Expression of Birt–Hogg–Dube´ gene mRNA in normal and ... · including brain, heart, placenta, testis, skin, lung and kidney, as well as fetal lung, liver, brain and kidney.10

Expression of Birt–Hogg–Dube gene mRNA innormal and neoplastic human tissues

Michelle B Warren1, Carlos A Torres-Cabala2, Maria L Turner3, Maria J Merino2,Vera Y Matrosova4, Michael L Nickerson1, Wenbin Ma1, W Marston Linehan5,Berton Zbar1 and Laura S Schmidt6

1Laboratory of Immunobiology, Center for Cancer Research, NCI Frederick, Frederick, MD, USA; 2Laboratoryof Pathology; 3Dermatology Branch, Center for Cancer Research, NCI, NIH, Bethesda, MD, USA; 4Departmentof Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA; 5Urologic OncologyBranch, Center for Cancer Research, NCI, NIH, Bethesda, MD, USA and 6Basic Research Program, SAIC-Frederick, Inc., NCI Frederick, Frederick, MD, USA

Birt–Hogg–Dube (BHD) syndrome is an inherited autosomal genodermatosis characterized by hamartomas ofthe hair follicle called fibrofolliculomas and an increased risk for developing spontaneous pneumothorax, lungcysts and renal neoplasia. BHD was localized to chromosome 17p11.2 by linkage analysis in BHD families, andgermline insertion/deletion and nonsense mutations in a novel gene were identified which were predicted toprematurely truncate the BHD protein, folliculin. No homology to other human proteins was found althoughfolliculin was conserved across species. As a first step toward understanding the function of BHD in the celland how BHD mutations can lead to the BHD phenotype, we measured the expression of BHD mRNA in normaland neoplastic human tissues by fluorescent in situ hybridization. BHD mRNA was expressed in a variety oftissues, including the skin and its appendages, the distal nephron of the kidney, stromal cells and type 1pneumocytes of the lung, acinar cells of the pancreas and parotid gland, and epithelial ducts of the breast andprostate. In the brain, BHD mRNA was expressed in neurons of the cerebrum, and Purkinje cells in thecerebellum. BHD mRNA was also expressed in macrophage and lymphocytes in the tonsils and spleen. Tissueswith reduced expression of BHD mRNA included heart, muscle and liver. BHD mRNA was expressed strongly inthe proliferating epithelial strands of fibrofolliculomas, the cutaneous lesions characteristic of BHD, but not inrenal tumors from BHD patients. These results indicate a wide expression pattern for BHD mRNA in manytissues, including skin, lung and kidney, which are involved in the BHD phenotype, and support a tumorsuppressor role for BHD in renal cancer.Modern Pathology (2004) 17, 998–1011, advance online publication, 14 May 2004; doi:10.1038/modpathol.3800152

Keywords: Birt–Hogg–Dube syndrome; BHD gene; renal carcinoma; tumor suppressor gene; fluorescent in situhybridization; mRNA expression

Birt–Hogg–Dube (BHD) syndrome is a genodermato-sis inherited as an autosomal dominant trait. Thedisorder, first recognized in 1977,1 is characterizedby the presence of multiple skin papules on thehead and neck accompanied by acrochordons (skintags) and trichodiscomas. Histologically, the cuta-neous papules consist of thin, epithelial strandsoriginating from a central hair follicle with promi-nent associated connective tissue. These skin le-sions were named fibrofolliculomas and considered

to be hamartomas of the hair follicle. Since theoriginal description of the disorder by Drs Birt, Hoggand Dube, BHD families with characteristic derma-tologic lesions and other phenotypic features havebeen reported including renal neoplasia,2 lung cysts,and/or spontaneous pneumothorax.3,4 Patients withfibrofolliculomas have a 50-fold increased riskfor developing spontaneous pneumothorax, and aseven-fold increased risk for renal carcinoma.5

Patients affected with BHD syndrome are predis-posed to develop several different histologic types ofrenal carcinoma including clear cell, papillary andchromophobe renal carcinomas, renal oncocytomasand a hybrid tumor containing elements of bothchromophobe renal carcinoma and renal oncocyto-ma.6,7 The oncocytic hybrid renal tumor is the mostcommon renal neoplasm found in BHD patients.

