysplasia verruciformis Ectodermal dysplasia syndromes Disorders of ectodermal appendages Hypotrichosis syndromes Nail-patella-elbow syndrome Cutis laxa Connective tissue defects Ehlers-Danlos syndrome Pseudoxanthoma elasticum Vascular and lymphatic disorders Osler-Rendu-Weber syndrome Metabolic disorders Porphyrias Acrodermatitis enteropathica Wiscott-Aldrich syndrome Primary immunodeficiency disorders Omenn syndrome Hyper-IgE syndrome 9

10 Introduction 1.2. Principles of diagnostics A multistep diagnostic algorithm designated as onion skin approach, which sequentially takes into account phenotypic characteristics, mode of inheritance, targeted proteins and gene mutations has been recently implemented for EB (Fig. 1) [9]. In our opinion, this approach could be applied to nearly all genodermatoses. In some disorders, such as Hailey-Hailey disease and Darier s disease, clinical features combined with a positive family history and/ or specific histomorphology suffice for diagnosis. For diseases with high genetic heterogeneity like EB and ichthyosis, electron microscopy (EM) and immunofluorescence mapping (IFM) help in identifying the targeted proteins and thus narrow the panel of genes to be examined. Molecular testing is important for confirming the diagnosis, genetic counselling, identifying new genes and sometimes even new diseases as well as comprehending pathomechanisms [6]. Figure 1: Onion skin approach for the evaluation of patients with a genetic skin disorder 10

11 Introduction Clinical evaluation and history When evaluating a patient suspected to have a genodermatosis, it is important to begin with the basics, namely to perform a thorough clinical examination as well as to obtain a complete medical and a three-generation family history Clinical Evaluation Sometimes a quick look will prompt the right question and provide red flags for recognizing a hereditary skin disorder or a syndrome (Table 2) [6]. For example, persistent neonatal erythroderma is often due to hereditary disorders like ichthyoses, immunodeficiencies and metabolic diseases. In a second step, complete examination including hair, nails, teeth and genital/ oral mucosa helps to identify additional, sometimes minor, changes which might point to the final diagnosis, e.g. trichorrhexis invaginata bamboo hair is the key diagnostic feature of Comèl- Netherton syndrome when found in a neonate or infant with erythroderma and failure to thrive. Early diagnosis of a genetic disorder would prompt further surveillance and early treatment of systemic manifestations such as growth delay, proneness to food allergies, and recurrent infections in Comèl-Netherton syndrome. Vice versa, congenital anomalies and extracutaneous findings may indicate a specific diagnosis. For example, cardiac rhabdomyomas are one of the earliest and most specific manifestations of tuberous sclerosis complex (TSC) these lesions are present in >80% of infants with TSC and are often detected prenatally. 11

12 Introduction Table 2: Red Flags in the diagnostics of genodermatoses [6] Age Red Flag / Clinical Feature Condition Ichtyosis e.g. ARCI, Netherton syndrome Erythroderma Immunodeficiency e.g. Omenn syndrome, Metabolic disorders e.g. Biotinidase deficiency Collodion Baby Ichthyosis e.g. ARCI At birth Generalized blistering or blisters at fraction sites Hanging skin and wrinkling in a newborn Association with other congenital abnormalities Association with extracutaneous manifestations especially CNS, skeleton, teeth, eyes and ears Epidermolysis bullosa Cutis laxa congenita e.g. Multiple milia and cleft lip/ palate or pseudocleft of the palate and lip in Orofacial-digital syndrome type I Ectodermal dysplasia syndromes Neurocutaneous syndromes Epidermal nevus syndromes Childhood Therapy-resistant eczema associated with recurrent infections, multiple allergies and growth failure Generalized scaling with / without erythroderma and /or blisters Palmo-plantar keratoderma Photosensitivity Poikiloderma Immunodeficiency disorders e.g. Hyper- IgE syndrome and SAM syndrome Ichthyosis Palmo-plantar keratoderma Ichthyosis DNA repair disorders e.g. XP, Rothmund-Thomson syndrome, Cockayne syndrome Porphyrias Kindler syndrome DNA repair disorders Kindler syndrome 12

13 Introduction Adolescence / Adulthood Multiple (mostly benign) adnexal tumors Brownish keratotic papules in the seborrheic areas Recurrent erosive dermatitis of the main folds Cutaneous lesions in a linear or segmental distribution respecting the midline Familial tumor syndromes e.g. Brooke- Spiegler syndrome, Gardner syndrome, Muir-Torre syndrome Darier disease Hailey-Hailey disease Systematized epidermal nevi Incontinentia pigmenti Focal dermal hypoplasia At any age Multiple hypo- and hypermelanotic macules Hypomelanotic macules e.g. piebaldism, Waardenburg syndrome, TSC Multiple café-au-lait macules e.g. neurofibromatosis, Legius syndrome, McCune-Albright syndrome, LEOPARD syndrome Positive family history Cave: A negative familiy history does not rule out a genetic skin disorder. 13

14 Introduction Family history Family history has been called the first genetic tool because it helps to recognize rare Mendelian (single-gene) disorders and to establish their mode of inheritance, namely autosomal dominant/ recessive or X-linked dominant/ recessive [10, 11]. Y- linked and mitochondrial patterns of inheritance are very rare among genodermatoses. It is important to note that genodermatoses are not necessarily always inherited. In fact, many of them exclusively occur sporadically or appear to be sporadic due to the following confounding factors [12, 13]: Variable expressivity and incomplete penetrance of the disorder Small families (with one child) in case of autosomal-recessive disorders Asymptomatic female carriers of X-linked recessive disorders Early death prior to phenotype manifestation Incomplete family history False paternity Phenocopies Mosaicism, germline or somatic (postzygotic) Anticipation, imprinting, uniparental disomy, paradominance and pseudodominance are other factors that can modify a recurrence risk [14, 15]. If possible, the physician should examine the first- and second-degree relatives in person, or obtain clinical pictures and medical documents, in order to recognize subtle phenotypic features. Repeated questioning might help in identifying newly discovered or forgotten information. Clinical pictures from the newborn period and early childhood are very helpful in narrowing the differential diagnoses as many genodermatoses show an age-dependent phenotypic shift. For example, in a patient with generalized mild scaling, a history of collodion membrane at birth would point to 14

15 Introduction the diagnosis of an autosomal recessive congenital ichthyosis (ARCI) (please refer to ). For practical reasons, a symptom-targeted and age-tailored family history might offer an alternative and easily applicable diagnostic tool during a short visit [11]. A more extensive history may be established later when the patient is referred to a specialized center and/ or a human geneticist Laboratory tests Laboratory diagnostics is required in most cases to confirm a clinical diagnosis. Morphologic studies, biochemical and enzymatic assays as well as molecular tests have to be conducted depending on the disorder suspected Morphologic studies Skin biopsies should be obtained for histopathology, including special stainings and immunochemistry, electron microscopy and/ or cell culture (keratinocytes and/or fibroblasts). Histopathology is important for determining the morphology of cutaneous features, and often enables recognition of diagnosis, e. g. distinguishing specific subtypes of nevi such as keratinocytic or sebaceous nevus in an infant with a linear epidermal nevus on the face. It is required to detect malignant transformation as a complication of certain genodermatoses i.e. the development of squamous cell carcinoma in dystrophic epidermolysis bullosa or a malignant transformation in spiradenocylindromas in Brooke-Spiegler syndrome [16, 17]. Dermatopathologists may often be the first to recognize a family cancer syndrome due to the diagnosis of multiple, mostly benign adnexal tumors occurring at a young age in a person without a positive family history. 15

16 Introduction Transmission electron microscopy is a valuable tool in the evaluation of ichthyoses, connective tissue disorders like Ehlers-Danlos syndrome [18] and hair shaft disorders [19]. For example, about 30-40% of patients with ichthyoses can be successfully classified based on ultrastructural criteria of the epidermis [8]. Immunofluorescence mapping (IFM) has currently replaced electron microscopy in the diagnostics of epidermolysis bullosa. Using IFM, the level of split formation as well as the normal expression, reduction or absence of structural proteins can be detected depending on the antibodies applied in the test. IFM allows thus fast classification of EB subtypes, defines candidate genes for mutation screening and represents the first method for counseling the patient and the parents about the disease outcome [20]. Other examples for useful immunohistochemical stainings of paraffin sections include the detection of the lack of epidermal LEKTI and fillagrin in the diagnostics of Netherton syndrome and ichthyosis vulgaris, respectively [21, 22]. These markers can be used as a cheaper screening test or as an alternative to molecular testing in case that genetic testing is not available Biochemical and enzymatic tests Biochemical and enzymatic tests have been for years the most important tools in the diagnostics of inherited metabolic diseases. The latter are in general classified according to the organelle or pathway involved [23]. Some examples of inherited metabolic disorders that show cutaneous manifestations as well as the tests that serve to detect them are listed in Table 3. 16

17 Introduction Table 3: Examples of inherited metabolic diseases with cutaneous manifestations and detection tests Cutaneous Disorder Example Defect Detection test Manifestations Amino acid disorders Phenylketonuria Phenylalanine hydroxylase deficiency 90% blond hair, blue eyes and fair skin, eczema Newborn screening Hyperlipidaemias Disorders of lipoprotein Xanthomas Blood lipid profile Lipid disorders Refsum metabolism Deficiency in Syndromic Increased Syndrome oxidation of ichthyosis (refer to phytanic acid phytanic acid Table 7) levels in blood Cutis laxa, Glycosylation disorders Autosomal recessive cutis laxa, Type 2A Combined N- and O-glycosylation defect delayed closure of the anterior fontanelle, facial dysmorphism, neuromotor delay Abnormal apolipoprotein C- III and transferrin isofocusing profile (refer to ) Enzyme test in plasma, Lysosomal disorders Fabry disease α-galactosidase A deficiency Angiokeratomas leucocytes or cultured fibroblasts; Specific skin histology Haem disorders Porphyrias A partial deficiency of one of the enzymes required for the biosynthesis of haem Fragility and blistering of lightexposed skin due to perphyrin phototoxicity Porphyrin analysis of plasma, urine and faeces Mineral disorders Acrodermatitis enteropathica Defects in the main intestinal zink transporter ZIP4 Symmetric periorificial dermatitis, alopecia Plasma or serum zink levels before breakfast 17

