BHD-associated kidney cancer exhibits unique molecular characteristics and a wide variety of variants in chromatin remodeling genes.

Birt-Hogg-Dubé (BHD) syndrome is a hereditary kidney cancer syndrome, which predisposes patients to develop kidney cancer, cutaneous fibrofolliculomas and pulmonary cysts. The responsible gene FLCN is a tumor suppressor for kidney cancer, which plays an important role in energy homeostasis through the regulation of mitochondrial oxidative metabolism. However, the process by which FLCN-deficiency leads to renal tumorigenesis is unclear. In order to clarify molecular pathogenesis of BHD-associated kidney cancer, we conducted whole-exome sequencing analysis using next-generation sequencing technology as well as metabolite analysis using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Whole-exome sequencing analysis of BHD-associated kidney cancer revealed that copy number variations of BHD-associated kidney cancer are considerably different from those already reported in sporadic cases. In somatic variant analysis, very few variants were commonly observed in BHD-associated kidney cancer; however, variants in chromatin remodeling genes were frequently observed in BHD-associated kidney cancer (17/29 tumors, 59%). Metabolite analysis of BHD-associated kidney cancer revealed metabolic reprogramming toward upregulated redox regulation which may neutralize reactive oxygen species potentially produced from mitochondria with increased respiratory capacity under FLCN-deficiency. BHD-associated kidney cancer displays unique molecular characteristics that are completely different from sporadic kidney cancer, providing mechanistic insight into tumorigenesis under FLCN-deficiency as well as a foundation for development of novel therapeutics for kidney cancer.

H255Y and K508R missense mutations in tumour suppressor folliculin (FLCN) promote kidney cell proliferation.

Germline H255Y and K508R missense mutations in the folliculin (FLCN) gene have been identified in patients with bilateral multifocal (BMF) kidney tumours and clinical manifestations of Birt-Hogg-Dubé (BHD) syndrome, or with BMF kidney tumours as the only manifestation; however, their impact on FLCN function remains to be determined. In order to determine if FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation leading to pathogenicity, we generated mouse models expressing these mutants using BAC recombineering technology and investigated their ability to rescue the multi-cystic phenotype of Flcn-deficient mouse kidneys. Flcn H255Y mutant transgene expression in kidney-targeted Flcn knockout mice did not rescue the multi-cystic kidney phenotype. However, expression of the Flcn K508R mutant transgene partially, but not completely, abrogated the phenotype. Notably, expression of the Flcn K508R mutant transgene in heterozygous Flcn knockout mice resulted in development of multi-cystic kidneys and cardiac hypertrophy in some mice. These results demonstrate that both FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation, but to different degrees. Based on the phenotypes of our preclinical models, the FLCN H255Y mutant protein has lost it tumour suppressive function leading to the clinical manifestations of BHD, whereas the FLCN K508R mutant protein may have a dominant negative effect on the function of wild-type FLCN in regulating kidney cell proliferation and, therefore, act as an oncoprotein. These findings may provide mechanistic insight into the role of FLCN in regulating kidney cell proliferation and facilitate the development of novel therapeutics for FLCN-deficient kidney cancer.

Folliculin-interacting proteins Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn.

Folliculin (FLCN)-interacting proteins 1 and 2 (FNIP1, FNIP2) are homologous binding partners of FLCN, a tumor suppressor for kidney cancer. Recent studies have revealed potential functions for Flcn in kidney; however, kidney-specific functions for Fnip1 and Fnip2 are unknown. Here we demonstrate that Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn. We observed no detectable phenotype in Fnip2 knockout mice, whereas Fnip1 deficiency produced phenotypes similar to those seen in Flcn-deficient mice in multiple organs, but not in kidneys. We found that absolute Fnip2 mRNA copy number was low relative to Fnip1 in organs that showed phenotypes under Fnip1 deficiency but was comparable to Fnip1 mRNA copy number in mouse kidney. Strikingly, kidney-targeted Fnip1/Fnip2 double inactivation produced enlarged polycystic kidneys, as was previously reported in Flcn-deficient kidneys. Kidney-specific Flcn inactivation did not further augment kidney size or cystic histology of Fnip1/Fnip2 double-deficient kidneys, suggesting pathways dysregulated in Flcn-deficient kidneys and Fnip1/Fnip2 double-deficient kidneys are convergent. Heterozygous Fnip1/homozygous Fnip2 double-knockout mice developed kidney cancer at 24 mo of age, analogous to the heterozygous Flcn knockout mouse model, further supporting the concept that Fnip1 and Fnip2 are essential for the tumor-suppressive function of Flcn and that kidney tumorigenesis in human Birt-Hogg-Dubé syndrome may be triggered by loss of interactions among Flcn, Fnip1, and Fnip2. Our findings uncover important roles for Fnip1 and Fnip2 in kidney tumor suppression and may provide molecular targets for the development of novel therapeutics for kidney cancer.

