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1.  Birt–Hogg–Dubé syndrome is a novel ciliopathy 
Human Molecular Genetics  2013;22(21):4383-4397.
Birt–Hogg–Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.
PMCID: PMC3792695  PMID: 23784378
2.  Report of the tenth annual International Pachyonychia Congenita Consortium meeting 
The International Pachyonychia Congenita Consortium (IPCC) was founded in 2004 in Park City, Utah, USA. Its goal is to find a cure for pachyonychia congenita, a rare keratinizing disorder. From February 14th–17th, 2013, the group convened in Park City for their tenth annual meeting. The 2013 meeting focused on how to best move forward with clinical trials and on learning from work in other scientific areas, with an emphasis on understanding mechanisms of pain and hyperkeratosis. Considerable time was spent on discussing the best way to move forward with development of new treatments and how to obtain or develop tools that can measure treatment outcomes in PC.
PMCID: PMC3930927  PMID: 24518109
Pachyonychia congenita; keratin; palmoplantar keratoderma; nail; pain
3.  The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation 
The Journal of Clinical Investigation  2014;124(6):2640-2650.
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.
PMCID: PMC4038567  PMID: 24762438
4.  Expression and knockdown of zebrafish folliculin suggests requirement for embryonic brain morphogenesis 
Birt-Hogg-Dubé syndrome (BHD) is a dominantly inherited familial cancer syndrome characterised by the development of benign skin fibrofolliculomas, multiple lung and kidney cysts, spontaneous pneumothorax and susceptibility to renal cell carcinoma. BHD is caused by mutations in the gene encoding Folliculin (FLCN). Little is known about what FLCN does in a healthy individual and how best to treat those with BHD. As a first approach to developing a vertebrate model for BHD we aimed to identify the temporal and spatial expression of flcn transcripts in the developing zebrafish embryo. To gain insights into the function of flcn in a whole organism system we generated a loss of function model of flcn by the use of morpholino knockdown in zebrafish.
flcn is expressed broadly and upregulated in the fin bud, somites, eye and proliferative regions of the brain of the Long-pec stage zebrafish embryos. Together with knockdown phenotypes, expression analysis suggest involvement of flcn in zebrafish embryonic brain development. We have utilised the zFucci system, an in vivo, whole organism cell cycle assay to study the potential role of flcn in brain development. We found that at the 18 somite stage there was a significant drop in cells in the S-M phase of the cell cycle in flcn morpholino injected embryos with a corresponding increase of cells in the G1 phase. This was particularly evident in the brain, retina and somites of the embryo. Timelapse analysis of the head region of flcn morpholino injected and mismatch control embryos shows the temporal dynamics of cell cycle misregulation during development.
In conclusion we show that zebrafish flcn is expressed in a non-uniform manner and is likely required for the maintenance of correct cell cycle regulation during embryonic development. We demonstrate the utilisation of the zFucci system in testing the role of flcn in cell proliferation and suggest a function for flcn in regulating cell proliferation in vertebrate embryonic brain development.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-016-0119-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4939010  PMID: 27391801
Birt-Hogg-Dubé syndrome (BHD); Zebrafish; flcn; Folliculin; zFucci; Cell cycle; Brain
5.  Neurocutaneous Manifestations of Genetic Mosaicism 
Journal of Pediatric Genetics  2015;4(3):144-153.
Genetic mosaicism is defined as the presence of two or more genetically distinct cell populations in a single individual. Ever more disorders are found to be manifestations of mosaicism and together constitute a significant proportion of the morbidity confronting pediatric specialists. An emerging category is that of overgrowth syndromes with skin manifestations and neurological or developmental abnormalities, such as the well-known Proteus syndrome. In recent years, we have seen dramatic advances in our understanding of these disorders and we now know the genetic basis of many of them. This has profound consequences for diagnosis, counselling, and even treatment, with therapies targeted to specific pathways becoming available for clinical use. Recognizing such overgrowth syndromes, therefore, is more important than ever. Fortunately, their skin manifestations can provide important diagnostic clues when evaluated in the entire phenotypic context. In this review, I provide an overview of the most frequently seen mosaic neurocutaneous phenotypes and discuss their molecular basis.
PMCID: PMC4918718  PMID: 27617125
mosaicism; skin; brain; overgrowth; intellectual disability
6.  Immunohistochemical Analysis of the Mechanistic Target of Rapamycin and Hypoxia Signalling Pathways in Basal Cell Carcinoma and Trichoepithelioma 
PLoS ONE  2014;9(9):e106427.
Basal cell carcinoma (BCC) is the most common cancer in Caucasians. Trichoepithelioma (TE) is a benign neoplasm that strongly resembles BCC. Both are hair follicle (HF) tumours. HFs are hypoxic microenvironments, therefore we hypothesized that hypoxia-induced signalling pathways could be involved in BCC and TE as they are in other human malignancies. Hypoxia-inducible factor 1 (HIF1) and mechanistic/mammalian target of rapamycin (mTOR) are key players in these pathways.