18 Introduction Newborn screening (NBS) as one of the current most effective public-health programs is largely based on biochemical tests that are conducted shortly after birth and measure the levels of various metabolites in blood collected from a heel prick on filter paper. In the early sixties, phenylketonuria became the first disease targeted by NBS with the help of the Guthrie test and presently serves as a gold standard for NBS. Early detection of high plasma phenylalanine levels followed by dietary modification within the first four weeks of life would prevent the development of irreversible neurodevelopmental disability in the child. At present, the NBS panel in Germany includes twelve metabolic disorders [24] and was recently expanded by measuring immunoreactive trypsinogen for cystic fibrosis. All positive screening results need to be accordingly confirmed. Acrodermatitis enteropathica provides another example for a metabolic disorder where cutaneous manifestations typically lead to diagnosis. The infants suffer from symmetric periorificial dermatitis, alopecia and paronychia as well as frothy and foulsmelling diarrhea. In addition, they may be listless, anorexic, and apathetic. The basic screening test detects elevated zinc levels in plasma or serum due to a defect in the intestinal zinc transporter ZIP4 [25]. Recessive X-linked ichthyosis as one of the most common genodermatoses can be confirmed by low steroid sulfatase activity (the test is performed on EDTA blood sample) or quantification of cholesterol sulfate serum level [26]. 18

19 Introduction Molecular genetic diagnostics Molecular genetic testing is indicated under following circumstances [27]: in patients with unclear phenotypes when molecular testing provides additional benefits in respect to the patient s prognosis (e.g., more accurate prediction of the patient s outcome based on an already identified genotype-phenotype correlation), recurrence risks (identification of de novo mutations) and his/her management in asymptomatic family members at risk of the condition, i. e. family cancer syndromes (predictive testing) in adult asymptomatic family members who could be carriers for a recessive disorder (heterozygosity testing) for prenatal and preimplantation genetic diagnosis Whereas DNA is traditionally obtained from EDTA blood or buccal epithelium for germline mutations, affected skin tissue is required for patients with mosaic conditions where a heterozygous postzygotic mutation occurs on a normal background. In mosaic disorders, a built-in control lies in the unaffected tissue of the patient [27, 28]. Molecular testing approaches can include (i) single-gene testing, (ii) use of multigene panels, and (iii) more comprehensive genomic testing like whole-exome sequencing (WES) and whole-genome sequencing (WGS). For patients with certain phenotypic features or ethnic backgrounds, specific hot-spot mutations or selected high-yield exons may be sequenced as first. This so-called traditional (single-gene) testing uses a bidirectional sequencing (Sanger method) which can identify missence, nonsence, splice-site mutations as well as small intragenic deletions or 19

20 Introduction insertions. For large deletions or duplications involving one or more exons, deletion/ duplication analysis is required that provides a copy number assessment. Multigene panels enable the targeted analysis of multiple genes that can cause a particular phenotype such as autosomal recessive congenital ichthyosis, EB, albinism and/ or periodic fever syndromes. These tests use next-generation sequencing (NGS), in which millions of small DNA segments are sequenced at the same time. NGS technology also enabled the development of whole-exome (WES) and wholegenome (WGS) sequencing which are presently the most advanced molecular methods. WES and WGS are indicated: in patients with atypical presentations suggestive of a Mendelian disorder; in Mendelian phenotypes with unknown genetic causes; in patients where standard genetic tests failed. While WGS is supposed to examine the whole genome, WES examines only the exome that represents the protein-coding region of the human genome ( 2% of genetic material) and includes ca. 85% of the disease-mutating genes. Compared to WES, WGS allows in addition the detection of mutations in noncoding regions, copy number variations and complex chromosomal rearrangements. On the other hand, WGS generates an even greater amount of data than WES meaning that while WES may identify up to single nucleotide variants per DNA sample (95% of which represent genetic polymorphisms), WGS reveals > 2-3 million variants per DNA sample [29]. The challenge these days is therefore considered, not so much the 20

21 Introduction DNA sequencing itself, but the correct interpretation of the generated molecular data in the context of clinical data. Comparing the results of the patient with those of his/ her parents (trio NGS analysis), setting up large databanks including precise and comprehensive information on both molecular and clinical data (deep phenotyping), functional protein studies and recent advances in bioinformatics are important steps on the way of sorting out clinically relevant information. Currently, the success rate of WES/ WGS in diagnosing single-gene disorders is about 20-50% [27, 30, 31, 32] Ethical issues Due to its tremendous impact across all age-groups and spheres of medicine, the rapid advances in human genetics raise important ethical, legal and social considerations. The latter have been recently addressed by the American Society of Human Genetics [33] and the American College of Medical Genetics and Genomics [34]. Major issues outlined in these recommendations relate to: prenatal and preimplantation diagnostics, especially for nondisease traits or sex testing asymptomatic family members for predisposition to disease reporting of incidental and secondary findings such as the discovery of targetoff mutations that can cause serious diseases which could be ameliorated or prevented privacy of genetic information parental decision-making about NBS Respect for individual autonomy, beneficence, avoidance of maleficence ( primum non nocere ) and justice keep being the four cardinal principles guiding medical 21

22 Introduction decisions in respect to the diagnostics and management of patients with hereditary skin disorders [35]. Genetic counselling of affected individuals and their families is required before initiating molecular diagnostics as well as thereafter (please also refer to the Discussion ad ) Correlations of clinical and laboratory data Clinico-pathological correlation A close clinico-pathological correlation is crucial for all sub-areas of dermatology including inflammatory dermatoses and skin tumors like melanocytic lesions and lymphomas [36]. One histological pattern is often associated with an array of disorders e.g. the phenomenon of eosinophilic spongiosis can be seen in conditions like incontinentia pigmenti, allergic contact dermatitis, autoimmune bullous diseases, drug reactions and insect bites. On the other hand, one disease can manifest itself with different patterns depending on how old the lesions. For example, incontinentia pigmenti presents in the acute (vesicular) stage in childhood with eosinophilic spongiosis and intraepidermal vesicles, and microabscesses. Atrophic and pale epidermis with apoptotic keratinocytes is to be seen in the end hypopigmented stage [37]. In cases of histologically normal skin, accurate clinical information allows further diagnostic steps with a better interpretation of microscopic findings. For example, broad and interlacing elastic fibers in the Vorhoeff-van Gieson staining are diagnostic for elastomas which might be associated with Buschke-Ollendorf syndrome in a child with multiple flesh-colored or yellowish papules on the proximal buttocks and limbs. In addition, the patients often show on X-ray small round radiodensities in the 22

23 Introduction epiphyses and metaphyses of the long bones, pelvis and scapulae which should be recognized as osteopoikilosis and not misdiagnosed as bone metastases Genotype-Phenotype Correlation Careful correlation of clinical and molecular features plays a crucial role in confirming the final diagnosis and determining the causality of novel variants in patients with complex phenotypes (refer to ). Clinical and genetic heterogeneity are two other concepts underscoring the necessity of genotype-phenotype correlations in the diagnostics of genodermatoses. Xeroderma pigmentosum (XP), Cockayne syndrome and trichothiodystrophy illustrate the phenomenon of clinical heterogeneity, in which different mutations in one gene (e. g. XPD) may result in distinct diseases or disease variants. Genetic heterogeneity represents the fact that identical clinical phenotypes are caused by mutations in different genes as in ARCI, in which mutations in nine different genes have been identified [38] Principles of management Genetic skin disorders represent rare chronic diseases that often manifest in childhood and - besides skin - may involve different organs and systems. They have a significant impact on the patient s quality of life, and that of his or her family. The overall goal of management is, therefore, to improve health-related quality of life, enhance the patient s autonomy and provide early recognition, and if possible, prevention of complications connected to the disease. For example, growth retardation, anemia, poor wound healing, joint contractures, infections, ocular, dental, social and psychological problems are important complications, with which EB 23

24 Introduction patients have to deal with. Therefore, depending on the severity and type of EB, individual patients demand an interdisciplinary management including the following specialties: dermatologists, pediatricians, human geneticists, plastic (hand) surgeons, gastroenterologists, dentists, ophthalmologists, wound and pain management, dieticians, psychologists and physiotherapists. To meet the specific demands of each patient in a coordinated manner, current attempts include the establishment of Interdisciplinary Centers as well as National and International Networks for Rare Genetic Skin Disorders. At present, most genodermatoses are treated symptomatically. Due to the latest achievements in understanding the molecular mechanisms behind, novel therapeutic approaches including cell-based therapies, gene replacement and repair technologies, and direct protein replacement therapies are on their way (refer to Discussion ad 4.6.). Epidermolysis bullosa has become a paradigm for the development of molecular and cell therapies, some of which have already entered early clinical trials [39, 40] German networks for rare genetic skin disorders Currently there are two Networks for Rare Genetic Skin Disorders functioning in Germany, namely the Network Epidermolysis Bullosa (www. netzwerk-eb.de), coordinated by the Department of Dermatology, University Medical Center Freiburg, and the German Network for Ichthyoses (NIRK) ( coordinated by the Department of Dermatology, University of Münster. The primary goals of these Networks comprise steady improvement of standardized clinical care and intensive collaboration between basic scientists and physicians from different 24

25 Introduction centers and disciplines. Both networks have operated since 2003 with the support of the Federal Ministry of Education and Research. The Cologne Department of Dermatology is an associate partner of both Networks. 25

26 Objectives II. Objectives While NGS has been successfully introduced into clinical practice, isolated molecular testing neither answers all questions with respect to disease classification nor does it address patients worries [41, 42]. As the functions of most genes are still unknown, only precise correlation of phenotypic and genotypic results will allow the stratification of diseases into subtypes with common pathomechanisms that have in turn prognostic and therapeutic implications [43]. Deep phenotyping with comprehensive analysis of clinical features is required on the way to precision medicine which aims at providing the best available care for individual patient based on such a stratification [41]. Most of the hereby presented genodermatoses are very rare with only a few cases reported so far e.g. epidermolysis bullosa Ogna (ca. 20 cases published so far including ours), nevus trichilemmocysticus (8 cases), oculo-ectodermal syndrome (ca. 20 cases). We are going to discuss these genodermatoses in the light of our own experience and, in particular, focus on the relevance of matching phenotypic, histomorphologic and molecular findings in the diagnostic process. The following major objectives are at the center of our Discussion: Better understanding of pathogenesis, e.g. epidermolysis bullosa simplex Ogna (EBS-Ogna) and autososomal recessive cutis laxa, type 2A (ARCL2A) and ALOX-related autosomal recessive congenital ichthyosis (ARCI) Development of pathogenesis-based disease classifications as illustrated by oculo-ectodermal syndrome/ encephalocraniocutaneous lipomatosis (OES/ 26

