WO2006135762A1 - Identification of a folliculin-binding protein fnip1 - Google Patents

Abstract

This disclosure provides a new nucleic acid molecule, FNIPl, and the protein encoded thereby, along with several specific alternate transcript FNIPl sequences and proteins (isoforms). The disclosure further provides methods for identifying mutant FNIPl proteins or nucleic acids in a subject, and using them to determine or predict a subject's BHD disease or hamartomatous syndrome state. Also disclosed are methods, including methods of screening for compounds for the treatment of BHD syndrome and other hamartomatous syndromes, such as tuberous sclerosis complex, Peutz- Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome, and methods of treatment.

Description

IDENTIFICATION OF A NOVEL BINDING PROTEIN FNIPl

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/689,749, filed June 9, 2005, and No. 60/697,685, filed My 8, 2005, both of which are incorporated herein by reference in their entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to methods and compositions that influence hamartomatous syndromes, for example Birt-Hogg-Dube syndrome. In particular, the present disclosure relates to nucleic acids encoding the folliculin binding protein-1 (FNIPl), methods of using the nucleic acids and proteins encoded thereby, and methods of screening for compounds that influence FNIPl binding to folliculin and AMPK.

BACKGROUND

Renal cell carcinoma is an important health problem in the United States, affecting 32,000 individuals each year and resulting in 12,000 deaths annually (Jemal et al, Cancer J Clin. 54(l):8-29, 2004). Although the majority of cases (~96%) are sporadic, studies of inherited renal cancer syndromes that predispose individuals to renal epithelial tumors have provided us with a growing understanding of the genetic basis of renal cancer. Several familial cancer disorders with a renal epithelial tumor phenotype have been well characterized and the causative genes have been identified (Pavlovich and Schmidt, Nat Rev Cancel- 4(5):381-93, 2004), including the Birt-Hogg-Dube (BHD) syndrome.

The triad of dermatologic lesions, including fibrofolliculomas, trichodiscomas and achrocordons, known as the Birt-Hogg-Dube (BHD) syndrome, was originally described in a

Canadian kindred in 1977 (Birt ^ α/., ^τ-cA. Dermatol. 113:1674-1677, 1977). Other phenotypic features were found to be associated with BHD syndrome, including renal neoplasia (Roth et ah, J. Amer. Acad. Derm. 29:1055-1056, 1993) and lung cysts and/or spontaneous pneumothorax (Toro et ah, Arch Dermatol. 135:1195-1202, 1999). When adjusted for age, patients with fibrofolliculomas (benign tumors of the hair follicle) have about a seven-fold increased risk for developing renal neoplasms and a 50-fold increased risk for developing spontaneous pneumothorax compared with their unaffected siblings. Lung cysts develop frequently (83%) in affected members of BHD families (Zbar et a , Cancer Epidem. Bio. Prev. 11 :393-400, 2002). Although colon polyps have been reported in BHD patients (Hornstein et ah, Hum. Genet. 33:193-197, 1976; Hornstein et ah, Arch. Derm. Res. 253:161-175, 1975), the frequency is not statistically significant compared to unaffected siblings (Zbar et ah, Cancer Epidem. Bio. Prev. 11:393-400, 2002). SUMMARY OF THE DISCLOSURE

It has now been found that a protein referred to as folliculin interacting protein 1 (FNIPl) specifically binds to the BHD protein, folliculin. The FNIPl full-length protein and cDNA are disclosed herein, as well as additional naturally-occurring FNIPl isoforms and the nucleic acids that encode them. Also disclosed are purified antibodies that selectively bind to an epitope of FNIPl protein. Without being bound by theory, it is believed that mutations that alter FNIPl expression or change its binding to folliculin or AMP-activated protein kinase (AMPK) are involved in, and in some cases causative of, BHD syndrome because mutations that alter folliculin or folliculin expression lead to BHD syndrome. Also disclosed is a method of identifying an agent having potential to treat a hamartomatous condition, of which BHD syndrome is an example. The method includes contacting with at least one test agent a cell comprising a nucleic acid sequence encoding a FNIPl protein or a reporter gene operably linked to a FNIPl transcription regulatory sequence, and detecting a change in expression or activity of the FNIPl protein or the reporter gene in the cell. In this method, an agent that alters the expression or activity of the FNIPl protein or the reporter gene in the cell is identified as an agent having potential to treat the hamartomatous condition.

Another embodiment is an assay to detect a change in binding of a binding partner of FNIPl. This method includes contacting a FNIPl polypeptide together with at least one binding partner polypeptide (folliculin or AMPK) with at least one test agent under conditions that would permit the FNIPl polypeptide and the binding partner polypeptide to bind to each other in the absence of the test agent, and determining whether the test agent affects the binding of the FNIPl polypeptide with the binding partner polypeptide(s). A change in the binding of the FNIP 1 polypeptide with the binding partner polypeptide identifies the test agent as an agent having potential to treat the hamartomatous condition. Further embodiments are methods for identifying an agent having the potential to be a FNIPl peptidomimetic. The method includes contacting at least one test agent with an antibody specific for a FNIPl polypeptide, and a test agent that is specifically bound by the antibody is identified as an agent having potential to be a FNIPl peptidomimetic.

Still other embodiments include a method for treating a hamartomatous condition, which includes administering to a subject a therapeutically effective amount of a FNIP 1 protein or a nucleic acid encoding the FNIPl protein or a FNIPl peptidomimetic In other embodiments, the method is a method of treating a hamartomatous condition in a subject by administering a small molecule inhibitor of FNBPl to the subject, thereby treating the hamartomatous condition.

Yet still other embodiments are methods of detecting a biological condition associated with a mutant FNIPl nucleic acid in a subject. Such methods include determining whether the subject has mutant FNIPl nucleic acid.

The foregoing and other features and advantages will become more apparent from the following detailed description of a several embodiments. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is set of schematics and digital images demonstrating that FNIPl binds to folliculin, and showing a sequence alignment, structural diagram, and expression pattern for FNIPl. FIG. IA is a digital image of a gel showing that folliculin binds to FNIP-I . Stably transfected HEK293 cells that expressed HA-tagged wild type or mutant folliculin in a doxycycline dependent manner were lysed and itnmunoprecipitated with anti-HA antibody. Immunoprecipitated proteins were eluted with HA peptide and separated by SDS-PAGE and transferred to PVDF membranes followed by colloidal gold staining. The FNIPl band was excised and digested followed by mass spectrometry. Arrows in doxy÷ lanes indicate FNIPl and folliculin. FIG. IB is a diagram showing the sequence alignment of FNIPl from several mammalian species. Highly conserved residues are indicated by asterisks (*), and residues from a conserved amino acid class are indicated by double dots. Five sequence blocks were identified with at least 35% similarity among species (FIG. 1C). FNIPl was shown to be a novel protein that does not share any homology to known proteins. FIG. ID is a schematic diagram showing the structure of FNIPl. Full length FNIPl cDNA (C16, C17, C48) and four alternative transcripts (C41; SEQ ID NO: 3, C20; SEQ ID NO: 5, C5; SEQ ID NO: 7, and ClO; SEQ ID NO: 9) were PCR-amplified from a human pooled tissue cDNA library. The verified exon-intron structures are shown. Mammals have another isoform (KIAA1450). Drosophila and C. elegans have a single isoform. FIG. IE is a digital image of an autoradiogram of a gel showing the FNIPl expression pattern in mammalian adult tissues. A cDNA probe was generated by PCR using Forward (SEQ ID NO: 21) and Reverse (SEQ ID NO: 22) primers and hybridized to a Clontech normal tissue Northern blot. FNIPl expression was seen in most human tissues in a pattern similar to BHD, with strongest expression in peripheral blood leukocytes, placenta, heart, and brain.

FIG 2 is a set of digital images showing that folliculin interacts with FNIP 1. FIG. 2A is a digital image showing that exogenous folliculin interacts with exogenous FNIP 1. HA-folliculin was cotransfected with FLAG-FNIPl into 293HEK cells. Cell lysates were immunoprecipitated with either anti-FLAG or anti HA antibodies and blotted with anti-FLAG and anti HA antibodies. Empty vector controls were transfected as indicated. FIG. 2B is a digital image showing further confirmation of the interaction between folliculin and FNIPl in vivo. HEK293 lysates were immunoprecipitated with anti-folliculin antibody 104 or anti-FNIPl antibody 181. Immunoprecipitates were blotted with anti-folliculin antibody 102 or anti-FNIPl antibody 181. The arrows indicate endogenous folliculin and endogenous FNIPl proteins. FIG. 2C is a digital image showing the colocalization of FNIPl and folliculin in the cytoplasm of HeLa cells. HeLa cells were cotransfected with HA-FNIPl and FLAG folliculin. After fixation, cells were incubated with anti HA antibody and anti FLAG antibody followed by incubation with anti-mouse Cye3 (red) labeled antibody and anti-rabbit Alexa 488

(green) labeled antibody, respectively, and viewed by confocal microscopy. FNIPl and folliculin colocalize with a reticular pattern in the cytoplasm with strong signal in the Golgi apparatus; nuclear localization is seen only for folliculin. FIG. 2D is a digital image showing that the carboxy-terminal - A -

region of folliculin interacts with FNIPl in vivo. HA-FNIPl and a series of FLAG tagged folliculin deletion mutants were cotransfected into HEK293 cells. Cell lysates were immunoprecipitated with anti-HA antibody followed by Western blotting with anti-FLAG or anti-HA antibody (right panel). Relative expression of the folliculin deletion mutants in the cell lysate is shown by Western blotting with anti-FLAG antibody (left panel). Lower inserts show HA-FNIPl expression. Residues 246-579 of folliculin gave the most productive FNIPl binding relative to full length protein. FIG. 2E is a digital image showing that FNIPl directly interacts with the carboxy-terminal region of folliculin in vitro. Recombinant GST fusion full-length folliculin and deletion mutants were expressed by transduction of baculovirus expression constructs into SF9 insect cells. Crude insect cell lysates were used as the source of the GST-proteins. Radiolabeled in vitro transcribed and translated (IVT) FNIPl was generated, mixed with the GST fusion proteins immobilized on glutathione-Sepharose beads, then run on SDS-PAGE and visualized by autoradiography. Coomassie staining shows the relative expression of the GST-fusion folliculin fragments and full length protein. Residues 246-579 and 345- 579 gave the most productive binding with IVT-FNIPl relative to full-length folliculin. FIG. 3 is a set of digital images showing that FNIPl interacts with the γ 1 subunit of AMP kinase. FIG. 3 A shows that FNIPl interacting proteins were immunoprecipitated with anti HA antibody from HEK 293 cells stably expressing doxycycline-inducible HA-FNIPl . The proteins were separated by SDS-PAGE, transferred to PVDF membrane and analyzed by mass spectrometry. The 40KDa protein was identified as the γ 1 subunit of AMP-activated protein kinase. Other interacting proteins were identified as folliculin (67Kda), Hsp 90 (90Kda), α-tubulin (55 Kda) and 14-3-3Θ

(33Kda). The 130Kda protein was confirmed as FNIPl. FIG. 3B shows that the AMPK heterotrimer binds directly to the folliculin:FNIPl complex in a FNIPl -dependent manner, but not to folliculin alone. HEK293 cells were co-transfected with HA-folliculin-expressing constructs with and without FLAG-FNIPl -expressing constructs. The immunoprecipitates were evaluated by Western blotting with antibodies to the three AMPK subunits and to HA-folliculin and FLAG-FNIP 1. The cell lysates fromHEK293 cells overexpressing HA-folliculin showed significant endogenous levels of the AMPK α, β and γ subunits independent of FNIPl overexpression (FIG. 3B, lanes 1 and T). All three AMPK subunits immunoprecipitated with HA-folliculin in a FLAG-FNIPl -dependent manner (FIG. 3B, lane 4). These data indicate that the AMPK heterotrimer, consisting of α, β and γ subunits, binds directly to the folliculin:FNIPl complex in a FNIPl -dependent manner, but not to folliculin alone. FIG. 3C shows that Folliculin exists in at least three electrophoretically distinct, phosphorylated forms when overexpressed in untreated HEK293 cells: a single, fast migrating form upon treatment with calf alkaline phosphatase (general phosphatase) or protein phosphatase 1 (serine/threonine-specific phosphatase) and at least two slower migrating forms upon treatment of cells with the serine/threonine specific phosphatase inhibitor, Calyculin A. FIG. 3D shows that, using a monoclonal antibody to folliculin, at least three electrophoretically distinct forms of endogenous folliculin were detected in HEK 293 cells, which shifted to the slower migrating (phosphorylated) species upon immunoprecipitation with anti FNIPl antibody Abl81 to endogenous FNIPl. FIG. 3E shows that, in the UOK257 renal tumor cell line with a BHD (-/-)genotype, endogenous FNIPl was able to bind to the AMPKβ sύbunit, and productive AMPK-FNIPl binding was not affected by restoration of folliculin expression by stable lentiviral transduction of wild type BHD (UOK257-2 and UOK257-6). These data support the observation in cells overexpressing folliculin and FNIPl, that AMPK-FNIPl interaction is folliculin-independent.

FIG.4 is a parr of digital images showing that endogenous AMPK activity is detected in FNIPl:folliculin immunoprecipitates from FNIPl- and folliculin-over-expressing cells. FIG. 4A shows that AMPK activity was measured in an in vitro kinase assay with SAMS peptide, a specific substrate for AMPK, with and without a functional FNIP:folliculin complex. Stably expressing, doxycyclrne-inducible HA-folliculin HEK 293 cells were transfected with FLAG-FNIP 1 , and anti HA immunoprecipitates were assayed for endogenous AMPK activity with SAMS peptide and γ-³²P-ATP. Ten-fold greater γ-³²P-ATP incorporation into SAMS peptide was observed in anti-HA immunoprecipitates from cells expressing wild-type HA-folliculin which binds to FNIPl compared with cells expressing a truncated mutant form of folliculin (c.C2034T, R527X) that does not bind FNIPl. FIG. 4B shows that HEK293 cells cotransfected with FLAG-FNIPl (pl30) and HA-folliculin revealed the presence of a slower migrating HA-folliculin form in immunoprecipitates from cells overexpressing FLAG-FNIPl, but not in those lacking FNIP.

FIG. 5 is a pair of diagrams showing the AMPK phosphorylation sites in human folliculin (FIG. 5A) and conservation of these sites within folliculin sequences from a variety of species (FIG. 5B).

FIG. 6 is a diagram showing four AMPK phosphorylation sites in human FNIP 1 and conservation of these sites in FNIPl from a number of species.

FIG. 7 is a digital image of an autoradiogram of a gel showing that recombinant GST- folliculin (but not GST alone) was phosphorylated in an in vitro kinase assay with partially purified AMPK from rat liver and Y³²P-ATP, that the phosphorylation was activated by 200μM AMP, an allosteric effector of AMPK, and partially inhibited by competition with SAMS peptide, specific substrate for AMPK, at lOOμM.

FIG 8 is a digital image of an autoradiogram of a gel showing that GST-folliculin, immobilized on glutathione-Sepharose beads, was phosphorylated by a partially purified preparation of AMPK in an in vitro kinase assay with γ³²P-ATP , and that the phosphorylation was activated by AMP. Furthermore, GST-FNIPl, in a GST-folliculin:GST-FNIPl complex, was also phosphorylated by AMPK in an in vitro kinase assay, and this phosphorylation was also activated by AMP. The lower panel shows Coomassie Blue staining of the GST-folliculin and GST-FNIPl proteins.

FIG. 9 is a digital image of an autoradiogram of a gel showing folliculin phosphorylation facilitated by FNIPl expression is partially regulated through mTOR signaling. FIG. 9A shows that Folliculin phosphorylation is affected by mTOR activity. UOK257-2 BHD restored cells were cultured under different culture conditions: S(+), with serum; S(-), serum starvation for 24 hours; S(- )-> S(+), 20% dialyzed serum stimulation for 30 minutes after 24 hours serum starvation; a.a.(-), amino acid starvation for 4 hours with Earl's balanced solution containing vitamins, pyruvate and glucose; a.a.(-)-> a.a.(+), 2X amino acid stimulation for 30 minutes after 4 hours amino acid starvation. Cells were pretreated before harvest or stimulation with various inhibitors: N/T, non treatment; CaIA (CalyculinA) 100 nM for 30 minutes; U0126, 5 μM for 2 hours; Wort (Wortmannin), 1 μM for 2 hours; Rapa (Rapamycin), 20 nM for 2 hours. FIG. 9B shows that Folliculin phosphorylation facilitated by FNIPl expression is partially regulated by mTOR activity. HA-FNIPl- inducible HEK293 cells were cultured ± doxycycline. For starvation conditions, cells were cultured with Earl's balanced solution containing vitamins, pyruvate and glucose for 16 hours ± doxycycline, followed by stimulation with 2X amino acids.

FIG. 10 is a digital image of an exposed x-ray film of a gel showing that BHD-null cells are more sensitive to extracellular amino acid starvation than 5i/Z)-restored cells. UOK257 (BHD-null), UOK257-2 and UOK257-6 (5HD-restored) cells were cultured with complete DMEM. After 24 hours culture, cells were starved for amino acids with Earl's balanced solution containing dialyzed FBS, vitamins, pyruvate and glucose for different time periods.

FIG. 11 is a digital image showing that expression levels of FNIP I are high in sporadic renal tumors derived from the distal nephron compared with normal kidney. FNIPl mRNA expression were measured by RQ-PCR normalized to β-actin mRNA expression in samples extracted from sporadic RCC. Note subpopulation of outlier clear cell renal tumor samples that expressed higher levels of FNIPl. BHD expression was strongest in the distal nephron of normal kidney and absent from BHD-associated chromophobe renal tumors. Cl, clear-cell RCC; Pap, papillary RCC; Chr, chromophobe RCC; Onco, oncocytoma; CD, collecting duct tumor; NK, normal kidney tissue; N, number of samples; CI, confidence interval.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

The nucleic acid and protein sequences listed in the application and/or the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and triple letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

SEQ ID NO: 1 shows the nucleotide sequence of human FNIPl encoding sequence.

SEQ ID NO: 2 shows the amino acid sequence of human FNIPl.

SEQ ID NO: 3 shows the nucleotide sequence of human FNIPl alternate transcript C41.

SEQ ID NO: 4 shows the amino acid sequence of human FNIPl isoform C41. SEQ ID NO: 5 shows the nucleotide sequence of human FNIPl alternate transcript C20.

SEQ ID NO: 6 shows the amino acid sequence of human FNIPl isoform C20.

SEQ ID NO: 7 shows the nucleotide sequence of human FNIPl alternate transcript C5.

SEQ ID NO: 8 shows the amino acid sequence of human FNIPl isoform C5. SEQ ID NO: 9 shows the nucleotide sequence of human FNIPl alternate transcript ClO. SEQ ID NO: 10 shows the amino acid sequence of human FNIPl isoformClO. SEQ ID NO: 11 shows the nucleotide sequence of the human FLCN gene. SEQ BD NO: 12 shows the amino acid sequence of human folliculin. SEQ DD NOs: 13 and 14 show the nucleotide sequence of FNIPl cDNA sense and antisense primers, respectively.

SEQ ID NOs: 15 and 16 show the nucleotide sequence of FNIPl N-terminal fragment antisense and sense primers, respectively.

SEQ ID NOs: 17 and 18 show the nucleotide sequence of FNIPl attB sense and antisense primers, respectively.

SEQ DD NO: 19 shows the amino acid sequence of an immunogenic FNIPl peptide. SEQ ID NO: 20 shows the amino acid sequence of the SAMS substrate peptide. SEQ ID NOs: 21 and 22 show the nucleotide sequence of the FNIPl Forward and reverse primers (respectively) used herein for Northern Blots. SEQ ID NO: 23 shows the nucleotide sequence of human FNIPl alternate transcript ClO; it is identical to SEQ ID NO: 9, and is included for the purpose of illustrating the amino acid sequence shown in SEQ ID NO: 24.

SEQ ID NO: 24 shows the amino acid sequence of a protein generated from an alternate reading frame of human FNIPl isoform ClO nucleic acid. SEQ ID NO: 25 shows the nucleotide sequence of the FNIPl N-terminal sense primer.

SEQ ID NO: 26 shows the nucleotide sequence of the FNIPl C-terminal fragment antisense primer.

SEQ DD NO: 27 shows the amino acid sequence of a folliculin peptide from the N- terminus, that was used to generate Antibody 102.

DETAILED DESCRIPTION I. Abbreviations

AMPK: AMP-activated protein kinase

BHD: Birt-Hogg-Dube bp: base ρair(s)

BRRS: Bannayan-Riley-Ruvalcaba syndrome

BSA: bovine serum albumin

DMEM: Dulbecco's modified Eagle's medium

DNA: deoxyribonucleic acid ELISA: enzyme-linked immunosorbant assay

FBS: fetal bovine serum

FLCN: folliculin protein

FNIPl : folliculin interacting tmrtein 1

GS: glutathione-Sepharose HPLC: high performance liquid chromatography

HRP: horseradish peroxidase

IVT: in vitro transcription/translation

MALDI-TOF/MS: matrix-assisted laser desoxption/ionization time-of-flight mass spectrometry

PBS: phosphate buffered saline

PCR: polymerase chain reaction

PJS: Peutz-Jeghers syndrome PVDF: polyvinylidene difluoride

TSC: tuberous sclerosis complex

//. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et at (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182- 9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitate review of the various embodiments of the invention, the following explanations of specific terms are provided:

Agent: Any substance (such as, an atom, molecule, molecular complex, chemical, peptide, protein, protein complex, nucleic acid, or drug) or any combination of substances that is useful for achieving an end or result; for example, a substance or combination of substances useful for inhibiting gene expression or inhibiting protein activity, or useful for modifying or interfering with protein-protein interactions. Similarly, a "component" is any substance (such as, an atom, molecule, molecular complex, chemical, peptide, protein, protein complex, nucleic acid, or drug) that is useful for achieving an end or result.

