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WO2001078575A2 - Ext2 as a predictive marker for osteoporosis - Google Patents

Ext2 as a predictive marker for osteoporosis Download PDF

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Publication number
WO2001078575A2
WO2001078575A2 PCT/US2001/012447 US0112447W WO0178575A2 WO 2001078575 A2 WO2001078575 A2 WO 2001078575A2 US 0112447 W US0112447 W US 0112447W WO 0178575 A2 WO0178575 A2 WO 0178575A2
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WO
WIPO (PCT)
Prior art keywords
ext2
polymoφhism
osteoporosis
subject
sequence
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PCT/US2001/012447
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French (fr)
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WO2001078575A3 (en
Inventor
Robert F. Klein
Eric S. Orwoll
John K. Belknap
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Oregon Health & Science University
United States Department Of Veterans Affairs
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Application filed by Oregon Health & Science University, United States Department Of Veterans Affairs filed Critical Oregon Health & Science University
Priority to AU2001257071A priority Critical patent/AU2001257071A1/en
Publication of WO2001078575A2 publication Critical patent/WO2001078575A2/en
Publication of WO2001078575A3 publication Critical patent/WO2001078575A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present disclosure is generally related to prediction and diagnosis of disease states, for instance prediction of a predisposition to osteoporosis and/or low bone mineral density in a subject.
  • Osteoporosis is a multifactorial disease that leads to an increased risk of bone fracture. It is most prevalent in post-menopausal women or the elderly of either gender, and is becoming a major public health problem due to extended life expectancy. Its major consequence, hip fracture, has major health consequences. Research on osteoporosis emphasizes finding new therapeutic approaches to treat the condition and characterization genetic markers that may prove useful in early identification of people, particularly women, at risk of developing osteoporosis. Since therapy of established osteoporosis remains unsatisfactory, prevention of the condition is the best choice.
  • the acquisition of bone mass results from bone modeling and linear growth during skeletal development, whereas its maintenance in adults results from a coupling mechanism between the activities of bone formation and bone resorption.
  • the processes governing acquisition of bone mineral have received far less attention than those related to the maintenance of adult bone density. With new emphasis on maximizing peak bone mass as a strategy for reducing fracture risk during the course of adult life, it is of increasing importance that the factors governing acquisition of bone mineral density be understood.
  • the factors known to influence bone mass accumulation during growth include heredity, gender, dietary components (calcium, proteins), endocrine factors (sex steroids, calcitriol, insulin growth factor I), and mechanical forces. Quantitatively, the most prominent determinant appears to be heredity.
  • Past research to identify specific genes that influence peak bone mass has focused mainly on evaluation of candidate genes with identifiable polymorphisms. For example, polymorphisms of the gene for vitamin D receptor have been associated with low bone mass in studies for some, but not all, populations.
  • Bone mineral density is determined by both environmental influences and polygenic inheritance (Eisman, Endocr. Rev. 20:788-804, 1999). The extreme difficulty of dissecting out environmental factors from genetic ones in humans has motivated the investigation of animal models (Beamer e. ⁇ /., //n/w. Genome 10: 1043-1049, 1999; Shimizu e/ ⁇ t, Mamm. Genome 10:81-87, 1999). Peak BMD has been examined in 24 recombinant inbred (RI) mouse strains, derived from a cross between C57BL/6J (B6) and DBA/2J (D2) progenitors (BXD RI) (Klein et al., J. Bone Miner. Res.
  • RI recombinant inbred
  • the inventors have found a strong link between predisposition to osteoporosis, or an increased likelihood of having low bone mineral density, and polymo ⁇ hisms in the Ext2 gene, for example polymo ⁇ isms in the coding or untranslated regions of Ext2.
  • the polymorphism may be, for example, at position 2055 of the coding region or position 2707 of the uncoding region of Ext2.
  • Polymo ⁇ hisms in this gene can be used to detect such predisposition or increased likelihood in subjects, as well as to diagnose and prognose osteoporosis.
  • aspects of the disclosure include methods for predicting a predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject.
  • Such methods can include determining whether the subject has a polymorphism in a Ext2 sequence (for instance, a Ala622Thr or 3'UTR stem-loop mutation), wherein presence of the polymo ⁇ hism indicates the predisposition to osteoporosis or the increased likelihood of having low bone mineral density.
  • Specific embodiments will further include determining whether the subject has one or more other alleles associated with predisposition to osteoporosis or increased likelihood of having low bone mineral density. Examples of such other alleles include a polymorphism at position 1245 of a Col 1 alpha gene; a polymo ⁇ hism of an interleukin-1 receptor antagonist gene; and a polymo ⁇ hism in a first intron of a human estrogen receptor gene.
  • Determining whether the subject has the polymo ⁇ hism will in some methods include providing DNA from the subject, and assessing the DNA for the presence of the Ala622Thr or 3'UTR stem-loop mutation polymo ⁇ hism, or both. Certain methods may further include determining whether the subject is homozygous or heterozygous for the polymo ⁇ hism. Assessing the DNA of the subject for the presence of a polymo ⁇ hism may be performed by a process that includes subjecting the DNA or RNA to amplification using oligonucleotide primers flanking the polymo ⁇ hism, such as an oligonucleotide ligation assay.
  • Further embodiments include methods of predicting predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject, which methods include obtaining a test sample of DNA containing a Ext2 sequence of the subject; and determining whether the subject has a polymo ⁇ hism in the Ext2 sequence, wherein the presence of the polymo ⁇ hism indicates the predisposition to osteoporosis or the increased likelihood of having low bone mineral density in a subject.
  • determining whether the subject has the polymo ⁇ hism can include using restriction digestion, probe hybridization (e.g., using a nucleic acid probe to a Ext2 polymo ⁇ hism), nucleic acid amplification (e.g., PCR or another amplification technique), and/or nucleotide sequencing.
  • probe hybridization e.g., using a nucleic acid probe to a Ext2 polymo ⁇ hism
  • nucleic acid amplification e.g., PCR or another amplification technique
  • nucleotide sequencing e.g., PCR or another amplification technique
  • Further methods include methods for predicting predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject, which method includes obtaining from the subject a test sample of DNA comprising an Ext2 sequence; contacting the test sample with at least one nucleic acid probe for an Ext2 sequence polymo ⁇ hism that is associated with increased predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject to form a hybridization sample; maintaining the hybridization sample under conditions sufficient for specific hybridization of the Ext2 sequence with the nucleic acid probe; and detecting whether there is specific hybridization of the Ext2 sequence with the nucleic acid probe, wherein specific hybridization of the Ext2 sequence with the nucleic acid probe indicates increased predisposition to osteoporosis or an increased likelihood of having low bone mineral density in the subject.
  • Representative polymo ⁇ hisms that can be detected using these methods include the
  • the probe is present on a substrate, such as a nucleotide array.
  • kits for use in diagnosing an increased predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject include a probe that specifically hybridizes to an Ext2 sequence polymo ⁇ hism that is associated with the increased predisposition to osteoporosis or an increased likelihood of having low bone mineral density, such as an Ext2 Ala622Thr or 3'UTR stem-loop mutation polymo ⁇ hism.
  • kits can also include instructions.
  • nucleic acid probes that specifically hybridize to a human Ext2 Ala622Thr or 3'UTR stem-loop mutation polymorphism.
  • the disclosure further includes methods of osteoporosis therapy.
  • Such methods can include screening an individual for a genetic predisposition to osteoporosis; and, if such a predisposition is identified, treating (e.g., by hormone replacement therapy) that individual to prevent or reduce osteoporosis or to delay the onset of osteoporosis, wherein predisposition to osteoporosis is correlated with a polymo ⁇ hism in a Ext2 sequence.
  • FIG. 2 is a LOD plot of chromosome 2 for peak whole body BMD as determined by an interval mapping approach (Map Manager QT). Two of the nine markers used in the linkage analysis were found to differ substantially from the consensus map (Blake et al., Nucleic Acids Res. 27:95-98, 1999). Thus, the resultant primary linkage map is shown with markers D2MU80 and D2MH200 as anchors to the consensus map at 10 cM and 107 cM distance from the centromere, respectively.
  • the QTL accounts for -5% of the phenotypic variance in peak whole body BMD and the LOD curve exceeded the Lander and Kruglyak (Nat. Genet.
  • FIG. 3 is a schematic representation of the recombinant inbred (RI) segregation test strategy.
  • FIG. 3A is a schematic drawing illustrating that a RI strain that possesses a recombination or cross-over point in the region of a QTL can be used to generate two F 2 populations - one crossed with each parental strain. Analysis of the two populations will detect the population in which the
  • QTL is segregating, and accordingly locate the QTL above or below the recombination point.
  • linkage between phenotype and markers below the cross-over point is examined. If marker genotype correlates with phenotype in this population, then the QTL must reside below the recombination point.
  • linkage between phenotype and markers above the cross-over point is examined. If marker genotype correlates with phenotype in this population, then the QTL must reside above the recombination point. If linkage is observed in both F 2 populations, then two closely linked QTLs are likely to be present.
  • FIG. 4 shows bar graphs of mean peak whole body BMD Z scores of animals from the two RIST populations.
  • FIG. 5 shows an alignment of portions of the Ext2 B6 and D2 allele sequences; variations between the two sequences are indicated by stars.
  • total RNA was isolated from cardiac tissue of B6 and D2 mice.
  • the Ext2 cDNAs were amplified by RT-PCR, subcloned into pBluesc ⁇ t and separately sequenced. To exclude artifacts, five animals of each inbred strain were examined and both DNA strands were sequenced.
  • the coding polymorphism is a G ⁇ A transition at cDNA position 2055. This polymo ⁇ hism predicts an amino acid sequence variant at position 622 in the COOH-terminal region of the mature Ext2 protein with threonine at this position in the B6 allele and alanine at this position in the D2 allele.
  • the 3 '-untranslated region polymorphism is a G -> C transversion at cDNA position 2707.
  • a short palindromic sequence (underlined) is evident in the B6 allele, which is disrupted by the sequence variant in the D2 allele.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three 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 Nos: 1 and 2 are the forward and backward primers used to amplify the D2Mit80 sequence.
  • SEQ ID NOs: 3 and 4 are the forward and backward primers used to amplify the D2Mitl52 sequence.
  • SEQ ID NOs: 5 and 6 are the forward and backward primers used to amplify the D2Mit91 sequence.
  • SEQ ID Nos: 7 and 8 are the forward and backward primers used to amplify the D2Mit94 sequence.
  • SEQ ID Nos: 9 and 10 are the forward and backward primers used to amplify the D2Mitl2 sequence.
  • SEQ ID NOs: 1 1 and 12 are the forward and backward primers used to amplify the D2Mitl64 sequence.
  • SEQ ID NOs: 13 and 14 are the forward and backward primers used to amplify the
  • SEQ ID Nos: 15 and 16 are the forward and backward primers used to amplify the D2Mit59 sequence.
  • SEQ ID Nos: 17 and 18 are the forward and backward primers used to amplify the D2Mit200 sequence.
  • SEQ ID NOs: 19 and 20 are the forward and backward primers used to amplify the Ext2 gene.
  • SEQ ID NO: 21 is an allele-specific oligonucleotide (ASO) specific for the position 2707 3'UTR stem-loop sequence (G allele).
  • SEQ ID NO: 22 is an allele-specific oligonucleotide (ASO) specific for the position 2707
  • SEQ ID NOs: 23 and 25 are oligonucleotide sequences useful for detection of the Ala622Thr polymo ⁇ hism, in the human Ext2 gene.
  • SEQ ID NOs: 24 and 26 are oligonucleotide sequences useful for detection of a stem-loop polymo ⁇ hism, in the human Ext2 gene.
  • ASO allele-specific oligonucleotide
  • ASOH allele-specific oligonucleotide hybridization
  • BMD bone mineral density
  • BMP-2 bone morphogenetic protein-2
  • FSH ⁇ follicle stimulating hormone
  • ILl ⁇ interleukin loc
  • ILl ⁇ interleukin l ⁇
  • LOD likelihood of linkage
  • RT-PCR reverse-transcription polymerase chain reaction
  • TMAC tetramethyl ammonium chloride
  • Deviation from normal characteristics can be found in a control, a standard for a population, etc.
