Maurice A.M. van Steensel

Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema

We recently reported two common filaggrin (FLG) null mutations that cause ichthyosis vulgaris and predispose to eczema and secondary allergic diseases. We show here that these common European mutations are ancestral variants carried on conserved haplotypes. To facilitate comprehensive analysis of other populations, we report a strategy for full sequencing of this large, highly repetitive gene, and we describe 15 variants, including seven that are prevalent. All the variants are either nonsense or frameshift mutations that, in representative cases, resulted in loss of filaggrin production in the epidermis. In an Irish case-control study, the five most common European mutations showed a strong association with moderate-to-severe childhood eczema (χ2 test: P = 2.12 × 10−51; Fishers exact test: heterozygote odds ratio (OR) = 7.44 (95% confidence interval (c.i.) = 4.9–11.3), and homozygote OR = 151 (95% c.i. = 20–1,136)). We found three additional rare null mutations in this case series, suggesting that the genetic architecture of filaggrin-related atopic dermatitis consists of both prevalent and rare risk alleles.

Mutations in the transcription factor gene SOX18 underlie recessive and dominant forms of hypotrichosis-lymphedema-telangiectasia.

Hereditary lymphedema is a developmental disorder characterized by chronic swelling of the extremities due to dysfunction of the lymphatic vessels. Two responsible genes have been identified: the vascular endothelial growth factor receptor 3 (VEGFR3) gene, implicated in congenital lymphedema, or Milroy disease, and the forkhead-related transcription factor gene FOXC2, causing lymphedema-distichiasis. We describe three families with an unusual association of hypotrichosis, lymphedema, and telangiectasia. Using microsatellite analysis, we first excluded both VEGFR3 and FOXC2 as causative genes; we then considered the murine ragged phenotype, caused by mutations in the Sox18 transcription factor, as a likely counterpart to the human disease, because it presents a combination of hair and cardiovascular anomalies, including symptoms of lymphatic dysfunction. Two of the families were consanguineous; in affected members of these families, we identified homozygous missense mutations in the SOX18 gene, located in 20q13. The two amino acid substitutions, W95R and A104P, affect conserved residues in the first alpha helix of the DNA-binding domain of the transcription factor. In the third family, the parents were nonconsanguineous, and both the affected child and his brother, who died in utero with hydrops fetalis, showed a heterozygous nonsense mutation that truncates the SOX18 protein in its transactivation domain; this substitution was not found in genomic DNA from either parent and hence constitutes a de novo germline mutation. Thus, we show that SOX18 mutations in humans cause both recessive and dominant hypotrichosis-lymphedema-telangiectasia, suggesting that, in addition to its established role in hair and blood vessel development, the SOX18 transcription factor plays a role in the development and/or maintenance of lymphatic vessels.

Birt-Hogg-Dubé syndrome: diagnosis and management

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant condition characterised clinically by skin fibrofolliculomas, pulmonary cysts, spontaneous pneumothorax, and renal cancer. The condition is caused by germline mutations in the FLCN gene, which encodes folliculin; the function of this protein is largely unknown, although FLCN has been linked to the mTOR pathway. The availability of DNA-based diagnosis has allowed insight into the great variation in expression of FLCN, both within and between families. Patients can present with skin signs and also with pneumothorax or renal cancer. Preventive measures are aimed mainly at early diagnosis and treatment of renal cancer. This Review gives an overview of current diagnosis and management of BHD.

Loss-of-function mutations of an inhibitory upstream ORF in the human hairless transcript cause Marie Unna hereditary hypotrichosis

Marie Unna hereditary hypotrichosis (MUHH) is an autosomal dominant form of genetic hair loss. In a large Chinese family carrying MUHH, we identified a pathogenic initiation codon mutation in U2HR, an inhibitory upstream ORF in the 5′ UTR of the gene encoding the human hairless homolog (HR). U2HR is predicted to encode a 34–amino acid peptide that is highly conserved among mammals. In 18 more families from different ancestral groups, we identified a range of defects in U2HR, including loss of initiation, delayed termination codon and nonsense and missense mutations. Functional analysis showed that these classes of mutations all resulted in increased translation of the main HR physiological ORF. Our results establish the link between MUHH and U2HR, show that fine-tuning of HR protein levels is important in control of hair growth, and identify a potential mechanism for preventing hair loss or promoting hair removal.

Familial Pityriasis Rubra Pilaris Is Caused by Mutations in CARD14

Pityriasis rubra pilaris (PRP) is a papulosquamous disorder phenotypically related to psoriasis. The disease has been occasionally shown to be inherited in an autosomal-dominant fashion. To identify the genetic cause of familial PRP, we ascertained four unrelated families affected by autosomal-dominant PRP. We initially mapped PRP to 17q25.3, a region overlapping with psoriasis susceptibility locus 2 (PSORS2 [MIM 602723]). Using a combination of linkage analysis followed by targeted whole-exome sequencing and candidate-gene screening, we identified three different heterozygous mutations in CARD14, which encodes caspase recruitment domain family, member 14. CARD14 was found to be specifically expressed in the skin. CARD14 is a known activator of nuclear factor kappa B signaling, which has been implicated in inflammatory disorders. Accordingly, CARD14 levels were increased, and p65 was found to be activated in the skin of PRP-affected individuals. The present data demonstrate that autosomal-dominant PRP is allelic to familial psoriasis, which was recently shown to also be caused by mutations in CARD14.