Altered expression: Expression of a biological molecule (for example, mKNA or protein) in a subject or biological sample from a subject that deviates from expression if the same biological molecule in a subject or biological sample from a subject having normal characteristics for the biological condition associated with the molecule, for example in the absence of characteristics of BHD syndrome, such as fibrofolliculoma, renal neoplasia, or spontaneous pneumothorax. Normal expression can be found in a control, a standard for a population, etc. For instance, characteristics of normal expression might include an individual who is not suffering from BHD syndrome or other hamartomatous syndromes, a population standard of individuals believed not to be suffering from BHD syndrome or other hamartomatous syndromes, etc.

Altered expression of a biological molecule may be associated with a disease. The term "associated with" includes an increased risk of developing the disease as well as the disease itself. For instance, certain altered expression, such as altered FNIPl nucleic acid or protein expression, may be associated with BHD syndrome and other hamartomatous syndromes, such as tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley- Ruvalcaba syndrome.

Altered protein expression, such as altered FNIPl protein expression, refers to expression of a protein that is in some manner different from expression of the protein in a normal (wild type) situation. This includes but is not necessarily limited to: (1) a mutation in the protein such that one or more of the amino acid residues is different; (2) a short deletion or addition of one or a few amino acid residues to the sequence of the protein; (3) a longer deletion or addition of amino acid residues, such that an entire protein domain or sub-domain is removed or added; (4) expression of an increased amount of the protein, compared to a control or standard amount; (5) expression of an decreased amount of the protein, compared to a control or standard amount; (6) alteration of the subcellular localization or targeting of the protein; (7) alteration of the temporally regulated expression of the protein (such that the protein is expressed when it normally would not be, or alternatively is not expressed when it normally would be); and (8) alteration of the localized (for example, organ or tissue specific) expression of the protein (such that the protein is not expressed where it would normally be expressed or is expressed where it normally would not be expressed), each compared to a control or standard.

Controls or standards appropriate for comparison to a sample, for the determination of altered expression, include samples believed to express normally as well as laboratory values, even though possibly arbitrarily set, keeping in mind that such values may vary from laboratory to laboratory. Laboratory standards and values may be set based on a known or determined population value and may be supplied in the format of a graph or table that permits easy comparison of measured, experimentally determined values.

Analog, derivative or mimetic: An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28). A derivative is a biologically active molecule derived from the base structure. A mimetic is a molecule that mimics the activity of another molecule, such as a biologically active molecule. Biologically active molecules can include chemical structures that mimic the biological activities of a compound. Animal: Living multi-cellular vertebrate organisms, a category that includes for example, mammals and birds. Antibody: An intact immunoglobulin or an antigen-binding portion thereof.

Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antigen-binding portions include, inter alia, Fab, Fab', F(ab')_2> Fv, dAb (Fd), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides (including fusion proteins) that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. A Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CHl domains; an F(ab')₂ fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consists of the VH and CHI domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment consists of a VH domain (see, for example, Ward et al, Nature, 341:544-546, 1989).

The terms "bind specifically" and "specific binding" refer to the ability of a specific binding agent (such as, an antibody) to bind to a target molecular species in preference to binding to other molecular species with which the specific binding agent and target molecular species are admixed. A specific binding agent is said specifically to "recognize" a target molecular species when it can bind specifically to that target.

A "single-chain antibody" (scFv) is a genetically engineered molecule containing the VH and VL domains of one or more antibody(ies) linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, for example, Bird et al, Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Set, 85:5879-5883, 1988). Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, for example, Holliger et al, Proc. Natl. Acad. Set, 90:6444-6448, 1993; Poljak et al, Structure,

2: 1121-1123, 1994). One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make the resultant molecule an immunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to specifically bind to a particular antigen of interest. A chimeric antibody is an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies.

An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a "bispecific" or "bifunctional" antibody has two different binding sites.

A "neutralizing antibody" or "an inhibitory antibody" is an antibody that inhibits at least one activity of a polypeptide, such as by blocking the binding of the polypeptide to a ligand to which it normally binds, or by disrupting or otherwise interfering with a protein-protein interaction of the polypeptide with a second polypeptide. An "activating antibody" is an antibody that increases an activity of a polypeptide.

Aptamer: A single-stranded nucleic acid molecule (such as DNA or RNA) that assumes a specific, sequence-dependent shape and binds to a target protein with high affinity and specificity. Aptamers generally comprise fewer than 100 nucleotides, fewer than 75 nucleotides, or fewer than 50 nucleotides. "Mirror-image aptamer(s)" (also called Spiegelmers™) are high-affinity L-enantionieric nucleic acids (for example, L-ribose or L-2'-deoxyribose units) that display high resistance to enzymatic degradation compared with D-oligonucleotides (such as aptamers). The target binding properties of mirror-image aptamers are designed by an in vzYrø-selection process starting from a randompool of oligonucleotides, as described for example, in Wlotzka et ah, Proc. Natl. Acad. Sd. 99(13):8898-8902, 2002. Applying this method, high affinity mirror-image aptamers specific for a polypeptide can be generated.

Bannayan-Riley-Ruvalcaba syndrome (BRRS): A rare inherited disorder characterized by excessive growth before and after birth, an abnormally large head (macrocephaly) that is often long and narrow (scaphocephaly), normal intelligence or mild mental retardation, and benign tumor- like growths (hamartomas) that, in most cases, occur below the surface of the skin (subcutaneously). Bannayan-Riley-Ruvalcaba syndrome is inherited as an autosomal dominant genetic trait, and is the name used to denote the combination of three conditions formerly recognized as separate disorders: Bannayan-Zonana syndrome, Riley-Smith syndrome, and Ruvalcaba-Myhre-Smith syndrome. The symptoms of this disorder vary greatly from case to case.

In most cases, infants with Bannayan-Riley-Ruvalcaba syndrome exhibit increased birth weight and length. As affected infants age, the growth rate slows and adults with this disorder often attain a height that is within the normal range. Additional findings associated with Bannayan-Riley- Ruvalcaba syndrome include eye (ocular) abnormalities such as crossed eyes (strabismus), widely spaced eyes (ocular hypertelorism), deviation of one eye away from the other (exotropia), and/or abnormal elevation of the optic disc so that it appears swollen (pseudopapilledema). In addition, affected infants may also have diminished muscle tone (hypotonia), excessive drooling, delayed speech development, and/or a significant delay in the attainment of developmental milestones such as the ability to sit, stand, and walk. In some cases, multiple growths (hamartomatous polyps) may develop within the intestines (intestinal polyposis), and in rare cases, the back wall of the throat (pharynx) and/or tonsils.

Additional abnormalities associated with this disorder may include abnormal skin coloration (pigmentation) such as areas of skin that may appear "marbled" (cutis marmorata) and/or the development of freckle-like spots (pigmented macules) on the penis in males or the vulva in females. In some cases, affected individuals may also have skeletal abnormalities and/or abnormalities affecting the muscles (myopathy).

Binding or stable binding to a nucleic acid: An oligonucleotide binds or stably binds to a target nucleic acid if a sufficient amount of the oligonucleotide forms base pairs or is hybridized to its target nucleic acid, to permit detection of that binding. Binding can be detected by either physical or functional properties of the target:oligonucleotide complex. Binding between a target and an oligonucleotide can be detected by any procedure known to one skilled in the art, including both functional and physical binding assays. Binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation and the like.

Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures. For example, one method that is widely used, because it is so simple and reliable, involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at 220 to 300 nm as the temperature is slowly increased. If the oligonucleotide or analog has bound to its target, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) and the target disassociate from each other, or melt.

The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (T_m) at which 50% of the oligomer is melted from its target. A higher (T_n,) means a stronger or more stable complex relative to a complex with a lower (T_n,).

Biological sample: Any sample in which the presence of a protein and/or ongoing expression of a protein may be detected. Suitable biological samples include samples containing genomic DNA or RNA (including mRNA), obtained from body cells of a subject, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material.

BHD Protein: (see Folliculin). cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments

(introns) and transcriptional regulatory sequences. cDNA can also contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.

DNA (deoxyribonucleic acid): A long chain polymer that comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid (RNA)). The repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule. Except where single-strandedness is required by the text herein, DNA molecules, though written to depict only a single strand, encompass both strands of a double-stranded DNA molecule. Thus, a reference to the nucleic acid molecule that encodes a specific protein, or a fragment thereof, encompasses both the sense strand and its reverse complement. Thus, for instance, it is appropriate to generate probes or primers from the reverse complement sequence of the disclosed nucleic acid molecules.

Deletion: The removal of a sequence of DNA, the regions on either side being joined together. Effective amount of a compound: A quantity of compound sufficient to achieve a desired effect in a subject being treated. An effective amount of a compound can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of the compound will be dependent on the compound applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the compound. The general term "administering to the subject" is understood to include all animals (for example, humans, apes, dogs, cats, horses, and cows) that have or may develop a tumor. Encode: A polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof. For example, the full-length FNIPl cDNA sequence (SEQ ID NO: 1) encodes the full-length FNIPl polypeptide. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

FNIPl protein: Folliculin interacting protein 1 (FNIPl) specifically binds to the BHD protein, folliculin. The FNIPl full-length protein and cDNA are disclosed herein (SEQ ID NOs: 1 and 2), as well as additional naturally-occurring FNIPl isoforms and the nucleic acids that encode them (SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10). Without being bound by theory, it is believed that mutations that alter FNIPl expression or change its binding to folliculin are involved in, and in some cases causative of, BHD syndrome because mutations that alter folliculin or folliculin expression lead to BHD syndrome.

As disclosed herein, FNIP 1 also binds to AMP-activated protein kinase (AMPK), which is a member of the mTOR pathway that has been implicated in a number of different hamartomatous conditions. Thus, without being bound by theory, it is believed that mutations that alter FNIP 1 expression or change its binding to AMPK are involved in, and in some cases causative of, hamartomatous syndromes such as BHD syndrome.

Folliculin: The protein encoded by the BHD gene, mutation in which causes BHD syndrome (Schmidt et al, Am J Hum Genet. 76(6): 1023-33, 2005). Folliculin (also known as the BHD protein) has a coiled-coil domain, three myristylation sites, and an N-glycosylation site.

Identification, characterization, and uses of folliculin are described in U.S. Patent Application No. 10/514,744

Homologs of folliculin have been identified in a number of non-human species. Mouse folliculin is 92% identical to human folliculin. Drosophila melanogaster folliculin (CG8616 gene product) is 22-36% identical (44-56% positive) to human folliculin. Caenorhabditis elegans folliculin (F22D3.2 gene product, AAK31497 protein) is 27-28% identical (44-52% positive) to human folliculin. Mutations in the BHD gene, for example mutations that produce truncated folliculin proteins, lead to BHD syndrome.

Functional fragments and variants of a polypeptide: Included are those fragments and variants that maintain at least one function of the parent polypeptide. It is recognized that the gene or cDNA encoding a polypeptide can be considerably mutated without materially altering one or more of the polypeptide's functions. First, the genetic code is well known to be degenerate, and thus different codons encode the same amino acids. Second, even where an amino acid substitution is introduced, the mutation can be conservative and have no material impact on the essential functions of a protein (see Stryer, Biochemistry 4th Ed., (c) W. Freeman & Co., New York, NY, 1995). Third, part of a polypeptide chain can be deleted without impairing or eliminating all of its functions. For example, sequence variants in a protein, such as a 5 ' or 3' variant, may retain the full function of an entire protein. Fourth, insertions or additions can be made in the polypeptide chain for example, adding epitope tags, without impairing or eliminating its functions (Ausubel et ah, Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley-Intersciences, 1998). Other modifications that can be made without materially impairing one or more functions of a polypeptide include, for example, in vivo or in vitro chemical and biochemical modifications or the incorporation of unusual amino acids. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquination, sumoylation, labeling, for example, with radionucleides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art. A variety of methods for labeling polypeptides and labels useful for such purposes are well known in the art, and include radioactive isotopes such as ³²P, ligands that bind to or are bound by labeled specific binding partners (for example, antibodies), fiuorophores, chemiluminescent agents, enzymes, and antiligands. Functional fragments and variants can be of varying length. For example, a fragment may consist of 10 or more, 25 or more, 50 or more, 75 or more, 100 or more, or 200 or more amino acid residues. In some embodiments, a function of a FNIPl functional fragment is binding to folliculin or AMPK. Gene expression: The process by which the coded information of a nucleic acid transcriptional unit (including, for example, genomic DNA or cDNA) is converted into an operational, non-operational, or structural part of a cell, often including the synthesis of a protein. Gene expression can be influenced by external signals; for instance, exposure of a subject to an agent that inhibits gene expression. Expression of a gene also may be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for instance, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression may be measured at the RNA level or the protein level and by any method known in the art, including Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s). The expression of a nucleic acid may be modulated compared to a control state, such as at a control time (for example, prior to adrninistration of a substance or agent that affects regulation of the nucleic acid under observation) or in a control cell or subject, or as compared to another nucleic acid. Such modulation includes but is not necessarily limited to overexpression, underexpression, or suppression of expression. In addition, it is understood that modulation of nucleic acid expression may be associated with, and in fact may result in, a modulation in the expression of an encoded protein or even a protein that is not encoded by that nucleic acid.

"Interfering with or inhibiting gene expression" refers to the ability of an agent to measurably reduce the expression of a target gene. Expression of a target gene may be measured by any method known to those of skill in the art, including for example measuring mRNA or protein levels. It is understood mat interfering with oτ inhibiting gene expression is relative, and does not require absolute suppression of the gene. Thus, in certain embodiments, interfering with or inhibiting gene expression of a target gene requires that, following application of an agent, the gene is expressed at least 5% less than prior to application, at least 10% less, at least 15% less, at least 20% less, at least 25% less, or even more reduced. Thus, in some particular embodiments, application of an agent reduces expression of the target gene by about 30%, about 40%, about 50%, about 60%, or more. In specific examples, where the agent is particularly effective, expression is reduced by 70%, 80%, 85%, 90%, 95%, or even more. Gene expression is "substantially eliminated" when expression of the gene is reduced by 90%, 95%, 98%, 99% or even 100%.

Hamartomas and hamartomatous conditions: Hamartomatous lesions, or hamartomas, are tumor-like growths which are referred to as tubers. The most common tuber forms of the internal organs are cerebral hamartias and subependymal giant cell astrocytomas of the brain, rhabdomyomas of the heart, and angiomyolipomas of the kidneys. Hamartomas also affect the skin, for example in Birt-Hogg-Dube syndrome. BHD syndrome is characterized by multiple, skin-colored, cutaneous papules located mainly on the face, neck, and upper part of the thorax. These skin changes are hamartomas with folliculo-sebaceous differentiation, called fibrofolliculomas and trichodiscomas. Specific, non-limiting examples of hamartomatous conditions include BHD syndrome, tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, and Bannayan-Riley-Ruvalcaba syndrome.

Heterologous: A type of sequence that is not normally (for example, in the wild-type sequence) found adjacent to a second sequence. In one embodiment, the sequence is from a different genetic source, such as a virus or organism, than the second sequence.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as "base pairing." More specifically, A will hydrogen bond to T or U, and G will bond to C. "Complementary" refers to the base pairing that occurs between to distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence.

Inhibiting protein activity: To decrease, limit, or block an action, function or expression of a protein. The phrase "inhibiting protein activity" is not intended to be an absolute term. Instead, the phrase is intended to convey a wide-range of inhibitory effects that various agents may have on the normal (for example, uninhibited or control) protein activity. Inhibition of protein activity may, but need not, result in an increase in the level or activity of an indicator of the protein's activity. By way of example, this can happen when the protein of interest is acting as an inhibitor or suppressor of a downstream indicator. Thus, protein activity may be inhibited when the level or activity of any direct or indirect indicator of the protein's activity is changed (for example, increased or decreased) by at least 10%, at least 20%, at least 30%, at least 50%, at least 80%, at least 100% or at least 250% or more as compared to control measurements of the same indicator.

Inhibition of protein activity may also be effected, for example, by inhibiting expression of the gene encoding the protein or by decreasing the half-life of the mRNA encoding the protein.

In vitro amplification: When used in reference to a nucleic acid, techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen. An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of in vitro amplification can be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques. Other examples of in vitro amplification techniques include strand displacement amplification (see U.S. Patent No. 5,744,311); transcription-free isothermal amplification (see U.S. Patent No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320308); gap filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S. Patent No. 6,027,889); and NASBA™ RNA transcription-free amplification (see U.S. Patent No. 6,025,134).

Isolated: A biological component (such as a nucleic acid molecule, protein or organelle) that has been substantially completely separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for example, other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

Labeled: A biomolecule attached covalently or noncovalently to a detectable label or reporter molecule. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example, in Sambrook et ah, Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989 and Ausubel et ah, Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley-Intersciences, 1998. For example, ATP can be labeled in any one of its three phosphate groups with radioisotopes such as ³²P or ³³P, or in its sugar moiety with a radioisotope such as ³⁵S.

Lhermitte-Duclos disease: Also known as diffuse cerebellar hypertrophy, gangliocytoma dysplasticum, dysplastic cerebellar gangliocytoma, granular-cell hypertrophy, granule cell hypertrophy of the cerebellum, hamartoma of the cerebellum, purkinjoma, Lhermitte-Duclos disease is a rare pathologic hamartomatous condition with progrediating, diffuse hypertrophy chiefly of the stratum granulosum of the cerebellum. The main clinical signs are headache, movement disorders and tremor, visual disturbances, enlarged head suggesting hydrocephalus, and abnormal EEG. Autosomal dominant inheritance has been demonstrated in some families. Mammal: This term includes both human and non-human mammals. Similarly, the term subject includes both human and veterinary subjects.

Modulator: An agent that increases or decreases (modulates) the activity of a protein as measured by the change in an experimental biological parameter. A modulator can be essentially any compound or mixture (for example, two or more proteins), such as a chemotherapeutic agent, a polypeptide, a hormone, a nucleic acid, a sugar, a lipid and the like.

Mutation: Any change of the DNA sequence within a gene or chromosome. In some instances, a mutation will alter a characteristic or trait (phenotype), but this is not always the case. Types of mutations include base substitution point mutations (for example, transitions or transversions), deletions, and insertions. Missense mutations are those that introduce a different amino acid into the sequence of the encoded protein; nonsense mutations are those that introduce a new stop codon. In the case of insertions or deletions, mutations can be in-frame (not changing the frame of the overall sequence) or frame shift mutations, which may result in the misreading of a large number of codons (and often leads to abnormal termination of the encoded product due to the presence of a stop codon in the alternative frame). This term specifically encompasses variations that arise through somatic mutation, for instance those that are found only in disease cells, but not constitutionally, in a given individual. Examples of such somatically-acquired variations include the point mutations that frequently result in altered function of various genes that are involved in development of cancers. This term also encompasses DNA alterations that are present constitutionally, that alter the function of the encoded protein in a readily demonstrable manner, and that can be inherited by the children of an affected individual. In this respect, the term overlaps with "polymorphism," as defined below, but generally refers to the subset of constitutional alterations that have arisen within the past few generations in a kindred and that are not widely disseminated in a population group. In particular embodiments, the term is directed to those constitutional alterations that have major impact on the health of affected individuals.

Nucleic acid molecule: A polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. A "nucleic acid molecule" as used herein is synonymous with "nucleic acid" and "polynucleotide." A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. Nucleic acid molecules may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendent moieties (for example, polypeptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular and padlocked conformations. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Unless specified otherwise, the left hand end of a polynucleotide sequence written in the sense orientation is the 5' end and the right hand end of the sequence is the 3' end. In addition, the left hand direction of a polynucleotide sequence written in the sense orientation is referred to as the 5' direction, while the right hand direction of the polynucleotide sequence is referred to as the 3' direction. Further, unless otherwise indicated, each nucleotide sequence is set forth herein as a sequence of deoxyribonucleotides. It is intended, however, that the given sequence be interpreted as would be appropriate to the polynucleotide composition: for example, if the isolated nucleic acid is composed of RNA, the given sequence intends ribonucleotides, with uridine substituted for thymidine.

An "anti-sense nucleic acid" is a nucleic acid (such as, an RNA or DNA oligonucleotide) that has a sequence complementary to a second nucleic acid molecule (for example, an mRNA molecule). An anti-sense nucleic acid will specifically bind with high affinity to the second nucleic acid sequence. If the second nucleic acid sequence is an mRNA molecule, for example, the specific binding of an anti-sense nucleic acid to the mRNA molecule can prevent or reduce translation of the mRNA into the encoded protein or decrease the half life of the mRNA, and thereby inhibit the expression of the encoded protein.

Oligonucleotide: A plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide. Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid (PNA) molecules. Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as DNA or RNA) that is at least 6 bases, for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long, or from about 6 to about 50 bases, for example about 10-25 bases, such as 12, 15 or 20 bases. Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Open reading frame: A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide. Ortholog: Two nucleic acid or amino acid sequences are orthologs of each other if they share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are also homologous sequences.

Peutz-Jeghers syndrome (PJS): A hamartomatous, autosomal dominant inherited disorder characterized by intestinal hamartomatous polyps in association with mucocutaneous melanocytic macules. The syndrome confers a 15-fold elevated relative risk of developing cancer over that of the general population. Associated cancers primarily affect the GI tract, including the pancreas and luminal organs, the lung, and the female and male reproductive tracts. The characteristic pathology of Peutz-Jeghers polyps includes extensive smooth muscle arborization throughout the polyp with the appearance of pseudoinvasion because some of the epithelial cells, usually from benign glands, are surrounded by the smooth muscle.