  • the abnormal condition is a disease condition, such as osteoporosis
  • a few appropriate sources of normal characteristics might include an individual who is not suffering from the disease (e.g., osteoporosis), a population standard of individuals believed not to be suffering from the disease, etc.
  • abnormal may refer to a condition that is associated with a disease.
  • the term "associated with” includes an increased risk of developing the disease as well as the disease itself.
  • a certain abnormality such as an abnormality in an Ext2 nucleic acid or Ext2 protein expression
  • An abnormal nucleic acid such as an abnormal Ext2 nucleic acid, is one that is different in some manner to a normal (wildtype) nucleic acid.
  • Such abnormality includes but is not necessarily limited to: (1) a mutation in the nucleic acid (such as a point mutation (e.g., a single nucleotide polymo ⁇ hism) or short deletion or duplication of a few to several nucleotides); (2) a mutation in the control sequence(s) associated with that nucleic acid such that replication or expression of the nucleic acid is altered (such as the functional inactivation of a promoter); (3) a decrease in the amount or copy number of the nucleic acid in a cell or other biological sample (such as a deletion of the nucleic acid, either through selective gene loss or by the loss of a larger section of a chromosome or under expression of the mRNA); and (4) an increase in the amount or copy number of the nucleic acid in a cell or sample (such as a genomic amplification of part
  • a genomic-amplified nucleic acid sequence may also contain one or more point mutations.
  • an abnormality in a nucleic acid may be associated with, and in fact may cause, an abnormality in expression of the corresponding protein.
  • Abnormal protein expression refers to expression of a protein that is in some manner different to expression of the protein in a normal (wildtype) 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 (e.g., organ or tissue specific) expression of the protein (such that the protein is not
  • Controls or standards appropriate for comparison to a sample, for the determination of abnormality include samples believed to be normal 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.
  • Double-stranded DNA has two strands, a 5'— > 3' strand, referred to as the plus strand, and a 3' - 5' strand (the reverse compliment), referred to as the minus strand. Because RNA polymerase adds nucleic acids in a 5' — » 3' direction, the minus strand of the DNA serves as the template for the RNA during transcription. Thus, the RNA formed will have a sequence complementary to the minus strand and identical to the plus strand (except that U is substituted for T).
  • Antisense molecules are molecules that are specifically hybridizable or specifically complementary to either RNA or the plus strand of DNA.
  • Sense molecules are molecules that are specifically hybridizable or specifically complementary to the minus strand of DNA. Antigene molecules are either antisense or sense molecules directed to a dsDNA target.
  • Binding or stable binding (of an oligonucleotide): 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 may 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 abso ⁇ tion detection procedures.
  • DNAse I or chemical footprinting gel shift and affinity cleavage assays
  • Northern blotting dot blotting
  • light abso ⁇ tion detection procedures For example, one method that is widely used, because it is so simple and reliable, involves observing a change in light abso ⁇ tion 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 abso ⁇ tion at a characteristic temperature as the oligonucleotide (or analog) and target disassociate from each other, or melt.
  • T m temperature at which 50% of the oligomer is melted from its target.
  • T m temperature at which 50% of the oligomer is melted from its target.
  • T m temperature at which 50% of the oligomer is melted from its target.
  • T m temperature at which 50% of the oligomer is melted from its target.
  • T m temperature at which 50% of the oligomer is melted from its target.
  • T m means a stronger or more stable complex relative to a complex with a lower (T m ).
  • cDNA may also contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule.
  • UTRs untranslated regions
  • cDNA is usually synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells. Complementarity and percentage complementarity: Molecules with complementary nucleic acids form a stable duplex or triplex when the strands bind, (hybridize), to each other by forming Watson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when an oligonucleotide remains detectably bound to a target nucleic acid sequence under the required conditions.
  • Complementarity is the degree to which bases in one nucleic acid strand base pair with the bases in a second nucleic acid strand. Complementarity is conveniently described by percentage, i.e. the proportion of nucleotides that form base pairs between two strands or within a specific region or domain of two strands. For example, if 10 nucleotides of a 15-nucleotide oligonucleotide form base pairs with a targeted region of a DNA molecule, that oligonucleotide is said to have 66.67% complementarity to the region of DNA targeted.
  • DNA deoxyribonucleic acid
  • DNA is a long chain polymer which 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, or for a stop signal.
  • codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
  • 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 Ext2, 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
  • Genomic target sequence A sequence of nucleotides located in a particular region in the human genome that corresponds to one or more specific genetic abnormalities, such as a nucleotide polymo ⁇ hism, a deletion, or an amplification
  • the target can be for instance a coding sequence, it can also be the non-coding strand that corresponds to a coding sequence
  • nucleic acid consists of nitrogenous bases that are either py ⁇ midines (cytosine (C), uracil (U), and thymine (T)) or pu ⁇ nes (adenine (A) and guanine (G))
  • C cytosine
  • U uracil
  • T thymine
  • G guanine
  • base pairing More specifically, A will hydrogen bond to T or U, and G will bond to C
  • 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
  • an oligonucleotide can be complementary to an Ext2 encoding mRNA, or an Ext2- encoding
  • oligonucleotide and oligonucleotide analog 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 1 1, herein inco ⁇ orated by reference.
  • stringent conditions encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence.
  • Stringent conditions may be broken down into particular levels of stringency for more precise definition.
  • “moderate stringency” conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of “medium stringency” are those under which molecules with more than 15% mismatch will not hybridize, and conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize.
  • Conditions of "very high stringency” are those under which sequences with more than 6% mismatch will not hybridize.
  • Isolated An "isolated" biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., 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.
  • Nucleotide includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA).
  • a nucleotide is one monomer in a polynucleotide.
  • a nucleotide sequence refers to the sequence of bases in a polynucleotide.
  • Oligonucleotide is 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.
  • 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.
  • PNA peptide nucleic acid
  • 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.
  • a sequence such as DNA or RNA
  • Operabiy linked A first nucleic acid sequence is operabiy 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.
  • a promoter is operabiy linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operabiy 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
  • Osteoporosis A bone disorder characterized by increased b ⁇ ttleness due to a reduction in bone density See the following references for further detail on recognized symptoms and other aspects of this disorder Melton and Riggs, Osteoporosis Etiology, Diagnosis, and Management, 2nd ed , Lippmcott-Raven Publishers, 1995 (ISBN 0781702755), Peel and Eastell, J Bone Miner Res 8(suppl 2) S505-510, 1994, Peel and Eastell, BMJ 310 989-992, 1995, Eastell and Riggs, Endocrmol Metab Clin NA 17 547-571,1988, Eastell and Riggs, Clin Obstet Gynecol 30 860-870, 1987, and Eastell and Riggs, Obstet Gynecol Clin NA 14 77-88, 1987)
  • the term osteoporosis encompasses both post-menopausal and senile osteoporosis, unless context indicates otherwise
  • 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
  • parenteral Administered outside of the intestine, e g , not via the alimentary tract
  • parenteral formulations are those that will be administered through any possible mode except ingestion This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance
  • PNA Peptide Nucleic Acid
  • 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
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate
  • 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 Polymorphism: Variant in a sequence of a gene Polymo ⁇ hisms 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
  • polymo ⁇ hism also encompasses variations that produce gene products with altered function, / e , 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 increased gene product
  • polymorphism may be used interchangeably with allele or mutation, unless context clearly dictates otherwise
  • Polymo ⁇ hisms 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 that is linked to the variation (e , 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, RNases, and so forth)
  • the polymo ⁇ hism disclosed herein m the 3' untranslated region of the Ext2 gene can be referred to by its location (e g , 3'UTR 2707, based on the numerical position of the variant residue) or by the effect it has on the secondary structure of the Ext2 mRNA (e g , the 3'UTR stem-loop mutation, because this mutation disrupts a stem-loop structure that is conserved in the Ext2 family of encoding sequences)
  • Probes and primers A probe comprises an isolated nucleic acid attached to a detectable label or other 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 pu ⁇ oses are discussed, e g , in Sambrook et al (In Molecular Cloning A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998)
  • Primers are short nucleic acid molecules, for instance DNA oligonucleotides 10 nucleotides or more in length, for example that hybridize to contiguous complementary nucleotides or a sequence to be amplified Longer DNA oligonucleotides may be about 15, 20, 25, 30 or 50 nucleotides or more in length
  • Primers can be anne
  • Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided in this disclosure It is also appropriate to generate probes and primers based on fragments or portions of these disclosed nucleic acid molecules, for instance regions that encompass the identified polymo ⁇ hisms at nucleotide 2707 in the untranslated region, and nucleotide 2055 within the Ext2 coding sequence
  • Amplification primer pairs can be derived from a known sequence, for example, by using computer programs intended for that pu ⁇ ose such as Primer (Version 0.5, ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).
  • probes and primers can be selected that comprise at least 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of Ext2 locus nucleotide sequences.
  • the disclosure thus includes isolated nucleic acid molecules that comprise specified lengths of the Ext2 encoding sequence and/or flanking regions. Such molecules may comprise at least 10, 15, 20, 23, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of these sequences or more, and may be obtained from any region of the disclosed sequences.
  • the human Ext2 locus, cDNA, ORF, coding sequence and gene sequences may be apportioned into about halves or quarters based on sequence length, and the isolated nucleic acid molecules (e.g., oligonucleotides) may be derived from the first or second halves of the molecules, or any of the four quarters.
  • the cDNA also could be divided into smaller regions, e.g. about eighths, sixteenths, twentieths, fiftieths and so forth, with similar effect.
  • isolated nucleic acid molecules comprise or overlap at least one residue position designated as being associated with a polymo ⁇ hism that is predictive of osteoporosis and/or bone mineral density.
  • polymo ⁇ hism sites include position 2055 (corresponding to the Ala622Thr polymo ⁇ hism) and position 2707 (corresponding to the 3'UTR stem-loop polymo ⁇ hism).
  • Protein A biological molecule expressed by a gene and comprised of amino acids. Purified: 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 or within a production reaction chamber (as appropriate).
  • a recombinant nucleic acid is one 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, e.g., by genetic engineering techniques.
  • Representational difference analysis A PCR-based subtractive hybridization technique used to identify differences in the mRNA transcripts present in closely related cell lines.
  • Serial analysis of gene expression The use of short diagnostic sequence tags to allow the quantitative and simultaneous analysis of a large number of transcripts in tissue, as described in Velculescu et al (Science 270 484-487, 1995)
  • Ext2 protein-specific binding agent An agent that binds substantially only to a defined target Thus an Ext2 protein-specific binding agent binds substantially only the Ext2 protein
  • Ext2 protein specific binding agent includes ant ⁇ -Ext2 protein antibodies (and functional fragments thereof) and other agents (such as soluble receptors) that bind substantially only to the Ext2 protein
  • Ant ⁇ -Ext2 protein antibodies may 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 the Ext2 protein may 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 may be used to determine that a given Ext2 protein binding agent, such as an ant ⁇ -Ext2 protein monoclonal antibody, binds substantially only to the Ext2 protein
  • FAbs, Fvs, and single-chain Fvs (SCFvs) that bind to Ext2 would be Ext2-spec ⁇ fic binding agents
  • 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
  • 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
  • (FAb')2 the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(Ab')2 a dimer of two FAb' fragments held together by two disulfide bonds
  • Fv a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as
  • Subject Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals
  • 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
  • oligonucleotide hybridizes therapeutically effectively oligonucleotide to an Ext2 target sequence results in inhibition of Ext2 expression
  • an antisense or a sense molecule can be used to target a portion of dsDNA, since 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
  • target sequences can be ssDNA, dsDNA, and RNA
  • a transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques
  • transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, hpofection, and particle gun acceleration
  • a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication A vector may also include one or more selectable marker genes and other genetic elements known in the art
  • the inventors have determined that polymo ⁇ hisms in the multiple exostoses 2 (Ext2) gene are linked to bone mineral density (BMD), and thereby can be used to predict the risk of a subject suffering from low bone mineral density and/or developing osteoporosis
  • BMD bone mineral density
  • the following examples illustrate this by showing particular examples of polymo ⁇ hisms that are associated with BMD
  • guidance is provided about finding other polymo ⁇ hisms associated with altered BMD
  • the disclosure is not limited to particular polymo ⁇ hisms, but is instead premised on the finding that Ext2 polymorphisms, and more broadly changes in Ext2 expression and/or activity, are associated with altered BMD
  • mice three complementary mapping populations of mice were utilized to narrow the BMD-related QTL to the region of the mouse chromosome 2 in which Ext2 resides
  • the RIST strategy was then used to significantly narrow the region of interest, which enabled isolation and definitive identification of the osteoporosis-linked gene, Ext2
  • mice and human (gi l 518042) Ext2 proteins are the same length, and share 95% sequence identity; numbering of amino acid positions within these two molecules is identical.