Loss of Folliculin Disrupts Hematopoietic Stem Cell Quiescence and Homeostasis Resulting in Bone Marrow Failure.

Folliculin (FLCN) is an autosomal dominant tumor suppressor gene that modulates diverse signaling pathways required for growth, proliferation, metabolism, survival, motility, and adhesion. FLCN is an essential protein required for murine embryonic development, embryonic stem cell (ESC) commitment, and Drosophila germline stem cell maintenance, suggesting that Flcn may be required for adult stem cell homeostasis. Conditional inactivation of Flcn in adult hematopoietic stem/progenitor cells (HSPCs) drives hematopoietic stem cells (HSC) into proliferative exhaustion resulting in the rapid depletion of HSPC, loss of all hematopoietic cell lineages, acute bone marrow (BM) failure, and mortality after 40 days. HSC that lack Flcn fail to reconstitute the hematopoietic compartment in recipient mice, demonstrating a cell-autonomous requirement for Flcn in HSC maintenance. BM cells showed increased phosphorylation of Akt and mTorc1, and extramedullary hematopoiesis was significantly reduced by treating mice with rapamycin in vivo, suggesting that the mTorc1 pathway was activated by loss of Flcn expression in hematopoietic cells in vivo. Tfe3 was activated and preferentially localized to the nucleus of Flcn knockout (KO) HSPCs. Tfe3 overexpression in HSPCs impaired long-term hematopoietic reconstitution in vivo, recapitulating the Flcn KO phenotype, and supporting the notion that abnormal activation of Tfe3 contributes to the Flcn KO phenotype. Flcn KO mice develop an acute histiocytic hyperplasia in multiple organs, suggesting a novel function for Flcn in macrophage development. Thus, Flcn is intrinsically required to maintain adult HSC quiescence and homeostasis, and Flcn loss leads to BM failure and mortality in mice.

Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation.

Cardiac hypertrophy, an adaptive process that responds to increased wall stress, is characterized by the enlargement of cardiomyocytes and structural remodeling. It is stimulated by various growth signals, of which the mTORC1 pathway is a well-recognized source. Here, we show that loss of Flcn, a novel AMPK-mTOR interacting molecule, causes severe cardiac hypertrophy with deregulated energy homeostasis leading to dilated cardiomyopathy in mice. We found that mTORC1 activity was upregulated in Flcn-deficient hearts, and that rapamycin treatment significantly reduced heart mass and ameliorated cardiac dysfunction. Phospho-AMP-activated protein kinase (AMPK)-alpha (T172) was reduced in Flcn-deficient hearts and nonresponsive to various stimulations including metformin and AICAR (5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide). ATP levels were elevated and mitochondrial function was increased in Flcn-deficient hearts, suggesting that excess energy resulting from up-regulated mitochondrial metabolism under Flcn deficiency might attenuate AMPK activation. Expression of Ppargc1a, a central molecule for mitochondrial metabolism, was increased in Flcn-deficient hearts and indeed, inactivation of Ppargc1a in Flcn-deficient hearts significantly reduced heart mass and prolonged survival. Ppargc1a inactivation restored phospho-AMPK-alpha levels and suppressed mTORC1 activity in Flcn-deficient hearts, suggesting that up-regulated Ppargc1a confers increased mitochondrial metabolism and excess energy, leading to inactivation of AMPK and activation of mTORC1. Rapamycin treatment did not affect the heart size of Flcn/Ppargc1a doubly inactivated hearts, further supporting the idea that Ppargc1a is the critical element leading to deregulation of the AMPK-mTOR-axis and resulting in cardiac hypertrophy under Flcn deficiency. These data support an important role for Flcn in cardiac homeostasis in the murine model.