The cause of Peutz-Jeghers syndrome appears to be a germline mutation of the STKIl (serine threonine kinase 11) gene (also known as LKBl or AMPK kinase) in most cases, located on band 19pl3.3. Penetrance of the gene is variable, causing varied phenotypic manifestations among patients with Peutz-Jeghers syndrome (inconsistent number of polyps, differing presentation of the macules) and allowing for a variable presentation of cancer.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful with the compositions provided herein are conventional. Martin, Remington 's Pharmaceutical

Sciences, published by Mack Publishing Co., Eastern, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the nucleotides and proteins herein disclosed.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Pharmaceutical agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell. Incubating includes exposing a target to an agent for a sufficient period of time for the agent to interact with a cell. Contacting includes incubating an agent in solid or in liquid form with a cell. Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The term polypeptide or protein as used herein encompasses any amino acid sequence and includes modified sequences such as glycoproteins. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

The term polypeptide fragment refers to a portion of a polypeptide that exhibits at least one useful epitope. The phrase "functional fragments of a polypeptide" refers to all fragments of a polypeptide that retain an activity, or a measurable portion of an activity, of the polypeptide from which the fragment is derived. Fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell. An epitope is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen. Thus, smaller peptides containing the biological activity of insulin, or conservative variants of the insulin, are thus included as being of use.

The term soluble refers to a form of a polypeptide that is not inserted into a cell membrane. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

In some circumstances, variations in the cDNA sequence that result in amino acid changes, whether conservative or not, are minimized in order to preserve the functional and immunologic identity of the encoded protein. The immunologic identity of the protein may be assessed by determining whether it is recognized by an antibody; a variant that is recognized by such an antibody is immunologically conserved. Any cDNA sequence variant will preferably introduce no more than twenty, and preferably fewer than ten amino acid substitutions into the encoded polypeptide. Variant amino acid sequences may, for example, be 80%, 90%, or even 95% or 98% identical to the native amino acid sequence. Programs and algorithms for determining percentage identity can be found at the NCBI website.

Polymorphism: Variant in a sequence of a gene; usually carried from one generation to another in a population. Polymorphisms can be those variations (nucleotide sequence differences) that, while having a different nucleotide sequence, produce functionally equivalent gene products, such as those variations generally found between individuals, different ethnic groups, geographic locations. The term polymorphism also encompasses variations that produce gene products with altered function, for example, variants in the gene sequence that lead to gene products that are not functionally equivalent. This term also encompasses variations that produce no gene product, an inactive gene product, or decreased or increased activity of the gene product.

Polymorphisms can be referred to, for instance, by the nucleotide position at which the variation exists, by the change in amino acid sequence caused by the nucleotide variation, or by a change in some other characteristic of the nucleic acid molecule or protein that is linked to the variation (for example, an alteration of a secondary structure such as a stem-loop, or an alteration of the binding affinity of the nucleic acid for associated molecules, such as polymerases, KNases, and so forth).

Probes and primers: Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided in this disclosure. A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example, in Sambrook et al. (in Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (in Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley-Intersciences, 1992). Primers are short nucleic acid molecules, preferably DNA oligonucleotides 10 nucleotides or more in length. More preferably, longer DNA oligonucleotides can be about 15, 17, 20, or 23 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example, by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.

Methods for preparing and using probes and primers are described, for example, in Sambrook et al. (in Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (in Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley-Intersciences, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, © 1991 , Whitehead Institute for Biomedical Research, Cambridge, MA). One of ordinary skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 30 consecutive nucleotides of FNIPl -encoding nucleotide sequence will anneal to a target sequence, such as a FNIPl-encoding sequence homolog from the gene family contained within a human genomic DNA library, with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers can be selected that comprise at least 17, 20, 23, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of FNIPl nucleotide sequences.

The disclosure thus includes isolated nucleic acid molecules that comprise specified lengths of the disclosed FNIPl cDNA sequences, including naturally occurring and variant transcripts. Such molecules can comprise at least 17, 20, 23, 25, 30, 35, 40, 45, or 50 consecutive nucleotides of these sequences, and can be obtained from any region of the disclosed sequences. By way of example, the FNIPl cDNA sequences can be apportioned into halves, thirds or quarters based on sequence length, and the isolated nucleic acid molecules can be derived from the first or second halves of the molecules, from any of the three thirds or any of the four quarters. By way of example, the human FNIPl cDNA, ORF, coding sequence and gene sequences can be apportioned into about halves, thirds or quarters based on sequence length, and the isolated nucleic acid molecules (for example, oligonucleotides) can be derived from the first or second halves of the molecules, from any of the three thirds, or any of the four quarters. The cDNA or protein also could be divided into smaller regions, for example about eighths, sixteenths, twentieths, fiftieths and so forth, with similar effect. For more detailed information, see Section H of the Detailed Description.

Another mode of division is to select the 5' (upstream) and/or 3' (downstream) region associated with a FNIPl encoding sequence, or to select an intron or portion thereof.

In addition, several alternative transcripts lacking one or two of the 18 FNIPl coding exons are disclosed herein (SEQ ID NOs: 3, 5, 7, and 9). Thus, primers and probes can readily be selected that distinguish between these transcripts.

Proteus syndrome: A rare hamartomatous condition. It is a complex disorder with multisystem involvement and great clinical variability. This condition is characterized by a variety of cutaneous and subcutaneous lesions including vascular malformations, lipomas, hyperpigmentation, and several types of nevi. Partial gigantism with limb or digital overgrowth is pathognomonic with an unusual body habitus and, often, cerebriform thickening of the soles of the feet. Orthopedic complications often pose the most challenging medical problems, although vascular complications also contribute to overall morbidity. Severe disfigurement and social stigmatization are additional challenges that must be addressed.

Manifestations of the syndrome probably result from somatic mosaicism for a dominant lethal gene, but the gene locus has yet to be identified. Reports of parents possibly transmitting mild cases to their children make this hypothesis questionable. Because hyperplasia and hypoplasia often occur together, another hypothesis suggests that the postzygotic event resulting in these clinical manifestations is embryonic somatic recombination leading to at least three subsets of cells. These subsets include normal, overgrowth (pleioproteus), and atrophy (elattoproteus) cells.

Proteus syndrome is believed to be exceedingly rare, with about 100-200 individuals affected worldwide. This suggests that prevalence is less than 1 per 1,000,000 live births. Purified: In a more pure form than is found in nature. The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell.

The term substantially purified as used herein refers to a molecule (for example, a nucleic acid, polypeptide, oligonucleotide, etc.) that is substantially free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated. In one embodiment, a substantially purified molecule is a polypeptide that is at least 50% free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated. In another embodiment, the polypeptide is at least at least 80% free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated. In yet other embodiments, the polypeptide is at least 90% or at least 95% free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated.

Recombinant: A nucleic acid that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

Ribozyme: RNA molecules with enzyme-like properties, which can be designed to cleave specific RNA sequences. Ribozymes are also known as RNA enzymes or catalytic RNAs. RNA interference (or, RNA silencing or RNAi): A gene-silencing mechanism whereby specific double-stranded RNA (dsRNA) trigger the degradation of homologous mRNA (also called target RNA). Double-stranded RNA is processed into small interfering RNAs (siRNA), which serve as a guide for cleavage of the homologous mRNA in the RNA-induced silencing complex (RISC). The remnants of the target RNA may then also act as siRNA; thus resulting in a cascade effect. Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. By way of example, homologs or orthologs of the FNIPl protein or isomers disclosed herein, and the corresponding cDNA sequences, will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous proteins or cDNAs are derived from species that are more closely related (for example, human and chimpanzee sequences), compared to species more distantly related (for example, human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman J. MoI. Biol. 147(1):195- 197, 1981; Needleman and Wunsch J. MoI. Biol. 48: 443-453, 1970; Pearson and LipmanProc. Natl. Acad. Sci. USA 85: 2444-2448, 1988; Higgins and Sharp Gene, 73: 237-244, 1988; Higgins and Sharp CABIOS 5: 151-153, 1989; Corpet et al. Nuc. Acids Res. 16, 10881-10890, 1988; Huang et al Computer Appls. in the Biosciences 8, 155-165, 1992; and Pearson et al Meth. MoI. Bio. 24, 307- 331, 1994. Furthermore, Altschul et al. (J. MoI. Biol. 215:403-410, 1990) present a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J. MoI. Biol. 215: 403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. The Search Tool can be accessed at the NCBI website, together with a description of how to determine sequence identity using this program.

An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence- dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5° C to 20° C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_n, is the temperature (under defined ionic strength and pH) at which 50% of the target sequence remains hybridized to a perfectly matched probe or complementary strand. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, CSHL, New York and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2, Elsevier, New York. Nucleic acid molecules that hybridize under stringent conditions to a human FNIPl encoding sequence will typically hybridize to a probe based on either an entire human FNIPl encoding sequence or selected portions of the gene under wash conditions of 2x SSC at 50° C. A more detailed discussion of hybridization conditions is presented below. Nucleic acid sequences that do not show a high degree of identity can nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.

Small interfering RNAs: Synthetic or naturally-produced small double stranded RNAs (dsRNAs) that can induce gene-specific inhibition of expression in invertebrate and vertebrate species are provided. These RNAs are suitable for interference or inhibition of expression of a target gene and comprise double stranded RNAs of about 15 to about 40 nucleotides containing a 3' and/or 5' overhang on each strand having a length of 0- to about 5-nucleotides, wherein the sequence of the double stranded RNAs is essentially identical to a portion of a coding region of the target gene for which interference or inhibition of expression is desired. The double stranded RNAs can be formed from complementary ssRNAs or from a single stranded RNA that forms a hairpin or from expression from a DNA vector. Specific binding agent: An agent that binds substantially only to a defined target. Thus a

FNIPl -specific binding agent binds substantially only the FNIPl protein. As used herein, the phrase FNIPl-specific binding agent includes anti-FNIPl antibodies (such as monoclonal antibodies) and other agents (such as soluble receptors) that bind substantially only to FNIP 1. FNIP 1 -specific binding agents can also be produced that bind substantially only to mutant FNIPl and not to wild-type FNIP 1 , or that bind substantially only to wild-type FNIP 1 and not to mutant FNIP 1. Such specific binding agents are described in greater detail below.

Anti-FNIPl antibodies can be produced using standard procedures described in a number of texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). The determination that a particular agent binds substantially only to FNIPl can readily be made by using or adapting routine procedures. One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane, Antibodies, A Laboratory Manual, CSHL, New York, 1988). Western blotting can be used to determine that a given FNIPl binding agent, such as an anti- FNIPl monoclonal antibody, binds substantially only to FNIPl. A phosphospecific binding agent specifically binds to a peptide containing a phosphorylated residue. Shorter fragments of antibodies can also serve as specific binding agents. For instance, Fabs,

Fvs, and single-chain Fvs (SCFvs) that bind to FNIPl would be FNIPl-specific binding agents. These antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (FaV)₂, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')₂, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody (SCA), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine.

Specifically hybridizable and specifically complementary are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide (or its analog) and the DNA or RNA target. The oligonucleotide or oligonucleotide analog need not be 100% complementary to its target sequence to be specifically hybridizable. An oligonucleotide or analog is specifically hybridizable when binding of the oligonucleotide or analog to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide or analog to non- target sequences under conditions where specific binding is desired, for example under physiological conditions in the case of in vivo assays or systems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization, though waste times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, chapters 9 and 11, herein incorporated by reference. The following is an exemplary set of hybridization conditions: Very High Stringency (detects sequences that share 90% identity)

Hybridization: 5x SSC at 65°C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each High Stringency (detects sequences that share 80% identity or greater)

Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: Ix SSC at 55°C-70°C for 30 minutes each Low Stringency (detects sequences that share greater than 50% identity)

Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals.

Target sequence: A "target sequence" is a portion of ssDNA, dsDNA, or RNA that, upon hybridization to a therapeutically effective oligonucleotide or oligonucleotide analog, results in the inhibition of expression of the target. For example, hybridization of therapeutically effectively oligonucleotide to a FNIPl target sequence results in inhibition of FNIPl expression. Either an antisense or a sense molecule can be used to target a portion of dsDNA, as both will interfere with the expression of that portion of the dsDNA. The antisense molecule can bind to the plus strand, and the sense molecule can bind to the minus strand. Thus, target sequences can be ssDNA, dsDNA, and RNA.

Test compound: A test compound can be essentially any compound, such as a chemotherapeutic, a polypeptide, a hormone, a nucleic acid, a sugar, a lipid and the like. Test compounds are used, for example, when screening for compounds with FNIPl -like activity, or for compounds that affect FNIPl binding to folliculin or AMPK. Therapeutically effective amount (e.g., of a FNIPl or a FNIPl antisense oligonucleotide or small molecule inhibitor): A quantity of compound or composition, for instance, FNIPl protein or antisense oligonucleotide or small molecule, sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or to measurably reduce a skin lesion or other symptom associated with BHD syndrome or another hamartomatous syndrome, such as tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome.

An effective amount of a FNIPl protein maybe administered in a single dose, or in several doses, for example daily or more often, during a course of treatment. However, the effective amount of FNIPl or a fragment thereof will be dependent on the FNIPl protein applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the protein.

Alternately, a FNIPl antisense oligonucleotide or FNIPl small inhibitory molecule may be administered in an amount necessary to inhibit or to measurably reduce a skin lesion or other symptom associated with BHD syndrome or another hamartomatous syndrome, such as tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or

Bannayan-Riley-Ruvalcaba syndrome, in a single dose, or in several doses, for example daily or more often, during a course of treatment. However, the effective amount FNIPl antisense oligonucleotide or FNIPl small inhibitory molecule will be dependent on the molecule applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the protein. The proteins disclosed in the present invention have equal application in medical and veterinary settings. Therefore, the general term "subject being treated" is understood to include all animals (for example humans, apes, dogs, cats, horses, and cows) that are or may display a symptom of BHD syndrome (or another hamartomatous syndrome, such as tuberous sclerosis complex, Peutz- Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome) that is susceptible to FNIPl protein-mediated amelioration.

Transfected: A process by which a nucleic acid molecule is introduced into cell, for instance by molecular biology techniques, resulting in a transfected cell. As used herein, the term transfection encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transfection with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

Treating a disease: Includes inhibiting or preventing the partial or full development or progression of a disease, for example in a person who is known to have a predisposition to a disease. Furthermore, treating a disease refers to a therapeutic intervention that ameliorates at least one sign or symptom of a disease or pathological condition, or interferes with a pathophysiological process, after the disease or pathological condition has begun to develop.

Tuberous sclerosis complex (TSC): Also known as Bourneville's Disease and Epilola, TSC is a multisystem hamartomatous disorder which can have a wide range of effects. Approximately one in 8,000 adults and one in 6,000 newborns are affected by TSC. Although TSC is often inherited, new mutations have been implicated in up to 75% of all cases. Males and females of equally likely to have Tuberous Sclerosis, and the chance of passing it on to offspring is 50%.

The physical symptoms of TSC which often lead to its diagnosis include hamartomatous lesions of the brain, heart, kidneys, skin, lungs, and eyes, mental retardation, seizures, autism, fibromas of the finger and toenails, pitted teeth, and dermatological lesions. Three symptoms which constitute a positive diagnosis of TSC are seizures, mental retardation, and adenoma sebaceum, which are known as the clinical triad. People with TSC may exhibit only a few or many of these symptoms.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transfected host cell. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. "Comprises" means "includes." Hence "comprising A or B" means include A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

///. Detailed Description

A. Overview of several embodiments Disclosed herein is the surprising finding that a previously uncharacterized protein, referred to as folliculin interacting protein 1 (FNIPl) herein, specifically binds to the BHD protein, folliculin. The FNIPl full-length protein and cDNA are disclosed herein, as well as additional naturally- occurring FNIPl isoforms and the nucleic acids that encode them. Mutations in the gene encoding folliculin, BHD, have been shown to cause BHD syndrome, which is characterized by multiple, skin- colored, cutaneous papules located mainly on the face, neck, and upper part of the thorax

(hamartomas; Published PCT Application WO 03/102149, which is incorporated by reference herein). It is expected that mutations in FNIPl also cause or influence BHD syndrome. Also disclosed herein is the discovery that FNIPl binds to AMP-activated protein kinase (AMPK), which is an energy-sensing molecule that regulates the mTOR pathway implicated in several familial renal cancer syndromes (see below), as well as a number of hamartomatous diseases. Thus, mutations in FNIPl may also cause or influence hamartomatous diseases such as tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, and Bannayan-Riley-Ruvalcaba syndrome.

Thus, disclosed herein is a purified polypeptide having an amino acid sequence that includes the sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 24. In some embodiments, the sequence includes s sequence having at least 95% or at least 98% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. In particular embodiments, the purified polypeptide includes SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 24 with 1 to 10 conservative amino acid substitutions.

Other embodiments are purified antibodies that selectively bind to an epitope of FNIPl protein, while still others are purified polypeptides that bind specifically to these antibodies. In some examples, the epitope of such an antibody is a region of the FNIPl protein that is truncated in a mutant FNIPl protein associated with BHD syndrome. In particular examples, the antibody has measurably stronger binding to the mutant form of FNIPl protein as compared to a wild-type form of FNIPl protein. Also disclosed herein is an isolated nucleic acid molecule encoding a purified FNIPl polypeptide as described above. In some examples, the nucleic acid molecule includes the sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 or in sequences having at least 90% , at least 95%, or at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. In particular examples, the nucleic acid molecule consists of the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. Also contemplated are recombinant nucleic acid molecules that include a promoter sequence operably linked to these FNIPl nucleic acid sequences, as well as cells transformed with these nucleic acid sequences.

Also disclosed herein is a method of identifying an agent having potential to treat a hamartomatous condition. The method includes contacting with at least one test agent a cell that includes a nucleic acid sequence encoding the FNIPl described above, or a reporter gene operably linked to a FNIPl transcription regulatory sequence, and detecting a change in expression of the FNIPl protein or the reporter gene in the cell. An agent that changes the expression of the FNIPl protein or the reporter gene in the cell is identified as an agent having potential to treat the hamartomatous condition. In some embodiments, the cell is an epithelial cell, a kidney cell, or an immortalized cell, and in other embodiments, detecting the change in the FNIPl protein expression includes analysis by Northern blot, Western blot, RT-PCR, immunohistochemistry, or a combination of two or more thereof. In certain embodiments, the nucleic acid sequence encoding the FNIPl protein is a FNIPl gene in the genome of the cell, and in other embodiments, the method also includes contacting each of a plurality of cells with a member of a library of test agents, wherein each cell includes a nucleic acid sequence encoding the FNIPl described above. In certain examples, the library of test agents includes at least about 100 different agents, and in particular examples, the library of compositions includes one or more natural products, chemical compositions, biochemical compositions, polypeptides, peptides, or antibodies. The hamartomatous condition is BHD syndrome, tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome in some examples of the method. Another embodiment is a method of detecting a change in binding of a binding partner of

FNIPl. The method includes contacting a FNIPl polypeptide and a binding partner polypeptide (either folliculin or AMPK) with at least one test agent under conditions that would permit the FNIPl polypeptide and the binding partner polypeptide to bind to each other in the absence of the test agent, and determining whether the test agent affects the binding of the FNIPl polypeptide and the binding partner polypeptide to each other. The effect on the binding of the FNIPl polypeptide and the binding partner polypeptide to each other identifies the test agent as an agent having potential to treat the hamartomatous condition. In some examples, the method also includes determining whether the test agent specifically binds to the FNIPl polypeptide, and in other examples, the method also includes determining whether the test agent specifically binds to the folliculin polypeptide or the AMPK polypeptide.

In some embodiments, the FNIPl polypeptide comprises at least 15 consecutive amino acids of SEQ ID NO: 2 or at least 15 consecutive amino acids of a polypeptide having 90% sequence identity with SEQ ID NO: 2. In certain embodiments, the folliculin polypeptide includes (a) at least 15 consecutive amino acids of SEQ ID NO: 12, (b) at least 15 consecutive amino acids of a polypeptide having 90% sequence identity with SEQ ID NO: 12, or (c) at least 15 consecutive amino acids of residues 246-579 of SEQ ID NO: 12. In particular embodiments, the effect on the binding of the FNIPl polypeptide and the binding partner polypeptide to each other is either an increase in binding affinity, or a decrease in binding affinity. In certain examples, the FNIPl polypeptide or the binding partner polypeptide is bound to a solid substrate or a soluble support, and in other particular examples, the hamartomatous condition is BHD syndrome, tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome.

Also disclosed herein is a method for identifying an agent having the potential to be a FNIPl peptidomimetic, comprising contacting at least one test agent with an antibody specific for a FNIPl polypeptide, wherein a test agent that is specifically bound by the antibody is identified as an agent having potential to be a FNEPl peptidomimetic. In some examples, the method further includes determining whether the agent having potential to be a FNIPl peptidomimetic can specifically bind a folliculin polypeptide or an AMPK polypeptide. Yet another embodiment is a method for treating a hamartomatous condition that includes administering to a subject a therapeutically effective amount of a FNIPl protein or a nucleic acid encoding the FNIPl protein. In certain examples, the FNIPl protein includes an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 24, and in other embodiments, the hamartomatous condition is BHD syndrome, tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome. In certain examples, the subject is a mammal, for instance a human. In particular examples, administration includes topical administration, intravenous administration, intra-arterial administration, or intraperitoneal administration. Some examples of the method involve administering the FNIPl protein or nucleic acid encoding the FNIPl protein in combination with a folliculin protein or a nucleic acid encoding the folliculin protein, and the nucleic acid encoding the FNIPl protein is a viral vector, a naked DNA, or a liposome-encapsulated DNA in certain examples.

Yet still other methods are methods of detecting a biological condition associated with a mutant FNIPl nucleic acid in a subject, wherein the method includes determining whether the subject has mutant FNIPl nucleic acid. In some examples, the mutant FNIPl nucleic acid encodes a truncated FNIPl protein and the method includes detecting the truncated FNIPl protein. In some examples, this method is a method of detecting BHD syndrome.