  • polymo ⁇ hisms and other nucleotide sequence designations herein are numbered as in the mouse Ext2 mRNA sequence, GenBank Accession Number U67837. These designations readily can be correlated to the numbering of the human Ext2 mRNA sequence (Accession number U62740) or human Ext2 gene sequence (Accession number AH00672) by comparing the sequences.
  • the coding sequence polymo ⁇ hism referred to herein as occurring at position 2055 is directly correlated with the human mRNA sequence (Accession number U62740) at position 2198.
  • the 3'UTR stem-loop, at position 2702 through 2714 on the mouse mRNA is found at position 14058 through 14070 on the human gene sequence (Accession number AH006672).
  • this stem-loop structure is conserved between mice and humans (see SEQ ID NO: 23), where the surrounding 3'UTR is substantially different.
  • this stem-loop structure is important in influencing the involvement of Ext2 in bone mineral density and thereby osteoporosis.
  • IL-l ra interleukin-1 receptor antagonist
  • Col 1 alpha gene U.S. Patent No. 5,922,542
  • human estrogen receptor gene U.S. Patent No. 5,834,200
  • This example provides the techniques that were used to identify the linkage between Ext2 and bone mineral density/osteoporosis, as well as to identify and characterize specific polymo ⁇ hisms in the Ext2 sequence.
  • mice used in these experiments were bred under identical conditions at the Portland VA Veterinary Medical Unit from stock originally obtained from The Jackson Laboratory (Bar Harbor, ME). Breeding mice were maintained for no more than three generations from stock obtained from The Jackson Laboratory. At the time of weaning, mice were group-housed (2-5 animals per cage) and provided with ad libitum water and laboratory rodent chow (Diet 5001 ; PMI Feeds, Inc., St. Louis, MO) in a 12 hour light/dark cycle at 21 ⁇ 2° C. All procedures were approved by the VA Institutional Animal Care and Use Committee and performed in accordance with National Institutes of Health guidelines for the care and use of animals in research. Bone Densitometry.
  • Bone mineral measurements were determined by dual energy X-ray abso ⁇ tiometry (DEXA). All studies were performed with a pencil beam Hologic QDR 1500 densitometer (Hologic Inc., Waltham, MA). Densitometric analyses were performed on anesthetized mice that were four months of age, the point at which the acquisition of adult bone mass is complete (Brodt et al., J. Bone Miner. Res. 14:2159-2166, 1999). The "global window” was defined as the whole body image minus the calvarium, mandible, and teeth.
  • the six highest-scoring mice of each gender were mated to form the high BMD line (six breeder pairs), while the six lowest scoring of each gender were mated to form the low BMD line.
  • Individual (or mass) selection was used to gain a more rapid and efficient selection response, with sib matings excluded.
  • the highest scoring of the high BMD line and lowest scoring of the low BMD line were selected to serve as breeders of the next generation, with ten breeder pairs per line.
  • Genomic DNA was isolated from individual mouse spleens using a salting-out method (Buck et al, J. Neurosci. 17:3946-3955, 1997). Mice were genotyped with microsatellite markers chosen from the MIT database and the Mouse Genome Database, both available on-line. All PCR primers (see Table 1) were purchased from Research Genetics (Huntsville, AL). Table 1
  • Amplification was performed on a Perkin-Elmer 9700 thermocycler (Branchburg, NJ). Each reaction mixture was in a total volume of 25 ⁇ l, consisting of approximately 150 ng of genomic DNA, 264 nM of both forward and reverse primers, 0.2 mM of each dNTP, 1 unit of Tag polymerase (Perkin-Elmer Cetus, Branchburg, NJ), and 2.5 ⁇ L of GeneAmp 10X PCR Buffer containing 100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2 , 500 mM KC1, and 0.01% (w/v) gelatin.
  • Thermal cycling included two, five-minute denaturation steps at 95° C then at 80° C, followed by 40 cycles of 30 seconds at 94° C, and 30 seconds at 53° C, 30 seconds at 72° C, and a final extension step for 10 minutes at 72° C. Amplified nucleic acid products were separated on 4% agarose gels and visualized with ethidium bromide staining.
  • mice were genotyped based on a known sequence polymo ⁇ hism (the 2707 stem-loop mutation) in the 3' untranslated region of the Ext2 gene (Clines et al, Genome Res. 7:359-367, 1997).
  • a 215 bp target region of the 3' UTR of the Ext2 gene was amplified in a 50 ⁇ l total reaction volume containing 100 ng genomic DNA, 210 nM of each primer (SEQ ID NO: 19 and SEQ ID NO: 20), 3 mM MgCl 2 , 50 mM K.C1, 10 mM Tris HC1 (pH 8.3), 0.2 mM dNTPs and 4 units of Tag Gold polymerase (PE Biosystems, Foster City, CA).
  • Amplification was performed on a Perkin-Elmer GeneAmp 9700 thermocycler and cycling included a 10 minute "hot start” to activate the enzyme, followed by 40 cycles of 30 seconds at 94° C, 30 seconds at 51.5° C, and 30 seconds at 72° C, and a final extension step for eight minutes at 72° C.
  • a 25 ⁇ l aliquot of this PCR reaction was loaded onto each of two 2% agarose gels, electrophoresed, and transferred onto Hybond-N + nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ). Allele-specific oligonucleotides (ASO) were labeled with digoxygenin (DIG) using a kit (Boehringer Mannheim, Indianapolis, IN) and 100 ⁇ M of ASO. The specific sequences of these ASO's (corresponding to residues 2700-2712 of the Ext2 3'UTR) were as follows:
  • AATACCTGTGAGGT (SEQ ID NO: 21) AATACCTCTGAGGT (SEQ IDNO: 22) Blots were hybridized with both DIG-labeled ASO (2 pmol/ml) and unlabeled ASO (40 pmol/ml) for three hours at 39° C. Blots were then washed (3 M TMAC, 50 mM Tris (pH 8.0), 1 mM EDTA (pH 8.0) and 0.1% SDS) twice for 10 minutes each at 47° C.
  • DIG detection was performed overnight with anti-DIG-AP Fab fragments (Boehringer Mannheim, Indianapolis, IN) at a 1 : 10,000 dilution in 1% blocking buffer (Roche Molecular Biochemicals, Indianapolis, IN) at 4° C. Blots were washed (0.1 M maleic acid, 0.15 M NaCl pH 7.5) and then developed using nitroblue tetrazolium/5-bromo-4- chloro-3-indoyl-phosphate (NBT/BCIP) for approximately 30 minutes.
  • NBT/BCIP nitroblue tetrazolium/5-bromo-4- chloro-3-indoyl-phosphate
  • RNA and cDNA Analysis Poly(A) + RNA was isolated directly from mouse heart tissue using a Micro-Fast Track mRNA Isolation Kit (Invitrogen Co ⁇ ., Carlsbad, CA). For RT-PCR, 500 ng of poly(A) + RNA was reverse-transcribed using a Superscript kit (Life Technologies, Inc., Grand Island, NY).
  • target sequences of the Ext2 gene from five B6 and five D2 mice were amplified, gel-purified and subcloned directly into a p-GEM-T Easy vector (Promega Co ⁇ ., Madison, WI) using a Fast-Link DNA Ligation Kit (Epicentre Technologies, Madison, WI). The plasmids were then transfected into ElectroMAX DH 10B cells (Life Technologies, Madison, WI).
  • B6D2F 2 genotyping data was accomplished by carrying out a 2 x 2 ⁇ 2 test for each marker between the tails of the distribution (low and high) and allele frequencies (B6 or D2). Individual p values for the phenotypic selection and RIST experiments were determined using linear least squares statistical analyses. For each marker tested, individual mice were assigned a genotypic score of zero, one, or two based on gene dosage (the number of D2 alleles at that locus). Correlation coefficients (Pearson's r) were determined and are equivalent to regressing BMD on gene dosage for each marker.
  • FIG. 1 illustrates the average BMD of animals with the three possible genotypes for the D2MU94 marker. The large difference between mice homozygous for the D2 allele versus those homozygous for the B6 allele is apparent, as is the intermediate BMD of heterozygous mice.
  • the allelic frequencies for D2MU94 in the third generation of mice selectively bred for low BMD was 70% B6 and 30% D2, while in the high BMD mice the allelic frequency was 100% D2 (fixation).
  • This region of the mouse genome is homologous to the centrome ⁇ c region of human chromosome 1 1 , a region that has been previously linked to BMD in both large kindred (Johnson et al , Am J Hum Genet 60 1326-1332, 1997, U S Patent No 5,691 , 153) and sib-pair QTL analyses (Koller et al , J Bone Miner Res 13 1903- 1908, 1998) None of these studies pinpointed the Ext2 gene as being involved in BMD
  • ttv protein appears to be required for the diffusion of hedgehog (Hh) signals in Drosophda (The et al , Mol Cell 4 633-639, 1999) Considering its similarity to Ext proteins, ttv could be responsible for the synthesis of GAGs that specifically enhance Hh diffusion (The et al , Mol Cell 4 633-639, 1999, Toyoda et al , J Biol Chem 275 2269-2275, 2000)
  • the mammalian homolog of Hh is Indian hedgehog (Ihh) (Iwamoto e/ -./ Crit Rev Oral Biol Med 10 477-486, 1999) Ihh is an important regulator of chondrocyte differentiation (Vortkamp et al , Science 273 613-622, 1996, St-Jacques et al , Genes Dev 13 2072-2086, 1999) Based on these observations, a plausible model for the effects of Ext proteins on bone growth in humans is that Ex
  • Ext2 gene polymorphisms With the provision herein of the correlation between Ext2 gene polymorphisms and predisposition to bone mineral density and/or osteoporosis, the isolation and identification of additional Ext2 polymo ⁇ hisms is enabled Any conventional method for the identification of genetic polymorphisms in a population can be used to identify such additional polymo ⁇ hisms
  • Ext2 sequences are then compared to a reference
  • Ext2 sequence such as the D2 high BMD allele described herein, 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 BMD and/or osteoporosis
  • a suitable genomic DNA-containing sample from a subject is obtained and the DNA extracted using conventional techniques Most typically, a blood sample, a buccal swab, a hair follicle preparation, or a nasal aspirate is used as a source of cells to provide the DNA sample.
  • the extracted DNA is then subjected to amplification, for example according to standard procedures.
  • the allele of the single base-pair polymo ⁇ hism is determined by conventional methods including manual and automated fluorescent DNA sequencing, primer extension methods (Nikiforov, et al, Nucl Acids Res.
  • OLA oligonucleotide ligation assay
  • Patents for descriptions of methods or applications of polymo ⁇ hism analysis to disease prediction and/or diagnosis 4,666,828 (RFLP for Huntington's); 4,801,531 (prediction of atherosclerosis); 5,1 10,920 (HLA typing); 5,268,267 (prediction of small cell carcinoma); and 5,387,506 (prediction of dysautonomia).
  • polymo ⁇ hisms associated with predisposition to osteoporosis and/or an increased likelihood of having low bone mineral density are the Ala622Thr and 3'UTR "stem-loop mutation" polymo ⁇ hisms of Ext2. The absence of these polymo ⁇ hisms indicates a relative resistance to osteoporosis and a relatively decreased likelihood of having low bone mineral density.
  • other alleles that may be associated with variable predisposition to osteoporosis or likelihood of having low bone mineral density can also be detected, and used in combination with the disclosed Ext2 polymo ⁇ hisms to predict the probability that a subject will tend to develop osteoporosis or be likely to display low bone mineral density.
  • IL-l ra interleukin- 1 receptor antagonist
  • Col 1 alpha gene U.S. Patent No. 5,922,542
  • human estrogen receptor gene U.S. Patent No. 5,834,200
  • the markers of the present disclosure can be utilized for the detection of, and differentiation of, individuals who are homozygous and heterozygous for the Ala622Thr or 3'UTR "stem-loop mutation" polymo ⁇ hisms.