Birt-Hogg-Dubé syndrome: Clinical and molecular aspects of recently identified kidney cancer syndrome.

Birt-Hogg-Dubé syndrome is an autosomal dominantly inherited disease that predisposes patients to develop fibrofolliculoma, lung cysts and bilateral multifocal renal tumors, histologically hybrid oncocytic/chromophobe tumors, chromophobe renal cell carcinoma, oncocytoma, papillary renal cell carcinoma and clear cell renal cell carcinoma. The predominant forms of Birt-Hogg-Dubé syndrome-associated renal tumors, hybrid oncocytic/chromophobe tumors and chromophobe renal cell carcinoma are typically less aggressive, and a therapeutic principle for these tumors is a surgical removal with nephron-sparing. The timing of surgery is the most critical element for postoperative renal function, which is one of the important prognostic factors for Birt-Hogg-Dubé syndrome patients. The folliculin gene (FLCN) that is responsible for Birt-Hogg-Dubé syndrome was isolated as a novel tumor suppressor for kidney cancer. Recent studies using murine models for FLCN, a protein encoded by the FLCN gene, and its two binding partners, folliculin-interacting protein 1 (FNIP1) and folliculin-interacting protein 2 (FNIP2), have uncovered important roles for FLCN, FNIP1 and FNIP2 in cell metabolism, which include AMP-activated protein kinase-mediated energy sensing, Ppargc1a-driven mitochondrial oxidative phosphorylation and mTORC1-dependent cell proliferation. Birt-Hogg-Dubé syndrome is a hereditary hamartoma syndrome, which is triggered by metabolic alterations under a functional loss of FLCN/FNIP1/FNIP2 complex, a critical regulator of kidney cell proliferation rate; a mechanistic insight into the FLCN/FNIP1/FNIP2 pathway could provide us a basis for developing new therapeutics for kidney cancer.

The folliculin-FNIP1 pathway deleted in human Birt-Hogg-Dubé syndrome is required for murine B-cell development.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder characterized by cutaneous fibrofolliculomas, pulmonary cysts, and kidney malignancies. Affected individuals carry germ line mutations in folliculin (FLCN), a tumor suppressor gene that becomes biallelically inactivated in kidney tumors by second-hit mutations. Similar to other factors implicated in kidney cancer, FLCN has been shown to modulate activation of mammalian target of rapamycin (mTOR). However, its precise in vivo function is largely unknown because germ line deletion of Flcn results in early embryonic lethality in animal models. Here, we describe mice deficient in the newly characterized folliculin-interacting protein 1 (Fnip1). In contrast to Flcn, Fnip1(-/-) mice develop normally, are not susceptible to kidney neoplasia, but display a striking pro-B cell block that is entirely independent of mTOR activity. We show that this developmental arrest results from rapid caspase-induced pre-B cell death, and that a Bcl2 transgene reconstitutes mature B-cell populations, respectively. We also demonstrate that conditional deletion of Flcn recapitulates the pro-B cell arrest of Fnip1(-/-) mice. Our studies thus demonstrate that the FLCN-FNIP complex deregulated in BHD syndrome is absolutely required for B-cell differentiation, and that it functions through both mTOR-dependent and independent pathways.

HLA-DRB1*04:05 is involved in the development of Vogt-Koyanagi-Harada disease-like immune-related adverse events in patients receiving immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) activate anti-tumor activity by inhibiting immune checkpoint molecules that suppress inflammatory T-cell activity. However, ICIs can initiate excessive immune responses, thereby causing immune-related adverse events (irAEs). ICI-associated uveitis (ICIU) is an irAE that affects the eyes. Although Vogt-Koyanagi-Harada disease (VKH)-like uveitis is a common form of ICIU, it is unclear which factors determine the ICIU form. We retrospectively reviewed the medical records of nine ICIU cases treated with ICIs for malignancies. We also performed HLA typing in seven cases to investigate the association between HLA and disease type. Fisher's exact test was used for the statistical analysis. Five of the ICIU cases were VKH-like ICIUs, and four were non-VKH-like ICIUs. No association was found between mean age, sex, primary disease, ICI, time to onset, and disease type. Four patients with VKH-like uveitis underwent HLA genotyping and were all positive for HLA-DRB1*04:05. All 3 patients with non-VKH-like uveitis were negative for HLA-DRB1*04:05. Statistical analysis showed that HLA-DRB1*04:05 was significantly associated with developing VKH-like ICIU (P = 0.029). In ICIU, HLA-DRB1*04:05 was associated with the pathogenesis of VKH-like uveitis, suggesting that ICI-associated VKH-like uveitis has a similar pathogenesis to VKH.