Still other embodiments are pharmaceutical compositions that include the isolated FNIPl polypeptide described above and a pharmaceutically acceptable carrier or diluent. Yet still other embodiments are methods of treating a hamartomatous condition, including administering to a subject with the hamartomatous condition an effective amount of the composition of this pharmaceutical composition. In certain examples, administration includes topical administration, intravenous administration, intra-arterial administration, or intraperitoneal administration, and in other examples the hamartomatous condition is BHD syndrome, tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome.

Yet still another embodiment is an antisense oligonucleotide that inhibits expression of the FNIPl polypeptide described above. Other embodiments are methods of diagnosis that include obtaining a sample of nucleic acid from a subject, and determrning a presence of a nucleotide that encodes the truncated FNIPl protein described above. In some examples, the determining step includes amplifying at least a portion of a nucleic acid molecule that includes the FNIPl gene, sequencing at least a portion of a nucleic acid molecule that the FNIPl gene, or a combination thereof. In certain examples, the method includes determining a propensity to develop a condition associated with BHD syndrome, and in particular examples, the condition comprises fϊbrofolliculoma, renal neoplasia, or spontaneous pneumothorax. In some examples, the method is a method for screening for an agent that inhibits binding of FNIPl to a binding partner, and the method further includes determining whether each member of a library of test agents affects the binding of the FNIPl polypeptide and the binding partner polypeptide to each other. In particular examples, the library includes small molecule inhibitors.

Finally, yet another method is method of treating a hamartomatous condition in a subject that includes administering to the subject a therapeutically effective amount of a small molecule inhibitor identified as described above, thus treating the hamartomatous condition in the subject. In particular examples, administration includes topical administration, intravenous administration, intra-arterial administration, or intraperitoneal administration. B. FNIPl and hamartomatous conditions and familial renal cancer disorders

Although the majority of renal cancer cases (~96%) are sporadic, studies of inherited renal cancer syndromes that predispose individuals to renal epithelial tumors have provided us with a growing understanding of the genetic basis of renal cancer. Four familial cancer disorders with a renal epithelial tumor phenotype have been well characterized and the causative genes have been identified (Pavlovich and Schmidt (2004) Nat Rev Cancer 4(5):381-93).

Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited multisystem neoplastic disorder that predisposes patients to develop clear cell renal cell carcinoma (CCRCC), the most common histologic class of renal tumors. Germline mutations in the VHL tumor suppressor gene accompanied by somatic "second hit" mutations lead to loss of function of VHL in renal tumors of VHL patients. pVHL acts as the substrate recognition member of an E3 ubiquitin ligase that targets members of the hypoxia-inducible factor-α transcription factor family (HIF- lα and HIF-2α) for ubiquitin-mediated proteasomal degradation in an oxygen-dependent manner. In the absence of functional pVHL, HIF-α accumulates, independent of oxygen levels, upregulating hypoxia-inducible genes (VEGF, GLUT 1, PDGF-β) that support tumor growth and neo-vascularization (for review, see Kaelin, Nat Rev Cancer. 2:673-682, 2002).

Hereditary papillary renal cell carcinoma (HPRC) is caused by germline activating mutations in the hepatocyte growth factor tyrosine kinase receptor, MET proto-oncogene, and predisposes patients to develop late-onset bilateral, multifocal papillary renal tumors with type I histology (Zbar et ah, J Urol. 151(3):561-566, 1994); Zbar et al, J Urol. 153(3 Pt 2):907-912, 1995); Schmidt et al, Nat Genet. 16(l):68-73, 1997); Schmidt et al, Contrib Nephrol. 128:11-27, 1999). Duplication of the chromosome 7 bearing the mutant MET allele in the affected individual's renal epithelial cell may prime the cell to become neoplastic and provide the second event leading to development of renal tumors in HPRC patients (Zhuang et al, Nat Genet. 20(l):66-69, 1998).

Inheritance of mutations in the fumarate hydratase (FH) gene lead to the development of a more aggressive renal tumor phenotype with papillary type II histology in patients with the dermatologic disorder, hereditary leiomyomatosis and renal cell carcinoma (HLRCC) (Launonen et al, Proc Natl Acad Sd USA 98(6):3387-3392, 2001; Tomlinson et al, Nat Genet. 30(4):406-410, 2002; Alam et al, Hum MoI Genet. 12(11):1241-1252, 2003); Toro et al, Am J Hum Genet. 73(l):95-106, 2003). These patients also may develop smooth muscle tumors (leiomyomata) of the skin and, in women, uterine leiomyomata. HLRCC-associated FH mutations lead to severe reduction in fumarase activity (Tomlinson et ah, Nat Genet. 30(4):406-410, 2002) and accumulation of fumarate, which may interfere with Kreb's cycle function. Recent evidence suggests high levels of fumarate inhibit HIF prolyl hydroxylase (HPH), which is important for oxygen-dependent hydroxylation of critical prolines in the VHL substrate recognition site of HIF-oc (Isaacs et ah, Cancer Cell, 8(2):143-153, 2005). Inactivation of HPH inhibits modification of HIF-α for pVHL recognition and degradation, which results in HIF-α stabilization under normoxic conditions, potentially contributing to the development of HLRCC-associated renal tumors.

The Birt-Hogg-Dube '(BHD) syndrome is an autosomal dominantly inherited genodermatosis that predisposes patients to develop hamartomas of the hair follicle, spontaneous pneumothorax/lung cysts and an increased risk for renal cancer (Birt et ah, Arch Dermatol 113:1674-1677, 1977; Roth et ah, J. Amer. Acad. Derm. 29:1055-1056, 1993; Binet et al., Ann. Dermatol. Venereol. 113:928-930, 1986; Toro et ah, Arch Dermatol 135: 1195-1202, 1999; Zbar et ah, Cancer Epidemiol Biomarkers Prev 11:393-400, 2002). BHD patients develop renal tumors with a variety of histologies (Pavlovich and Schmidt, Nat Rev Cancer. 4(5):381-93, 2004). The majority of BHD-associated renal neoplasms are oncocytic hybrid tumors comprised of features of both chromophobe renal carcinoma and renal oncocytoma (Tickoo et ah, Amer. J. Surg. Pathol. 23:1094-1101,1999). Recently the BHD disease locus was localized to chromosome 17p 11.2 by linkage analysis in BHD kindreds (Khoo et a , Oncogene 20:5239-5242, 2001; Schmidt et ah, Am J Hum Genet 69: 876-882, 2001) and germline mutations were identified in a novel gene with no homology to other human genes but highly conserved across species (Nickerson et ah, Cancer Cell 2:157-16, 2002; Khoo et ah, J Med Genet. 39(12):906-912, 2002). Over half of the BHD kindreds carry a germline cytosine insertion/deletion mutation in a C8 tract "hot spot" within exon 11 of BHD predicted to truncate the BHD protein, folliculin.

The mutation analysis of a large BHD cohort revealed a total of 22 unique mutations predicted to truncate folliculin, including 16 insertion/deletion, three nonsense and three splice-site mutations, in 51 of 61 BHD kindreds (Schmidt et ah, Am J Hum Genet. 76(6): 1023-33, 2005). Either somatic "second hit" mutations predicted to truncate the protein or loss of heterozygosity at the BHD locus have been identified in 70% of 77 renal tumors from BHD patients (Vocke et ah, J. Natl Cancer Inst, 97(12):931-5, 2005; erratum in JNatl Cancer Inst. 97(14):1096, 2005), supporting a tumor suppressor function for folliculin. In further support of these molecular genetic analyses, BHD mRNA was not detected by in situ hybridization in renal tumors from patients affected with BHD (Warren et ah, Modern Pathology vol. 1-14, 2004). Notably, few sporadic renal tumors have been found to harbor BHD mutations (Khoo et ah, Cancer Res. 63(15):4583-4587, 2003); Kovacs et ah, Pathol Oncol Res. 10(3):169-171, 2004). Recent studies have suggested a link among several hamartomatous syndromes including tuberous sclerosis complex (TSC), Peutz-Jeghers syndrome (PJS), Cowden syndrome, Proteus syndrome, Lhermitte-Duclos disease and Bannayan-Riley-Ruvalcaba syndrome (BRRS), through convergent energy- and nutrient-sensing cascades, LKBl-AMPK-TSCl/2-mTOR and PI3K-AKT- TSCl/2-mTOR (Inoki et al , Nat. Gen. 37:19-24, 2005). In response to an increased AMP/ATP ratio in the cell, LKBl (also known as STKIl), a serine-threonine kinase, phosphorylates and activates AMP-activated protein kinase (AMPK), which in turn phosphorylates and activates the TSCl/2 complex (Inoki et al, Cell 115:577-590, 2003; Corradetti et al, Genes Dev. 118:1533-1538, 2004; Shaw et al, Cancer Cell 6:91-99, 2004). mTOR (mammalian target of rapamycin), which serves as the master "on-off switch for control of cell growth and size through stimulation of protein synthesis, is negatively regulated by the activated TSCl/2 complex acting as a GAP (GTPase-activating protein) toward the small GTPase, Rheb (Inoki et al, Genes Dev. 17:1829-1834, 2003). Mutations in LKBl associated with the hamartomatous phenotype of PJS, and mutations in either TSCl or TSC2, which result in the phenotypic features of the tuberous sclerosis complex, are the result of mTOR-dependent dysregulation of protein synthesis.

In a parallel pathway, growth factors signaling through their receptors at the cell membrane activate phospho-inositide-3-kinase (PI3K) which converts PIP2 to PIP3 (reversed by the lipid phosphatase, PTEN) and activates the serine/threonine kinase, AKT. Subsequent phosphorylation of TSC2 by activated AKT negatively regulates the TSCl/2 complex, resulting in activation of mTOR, which triggers increased protein synthesis. Mutations in the negative regulator of this cascade, PTEN, lead to multiple hamartomatous syndromes including Cowden syndrome, BRRS, Proteus syndrome, and Lhermitte-Duclos disease. Birt-Hogg-Dube' syndrome, a hamartomatous syndrome in which patients develop hamartomas of the hair follicle, displays phenotypic similarities with TSC (cutaneous lesions and lung malformations, renal cysts and renal cell carcinomas with variable histologies) that has led to speculation that BHD may function in the pathway(s) signaling through mTOR (Inoki et al, Nature Genetics 37: 19-24, 2005).

To further characterize the function of folliculin, co-immunoprecipitation and mass spectrometric analysis were used to search for proteins that interacted with folliculin. Described herein is a 130 kDa folliculin-interacting protein, FNIPl, which was also found to interact with the γ-subunit of AMPK. Also disclosed are several FNIPl isoforms, each of which is missing one or two of the 18 exons of the full-length FMPi cDNA. In addition, the folliculin-FNIPl-AMPK interaction is described, which may play a role in energy-sensing pathways in normal cells. Dysregulation of this complex may contribute to development of hamartomas of the hair follicle and renal tumors found in BHD patients, and may contribute to the development of other hamartomatous conditions, such as tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, and Bannayan-Riley-Ruvalcaba syndrome. C. Methods of Making Human FNIPl cDNA

This example recites a number of different methods for making FNIPl cDNA. The original means by which the wildtype and mutant FNIPl cDNAs were identified and obtained is described above. With the provision of the sequence of the FNIPl proteins (SEQ ID NOs: 2, 4, 6, 8, and 10) and cDNA (SEQ ID NOs: 1, 3, 5, 7, and 9), in vitro nucleic acid amplification (such as polymerase chain reaction (PCR)) now may be utilized in a simple method for producing FNIPl cDNA. The following example provides techniques for preparing cDNA in this manner.

Total RNA is extracted from human cells by any one of a variety of methods well known to those of ordinary skill in the art. Sambrook et al. (in Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (in Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley-Intersciences, 1992) provide descriptions of methods for RNA isolation. Because FNIPl is expressed in normal tissue and may be expressed in tumors, human cell lines derived from tumors or normal tissue may be used as a source of such RNA. The extracted RNA is then used as a template for performing reverse transcription-polymerase chain reaction (RT-PCR) amplification of cDNA. Methods and conditions for RT-PCR are described in Kawasaki et al. (in PCR Protocols, A Guide to Methods and Applications, Innis et al. (eds.), 21-27, Academic Press, Inc., San Diego, California, 1990).

The selection of amplification primers will be made according to the portion(s) of the cDNA that is to be amplified. Primers may be chosen to amplify a segment of a cDNA or the entire cDNA molecule. Variations in amplification conditions may be required to accommodate primers and amplicons of differing lengths and composition; such considerations are well known in the art and are discussed for instance in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990). By way of example, the portions of the human FNIPl cDNA molecule may be amplified using the combination of primers discussed above, in Example 1. These primers are illustrative only; one skilled in the art will appreciate that many different primers may be derived from the provided cDNA sequence in order to amplify particular regions of FNIPl cDNA, as well as the complete sequence of the human FNIPl cDNA.

Re-sequencing of PCR products obtained by these amplification procedures is advantageous to facilitate confirmation of the amplified sequence and provide information about natural variation of this sequence in different populations or species. Oligonucleotides derived from the provided FNIPl sequences may be used in such sequencing methods.

Orthologs of human FNIPl can be cloned in a similar manner, where the starting material consists of cells taken from a non-human species. Orthologs will generally share at least 20% sequence identity with the disclosed human FNIPl cDNA, while exhibiting substantially greater sequence identity at the protein level due to the wobble effect. Where the non-human species is more closely related to humans, the sequence identity will in general be greater. Closely related orthologous FNIPl molecules may share at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 91%, at least 93%, at least 95%, or at least 98% sequence identity with the disclosed human sequences. Oligonucleotides derived from the human FNIPl cDNA, or fragments of this cDNA, are encompassed within the scope of the present disclosure. Such oligonucleotides may comprise a sequence of at least 15 consecutive nucleotides of the FNIPl nucleic acid sequence. If these oligonucleotides are used with an in vitro amplification procedure (such as PCR), lengthening the oligonucleotides may enhance amplification specificity. Thus, oligonucleotide primers comprising at least 25, 30, 35, 40, 45, or 50 consecutive nucleotides of these sequences may be used. These primers for instance may be obtained from any region of the disclosed sequences. By way of example, the human FNIPl cDNA, ORF and gene sequences may be apportioned into about halves or quarters based on sequence length, and the isolated nucleic acid molecules (for example, oligonucleotides) may be derived from the first or second halves of the molecules, or any of the four quarters.

Nucleic acid molecules may be selected that comprise at least 15, 20, 23, 25, 30, 35, 40, 50, or 100 consecutive nucleotides of any of these or other portions of the human FNIPl cDNA. Thus, representative nucleic acid molecules might comprise at least 15 consecutive nucleotides of the human FNIPl cDNA (SEQ ID NO: 1). D. FNIPl Sequence Variants

With the provision of human FNEP 1 protein and corresponding nucleic acid sequences herein, both wildtype and various mutants, the creation of variants of these sequences is now enabled. Variant FNIPl proteins include proteins that differ in amino acid sequence from the human FNIPl sequences disclosed but that share at least 60% amino acid sequence identity with the provided human FNIPl protein. Other variants will share at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity. Manipulation of the nucleotide sequence of FNIPl using standard procedures, including for instance site-directed mutagenesis or PCR mutagenesis, can be used to produce such variants. The simplest modifications involve the substitution of one or more amino acids for amino acids having similar biochemical properties. These conservative substitutions are likely to have minimal impact on the activity of the resultant protein. Table 2 shows amino acids that may be substituted for an original amino acid in a protein, and which are regarded as conservative substitutions.

Table 2 Original Residue Conservative Substitutions

Ala ser

Arg lys Asn gin; his

Asp glu

Cys ser

GIn asn

Glu asp GIy pro

His asn; gin

He leu; val

Leu ile; val

Lys arg; gin; glu Met leu; ile

Phe met ; leu; tyr

Ser thr

Thr ser

Trp tyr Tyr up; phe

Val ile; leu

More substantial changes in enzymatic function or other protein features may be obtained by selecting amino acid substitutions that are less conservative than those listed in Table 2. Such changes include changing residues that differ more significantly in their effect on maintaining polypeptide backbone structure (for example, sheet or helical conformation) near the substitution, charge or hydrophobicity of the molecule at the target site, or bulk of a specific side chain. The following substitutions are generally expected to produce the greatest changes in protein properties: (a) a hydrophilic residue (for example, seryl or threonyl) is substituted for (or by) a hydrophobic residue (for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl); (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain (for example, lysyl, arginyl, or histadyl) is substituted for (or by) an electronegative residue (for example, glutamyl or aspartyl); or (d) a residue having a bulky side chain (for example, phenylalanine) is substituted for (or by) one lacking a side chain (for example, glycine). Variant FNIP 1 -encoding sequences may be produced by standard DNA mutagenesis techniques, for example, M13 primer mutagenesis. Details of these techniques are provided in Sambrook et al. (in Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ch. 15. By the use of such techniques, variants may be created that differ in minor ways from the human FNIPl sequences disclosed. DNA molecules and nucleotide sequences that are derivatives of those specifically disclosed herein, and which differ from those disclosed by the deletion, addition, or substitution of nucleotides while still encoding a protein that has at least 60% sequence identity with the human FNIPl -encoding sequence disclosed (SEQ ID NO: 1) or with one of the isoforms disclosed herein (SEQ ID NOs: 3, 5, 7, and 9) are comprehended by this disclosure. Also comprehended are more closely related nucleic acid molecules that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% nucleotide sequence identity with the disclosed FNIPl sequences. In their most simple form, such variants may differ from the disclosed sequences by alteration of the coding region to fit the codon usage bias of the particular organism into which the molecule is to be introduced.

Alternatively, the coding region may be altered by taking advantage of the degeneracy of the genetic code to alter the coding sequence such that, while the nucleotide sequence is substantially altered, it nevertheless encodes a protein having an amino acid sequence substantially similar to the disclosed human FNIPl protein sequences. For example, because of the degeneracy of the genetic code, four nucleotide codon triplets - GCT, GCG, GCC and GCA - code for alanine. The coding sequence of any specific alanine residue within the human folliculin protein, therefore, could be changed to any of these alternative codons without affecting the amino acid composition or characteristics of the encoded protein. Based upon the degeneracy of the genetic code, variant DNA molecules may be derived from the cDNA and gene sequences disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences. Thus, this disclosure also encompasses nucleic acid sequences that encode a FNIPl protein, but which vary from the disclosed nucleic acid sequences by virtue of the degeneracy of the genetic code.

Variants of the FNIPl protein may also be defined in terms of their sequence identity with the prototype human FNIPl protein (SEQ ID NO: 2). As described above, FNIPl proteins share at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with the human FNIPl protein (SEQ ID NO: 2). Nucleic acid sequences that encode such proteins/fragments readily may be determined simply by applying the genetic code to the amino acid sequence of a FNIPl protein or fragment, and such nucleic acid molecules may readily be produced by assembling oligonucleotides corresponding to portions of the sequence.

Nucleic acid molecules that are derived from the human FNIPl cDNA nucleic acid sequences include molecules that hybridize under stringent conditions to the disclosed prototypical FNIPl nucleic acid molecules, or fragments thereof. In particular embodiments, the nucleic acid molecule or fragments hybridize under conditions of low stringency, high stringency, or very high stringency as defined above.

Human FNIPl nucleic acid encoding molecules (including the cDNA shown in SEQ ID NOs: 1, 3, 5, 7, and 9, and nucleic acids comprising this sequence), and orthologs and homologs of these sequences, may be incorporated into transformation or expression vectors. E. Expression of FNIPl proteins

This example details several methods for expressing FNIPl proteins and polypeptides. The expression and purification of proteins, such as the FNIPl protein, can be performed using standard laboratory techniques. After expression, purified FNTPl protein may be used for functional analyses, antibody production, diagnostics, and patient therapy. Furthermore, the DNA sequence of the FNIPl cDNA can be manipulated in studies to understand the expression of the gene and the function of its product. Mutant forms of the human FNIPl gene may be isolated based upon information contained herein, and may be studied in order to detect alteration in expression patterns in terms of relative quantities, tissue specificity, and functional properties of the encoded mutant FNIPl protein. Partial or full-length cDNA sequences, which encode for the subject protein, may be ligated into bacterial expression vectors. Methods for expressing large amounts of protein from a cloned gene introduced into Escherichia coli (E. coli) may be utilized for the purification, localization, and functional analysis of proteins. For example, fusion proteins consisting of amino terminal peptides encoded by a portion of the E. coli lacZ or trpE gene linked to FNIPl proteins may be used to prepare polyclonal and monoclonal antibodies against these proteins. Thereafter, these antibodies may be used to purify proteins by immunoaffinity chromatography, in diagnostic assays to quantitate the levels of protein and to localize proteins in tissues and individual cells by immunofluorescence. Similarly, fusion proteins comprising FNIPl or a fragment thereof may also be generated for use as fusion proteins, depending on the peptide or protein to which the FNIP In is linked. The construction and use of fusion proteins is generally known to those of ordinary skill.