  • the value of identifying individuals who carry a low bone mineral density allele of Ext2 i.e., individuals who are heterozygous or homozygous for the an allele that contains a low bone mineral density Ext2 polymo ⁇ hism, such as the G to C transversion at nucleotide position 2707) is that these individuals can then initiate or customize therapy (such as calcium supplementation, hormone therapy, and so forth) to reduce the occurrence of or reverse osteoporosis or improve bone mineral density, or undergo more aggressive treatment of the condition, and thereby beneficially alter its course.
  • therapy such as calcium supplementation, hormone therapy, and so forth
  • Sequences surrounding and overlapping single base-pair polymo ⁇ hisms in the Ext2 gene can be useful for a number of gene mapping, targeting, and detection procedures.
  • genetic probes can be readily prepared for hybridization and detection of the Ala622Thr or 3'UTR "stem-loop mutation" polymo ⁇ hism.
  • probe sequences may be greater than about 12 or more oligonucleotides in length and possess sufficient complementarity to distinguish between the Alanine (at amino acid residue 622 in the D2 high BMD allele) and Threonine (in the B6 low BMD Ala622Thr polymo ⁇ hism), or between the G (in the D2 high BMD allele) and the C (in the B6 low BMD 3'UTR "stem-loop mutation" polymo ⁇ hism).
  • sequences surrounding and overlapping either of the specifically disclosed single base-pair polymo ⁇ hisms (or other polymo ⁇ hisms found in accordance with the present teachings), or longer sequences encompassing both specifically disclosed polymo ⁇ hisms, can be utilized in allele specific hybridization procedures.
  • a similar approach can be adopted to detect other Ext2 polymo ⁇ hisms.
  • Sequence surrounding and overlapping a Ext2 polymo ⁇ hism, or any portion or subset thereof that allows one to identify the polymo ⁇ hism, are highly useful.
  • another embodiment provides a genetic marker predictive of the Ala622Thr polymo ⁇ hism of Ext2, comprising a partial sequence of the human genome including at least about 10 contiguous nucleotide residues including "N" in the following nucleotide sequence: AACTGGGTAGATNCTCATATGAACTGTGAA (SEQ ID NO: 23), and sequences complementary therewith, wherein "N” represents A or a single base-pair polymo ⁇ hism of the G that is present at N in a human allele analogous to the murine D2 (high BMD) allele.
  • One example polymo ⁇ hism is a G to A transition, but can also include a G to T transversion or G to C transversion.
  • another specific embodiment is a genetic marker predictive of a 3'UTR "stem- loop mutation" polymo ⁇ hism of Ext2, comprising a partial sequence of the human genome including at least about 10 contiguous nucleotide residues in the following nucleotide sequence: CAGAGAAAAACAGAGGGTCTGTACTAGCCAT (SEQ ID NO: 24), and sequences complementary therewith.
  • mutations in the 10 th through 15 th or 19 lh through 23 rd residues of this sequence which destabilize the stem of the stem-loop structure (underlined), are of particular interest.
  • the murine Ext2 it is a G to C transversion that results in the D2 (high BMD) allele.
  • oligonucleotide molecules including at least about 10 contiguous nucleotide residues of SEQ ID NO: 24, with at least one residue changed in the sequence, in particular in the 10 th through 15 th residue or the 19 th through 23 rd residues of SEQ ID NO: 24.
  • the SNPs at nucleotide residue 2055 (the first position encoding amino acid residue 622) and/or nucleotide residue 2707 can be detected by a variety of techniques. These techniques include 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 new methods include techniques that incorporate more robust scoring of hybridization.
  • ASOH allele-specific oligonucleotide hybridization
  • ASOH ligation chain reaction
  • ASOH selective ligation and amplification
  • mini-sequencing ASOH plus a single base extension
  • Syvanen Method of Synchronization
  • DNA chips miniaturized ASOH with multiple oligonucleotide arrays
  • 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).
  • DASH dynamic allele-specific hybridization
  • 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.
  • the 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.
  • T m melting temperature
  • Example 6 Detection of Ext2 Nucleic Acid Level(s) Individuals carrying mutations in the Ext2 gene, or having amplifications or heterozygous or homozygous deletions of the Ext2 gene, may be detected at the DNA or RNA level with the use of a variety of techniques. The detection of point mutations was discussed above; in the following example, techniques are provided for detecting the level of Ext2 nucleic acid molecules in a sample.
  • a biological sample of the subject an animal, such as a mouse or a human
  • a biological sample is assayed for a mutated, amplified or deleted Ext2 encoding sequence, such as a genomic amplification of the Ext2 gene or an over- or under-abundance of a Ext2 mRNA.
  • Suitable biological samples include samples containing genomic DNA or mRNA obtained from 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 Ext2 gene, a mutant Ext2 RNA, or an amplified or homozygously or heterozygously deleted Ext2 gene may be performed by a number of methodologies.
  • Gene dosage 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 Ext2 nucleic acids in samples of tissue.
  • Probes generated from the encoding sequence of Ext2 can be used to investigate and measure genomic dosage of the Ext2 gene.
  • Ext2 amplification in immortalized cell lines as well as uncultured cells taken from a subject can be carried out using bicolor FISH analysis
  • inte ⁇ hase 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 fluorescence microscope By way of example, approximately 20 non- overlapping nuclei with intact mo ⁇ hology based on DAPI counterstain are scored to determine the mean number of hybridization signals for each test and reference probe
  • FISH 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% formam ⁇ de/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 WO9944063A2 and WO9944062A1
  • Overexpression of the Ext2 gene can also be detected by measuring the cellular level of Exf2-spec ⁇ fic mRNA mRNA can be measured using techniques well known in the art, including for instance Northern analysis, RT-PCR and mRNA in situ hybridization
  • Ext2 protein proteins
  • DNA sequence of the Ext2 cDNA can be manipulated in studies to understand the expression of the gene and the function of its product
  • Mutant forms of the human Ext2 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 Ext2 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 Eschenchia coll (E cob) may be utilized for the purification, localization and functional analysis of proteins
  • fusion proteins consisting of amino terminal peptides encoded by a portion of the E coli lacZ or trpE gene linked to Ext2 proteins may be used to
  • 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 Set USA 78 2072-2076, 1981), and introduced into cells, such as monkey COS-1 cells (Gluzman, Cell 23 175-182, 1981), to achieve transient or long-term expression
  • heterologous promoters such as the simian virus (SV) 40 promoter in the pSV2 vector (Mulligan and Berg, Proc Natl Acad Set USA 78 2072-2076, 1981)
  • SV simian virus
  • pSV2 vector simian virus 40 promoter in the pSV2 vector
  • cells such as monkey COS-1 cells (Gluzman, Cell 23 175-182, 1981)
  • the stable integration of the chime ⁇ c gene construct may be maintained in mammalian cells by biochemical selection, such as neomycin (Southern and Berg, ./ Mol
  • 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 bacte ⁇ ophage 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 SV
  • 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 Mol 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 , Mol Cell Biol 1 486, 1981) or Epstein-Barr (Sugden et al , Mol Cell Biol 5 410, 1985)
  • 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
  • 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 , Mol Cell Biol 7 2013, 1987), electroporation (Neumann et al , EMBO J 1 841 , 1982), hpofection (Feigner et al , Proc Natl Acad Sci USA 84 7413, 1987), DEAE dextran (McCuthan et al , J Natl Cancer Inst 41 351, 1968), microinjection (Mueller et al , Cell 15 579, 1978), protoplast fusion (Schafner, Proc Natl Acad Sci USA 11 2163-2167, 1980), or pellet guns (Klein et al , Nature 327 70, 1987)
  • eukaryotic expression systems can be used for studies of Ext2 encoding nucleic acids and mutant forms of these molecules, the Ext2 protein and mutant forms of this protein Such uses include, for example, the identification of regulatory elements located in the 5' region of the Ext2 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 Ext2 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 pu ⁇ ose 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
  • the present disclosure thus encompasses recombinant vectors that comprise all or part of the Ext2 gene or cDNA sequences, for expression in a suitable host
  • the Ext2 DNA is operatively linked in the vector to an expression control sequence in the recombinant DNA molecule so that the Ext2 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 phosphata
  • the host cell which may be transfected with the vector of this disclosure, may be selected from the group consisting of £ coli, Pseudomonas, Bacillus subt is, Bacillus stearothermophilus or other bacilli, other bacteria, yeast, fungi, insect, mouse or other animal, or plant hosts, or human tissue cells
  • Ext2 protein can be expressed essentially as detailed above Such fragments include individual Ext2 protein domains or sub-domains, as well as shorter fragments such as peptides Ext2 protein fragments having therapeutic properties may be expressed in this manner also
  • Example 8 Production of Ext2 Protein Specific Binding Agents
  • Monoclonal or polyclonal antibodies may be produced to either the normal Ext2 protein or mutai forms of this protein. Optimally, antibodies raised against these proteins or peptides would specifically dete the protein or peptide with which the antibodies are generated. That is, an antibody generated to the Exi protein or a fragment thereof would recognize and bind the Ext2 protein and would not substantially recogni; or bind to other proteins found in human cells.
  • an antibody specifically detects the Ext2 protein is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al, In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989).
  • Western blotting technique Standard Immunoblotting technique
  • 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.
  • an anti-mouse antibody conjugated to an enzyme such as alkaline phosphatase.
  • an enzyme such as alkaline phosphatase.
  • 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 Ext2 protein will, by this technique, be shown to bind to the Ext2 protein band (which will be localized at a given position on the gel determined by its molecular weight). Nonspecific binding of the antibody to other proteins may occur and may be detectable as a weak signal on the Western blot.
  • Ext2 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:
  • Monoclonal antibody to epitopes of the Ext2 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).
  • HAT media aminopterin
  • 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. En ⁇ ymol. 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).
  • polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein (Example 7), 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.
  • 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.
  • 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 el 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 M). Affinity 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).
  • a third approach to raising antibodies against the Ext2 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 Ext2 protein or peptide.
  • Polyclonal antibodies can be generated by injecting these peptides into, for instance, rabbits.
  • Antibodies may be raised against Ext2 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 Sci. Technol. 5:27-37, 1987) as described by Tang et al. (Nature 356: 152-154, 1992).