Homozygous loss of BHD causes early embryonic lethality and kidney tumor development with activation of mTORC1 and mTORC2.

Germline mutations in the BHD/FLCN tumor suppressor gene predispose patients to develop renal tumors in the hamartoma syndrome, Birt-Hogg-Dubé (BHD). BHD encodes folliculin, a protein with unknown function that may interact with the energy- and nutrient-sensing AMPK-mTOR signaling pathways. To clarify BHD function in the mouse, we generated a BHD knockout mouse model. BHD homozygous null (BHD(d/d)) mice displayed early embryonic lethality at E5.5-E6.5, showing defects in the visceral endoderm. BHD heterozygous knockout (BHDd(/+)) mice appeared normal at birth but developed kidney cysts and solid tumors as they aged (median kidney-lesion-free survival = 23 months, median tumor-free survival = 25 months). As observed in human BHD kidney tumors, three different histologic types of kidney tumors developed in BHD(d/+) mice including oncocytic hybrid, oncocytoma, and clear cell with concomitant loss of heterozygosity (LOH), supporting a tumor suppressor function for BHD in the mouse. The PI3K-AKT pathway was activated in both human BHD renal tumors and kidney tumors in BHD(d/+) mice. Interestingly, total AKT protein was elevated in kidney tumors compared to normal kidney tissue, but without increased levels of AKT mRNA, suggesting that AKT may be regulated by folliculin through post translational or post-transcriptional modification. Finally, BHD inactivation led to both mTORC1 and mTORC2 activation in kidney tumors from BHD(d/+) mice and human BHD patients. These data support a role for PI3K-AKT pathway activation in kidney tumor formation caused by loss of BHD and suggest that inhibitors of both mTORC1 and mTORC2 may be effective as potential therapeutic agents for BHD-associated kidney cancer.

An open-label, prospective, single-arm study of switching from infliximab to cyclosporine for refractory uveitis in patients with Behçet's disease in long-term remission.

PURPOSE: Although infliximab (IFX) decreases the risk of blindness due to refractory uveitis in patients with Behçet's disease (BD), there are no standard criteria for IFX switching or withdrawal. To evaluate the effect of IFX switching in patients with BD in long-term remission, a prospective, single-arm intervention trial was conducted, switching from IFX to cyclosporine A (CYA). STUDY DESIGN: A prospective open-label study. METHODS: Eligible patients met the following criteria: administration of IFX without concomitant immunosuppressants for more than 5 years with no episodes of ocular attacks, no retinal vasculitis on fluorescein fundus angiography, negative C-reactive protein in serum, and no extraocular lesions at the time of IFX withdrawal. CYA 5 mg/kg/day was administered from 6 weeks after IFX withdrawal. The primary outcome was the rate of readministration of tumor necrosis factor inhibitors at 1 year after IFX withdrawal. RESULTS: Three of 45 BD patients treated with IFX for refractory uveitis were included in the study. At 1 year after withdrawal of IFX, no patient had experienced any ocular attacks or needed readministation of IFX. However, extraocular lesions, such as recurrent oral ulcers, folliculitis, and recurrent fevers, occurred in all patients. Liver or renal dysfunction, which may have been caused by CYA, was also observed in all patients. CONCLUSIONS: Although no ocular attacks were observed for at least 1 year after IFX withdrawal, this prospective study indicates that IFX withdrawal should be considered carefully, even for patients in long term remission of ocular and extraocular lesions.

Blood and lymphatic systems are segregated by the FLCN tumor suppressor.