To determine whether HIF1/mTOR signalling is involved in BCC and TE.
We used immunohistochemical staining of formalin-fixed paraffin-embedded BCC (n = 45) and TE (n = 35) samples to assess activity of HIF1, mTORC1 and their most important target genes. The percentage positive tumour cells was assessed manually in a semi-quantitative manner and categorized (0%, <30%, 30–80% and >80%).
Among 45 BCC and 35 TE examined, expression levels were respectively 81% and 57% (BNIP3), 73% and 75% (CAIX), 79% and 86% (GLUT1), 50% and 19% (HIF1α), 89% and 88% (pAKT), 55% and 61% (pS6), 15% and 25% (pMTOR), 44% and 63% (PHD2) and 44% and 49% (VEGF-A). CAIX, Glut1 and PHD2 expression levels were significantly higher in TE when only samples with at least 80% expression were included.
HIF and mTORC1 signalling seems active in both BCC and TE. There are no appreciable differences between the two with respect to pathway activity. At this moment immunohistochemical analyses of HIF, mTORC1 and their target genes does not provide a reliable diagnostic tool for the discrimination of BCC and TE.
PMCID: PMC4152244  PMID: 25181405
7.  Topical Rapamycin as a Treatment for Fibrofolliculomas in Birt-Hogg-Dubé Syndrome: A Double-Blind Placebo-Controlled Randomized Split-Face Trial 
PLoS ONE  2014;9(6):e99071.
Birt-Hogg-Dubé syndrome (BHD) is a rare autosomal dominant disorder characterised by the occurrence of benign, mostly facial, skin tumours called fibrofolliculomas, multiple lung cysts, spontaneous pneumothorax and an increased renal cancer risk. Current treatments for fibrofolliculomas have high rates of recurrence and carry a risk of complications. It would be desirable to have a treatment that could prevent fibrofolliculomas from growing. Animal models of BHD have previously shown deregulation of mammalian target of rapamycin (mTOR). Topical use of the mTOR inhibitor rapamycin is an effective treatment for the skin tumours (angiofibromas) in tuberous sclerosis complex, which is also characterised by mTOR deregulation. In this study we aimed to determine if topical rapamycin is also an effective treatment for fibrofolliculomas in BHD.
We performed a double blinded, randomised, facial left-right controlled trial of topical rapamycin 0.1% versus placebo in 19 BHD patients. Trial duration was 6 months. The primary outcome was cosmetic improvement as measured by doctors and patients. Changes in fibrofolliculoma number and size were also measured, as was occurrence of side effects.
No change in cosmetic status of fibrofolliculomas was reported in the majority of cases for the rapamycin treated (79% by doctors, 53% by patients) as well as the placebo treated facial sides (both 74%). No significant differences between rapamycin and placebo treated facial halves were observed (p = 1.000 for doctors opinion, p = 0.344 for patients opinion). No significant difference in fibrofolliculoma number or change in size of the fibrofolliculomas was seen after 6 months. Side effects occurred more often after rapamycin treatment (68% of patients) than after placebo (58% of patients; p = 0.625). A burning sensation, erythema, itching and dryness were most frequently reported.
This study provides no evidence that treatment of fibrofolliculomas with topical rapamycin in BHD results in cosmetic improvement.
Trial Registration +NCT00928798
PMCID: PMC4049818  PMID: 24910976
8.  Epigenetic Changes in Basal Cell Carcinoma Affect SHH and WNT Signaling Components 
PLoS ONE  2012;7(12):e51710.
The genetic background of Basal Cell Carcinoma (BCC) has been studied extensively, while its epigenetic makeup has received comparatively little attention. Epigenetic alterations such as promoter hypermethylation silence tumor suppressor genes (TSG) in several malignancies.
We sought to analyze the promoter methylation status of ten putative (tumor suppressor) genes that are associated with Sonic Hedgehog (SHH), WNT signaling and (hair follicle) tumors in a large series of 112 BCC and 124 healthy control samples by methylation-specific PCR.
Gene promoters of SHH (P = 0.016), adenomatous polyposis coli (APC) (P = 0.003), secreted frizzled-related protein 5 (SFRP5) (P = 0.004) and Ras association domain family 1A (RASSF1A) (P = 0.023) showed significantly more methylation in BCC versus normal skin. mRNA levels of these four genes were reduced for APC and SFRP5 in BCC (n = 6) vs normal skin (n = 6). Down regulation of SHH, APC and RASSF1A could be confirmed on protein level as well (P<0.001 for all genes) by immunohistochemical staining. Increased canonical WNT activity was visualized by β-catenin staining, showing nuclear β-catenin in only 28/101 (27.7%) of BCC. Absence of nuclear β-catenin in some samples may be due to high levels of membranous E-cadherin (in 94.1% of the samples).