  • Expression vectors suitable for this pu ⁇ ose may include those that express the Ext2 encoding sequence under the transcriptional control of either the human -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 Ext2 protein
  • antibodies for administration to human patients, antibodies, e g , Ext2 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)
  • An alternative method of diagnosing Ext2 gene amplification, deletion or mutation, as well as abnormal Ext2 expression is to quantitate the level of Ext2 protein in the cells of an individual
  • This diagnostic tool would be useful for detecting reduced levels of the Ext2 protein that result from, for example, mutations in the promoter regions of the Ext2 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 Ext2 gene
  • duplications of a Ext2 encoding sequence may be detected as an increase in the expression level of Ext2 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 Ext2 gene
  • Ext2 expression (temporally or spatially) can also be examined using known techniques, such as isolation and comparison Ext2 from cell or tissue specific, or time specific, samples
  • the determination of reduced or increased Ext2 protein levels, in comparison to such expression in a control cell would be an alternative or supplemental approach to the direct determination of Ext2 gene deletion, amplification or mutation status by the methods outlined above and equivalents
  • any standard immunoassay format e , ELISA, Western blot, or RIA assay
  • ELISA ELISA
  • Western blot or RIA assay
  • an alteration in Ext2 polypeptide may be indicative of an abnormal biological condition altered BMD and/or a predilection to development of osteoporosis
  • Immunohistochemical techniques may also be utilized for Ext2 polypeptide or protein detection
  • a tissue sample may be obtained from a subject, and a section stained for the presence of Ext2 using a Ext2 specific binding agent (e g , ant ⁇ -Ext2 antibody) and any standard detection system (e , one which includes a secondary antibody conjugated to horseradish peroxidase)
  • Ext2 specific binding agent e g , ant ⁇ -Ext2 antibody
  • any standard detection system e , one which includes a secondary antibody conjugated to horseradish peroxidase
  • a biological sample of the subject (which can be any animal, for instance a mouse or a human), which sample includes cellular proteins, is required
  • 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 breast cells
  • Quantitation of Ext2 protein can be achieved by immunoassay and compared to levels of the protein found in control cells (e , healthy, as in from a patient known not to have osteoporosis)
  • a significant (e , 10% or greater) reduction in the amount of Ext2 protein in the cells of a subject compared to the amount of Ext2 protein found in normal human cells could be taken as an indication that the subject may have deletions or mutations in the Ext2 gene, whereas a significant (e , 10% or greater) increase would indicate that a duplication (amplification), or mutation that increases the stability of the Ext2 protein or mRNA, may
  • the oligonucleotide ligation assay (OLA), as described at Nickerson et al (Proc Natl Acad Sci USA 87 8923-8927, 1990), allows the differentiation between individuals who are homozygous versus heterozygous for either the Ala622Thr or the 3'UTR polymorphisms This feature allows one to rapidly and easily determine whether an individual is homozygous for at least one B6-l ⁇ nked polymo ⁇ hism, which condition is linked to a relatively high predisposition to developing osteoporosis and/or an increased likelihood of having low bone mineral density Alternatively, OLA can be used to determine whether a subject is homozygous for either of these polymo ⁇ hisms
  • the OLA assay when carried out in microtiter plates, one well is used for the determination of the presence of the Ext2 allele that contains a G at nucleotide position 2707 and a second well is used for the determination of the presence of the Ext2 allele that contains a C at nucleotide position 2707
  • the results for an individual who is heterozygous for the polymo ⁇ hism will show a signal in each of the G and C wells
  • an individual who is homozygous for the 3'UTR "stem-loop mutation" polymo ⁇ hism will show a signal in only the C well
  • a reduction of Ext2 protein expression in a transgenic cell may be obtained by introducing into cells an antisense construct based on the Ext2 encoding sequence, including the human Ext2 cDNA or gene sequence (Accession number U62740 or AH00672, respectively) or flanking regions thereof
  • a nucleotide sequence from an Ext2 encoding sequence e g all or a portion of the Ext2 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 above (Example 7)
  • the introduced sequence need not be the full length human Ext2 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 Ext2 sequence will be needed for effective antisense suppression
  • the introduced antisense sequence in the vector may be at least 30 nucleotides in length, and
  • Ribozymes are synthetic RNA molecules that possess highly specific endo ⁇ bonuclease activity
  • the production and use of ribozymes are disclosed in U S Patent No 4,987,071 to Cech and U S Patent No 5,543,508 to Haselhoff
  • the inclusion of ⁇ bozyme 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
  • dominant negative mutant forms of Ext2 may be used to block endogenous Ext2 activity
  • Retroviruses have been considered a preferred vector for experiments in gene therapy, with a high efficiency of infection and stable integration and expression (Orkin et al , Prog Med Genet 1 130-142, 1988)
  • the full-length Ext2 gene or cDNA can be cloned into a retroviral vector and driven from either its endogenous promoter or from the retroviral LTR (long terminal repeat)
  • Other viral transfection systems may also be utilized for this type of approach, including adenovirus, adeno- associated virus (AAV) (McLaughlin et al J Virol 62 1963- 1973, 1988), Vaccinia virus (Moss et al , Annu Rev Immunol 5 305-324, 1987), Bovine Papilloma virus (Rasmussen et al , Methods Enzymol 139 642-654, 1987) or members of the he ⁇ esvirus group such as Epstein-Barr virus (Margolskee / ⁇ / , Mol
  • Kits which contain the necessary reagents for determining the presence or absence of polymo ⁇ h ⁇ sm(s) in an Ext2-encod ⁇ ng sequence, such as probes or primers specific for the Ext2 gene
  • Such kits can be used with the methods described herein to determine whether a subject is predisposed to osteoporosis and/or low bone mineral density
  • kits may also include written instructions
  • the instructions can provide calibration curves or charts to compare with the determined (e g , experimentally measured) values
  • Kits are also provided to determine elevated or depressed expression of mRNA (/ e , containing probes) or Ext2 protein (/ e , containing antibodies or other Ext2 -protein specific binding agents)
  • the oligonucleotide probes and primers disclosed herein can be supplied in the form of a kit for use in detection of a predisposition to osteoporosis or low bone mineral density in a subject
  • an appropriate amount of one or more of the oligonucleotide primers is provided in one or more containers
  • the oligonucleotide primers may be provided suspended in an aqueous solution or as a freeze-d ⁇ ed or lyophi zed powder, for instance
  • the conta ⁇ ner(s) in which the ohgonucleot ⁇ de(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microftige tubes, ampoules, or bottles
  • pairs of primers may be provided in pre-measured single use amounts in individual, typically disposable, tubes or equivalent containers With such an arrangement, the sample to be tested for the presence of an Ext2 polymo ⁇ hism can be added to the individual tubes and amplification carried out directly The amount of each oli
  • the amount of each oligonucleotide primer provided would likely be an amount sufficient to prime several PCR amplification reactions.
  • Those of ordinary skill in the art know the amount of oligonucleotide primer that is appropriate for use in a single amplification reaction. General guidelines may for instance be found in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990), Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989), and Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992).
  • a kit may include more than two primers, in order to facilitate the in vitro amplification of Ext2 sequences, for instance the Exl2 gene or the 5' or 3' flanking region thereof.
  • kits may also include the reagents necessary to carry out nucleotide amplification reactions, including, for instance, DNA sample preparation reagents, appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesium chloride), and deoxyribonucleotides (dNTPs).
  • appropriate buffers e.g., polymerase buffer
  • salts e.g., magnesium chloride
  • dNTPs deoxyribonucleotides
  • Kits may in addition include either labeled or unlabeled oligonucleotide probes for use in detection of Ext2 polymo ⁇ hism(s).
  • these probes will be specific for a potential polymo ⁇ hism that may be present in the target amplified sequences.
  • the appropriate sequences for such a probe will be any sequence that includes one or more of the identified polymo ⁇ hic sites, particularly nucleotide positions 2055 and 2707, such that the sequence the probe is complementary to a polymo ⁇ hic site and the surrounding Ext2 sequence.
  • Oligonucleotides GTAGATGCTCATA (SEQ ID NO: 25) and ACAGAGGGTCTGT (SEQ ID NO: 26) exemplify such sequences, and an appropriate probe could comprise either (or both) of these sequences. It may also be advantageous to provide in the kit one or more control sequences for use in the amplification reactions. The design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art.
  • Kits similar to those disclosed above for the detection of Ext2 polymo ⁇ hisms directly can be used to detect Ext2 mRNA expression, such as over- or under-expression.
  • Such kits include an appropriate amount of one or more oligonucleotide primers for use in, for instance, reverse transcription PCR reactions, similarly to those provided above with art-obvious modifications for use with RNA amplification.
  • kits for detection of altered expression of Ext2 mRNA may also include some or all of the reagents necessary to carry out RT-PCR in vitro amplification reactions, including, for instance, RNA sample preparation reagents (including e.g., an RNase inhibitor), appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesium chloride), and deoxyribonucleotides (dNTPs).
  • RNA sample preparation reagents including e.g., an RNase inhibitor
  • appropriate buffers e.g., polymerase buffer
  • salts e.g., magnesium chloride
  • dNTPs deoxyribonucleotides
  • kits may in addition include either labeled or unlabeled oligonucleotide probes for use in detection of the in vitro amplified target sequences.
  • the appropriate sequences for such a probe will be any sequence that falls between the annealing sites of the two provided oligonucleotide primers, such that the sequence the probe is complementary to is amplified during the PCR reaction.
  • these probes will be specific for a potential polymo ⁇ hism that may be present in the target amplified sequences, for instance specific for the B6 allele at the 3'UTR site (e.g., capable of detecting a C residue at position 2707 of the Ext2 sequence).
  • control sequences for use in the RT-PCR reactions.
  • the design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art.
  • kits may be provided with the necessary reagents to carry out quantitative or semi-quantitative Northern analysis of Ext2 mRNA.
  • Such kits include, for instance, at least one Ext2- specific oligonucleotide for use as a probe. This oligonucleotide may be labeled in any conventional way, including with a selected radioactive isotope, enzyme substrate, co-factor, ligand, chemiluminescent or fluorescent agent, hapten, or enzyme.
  • such probes will be specific for a potential polymo ⁇ hism that may be present in the target amplified sequences, for instance specific for the B6 allele at the 3'UTR site (e.g., capable of detecting a C residue at position 2707 of the Ext2 sequence).
  • kits for the detection of Ext2 protein expression are also encompassed.
  • Such kits may include at least one target protein specific binding agent (e.g., a polyclonal or monoclonal antibody or antibody fragment that specifically recognizes the Ext2 protein) and may include at least one control (such as a determined amount of Ext2 protein, or a sample containing a determined amount of Ext2 protein).
  • the Ext2-protein specific binding agent and control may be contained in separate containers.
  • the Ext2 protein expression detection kits may also include a means for detecting Ext2:binding agent complexes, for instance the agent may be detectably labeled. If the detectable agent is not labeled, it may be detected by second antibodies or protein A for example which may also be provided in some kits in one or more separate containers. Such techniques are well known. Additional components in specific kits may include instructions for carrying out the assay.
  • Reaction vessels and auxiliary reagents such as chromogens, buffers, enzymes, etc. may also be included in the kits.
  • kits for Detection of Homozygous versus Heterozygous Allelism are kits that allow differentiation between individuals who are homozygous versus heterozygous for either the Ala622Thr or the 3'UTR polymo ⁇ hisms of Ext2.
  • kits provide the materials necessary to perform oligonucleotide ligation assays (OLA), as described at Nickerson et al. (Proc. Natl. Acad. Sci USA 87:8923-8927, 1990).
  • these kits contain one or more microtiter plate assays, designed to detect polymo ⁇ hism(s) in the Ext2 sequence of a subject, as described herein.
  • kits may include instructions for carrying out the assay. Instructions will allow the tester to determine whether an Ext2 allele is homozygous or heterozygous. Reaction vessels and auxiliary reagents such as chromogens, buffers, enzymes, etc. may also be included in the kits.
  • control sequences for use in the OLA reactions.
  • the design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art.
  • Mutant organisms that under-express or over-express Ext2 protein are useful for research. Such mutants allow insight into the physiological and/or pathological role of Ext2 in a healthy and/or pathological organism.
  • These mutants are "genetically engineered,” 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-£x.2 promoter inserted upstream of a native Ext2 encoding sequence would be non-native. An extra copy of an Ext2 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 Ext2 or under-express Ext2, or that do not express Ext2 at all.
  • Over-expression mutants are made by increasing the number of Ext2 genes in the organism, or by introducing an Ext2 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.
  • MMTV mouse mammary tumor virus
  • WAP whey acidic protein
  • Mutants that under-express Ext2 may be made by using an inducible or repressible promoter, or by deleting the Ext2 gene, or by destroying or limiting the function of the Ext2 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 Ext2 expression, as discussed above in Example 1 1.
  • a gene is "functionally deleted" when genetic engineering has been used to negate or reduce gene expression to negligible levels.
  • a mutant is referred to in this application as having the Ext2 gene altered or functionally deleted, this refers to the Ext2 gene and to any ortholog of this gene.
  • 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, e.g., in the diploid mouse or human.
  • a mutant mouse over-expressing Ext2 may be made by constructing a plasmid having an Ext2 encoding sequence driven by a promoter, such as the mouse mammary tumor virus (MMTV) promoter or the whey acidic protein (WAP) promoter.
  • MMTV mouse mammary tumor virus
  • WAP whey acidic protein
  • 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, e.g., 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.
  • an agent that can be fed to the mouse, such as tetracycline.
  • a mutant knockout animal e.g., mouse
  • an Ext2 gene can be made by removing all or some of the coding regions of the Ext2 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).
  • This disclosure provides specific polymo ⁇ hisms in a gene, Ext2, that are linked to differential bone mass density, and more particularly to predisposition to or the condition of osteoporosis.
  • the disclosure further provides methods for identifying these polymo ⁇ hisms in a subject, and using them to determine or predict a subject's osteoporosis state. It will be apparent that the precise details of the methods described 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.

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Abstract

This disclosure provides the identification of polymorphisms of the Ext2 gene that are linked to a predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject. Also provided are methods for diagnosing and/or treating osteoporosis and low bone mineral density in a subject.

Description

Ext2 AS A PREDICTIVE MARKER FOR OSTEOPOROSIS
STATEMENT OF GOVERNMENT SUPPORT This work was supported by funds from the National Institutes of Health (AA 10760 and
AR 44659) and the Medical Research Service of the Veterans Administration. The government may have certain rights in this invention.