Intact native protein may also be produced in E. coli in large amounts for functional studies. Methods and plasmid vectors for producing fusion proteins and intact native proteins in bacteria are described in Sambrook et al (in Molecular Cloning: A Laboratory Manual, Ch. 17, CSHL, New

York, 1989). Such fusion proteins may be made in large amounts, are easy to purify, and can be used to elicit antibody response. Native proteins can be produced in bacteria by placing a strong, regulated promoter and an efficient ribosome-binding site upstream of the cloned gene. If low levels of protein are produced, additional steps may be taken to increase protein production; if high levels of protein are produced, purification is relatively easy. Suitable methods are presented in Sambrook et al. (in Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and are well known in the art. Often, proteins expressed at high levels are found in insoluble inclusion bodies. Methods for extracting proteins from these aggregates are described by Sambrook et al. (in Molecular Cloning: A Laboratory Manual, Ch. 17, CSHL, New York, 1989). Vector systems suitable for the expression of lacZ fusion genes include the pUR series of vectors (Ruther and Muller-Hill, EMBO J. 2:1791, 1983), pEXl-3 (Stanley and Luzio, EMBO J. 3: 1429, 1984) and pMRlOO (Gray et al, Proc. Natl. Acad. ScI USA 79:6598, 1982). Vectors suitable for the production of intact native proteins include pKC30 (Shimatake and Rosenberg, Nature 292:128, 1981), pKK177-3 (Amann and Brosius, Gene 40:183, 1985) andpET-3 (Studiar and Moffatt, J. MoI. Biol. 189:113, 1986). FNIPl fusion proteins may be isolated from protein gels, lyophilized, ground into a powder, and used as an antigen. The DNA sequence can also be transferred from its existing context to other cloning vehicles, such as other plasmids, bacteriophages, cosmids, animal viruses and yeast artificial chromosomes (YACs) (Burke et al, Science 236:806-812, 1987). These vectors may then be introduced into a variety of hosts including somatic cells, and simple or complex organisms, such as bacteria, fungi (Timberlake and Marshall, Science 244:1313-1317, 1989), invertebrates, plants (Gasser and Fraley, Science 244:1293, 1989), and animals (Pursel et al, Science 244:1281-1288, 1989), which cell or organisms are rendered transgenic by the introduction of the heterologous FNIPl cDNA. For expression in mammalian cells, the cDNA sequence may be ligated to heterologous promoters, such as the simian virus (SV) 40 promoter in the pSV2 vector (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981), and introduced into cells, such as monkey COS-I cells (Gluzman, Cell 23:175-182, 1981), to achieve transient or long-term expression. The stable integration of the chimeric gene construct may be maintained in mammalian cells by biochemical selection, such as neomycin (Southern and Berg, J. MoI. Appl. Genet. 1:327-341, 1982) and mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981).

DNA sequences can be manipulated with standard procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence- alteration via single-stranded bacteriophage intermediate or with the use of specific oligonucleotides in combination with PCR.

The cDNA sequence (or portions derived from it) or a mini gene (a cDNA with an intron and its own promoter) may be introduced into eukaryotic expression vectors by conventional techniques. These vectors are designed to permit the transcription of the cDNA in eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription of the cDNA and ensure its proper splicing and polyadenylation. Vectors containing the promoter and enhancer regions of the SV40 or long terminal repeat (LTR) of the Rous Sarcoma virus and polyadenylation and splicing signal from SV40 are readily available (Mulligan et al, Proc. Natl. Acad. Sci. USA 78:1078-2076, 1981; Gorman et al, Proc. Natl. Acad. Sci USA 78:6777-6781, 1982). The level of expression of the cDNA can be manipulated with this type of vector, either by using promoters that have different activities (for example, the baculovirus pAC373 can express cDNAs at high levels in S. frugiperda cells (Summers and Smith, in Genetically Altered Viruses and the Environment, Fields et al. (Eds.) 22:319-328, CSHL Press, Cold Spring Harbor, New York, 1985) or by using vectors that contain promoters amenable to modulation, for example, the glucocorticoid-responsive promoter from the mouse mammary tumor virus (Lee et al, Nature 294:228, 1982). The expression of the cDNA can be monitored in the recipient cells 24 to 72 hours after introduction (transient expression).

In addition, some vectors contain selectable markers such as the gpt (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981) or neo (Southern and Berg, J. MoI. Appl. Genet. 1 :327-341 , 1982) bacterial genes. These selectable markers permit selection of transfected cells that exhibit stable, long-term expression of the vectors (and therefore the cDNA). The vectors can be maintained in the cells as episomal, freely replicating entities by using regulatory elements of viruses such as papilloma (Sarver et al, MoI. Cell Biol. 1 :486, 1981) or Epstein-Barr (Sugden et al, MoI Cell Biol. 5:410, 1985). Alternatively, one can also produce cell lines that have integrated the vector into genomic DNA. Both of these types of cell lines produce the gene product on a continuous basis. One can also produce cell lines that have amplified the number of copies of the vector (and therefore of the cDNA as well) to create cell lines that can produce high levels of the gene product (Alt et al, J. Biol. Chem. 253:1357, 1978). The transfer of DNA into eukaryotic, in particular human or other mammalian cells, is now a conventional technique. The vectors are introduced into the recipient cells as pure DNA (transfection) by, for example, precipitation with calcium phosphate (Graham and vander Eb, Virology 52:466, 1973) or strontium phosphate (Brash et al, MoI. Cell Biol. 7:2013, 1987), electroporation (Neumann et al, EMBOJ 1:841, 1982), lipofection (Feigner et al., Proc. Natl. Acad. Sd USA 84:7413, 1987), DEAE dextran (McCuthan ef α/., J Natl. Cancer Inst. 41:351, 1968), microinjection (Mueller et al, Cell 15:579, 1978), protoplast fusion (Schafher, Proc. Natl. Acad. ScL USA 77:2163-2167, 1980), or pellet guns (Klein et al, Nature 327:70, 1987). Alternatively, the cDNA, or fragments thereof, can be introduced by infection with virus vectors. Systems are developed that use, for example, retroviruses (Bernstein et al, Gen. Engr'g 7:235, 1985), adenoviruses (Ahmad et al, J. Virol. 57:267, 1986), or Herpes virus (Spaete et al, Cell 30:295, 1982). FNIPl -encoding sequences can also be delivered to target cells in vitro via non-infectious systems, for instance liposomes.

These eukaryotic expression systems can be used for studies of FNIPl -encoding nucleic acids and mutant forms of these molecules, the FNIPl protein, and mutant forms of this protein. Such uses include, for example, the identification of regulatory elements located in the 5' region of the FNIPl gene on genomic clones that can be isolated from human genomic DNA libraries using the information contained in the present disclosure. The eukaryotic expression systems may also be used to study the function of the normal complete protein, specific portions of the protein, or of naturally occurring or artificially produced mutant proteins.

Using the above techniques, the expression vectors containing the FNIPl gene sequence or cDNA, or fragments or variants or mutants thereof, can be introduced into human cells, mammalian cells from other species, or non-mammalian cells as desired. The choice of cell is determined by the purpose of the treatment. For example, monkey COS cells (Gluzman, Cell 23:175-182, 1981) that produce high levels of the SV40 T antigen and permit the replication of vectors containing the SV40 origin of replication may be used. Similarly, Chinese hamster ovary (CHO), mouse NIH 3T3 fibroblasts or human fibroblasts or lymphoblasts may be used.

The present disclosure thus encompasses recombinant vectors that comprise all or part of the FNIPl gene or cDNA sequences, for expression in a suitable host. The FNIPl DNA is operatively linked in the vector to an expression control sequence in the recombinant DNA molecule so that the FNIPl polypeptide can be expressed. The expression control sequence may be selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof. The expression control sequence may be specifically selected from the group consisting of the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the early and late promoters of SV40, promoters derived from polyoma, adenovirus, retrovirus, baculovirus and simian virus, the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, the promoter of the yeast alpha-mating factors and combinations thereof. The host cell, which may be transfected with the vector of this disclosure, may be selected from the group consisting of E. coli, Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus or other bacilli; other bacteria; yeast; fungi; insect; mouse or other animal; or plant hosts; or human tissue cells. It is appreciated that for mutant or variant FNIPl DNA sequences, similar systems are employed to express and produce the mutant product. In addition, fragments of the FNIPl protein can be expressed essentially as detailed above. Such fragments include individual FNIPl protein domains or sub-domains, as well as shorter fragments such as peptides. FNIPl protein fragments having therapeutic properties may be expressed in this manner also. F. Production of FNIPl Protein Specific Binding Agents

This example describes methods of making FNIPl specific binding agents, for example monoclonal or polyclonal antibodies, antibodies raised against synthetic peptides, antibodies raised by injection of FNIPl -encoding sequence, and antibodies specific for mutant FNIPl. Monoclonal or polyclonal antibodies may be produced to either the normal FNIPl protein or mutant forms of this protein. For instance, antibodies may be produced that recognize a mutant FNIPl protein but fail to recognize a wild-type FNIPl protein, or which recognize a wild-type FNIPl protein, but fail to recognize a mutant FNIPl protein (see below). Optimally, antibodies raised against these proteins or peptides would specifically detect the protein or peptide with which the antibodies are generated. That is, an antibody generated to the FNIPl protein or a fragment thereof would recognize and bind the FNIP 1 protein and would not substantially recognize or bind to other proteins found in human cells.

The determination that an antibody specifically detects the FNIPl protein is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sanibrook et al, in Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989). To determine that a given antibody preparation (such as one produced in a mouse) specifically detects the FNIPl protein by Western blotting, total cellular protein is extracted from human cells (for example, lymphocytes) and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. The proteins are then transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane. After washing the membrane to remove non-specifically bound antibodies, the presence of specifically bound antibodies is detected by the use of an anti- mouse antibody conjugated to an enzyme such as alkaline phosphatase. Application of an alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immunolocalized alkaline phosphatase. Antibodies that specifically detect the FNIPl protein will, by this technique, be shown to bind to the FNIPl protein band (which will be localized at a given position on the gel determined by its molecular weight). Non-specific binding of the antibody to other proteins may occur and may be detectable as a weak signal on the Western blot. The non-specific nature of this binding will be recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific antibody-FNEPl protein binding.

Substantially pure FNIPl protein or protein fragment (peptide) suitable for use as an immunogen may be isolated from the transfected or transformed cells as described above. Concentration of protein or peptide in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms per milliliter. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:

1. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of the FNIPl protein identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler and Milstein

{Nature 256:495-497, 1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess un-fused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Meth. Enzymol. 70:419-439, 1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane {Antibodies, A Laboratory Manual, CSHL, New York, 1988).

2. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein (Example 19), which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with either inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appear to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis et al. {J. Clin. Endocrinol. Metab. 33:988-991, 1971). Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony et al. (in Handbook of Experimental Immunology, Wier, D. (ed.) chapter 19. Blackwell, 1973). Plateau concentration of antibody is usually in the range of about 0.1 to 0.2 mg/ml of serum (about 12 μM). Affϊnity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (Manual of Clinical Immunology, Ch. 42, 1980).

3. Antibodies Raised against Synthetic Peptides

A third approach to raising antibodies against the FNIPl protein or peptides is to use one or more synthetic peptides synthesized on a commercially available peptide synthesizer based upon the predicted amino acid sequence of the FNIPl protein or peptide. Polyclonal antibodies can be generated by injecting these peptides into, for instance, rabbits.

4. Antibodies Raised by Injection of FNIPl Encoding Sequence

Antibodies may be raised against FNIPl proteins and peptides by subcutaneous injection of a DNA vector that expresses the desired protein or peptide, or a fragment thereof, into laboratory animals, such as mice. Delivery of the recombinant vector into the animals may be achieved using a hand-held form of the Biolistic system (Sanford et al, Paniculate ScL Technol. 5:27-37, 1987) as described by Tang et al. (Nature 356:152-154, 1992). Expression vectors suitable for this purpose may include those that express the FNIPl encoding sequence under the transcriptional control of either the human beta-actin promoter or the cytomegalovirus (CMV) promoter.

Antibody preparations prepared according to these protocols are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample; or for immunolocalization of the FNIPl protein. For administration to human patients, antibodies, for example, FNIPl -specific monoclonal antibodies, can be humanized by methods known in the art. Antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland, UK; Oxford Molecular, Palo Alto, CA).

5. Antibodies Specific for Mutant FNIPl The production of antibodies (and fragments and engineered versions thereof) that recognize at least one FNIPl variant with a higher affinity than they recognize wild type FNIPl is beneficial, as the resultant antibodies can be used in diagnosis and treatment, as well as in study and examination of the FNIPl proteins themselves.

In particular embodiments, it is beneficial to generate antibodies from a peptide taken from a mutation or variation-specific region of the FNIPl protein. More particularly, it is beneficial to raise antibodies against peptides of four or more contiguous amino acids that overlap the mutations. Longer peptides also can be used, and in some instances will produce a stronger or more reliable immunogenic response. Thus, it is contemplated in some embodiments that more than 4 amino acids are used to elicit the immune response, for instance, at least 5, at least 6, at least 8, at least 10, at least 12, at least 15, at least 18, at least 20, at least 25, or more, such as 30, 40, 50, or even longer peptides. Also, it will be understood by those of ordinary skill that it is beneficial in some instances to include adjuvants and other immune response enhancers, including passenger peptides or proteins, when using peptides to induce an immune response for production of antibodies. Embodiments are not limited to antibodies that recognize epitopes containing the actual mutation identified in each variant. Instead, it is contemplated that variant-specific antibodies also may each recognize an epitope located anywhere throughout the folliculin variant molecule, which epitopes are changed in conformation and/or availability because of the activating mutation. Antibodies directed to any of these variant-specific epitopes are also encompassed herein.

By way of example, the following references provide descriptions of methods for making antibodies specific to mutant proteins: Hills et al, {Int. J. Cancer, 63: 537-543, 1995); Reiter & Maihle {Nucleic Acids Res., 24: 4050-4056, 1996); Okamoto et al. {Br. J. Cancer, 73: 1366-1372, 1996); Nakayashiki etal. {Jpn. J. Cancer Res. 91: 1035-1043, 2000); Gannon et al {EMBO J. 9: 1595-1602, 1990); Wong et al {Cancer Res. 46: 6029-6033, 1986); and Carney et al. (7. Cell

Biochem., 32: 207-214, 1986). Similar methods can be employed to generate antibodies specific to specific FNIPl protein variants. G. FNIPl Mutations

Mutation of FNIPl is expected to play a causative role in some cases of BHD syndrome, in addition to other hamartomatous syndromes, such as tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome. Thus, it is desirable to isolate and identify FNIPl mutations. Any conventional method for the identification of generic mutations in a population can be used to identify such mutations.

For instance, existing populations (such as mouse or human populations) are assessed for symptoms of BHD syndrome, renal neoplasia, and/or spontaneous pneumothorax, and individuals within the population are genotyped as relates to a FNIPl sequence. These FNIPl sequences are then compared to a reference FNIPl sequence, such as the wild-type FNIPl sequence (SEQ ID NO: 1) or one of the alternative transcripts disclosed herein (SEQ ID NOs: 3, 5, 7, and 9) to determine the presence of one or more variant nucleotide positions. Once variant nucleotides are identified, statistical analysis of the population is used to determine whether these variants are correlated with BHD syndrome and/or associated symptoms or with another hamartomatous syndrome.

FNIPl mutations, for example single nucleotide alterations, can be detected by a variety of techniques. The techniques used in evaluating either somatic or germline single nucleotide alterations include allele-specifϊc oligonucleotide hybridization (ASOH) (Stoneking et al, Am. J. Hum. Genet. 48:370-382, 1991) which involves hybridization of probes to the sequence, stringent washing, and signal detection. Other methods include techniques that incorporate more robust scoring of hybridization. Examples of these procedures include the ligation chain reaction (ASOH plus selective ligation and amplification), as disclosed in Wu and Wallace {Genomics 4:560-569, 1989); mini-sequencing (ASOH plus a single base extension) as discussed in Syvanen {Meth. MoI. Biol. 98:291-298, 1998); and the use of DNA chips (miniaturized ASOH with multiple oligonucleotide arrays) as disclosed in Lipshutz et al. {BioTechniques 19:442-447, 1995). Alternatively, ASOH with single- or dual- labeled probes can be merged with PCR, as in the 5'- exonuclease assay (Heid et ah, Genome Res. 6:986-994, 1996), or with molecular beacons (as in Tyagi and Kramer, Nat. Biotechnol. 14:303-308, 1996).

Another technique is dynamic allele-specific hybridization (DASH), which involves dynamic heating and coincident monitoring of DNA denaturation, as disclosed by Howell et a (Nat. Biotech. 17:87-88, 1999). A target sequence is amplified by PCR in which one primer is biotinylated. The biotinylated product strand is bound to a streptavidin-coated microliter plate well, and the non-biotinylated strand is rinsed away with alkali wash solution. An oligonucleotide probe, specific for one allele, is hybridized to the target at low temperature. This probe forms a duplex DNA region that interacts with a double strand-specific intercalating dye. When subsequently excited, the dye emits fluorescence proportional to the amount of double-stranded DNA (probe-target duplex) present. The sample is then steadily heated while fluorescence is continually monitored. A rapid fall in fluorescence indicates the denaturing temperature of the probe-target duplex. Using this technique, a single-base mismatch between the probe and target results in a significant lowering of melting temperature (T_n,), which can be readily detected. A variety of other techniques can be used to detect mutations in FNIPl DNA. Merely by way of example, see U.S. Patents No. 4,666,828; 4,801,531; 5,110,920; 5,268,267; 5,387,506; 5,691,153; 5,698,339; 5,736,330; 5,834,200; 5,922,542; and 5,998,137 for such methods.

FNIPl nucleotide variants can also be detected using an array of nucleic acid molecules attached to a solid support, in which the array includes an oligonucleotide that hybridizes to a nucleic acid molecule that contains a mutation associated with abnormal expression of the FNIPl molecule. Hybridization is performed under conditions in which the oligonucleotide will hybridize to the mutant sequence but not to the wild-type sequence (SEQ ID NO: 1) or one of the isoforms disclosed herein (SEQ ID NOs: 3, 5, 7, and 9). Examples of patents that disclose how to make and use such arrays include US Patent Nos. 6,344,316 and 6,551,784. H. Gene Probes and Markers

Sequences surrounding and overlapping one or more mutations in the FNIPl gene can be useful for a number of gene mapping, targeting, and detection procedures. For example, genetic probes can be readily prepared for hybridization and detection of a FNIPl mutation. As will be appreciated, probe sequences may be greater than about 10 or more oligonucleotides in length and possess sufficient complementarity to distinguish between the mutant and wild-type sequences.

Similarly, sequences surrounding and overlapping any mutations, or longer sequences encompassing more than one mutation, can be utilized in allele specific hybridization procedures. A similar approach can be adopted to detect other FNIPl mutations.

In each embodiment, longer oligonucleotides are contemplated, that have at least 11, at least 12, at least 13, at least 14, at least 15, at least 17, at least 18, at least 20, at least 25, or more contiguous nucleotides. Specific oligonucleotides are about 30, 35, or 40 nucleotides in length, or longer. A skilled practitioner will understand how to select specific oligonucleotide sequences from the provided sequences and the guidance provided herein, in order to generate probes for determining the presence or absence of any of these markers in a biological sample from a subject, which subject includes nucleic acids from the subjects (either genomic of mRNA nucleic acids, or both). I. Detecting Nucleotide Variants/Mutations

Mutations in the FNIPl gene, such as truncation mutations or activating mutations, may be linked to BHD syndrome and related symptoms, such as spontaneous pneumothorax and/or renal neoplasia, or with other hamartomatous syndromes, such as tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome. These mutations can be detected by a variety of techniques, for example allele-specific oligonucleotide hybridization (ASOH) (Stoneking et al, Am. J. Hum. Genet. 48:370-382, 1991), which involves hybridization of probes to the sequence, stringent washing, and signal detection. Other suitable methods include techniques that incorporate more robust scoring of hybridization. Examples of these procedures include the ligation chain reaction (ASOH plus selective ligation and amplification), as disclosed in Wu and Wallace {Genomics 4:560-569, 1989); mini-sequencing (ASOH plus a single base extension) as discussed in Syvanen (Meth. MoI Biol. 98:291-298, 1998); and the use of DNA chips (miniaturized ASOH with multiple oligonucleotide arrays) as disclosed in Lipshutz et al. {BioTechniques 19:442-447, 1995). Alternatively, ASOH with single- or dual-labeled probes can be merged with PCR, as in the 5'-exonuclease assay (Heid et al., Genome Res. 6:986-994, 1996), or with molecular beacons (as in Tyagi and Kramer, Nat. Biotechnol. 14:303-308, 1996).

Another technique is dynamic allele-specific hybridization (DASH), which involves dynamic heating and coincident monitoring of DNA denaturation, as disclosed by Howell et al. (Nat. Biotech. 17:87-88, 1999). A target sequence is amplified by PCR in which one primer is biotinylated. The biotinylated product strand is bound to a streptavidin-coated microtiter plate well, and the non- biotinylated strand is rinsed away with alkali wash solution. An oligonucleotide probe, specific for one allele, is hybridized to the target at low temperature. This probe forms a duplex DNA region that interacts with a double strand-specific intercalating dye. When subsequently excited, the dye emits fluorescence proportional to the amount of double-stranded DNA (probe-target duplex) present. The sample is then steadily heated while fluorescence is continually monitored. A rapid fall in fluorescence indicates the denaturing temperature of the probe-target duplex. Using this technique, a single-base mismatch between the probe and target results in a significant lowering of melting temperature (T_1n) that can be readily detected.

A variety of other techniques can be used to detect point mutations in DNA, which will be appreciated by those of ordinary skill in the art. Merely by way of example, see U.S. Patents No. 4,666,828; 4,801,531; 5,110,920; 5,268,267; 5,387,506; 5,691,153; 5,698,339; 5,736,330; 5,834,200; 5,922,542; and 5,998,137 for such methods. Nucleotide variants can also be detected using an array of nucleic acid molecules attached to a solid support, in which the array includes an oligonucleotide that hybridizes to a nucleic acid molecule that contains a mutation associated with abnormal expression of FNIPl . Hybridization is performed under conditions in which the oligonucleotide will hybridize to the mutant sequence but not to the wild-type sequence (SEQ ID NO: 1) or to one of the isoforms disclosed herein (SEQ ID NOs: 3, 5, 7, and 9). Examples of patents that disclose how to make and use such arrays include US Patent Nos: 6,344,316 and 6,551,784. J. Detection of FNIPl Nucleic Acid Level(s) Individuals carrying mutations in the FNIPl gene, or having amplifications or heterozygous or homozygous deletions of the FNIPl gene, may be detected at the DNA or RNA level with the use of a variety of techniques. The detection of mutations was discussed above; in the following example, techniques are provided for detecting the level of FNIPl nucleic acid molecules in a sample.