Received 23 December 2003; revised 15 March 2004; accepted23 March 2004; published online 14 May 2004

Correspondence: Dr LS Schmidt, PhD, Laboratory of Immuno-biology, National Cancer Institute Frederick, Bldg 560, Rm 12-69,Frederick, MD 21702, USA.E-mail: [email protected]

Modern Pathology (2004) 17, 998–1011& 2004 USCAP, Inc All rights reserved 0893-3952/04 $30.00

www.modernpathology.org

The human BHD gene was localized to chromo-some 17p11.2 by linkage analysis8,9 and identifiedby positional cloning.10 Nearly all BHD gene muta-tions identified in the germ line of BHD patients arepredicted to result in frameshifts, which produce atruncated protein. Almost half of BHD patientsharbor an insertion/deletion mutation in a tract ofeight cytosines leading to premature protein termi-nation. The full-length BHD sequence encodes anovel protein, folliculin, with an open reading frameof 579 amino acids and no significant homology toknown human proteins by BLAST alignment againstSwissPro and nonredundant NCBI protein data-bases. The amino-acid sequence of folliculin pre-dicts a 64 kDa protein with a short hydrophobicN-terminal sequence, one N-glycosylation site, threemyristoylation sites and a glutamic acid-rich coiled-coil domain centrally located in the BHD protein.No transmembrane domains or organelle localiza-tion signals were identified by computer-assistedsearches (Expert Protein Analysis System proteo-mics server of the Swiss Institute of Bioinformaticsat http://us.expasy.org). Homologs of the BHDprotein have been identified in the mouse, Droso-phila and Caenorhabditis elegans. Interestingly, twoinherited forms of renal cancer in the dog and the ratmay be caused by mutations in homologs of humanBHD. A naturally occurring form of inherited renalcancer and nodular dermatofibromatosis in theGerman Shepherd dog11,12 has been shown tocosegregate with a missense mutation in the dogBHD gene;13 the Nihon rat cancer model, a sponta-neously occurring inherited renal cancer in theSprague–Dawley rat, has been linked to a region ofthe rat chromosome 10 which is syntenic withhuman chromosome 17p11.2.14,15 Homozygosity ofthe BHD homolog alleles in rat and dog is embryoniclethal.

By Northern blot analysis a 3.8 kb BHD transcriptwas detected in a wide variety of adult tissues,including brain, heart, placenta, testis, skin, lungand kidney, as well as fetal lung, liver, brain andkidney.10 As a first step toward understanding thebiological function of this novel renal cancer-associated gene, we evaluated the specific cell typeand tissue distribution of BHD mRNA by fluorescentin situ hybridization in normal and neoplastichuman tissues, and selected abnormal and neoplas-tic tissues from BHD patients.

Materials and methods

Tissue Specimens

Both fresh frozen tissue and paraffin-embeddedtissue were used in this study. The in situ hybridiza-tion protocol was optimized by hybridizing probe tofresh frozen and paraffin-embedded human scalpsamples (voluntarily obtained through the NIHDermatology Service at the NIH Clinical Center withpatient informed consent). Paraffin-embedded nor-

mal and neoplastic tissue was obtained from thearchives of the NIH Laboratory of Pathology.

Two tissue arrays were used to study the expres-sion of BHD mRNA in normal and neoplastictissues. A low-density Landmark Tissue MicroArray(TMA) was obtained from Ambion with 22 normaltissues each represented by samples from fourindividuals. For comparison of normal tissue ex-pression to neoplastic tissue, a low-density TissueMicroArray of 34 cancerous tissue samples (Am-bion) with matched benign specimens was alsohybridized. To specifically examine renal carcinomasamples, a custom tissue array was produced by theTissue Array Research and Production Lab of theNational Cancer Institute with samples of oncocy-toma, oncocytic hybrid, papillary and clear cellhistologic types obtained from patients with BHD,hereditary papillary renal carcinoma or von Hippel–Lindau disease.