27 Objectives ECCL) and Brooke-Spiegler syndrome (BSS), where seemingly different phenotypes showed common etiology Implementation of new diagnostic markers and methods, e.g. in hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC) and ALOX-related ARCI Better phenotypic and genotypic disease characterization of some very rare subtypes such as EBS-Ogna, junctional EB-late onset (JEB-lo) and ARCL2A Improved interdisciplinary management of genodermatoses patients with more accurate genetic counselling and development of causal therapeutic approaches 27

28 Selected clinical cases and case studies III. Selected clinical cases and case studies 3.1. Inherited epidermolysis bullosa (EB) Inherited epidermolysis bullosa (EB) represents a heterogeneous group of disorders characterized by marked mechanical fragility of the skin leading to blistering, erosions and chronic ulcers. EB results from mutations in at least 18 genes encoding proteins involved in the epidermal cell-cell and cell basement membrane adhesions, namely the desmosomes and the epidermal-dermal junction. Recent advances in understanding the pathogenesis of EB are reflected in the latest classification which stratifies EB into four main types, including more than 30 subtypes, based on the anatomic level of blister formation in the skin as determined by immunofluorescence mapping or transmission electron microscopy [7, 9] (Table 4). Thanks to modern sequencing methods, some new very rare autosomal recessive forms of EB have been recently recognized and mapped to DST-e (coding for epidermal dystonin), EXPH5 (coding for the exophilin.5) and ITGA3 (coding for the integrin alpha-3 subunit) [44]. The clinical EB spectrum is broad ranging from mild forms, which usually do not require medical care to more severe types and subtypes with significant mortality and morbidity. Some subtypes like generalized junctional EB (JEB) and recessive dystrophic EB (RDEB) are associated with premature death due to cutaneous and extracutaneous complications. It is, however, mostly impossible to predict the exact EB type only based on clinical features at birth. Therefore, early diagnosis is critical and a multistep onion skin approach, which sequentially takes into account phenotypic characteristics, mode of inheritance, targeted proteins, and gene mutations, has been recently developed [9] (refer also to the Introduction ad 1.2.). 28

29 Selected clinical cases and case studies Table 4: Major EB types and subtypes (modified after Fine 2008 [7]) Major EB type/ level of skin cleavage Major EB subtypes Targeted proteins EB simplex (EBS)/ Intraepidermal Suprabasal EBS Basal EBS Transglutaminase 5; plakophilin 1; desmoplakin; plakoglobin Keratin 5 and 14; plectin; exophilin 5; bullous pemphigoid antigen 1 Junctional EB (JEB)/ Intralamina lucida JEB, generalized JEB, localized Laminin-332; collagen XVII ; α6β4 integrin ; α3 integrin subunit Collagen XVII ; laminin-332 ; α6β4 integrin Dystrophic EB (DEB)/ Sublamina densa Kindler Syndrome/ Mixed DEB, dominant DEB, recessive Collagen VII Collagen VII Kindlin-1 To illustrate the advantages of having such a flexible algorithm incorporating clinical (phenotype, mode of inheritance) and laboratory (immunohistochemical and molecular) findings, we present three very rare EB subtypes which we studied in detail: EB simplex Ogna (EBS-Ogna) p.r1303q-related junctional EB/ junctional EB late-onset (JEB-lo) A mild phenotype of recessive dystrophic EB with two compound heterozygous COL7A1 mutations. 29

30 Selected clinical cases and case studies In a close collaboration with the German Network for EB, we further characterized EBS-Ogna and p.r1303q-related junctional EB. EBS-Ogna offers an interesting example of clinical heterogeneity where different mutations in the plectin gene result in very dissimilar phenotypes depending on the domain involved, namely EBS with muscular dystrophy (EBS-MD), EBS with pyloric atresia (EBS-PA) and EBS-Ogna [45]. A relatively mild phenotype of JEB was associated with specific p.r1303q mutations in the COL17A1 gene i.e. junctional EB late-onset (JEB-lo). In our patient with recessive dystrophic EB, the relatively mild phenotype was attributed to two compound heterozygous COL7A1 mutations thus underlying the significance of genotype-phenotype correlations in predicting the patients outcome EB simplex Ogna (MIM #131950) Own Publication: [46] In epidermolysis bullosa simplex (EBS), the split formation occurs within the epidermis by cytolysis of basal keratinocytes. In about 75% of the cases, it is associated with dominantly inherited mutations in the genes for keratin 5 (KRT5) or 14 (KRT14) [47, 48]. Mutations in some recently identified genes encoding plectin, BPAG1e (dystonin) and exophilin 5 may explain some of the molecularly unsolved EBS cases [49, 50, 51]. EBS-Ogna is caused by the specific PLEC mutation p.arg2000trp, and has been reported in fewer than 20 patients to date [46, 49, 52]. Plectin is an intracellular linker protein which is localized in the hemidesmosomal inner plaque that interacts with keratin intermediate filaments [53] (please, also refer to the Discussion ad 4.1.). Plectin mutations result in distinct phenotypes, including EBS-MD, EBS-PA and EBS- Ogna [45]. EBS-MD is characterized by congenital blistering and progressive 30

31 Selected clinical cases and case studies muscular weakness, whereas EBS-PA comprises neonatal blistering, pyloric atresia, and often early demise [45]. In contrast to the severe recessive subtypes EBS-MD and EBS-PA, the dominant EBS-Ogna has a mild course. Patients: In the index patient, onset of posttraumatic erosions was seen soon after birth, but blistering started at the age of 8 years. Clinical findings included small erosions on the extremities, which healed with violaceous macules (Fig. 2a, b), and spotty and linear hypopigmented macules on the trunk. Other features encompassed acral blisters, focal palmoplantar keratoderma, partly dystrophic toe nails and pitting of the teeth. The mother reported to have had acral blisters and erosions in her childhood, with predominance of erosions with age. The brother had a much milder phenotype. Two other families with EBS-Ogna, designated as A and B (Table 5), were identified through revision of 40 cases from the German EB registry. The survey included clinically suspected EBS, in which KRT5 and KRT14 mutations had been excluded. Methods: Diagnostic skin biopsies were subjected to indirect immunofluorescence staining (IIF) using a panel of antibodies to components of the dermal-epidermal junction, including the domain specific plectin antibodies 31/Plectin (directed against amino acids , BD Biosciences, Heidelberg, Germany) and 5B3 (directed against the rod domain, a kind gift of Dr. G. Wiche, Vienna, Austria) (Fig. 2 c-f). For mutation analyses, EDTA-blood was obtained from patients and family members following informed consent in adherence to the Declaration of Helsinki principles. The primers for amplification of PLEC exon 31 and KRT5 / KRT14 have been described previously [47, 54]. 31

32 Selected clinical cases and case studies Results: H&E staining of cryosections revealed intraepidermal (micro) blisters. The signal obtained with the plectin rod domain-specific antibodies 5B3 and 10F6 was reduced, whereas the C-terminus-specific 31/Plectin and GP21 antibodies generated a signal similar to control skin. The hemidesmosomal binding partners of plectin, namely dystonin, collagen XVII, and the β4 integrin subunit showed a reduced and interrupted IIF staining with small, cell-sized gaps. All other markers of the dermalepidermal junction zone showed no abnormalities (Figure 2 c-f). Similar skin findings had been reported in the mouse model for EBS-Ogna, which had no overt skin lesions, but microblisters on microscopic examination [55]. In all patients, mutation analysis disclosed the heterozygous c.5998c>t in exon 31(p.Arg2000Trp) mutation which was in concordance with previous studies [49]. The pathogenicity of the p.arg2000trp mutation was supported by the immunofluorescence studies in our patients which were comparable with the findings reported in the EBS-Ogna mouse model. In our study, the phenotype of EB-Ogna was outlined based on the clinical features of eight patients from three families. Mild skin fragility manifesting predominantly with erosions and healing with violaceous macules, seems to be the main feature that may distinguish this rare subtype from the frequent keratin-associated EBS. 32

33 Selected clinical cases and case studies Table 5 Clinical features of the EBS-Ogna patients in this study and in the literature [46] Family Individual Age of onset Skin fragility 2 Age 1 Distribution of blisters and erosions Focal palmoplantar Other skin features Nails keratoderma 2 Other features A.III.6 11 years 3 years predominantly erosions; blisters (2+) disseminated 1+ spotty hypopigmentation on the trunk, violaceous macules, hyperhidrosis normal enamel pits A.II.4 45 years early adulthood predominantly erosions; blisters (1+) lower legs, arms, back 1+ / 2+ spotty hypopigmentation on the trunk, violaceous macules normal discrete enamel pits A.III.1 27 years 20 years predominantly erosions; blisters (1+) lower legs, arms, back 1+ spotty hypopigmentation on the trunk, violaceous macules normal discrete enamel pits A.III.4 21 years 16 years blisters and erosions (1+) lower legs, arms, back 1+ spotty hypopigmentation on the trunk, violaceous macules normal enamel pits B.III.1 34 years years blisters (1+) hands and feet 1+ / 2+ NA normal absent C.II.1 14 years erosions after birth, blisters at age of 9 years blisters and erosions (1+) hands and feet, lower legs absent spotty hypopigmentation on the trunk, violaceous macules normal absent C.I.1 47 years blisters in childhood blisters and erosions not applicable NA NA NA NA Reference Gedde- Dahl, 1971 erosions in first months of life, blisters in early childhood traumatic erosions and blisters; small, haemorrhagic blisters NA not described tendency to bruise inconstant onycho gryphosis Fine et al, onycho 2008 birth 3+ NA absent tendency to bruise gryphosis absent Legend: 1, age at last examination; 2, scale as defined in Fine et al, 2008: absent, 1+, 2+, 3+, 4+; NA, information not available; bold, index cases absent 33