For such diagnostic procedures, a biological sample of the subject (an animal, such as a mouse or a human), which biological sample contains either DNA or RNA derived from the subject, is assayed for a mutated, amplified or deleted FNIPl -encoding sequence, such as a genomic amplification of the FNIPl gene or an over- or under-abundance of a FNIPl mRNA. Suitable biological samples include samples containing genomic DNA or mRNA obtained from, for instance, subject body cells, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material. The detection in the biological sample of a mutant FNIPl gene, a mutant or truncated FNIPl RNA, or an amplified or homozygously or heterozygously deleted FNIPl gene, may be performed by a number of methodologies well known to those of ordinary skill in the art.

Gene dosage (copy number) can be important in disease states, and can influence mRNA and thereby protein level; it is therefore advantageous to determine the number of copies of FNIPl nucleic acids in samples of tissue. Probes generated from the encoding sequence of FNIPl (FNIPl probes or primers) can be used to investigate and measure genomic dosage of the FNIPl gene.

Techniques for measuring gene dosage are known in the art; see for instance, US Patent No. 5,569,753 ("Cancer Detection Probes") and Pinkel et αl. (Nat. Genet. 20:207-211, 1998). Determination of gene copy number in cells of a patient-derived sample using other techniques is known in the art. For example, FNIPl amplification in immortalized cell lines as well as uncultured cells taken from a subject can be carried out using bicolor FISH analysis. By way of example, interphase FISH analysis of immortalized cell lines can be carried out as previously described (Barlund et al, Genes Chromo. Cancer 20:372-376, 1997). The hybridizations can be evaluated using a Zeiss or other fluorescence microscope. By way of example, approximately 20 non- overlapping nuclei with intact morphology based on DAPI counterstain are scored to determine the mean number of hybridization signals for each test and reference probe.

Likewise, FISH can be performed on tissue microarrays, as described in Kononen et al. (Nat. Med. 4:844-847, 1998). Briefly, consecutive sections of the array are deparaffinized, dehydrated in ethanol, denatured at 74° C for 5 minutes in 70% formamide/2 x SSC, and hybridized with test and reference probes. The specimens containing tight clusters of signals or >3-fold increase in the number of test probe as compared to chromosome 17 centromere in at least 10% of the tumor cells may be considered as amplified. Microarrays using various tissues can be constructed as described in WO 9944063A2 and WO 9944062A1.

Overexpression of the FNIPl gene can also be detected by measuring the cellular level of FNIPl -specific mRNA. mRNA can be measured using techniques well known to those of ordinary skill in the art, including for instance Northern analysis, RT-PCR and mRNA in situ hybridization. K. Protein-Based Diagnosis

An alternative method of detecting FNIPl gene amplification, deletion or mutation, as well as abnormal FNIPl expression, is to quantitate the level of FNIPl protein in the cells of an individual, or to quantitate the level of truncated FNIPl protein and/or the full length FNIPl protein. This diagnostic tool would be useful for detecting reduced levels of the FNIPl protein that result from, for example, mutations in the promoter regions of the FNIPl gene or mutations within the coding region of the gene that produced truncated, non-functional or unstable polypeptides, as well as from deletions of a portion of or the entire FNIPl gene. Alternatively, duplications of a FNIPl-encoding sequence may be detected as an increase in the expression level of FNIPl protein. Such an increase in protein expression may also be a result of an up-regulating mutation in the promoter region or other regulatory or coding sequence within the FNIPl gene.

Localization and/or coordinated FNIPl expression (temporally or spatially) can also be examined using known techniques, such as isolation and comparison of FNIPl from cell or tissue specific, or time specific, samples. The determination of reduced or increased FNIPl protein levels, in comparison to such expression in a control cell (for example, normal, as in taken from a subject not suffering from BHD syndrome or related symptoms), would be an alternative or supplemental approach to the direct determination of FNIPl gene deletion, amplification or mutation status by the methods disclosed herein and equivalents.

The availability of antibodies specific to the FNIPl protein facilitates the detection and quantitation of cellular FNIP 1 by one of a number of immunoassay methods which are well known in the art and are presented in Harlow and Lane {Antibodies, A Laboratory Manual, CSHL, New York, 1988). Methods of constructing such antibodies are discussed above, in Section I, above.

Any standard immunoassay format (for example, ELISA, Western blot, or RIA assay) can be used to measure FNIPl polypeptide or protein levels and/or size; comparison is to wild-type (normal) FNIPl levels and/or size, and an alteration in FNIPl polypeptide may be indicative of an abnormal biological condition such as BHD syndrome or another hamartomatous syndrome, such as tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome, and/or a predilection to develop spontaneous pneumothorax and/or renal neoplasia. Immunohistochemical techniques may also be utilized for FNIPl polypeptide or protein detection. For example, a tissue sample may be obtained from a subject, and a section stained for the presence of FNIPl using a FNIPl -specific binding agent (for example, anti-FNIPl antibody) and any standard detection system (for example, one which includes a secondary antibody conjugated to horseradish peroxidase). General guidance regarding such techniques can be found in, for example, Bancroft and Stevens (Theory and Practice of Histological Techniques, Churchill Livingstone, 1982) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).

For the purposes of quantitating a FNIPl protein, a biological sample of the subject (which can be any animal, for instance a mouse or a human), which sample includes cellular proteins, is used. Such a biological sample may be obtained from body cells, such as those present in peripheral blood, urine, saliva, tissue biopsy, amniocentesis samples, surgical specimens and autopsy material, particularly kidney or skin cells. Quantitation of FNIPl protein can be achieved by immunoassay and compared to levels of the protein found in control cells (for example, healthy, as in from a subject known not to have BHD syndrome or related symptoms). A significant (for example, 10% or greater) reduction in the amount of FNIPl protein in the cells of a subject compared to the amount of FNIPl protein found in normal human cells could be taken as an indication that the subject may have deletions or mutations in the FNIPl gene, whereas a significant (for example, 10% or greater) increase would indicate that a duplication (amplification), or mutation that increases the stability of the FNIPl protein or mRNA, may have occurred. Deletion, mutation, and/or amplification of or within the FNIPl -encoding sequence, and substantial under- or over-expression of FNIPl protein, is indicative of BHD syndrome or another hamartomatous syndrome, such as tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley- Ruvalcaba syndrome, and/or a predilection to develop spontaneous pneumothorax and/or renal neoplasia.

L. Suppression of FNIPl Protein Expression

In some embodiments, it may be desirable to reduce or suppress FNIPl protein expression, for example in various experimental conditions or in the treatment of a hamartomatous condition that is associated with an overexpression of FNIPl. A reduction of FNIPl protein expression in a transgenic cell may be obtained by introducing into cells an antisense construct based on the FNIPl encoding sequence, including the human FNIPl cDNA (SEQ ID NO: 1) or gene sequence or flanking regions thereof. For antisense suppression, a nucleotide sequence from a FNIPl -encoding sequence, for example all or a portion of the FNIPl cDNA or gene, is arranged in reverse orientation relative to the promoter sequence in the transformation vector. Other aspects of the vector may be chosen as discussed herein (Example 19).

The introduced sequence need not be the full length human FNIPl cDNA or gene or reverse complement thereof, and need not be exactly homologous to the equivalent sequence found in the cell type to be transformed. Generally, however, where the introduced sequence is of shorter length, a higher degree of homology to the native FNIPl sequence will be needed for effective antisense suppression. The introduced antisense sequence in the vector may be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. The length of the antisense sequence in the vector advantageously may be greater than 100 nucleotides. For suppression of the FNIPl gene itself, transcription of an antisense construct results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous FNIPl gene in the cell.

Although the exact mechanism by which antisense RNA molecules interfere with gene expression has not been elucidated, it is believed that antisense RNA molecules bind to the endogenous mRNA molecules and thereby inhibit translation of the endogenous mRNA.

Suppression of endogenous FNIPl expression can also be achieved using ribozymes. Ribozymes are synthetic FNIPl molecules that possess highly specific endoribonuclease activity. The production and use of ribozymes are disclosed in U.S. Patent No. 4,987,071 and U.S. Patent No. 5,543,508. The inclusion of ribozyme sequences within antisense RNAs may be used to confer RNA cleaving activity on the antisense RNA, such that endogenous mRNA molecules that bind to the antisense RNA are cleaved, which in turn leads to an enhanced antisense inhibition of endogenous gene expression.

Suppression can also be achieved using RNA interference, using known and previously disclosed methods. Several models have been put forward to explain RNAi, in particular the mechanisms by which the cleavage derived small dsRNAs or siRNAs interact with the target mRNA and thus facilitate its degradation (Hamilton et al, Science 286, 950, 1999; Zamore et al, Cell 101, 25, 2000; Hammond et al, Nature 404, 293, 2000; Yang et al, Curr. Biol. 10, 1191, 2000; Elbashir et al, Genes Dev. 15, 188, 2001; Bass Cell 101, 235, 2000). It has been proposed that the cleavage derived small dsRNAs or siRNAs act as a guide for the enzymatic complex required for the sequence specific cleavage of the target mRNA. Evidence for this includes cleavage of the target mRNA at regular intervals of ~21-23 nts in the region corresponding to the input dsRNA (Zamore et al, Cell 101, 25, 2000), with the exact cleavage sites corresponding to the middle of sequences covered by individual 21- or 22 nt small dsRNAS or siRNAs (Elbashir et al, Genes Dev. 15, 188, 2001). Although mammals and lower organisms appear to share dsRNA-triggered responses that involve a related intermediate (small dsRNAs), it is likely that there will be differences as well as similarities in the underlying mechanism. dsRNAs can be formed from RNA oligomers produced synthetically (for technical details see material from the companies Xeragon and Dharmacon, both available on the internet). Small dsRNAs and siRNAs can also be manufactured using standard methods of in vitro RNA production. In addition, the Silencer™ siRNA Construction kit (and components thereof) available from Ambion (Catalog # 1620; Austin, TX), which employs a T7 promoter and other well known genetic engineering techniques to produce dsRNAs. Double stranded RNA triggers could also be expressed from DNA based vector systems.

Finally, dominant negative mutant forms of FNIPl may be used to block endogenous FNIPl activity. M. Incorporation of FNIPl Protein into Pharmaceutical Compositions

Administration of FNIPl to subjects, either alone or in combination with AMPK, may be useful in the treatment of a hamartomatous condition in a subject. Pharmaceutical compositions that comprise at least one FNTPl protein or fragment thereof as an active ingredient will normally be foπnulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen. The pharmaceutically acceptable carriers and excipients useful in this invention are conventional. For instance, parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. For instance, in addition to injectable fluids, topical and oral formulations can be employed. Topical preparations can include eye drops, ointments, sprays and the like. Oral formulations may be liquid (for example, syrups, solutions or suspensions), or solid (for example, powders, pills, tablets, or capsules). For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

The pharmaceutical compositions that comprise FNIPl protein will preferably be formulated in unit dosage form, suitable for individual administration of precise dosages. One possible unit dosage contains approximately 100 μg of protein. The amount of active compound administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in an amount effective to achieve the desired effect in the subject being treated.

In some embodiments, an FNIPl protein or polypeptide is administered to the subject in combination with an AMPK protein or polypeptide, for example as a cream or ointment applied topically to the face or other region of the body affected by BHD-related skin conditions or skin conditions related to other hamartomatous conditions, such as tuberous sclerosis complex, Peutz- Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome. The dosage of FNIPl and AMPK protein applied will be dependent on the subject being treated, the severity of the affliction, and may range from, for example, 1 μg to 1000 μg of each of FNIPl and AMPK. Administration may be parenteral or topical, and may be repeated, for example, monthly, weekly, daily, twice daily, or three times daily or more. N. FNIPl Knockout and Overexpression Transgenic Animals

Mutant organisms that under-express or over-express the FNIPl protein are useful for research, for instance. Such mutants allow insight into the physiological and/or pathological role of FNIPl in a healthy and/or pathological organism, as well as providing model systems useful, for instance, for testing the ability of the AMPK and/or mTOR pathways to support tumor regression. These mutant animals are "genetically engineered" or "transgenic", meaning that information in the form of nucleotides has been transferred into the mutant's genome at a location, or in a combination, in which it would not normally exist. Nucleotides transferred in this way are said to be "non-native." For example, a non-FNIPl promoter inserted upstream of a native FNIPl -encoding sequence would be non-native. An extra copy of a FNIPl gene on a plasmid, transformed into a cell, would be non- native.

Mutants may be, for example, produced from mammals, such as mice, that either over- express FNIPl or under-express FNIPl, or that do not express FNIPl at all. Over-expression mutants are made by increasing the number of FNIPl genes in the organism, or by introducing a FNIPl gene into the organism under the control of a constitutive or inducible or viral promoter such as the mouse mammary tumor virus (MMTV) promoter or the whey acidic protein (WAP) promoter or the metallothionein promoter. Mutants that under-express folliculin may be made by using an inducible or repressible promoter, or by deleting the FNIPl gene, or by destroying or limiting the function of the FNIPl gene, for instance by disrupting the gene by transposon insertion.

Antisense genes may be engineered into the organism, under a constitutive or inducible promoter, to decrease or prevent FNIPl expression, as discussed above.

A gene is "functionally deleted" when genetic engineering has been used to negate or reduce gene expression to negligible levels. When a mutant is referred to in this application as having the FNIPl gene altered or functionally deleted, this refers to the FNIPl gene and to any ortholog of this gene. When a mutant is referred to as having "more than the normal copy number" of a gene, this means that it has more than the usual number of genes found in the wild-type organism, for example, in the diploid mouse or human.

A mutant (transgenic) mouse or other mammal over-expressing FNIP 1 may be made by constructing a plasmid having a FNIPl -encoding sequence driven by a promoter, such as the mouse mammary tumor virus (MMTV) promoter or the whey acidic protein (WAP) promoter. This plasmid may be introduced into mouse oocytes by microinjection. The oocytes are implanted into pseudopregnant females, and the litters are assayed for insertion of the transgene. Multiple strains containing the transgene are then available for study.

WAP is quite specific for mammary gland expression during lactation, and MMTV is expressed in a variety of tissues including mammary gland, salivary gland, and lymphoid tissues. Many other promoters might be used to achieve various patterns of expression, for example, the metallothionein promoter.

An inducible system may be created in which the subject expression construct is driven by a promoter regulated by an agent that can be fed to the mouse, such as tetracycline. Such techniques are well known in the art. A mutant knockout animal (for example, mouse) from which a FNIPl gene is deleted can be made by removing all or some of the coding regions of the FNIPl gene from embryonic stem cells. The methods of creating deletion mutations by using a targeting vector have been described (Thomas and Capecch, Ce// 51:503-512, 1987). A mutant knockout animal (for example mouse) can be made by conditional FNIPl gene targeting using Cre/lox or other site-specific recombination technology and deleting the FNIPl gene in a tissue-(for example, skin) or time- or development-dependent manner. O. Knock-in Organisms In addition to knock-out systems, it is also beneficial to generate "knock-ins" that have lost expression of the wildtype protein but have gained expression of a different, usually mutant form of the same protein. By way of example, the FNIPl proteins provided herein (for example, in SEQ ID NOs: 2, 4, 6, 8, and 10) can be expressed in a knockout background in order to provide model systems for studying the effects of these mutants. In particular embodiments, the resultant knock-in organisms provide systems for studying neoplasia, such as renal neoplasia.

Those of ordinary skill in the relevant art know methods of producing knock-in organisms. See, for instance, Rane et al (MoI. Cell Biol, 22: 644-656, 2002); Sotillo et al. (EMBOJ., 20: 6637- 6647, 2001); Luo et al. (Oncogene, 20: 320-328, 2001); Tomasson et al. (Blood, 93: 1707-1714, 1999); Voncken et al. (Blood, 86: 4603-4611, 1995); Andrae et al. (Mech.Dev., 107: 181-185, 2001); Reinertsen et al (Gene Expr., 6: 301-314, 1997); Huang et al (MoI Med., 5: 129-137, 1999);

Reichert et al. (Blood, 97: 1399-1403, 2001); and Huettner et al. (Nat. Genet, 24: 57-60, 2000), by way of example.

P. Screening for Agents that Affect FNIPl Activity

In some embodiments, it may be useful to treat a subject with a hamartomatous condition with an agent that mimics or augments FNIPl activity (for example binding to folliculin or AMPK), or with an agent that inhibits a FNIPl activity. Such agents also may be useful in a variety of experimental models. Thus, this example describes methods for identifying agents with FNIPl inhibitory activity, methods of identifying agents that interfere with an interaction between a FNIPl polypeptide and a folliculin polypeptide or between a FNIPl polypeptide and an AMPK polypeptide, and methods for the identification of agents that mimic FNIP 1 's folliculin- or AMPK-binding activity.

The compounds which may be screened in accordance with this disclosure include, but are not limited to peptides, antibodies and fragments thereof, and other organic compounds (for example, peptidomimetics, small molecules) that inhibit FNIPl activity as described herein or interfere with an interaction between FNIPl and folliculin or FNIPl and AMPK. Such compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to members of random peptide libraries; (see, for example, Lam et aL, Nature, 354:82-84, 1991; Houghten et al, Nature, 354:84-86, 1991), and combinatorial chemistry-derived molecular library made of D- and/or L-configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; see, for example, Songyang et al, Cell, 72:767-778, 1993), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab')₂ and Fab expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules. Other compounds which can be screened in accordance with this disclosure include but are not limited to small organic molecules that are able to gain entry into an appropriate cell and affect the expression of FNIPl gene or some other gene involved in a FNIPl -mediated pathway (for example, by interacting with the regulatory region or transcription factors involved in FNIPl gene expression); or such compounds that affect an activity of a FNIP 1 isoform or the activity of some other intracellular factor involved in a FNIPl -mediated pathway, such as folliculin or AMPK.

Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds that can modulate expression or activity of a FNIPl isoform. Examples of molecular modeling systems are the CHARMM (Chemistry at HARvard Molecular Mechanics) and QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMM performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other. A number of articles review computer modeling of drugs interactive with specific-proteins, such as Rotivinen et ah, Acta Pharmaceutical Fennica 97:159-166, 1988; Ripka, New Scientist 54- 57, 1988; McKinaly and Rossmann, Annu Rev Pharmacol Toxicol 29:111-122, 1989; Perry and Davies, OSAR: Quantitative Structure-Activity Relationships in Drug Design pp. 189-193, 1989, (Alan R. Liss, Inc.); Lewis and Dean, Proc R Soc Lond 236:125-140 and 141-162, 1989; and, with respect to a model receptor for nucleic acid components, Askew et ah, J Am Chem Soc 111 : 1082- 1090, 1989. Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these are primarily designed for application to drugs specific to particular proteins, they can be adapted to design of drugs specific to regions of DNA or RNA, once that region is identified. 1. Screening for FNIPl inhibitory agents

Disclosed herein are methods of identifying agents with potential for inhibition of FNIPl. Any agent capable of inhibiting any biological activity of FNIPl is contemplated. In some embodiments, a FNIPl inhibitory agent interferes with an interaction between FNIPl and AMPK, or with an interaction between FNIPl and folliculin, which is discussed below. Screening assays may be conducted in a variety of ways. For example, one method would involve transiently transfecting cells with a FNIPl expression vector and separating FNIPl -expressing cells for use in kinase assays. Any eukaryotic cells or cell line may be used for transfections, such as 293T, NIH373, Wehi 7.2, 293F, or Cos7 cell lines. In one embodiment, cells may be transfected with an EGFP-FNIPl expression vector as described, in which case FNIPl transfectants could be identified by EGFP fluorescence and, optionally, could be separated or analyzed by fluorescence activated cell sorting (FACS; also called flow cytometry). Test compounds would be applied to FNIPl -transfected cells and kinase activity evaluated using a kinase assay. FNIPl inhibitory compounds would be identified by a decrease in kinase activity, for example AMPK activity, as compared to control. Animal models, for instance based on transgenic animals, are also contemplated.

2. Screening for compounds that affect FNIPl/Folliculin or FNIPl /AMPK interaction

In vitro systems may be designed to identify compounds capable of affecting an interaction between FNIPl and folliculin or FNIPl and AMPK. Compounds identified may be useful, for example, in modulating an activity of FNIPl isoforms or increasing or decreasing a binding affinity between FNIPl and folliculin or FNIPl and AMPK, thereby treating a hamartomatous condition, such as BHD syndrome, tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome. The principle of assays used to identify compounds that affect an interaction between FNIP 1 and folliculin or FNIPl and AMPK involves preparing a reaction mixture of a FNIPl polypeptide, fragment, or functional variant and a folliculin polypeptide, fragment, or functional variant or an AMPK polypeptide, fragment, or functional variant under conditions and for a time sufficient to allow the two components to interact and form a complex. Thereafter, a test compound is added to the reaction mixture and various means are used to determine if the FNIPl /folliculin or FNIP1/AMPK complex is affected by the test compound.

The screening assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring a FNIPl polypeptide, peptide, or fusion protein onto a solid surface or a soluble support, adding a folliculin or AMPK polypeptide, peptide, or fusion protein to the reaction vessel, and adding the test substance and detecting FNIPl /folliculin or

FNIP1/AMPK complexes anchored on the solid phase or soluble support at the end of the reaction. In one embodiment of such a method, FNIPl maybe anchored onto a solid surface, and folliculin or AMPK, which is not anchored, may be labeled, either directly or indirectly.

In practice, microliter plates may conveniently be utilized as the solid phase. The anchored component may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored. In order to conduct the assay, the non-immobilized component and test compound are added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (for example, by washing) under conditions such that any FNIPl /folliculin or FNIPl/AMPK complexes formed (or a substantial portion thereof) will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non- immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; for example, using a labeled antibody specific for the previously non-immobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; for example, using an immobilized antibody specific for a FNIPl protein, polypeptide, peptide, or fusion protein or a folliculin or AMPK protein, polypeptide, peptide, or fusion protein to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes. 3. Screening for Agents Having FNIPl -like Activity

Other methods contemplated herein include identifying agents that mimic or enhance FNIPl activity, for example to increase AMPK activity.