In Situ Hybridization Probes

In situ hybridization was performed using a ribop-robe designed from the cDNA sequence of the BHDgene. The primers for the BHD probe were designedand synthesized from exons 11, 12 and 13 of theBHD mRNA sequence (F 50-CTG TGT TGC CAGAGA GTA CAG AAG G-30 and R 50-CCA CAG ACAGGT TCT GGT TGG TC-30). The primers were usedto PCR amplify a 561 bp product from BHD cDNAisolated from fetal heart (Clonetech) using standardconditions with a 641C annealing temperature. Sp6adapter ends containing a primer sequence wereligated directly to the PCR product using the Lig-n-Scribe kit (Ambion) following the manufacturer’sprotocol, and PCR-amplified to produce sense andantisense templates. The templates were in vitrotranscribed with fluorescein-labeled dUTP (Roche)using the MAXIscript in vitro transcription kit(Ambion) and treated with DNase I per manufac-turer’s protocol.

A riboprobe to a keratin known to be expressed inthe inner root sheath of the hair follicle, Ker6IRS1,16

was used as a positive control and to optimize the insitu hybridization method. PCR primers for Ker6-IRS1 were designed (F 50-CAT CAG CAG CAC CAGTGG CGG-30 and R 50-CAG GAA CTA TGC TAG GTCCCA G-30) and the probe of interest was amplified byPCR at a 641C annealing temperature using standardconditions. Ker6IRS1 was designed as a 589 bp PCRprobe amplified from the 30UTR of the KER6 geneusing genomic DNA as the template.

Tissue Preparation, Pretreatment, Hybridization andSignal Detection

The tissue samples were flash frozen in OCT mediaon dry ice and then sectioned using a cryostat at athickness of 8–10 mm. Paraffin sections (5 mm) weredeparaffinized by soaking in xylene and rehydrated

BHD expression in normal and neoplastic tissuesMB Warren et al

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Modern Pathology (2004) 17, 998–1011

in gradient alcohol washes. Frozen sections anddewaxed paraffin sections on slides were fixed atroom temperature in 4% paraformaldehyde pre-pared in phosphate-buffered saline. Pretreatment ofslides included quenching endogenous peroxidasewith 1–3% hydrogen peroxide for 10 min, 25 mg/mlproteinase K for 7 min and an acetic anhydridetreatment in 0.1 M triethanolamine with 25 mMacetic anhydride (added right before slide addition)for 10 min. The slides were incubated with prehy-bridization solution containing 0.3 M NaCl, 20 mMTris pH 7.5, 5 mM EDTA, 1�Denhardt’s solution,0.5 mg/ml yeast tRNA, and 60% formamide for 2 hat 371C. Hybridization solution was preparedsame as the prehybridization solution with theaddition of 10% dextran sulfate. The hybridizationsolution was heated to 701C, and then probe (1 ng/mlfinal concentration) was added and heated for anadditional 5 min. The probe and hybridizationsolution were cooled on ice, applied to the slides,and incubated overnight at 371C. Serial tissuesections were probed with the antisense and senseprobes separately, and a third section was stainedwith hematoxylin and eosin to visualize tissuemorphology.

Following hybridization, the slides were washedin 1�SSC and subjected to RNase A treatment(0.131–1.0mg/ml in PBS, Ambion) for 10 min at371C. Slides were soaked twice for 8 min in a 501Csolution of 50% formamide in 2�SSC. Next, slideswere run through a series of SSC-containing washes(1� , 0.1� , 4� /0.1% Tween20) at 501C whileshaking. The hybridized signal was detected usingthe Tyramide System Amplification (TSA) Plus kitfor fluorescein from Ambion using the manufac-turer’s suggested protocol. Mounting media contain-ing DAPI nuclei stain (Vector Laboratories) wasadded to the slides and slides were examined usinga Zeis Axioplan epifluorescence microscope withDAPI and FITC filters from Chroma. The slides werephotographed using a black and white PhotometricsCool Snap camera and IP-Lab 3.6.3-imaging softwarewas used to layer and pseudocolor the images.

Results

Optimization of the In Situ Hybridization Protocol

Our initial experiments utilized standard publishedfluorescent in situ hybridization methods, but didnot produce satisfactory results. Therefore, modifi-cations to the basic procedure were necessary tooptimize the in situ hybridization protocol (Figure1). We found that paraffin-embedded tissue sectionsprovided less background signal than frozen sec-tions and better tissue morphology. Proteinase K wasused at the highest concentration possible whichdid not damage the tissue section. Addition ofdextran sulfate to the hybridization buffer produceda uniform signal and the increased viscosity helpedprevent the slide from drying during the overnight

incubation. Higher hybridization temperatures da-maged the tissue sample; it was found that a lowerhybridization temperature of 371C preserved tissueintegrity, and increased wash stringency helped toreduce background. Pretreatment with acetic anhy-dride and treatment of the slides with RNase A afterhybridization were key steps in reducing the back-ground signal.