FIELD The present disclosure is generally related to prediction and diagnosis of disease states, for instance prediction of a predisposition to osteoporosis and/or low bone mineral density in a subject.
BACKGROUND
Osteoporosis is a multifactorial disease that leads to an increased risk of bone fracture. It is most prevalent in post-menopausal women or the elderly of either gender, and is becoming a major public health problem due to extended life expectancy. Its major consequence, hip fracture, has major health consequences. Research on osteoporosis emphasizes finding new therapeutic approaches to treat the condition and characterization genetic markers that may prove useful in early identification of people, particularly women, at risk of developing osteoporosis. Since therapy of established osteoporosis remains unsatisfactory, prevention of the condition is the best choice.
A major determinant of osteoporotic fracture risk, independent of other factors such as falls and aging per se, is bone mass. The acquisition of bone mass results from bone modeling and linear growth during skeletal development, whereas its maintenance in adults results from a coupling mechanism between the activities of bone formation and bone resorption. The processes governing acquisition of bone mineral have received far less attention than those related to the maintenance of adult bone density. With new emphasis on maximizing peak bone mass as a strategy for reducing fracture risk during the course of adult life, it is of increasing importance that the factors governing acquisition of bone mineral density be understood.
The factors known to influence bone mass accumulation during growth include heredity, gender, dietary components (calcium, proteins), endocrine factors (sex steroids, calcitriol, insulin growth factor I), and mechanical forces. Quantitatively, the most prominent determinant appears to be heredity. Past research to identify specific genes that influence peak bone mass has focused mainly on evaluation of candidate genes with identifiable polymorphisms. For example, polymorphisms of the gene for vitamin D receptor have been associated with low bone mass in studies for some, but not all, populations. These inconsistent findings are not surprising, considering the genetic heterogeneity and variations in gene frequency and penetrance that can exist between different populations. Moreover, it is very likely that other as yet unknown genes also contribute to the determination of bone mass. Bone mineral density (BMD) is determined by both environmental influences and polygenic inheritance (Eisman, Endocr. Rev. 20:788-804, 1999). The extreme difficulty of dissecting out environmental factors from genetic ones in humans has motivated the investigation of animal models (Beamer e. α/., //n/w. Genome 10: 1043-1049, 1999; Shimizu e/ αt, Mamm. Genome 10:81-87, 1999). Peak BMD has been examined in 24 recombinant inbred (RI) mouse strains, derived from a cross between C57BL/6J (B6) and DBA/2J (D2) progenitors (BXD RI) (Klein et al., J. Bone Miner. Res. 13: 1648-1656, 1998). The distribution of BMD values among the RI strains indicated strong genetic influences and quantitative trait locus (QTL) analysis provisionally identified ten chromosomal sites linked to BMD. To that point, however, no particular gene linkage had been identified. It would be of great practical importance to identify one or more gene loci, and more particularly alleles at each locus, that are strongly linked to bone mineral density and/or predisposition of a subject to develop osteoporosis.
SUMMARY The inventors have found a strong link between predisposition to osteoporosis, or an increased likelihood of having low bone mineral density, and polymoφhisms in the Ext2 gene, for example polymoφisms in the coding or untranslated regions of Ext2. In particular disclosed examples, the polymorphism may be, for example, at position 2055 of the coding region or position 2707 of the uncoding region of Ext2. Polymoφhisms in this gene can be used to detect such predisposition or increased likelihood in subjects, as well as to diagnose and prognose osteoporosis. Aspects of the disclosure include methods for predicting a predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject. Such methods can include determining whether the subject has a polymorphism in a Ext2 sequence (for instance, a Ala622Thr or 3'UTR stem-loop mutation), wherein presence of the polymoφhism indicates the predisposition to osteoporosis or the increased likelihood of having low bone mineral density. Specific embodiments will further include determining whether the subject has one or more other alleles associated with predisposition to osteoporosis or increased likelihood of having low bone mineral density. Examples of such other alleles include a polymorphism at position 1245 of a Col 1 alpha gene; a polymoφhism of an interleukin-1 receptor antagonist gene; and a polymoφhism in a first intron of a human estrogen receptor gene.
Determining whether the subject has the polymoφhism will in some methods include providing DNA from the subject, and assessing the DNA for the presence of the Ala622Thr or 3'UTR stem-loop mutation polymoφhism, or both. Certain methods may further include determining whether the subject is homozygous or heterozygous for the polymoφhism. Assessing the DNA of the subject for the presence of a polymoφhism may be performed by a process that includes subjecting the DNA or RNA to amplification using oligonucleotide primers flanking the polymoφhism, such as an oligonucleotide ligation assay.
Further embodiments include methods of predicting predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject, which methods include obtaining a test sample of DNA containing a Ext2 sequence of the subject; and determining whether the subject has a polymoφhism in the Ext2 sequence, wherein the presence of the polymoφhism indicates the predisposition to osteoporosis or the increased likelihood of having low bone mineral density in a subject. In certain of these methods, determining whether the subject has the polymoφhism can include using restriction digestion, probe hybridization (e.g., using a nucleic acid probe to a Ext2 polymoφhism), nucleic acid amplification (e.g., PCR or another amplification technique), and/or nucleotide sequencing. Representative examples of polymoφhisms that can be detected using these methods include the Ala622Thr or a 3'UTR stem-loop mutation of the Ext2 gene. Further methods include methods for predicting predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject, which method includes obtaining from the subject a test sample of DNA comprising an Ext2 sequence; contacting the test sample with at least one nucleic acid probe for an Ext2 sequence polymoφhism that is associated with increased predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject to form a hybridization sample; maintaining the hybridization sample under conditions sufficient for specific hybridization of the Ext2 sequence with the nucleic acid probe; and detecting whether there is specific hybridization of the Ext2 sequence with the nucleic acid probe, wherein specific hybridization of the Ext2 sequence with the nucleic acid probe indicates increased predisposition to osteoporosis or an increased likelihood of having low bone mineral density in the subject. Representative polymoφhisms that can be detected using these methods include the
Ala622Thr mutation and 3'UTR stem-loop mutation. In certain examples of these methods, the probe is present on a substrate, such as a nucleotide array.
Other embodiments are kits for use in diagnosing an increased predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject. Such kits include a probe that specifically hybridizes to an Ext2 sequence polymoφhism that is associated with the increased predisposition to osteoporosis or an increased likelihood of having low bone mineral density, such as an Ext2 Ala622Thr or 3'UTR stem-loop mutation polymoφhism. Optionally, kits can also include instructions. Also encompassed in the disclosure are nucleic acid probes that specifically hybridize to a human Ext2 Ala622Thr or 3'UTR stem-loop mutation polymorphism. The disclosure further includes methods of osteoporosis therapy. Such methods can include screening an individual for a genetic predisposition to osteoporosis; and, if such a predisposition is identified, treating (e.g., by hormone replacement therapy) that individual to prevent or reduce osteoporosis or to delay the onset of osteoporosis, wherein predisposition to osteoporosis is correlated with a polymoφhism in a Ext2 sequence. The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a bar graph showing mean peak whole body BMD (expressed as standard deviations away from the B6D2F2 population mean or Z score) of animals with the three different possible genotypes (homozygous B6, heterozygous or homozygous D2) for the D2MH94 microsatellite marker. There were 71, 146 and 82 mice of the D2/D2, D2/B6, and B6/B6 genotypes, respectively. Gene dosage at D2MU94 had a significant influence on the attainment of peak bone mass (F(2j29-) = 23.8, p = 1.7 x 10'6).
FIG. 2 is a LOD plot of chromosome 2 for peak whole body BMD as determined by an interval mapping approach (Map Manager QT). Two of the nine markers used in the linkage analysis were found to differ substantially from the consensus map (Blake et al., Nucleic Acids Res. 27:95-98, 1999). Thus, the resultant primary linkage map is shown with markers D2MU80 and D2MH200 as anchors to the consensus map at 10 cM and 107 cM distance from the centromere, respectively. The QTL accounts for -5% of the phenotypic variance in peak whole body BMD and the LOD curve exceeded the Lander and Kruglyak (Nat. Genet. 1 1 :241-247, 1995) significance threshold of 4.3 (df = 2) for F2 data. Since the results for the "additive" and "free" QTL models were virtually identical, only the "free" model is shown. The 1 LOD confidence interval for this QTL is represented by the shaded area and is bounded by the microsatellite markers D2MH91 and D2M 166. A schematic representation of the allele distribution in recombinant inbred strain BXD-8 is superimposed over the LOD plot for comparison puφoses.
FIG. 3 is a schematic representation of the recombinant inbred (RI) segregation test strategy.
FIG. 3A is a schematic drawing illustrating that a RI strain that possesses a recombination or cross-over point in the region of a QTL can be used to generate two F2 populations - one crossed with each parental strain. Analysis of the two populations will detect the population in which the
QTL is segregating, and accordingly locate the QTL above or below the recombination point. In the B6 cross shown here, linkage between phenotype and markers below the cross-over point is examined. If marker genotype correlates with phenotype in this population, then the QTL must reside below the recombination point. Conversely, in the D2 cross, linkage between phenotype and markers above the cross-over point is examined. If marker genotype correlates with phenotype in this population, then the QTL must reside above the recombination point. If linkage is observed in both F2 populations, then two closely linked QTLs are likely to be present.
FIG. 3B is a schematic representation of the chromosome 2 QTL region in RI BXD-8 mice. Positions of microsatellite markers (PCR genotyping) and Ext2 (allele-specific oligonucleotide hybridization) were determined by linkage analysis of 300 B6D2F2 mice. Location of the potential candidate genes (FSHβ = follicle stimulating hormone β; ILlα = interleukin l α; ILl β = interleukin l β; BMP-2 = bone moφhogenetic protein-2) was determined by relation to the marker assignment and comparison with the Mouse Genome Database (Blake et al., Nucleic Acids Res. 27:95-98, 1999). FIG. 4 shows bar graphs of mean peak whole body BMD Z scores of animals from the two RIST populations.
FIG. 4A provides results for the BXD-8 X D2 F2 population (n = 200). FIG. 4B provides results for the B6 X BXD-8 F2 population (n = 200). Gene dosage at
D2MU94 had a significant influence on the attainment of peak bone mass by BXD-8 X D2 F2 mice (^(2, 199) = 20.0, p = 1.3 x 10'5). In contrast, gene dosage at D2MU249 did not correlate with peak BMD ( (2i,99) = 0.16, p = NS).
FIG. 5 shows an alignment of portions of the Ext2 B6 and D2 allele sequences; variations between the two sequences are indicated by stars. To generate this sequence information, total RNA was isolated from cardiac tissue of B6 and D2 mice. The Ext2 cDNAs were amplified by RT-PCR, subcloned into pBluesc ψt and separately sequenced. To exclude artifacts, five animals of each inbred strain were examined and both DNA strands were sequenced. In FIG. 5 A, the coding polymorphism is a G → A transition at cDNA position 2055. This polymoφhism predicts an amino acid sequence variant at position 622 in the COOH-terminal region of the mature Ext2 protein with threonine at this position in the B6 allele and alanine at this position in the D2 allele.
In FIG. 5B, the 3 '-untranslated region polymorphism is a G -> C transversion at cDNA position 2707. A short palindromic sequence (underlined) is evident in the B6 allele, which is disrupted by the sequence variant in the D2 allele.
SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three 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. In the accompanying sequence listing:
SEQ ID NOs: 1 and 2 are the forward and backward primers used to amplify the D2Mit80 sequence.
SEQ ID NOs: 3 and 4 are the forward and backward primers used to amplify the D2Mitl52 sequence.
SEQ ID NOs: 5 and 6 are the forward and backward primers used to amplify the D2Mit91 sequence.
SEQ ID NOs: 7 and 8 are the forward and backward primers used to amplify the D2Mit94 sequence. SEQ ID NOs: 9 and 10 are the forward and backward primers used to amplify the D2Mitl2 sequence.
SEQ ID NOs: 1 1 and 12 are the forward and backward primers used to amplify the D2Mitl64 sequence. SEQ ID NOs: 13 and 14 are the forward and backward primers used to amplify the
D2Mitl 66 sequence.
SEQ ID NOs: 15 and 16 are the forward and backward primers used to amplify the D2Mit59 sequence.