Agents that mimic or enhance FNIPl activity can include, for example, agents that induce or increase FNIPl expression in one or more cells; or agents that interact with FNIPl and enhance its activity; or FNIPl peptides having a desired FNIPl activity; or molecules designed to have a FNIPl structure that mediates a particular FNIPl activity. In some embodiments, agents that induce or increase FNIPl expression in one or more cells may be identified by contacting a biological system (such as a cell) that expresses or is capable of expressing FNIPl with an agent. FNIPl expression or activity in the biological system may be measured in response to contact with the agent by methods well known in the art. For instance, trans-acting coactivators of the FNIPl gene regulatory region may be expected to increase FNIPl activity. In other embodiments, agents may increase the half-life of the FNIPl protein or its mRNA and thereby increase FNIPl activity.

In other embodiments, agents that interact with FNIPl and enhance its activity are contemplated. These agents may be identified, for example, by first identifying agents that interact with FNIPl . Biophysical methods of accomplishing this step are well known in the art and include, for example, co-immunoprecipitation, yeast two-hybrid system, and GST pulldown assay, cross-linking of small molecules to FNIPl, among other methods. Agents that interact with FNIPl are then screened for enhancement of FNIPl activity. In some embodiments, FNIPl activity may be increased by agents that enhance an interaction between FNIPl and folliculin, or between FNIPl and AMPK. In other embodiments, molecules can be designed to have a FNIPl structure that mediates a particular FNIPl activity using modeling analyses. Candidate agents designed, for example in silico, to assume a FNIPl structure may then be screened for desired FNIPl activity, for example binding to folliculin or AMPK, or increasing AMPK kinase activity. Agents with such activity may then be used to treat BHD syndrome. In addition, various screening methods can be used to test the importance of FNIP-AMPK binding or FNIP-FLCN binding on therapeutic efficacy. For example, a deletion mutant of one of the pair of proteins can be introduced into an animal as described above in Section O. If disruption of binding is important for therapeutic treatment, then mutant molecules are used as a test agent to compete for wild type protein, or antisense molecules. Alternatively, small molecule inhibitors that fit in the binding region/pocket of FNIPl and abrogate binding of the binding partner (either AMPK or FLCN) can serve as a suitable test agent. As described above, such small molecule inhibitors also can be used therapeutically in the treatment of hamartomatous syndromes, such as BHD syndrome and other hamartomatous syndromes, such as tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome.

EXAMPLES Example 1: General Materials and Methods This example describes general materials and methods used in the other Examples. These are example materials and methods; one of ordinary skill in the art will recognize what other materials and methods can be substituted.

Reagents

Reagents were obtained from the following sources: Anti-HA Affinity Matrix, anti HA antibody (3F 10), HA peptide and ATP Bioluminescence Assay Kit CLSII from Roche Applied Science; protein G-SEPHAROSE®, glutathione-SEPHAROSE® (GS) 4B, ECL, ECL plus, Cye3 conjugated anti-mouse antibody and [α-³²P]dATP from Amersham Biosciences; FIp-In T-Rex System, T-Rex-293 Cell Line, Blasticidin S, HygromicinB and Dulbecco's modified Eagle's medium (DMEM) from Invitrogen; qualified fetal bovine serum, dialyzed fetal bovine serum and Tet screened fetal bovine serum from Hyclone; normal rabbit IgG, HRP-labeled anti-mouse, anti-goat, and anti- rabbit secondary antibodies from Santa Cruz Biotechnology; phospho-p70 S6 Kinase(T389) antibody, p70 S6 Kinase antibody, ρhospho-AMPKα(T172) antibody, AMPKα antibody, and AMPKβl antibody from Cell Signaling; actin antibody from Biomedical Technology; AMPKγl antibody from Zymed; AMPKβ mouse monoclonal antibody from BD Biosciences; FLAG antibody, anti-FLAG M2 Affinity Gel from Sigma; Alexa Fluor® 488 conjugated anti-rabbit antibody and TO-PRO-3 iodide from Molecular Probes; AMP Kinase and SAMS peptide from Upstate Biotechnology; Benzonase from Novagen. Clone KIAA1961 (GenBank Accession #AB075841) was provided by The Kazusa DNA Research Institute, Chiba, Japan. Establishment of cell lines and cell culture A UOK257 (folliculin null) human renal cell cancer cell line was established from a clear cell renal carcinoma that was surgically removed from a BHD patient. Isolated genomic DNA was sequenced using ABI BIGD YE™ chemistry (Perkin-Elmer) to confirm the presence of a protein truncating germline BHD gene mutation and loss of the wild type BHD allele. BHD-restored UOK257 cells were established by transduction of a pLentiviral vector encoding the wild type BHD sequence and selection of stable transductants using the VIRAPOWER™ Lentiviral Expression

System according to manufacturer's protocol (Invitrogen). Tetracycline inducible HA-folliculin and FNIPl-HA expressing HEK 293 cells were established by using the FLP-IN™ T-REX™ System according to manufacturer's protocols (Invitrogen). Tetracycline-inducible RNAi HEK 293 cell lines were established by transfecting pSuperior vector encoding the wild type FNIP-I sequence into Tet repressor-expressing 293 cells. HEK 293 cells, UOK257 cells and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). Tetracycline-inducible HEK 293 cells were cultured in DMEM with Tet-screened 10% FBS, 15μg/ml Blasticidin S and 150μg/ml Hygromicin B. Tetracycline- inducible RNAi HEK 293 cell lines were cultured in DMEM with Tet-screened 10% FBS, 15μg/ml Blasticidin S and 0.2 μg/ml puromycin. BHD-restored UOK257 cells were cultured in DMEM with 10% FBS and 1.5μg/ml Blasticidin S. Identification offolliculin and FNIP-I interacting proteins HA-folliculin or FNIP-I-HA inducible HEK 293 cells were cultured in 15 cm diameter dishes with or without 1 μg/ml of Doxycycline for 36 hours. For each culture, 5 x 10⁷ cells were harvested and lysed in lysis buffer (20 mM Tris-HCl pH7.5, 135 mM NaCl, 5% glycerol, 0.1% TritonX-100, 50 mM NaF, 1 mM vanadate and Complete Protease Inhibitor cocktail) on ice for 30 minutes and vortexed. After centrifugation (15,000 x g for 30 minutes), lysates were immunoprecipitated at 4°C for 10 hours with anti-HA Affinity Matrix. The affinity matrix was washed seven times with lysis buffer.

The affinity purified proteins were eluted by incubating with HA peptide (2 mg/ml) at 30⁰C for 15 minutes. The eluted proteins were boiled with SDS-sample buffer and subjected to SDS- polyacrylamide gel electrophoresis. The separated proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. The blotted membranes were soaked in TBST

(20 mM Tris-HCl pH7.5, 500 mM NaCl, 0.3% Tween20 detergent) and stained with colloidal gold. The specific bands which were observed in Doxy(+) lanes were excised. The immobilized protein were reduced, S-carboxymethylated, and digested in situ with Achromobacter protease I (a Lys-C) (Iwamatsu, Electrophoresis 13(3):142-147, 1992). Molecular mass analyses of Lys-C fragments were performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) using a PerSeptive Biosystem Voyager-DE/RP (MALDI-TOF mass spectrometer) (Jensen et al, J Virol. 70(11):7485-7497, 1996). Identification of proteins was carried out by comparison between the molecular weights determined by MALDI-TOF/MS and theoretical peptide masses from the proteins registered in the NCBInr database (Jensen et al, J Virol. 70(ll):7485-7497, 1996).

Molecular Cloning of Human FNIPl cDNA and its splicing variants

The FNIPl polypeptide was identified as the protein product of the cDNA KIAA1961 (GenBank accession no. BAB85547). The KIAA961 mRNA encodes an open reading frame of 943 amino acids followed by poly (A) tail, but lacks an ATG start codon. BLAST analysis of the publicly available genomic databases identified several overlapping human expressed sequence tags (ESTs) corresponding to KIAA1961. The clone BC001956 encodes a 508 amino acid protein with an ATG start codon, which shares 284 amino acids from its C terminus with the N terminus of clone BAB85547. A genomic BLAST analysis was performed, and it confirmed that these two clones were located at the same genomic locus and shared coding sequences.

Full length FNIPl cDNA was amplified by PCR with the specific primer set (Sense, SEQ ID NO: 13, Antisense, SEQ ID NO: 14). Amplified fragments were cloned into a pCR-TOPO® vector (Invitrogen). Double-stranded sequencing reactions using BIGD YE® Terminators (Applied

Biosystems) were purified using Performa plates (Edge Biosystems) and electrophoresed on an ABI 3700 genetic analyzer. The full length FNIPl cDNA, which has 18 coding exons and is the major transcript, was obtained as follows: The N-terminal fragment was amplified with the specific primer sets (Sense: SEQ ID NO: 25; Antisense: SEQ ID NO: 15) by KOD hot start polymerase (Novagen) using cDNA clone BC001956 as template. The C-terminal fragment was amplified with the specific primer sets (Sense: SEQ ID NO: 16; Antisense: SEQ ID NO: 26) using cDNA clone BAB85547 as template. Then both fragments were gel purified, mixed and used as templates for a final PCR reaction with the specific primer sets (Sense: SEQ ID NO: 17; Antisense: SEQ ID NO: 18), which contain attB sequences, to produce full length FNIPl cDNA. The PCR product containing attB recombination sites was inserted into pDONR221 by BP CLONASE™ enzyme reaction (Invitrogen GATEWAY® Protein Expression System) and the sequence was verified. Antibodies

To generate rabbit polyclonal antibodies against FNIPl, a FNIPl peptide (SEQ ID NO: 19) was synthesized. The FNIPl peptide was conjugated to KLH, dialyzed against PBS and used as antigen to produce polyclonal antibody 181 in rabbits. Two different rabbit polyclonal antibodies against folliculin were produced. Antibody 102 was generated using a folliculin peptide from the N- terminus as antigen (SEQ ID NO: 27). Antibody 105 was generated using full length GST- folliculin expressed in E. coli as antigen. To generate mouse monoclonal antibodies against folliculin, full length GST-folliculin was used as antigen. Immunoprecipitation, Western Blotting and Northern Blotting

Cells were lysed in lysis buffer (20 mM Tris-HCl pH 7.5, 135 mM NaCl, 5% glycerol, 0.1% TritonX-100, 50 mM NaF, 1 mM vanadate and Complete Protease Inhibitor cocktail (Roche)) or (20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM vanadate, 50 mM NaF, 1.0% TritonX-100, 0.5% deoxycholate, 0.1% SDS (RIPA) and Complete Protease Inhibitor cocktail) on ice for 30 minutes and vortexed. The protein concentration of the cell lysate was measured and lysates containing equal amounts of protein were immunoprecipitated at 4⁰C overnight with various antibodies pre-fixed on protein G-SEPHAROSE® 4 Fast Flow resin (Pharmacia Biotech). The SEPHAROSE® resin was washed five times with lysis buffer, and the immunoprecipitated proteins were eluted with SDS-sample buffer for Western-blot analysis. Western blotting was performed by standard methods. For Northern blotting, the FNIPl sequence was amplified with the following primers (Forward: SEQ ID NO: 21; Reverse: SEQ ID NO: 22), and was used as a probe. Northern blotting was performed as previously described (Nickerson et ah, Cancer Cell 2:157-164, 2002). Immunofluorescence Microscopy

HeLa cells transfected with FNIPl-HA and FLAG-folliculin were cultured on Chamber Slides for 12 hours. Cells were washed three times with ice cold PBS and fixed with 2% paraformaldehyde in PBS for 15 minutes at room temperature. Cells were then permeabilized with PBS containing 0.5 % (v/v) Triton X-IOO for 10 minutes and blocked in PBS containing 10% calf serum for one hour at room temperature. Antibody incubations were performed at room temperature for one hour in buffer containing 10 mM Tris/HCl, pH7.5, 150 mM NaCl, 0.01% (v/v) Tween 20, and 0.1% (w/v) BSA. The secondary antibodies used were Alexa 488-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse antibody. TO-PRO-3 iodide was used to stain nuclei. The samples were mounted with a Slow Fade Antifade kit (Molecular Probes) and were viewed with a confocal microscope system (LSM 510; Carl Zeiss). Plasmids, transfection, recombinant protein expression and purification

The Invitrogen GATEWAY® Protein Expression System was used to produce a variety of mammalian expression vectors. The full length and partial fragment cDNAs encoding folliculin and FNIPl were generated by PCR with specific primers containing attB sequences (described above), then inserted into pDONR221 by the BP CLONASE™ in vitro recombination enzyme reaction and sequence verified (Entry clones). The sequence verified Entry clones were recombined into a series of Destination expression vectors using the LR Clonase enzyme reaction according to manufacturer's protocols and miniprep DNA was generated. GST-folliculin fragments and GST-FNIPl fragments were expressed in baculovirus Destination vector infected Sf9 insect cells, and released by sonication of cell pellets. Transfections of 293 HEK cells were performed by the lipofection method using LIPOFECTAMINE™ 2000 (Invitrogen) according to the manufacturer's protocol. Recombinant GST-Folliculin was expressed in baculovirus GATEWAY® Destination vector-infected Sf9 insect cells and purified on a glutathione matrix. In vitro binding assay

A variety of GST-fusion protein constructs were transfected into SF9 insect cells and cultured for 72 hours at 27⁰C in Hyclone SFX-INSECT™ (protein-free) medium. The culture supernatants were clarified by centrifugation. Cell pellets were washed twice with PBS, resuspended in 8 ml sonication buffer (500 mM NaCl, 50 mM Tris-HCl pH 7.5, 5% glycerol, 1 mM β- mercaptoethanol, 5 mM MgC12, and Complete Protease Inhibitor cocktail) and lysed by sonication. Benzonase solution (1 U/ml) was added to the cell lysate and incubated on ice for 20 minutes. Lysates were centrifuged at 11,000 x g for 30 minutes at 4 ⁰C and supernatant was recovered and stored at -80⁰C until used for further analysis. For affinity purification, 50 μl equilibrated glutathione beads were preincubated in 0.3% BSA for 1 hour at 4°C, then mixed with cell extracts containing GST-fusion proteins on a rocking platform for one hour at 4°C. The beads were then washed three times with PBS and once with binding buffer (25 mM Tris-HCl pH 7.5, 135 mM NaCl, 5 mM DTT, 1% NP- 40 and Complete Protease Inhibitor cocktail). T7 promoter-driven expression plasmids encoding wild type FNIPl or wild type folliculin were used for in vitro transcription/translation (IVT) with the rabbit reticulocyte TNT® T7 Quick kit (Promega) and 35S -methionine according to the manufacturer's protocols. The reactions were incubated for 90 minutes at 3O⁰C. The IVT reactions containing ³⁵S labeled folliculin or FNIPl were diluted with binding buffer, mixed with GST- fusion protein immobilized on glutathione-

SEPHAROSE® beads, and incubated at 4°C for one hour. After washing four times with binding buffer, beads were boiled with SDS-sample buffer and subjected to SDS-PAGE, followed by Coomassie Blue staining and exposure to X-ray film.

To determine the AMPK binding domain in FNIPl, purified AMPK subunits or HEK293 cell extract were incubated with GST- FNIP 1 immobilized on glutathione-SEPHAROSE® beads. After incubation and washing, eluted proteins were subjected to SDS-PAGE followed by Western blotting with anti-AMPK subunit specific antibodies. AMPK in vitro kinase assay

To measure AMPK activity that co-immunoprecipitated with folliculin or FNIPl, anti-HA, anti-FLAG or anti-FNIP 1 immunoprecipitants were washed twice with AMPK reaction buffer

(20 mM HEPES-NaOH pH7.0, 0.4 mM DTT, 0.01 % Brij-35 (polyoxyethyleneglycol dodecyl ether; non-ionic detergent)). After washing, beads were resuspended in 23 μl of AMPK reaction buffer, 7 μl of SAMS substrate peptide (SEQ ID NO: 20; lμg/μl, UpState Cell Signaling Solutions) and 10 μl of 1 μCi/μl[γ-³²P]ATP (diluted with ATP dilution buffer containing 75 mM MgCl₂, 500 μM ATP, 20 mM HEPES pH7.2, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM Na₃VO₄, ImM DTT). The reaction mixture was incubated at 30⁰C for 15 minutes in a shaking incubator. After centrifugation, 35 μl of supernatant was spotted onto P81 filter paper. Filter paper was washed with 0.075% Phosphoric acid for 5 minutes twice and with acetone for 5 minutes once. Radioactivity was measured in a scintillation counter. To measure AMPK activity in UOK257 cells, cells cultured with or without glucose for 36 hours were washed once with ice cold PBS and lysed with lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1% Triton X-100) in situ on ice. After lysis, cells were scraped from dishes, and lysates were centrifuged for 30 minutes at 4⁰C. Supernatants were subjected to immunoprecipitation by anti AMPKβl antibody (Cell Signaling) for 4 hours. Immunoprecipitants were washed five times with lysis buffer and twice with AMPK reaction buffer. The beads were incubated in a reaction mixture as described above and radioactivity was measured.

Example 2: A novel 130 kDa protein, FNIPl, interacts with folliculin, the BHD protein Previously the BHD gene had been cloned and characterized and was localized at chromosome 17pll.2 (Schmidt et al., Am. J. Hum. Genet. 69:876-882, 2001) which is mutated in the germline of patients with the Birt-Hogg-Dube' syndrome (Nickerson et ah, Cancer Cell. 2(2): 157- 164, 2002). BHD encodes a novel, highly conserved protein, folliculin, with no previously characterized functional domains to suggest its role in normal cell pathways. To obtain clues to its biologic function, folliculin-interacting protein(s) were sought by co-immunoprecipitation from 293HEK cells stably expressing doxycycline-inducible, HA-tagged folliculin. Proteins bound to HA- tagged folliculin were immunoprecipitated with anti HA antibody, eluted with HA peptide, and separated on SDS-polyacrylamide gel electrophoresis. A major 130KDa protein was identified, along with several minor high molecular weight proteins (FIG. IA). Mass spectrometric analysis of the 130 KDa protein, designated FNIPl (folliculin interacting pjrotein D, revealed sequence identity to a polypeptide encoded by the cDNA KIAAl 961 (GenBank accession no. BAB85547), which lacks an ATG initiation codon but shares substantial sequence homology with an uncharacterized human EST in the public database containing a 5'-ATG (GenBank Accession No. BC001956). Using these cDNAs as templates, a full length FNIPl cDNA was amplified and sequence verified (SEQ ID NO: 1; see Methods, Example 1).

Primers designed from the 5' and 3' ends of the full length sequence were used to amplify and clone the full length 3498 nucleotide FNIPl cDNA transcript from a pooled tissue cDNA library, confirming its presence in normal tissues. Four alternative transcripts lacking one or two of the 18 coding exons were also identified from this pooled tissue source, indicating that alternate tissue- specific isoforms of FNIPl exist (SEQ ID NOs: 3, 5, 7, and 9; FIG. ID).

The largest encoded FNIPl protein of 1166 amino acids is highly conserved across species (FIG. IB) with 5 conserved blocks of sequence (>35% similarity) (FIG. 1C) but BLAST analysis revealed no previously characterized domains that might suggest its function. Northern blot analysis confirmed FNIPl mRNA expression was strongest in peripheral blood leukocytes, placenta, and heart, with moderate expression in brain, liver, small intestine and lung, and weak expression in skeletal muscle, spleen, and kidney (FIG. IE).

Example 3: Interaction of exogenously expressed folliculin and FNIPl is supported by reciprocal tagged co-immunoprecipitation experiments

To confirm the mass spectrometric results suggesting an interaction between a novel protein, FNIPl, and folliculin, reciprocal co-immunoprecipitations were performed in 293HEK cells cotransfected with HA-folliculin and FLAG-FNIPl -expressing constructs. Western analysis of cell lysates immunoprecipitated with anti-FLAG antibody and blotted with anti-HA antibody detected both FLAG-FNIPl and HA-folliculin in the immunoprecipitates. In the reciprocal experiment, immunoprecipitation with anti-HA antibody and blotting with anti-FLAG antibody revealed both HA- folliculin and FLAG-FNIPl in the immunoprecipitates (FIG. 2A), confirming the interaction of these exogenous proteins. Example 4: Folliculin and FNIPl interaction is confirmed by reciprocal co- immunoprecipitation of the endogenous proteins

Confirmation of binding of endogenous FNIPl to endogenous folliculin would strengthen the physiologic importance of this interaction. Thus, polyclonal antibodies were developed against peptides contained in folliculin (Ab 102) or full length GST-folliculin (Ab 104) and against peptides in FNIPl (AbI 81), and co-immunoprecipitation of endogenous FNIPl and folliculin was demonstrated by reciprocal imtnunoprecipitations from 293 HEK cells (FIG. 2B). AblO4 co-immunoprecipitated FNIPl with folliculin, as detected by blotting with Abl81; conversely, AbI 81 co-immunoprecipitated folliculin with FNIPl, as seen by blotting with AblO2.

Example 5: Colocalization of FNIPl and folliculin occurs in the cytoplasm

If folliculin-FNIPl interaction is biologically relevant, they would be expected to localize in the same cell organelle. To examine this, the cellular location of HA-FNIPl and FLAG-folliculin transiently cotransfected into HEK293 cells was evaluated by immunofluorescence microscopy using secondary antibodies conjugated to Cy3 to detect HA-FNIPl (red signal; upper left panel in both sets of four) and to Alexa 488 to detect FLAG-folliculin (green signal; upper right panel in both sets of four). FNIPl and folliculin were co-localized with a reticular pattern in the cytoplasm (yellow signal; lower right panel in both sets of four) with strongest signal in the Golgi apparatus (FIG. 2C). Folliculin was also located in the nucleus.