34 Selected clinical cases and case studies Figure 2 a-f: Clinical features and immunofluorescence mapping in our patients with EBS-Ogna Violaceous macules on the hands and lower legs after healing of erosions (a, b); Indirect immunofluorescence staining of plectin and other components of the dermal epidermal junction (c h). In the skin of the patient, the signal for the rod domain specific plectin antibody 5B3 is reduced (c), whereas the signal is positive and comparable to the control with the C-terminus-specific plectin antibody 31/ Plectin (d). The signal for the integrin α6 subunit remained unchanged (c, d). BPAG1e, collagen XVII, and the β4 integrin subunit show an interrupted staining with small, cell-sized gaps (white arrows) in the patient's skin. Keratin 5/6 and laminin 332 stain similar to control skin (e, f). Bar=20 μm. 34

35 Selected clinical cases and case studies p.r1303q-related junctional EB/ junctional EB late-onset (MIM #226650) Own publication: [56] In junctional epidermolysis bullosa (JEB), the cleavage plane occurs in the lamina lucida at the junction of the epidermis and dermis. Encompassing a spectrum from severe life-threatening subtypes (the most ominous one being JEB-Herlitz) to relatively mild involvement, various subtypes have been described, mostly transmitted in an autosomal recessive manner [7, 39]. Here, we present a distinct group of JEB patients with p.r1303q mutations in the collagen XVII gene (COL17A1), also known as junctional EB late-onset (JEB-lo). These patients showed a relatively mild phenotype (late-onset blistering and nail anomalies) with some clinical and morphological features resembling Kindler syndrome such as mild blistering and progressive skin atrophy. Collagen XVII is a type II-oriented transmembrane protein that represents a hemidesmosomal component with multiple intracellular and extracellular binding partners, e.g. BP230, integrinα6β4, and laminin-332. For Kindler syndrome, please, refer to the Discussion ad Patients: A 22-year-old patient was referred to our department with mild acral skin blistering, which started at the age of 6 years and resolved with mild atrophic scarring. Some finger nails were lost, whereas all toe nails were either lost or dystrophic (Fig. 3a-c). The younger sibling was similarly affected but occasionally had oral blisters. On the basis of genotype-phenotype correlations, 13 more patients with the COL17A1 mutation c.3908g>a (p.r1303q) were detected in the German EB Registry (Table 6). 35

36 Selected clinical cases and case studies Methods and Results: Immunofluorescence microscopy was performed which demonstrated abnormal labeling with extensive immunostaining for collagens XVII, IV and VII throughout the papillary dermis (Fig. 3d). There was focal reduplication of the basal membrane. In all 15 patients, a similar c.3908g>a (p.r1303q) mutation in the collagen XVII gene in a homozygous or a compound heterozygous compound state was detected. The present cohort allowed the characterization of the molecular and phenotypic features caused by this immune-acid substitution. The p.r1303q mutation determines namely a late-onset form of junctional EB where the patients suffer from a mild acral blistering in contrast to junctional EB-other (JEB-o, also known as non- Herlitz), which is due to COL17A1 loss-of-function mutations. JEB-o manifests at birth with generalized blistering. On the other hand, JEB-lo shares many similarities with Kindler syndrome at phenotypic (i.e. scleroderma-like fingers, acral skin atrophy and poikiloderma in adulthood) and ultrastructural (irregular broad staining pattern of collagen XVII, IV, and VII, laminin ϒ2 chain, and tenascin C, as well as the significant interruptions and duplications of the lamina densa.level) level. The exact pathogenesis of these common features has not been elucidated yet. 36

37 Selected clinical cases and case studies Table 6: Clinical features of patients with JEB-lo, in this study [56] Patient, Age (years), Origin COL17A1 mutations 1 Onset of cutaneous manifestations / Presentation Nails Mucous membranes Turkey c.3908g>a p.r1303q At the age of 5 years / Scarring, skin atrophy, milia Lost or dystrophic Mild oral blistering Turkey c.3908g>a p.r1303q At the age of 12 years / Scarring, skin atrophy, milia, loss of dermatoglyphs Lost or dystrophic No abnormality Germany c.3908g>a p.r1303q At the age of 6 years / Discreet atrophic scars on the dorsal aspects of the hands, palmoplantar hyperhidrosis Partially dystrophic or lost No abnormality Germany c.3908g>a p.r1303q At the age of 8 years / Discreet atrophic scars on the hands, feet, knees and elbows, palmoplantar keratoderma Dystrophic Occasional blistering in the mouth Switzerland c.3908g>a p.r1303q Childhood / Atrophy of skin, loss of dermatoglyphs, diffuse plantar keratoderma Lost Dry eyes Japan c.3908g>a p.r1303q At the age of 10 years / Skin atrophy, loss of dermatoglyphs, sclerosis and contracture of fingers, diffuse palmoplantar keratoderma Lost Leukokeratosis of oral mucosa and tongue At the age of 16 years / Atrophy and mottled Japan c.[2003-1g>c]; [3908G>A], p.r1303q depigmentation on the back of both hands, flat dermatoglyphs, contracture and scleroderma-like fingers, Dystrophic No abnormality diffuse palmoplantar keratoderma -Table 6 continued on next page- 37

38 Selected clinical cases and case studies -Continued table 6- Microstomia, Russian- German c.3908g>a p.r1303q Childhood / Skin atrophy, flat dermatoglyphs, sclerodermalike fingers and contracture of hands, diffuse palmoplantar keratoderma Lost oesophageal stenosis, corneal erosions and ectropion, lacrimal duct obstruction Russian- German c.3908g>a p.r1303q Childhood / Skin atrophy, scleroderma-like fingers and contracture of hands, partial loss of dermatoglyphs, diffuse palmoplantar keratoderma Lost Microstomia, oesophageal stenosis, corneal erosions and ectropion, lacrimal duct obstruction Russian- c.3908g>a p.r1303q Blistering starting in childhood NA NA German 11. At the age of 16 years / Fragile c.[433c>t];[3908g>a] Mostly acral, loss of 48 nails, Dry eyes p. [R145X];[R1303Q] dermatoglyphs, palmoplantar Switzerland dystrophic keratoderma 12. At the age of 17 years / Fragile Transient blisters c.[433c>t];[3908g>a] Mostly acral, flat 31 nails, of the oral p. [R145X];[R1303Q] dermatoglyphs, mild plantar Switzerland dystrophic mucosa keratoses 13. At the age of 17 years / Fragile c.[433c>t];[3908g>a] 30 Mostly acral, flat nails, No abnormality p. [R145X];[R1303Q] Switzerland dermatoglyphs dystrophic? / Blisters on buttocks and extremities, scleroderma-like 14. fingers, contracture of hands, c.3908g>a Lost or 73 palmoplantar keratoderma, Ectropion p.r1303q dystrophic Japan skin atrophy and dyspigmentation, flat dermatoglyphs Childhood / 15. Acral, scleroderma-like c.3908g>a Lost or 62. fingers, contracture of hands, No abnormality p.r1303q dystrophic Japan palmoplantar keratoderma, flat dermatoglyphs Legend: 1, if not otherwise mentioned, homozygous mutations; NA, not available. Patients 8-10, and 14-15, respectively were siblings. 38

39 Selected clinical cases and case studies Figure 3 a-d: Clinical features and immunofluorescence mapping in our patients with JEB-lo Acral skin blistering and loss of toenails (a-c); Immunofluorescence staining patterns with antibodies to collagens XVII (mab-123, gift of P. Marinkovich, Stanford, USA), IV (COL94, Sigma-Aldrich, Poole, UK), and VII (LH7.2, Sigma-Aldrich) (green) in a control and an index patient demonstrate an irregular, increased deposition of all markers in the papillary dermis of the patient, in contrast to the normal linear pattern. Nuclei are stained with 4,6-diamidino-2-phenylindole. 39

40 Selected clinical cases and case studies A mild phenotype of recessive dystrophic EB with two compound heterozygous COL7A1 mutations (MIM #226600) Own publication: [57] In dystrophic epidermolysis bullosa (DEB), the cleavage occurs immediately below the lamina densa. On the basis of clinical presentation and the mode of inheritance, DEB is currently classified into three major subtypes: dominant DEB (DDEB), severe generalized recessive DEB (RDEBsg) and RDEB generalized other (RDEB-O) [7]. All forms of DEB are caused by mutations in the collagen type VII-encoding gene COL7A, causing defective anchoring fibrils and disturbed dermal-epidermal adhesion. Here, we report on a woman with a relatively mild phenotype, harboring two compound heterozygous COL7A1 mutations, who we classified as a patient with RDEB-O. Patient: A 45-year-old Caucasian female presented with a mild phenotype of typical RDEB skin findings such as blisters, scars, milia and nail dystrophy (Figure 4a-c), and esophageal strictures. Other extracutaneous symptoms, in particular anemia, growth retardation, soft tissue abnormalities, caries, pseudosyndactyly, glomerulonephritis or cardiomyopathy, were not observed. The patient developed squamous cell carcinomas at the age of 41 years and 58 years which were treated by excision. In addition, she had repeated wound infections on her lower legs. During pregnancy and after menopause, the skin lesions had improved. Her family history was non-contributory. 40

41 Selected clinical cases and case studies Methods: Immunofluorescence mapping revealed reduced immunoreactivity for collagen VII, whereas stainings with antibodies to pankeratin, cytokeratin 5, cytokeratin 14, α6 integrin, β4 integrin, plakophilin 1, plectin, laminin-332, collagen IV, and collagen XVII were unaltered. The priority strategy was used to identify mutations, where, based on the frequency of previously reported mutations, COL7A1 exons were classified into three categories and each category was sequenced on a separate plate. Results: Two heterozygous mutations were identified, the intronic splice site mutation c a>g and the missense mutation c.4039g>t causing the glycine substitution p.g1347w (figure 4d). Validation of both mutations was confirmed by repetitive sequencing in both directions. Additionally, both mutations were verified in 100 control chromosomes by restriction endonuclease digestion. This case further validates the association of compound heterozygous mutations within the triple helical domain of COL7A1 with a moderate clinical phenotype in RDEB-O. For a detailed explanation of the functional defect that these mutations have, please, refer to the Discussion ad