Modifications to the procedure had to be made foreach tissue and probe to optimize the signal strengthand to reduce the background. During the pretreat-ment of tissues the endogenous peroxidase quench-ing step was increased from 1% hydrogen peroxidefor kidney (and most tissues) to 3% for skin samplesand fibrofolliculoma. Also, the optimal concentra-tion of RNase A ranged from 0.1 to 1mg/mldepending on the strength of the nonspecific signalfrom the sense probe and the binding strength of theantisense probe. For the Ker6IRS1 probe, the back-ground for the sense probe was still high at an RNaseconcentration of 0.625 mg/ml necessitating an in-crease to 1.0 mg/ml to obtain a signal-free sense slide.However, at an RNase A concentration of 0.625 mg/ml the BHD signal for the antisense and sense probeswas absent. The optimal RNase A concentration forthe BHD sense and antisense probes was 10-foldlower at 0.131 mg/ml.

Expression of BHD mRNA in Skin and SkinAppendages

A riboprobe from the Ker6IRS1 gene16 was used tooptimize the in situ hybridization protocol and as acontrol to visualize the different regions of the hairfollicle. The Ker6IRS1 riboprobe bound specificallyto all three layers of the inner root sheath (Henle,

Figure 1 Flowchart of the optimized in situ hybridizationprotocol.


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Huxley and inner cuticle) and sense probe slideswere void of signal (Figure 2a and b). This indicatedthat the in situ hybridization procedure was opti-

mized for signal strength and produced a negativesense control. The BHD riboprobe, designed fromthe 30 end of the BHD gene cDNA, hybridized to the

Figure 2 In situ hybridization of a riboprobe from Ker6IRS1 to a (a) longitudinal and (b) cross-section hair follicle from a human scalpsample demonstrated expression specific to the inner root sheath. Top panels, hematoxylin and eosin staining; middle panels, antisenseprobe (green); bottom panels, sense probe. Nuclei counterstained with DAPI (blue). Original magnification, � 100.


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three layers of the inner root sheath as well as to theouter root sheath (Figure 3a and b). Both long-itudinal and cross-sectioned hair follicles werehybridized and no signal was seen in the dermalpapilla, the fibrous root sheath, the arrector pilimuscle or the melanocytes. The sense probe controlslides were void of signal (Figure 3).

The epidermis exhibited strong positive signal inthe keratinocytes of the spinous layer, but a weakersignal in the basal cell layer directly above thedermis (Figure 4a). The stratum corneum and the

granular layer of the epidermis did not show BHDmRNA signal. In the dermis, discrete expression wasseen in a wide variety of cells within the collagenfibers including nerve cells, lymphocytes, macro-phage, mast cells and fibroblasts (below the epider-mis in Figure 4a). The collagen fibers themselves, aswell as the blood vessels, did not fluoresce. Thesebaceous glands branch from the outer root sheathof the hair follicle. The keratinocytes surroundingthe sebaceous glands of the hair follicle and theeccrine glands (sweat glands) in the skin were

Figure 3 In situ hybridization of a riboprobe to BHD mRNA to the inner and outer root sheaths of a (a) longitudinal and (b) cross-sectionhair follicle from a human scalp sample. Top panels, hematoxylin and eosin staining; middle panels, antisense probe (green); bottompanels, sense probe. Nuclei counterstained with DAPI (blue). Original magnification, � 100.


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Figure 4 In situ hybridization of a BHD mRNA probe to tissues involved in the BHD phenotype: skin, kidney and lung. (a) Skin. BHDmRNA signal was highest in the spinous layer of the epidermis with expression in discrete cells of the dermis. (b) Kidney. BHD mRNAexpression was seen in the distal nephron of the kidney. (c) Lung. BHD mRNA expression predominates in the stromal cells, but was alsofound in the type 1 pneumocytes. Left panels, hematoxylin and eosin staining; right panels, antisense probe. Nuclei counterstained withDAPI (blue). Original magnification, � 200 for (a) and (b) and � 200 for (c).