Repetitive disruptions of the nuclear envelope invoke temporary loss of cellular compartmentalization in laminopathies.

The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C. These ruptures, which were absent in normal fibroblasts, could be mimicked by selective knockdown as well as knockout of LMNA and were accompanied by the loss of cellular compartmentalization. This was demonstrated by the influx of cytoplasmic transcription factor RelA and regulatory protein Cyclin B1 into the nucleus, and efflux of nuclear transcription factor OCT1 and nuclear structures containing the promyelocytic leukemia (PML) tumour suppressor protein to the cytoplasm. While recovery of enhanced yellow fluorescent protein-tagged nuclear localization signal in the nucleus demonstrated restoration of nuclear membrane integrity, part of the mobile PML structures became permanently translocated to the cytoplasm. These satellite PML structures were devoid of the typical PML body components, such as DAXX, SP100 or SUMO1. Our data suggest that nuclear rupture and loss of compartmentalization may add to cellular dysfunction and disease development in various laminopathies.

A Homozygous Missense Mutation in TGM5 Abolishes Epidermal Transglutaminase 5 Activity and Causes Acral Peeling Skin Syndrome

Peeling skin syndrome is an autosomal recessive genodermatosis characterized by the shedding of the outer epidermis. In the acral form, the dorsa of the hands and feet are predominantly affected. Ultrastructural analysis has revealed tissue separation at the junction between the granular cells and the stratum corneum in the outer epidermis. Genomewide linkage analysis in a consanguineous Dutch kindred mapped the gene to 15q15.2 in the interval between markers D15S1040 and D15S1016. Two homozygous missense mutations, T109M and G113C, were found in TGM5, which encodes transglutaminase 5 (TG5), in all affected persons in two unrelated families. The mutation was present on the same haplotype in both kindreds, indicating a probable ancestral mutation. TG5 is strongly expressed in the epidermal granular cells, where it cross-links a variety of structural proteins in the terminal differentiation of the epidermis to form the cornified cell envelope. An established, in vitro, biochemical cross-linking assay revealed that, although T109M is not pathogenic, G113C completely abolishes TG5 activity. Three-dimensional modeling of TG5 showed that G113C lies close to the catalytic domain, and, furthermore, that this glycine residue is conserved in all known transglutaminases, which is consistent with pathogenicity. Other families with more-widespread peeling skin phenotypes lacked TGM5 mutations. This study identifies the first causative gene in this heterogeneous group of skin disorders and demonstrates that the protein cross-linking function performed by TG5 is vital for maintaining cell-cell adhesion between the outermost layers of the epidermis.

Connexin mimetic peptides improve cell migration rates of human epidermal keratinocytes and dermal fibroblasts in vitro

Nonhealing cutaneous wounds, a major cause of morbidity and mortality, are difficult to treat. Recent studies suggest that significant increases in skin wound‐healing rates occur by altering gap junction intercellular communication (GJIC). As migration of keratinocytes and fibroblasts is an important feature of wound healing, this study investigated whether migration rates in cultured normal human epidermal keratinocytes and dermal fibroblasts could be altered by modulating GJIC via connexin mimetic peptides. First, HeLa cells stably transfected with connexin43 (Cx43), Cx40, or Cx26 were used as a model to determine connexin specificity and the doses of connexin mimetic peptides required to attenuate GJIC. Gap26 and Gap26M inhibited GJIC dose dependently and were nonconnexin specific, whereas Gap27 was Cx43‐selective. Skin keratinocytes and fibroblasts expressed a variety of connexins, with Cx43 predominating. Cx43 protein expression was reduced at leading edges 3 hours after scraping confluent monolayers, resolving at 24 hours. Gap26M and Gap27 significantly increased migration rates across scrapes in keratinocytes and fibroblasts by blocking gap junction functionality. GJIC inhibition can thus directly influence keratinocyte and fibroblast migration. Furthermore, our results support the therapeutic potential of connexin mimetic peptides to aid wound closure, and provide a simple approach to screening new agents.

A New Locus-Specific Database (LSDB) for Mutations in the Folliculin (FLCN) Gene

Birt‐Hogg‐Dubé syndrome (BHD) is an autosomal dominant condition characterised by the presence of facial fibrofolliculomas, pulmonary cysts which may be associated with spontaneous pneumothorax and renal tumours. Germline mutations in the gene Folliculin (FLCN) were first identified in BHD patients in 2002. In addition FLCN mutations have also been described in families with isolated primary spontaneous pneumothorax (PSP) and also familial clear cell renal carcinomas (FcRCC). We have established a locus‐specific database based on the Leiden Open (source) Variation Database (LOVD) software. The version of the database contains 60 previously published mutations and 10 previously unpublished novel germline FLCN mutations. The mutations are comprised of deletions (44.3%), substitutions (35.7%), duplications (14.3%) and deletion/insertions (5.7%). The database is accessible online at http://www.lovd.nl/flcn.

The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPK-dependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.