SEQ ID NOs: 17 and 18 are the forward and backward primers used to amplify the D2Mit200 sequence.
SEQ ID NOs: 19 and 20 are the forward and backward primers used to amplify the Ext2 gene.
SEQ ID NO: 21 is an allele-specific oligonucleotide (ASO) specific for the position 2707 3'UTR stem-loop sequence (G allele). SEQ ID NO: 22 is an allele-specific oligonucleotide (ASO) specific for the position 2707
3'UTR stem-loop sequence (C allele).
SEQ ID NOs: 23 and 25 are oligonucleotide sequences useful for detection of the Ala622Thr polymoφhism, in the human Ext2 gene.
SEQ ID NOs: 24 and 26 are oligonucleotide sequences useful for detection of a stem-loop polymoφhism, in the human Ext2 gene.
DETAILED DESCRIPTION
/. Abbreviations
ASO: allele-specific oligonucleotide ASOH: allele-specific oligonucleotide hybridization
BMD: bone mineral density
BMP-2: bone morphogenetic protein-2
DASH: dynamic allele-specific hybridization
DEXA: dual energy X-ray absoφtiometry DIG: digoxygenin
Ext2: exostoses linked gene 2
FSHβ: follicle stimulating hormone β
ILlα: interleukin loc
ILlβ: interleukin l β LOD: likelihood of linkage
QTL: quantitative trait locus
RIST: recombinant inbred segregation testing
RT-PCR: reverse-transcription polymerase chain reaction TMAC: tetramethyl ammonium chloride
//. 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 al. (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, the following descriptions of terms are provided:
Abnormal: Deviation from normal characteristics. Normal characteristics can be found in a control, a standard for a population, etc. For instance, where the abnormal condition is a disease condition, such as osteoporosis, a few appropriate sources of normal characteristics might include an individual who is not suffering from the disease (e.g., osteoporosis), a population standard of individuals believed not to be suffering from the disease, etc.
Likewise, abnormal may refer to a condition that is associated with a disease. The term "associated with" includes an increased risk of developing the disease as well as the disease itself. For instance, a certain abnormality (such as an abnormality in an Ext2 nucleic acid or Ext2 protein expression) can be described as being associated with the biological conditions of altered BMD and tendency to develop osteoporosis.
An abnormal nucleic acid, such as an abnormal Ext2 nucleic acid, is one that is different in some manner to a normal (wildtype) nucleic acid. Such abnormality includes but is not necessarily limited to: (1) a mutation in the nucleic acid (such as a point mutation (e.g., a single nucleotide polymoφhism) or short deletion or duplication of a few to several nucleotides); (2) a mutation in the control sequence(s) associated with that nucleic acid such that replication or expression of the nucleic acid is altered (such as the functional inactivation of a promoter); (3) a decrease in the amount or copy number of the nucleic acid in a cell or other biological sample (such as a deletion of the nucleic acid, either through selective gene loss or by the loss of a larger section of a chromosome or under expression of the mRNA); and (4) an increase in the amount or copy number of the nucleic acid in a cell or sample (such as a genomic amplification of part or all of the nucleic acid or the overexpression of an mRNA), each compared to a control or standard. It will be understood that these types of abnormalities can co-exist in the same nucleic acid or in the same cell or sample; for instance, a genomic-amplified nucleic acid sequence may also contain one or more point mutations. In addition, it is understood that an abnormality in a nucleic acid may be associated with, and in fact may cause, an abnormality in expression of the corresponding protein.
Abnormal protein expression, such as abnormal Ext2 protein expression, refers to expression of a protein that is in some manner different to expression of the protein in a normal (wildtype) 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 (e.g., 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 abnormality, include samples believed to be normal 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.
Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA) has two strands, a 5'— > 3' strand, referred to as the plus strand, and a 3' - 5' strand (the reverse compliment), referred to as the minus strand. Because RNA polymerase adds nucleic acids in a 5' — » 3' direction, the minus strand of the DNA serves as the template for the RNA during transcription. Thus, the RNA formed will have a sequence complementary to the minus strand and identical to the plus strand (except that U is substituted for T). Antisense molecules are molecules that are specifically hybridizable or specifically complementary to either RNA or the plus strand of DNA. Sense molecules are molecules that are specifically hybridizable or specifically complementary to the minus strand of DNA. Antigene molecules are either antisense or sense molecules directed to a dsDNA target.
Binding or stable binding (of an oligonucleotide): 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 may 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 absoφtion detection procedures. For example, one method that is widely used, because it is so simple and reliable, involves observing a change in light absoφtion 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 absoφtion at a characteristic temperature as the oligonucleotide (or analog) and target disassociate from each other, or melt.
The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (Tm) at which 50% of the oligomer is melted from its target. A higher (Tm) means a stronger or more stable complex relative to a complex with a lower (Tm). cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments
(introns) and transcriptional regulatory sequences. cDNA may also contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule. cDNA is usually synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells. Complementarity and percentage complementarity: Molecules with complementary nucleic acids form a stable duplex or triplex when the strands bind, (hybridize), to each other by forming Watson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when an oligonucleotide remains detectably bound to a target nucleic acid sequence under the required conditions. Complementarity is the degree to which bases in one nucleic acid strand base pair with the bases in a second nucleic acid strand. Complementarity is conveniently described by percentage, i.e. the proportion of nucleotides that form base pairs between two strands or within a specific region or domain of two strands. For example, if 10 nucleotides of a 15-nucleotide oligonucleotide form base pairs with a targeted region of a DNA molecule, that oligonucleotide is said to have 66.67% complementarity to the region of DNA targeted.
A thorough treatment of the qualitative and quantitative considerations involved in establishing binding conditions that allow one skilled in the art to design appropriate oligonucleotides for use under the desired conditions is provided by Beltz et al. Methods Enzymol 100:266-285, 1983, and 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.
DNA (deoxyribonucleic acid): DNA is a long chain polymer which 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, or for a stop signal. 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 Ext2, 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 of the removed sequence being joined together Genomic target sequence: A sequence of nucleotides located in a particular region in the human genome that corresponds to one or more specific genetic abnormalities, such as a nucleotide polymoφhism, a deletion, or an amplification The target can be for instance a coding sequence, it can also be the non-coding strand that corresponds to a coding 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 pyπmidines (cytosine (C), uracil (U), and thymine (T)) or puπnes (adenine (A) and guanine (G)) These nitrogenous bases form hydrogen bonds between a pyπmidine and a puπne, and the bonding of the pyπmidine to the puπne 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 For example, an oligonucleotide can be complementary to an Ext2 encoding mRNA, or an Ext2- encoding dsDNA
"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 1 1, herein incoφorated by reference.
For puφoses of the present disclosure, "stringent conditions" encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence. "Stringent conditions" may be broken down into particular levels of stringency for more precise definition. Thus, as used herein, "moderate stringency" conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of "medium stringency" are those under which molecules with more than 15% mismatch will not hybridize, and conditions of "high stringency" are those under which sequences with more than 10% mismatch will not hybridize. Conditions of "very high stringency" are those under which sequences with more than 6% mismatch will not hybridize.
Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., 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.
Nucleotide: "Nucleotide" includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.
Oligonucleotide: An oligonucleotide is 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.
Operabiy linked: A first nucleic acid sequence is operabiy 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 operabiy linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operabiy 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
Osteoporosis A bone disorder characterized by increased bπttleness due to a reduction in bone density See the following references for further detail on recognized symptoms and other aspects of this disorder Melton and Riggs, Osteoporosis Etiology, Diagnosis, and Management, 2nd ed , Lippmcott-Raven Publishers, 1995 (ISBN 0781702755), Peel and Eastell, J Bone Miner Res 8(suppl 2) S505-510, 1994, Peel and Eastell, BMJ 310 989-992, 1995, Eastell and Riggs, Endocrmol Metab Clin NA 17 547-571,1988, Eastell and Riggs, Clin Obstet Gynecol 30 860-870, 1987, and Eastell and Riggs, Obstet Gynecol Clin NA 14 77-88, 1987) The term osteoporosis encompasses both post-menopausal and senile osteoporosis, unless context indicates otherwise
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
Parenteral: Administered outside of the intestine, e g , not via the alimentary tract Generally, parenteral formulations are those that will be administered through any possible mode except ingestion This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance
Peptide Nucleic Acid (PNA): An oligonucleotide analog with a backbone comprised of monomers coupled by amide (peptide) bonds, such as amino acid monomers joined by peptide bonds
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful with this disclosure are conventional Martin, Remington 's Pharmaceutical Sciences, published by Mack Publishing Co , Easton, 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 (e g , 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 Polymorphism: Variant in a sequence of a gene Polymoφhisms 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 polymoφhism also encompasses variations that produce gene products with altered function, / e , 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 increased gene product The term polymorphism may be used interchangeably with allele or mutation, unless context clearly dictates otherwise
Polymoφhisms 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 that is linked to the variation (e , 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, RNases, and so forth) By way of example, the polymoφhism disclosed herein m the 3' untranslated region of the Ext2 gene can be referred to by its location (e g , 3'UTR 2707, based on the numerical position of the variant residue) or by the effect it has on the secondary structure of the Ext2 mRNA (e g , the 3'UTR stem-loop mutation, because this mutation disrupts a stem-loop structure that is conserved in the Ext2 family of encoding sequences)
Probes and primers A probe comprises an isolated nucleic acid attached to a detectable label or other 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 puφoses are discussed, e g , in Sambrook et al (In Molecular Cloning A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998) Primers are short nucleic acid molecules, for instance DNA oligonucleotides 10 nucleotides or more in length, for example that hybridize to contiguous complementary nucleotides or a sequence to be amplified Longer DNA oligonucleotides may be about 15, 20, 25, 30 or 50 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, e g , by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art Other examples of amplification include strand displacement amplification, as disclosed in U S Patent No 5,744,31 1 , transcription-free isothermal amplification, as disclosed in U S Patent No 6,033,881 , repair chain reaction amplification, as disclosed in WO 90/01069, ligase chain reaction amplification, as disclosed in EP-A-320 308, gap filling ligase chain reaction amplification, as disclosed in 5,427,930, and NASBA™ RNA transcription-free amplification, as disclosed in U S Patent No 6,025, 134
Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided in this disclosure It is also appropriate to generate probes and primers based on fragments or portions of these disclosed nucleic acid molecules, for instance regions that encompass the identified polymoφhisms at nucleotide 2707 in the untranslated region, and nucleotide 2055 within the Ext2 coding sequence
Methods for preparing and using nucleic acid probes and primers are described, for example, in Sambrook et al (In Molecular Cloning A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990). Amplification primer pairs can be derived from a known sequence, for example, by using computer programs intended for that puφose 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 an Ext2- encoding nucleotide or flanking region thereof (an "Ext2 primer" or "Ext2 probe") will anneal to a target sequence 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 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of Ext2 locus nucleotide sequences.
The disclosure thus includes isolated nucleic acid molecules that comprise specified lengths of the Ext2 encoding sequence and/or flanking regions. Such molecules may comprise at least 10, 15, 20, 23, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of these sequences or more, and may be obtained from any region of the disclosed sequences. By way of example, the human Ext2 locus, cDNA, ORF, coding sequence and gene sequences (including sequences both upstream and downstream of the Ext2 coding sequence) may be apportioned into about halves or quarters based on sequence length, and the isolated nucleic acid molecules (e.g., oligonucleotides) may be derived from the first or second halves of the molecules, or any of the four quarters. The cDNA also could be divided into smaller regions, e.g. about eighths, sixteenths, twentieths, fiftieths and so forth, with similar effect.
In particular embodiments, isolated nucleic acid molecules comprise or overlap at least one residue position designated as being associated with a polymoφhism that is predictive of osteoporosis and/or bone mineral density. Such polymoφhism sites include position 2055 (corresponding to the Ala622Thr polymoφhism) and position 2707 (corresponding to the 3'UTR stem-loop polymoφhism).
Protein: A biological molecule expressed by a gene and comprised of amino acids. Purified: 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 or within a production reaction chamber (as appropriate).
Recombinant: A recombinant nucleic acid is one 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, e.g., by genetic engineering techniques.