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positive for BHD mRNA expression, but demon-strated less fluorescence than the hair follicle innerroot sheath or outer root sheath (data not shown).

The expression pattern of BHD mRNA in tissuesassociated with the BHD syndrome and othernormal and neoplastic tissues is summarized inTable 1. All sense control slides for tissues werevoid of signal (data not shown).

Expression of BHD mRNA in the Kidney and Lung,Tissues Involved in the BHD Phenotype

In the kidney, in situ hybridization localized BHDmRNA strongly to the distal nephron and collectingduct and showed a weak distribution of signal in theproximal tubules, but no signal in the glomeruli(Figure 4b).

The stromal cells (ie macrophage, fibroblasts,lymphocytes) within the connective tissue of thelung exhibited strong expression of BHD mRNA,while the type 1 pneumocytes showed a weak signalthroughout the lung tissue (Figure 4c). In addition,the macrophage within the spaces of the alveolarsepta showed very strong BHD expression (data notshown).

BHD mRNA Expression in Exocrine Glands and inOther Normal Human Tissues

Since the BHD protein, folliculin, is a novel proteinwith no known function, BHD mRNA expressionwas evaluated on a normal tissue array in order togain information about the role of folliculin in thecell based on the tissue expression pattern. BHDmRNA expression was seen in a wide variety oftissues including tonsils, lymph nodes, spleen,brain, breast, bladder, testis, prostate, ovary, myo-metrium, pancreas and parotid gland.

The serous (acinar) glands of the parotid glandhighly expressed BHD mRNA while the mucinousglands and intercalated ducts did not express BHD(Figure 5a). The enzyme-secreting acinar cells of thepancreas fluoresced, but not the insulin-regulatingislet cells (Figure 5b). In the cerebrum of the brainthe neurons strongly expressed BHD mRNA, but theglial cells did not. Purkinje cells in the cerebellumbordering the granular layer strongly expressed BHDmRNA (Figure 5c). The tonsils highly expressedBHD mRNA in the germinal centers where lympho-cytes proliferate and then progress out (Figure 5d).Signal from the lymphocytes was also seen in thewhite pulp of the spleen, but not in the red pulpwhere red blood cells are found (data not shown).

Tissues with no BHD mRNA Expression

No expression was seen in the heart, colon epithe-lium, mucinous glands of colon and parotid, adrenalgland, liver hepatocytes, stratified muscle, thyroid

and in areas surrounding blood vessels (data notshown).

BHD mRNA Expression Pattern in Fibrofolliculomasand Kidney Tumors from Patients Affected with BHD

Fibrofolliculomas, the dermatologic skin lesionsdiagnostic for the BHD syndrome, were evaluatedfor expression with the BHD riboprobe. Strongexpression of BHD mRNA was observed in theproliferating epithelial strands of fibrofolliculomas,as well as in keratinocytes within the spinous layerof the epidermis and surrounding the sebaceousglands (Figure 6a).

Several cases of renal tumors with chromophobe,clear cell and hybrid oncocytoma histology frompatients with BHD were evaluated for BHD mRNAexpression; almost no expression of BHD mRNA wasdetected in these renal tumors relative to adjacentnormal kidney parenchyma (Figure 6b and c). Lungblebs obtained from BHD patients who were treatedfor a spontaneous pneumothorax showed the sameexpression level of BHD mRNA in stromal cells andtype 1 pneumocytes as seen in the lung tissue ofunaffected individuals (data not shown).

BHD mRNA Expression in Neoplastic Tissues

Neoplastic tissues on a Cancer Tissue Array wereexamined for BHD mRNA expression and comparedwith adjacent normal tissue sections. Several typesof breast carcinomas including infiltrating ductaladenocarcinoma and lobular carcinoma were exam-ined. BHD mRNA was highly expressed in thesebreast carcinomas (Figure 7a) as well as in normalbreast tissue. In normal breast tissue, the epithelialmammary glands highly expressed BHD mRNA withminor expression in the fibroblasts of the surround-ing stroma (Figure 7b). Serous adenocarcinoma andcystadenocarcinoma of the ovary expressed BHD athigh levels, as did adjacent normal ovary tissue(data not shown). In adenocarcinoma of the prostate,the expression of the BHD mRNA in the epithelialcells was much higher than in the adjacent normalprostate tissue (Figure 7c). In normal tissue, thebasal cells lining the prostate glands showed highexpression, but only minor expression was seen inepithelial cells of the glands (Figure 7d).