42 Selected clinical cases and case studies Figure 4 a-d: Clinical features and sequencing in our patient with RDEB Blisters, erosions and atrophic scarring covering the back and the lower legs (a,b). Nail dystrophy (c); Sequencing revealing two heterozygous mutations, the intronic splice site mutation c a>g and the missense mutation c.4039g>t causing the glycine substitution p.g1347w (d). 42

43 Selected clinical cases and case studies 3.2. Mendelian disorders of cornification Ichthyoses Inherited ichthyoses belong to the large group of Mendelian Disorders of Cornification (MEDOC) (Table 7). They encompass a heterogenous group of diseases having an abnormal skin barrier which leads to transepidermal water loss and compensatory hyperproliferation. The skin changes are clinically characterized by hyperkeratosis, scaling, or both. They are often associated with xerosis, hypohidrosis, erythroderma, alopecia and palmoplantar keratoderma. Mutations in over 50 genes may cause ichthyoses by affecting different pathways such as lipid biosynthesis, DNA repair, adhesion, desquamation [60]. In 2009, the First Ichthyoses Consensus Conference established a clinically based classification for MEDOC which divided ichthyoses into two main groups [8]: Non-syndromic forms with clinical findings limited to the skin Syndromic forms with involvement of additional organ systems. Non-syndromic ichthyoses have been largely stratified into: Common ichthyoses, i.e. ichthyosis vulgaris Keratinopathic ichthyosis, i.e. epidermolytic ichthyosis Autosomal recessive congenital ichthyosis (ARCI) (please, refer to the text) And others, i.e. loricrin keratoderma. Syndromic Ichthyosis may be associated with a variety of extracutaneous abnormalities such as hair abnormalities (e.g. bamboo hait in Netherton syndrome), prominent neurological signs (e.g. Refsum syndrome) and deafness (e.g. Refsum syndrome and keratitis-ichthyosis-deafness syndrome/ KID-syndrome) (Table 7). 43

44 Selected clinical cases and case studies Table 7: Examples of non-syndromic and syndromic Ichthyoses according to the revised nomenclature and classification, 2009 [8] Major Ichthyosis Types Major Ichthyosis Subtypes Examples with phenotypic description Mode of Inheritance (Gene) Common ichthyoses Ichthyosis vulgaris (xerosis, scaling, eczema after ca 2-6 months) Autosomal semidominant (FLG) Keratinopathic ichthyosis Epidermolytic ichthyosis (erythroderma with erosions at AR (KRT10) birth, later on hyperkeratosis) Nonsyndromic ARCI LI/ CIE (refer to the text) Refer to the text Loricrin keratoderma (collodion baby at birth, Other generalized scaling with hyperkeratosis over joint, AD (LOR) honeycomb palmo-plantar keratoderma) Netherton syndrome With hair abnormalities (congenital erythroderma, failure to thrive, atopy, AR (SPINK5) infection susceptibility, bamboo hair) Syndromic With prominent Refsum syndrome (generalised scaling, AR (PHYH) neurologic signs development of night Refer also to table 4 blindness, anosmia, deafness, ataxia) With deafness KID syndrome (keratitis, ichthyosis, AD (GJB2, GJB6) deafness) 44

45 Selected clinical cases and case studies Understanding the pathogenesis of ichthyosis has significantly contributed to our better understanding of the epidermal barrier structure and functioning. In the horn layer of our ALOX12B- and ALOXE3-related ARCI patients, diminished ceramide levels were demonstrated for the first time with the help of a non-invasive stripping technique. These findings confirmed the important role of lipoxygenases in the sphingosine metabolism of the skin which constitutes an important step in the formation of the corneocyte lipid envelope [61] Further evidence for a disordered ceramide composition in the epidermis of ALOX-related ARCI patients (MIM PS242300) Accepted: [Br J Dermatol] Autosomal recessive congenital ichthyosis (ARCI; MIM PS242300) is a new umbrella term in the spectrum of the non-syndromic ichthyoses encompassing harlequin ichthyosis, lamellar ichthyosis (LI) and congenital ichthyosiform erythroderma (CIE). According to the Foundation for Ichthyosis and Related Skin Types, ARCI affects approximately 1: individuals in the US. Most ARCI children are born as collodion babies (Fig. 8a) who are encased in a shiny translucent membrane at birth. The membrane is shed within 3 to 4 weeks. Subsequently the patients develop a lamellar ichthyosis (LI) phenotype with large, thickened and dark gray or brown scales or congenital ichthyosiform erythroderma (CIE) phenotype. CIE patients suffer from erythroderma with fine, white scaling and often palmoplantar hyperlinearity and/ or keratoderma (Fig.7b, c, d). The following genes have been reported as causative for ARCI: TGM1, ALOX12B, ALOXE3, ABCA12, CERS3, CYP4F22, LIPN, NIPAL4/ ICHTHYIN, PNPLA1 and CERS3 (Fisher et al). In Germany, a transglutaminase 1 45

46 Selected clinical cases and case studies deficiency is found in about 30% of all patients with ARCI; the second most common cause are mutations in both lipoxygenase genes ALOXE3 and ALOX12B, which are found in about 10% of all patients [38]. ALOX patients usually show a mild to moderate CIE phenotype with ca. 76% of the patients being born as collodion babies, and 88% suffering from hypohidrosis [62]. ALOXE3 and ALOX12B encode the epidermal lipoxygenases elox-3 and 12R-LOX. LOXs catalyze the oxygenation of the lineate-containing ceramides which represent important constituents of the corneocyte lipid envelope in the epidermis [Krieg P 2014]. The linkage of the LOX pathway to ceramide processing, and consequently to skin barrier formation, has been so far demonstrated in mice and pig models [63, 64, 65]. Patients: We identified three ALOX patients among our ichthyosis children with CIEphenotype. The clinical and molecular data of our patients are presented in Table 8. All of them were born as collodion babies and suffered from relatively mild erythroderma with fine white scaling (Fig. 5 a, b). In addition, they showed mild palmo-plantar hyperlinearity (Fig. 5 c) and could not endure high temperatures due to hypohydrosis. Patient 1, who had a mutation in ALOX12B and suffered from more severe erythroderma, also had a mild palmo-plantar keratoderma (5 d). Four additional patients (two having ALOXE3 mutations, and one having ALOX12B mutations; age 6 months to 17 years old) from the centers in Münster and Münich were included. The study was conducted in collaboration with the Division of Molecular Pediatrics, Friedrich-Alexander University of Erlangen-Nürnberg and NIRK. 46

47 Selected clinical cases and case studies Methods: Using a tape stripping technique, material from stratum corneum of the patients volar forearms or thighs was obtained. Afterwards, the probes were analyzed for the composition of ω-hydroxyceramides performing a lipid extraction and using a liquid chromatography-tandem mass spectrometry. Horn material from healthy children and adolescents (n=8) as well as adult (n=7) were used as controls. Results: In comparison to their age-paired healthy controls, both ALOXE3- and ALOX12B-patients showed a decrease of protein bound ceramide species in stratum corneum. Interestingly, patients with ALOX12B mutations showed a more pronounced ceramide reduction compared to the ALOXE3 patients which was in line with the more severe phenotype which was also observed in our patients (Fig. 6). Thus, our results support previous findings on mouse models and reveal the essential link of the 12R-LOX/ elox-3 pathway to ceramide processing and consequently to the formation of the corneocyte lipid envelope [63, 64, 66, 67]. 47

48 Selected clinical cases and case studies Table 8: Clinical and molecular features of patients with ALOX-related ARCI in this study ID Mutation Age Sex Integument Scalp Palmo-plantar phenotype Hyperlinearity Keratoderma Scaling Erythema Scaling Alopecia Hypohidrosis ALOXE3 homozygous c.2357c>t (p.p786l) exon 14 ALOXE3 heterozygous c.506_509delacct (p.y169x) exon 1 c.1420dupc (p.l474pfsx58) exon 7 ALOXE3 heterozygous c.1168c>t (p.r390x) exon 6 c.2357c>t (p.p786l) exon 14 ALOXE3 homozygous c.2357c>t (p.p786l) exon 14 ALOXE3 homozygous c.1168c>t (p.r390x) exon 6 ALOX12B homozygous c.1562a>g (p.y521c) exon 12 ALOX12B heterozygous c.340c>t (p.r114x) exon 2 c.2036g>t (p.r679l) exon 14 - none, + mild, ++ medium, +++ severe 3 f f m -/+ -/+ -/ /+ 6 f /+ 7 m /++ 2 f f

49 Selected clinical cases and case studies Figure 5 a-d: Clinical features of our children with ARCI-phenotype All of them were born as collodion babies (a) and suffered from relatively mild erythroderma with fine white scaling (b). In addition, they showed palmo-plantar hyperlinearity (c); Mild palmo-plantar keratoderma in patient 1, who had a mutation in ALOX12B (d). Figure 6: Loss of protein-bound ω-hydroxylated ceramides in skin of ARCI patients Cer [OS(18)] species extracted from tape stripping samples of the upper epidermal layers of ARCI patients with ALOXE3 (n=5) or ALOX12B mutations (n=2), juvenile (n=8) and adult controls (n=7) were analyzed. Lipid analysis of Cer with fatty acid chains 28:0, 30:0, 32:0, 32:1, 34:0, and 34:1 was performed using LC MS/MS. The peak area was normalized internally on the peak area of short-chain free Cer [NS] and an internal standard (C17:0). Ceramide amounts are shown relatively to those of the juvenile control group. Data are displayed ±SEM. * = p 0,05, ** = p 0,01, *** = p 0,001. Cer [OS], ω- hydroxylated ceramide; Cer [NS], not-hydroxylated ceramide. 49