Blood and lymphatic vessels structurally bear a strong resemblance but never share a lumen, thus maintaining their distinct functions. Although lymphatic vessels initially arise from embryonic veins, the molecular mechanism that maintains separation of these two systems has not been elucidated. Here, we show that genetic deficiency of Folliculin, a tumor suppressor, leads to misconnection of blood and lymphatic vessels in mice and humans. Absence of Folliculin results in the appearance of lymphatic-biased venous endothelial cells caused by ectopic expression of Prox1, a master transcription factor for lymphatic specification. Mechanistically, this phenotype is ascribed to nuclear translocation of the basic helix-loop-helix transcription factor Transcription Factor E3 (TFE3), binding to a regulatory element of Prox1, thereby enhancing its venous expression. Overall, these data demonstrate that Folliculin acts as a gatekeeper that maintains separation of blood and lymphatic vessels by limiting the plasticity of committed endothelial cells.

TFE3 Xp11.2 Translocation Renal Cell Carcinoma Mouse Model Reveals Novel Therapeutic Targets and Identifies GPNMB as a Diagnostic Marker for Human Disease.

Renal cell carcinoma (RCC) associated with Xp11.2 translocation (TFE3-RCC) has been recently defined as a distinct subset of RCC classified by characteristic morphology and clinical presentation. The Xp11 translocations involve the TFE3 transcription factor and produce chimeric TFE3 proteins retaining the basic helix-loop-helix leucine zipper structure for dimerization and DNA binding suggesting that chimeric TFE3 proteins function as oncogenic transcription factors. Diagnostic biomarkers and effective forms of therapy for advanced cases of TFE3-RCC are as yet unavailable. To facilitate the development of molecular based diagnostic tools and targeted therapies for this aggressive kidney cancer, we generated a translocation RCC mouse model, in which the PRCC-TFE3 transgene is expressed specifically in kidneys leading to the development of RCC with characteristic histology. Expression of the receptor tyrosine kinase Ret was elevated in the kidneys of the TFE3-RCC mice, and treatment with RET inhibitor, vandetanib, significantly suppressed RCC growth. Moreover, we found that Gpnmb (Glycoprotein nonmetastatic B) expression was notably elevated in the TFE3-RCC mouse kidneys as seen in human TFE3-RCC tumors, and confirmed that GPNMB is the direct transcriptional target of TFE3 fusions. While GPNMB IHC staining was positive in 9/9 cases of TFE3-RCC, Cathepsin K, a conventional marker for TFE3-RCC, was positive in only 67% of cases. These data support RET as a potential target and GPNMB as a diagnostic marker for TFE3-RCC. The TFE3-RCC mouse provides a preclinical in vivo model for the development of new biomarkers and targeted therapeutics for patients affected with this aggressive form of RCC. IMPLICATIONS: Key findings from studies with this preclinical mouse model of TFE3-RCC underscore the potential for RET as a therapeutic target for treatment of patients with TFE3-RCC, and suggest that GPNMB may serve as diagnostic biomarker for TFE3 fusion RCC.

Identification and characterization of a novel folliculin-interacting protein FNIP2.

Birt-Hogg-Dube' syndrome characterized by increased risk for renal neoplasia is caused by germline mutations in the BHD/FLCN gene encoding a novel tumor suppressor protein, folliculin(FLCN), which interacts with FNIP1 and 5'-AMP-activated protein kinase(AMPK). Here we report the identification and characterization of a novel FNIP1 homolog FNIP2 that also interacts with FLCN and AMPK. C-terminally-deleted FLCN mutants, similar to those produced by naturally-occurring germline mutations in BHD patients, were unable to bind FNIP2. These data taken together with our previous results that demonstrated FNIP1 binding to the C-terminus of FLCN suggest that FLCN tumor suppressor function may be facilitated by interactions with both FNIP1 and FNIP2 through its C-terminus. Furthermore, we demonstrate that FNIP1 and FNIP2 are able to form homo- or heteromeric multimers suggesting that they may function independently or cooperatively with FLCN. Differential expression of FNIP1 and FNIP2 transcripts in some normal tissues may indicate tissue specificity for these homologs. Interestingly FNIP1 and FNIP2 were oppositely expressed in human clear cell renal cell carcinoma (RCC), and coordinately expressed in chromophobe RCC and oncocytoma, suggesting their differential function in different histologic variants of RCC.

Polymorphism of IFN-gamma gene and Vogt-Koyanagi-Harada disease.