A tissue array containing renal tumors of varioushistologic types was evaluated for BHD expression.All five histologic variants seen in renal tumors ofBHD patients including clear cell, papillary, onco-cytoma, chromophobe and oncocytic hybrid, wererepresented. These tumor samples were obtainedfrom patients with von Hippel–Lindau disease(clear cell), Hereditary Papillary Renal Carcinoma(papillary) or BHD (oncocytoma, chromophobe andoncocytic hybrid). All renal tumors showed severelyreduced expression of BHD regardless of histology;


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in fact, papillary renal tumors did not show anyBHD expression (data not shown).

Discussion

In an effort to learn more about the biologicalfunction of the BHD gene and its role in the skin,lung and kidney phenotype observed in patientswith BHD syndrome, we evaluated expression ofBHD mRNA in normal and neoplastic human tissuesby fluorescent in situ hybridization. We found thatBHD mRNA was widely, but not universally ex-pressed in the organs and tissues examined withselective expression in certain cell types. Forexample, in the pancreas, BHD mRNA was detected

in the acinar cells, but not in the islets ofLangerhans. In the kidney, BHD mRNA was ex-pressed strongly in the distal nephron, but onlyweakly in the proximal renal tubules, and wasundetectable in glomeruli. BHD mRNA was ex-pressed in keratinocytes of the spinous layer of theepidermis, but only weakly in cells of the basal layerof the epidermis. The expression of BHD mRNA incertain cell types within each tissue may suggestthat BHD participates in particular cellular pro-cesses rather than serving as a housekeeping gene.

Notably, BHD mRNA was expressed in manydiverse tissues and cell types such as the skin andits appendages, lymphocytes in the germinal centersof lymph nodes, neurons in the cerebrum andPurkinje cells in the cerebellum. This wide distribu-

Figure 5 In situ hybridization of a BHD mRNA probe to (a) serous cells of parotid tissue, (b) acinar cells of pancreas tissue, (c) Purkinjecells in the cerebellum of the brain, and (d) lymphocytes in the germinal center of the tonsil. Left panels, hematoxylin and eosin staining;right panels, antisense probe. Nuclei counterstained with DAPI (blue). Original magnification, �100 for (a), (c) and (d), and �200 for (b).


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tion of BHD mRNA expression suggests an impor-tant fundamental role for BHD in biological pro-cesses within a variety of tissues.

The strong expression of BHD in cell types with asecretory function, such as the acinar cells of thepancreas and parotid gland, may suggest a possiblerole of the BHD protein in secretion. Of interest is areport of a parotid oncocytoma in a BHD patient.17

Additionally, strong BHD expression in ductal cellsof the breast, collagen-producing fibroblasts, andantibody-producing lymphocytes lends support to apossible secretory function. Alternatively, BHDmRNA expression in type I pneumocytes, whichplay a role in surfactant turnover by pinocytosis,18

and expression in alveolar macrophage, may suggesta role for BHD protein in endocytosis or phagocy-tosis. Experiments designed to test these and other

possible functions for the BHD protein in cellularprocesses are currently underway.

The primary clinical characteristics of the BHDsyndrome are cutaneous skin lesions known asfibrofolliculomas, spontaneous pneumothorax, anda spectrum of histologic types of renal carcinoma.Examination of the tissues that are the targets of thedisease showed that BHD mRNA was expressedstrongly in the epithelial strands of the fibrofollicu-lomas, but not expressed or expressed weakly inrenal tumors from patients affected with the BHDsyndrome. Germline mutations in the BHD geneaccompanied by somatic inactivation of the remain-ing BHD allele, through LOH (20%)19 or somaticmutations (50%; C Vocke, manuscript in prepara-tion), were identified in renal tumors from BHDpatients. LOH on 17p has been reported in sporadic

Figure 5 (Continued).