50 Selected clinical cases and case studies 3.3. Heritable connective tissue disorders Heritable connective tissue disorders result from genetic defects affecting the extracellular matrix assembly and/ or homeostasis [68]. They mostly affect the skin, the eyes, and the musculoskeletal, cardiovascular, and pulmonary systems. Today, these disorders are divided into two large classes based on the major constituent of the connective tissue involved: collagenopathies (disorders connected to collagen) and elastinopathies (disorders connected to elastin). The most common collagenopathies include Ehlers-Danlos syndrome and osteogenesis imperfecta, while pseudoxanthoma elasticum and cutis laxa constitute the major elastinopathies. In co-operation with other centres we were able to study the phenotypic and genotypic features of 13 patients with autosomal recessive cutis laxa type 2A (ARCL2A) that had been characterized as a metabolic disorder of N- and O- glycosylation shortly before [69]. In 2008, ARCL2A had been mapped to the ATP6V0A2 gene, the mutational spectrum of which we expanded by 14 novel mutations [70]. Functional studies in our patients also suggested that, besides the known glycosylation defect, alterations in trafficking and signalling processes are potential key events in the pathogenesis of ARCL2A Cutis laxa congenita Cutis laxa (CL) represents an elastinopathy characterized by loose, redundant, inelastic skin. In the congenital forms, 11 genes have been so far identified causing pleiotropic manifestations including aortic root dilatation, pulmonary emphysema, and gastrointestinal and bladder diverticula. The products of these genes are: (1) either proteins involved in the assembly of elastic fibers and upregulation of TGFbeta signalling e.g. ELN, FBLN4, FBLN 5, and LTBP4; (2) Golgi proteins that 50

51 Selected clinical cases and case studies facilitate secretion of elastic network components e.g. ATP6V0A2, RIN2, and GORAB; or (3) mitochondrial proteins e.g. PYCR1, ALDH18A1, and SLC2A10 [68]. CL congenita can be inherited as autosomal dominant, X-linked recessive and autosomal recessive with the latter one usually being more severe. The autosomal recessive CL (ARCL) has been initially divided into three subgroups based on the clinical findings (Table 9). In the past few years, seven ARCL-causative genes have been identified. Based on our own experience, we are going to consider as next the autosomal recessive ATP6V0A2-related cutis laxa, presently defined as type 2A. Table 9: Classification of autosomal recessive cutis laxa AR Cutis laxa Gene Specific clinical features ARCL-1 ARCL-2 ARCL-3 (De Barsy syndrome) FBLN4, FBLN5, LTPB4 ATP6V0A2, PYCR1 PYCR1, ALDH18A1 Severe cardiopulmonary lesions with arterial tortuosity and aneurysms; Pulmonary emphysema Delayed neuromotor development Facial dysmorphism Skeletal malformations Intellectual deficiency with athetosis Progeria Cataracts Short stature Geroderma osteodysplastica GORAB Osteoporosis Normal neuromotor development 51

52 Selected clinical cases and case studies Autosomal recessive cutis laxa, type 2A (MIM #219200) Own Publications: [71, 72] ARCL2A is clinically characterized by cutis laxa, generalized wrinkling, delayed closure of the anterior fontanelle and neuromotor delay. Other phenotypic features include facial features, developmental delay, proportionate short stature, congenital hip dislocation, and inguinal hernias. ARCL2A represents a clinical spectrum ranging from the mild wrinkly skin syndrome to the more severe Debre-type with developmental retardation, seizures and neurologic regression [73, 74, 75]. Today ca 60 patients with ARCL2A mutations have been reported. Loss-of-function mutations in the ATP6V0A2 gene, encoding the a2 subunit of a v- type H+ -ATPase, were originally described by Kornak et al All ARCL2Apatients appear to have a combined N- and O-glycosylation defect that seems to be specific for this subtype [73]. Patients: A 7-month old boy of Turkish origin presented with into big folds hanging, inelastic skin with generalized wrinkling since birth (Fig. 7a, b). The boy was born prematurely (35 weeks of pregnancy) and suffered from congenital inguinal hernias, transient feeding problems and hypospadia. He also had a mild ventricular septal defect. Clinical examination revealed mild neuromotor delay and muscle hypotonia, a large anterior fontanel and facial dysmorphism with downslanting palpebral fissures and a broad nasal root. Pulmonary, ocular and cardiovascular abnormalities were excluded. The healthy parents were consanguineous. Data and material from our patient were included in a multi-centre study together with the clinical and molecular features of 12 additional patients. In each tested individual or the parents as obligate heterozygous carriers, sequencing of all exons and the 52

53 Selected clinical cases and case studies flanking intron regions of the ATP6V02 gene was performed as described previously [70]. In addition, fibroblast cultures were examined to analyse the role of ATP6A2 in ARCL pathogenesis. The study was conducted in close collaboration with the Institute of Human Genetics, Charite Berlin. Results: In our patient, genetic testing revealed homozygosity for a novel mutation in the ATP6V0A2 gene, namely c.1425dupa (p.val476serfsx23). Both parents were heterozygous for the same mutation. With this study, the genotypic and phenotypic spectrum of ARCL2A was expanded by 13 new patients (Table 10). 14 novel mutations were described which mostly caused truncations and thus supported the hypothesis that ARCL2A is due to loss-offunction mutations. In fibroblast cultures from three CL patients, a localization of the mutated ATP6V0A2 protein at the Golgi-apparatus was demonstrated with the help of immunostaining against the N-terminal domain of ATP6V0A2. Furthermore, the loss-of-function effect was confirmed by loss of the mutated protein in dermal fibroblasts from these patients. Upon treatment with brefeldin A, ATP6VA02-deficient skin fibroblasts showed a delayed collapse of the Golgi compartment which confirmed previous observations by the same group [70] and suggested that, besides glycosylation defects, alterations in trafficking and signalling processes may contribute to ARCL2A pathogenesis. Finally, patients fibroblasts displayed elevated TGF-beta signalling. 53

54 Selected clinical cases and case studies Figure 7a, b: Clinical features of our patient with ARCL2A A 7-month old boy with inelastic, into big folds hanging skin (b) and generalized wrinkling (b) since birth. 54

55 Selected clinical cases and case studies Table 10: Clinical and molecular features of patients with ARCL2A in this study Clinical features Patient Consan- Mutation Origin (Hom/Het) Wrinkled skin Joint laxity Large fontanel Mental guinity retardation 1. Poland No c.708_713delcaaggt* c.2015t>a* Het New Zealand No c.117+1delg* Het India nd c deltggcgtc* Hom India nd c.430_432delcag* Hom Kuwait No c dupt* c.2432t>c* Het a + 6. Turkey Yes c.100_101insa* Hom b nd 7. UK 8. India 9. Turkey 10. Afghanis tan 11. Turkey 12. Turkey 13. Italy nd Yes Yes Yes nd Yes No c.600delc* c.1246g>a* Het c.16_17dupc* Hom c.187c>t Hom c.1a>t* Hom c.187c>t Hom c.1936_2055del Hom c g>a* c.2287c>t Het nd nd +++ nd nd * novel mutation; - absent; +- very mild; + mild; ++ moderate; +++ severe; a closure of fontanel at 26 months; b closure of fontanel at 48 months; nd not determined 55

56 Selected clinical cases and case studies 3.4. Cancer-associated genodermatoses Cutaneous signs may serve as markers for the early recognition of some hereditary cancer syndromes. In general, these so-called cancer-associated genodermatoses are characterized by: An autosomal-dominant mode of inheritance Development of cancer at an early age Multiplicity of cutaneous, and often extra-cutaneous, tumors The majority of cutaneous tumors are benign such as spiradenomas and cylindromas in Brooke-Spiegler syndrome (BSS) or cutaneous leiomyomas in hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC). However, malignant neoplasms can also occur as in our BSS patient (patient 2) or leiomyosarcoma in HLRCC [76, 77]. The majority of cancer-associated genodermatoses involve tumor suppressor genes and their tumorigenesis is largely explained by the so-called Knudson s two-hit hypothesis [78, 79]. According to this hypothesis, a tumor suppressor gene requires two hits for its inactivation and initiation of tumor formation. The first hit is represented by a germline mutation, whereas the second one is generated somatically, for example, through loss of heterozygosity or mutations leading to a change in nucleotide sequence. Thus, germline mutations cause cancer predisposition as they are already present in each cell. Thereafter, for the tumor progression, somatic mutations in the at-risk organs are required. Meanwhile, this sequential cancer gene hypothesis has been extended to sporadic tumors where specific driver-gene alterations occur in somatic tissue and underlie specific steps, namely a breakthrough phase, an expansion phase and finally, an 56

57 Selected clinical cases and case studies invasive phase [80]. In other words, although individuals with a hereditary cancer syndrome represent probably less than 5% of all cancer patients, identification of the genetic basis for their disease has great importance for both clinical management of the families (e.g. tumor detection at a pre-symptomatic stage and identification of relatives with strong hereditary predispositions) as well as understanding the molecular mechanisms of malignancy. Elucidation of the oncogenic pathway is relevant for evolving new diagnostic and therapeutic options. In order to illustrate these concepts, BSS and HLRCC are going to be discussed based on our own experience Brooke-Spiegler syndrome (MIM #605041) Own publication: [17] Brooke-Spiegler syndrome (BSS) is a rare autosomal-dominant genetic disorder characterized by multiple adnexal tumors, including cylindromas, spiradenomas, spiradenocylindromas and trichoepitheliomas (cribriform trichoblastomas). The tumors are mostly localized in the head and neck area and may undergo a malignant transformation in 5 to 10% of cases [81, 82, 83]. In addition, BSS patients may very rarely develop tumors of the major (parotid) and minor salivary glands, and exceptionally in the breast [84, 85, 86, 87]. Based on their common genetic background, familial cylindromatosis and multiple familial trichoepitheliomas (MFT) are nowadays considered phenotypic variants of BSS: one patient may develop various types of tumors and/ or different phenotypes may occur in the same family [88, 89, 90, 91, 92, 93, 94, 95, 96]. All three conditions are caused by germline mutations in the same CYLD gene which represents a tumor suppressor gene located on chromosome 16q [97, 98, 99, 100]. CYLD contains 20 57

58 Selected clinical cases and case studies exons and encodes an ubiquitin-specific hydroxylase that negatively regulates NFkappa B and JNK pathways at several steps including TRAF2, TRAF6, TAK1 and BCL3 [101, 102]. Recent studies demonstrated an activation of the MYB protooncogene in both sporadic and inherited cylindromas although hereditary cylindromas did not harbor MYB-NFIB gene fusions as seen in sporadic cylindromas. This observation was in line with the hypothesis that MYB as a NF-kB target gene, having NF-kB binding sites within its promoter, might be activated as a consequence of increased NF-kB signaling due to loss of CYLD function [103]. Importantly, MYB might become an attractive therapeutic target for patients with both hereditary and sporadic cylindromas [103]. Patients: Patient 1, a 29-year-old woman, presented with a few papules on her face, favoring the nasolabial folds and eyelids (Fig. 8a). She started developing them at the age of 13. Shave biopsy of two papules revealed cribriform trichoblastoma (trichoepithelioma) in each of the specimens (Fig. 8 b). Family pedigree showed an autosomal dominant mode of inheritance (Fig. 8c). Figure 8 a-c: BSS, MFTtype Multiple skin-colored papules favoring the nasolabial folds of patient 1 (a); Cribriform trichoblastoma (trichoepithelioma) with basaloid cell aggregates resembling follicular germ structures surrounded by specific follicular stroma (b); Family pedigree of patient 1(c). 58