We provide evidence that promoter hypermethylation of key players within the SHH and WNT pathways is frequent in BCC, consistent with their known constitutive activation in BCC. Epigenetic gene silencing putatively contributes to BCC tumorigenesis, indicating new venues for treatment.
PMCID: PMC3524166  PMID: 23284750
Identification of the underlying genetic, cellular, and biochemical basis of lipid metabolic disorders provides an opportunity to deploy corrective, mechanism-targeted, topical therapy. We assessed this therapeutic approach in two patients with Congenital Hemidysplasia with Ichthyosiform Erythroderma and Limb Defects (CHILD) syndrome, an X-linked dominant disorder of distal cholesterol metabolism. Based upon the putative pathogenic role of both pathway-product deficiency of cholesterol and accumulation of toxic metabolic intermediates, we assessed the efficacy of combined therapy with lovastatin and cholesterol. We also evaluated the basis for the poorly understood, unique lateralization of the cutaneous and bone malformations of CHILD syndrome by analyzing gene activation in abnormal and unaffected skin. Ultrastructural analysis of affected skin showed evidence of both cholesterol depletion and toxic metabolic accumulation. Topical treatment with lovastatin/cholesterol (but not cholesterol alone) virtually cleared skin lesions by 3 months, accompanied by histologic and ultrastructural normalization of epidermal structure and lipid secretion. The unusual lateralization of abnormalities in CHILD syndrome reflects selective clearance of keratinocytes and fibroblasts that express the mutant allele from the unaffected side. These findings validate pathogenesis-based therapy that provides the deficient end-product and prevents accumulation of toxic metabolites, an approach of potential utility for other syndromic lipid metabolic disorders.
PMCID: PMC3193573  PMID: 21753784
ichthyosis; lipids; lateralization; X-inactivation; CHILD syndrome; statin; lovastatin
10.  Reorganization of the nuclear lamina and cytoskeleton in adipogenesis 
Histochemistry and Cell Biology  2011;135(3):251-261.
A thorough understanding of fat cell biology is necessary to counter the epidemic of obesity. Although molecular pathways governing adipogenesis are well delineated, the structure of the nuclear lamina and nuclear-cytoskeleton junction in this process are not. The identification of the ‘linker of nucleus and cytoskeleton’ (LINC) complex made us consider a role for the nuclear lamina in adipose conversion. We herein focused on the structure of the nuclear lamina and its coupling to the vimentin network, which forms a cage-like structure surrounding individual lipid droplets in mature adipocytes. Analysis of a mouse and human model system for fat cell differentiation showed fragmentation of the nuclear lamina and subsequent loss of lamins A, C, B1 and emerin at the nuclear rim, which coincides with reorganization of the nesprin-3/plectin/vimentin complex into a network lining lipid droplets. Upon 18 days of fat cell differentiation, the fraction of adipocytes expressing lamins A, C and B1 at the nuclear rim increased, though overall lamin A/C protein levels were low. Lamin B2 remained at the nuclear rim throughout fat cell differentiation. Light and electron microscopy of a subcutaneous adipose tissue specimen showed striking indentations of the nucleus by lipid droplets, suggestive for an increased plasticity of the nucleus due to profound reorganization of the cellular infrastructure. This dynamic reorganization of the nuclear lamina in adipogenesis is an important finding that may open up new venues for research in and treatment of obesity and nuclear lamina-associated lipodystrophy.
Electronic supplementary material
The online version of this article (doi:10.1007/s00418-011-0792-4) contains supplementary material, which is available to authorized users.
PMCID: PMC3052499  PMID: 21350821
LINC; Fat; Lamin; Adipose tissue; Nesprin-3; Vimentin
11.  Porokeratotic Eccrine Nevus May Be Caused by Somatic Connexin26 Mutations 
Porokeratotic eccrine ostial and dermal duct nevus, or porokeratotic eccrine nevus (PEN), is a hyperkeratotic epidermal nevus. Several cases of widespread involvement have been reported, including one in association with the keratitis–ichthyosis–deafness (KID) syndrome (OMIM #148210), a rare disorder caused by mutations in the GJB2 gene coding for the gap junction protein connexin26 (Cx26). The molecular cause is, as yet, unknown. We have noted that PEN histopathology is shared by KID. The clinical appearance of PEN can resemble that of KID syndrome. Furthermore, a recent report of cutaneous mosaicism for a GJB2 mutation associated with KID describes linear hyperkeratotic skin lesions that might be consistent with PEN. From this, we hypothesized that PEN might be caused by Cx26 mutations associated with KID or similar gap junction disorders. Thus, we analyzed the GJB2 gene in skin samples from two patients referred with generalized PEN. In both, we found GJB2 mutations in the PEN lesions but not in unaffected skin or peripheral blood. One mutation was already known to cause the KID syndrome, and the other had not been previously associated with skin symptoms. We provide extensive functional data to support its pathogenicity. We conclude that PEN may be caused by mosaic GJB2 mutations.
PMCID: PMC3422696  PMID: 22592158

Results 1-11 (11)