PURPOSE: Interferon-gamma (IFN-gamma) is a key cytokine in inflammatory disorders. Elevated aqueous and serum levels of IFN-gamma levels have been reported to be elevated in patients with Vogt-Koyanagi-Harada (VKH) disease. The aim of this study was to determine the IFN-gamma gene polymorphisms in VKH disease. METHODS: The study involved 136 VKH patients and 176 healthy controls, who were genotyped for functional single nucleotide polymorphism (SNP; rs2430561; A/T) and functional microsatellite (CA) repeats (rs3138557) in the first intron of the IFN-gamma gene. Moreover, clinical manifestations of the patients were also analyzed. RESULTS: Diffuse choroiditis/staining of fluorescein angiography was seen in all VKH patients in this study. Sunset glow fundus and nummular chorioretinal depigmented scars were observed in 83.9%, and 36.1% of the patients, respectively. Neurological and auditory disorders were observed in 90.1% of the patients: meningismus (79.8%), tinnitus (53.0%), and cerebrospinal fluid pleocytosis (70.0%). Dermatologic manifestations were observed in 22.9% of the patients, manifesting as alopecia (6.9%), poliosis (17.6%), and vitiligo (13.0%). In addition, 22.1% of the patients were classified as having complete VKH disease, while 65.4% as having incomplete VKH disease, and 12.5% as having probable VKH disease. There were no significant differences in the allele and genotype frequencies between VKH patients and healthy controls. In addition, we found no association between each clinical manifestation and SNP (re2430561) in the healthy control subject. A strong linkage disequilibrium (LD) was found in the functional SNP T allele and functional microsatellite 12 (CA) repeats (D'=0.96-0.99). CONCLUSIONS: The functional SNP T allele and microsatellite 12 (CA) repeats were found to have a strong LD, although a genetic susceptibility for the IFN-gamma gene could not be demonstrated among the Japanese VKH patients.

[Case of neurosarcoidosis with rapid visual field defect progression].

OBJECTIVE: To report a case of neurosarcoidosis with rapid progression of visual field defects. CASE: A 28-year-old woman presented with bilateral uveitis and was diagnosed as having sarcoidosis after skin and cervical lymph node biopsy. Since bilateral excavations of the optic nerve head and visual field defects were observed, endocranial lesion was suspected. However, a computed tomography (CT) scan of the head detected nothing abnormal. It was regarded as a case of sarcoidosisaccompanied by normal-tension glaucoma and treatment was initiated with latanoprost. Four months later, the patient's visual field deteriorated rapidly. A CT scan showed a pituitary mass. Neurologicalfindings and hypopituitarism were found which improved with systemic prednisolone therapy. Diabetes insipidus developed after the start of treatment, and was treated with intranasal desmopressin therapy. After 6 weeks, head magnetic resonance imaging (MRI) showed a remarkable reduction of the enhanced regions. CONCLUSIONS: Although ocular sarcoidosis is often accompanied by secondary glaucoma or optic nerve atrophy, the progression of neurosarcoidosis can lead to visual field defects. Central nervous system (CNS) sarcoidosis is rare, but a precise examination with enhanced MRI should be considered when the visual field defect progresses rapidly.

Tyrosinase gene family and Vogt-Koyanagi-Harada disease in Japanese patients.

PURPOSE: The aim of the present study was to examine the genetic background of Vogt-Koyanagi-Harada (VKH) disease in a Japanese population by analyzing the tyrosinase gene family (TYR, TYRP1, and dopachrome tautomerase (DCT)). METHODS: 87 VKH patients and 122 healthy controls were genotyped using seven microsatellite markers on the candidate loci. We analyzed microsatellite (MS) polymorphisms at regions within tyrosinase gene family loci. In addition, the haplotype frequencies were also estimated and statistical analysis was performed. HLA-DRB1 genotyping was performed by the PCR-restriction fragment length polymorphism (RFLP) method. RESULTS: No significant evidence for an association was found. HLA-DRB1*0405 showed a highly significant association with VKH disease compared with the healthy controls (Pc=0.000000079), as expected. CONCLUSIONS: We concluded that there is no genetic susceptibility or increased risk attributed to the tyrosinase gene family. Our results suggest the need for further genetic study and encourage a search for novel genetic loci and predisposing genes in order to elucidate the genetic mechanisms underlying VKH disease.

Regulation of mitochondrial oxidative metabolism by tumor suppressor FLCN.