59 Selected clinical cases and case studies Patient 2, a 60-year-old man, had multiple partly confluent tumors on his scalp (Fig.9 a) that first appeared at the age of 40. Some of the tumors grew faster in the last two years, with the largest one (right temporal area) reaching the size of 5x3x2.5cm. Two out of four specimens revealed malignant transformation of a preexisting spiradenocylindroma featuring p53-positive nests and sheets of medium-sized pleomorphic basaloid cells with an infiltrative growth pattern and many mitoses (Fig. 9 b-c). The other two specimens showed stereotypical features of a cylindroma, a spiradenoma and a spiradenocylindroma (Fig. 10 a-d). The mother of the patient suffered from similar tumors on her scalp. Fig. 9a-d: BSS, familial cylindromatosis Multiple variably sized red to bluish confluent nodules on the scalp of patient 2 (a); Malignant tumor evolving from a preexisting spiradenocylindroma with infiltrative nodules composed of pleomorphic medium-sized to large basaloid cells with plentiful mitoses and necrotic cells (b); Strong p53 expression in the malignant part of the tumor (c). 59

60 Selected clinical cases and case studies Figure 10 a-d: Stereotypical features of a spiradenocylindroma (Patient 2) Spiradenocylindroma composed of small nodules arranged in jigsaw pattern (left) corresponding to cylindromatous moiety in close proximity to large nodular aggregates representing spiradenomatous portion (right) (a); Close-up of the cylindromatous part: note conspicuous droplets of hyaline basement membrane material (b); Transitional area with cylindromatous jigsaw pattern but numerous intratumoral lymphocytes, an essential component of a spiradenoma (c); Area with sharp demarcation of spiradenomatous (upper right) and cylindromatous (lower left) components (d). 60

61 Selected clinical cases and case studies Patient 3, a 76-year-old woman, presented with multiple skin tumors on her face and scalp, which appeared at the age of 17. Two biopsies were taken, which revealed a spiradenocylindroma and cribriform trichoblastoma (trichoepithelioma) next to an incipient spiradenocylindroma. Additional family members were affected in an autosomal-dominant fashion. After obtaining the patients informed consent, analysis of germline CYLD mutations was performed as described previously [104]. Results: The main clinical data, histopathological and molecular findings are summarized in Table 11. Our patients had a variable phenotypic expression ranging from MFT-phenotype to familial cylindromatosis. Additionally, in patient 2, malignant transformation of spiradenocylindroma has occurred, and the tumor was classified as a salivary gland type basal cell adenocarcinoma-like pattern, high-grade (BCAC-HG). In a series of 24 malignant tumors evolving from preexisting benign spiradenoma, cylindroma and spiradenocylindroma, both sporadic and associated with BSS, this was the second most common type of a malignant pattern after low-grade salivary gland-type BCAC [105]. In the same study, a correlation between the histologic pattern and clinical course was found in the sense that in patients with BCAC-HG, a more aggressive course is to be expected. The malignant portion in our tumor was strikingly positive for p53, as is sometimes seen in these lesions [106]. Interestingly, both sporadic cylindromas and adenoid cystic carcinomas of the salivary glands express the aberrant MYB-NFIB fusion transcript, which might explain their common histologic features [107]. Furthermore, BSS patients with multiple cylindromas may also rarely develop tumors of the salivary gland [84, 85, 87]. 61

62 Selected clinical cases and case studies Three novel CYLD mutations in three unrelated patients were found and proteins were in-silico predicted. These included c.2666a T, c.2712delt and c.1821_ delinsCT. Prediction of protein functionality by PolyPhen-2 and by PROVEAN suggested a pathogenic role of the missense mutation c.1666a>t ( probably damaging (score 0.999) and deleterious, respectively). So far, genotype-phenotype correlations have not been established for BSS [108]. By extending the spectrum of CYLD mutations, better understanding of the molecular mechanisms can be obtained which, in future, may help to develop prognostic criteria and new treatment options. Table 11: Clinical, histomorphologic and molecular data of BSS patient in this study Case Sex/ Age Ethnicity Clinical Features Available Histology CYLD Mutation 1. F/ 29 Swiss Multiple paranasal papules 2 tricho- epitheliomas c.2666a T 1 BCAC-HG evolving from Multiple confluent scalp tumors, M/ spiradenocylindroma; 2. Czech the biggest one measuring 5 x cm, with recent rapid growth 1 spiradenoma; 1 cylindroma 2 spiradenocylindromas; F/ Multiple tumors on the face and 3. Slovak 76 scalp 1 trichoepithelioma BCAC-HG: salivary gland type basal cell adenocarcinoma-like pattern, high-grade c.2712delt c.1821_ delinsCT 62

63 Selected clinical cases and case studies Hereditary leiomyomatosis and renal cell cancer syndrome (MIM #150800) Submitted Hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome is a rare autosomal-dominant tumor predisposition syndrome characterized by multiple cutaneous and uterine leiomyomas occurring at an early age. Cutaneous leiomyomas are present in nearly all patients whereas uterine leiomyomas are found in up to 70% of affected females. Ca. 15% of patients develop renal cell cancer (RCC) that is particularly aggressive with early metastasis despite a small primary tumor size [109]. To date more than 200 families have been reported. Very rarely leiomyosarcomas, Leydig cell tumors, ovarian cystadenomas, gastrointestinal stromal tumours (GISTs) and adrenal gland tumors may occur [110]. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (FH) gene (1q ) [111] which acts as a tumor suppressor gene and catalyzes the conversion of fumarate into malate as part of the Krebs cycle. Patient: A 43-year-old patient of African origin presented with three painful cutaneous nodules on the back and the right gluteal area, the biggest one measuring 1cm (Fig.11a,). A leiomyoma on the left upper arm had been excised two years before. The patient had started developing the cutaneous lesions at the age of 30 years. Excisional biopsy of the tumors revealed well-circumscribed dermal nodules composed of SMA- and desmin-positive spindle cells with monomorphous cigarshaped nuclei and no mitoses (Fig.11b). One excised tumor was stained for FH and showed negative immunostaining (Fig.11c). 63

64 Selected clinical cases and case studies The family history indicated that the mother had undergone a hysterectomy at an early age. As HLRCC was suspected, a germline FH analysis was performed after having obtained the patient s informed consent. A novel heterozygous nonsense mutation (c.298a>t, p.lys100) in exon 3 was disclosed (Fig.11e). Computer tomography (CT) and magnetic resonance imaging (MRI) showed a cystic renal tumor in the upper pole of the left kidney with a diameter of approximately 1 cm. Histopathology revealed a strongly HMB45-positive fat-poor angiomyolipoma. In contrast to the FH-negative cutaneous leiomyomas, the renal angiomyolipoma showed strong intact FH expression (fig 11d). FH-negativity combined with anti-s-(2-succino)-cysteine immunostaining was shown to be a helpful tool for distinguishing HLRCC-related leiomyomas from sporadic leiomyomas [112, 113]. Early detection of the hereditary forms would allow the patients early involvement in screening programs before manifestation of the highly aggressive renal tumors. In addition, a novel nonsense mutation was found in our patient. 64

65 Selected clinical cases and case studies Figure 11 a-e: Clinical, histomorphologic and molecular findings in our patient with HLRCC A firm nodule on the right gluteal area of our patient histologically consistent with a leiomyoma (a); HE staining showing fascicles of spindle cells with monomorphous cigar-shaped nuclei and no mitoses (b); Negative FH-immunostaining of a leiomyoma compared to retained expression in normal vessels and stromal cells (c); The renal angiomyolipoma showed strong intact FH expression (d); Chromatograms of mutational analysis in FH exon 3. Heterozygous mutation (black arrows) pointing to nonsense mutation c.298a>t (CAG TAG), p.lys100*. A: Forward direction B: Reverse direction (e). 65

66 Selected clinical cases and case studies 3.5. Nevi as manifestations of somatic (postzygotic) mosaicism A mosaic is defined as an organism composed of at least two genetically different populations of cells that originate from a genetically homogenous zygote [27]. In this way, the skin provides a unique opportunity to study mosaicism, which clinically presents with congenital nevi on the skin. In fact, any individual with a single small congenital nevus is mosaic, and all mammalian organisms will develop during their lifetime various forms of genomic mosaicism, for example due to postzygotic (somatic) new mutations [114, 115]. In real life, however, clinicians delineate with the term mosaicim the distribution of cutaneous findings along Blaschko s lines or in a block-like pattern. The cell type affected by the mutation is central to the clinical phenotype, and nevi might be classified depending on their predominant composition as pigmentary, epidermal, vascular, connective tissue and fatty tissue [114] (Table 12). Table 12: Classification of congenital nevi based on clinical and histologic criteria Group of Nevi Pigmentary Nevi Epidermal (epithelial) Nevi Predominant histologic composition/ Origin Due to abnormal number of melanocytes Due to abnormal amount of melanin Keratinocytic nevi involving the interfollicular epidermis Organoid nevi involving the adnexal structures of the skin Example Congenital melanocytic nevus Linear hypo- and hypermelanosis Common (nonepidermolytic) type ; Epidermolytic type Nevus sebaceus Nevus comedonicus Nevus trichilemmocysticus Vascular Capillaries Nevus flammeus Connective Tissue Nevi Fatty Tissue Nevi Collagen nevi Elastin-rich nevi Subcutis Collagenoma of tuberous sclerosis Elastoma of Buschke-Ollendorff syndrome Nevus lipomatosis superficialis Nevus psiloliparus 66