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Table 1 BHD mRNA expression in normal and neoplastic tissues

Normal tissue Expressiona Abnormal/neoplastic tissue Expressiona

Skin FibrofolliculomaInner root sheath of hair follicle +++ Epithelial strands of fibrofolliculoma +++Outer root sheath of hair follicle +++ Mucin-rich stroma �Dermal papilla of hair follicle � Collagen bundles �Fibrous root sheath of hair follicle �Matrix cells of hair follicle �Keratinocytes around sebaceous gland +++Eccrine (sweat) glands +++Spinous layer keratinocytes of epidermis +++Basal cell layer of epidermis 7Discrete cells in dermis such as fibroblasts,macrophage and nerve cells

+++

Collagen fibrils of dermis �Kidney Renal neoplasms

Distal tubules and collecting duct +++ Renal oncocytoma 7Proximal tubules 7 Clear cell renal cancer 7Glomeruli � Chromophobe renal cancer 7

Papillary renal cancer �Oncocytic hybrid 7

Lung Lung blebb

Type 1 pneumocytes ++ Type 1 pneumocytes ++Stromal cells including fibroblasts andmacrophage

+++ Stromal cells including fibroblasts andmacrophage

+++

Parotid NAAcinar cells of the serous gland +++Intercalated ducts �Mucinous glands �

Pancreas NAAcinar cells +++Islet cells �

Breast Breast carcinomasAcinar cells of epithelial ducts +++ Adenocarcinoma +++

Lobular carcinoma +++Ductal carcinoma +++Mucinous adenocarcinoma +++Infiltrating ductal adenocarcinoma +++

Prostate Prostate adenocarcinomaBasal cells of the prostate glands +++ Epithelial cells +++Epithelial cells of the prostate gland 7

TonsilGerminal center +++ NA

Spleen NAWhite pulp +++Red pulp �

Brain NANeurons of the cerebrum +++Glial cells of the cerebrum �Purkinje cells of the cerebellum +++

BladderTransitional epithelial +++ NA

Ovary Ovarian cancerMesenchymal cells +++ Serous adenocarcinoma +++

Cystadenocarcinoma +++Mucinous cystadenocarcinoma +++

UterusMyometrium +++ NA

Colon � NAAdrenal gland � NALiver � NAStratified muscle � NAHeart � NAThyroid � NA

aExpression: +++ strong expression; ++ moderate expression; 7 low expression; � no expression.bLung surgical sample removed from BHD patient with pneumothorax.NA: not available.


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Figure 6 In situ hybridization of a BHD mRNA probe to sections from a (a) fibrofolliculoma, (b) chromophobe renal tumor, and a (c)oncocytic hybrid renal tumor. Note the BHD mRNA expression in normal tissue bordering the renal tumor sections (arrows). Left panels,hematoxylin and eosin staining; right panels, antisense probe. Nuclei counterstained with DAPI (blue). Original magnification, �100 for(a), and �200 for (b) and (c).


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renal tumors,20 with one case also carrying a somaticBHD gene mutation.21 These observations suggest atumor suppressor role for BHD, which is furthersupported by the reduced expression of the BHDtranscript in BHD renal tumors.

Studies are ongoing to evaluate tissue expressionof the BHD protein, folliculin, using polyclonalantibodies prepared against synthetic peptides. Theresults of these studies will contribute further to ourunderstanding of the role of the BHD gene product,folliculin, in regulating cell proliferation and growthin skin, lung and kidney.

Acknowledgements

We thank Drs Jorge Toro, George Cotsarelis and PaulDuray for helpful discussions and Dr Steven Hewittfor providing the kidney tumor tissue array. Thispublication has been funded in part with Federalfunds from the National Cancer Institute, NationalInstitutes of Health, under Contract No. N01-C0-12400. The content of this publication does notnecessarily reflect the views or policies of theDepartment of Health and Human Services, nordoes mention of trade names, commercial products,

Figure 7 In situ hybridization of a BHD mRNA probe to sections from (a) ductal carcinoma of the breast, (b) normal breast tissue, (c)adenocarcinoma of the prostate, and (d) normal prostate tissue. Left panels, hematoxylin and eosin staining; right panels, antisenseprobe. Nuclei counterstained with DAPI (blue). Original magnification, � 100.


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or organizations imply endorsement by the USGovernment. Research reported in this manuscriptwas part of a Master’s degree thesis for the GraduateSchool of Hood College in Frederick, Maryland(MBW).

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