BACKGROUND: Birt-Hogg-Dubé (BHD) syndrome is a hereditary hamartoma syndrome that predisposes patients to develop hair follicle tumors, lung cysts, and kidney cancer. Genetic studies of BHD patients have uncovered the causative gene, FLCN, but its function is incompletely understood. METHODS: Mice with conditional alleles of FLCN and/or peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), a transcriptional coactivator that regulates mitochondrial biogenesis, were crossbred with mice harboring either muscle creatine kinase (CKM) -Cre or myogenin (MYOG) -Cre transgenes to knock out FLCN and/or PPARGC1A in muscle, or cadherin 16 (CDH16)- Cre transgenes to knock out FLCN and/or PPARGC1A in kidney. Real-time polymerase chain reaction, immunoblotting, electron microscopy, and metabolic profiling assay were performed to evaluate mitochondrial biogenesis and function in muscle. Immunoblotting, electron microscopy, and histological analysis were used to investigate expression and the pathological role of PPARGC1A in FLCN-deficient kidney. Real-time polymerase chain reaction, oxygen consumption measurement, and flow cytometry were carried out using a FLCN-null kidney cancer cell line. All statistical analyses were two-sided. RESULTS: Muscle-targeted FLCN knockout mice underwent a pronounced metabolic shift toward oxidative phosphorylation, including increased mitochondrial biogenesis (FLCN ( f/f ) vs FLCN ( f/f ) /CKM-Cre: % mitochondrial area mean = 7.8% vs 17.8%; difference = 10.0%; 95% confidence interval = 5.7% to 14.3%; P < .001), and the observed increase in mitochondrial biogenesis was PPARGC1A dependent. Reconstitution of FLCN-null kidney cancer cells with wild-type FLCN suppressed mitochondrial metabolism and PPARGC1A expression. Kidney-targeted PPARGC1A inactivation partially rescued the enlarged kidney phenotype and abrogated the hyperplastic cells observed in the FLCN-deficient kidney. CONCLUSION: FLCN deficiency and subsequent increased PPARGC1A expression result in increased mitochondrial function and oxidative metabolism as the source of cellular energy, which may give FLCN-null kidney cells a growth advantage and drive hyperplastic transformation.

Guidelines for the diagnosis of ocular sarcoidosis.

PURPOSE: To delineate features of ocular sarcoidosis. METHODS: Comparison of the old and new Japanese guidelines for diagnosing ocular sarcoidosis by review of the medical records of 100 sarcoidosis patients and 147 patients with non-sarcoidosis uveitis. RESULTS: The overall diagnostic sensitivity and specificity for the old guidelines were 80.0% and 45.6%; sensitivity and specificity for the new guidelines were 84.0% and 83.0%. Positive and negative predictive values and the likelihood ratios for a positive and negative test result improved. The new guidelines achieved more than 80% specificity for all categories. CONCLUSION: The new guidelines allow easier distinction of ocular sarcoidosis.

Analysis of single nucleotide polymorphisms at 13 loci within the transforming growth factor-induced factor gene shows no association with high myopia in Japanese subjects.

A previous study in China first indicated that the transforming growth factor-induced factor (TGIF) is a probable candidate gene for high myopia. The purpose of our study was to investigate whether there are significant associations between high myopia and single nucleotide polymorphism (SNP) variants in the TGIF gene of Japanese subjects. Genomic DNA was collected from 330 Japanese subjects with high myopia and at a level refractive error was less than -9.25 Dsph and 330 randomized controls without high myopia. Thirteen SNPs were detected by polymerase chain reaction (PCR) and primer extension or by PCR and SNP-specific fluorogenic probes in all of the cases and controls. Thirteen SNPs were found within the TGIF genes of the cases and controls. Two of the SNPs were monomorphic and none of the 13 SNPs showed a significant result. The pairwise linkage disequilibrium (LD) mapping confirmed that these alleles have a comparatively strong LD index of >0.8 for D' and >0.4 for r(2). We found no statistical association between any of the 13 SNPs located on the TGIF gene and high myopia in Japanese subjects. Based on our study using Japanese subjects and the previous studies of TGIF gene polymorphism in Chinese and northern European subjects with myopia, there is no convincing evidence to prove a connection between nucleotide sequence variations in TGIF and high myopia.