Example 6: Folliculin interacts with FNIPl through its carboxy-terminus

Next, the FNIPl binding domain in folliculin was identified. A series of FLAG-tagged folliculin deletion mutants in mammalian expression constructs were transfected into doxycycline- induced HA-FNIPl expressing 293HEK cells. As seen in FIG. 2D, the expression level of folliculin mutant proteins is strong for all fragments except the construct expressing residues 1-516. The results support a requirement for the carboxy-terminal half of folliculin for productive binding to FNTPl.

Residues 1-344 showed no binding to FNIPl in an immunoprecipitation assay with anti HA antibody followed by Western blotting with anti FLAG antibody; residues 1-516 showed weak binding, and residues 246-579 showed the strongest binding relative to full length folliculin (residues 1-579; FIG. 2D). Reduced binding of the deletion mutant containing only residues 345-579, when compared to the longer mutant containing residues 246-579, indicated that the additional residues (246-344) may be important for maintaining proper tertiary structure in the region of the FNIPl binding domain. A germline missense mutation that produced a premature termination codon at residue 527 in folliculin was found in the proband and affected members of a family with BHD syndrome (Schmidt et ah, Am J Hum Genet. 76(6):1023-1033, 2005). When a mammalian expression construct carrying the C.C2034T mutation was transfected into the HA-FNIPl expressing cells, no binding of the R527X truncated folliculin protein was seen in HA-FNIPl co-immunoprecipitations with anti-HA antibody. Example 7: Binding of FNIPl to the carboxy-terminal region of folliculin is confirmed by in vitro GST-pull down experiments To confirm the binding results shown in FIG. 2D, in vitro experiments were performed in which the amount of radiolabeled in vitro transcribed and translated (IVT)-FNIPl, which bound to recombinant GST-folliculin and GST tagged deletion mutant proteins (expressed in insect cells and obtained from lysed cell pellets). These were immobilized on glutatbione-Sepharose beads were measured. As seen in the in vivo experiments, GST-folliculin deletion mutant protein containing the carboxy-terminal half of folliculin (residues 246-579 or 345-579) gave the most productive binding to FNIPl, whereas a deletion mutant protein produced from the ammo-terminal half of the protein (residues 1-344) or lacking the carboxy-terminus (residues 1-516) were unable to bind FNIPl. These results indicate a FNIPl binding domain in the carboxy-terminus of folliculin.

Example 8: AMP kinase γ-1 subunit, HSP90 and 14-3-3Θ were identified as FNIPl interacting proteins

Efforts to obtain clues to folliculin function by searching for interacting proteins led to title identification of FNIPl, a novel 130KDa. Thus, identifying FNIPl interacting proteins is expected to shed light on biologic relevance of folliculin-FNIPl association. To identify proteins interacting with FNIPl, the same general experimental approach was used that was described above.

HA-FNIPl was immunoprecipitated from stable, doxycycline-inducible, HA-FNIPl expressing HEK 293 cells with anti HA antibody. Interacting proteins were eluted with HA peptide, separated by SDS-PAGE, and the HA-FNIPl interacting proteins were identified by mass spectrometry. Identified proteins included the γ-1 subunit (40 kDa) of 5 '-AMP-activated protein kinase (AMPK), heat shock protein 90 (HSP90, 90 kDa), 14-3-3Θ (33 kDa) as well as folliculin (67 kDa) and FNIPl (130 kDa) (FIG. 3A). Other minor protein bands were detected in doxycycline- induced (but not uninduced) HA-FNIP 1 expressing cells as well.

Example 9: AMPK complex binds to FNIPl in a folliculin-independent manner

AMPK is known to play a critical role in energy sensing in cells, negatively regulating biosynthetic pathways during cellular stress such as nutrient deprivation, hypoxia, and low ATP levels (for review, see Carling, Trends Biochem. Sd. 29:18-24, 2004). This heterotrimeric protein kinase consists of a catalytic subunit (α), and two regulatory subunits (β and γ) (Neumann et ah, Prot Exp Purij ^'30:230-237, 2003). Since the α and β subunits of AMPK were not detected by mass spectrometric analysis of the HA-FNIPl immunoprecipitations (FIG. 3A), it was unknown whether all three subunits of AMPK were FNIPl -interacting proteins and if the interaction was folliculin- dependent.

HEK293 cells were co-transfected with HA-folliculin-expressing constructs with and without FLAG-FNIP 1-exρressing constructs. The immunoprecipitates were evaluated by Western blotting with antibodies to the three AMPK subunits and to HA-folliculin and FLAG-FNIP 1. The cell lysates from HEK293 cells overexpressing HA-folliculin showed significant endogenous levels of the AMPK α, β and γ subunits independent of FNIPl overexpression (FIG. 3B, lanes 1 and 2). AU three AMPK subunits immunoprecipitated with HA-folliculin in a FLAG-FNIP 1 -dependent manner (FIG. 3B, lane 4).

The catalytic α subunit of AMPK is phosphorylated on T 172 by an AMPK kinase, LKBl, (Carling, Trends Biochem ScI 29:18-24, 2004) and it is the phosphorylated form that was preferentially bound in the HA-folliculin: FLAG-FNIPl complex and detected by a phospho-T172 specific antibody (FIG. 3B, lane 4). These data indicate that the AMPK heterotrimer, consisting of α, β and γ subunits, binds directly to the folliculin:FNIPl complex in a FNIPl -dependent manner, but not to folliculin alone.

To confirm these data, folliculin-independent AMPK binding to endogenous FNIPl was sought. AMPKβ subunit binding to endogenous FNIPl was not dependent upon functional endogenous folliculin, as shown in experiments with UOK257 cells lacking a functional BHD gene. In the UOK257 renal rumor cell line with a BHD (-/-) genotype (established from a BHD patient), endogenous FNIPl was able to bind to the AMPKβ subunit, and productive AMPK-FNIPl binding was not affected by restoration of folliculin expression by stable lentiviral transduction of wild type BHD (UOK257-2 and UOK257-6; FIG. 3E). These data support the observation in cells overexpressing folliculin and FNIPl, that AMPK-FNIPl interaction is folliculin-independent.

Example 10: Folliculin can exist in a phosphorylated form and phosphorylated folliculin is preferentially bound to FNIPl

Folliculin exists in at least three electrophoretically distinct, phosphorylated forms when overexpressed in untreated HEK293 cells: a single, fast migrating form upon treatment with calf alkaline phosphatase (general phosphatase) or protein phosphatase 1 (serine/threonine-specific phosphatase) and at least two slower migrating forms upon treatment of cells with the phosphatase inhibitor, Calyculin A (FIG. 3C). Using a monoclonal antibody to folliculin, at least three electrophoretically distinct forms of endogenous folliculin were also detected in HEK 293 cells. These shifted to the slower migrating (phosphorylated) species upon immunoprecipitation with AbI 81 (which binds to endogenous FNIP 1 ; FIG. 3D), indicating that FNIP 1 preferentially binds phosphorylated folliculin. In this experiment, the AMPKβ subunit, representing the AMPK complex, was shown to co-immunoprecipitate with endogenous FNIPl and folliculin, raising the possibility that folliculin becomes phosphorylated by AMPK through their mutual interaction with FNIP 1. Similar experiments with HEK293 cells cotransfected with FLAG-FNIP 1 and HA-folliculin revealed the presence of a slower migrating HA-folliculin form in immunoprecipitates from cells overexpressing FLAG-FNIPl, but not in those lacking FNIPl (FIG. 4B). In this experiment, the endogenous AMPKα subunit protein in the immunoprecipitate was shown to be phosphorylated in a FNIP-dependent manner.

Example 11: Endogenous AMPK activity is detected in FNIP:folliculin immunoprecipitates from FNIP- and folliculin-over-expressing cells

Evidence from these above experiments suggested that folliculin might act as substrate for a kinase in a FNIPl -dependent manner and that FNIPl may be important interactor with AMPK. Thus AMPK activity was measured in an in vitro kinase assay with SAMS peptide, a specific substrate for AMPK, with and without a functional FNIP: folliculin complex. Stably expressing, doxycycline- inducible HA-folliculin HEK 293 cells were transfected with FLAG-FNIP, and anti HA immunoprecipitates were assayed for endogenous AMPK activity with SAMS peptide and γ-³²P-ATP. Ten-fold greater γ-³²P-ATP incorporation into SAMS peptide was observed in anti-HA immunoprecipitates from cells expressing wild-type HA-folliculin which binds to FNIPl compared with cells expressing a truncated mutant form of folliculin (c.C2034T, R527X) that does not bind FNIPl (FIG. 4A). Empty FLAG-vector controls not expressing FNIPl and controls lacking the SAMS peptide were inactive. These data indicate that an AMPK-FNIP-folliculin complex exists in the immunoprecipitates from cells expressing wild type folliculin (but not a FNIP -binding defective mutant folliculin) that catalyzes in vitro kinase activity toward an AMPK specific substrate.

Example 12: Folliculin and FNIPl both have consensus AMPK substrate sequences and are phosphorylated by AMPK in vitro

Since these proteins exist in a complex with AMPK, and at least folliculin shows evidence of phosphorylation, it was evaluated whether folliculin or FNIPl could act as a substrate for AMPK. Search of the literature identified a consensus sequence for the substrate binding site of AMPK present in known substrates for AMPK, for example, Acetyl CoA carboxylase, phospho-2- fructokinase, and SAMS. The consensus sequence is represented by H-X-B-X-X-S-X-X-X-H or H- B-X-X-S-X-X-X-H where H is a bulky hydrophobic amino acid and B is a basic amino acid. The phosphorylation site is S, serine. Both folliculin and FNIPl have several consensus phosphorylation sites for AMPK in regions that are highly conserved in other species (FIG. 5 and FIG. 6).

Phosphorylation of recombinant GST-folliculin (but not GST alone) was observed in an in vitro kinase assay with partially purified AMPK from rat liver and 7³²P-ATP, which is activated by 200μM AMP, an allosteric effector of AMPK, and partially inhibited by competition with SAMS peptide at 1 OOμM (FIG. 7). Additionally, GST-FNIP 1 from an insect cell pellet was also phosphorylated by 7³²P-ATP and partially purified AMPK and was also activated by AMP (FIG. 8). These results suggest that FNIPl and folliculin may serve as substrates for AMPK, since an AMPK consensus site was found in these two proteins, and AMP activated the phosphorylation of these two proteins in the AMPK in vitro kinase assays. Example 13: Folliculin phosphorylation, which is facilitated by FNIPl expression, is blocked by inhibitors of mTOR

In the presence of growth factors and nutrients, the mTOR pathway acts as a master regulator of cell growth and protein synthesis through increased protein translation. Under conditions of nutrient depletion (in particular, amino acid starvation), mTOR activity is downregulated, and autophagy, a protein scavenging pathway, is upregulated. BHD-null cells are exquisitely sensitive to amino acid deprivation and shut down mTOR activity within 30 minutes compared with BHD- restored cells (FIG. 10). FNIPl overexpression drives folliculin phosphorylation, which is abrogated by the mTOR inhibitor rapamycin, or by serum or amino acid starvation, both of which downregulate mTOR (FIG. 9). These data suggest that folliculin phosphorylation facilitated by FNIPl is regulated by mTOR, although it is not clear whether folliculin is a direct substrate of mTOR or a substrate of a downstream kinase that is activated by mTOR.

Example 14: FNIPl expression is high in BHD-associated renal tumors and sporadic renal tumors arising from the distal nephron.

Recent evidence, demonstrating "second hit" mutations in BHD-associated renal tumors from patients with germline mutations, indicates that BHD may act as a tumor suppressor gene (Vocke et ah, J Natl Cancer Inst 97:931-935, 2005); thus folliculin may serve a function in suppressing cell growth in response to cellular stress. BHD-associated renal tumor subtypes displayed very weak or undetectable expression of BHD mRNA by in situ hybridization (Warren et ah, Mod. Pathol. 17:998-1011, 2004) in support of the Knudson "two hit" tumor suppressor model. Since FNIPl interacts with folliculin in vivo, we evaluated FNIPl mRNA expression by real-time quantitative PCR in renal tumors to determine if FNIPl and BHD demonstrated coordinate or reciprocal expression patterns. FNIPl expression was higher in the majority of chromophobe RCC and collecting duct carcinoma when compared with expression levels in corresponding normal kidney tissue (FIG. l l;/?=0.01 and /7=0.004, respectively). Expression levels of FNIPl in clear cell RCC, papillary RCC and oncocytomas was comparable to normal kidney tissue. In support of the RT-PCR data, FNIPl mRNA expression measured by in situ hybridization was high in renal tumors from BHD patients in contrast to the low BHD expression reported in BHD-associated renal tumors. FNIPl overexpression may be useful as a diagnostic marker for renal tumors arising from the distal nephron, the most frequent tumors found in patients with BHD syndrome

Provided is a new nucleic acid molecule, FNIPl, and the protein encoded thereby, along with several specific alternate transcript FNIPl sequences and protein isoforms. Also provided are methods for identifying mutant FNIPl proteins in a subject, and using them to determine or predict a subject's BHD disease state. Methods of screening for a compound for the treatment of BHD syndrome or another hamartomatous syndrome, are also described. The precise details of the methods may be varied or modified without departing from the spirit of the described disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

1. A purified polypeptide having an amino acid sequence comprising the sequence as set forth in: (a) SEQ ID NO: 2;

(b) SEQ ID NO: 4; <c) SEQ ID NO: 6;

(d) SEQ ID NO: 8;

(e) SEQ ID NO: 10; (f) a sequence having at least 95% sequence identity to SEQ ID NO: 2,

SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; or

(g) a sequence having at least 98% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

2. The purified polypeptide of claim 1 which comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 with 1 to 10 conservative amino acid substitutions.

3. A purified antibody that selectively binds to an epitope of FNIPl protein.

4. A purified polypeptide that binds specifically to the antibody of claim 3.

5. The purified antibody of claim 3, wherein the epitope is a region of the FNIPl protein that is truncated in a mutant FNIPl protein associated with BHD syndrome.

6. The purified antibody of claim 5, wherein the antibody has measurably stronger binding to the mutant form of FNIPl protein as compared to a wild-type form of FNIPl protein.

7. An isolated nucleic acid molecule encoding the purified polypeptide of claim 1.

8. The isolated nucleic acid of claim 7, comprising the sequence as set forth in: (a) SEQ ID NO: 1;

(b) SEQ ID NO: 3; (c) SEQ ID NO: 5;

(d) SEQ ID NO: 7;

(e) SEQ ID NO: 9; (f) a sequence having at least 90% sequence identity to (a), (b), (c), (d), or (e);

(g) a sequence having at least 95% sequence identity to (a), (b), (c), (d), or (e); or (h) a sequence having at least 98% sequence identity to (a), (b), (c), (d), or (e).

9. The nucleic acid molecule of claim 8, wherein the nucleic acid sequence consists of the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

10. A recombinant nucleic acid molecule comprising a promoter sequence operably linked to a nucleic acid molecule according to claim 8.

11. A cell transformed with a recombinant nucleic acid molecule according to claim 10.

12. A method of identifying an agent having potential to treat a hamartomatous condition, the method comprising: contacting with at least one test agent a cell comprising a nucleic acid sequence encoding the FNIPl protein of claim 1, or a reporter gene operably linked to a FNIPl transcription regulatory sequence; and detecting a change in expression of the FNIPl protein or the reporter gene in the cell; wherein an agent that changes the expression of the FNIPl protein or the reporter gene in the cell is identified as an agent having potential to treat the hamartomatous condition.

13. The method of claim 12, wherein the cell is an epithelial cell, a kidney cell, or an immortalized cell.

14. The method of claim 12, wherein detecting the change in the FNIPl protein expression comprises analysis by Northern blot, Western blot, RT-PCR, immunohistochemistry, or a combination of two or more thereof.

15. The method of claim 12, wherein the nucleic acid sequence encoding the FNIPl protein is a FNIPl gene in the genome of the cell.

16. The method of claim 17, further comprising contacting each of a plurality of cells with a member of a library of test agents, wherein each cell comprises a nucleic acid sequence encoding the FNIPl protein of claim 1.

17. The method of claim 16, wherein the library of test agents comprises at least about 100 different agents.

18. The method of claim 16, wherein the library of compositions comprises one or more natural products, chemical compositions, biochemical compositions, polypeptides, peptides, or antibodies.

19. The method of claim 12, wherein the hamartomatous condition is BFfD syndrome, tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or

Bannayan-Riley-Ruvalcaba syndrome

20. A method of detecting a change in binding of a binding partner of FNIP 1 , the method comprising: contacting a FNIPl polypeptide and a binding partner polypeptide with at least one test agent under conditions that would permit the FNIPl polypeptide and the binding partner polypeptide to bind to each other in the absence of the test agent; and determining whether the test agent affects the binding of the FNIPl polypeptide and the binding partner polypeptide to each other; wherein the binding partner is folliculin or AMPK, and wherein an effect on the binding of the FNIPl polypeptide and the binding partner polypeptide to each other identifies the test agent as an agent having potential to treat the hamartomatous condition.

21. The method of claim 20, further comprising determining whether the test agent specifically binds to the FNIPl polypeptide.

22. The method of claim 20, further comprising determining whether the test agent specifically binds to the folliculin polypeptide or the AMPK polypeptide.

23. The method of claim 20, wherein the FNIP 1 polypeptide comprises at least 15 consecutive amino acids of SEQ ID NO: 2 or at least 15 consecutive amino acids of a polypeptide having 90% sequence identity with SEQ ID NO: 2.

24. The method of claim 20, wherein the folliculin polypeptide comprises:

(a) at least 15 consecutive amino acids of SEQ ID NO: 12;

(b) at least 15 consecutive amino acids of a polypeptide having 90% sequence identity with SEQ ID NO: 12; or (c) at least 15 consecutive amino acids of residues 246-579 of SEQ ID NO: 12.

25. The method of claim 20, wherein the effect on the binding of the FNIPl polypeptide and the binding partner polypeptide to each other comprises (1) an increase in binding affinity, or (2) a decrease in binding affinity.

26. The method of claim 24, wherein the FNIP 1 polypeptide or the binding partner polypeptide is bound to a solid substrate or a soluble support.

27. The method of claim 20, wherein the hamartomatous condition is BHD syndrome, tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome.

28. A method for identifying an agent having the potential to be a FNIPl peptidomimetic, comprising contacting at least one test agent with an antibody specific for a FNIPl polypeptide, wherein a test agent that is specifically bound by the antibody is identified as an agent having potential to be a FNIPl peptidomimetic.

29. The method of claim 28 further comprising determining whether the agent having potential to be a FNIPl peptidomimetic can specifically bind a folliculin polypeptide.

30. The method of claim 28 further comprising determining whether the agent having potential to be a FNIPl peptidomimetic can specifically bind an AMPK polypeptide.

31. A method for treating a hamartomatous condition comprising administering to a subject a therapeutically effective amount of a FNIPl protein or a nucleic acid encoding the FNIPl protein.

32. The method of claim 31, wherein the FNIPl protein comprises an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID

NO: 10;

33. The method of claim 31, wherein the hamartomatous condition is BHD syndrome, tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or Bannayan-Riley-Ruvalcaba syndrome.

34. The method of claim 31, wherein the subject is a mammal.

35. The method of claim 34, wherein the subject is a human.

36. The method of claim 31 , wherein administration comprises topical administration, intravenous administration, intra-arterial administration, or intraperitoneal administration.

37. The method of claim 31, wherein the FNIPl protein or nucleic acid encoding the FNIPl protein is administered in combination with a folliculin protein or a nucleic acid encoding the folliculin protein.

38. The method of claim 31 , wherein the nucleic acid encoding the FNIP 1 protein is a viral vector, a naked DNA, or a liposome-encapsulated DNA.

39. A method of detecting a biological condition associated with a mutant FNIPl nucleic acid in a subject, comprising determining whether the subject has mutant FNIPl nucleic acid.

40. The method of claim 39, wherein the mutant FNIPl nucleic acid encodes a truncated FNIPl protein and the method comprises detecting the truncated FNIPl protein.

41. The method of claim 39, which is a method of detecting BHD syndrome.

42. A pharmaceutical composition, comprising the isolated polypeptide of claim 1, and a pharmaceutically acceptable carrier or diluent.

43. A method of treating a hamartomatous condition, comprising administering to a subject with the hamartomatous condition an effective amount of the composition of claim 42.

44. The method of claim 43, wherein administration comprises topical administration, intravenous administration, intra-arterial administration, or intraperitoneal administration.

45. The method of claim 43, wherein the hamartomatous condition is BHD syndrome, tuberous sclerosis complex, Peutz-Jeghers syndrome, Proteus syndrome, Lhermitte-Duclos disease, or

Bannayan-Riley-Ruvalcaba syndrome.

46. An antisense oligonucleotide that inhibits expression of the FNIPl polypeptide of claim 1.

47. A method comprising: a. obtaining a sample of nucleic acid from a subject; and b. determining a presence of a nucleotide that encodes the truncated FNIPl protein of claim 40.

48. The method of claim 47, wherein the determining step comprises amplifying at least a portion of a nucleic acid molecule comprising the FNIPl gene, sequencing at least a portion of a nucleic acid molecule comprising the FNIPl gene, or a combination thereof.

49. The method of claim 47, wherein the method comprises determining a propensity to develop a condition associated with BHD syndrome.

50. The method of claim 49, wherein the condition comprises fibrofolliculoma, renal neoplasia, or spontaneous pneumothorax.

51. The method of claim 20, wherein the method is a method for screening for an agent that inhibits binding of FNIPl to a binding partner, and wherein the method further comprises determining whether each member of a library of test agents affects the binding of the FNIPl polypeptide and the binding partner polypeptide to each other.

52. The method of claim 51 , where the library comprises small molecule inhibitors.

53 A method of treating a hamartomatous condition in a subject comprising administering to the subject a therapeutically effective amount of a small molecule inhibitor identified according to the method of claim 21, thus treating the hamartomatous condition in the subject. 54. The method of claim 53, wherein administration comprises topical administration, intravenous administration, intra-arterial administration, or intraperitoneal administration