67 Selected clinical cases and case studies Congenital nevi may occur as isolated lesions or as a part of a syndrome, and most of them are caused by mutations of a gene that are compatible with survival only as mosaicism. In these instances, the skin findings provide not only a clue to the diagnosis, but also an accessible source of tissue for molecular analysis as in our patients with nevus psiloliparus. In these patients, molecular diagnostics conducted on tissue samples allowed for the approximation of the oculo-ectodermal syndrome (OES) and encephalocranio-cutaneous lipomatosis (ECCL), which were initially considered as distinct entities. In addition, we identified nevus trichilemmocysticus as a new sub-type in the group of organoid epidermal nevi based on the correlation of clinical and histologic criteria Fatty tissue nevi Fatty tissue nevi involve the subcutaneous fat and include two different types: nevus psiloliparus and nevus lipomatosus superficialis Nevus psiloliparus: a phenotypic link between oculo-ectodermal syndrome and encephalocraniocutaneous lipomatosis (MIM #613001) Own publication: [116] Nevus psiloliparus is a distinct fatty tissue nevus which manifests as a smoothly surfaced, slightly yellowish and hairless lesion involving the scalp. Histologically, it is characterized by an abundant amount of fatty tissue, absence or paucity of hair follicles and isolated erector pili muscles arranged in an Indian file [117]. It is regarded as the dermatological hallmark of encephalocraniocutaneous lipomatosis (ECCL) although it may also occur as an isolated lesion [118, 119, 120, 121]. The 67

68 Selected clinical cases and case studies etiological concept of a lethal mutation surviving by mosaicism has been postulated by Happle [122]. ECCL (MIM #613001) is a very rare (ca. 60 cases to date), sporadically occurring neurocutaneous disorder characterized by skin (nevus psiloliparus, non-scarring and scarring alopecia, subcutaneous fatty masses in the craniofacial region, skin tags), neurologic (intracranial and spinal lipomas, arachnoid and porencephalic cysts, cerebral asymmetry, dilated ventricles and calcifications) and ophthalmologic (choristomas) abnormalities [123]. Associated manifestations include skeletal defects (osteomas, odontomas, ossifying fibromas, multiple lytic lesions) and congenital heart defects, in particular aortic coarctation. ECCL shares many common features with oculo-ectodermal syndrome (OES) which is a very rare disorder with ca. 20 cases reported so far [123, 124]. Common manifestations include aplasia cutis congenita / focal alopecia, epibulbar dermoids, skin tags of the upper eyelids, and linear hyperpigmentation. It therefore has been assumed for a long time that OES and ECCL belong to the same spectrum of disorders with OES being the mild phenotype. In both, OES and ECCL, exclusively sporadic occurrence has been observed. Together with the obvious mosaic pattern of skin involvement, genetic mosaicism with postzygotic mutations in a gene that would be lethal as germline mutations has been assumed [123, 124]. Recently, Peacock et al., identified mutations in the KRAS (V-Ki-Ras2 Kirsten rat sarcoma viral oncogene homolog) gene in affected tissues from two patients with OES, thus suggesting that OES is a mosaic RASopathy [125]. Patients: Patient 1 was the first child of healthy non-consanguineous parents of German origin. At birth, small skin tags of the right upper eyelid and the right 68

69 Selected clinical cases and case studies eyebrow as well as an epibulbar dermoid on the right conjunctiva and cornea were noted (Fig. 12 a). At clinical genetic evaluation the boy presented as an active, friendly boy with normal psychomotor development. He had a triangular face with prominent zygomatic arches and mild frontal bossing, a frontal upsweep, as well as deeply set eyes with a relatively narrow intercanthal distance. He had a hairless patch with immovable, yellowish, leathery skin on his right fronto-parietal scalp, which measured approximately 8 x 4 cm (Fig. 12b). Due to the presentation of the three cardinal features: fibroma of the eyelids / eyebrows, epibulbar dermoid and the hairless scalp lesion in a boy with normal psychomotor development, a clinical diagnosis of OES was made. Figure 12 a-c: Clinical and histomorphologic findings in our patient with OES Right epibulbar dermoid and a hairless yellowish patch on the scalp (a, b); Histology from the scalp consistent with N. psiloliparus, namely the presence of isolated arrector pili muscles arranged in a line parallel to the surface (c). 69

70 Selected clinical cases and case studies The data of patient 2 and patient 3 are briefly presented in Table 13 as these patients came from other centres. For the clinical diagnosis of ECCL in patient 2, diagnostic criteria published by Moog 2009 were applied. Table 13: Clinical and molecular data of patients with OES/ ECCL in this study Case Ethnicity Sex/ Age Clinical Features Clinical Diagnosis Material examined for DNA analysis KRAS Mutation* 1. German M/ 5 N. psiloliparus Skin tags of the right upper eyelid and eyebrow Epibulbar dermoid on the right ocular surface OES Epibulbar dermoid Blood c.437c>t (p.ala146val) Bilateral epibulbar dermoids 2. Danish M/ 5 Hemiatrophy of left cerebral hemisphere and ventriculomegaly Aortic coarctation Cystic bone lesions Focal alopecia ECCL Biopsy from a scalp lesion Blood c.436g>a (p.ala146thr) Linear pigmentary changes on the neck and both arms 3. Slovak M/ 3.5 ACC of the scalp Unilateral epibulbar dermoid Pigmentary changes following Blaschko lines Giant cell granulomas of the mandible Non-ossifying fibromas of the humerus and clavicle OES Fibroblasts from a skin biopsy Blood Paraffin embedded bone tumor tissue c.437c>t (p.ala146val) Multiple alopecic lesions on the scalp 4. Turkish M/ 3 Bilateral epibulbar dermoids Pigmentary changes following Blaschko lines Intellectual disability, ADHS OES/ ECCL Biopsy from the scalp and hyperpigmented lesion c.436g>a (p.ala146thr) Ventriculomegaly and arachnoidal cysts Blood Subcutaneous masses within the scalp Tissue in which the KRAS mutations were identified 70

71 Selected clinical cases and case studies Methods: In patient 1, a blood sample and a native tissue sample from the resected epibulbar dermoid were available for DNA extraction and genetic analysis. In addition, routine histology was performed on a sample from the scalp lesion (Fig. 12c). In patients 2, 4 and 4, blood and tissue samples were examined for DNA analysis as shown in Table 13. Genomic DNA was isolated from peripheral leukocytes and tissue samples according to standard procedures. All coding exons with flanking introns of KRAS gene (NM_004985) were amplified by PCR and bidirectional Sanger sequencing was performed using Big Dye Terminator Cycle Sequencing Kit and a 3500xl Genetic Analyzer (Applied Biosystems, Foster City, Calif., USA). Sequences were aligned using the Seqpilot analysis software (JSI medical systems, Kippenheim, Germany) and compared with the reference sequences (KRASB; ENST ). Mosaic level for mutations were estimated by comparing the area under the curve of electropherograms for the wildtype and mutant peaks in the forward and reverse sequencing directions using the Seqpilot software. The study was conducted in close collaboration with our Department of Ophthalmology and Institute of Human Genetics as well as the Institute of Human Genetics, University Hospital Magdeburg, Otto-von-Guericke University Magdeburg. Results: Clinical and molecular data of our patients are presented in Table 13. In lesional tissues from all four patients KRAS mutations at various levels of mosaicism were identified, while the mutations were not detected in leukocytederived DNA at the detection threshold of Sanger sequencing. The observed 71

72 Selected clinical cases and case studies mutations affected exon 4, codon 146, and we defined codon 146 of KRAS as hotspot for mutations related to OES/ ECCL. Furthermore, these findings corroborate that OES consistently results from a mosaic status for specific KRAS mutations and confirm for the first time the common genetic etiology of OES and ECCL. Interestingly, the scalp lesion in patient 1 revealed clinical (a smooth, slightly yellowish and hairless scalp lesion) and histological (arrangement of isolated arrector pili muscles in a line parallel to the surface) features consistent with nevus psiloliparus which was initially considered as a cutaneous hallmark of ECCL (Fig. 12c). Thus, the common etiology of OES and ECCL was also shown on histo-morphologic grounds Epidermal nevi Epidermal nevi are divided into two large categories [126]: True epidermal nevi (keratinocytic nevi) that are due to abnormalities in the interfollicular epidermis Organoid nevi that involve the adnexal structures of the skin e.g. sebaceous nevus and nevus comedonicus. A syndromic manifestation has been reported for the different types of epidermal nevi most commonly affecting the central nervous system, the skeletal and ocular systems [127, 128]. 72

73 Selected clinical cases and case studies Nevus trichilemmocysticus Own publication: [129] Nevus trichilemmocysticus represents an organoid epidermal nevus, characterized by multiple trichilemmal cysts arranged along the Blaschko s lines which are usually intermingled with filiform hyperkeratosis and comedo-like plugs. This case is the first report of Nevus tricholemmocysticus. Retrospectively, we could identify four similar cases in the literature [130,131,132] and re-categorized them as nevus trichilemmocysticus. Three additional cases [133,134,135] have been recently described. Patient: A 31-year-old Macedonian woman complained of hyperkeratotic skin lesions since birth that showed a linear arrangement on her face and the right side of her body including the palm and the sole. At the age of 3 years multiple filiform hyperkeratoses began to emerge followed by the first lumps on her chest two years later. The lumps increased with age in number and size; some of them had to be excised because of inflammation and fistula formation. Her family history was noncontributory. Furthermore, our patient suffered from arthritis in her childhood and adolescence. In the last years she had slowly lost weight and at the time of presentation in our hospital she weighed 40 kg. Since the age of 26 years the patient was bound to a wheelchair due to severe arthrosis of the hips and bowing of the legs. Severe osteomalacia with multiple fractures was found. She also suffered from chronic headache. 73

74 Selected clinical cases and case studies Physical examination revealed multiple cystic lesions involving both sides of her face and scalp and the right side of her body. The cysts were arranged in a band-like distribution, following a fountain-like pattern characteristic of the lines of Blaschko. In addition, the involved streaks were covered by multiple filiform hyperkeratoses and comedo-like plugs (Fig. 13 a-c). Fig. 13 a-d: Clinical and histomorphologic findings in our patient with nevus trichilemmocysticus Multiple cystic tumors showing systematized arrangement along the Blaschko lines (a,b); The affected areas were in addition covered with filiform hyperkeratoses (simple arrow) and comedo-like lesions (double arrow) (c); Trichilemmal cysts containing homogenous horny material (d). 74