Representational difference analysis: A PCR-based subtractive hybridization technique used to identify differences in the mRNA transcripts present in closely related cell lines. Serial analysis of gene expression: The use of short diagnostic sequence tags to allow the quantitative and simultaneous analysis of a large number of transcripts in tissue, as described in Velculescu et al (Science 270 484-487, 1995)
Specific binding agent: An agent that binds substantially only to a defined target Thus an Ext2 protein-specific binding agent binds substantially only the Ext2 protein As used herein, the term "Ext2 protein specific binding agent" includes antι-Ext2 protein antibodies (and functional fragments thereof) and other agents (such as soluble receptors) that bind substantially only to the Ext2 protein
Antι-Ext2 protein antibodies may 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 the Ext2 protein may 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 may be used to determine that a given Ext2 protein binding agent, such as an antι-Ext2 protein monoclonal antibody, binds substantially only to the Ext2 protein
Shorter fragments of antibodies can also serve as specific binding agents For instance, FAbs, Fvs, and single-chain Fvs (SCFvs) that bind to Ext2 would be Ext2-specιfic 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) (FAb')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
Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals
Target sequence: "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 For example, hybridization of therapeutically effectively oligonucleotide to an Ext2 target sequence results in inhibition of Ext2 expression Either an antisense or a sense molecule can be used to target a portion of dsDNA, since 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
Transformed: A transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, hpofection, and particle gun acceleration
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication A vector may also include one or more selectable marker genes and other genetic elements known in the art
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise Although methods and materials similar or equivalent to those described herein can be used in the practice or testing, suitable methods and materials are described below 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
///. Identification of Polymorphisms Linked to Osteoporosis/Low Bone
Mineral Density
The inventors have determined that polymoφhisms in the multiple exostoses 2 (Ext2) gene are linked to bone mineral density (BMD), and thereby can be used to predict the risk of a subject suffering from low bone mineral density and/or developing osteoporosis The following examples illustrate this by showing particular examples of polymoφhisms that are associated with BMD Moreover, guidance is provided about finding other polymoφhisms associated with altered BMD Hence, in its broadest aspect, the disclosure is not limited to particular polymoφhisms, but is instead premised on the finding that Ext2 polymorphisms, and more broadly changes in Ext2 expression and/or activity, are associated with altered BMD
In specific embodiments, three complementary mapping populations of mice were utilized to narrow the BMD-related QTL to the region of the mouse chromosome 2 in which Ext2 resides The RIST strategy was then used to significantly narrow the region of interest, which enabled isolation and definitive identification of the osteoporosis-linked gene, Ext2
Sequence analysis has identified two examples of polymoφhisms in the low BMD (B6) Ext2 allele (see FIG 5) One polymoφhism predicts an amino acid change (Ala622Thr) corresponding to nucleotide position 2055 Mutations within this region of the protein may be associated with alterations in bone cartilage development in humans (Kivioja et al , J Bone Joint Surg. (Br) 82-B:261-266, 1999). The other polymoφhism is a G → C transversion that lies within the 3 '-untranslated region at residue 2707. The mouse (gi 1619954) and human (gi l 518042) Ext2 proteins are the same length, and share 95% sequence identity; numbering of amino acid positions within these two molecules is identical. Unless context clearly indicates otherwise, the polymoφhisms and other nucleotide sequence designations herein are numbered as in the mouse Ext2 mRNA sequence, GenBank Accession Number U67837. These designations readily can be correlated to the numbering of the human Ext2 mRNA sequence (Accession number U62740) or human Ext2 gene sequence (Accession number AH00672) by comparing the sequences. By way of example, the coding sequence polymoφhism referred to herein as occurring at position 2055 (on the mouse mRNA) is directly correlated with the human mRNA sequence (Accession number U62740) at position 2198. Likewise, the 3'UTR stem-loop, at position 2702 through 2714 on the mouse mRNA, is found at position 14058 through 14070 on the human gene sequence (Accession number AH006672). Suφrisingly, this stem-loop structure is conserved between mice and humans (see SEQ ID NO: 23), where the surrounding 3'UTR is substantially different. Thus, it is currently believed that this stem-loop structure is important in influencing the involvement of Ext2 in bone mineral density and thereby osteoporosis.
Prior linkage analysis for osteoporosis and/or peak bone mass density has implicated several other human loci whose alleles assert differential influence on predisposition to osteoporosis or increased likelihood of having low bone mineral density. These linkages include: interleukin-1 receptor antagonist (IL-l ra) (U.S. Patent No. 5,698,399); Col 1 alpha gene (U.S. Patent No. 5,922,542); and the human estrogen receptor gene (U.S. Patent No. 5,834,200).
Using the techniques discussed herein, associations were found between alleles of the Ext2 gene and low bond mineral density, which is correlated with osteoporosis. Single nucleotide polymoφhisms (SNPs) within the coding region and downstream untranslated region of Ext2 were demonstrated to be linked to increased likelihood of having low bone mineral density, and thereby predisposition to osteoporosis.
The discovery that polymoφhisms in the sequence of Ext2 predisposes a subject to osteoporosis and/or low bone mineral density also enables a variety of diagnostic, prognostic, and therapeutic methods that are further embodiments. The new appreciation of the role of Ext2 in osteoporosis and low bone mineral density enables detection of predisposition to these conditions in a subject. This disclosure also enables early detection of subjects at high risk of these conditions, and provides opportunities for prevention and/or early treatment.
The disclosure is further illustrated by the following non-limiting Examples. EXAMPLES
Example 1 : Identification of Low Bone Mineral Density-
Linked Polymorphisms in Ext2
This example provides the techniques that were used to identify the linkage between Ext2 and bone mineral density/osteoporosis, as well as to identify and characterize specific polymoφhisms in the Ext2 sequence.
General Methods
Animals.
All mice used in these experiments were bred under identical conditions at the Portland VA Veterinary Medical Unit from stock originally obtained from The Jackson Laboratory (Bar Harbor, ME). Breeding mice were maintained for no more than three generations from stock obtained from The Jackson Laboratory. At the time of weaning, mice were group-housed (2-5 animals per cage) and provided with ad libitum water and laboratory rodent chow (Diet 5001 ; PMI Feeds, Inc., St. Louis, MO) in a 12 hour light/dark cycle at 21 ± 2° C. All procedures were approved by the VA Institutional Animal Care and Use Committee and performed in accordance with National Institutes of Health guidelines for the care and use of animals in research. Bone Densitometry.
Bone mineral measurements were determined by dual energy X-ray absoφtiometry (DEXA). All studies were performed with a pencil beam Hologic QDR 1500 densitometer (Hologic Inc., Waltham, MA). Densitometric analyses were performed on anesthetized mice that were four months of age, the point at which the acquisition of adult bone mass is complete (Brodt et al., J. Bone Miner. Res. 14:2159-2166, 1999). The "global window" was defined as the whole body image minus the calvarium, mandible, and teeth.
Short-Term Selected Lines. Peak BMD was determined on all members of a foundation population (n = 183; male/female = 88/95) of genetically heterogeneous mice derived from the second filial generation of a cross between B6 and D2 mice. The six highest-scoring mice of each gender were mated to form the high BMD line (six breeder pairs), while the six lowest scoring of each gender were mated to form the low BMD line. Individual (or mass) selection was used to gain a more rapid and efficient selection response, with sib matings excluded. In subsequent generations, the highest scoring of the high BMD line and lowest scoring of the low BMD line were selected to serve as breeders of the next generation, with ten breeder pairs per line.
PCR Genotyping. Genomic DNA was isolated from individual mouse spleens using a salting-out method (Buck et al, J. Neurosci. 17:3946-3955, 1997). Mice were genotyped with microsatellite markers chosen from the MIT database and the Mouse Genome Database, both available on-line. All PCR primers (see Table 1) were purchased from Research Genetics (Huntsville, AL). Table 1
Figure imgf000020_0001
Amplification was performed on a Perkin-Elmer 9700 thermocycler (Branchburg, NJ). Each reaction mixture was in a total volume of 25 μl, consisting of approximately 150 ng of genomic DNA, 264 nM of both forward and reverse primers, 0.2 mM of each dNTP, 1 unit of Tag polymerase (Perkin-Elmer Cetus, Branchburg, NJ), and 2.5 μL of GeneAmp 10X PCR Buffer containing 100 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 500 mM KC1, and 0.01% (w/v) gelatin. Thermal cycling included two, five-minute denaturation steps at 95° C then at 80° C, followed by 40 cycles of 30 seconds at 94° C, and 30 seconds at 53° C, 30 seconds at 72° C, and a final extension step for 10 minutes at 72° C. Amplified nucleic acid products were separated on 4% agarose gels and visualized with ethidium bromide staining.
Alternatively, for the Ext2 locus, mice were genotyped based on a known sequence polymoφhism (the 2707 stem-loop mutation) in the 3' untranslated region of the Ext2 gene (Clines et al, Genome Res. 7:359-367, 1997). A 215 bp target region of the 3' UTR of the Ext2 gene was amplified in a 50 μl total reaction volume containing 100 ng genomic DNA, 210 nM of each primer (SEQ ID NO: 19 and SEQ ID NO: 20), 3 mM MgCl2, 50 mM K.C1, 10 mM Tris HC1 (pH 8.3), 0.2 mM dNTPs and 4 units of Tag Gold polymerase (PE Biosystems, Foster City, CA). Amplification was performed on a Perkin-Elmer GeneAmp 9700 thermocycler and cycling included a 10 minute "hot start" to activate the enzyme, followed by 40 cycles of 30 seconds at 94° C, 30 seconds at 51.5° C, and 30 seconds at 72° C, and a final extension step for eight minutes at 72° C.
A 25 μl aliquot of this PCR reaction was loaded onto each of two 2% agarose gels, electrophoresed, and transferred onto Hybond-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ). Allele-specific oligonucleotides (ASO) were labeled with digoxygenin (DIG) using a kit (Boehringer Mannheim, Indianapolis, IN) and 100 μM of ASO. The specific sequences of these ASO's (corresponding to residues 2700-2712 of the Ext2 3'UTR) were as follows:
AATACCTGTGAGGT (SEQ ID NO: 21) AATACCTCTGAGGT (SEQ IDNO: 22) Blots were hybridized with both DIG-labeled ASO (2 pmol/ml) and unlabeled ASO (40 pmol/ml) for three hours at 39° C. Blots were then washed (3 M TMAC, 50 mM Tris (pH 8.0), 1 mM EDTA (pH 8.0) and 0.1% SDS) twice for 10 minutes each at 47° C. DIG detection was performed overnight with anti-DIG-AP Fab fragments (Boehringer Mannheim, Indianapolis, IN) at a 1 : 10,000 dilution in 1% blocking buffer (Roche Molecular Biochemicals, Indianapolis, IN) at 4° C. Blots were washed (0.1 M maleic acid, 0.15 M NaCl pH 7.5) and then developed using nitroblue tetrazolium/5-bromo-4- chloro-3-indoyl-phosphate (NBT/BCIP) for approximately 30 minutes.
RNA and cDNA Analysis Poly(A)+ RNA was isolated directly from mouse heart tissue using a Micro-Fast Track mRNA Isolation Kit (Invitrogen Coφ., Carlsbad, CA). For RT-PCR, 500 ng of poly(A)+ RNA was reverse-transcribed using a Superscript kit (Life Technologies, Inc., Grand Island, NY). Using 2 μl of the 20 μl RT-PCR reaction as a template for PCR, target sequences of the Ext2 gene from five B6 and five D2 mice were amplified, gel-purified and subcloned directly into a p-GEM-T Easy vector (Promega Coφ., Madison, WI) using a Fast-Link DNA Ligation Kit (Epicentre Technologies, Madison, WI). The plasmids were then transfected into ElectroMAX DH 10B cells (Life
Technologies, Inc., Grand Island, NY) by electroporation, and the cells cultured on ampicillin plates. Single colonies were isolated and grown in 5 ml LB Broth. Plasmids were purified, restriction enzyme-digested to check for inserts, and then sequenced. DNA sequence analysis was performed with fluorescent-labeled dye primers and Taq ff cycle sequencing kits supplied by Applied Biosystems, Inc. (ABI; Foster City, CA), and was analyzed on an ABI model 373A Automatic Sequencing System .
Data Analysis. Analysis of B6D2F2 genotyping data was accomplished by carrying out a 2 x 2 χ2 test for each marker between the tails of the distribution (low and high) and allele frequencies (B6 or D2). Individual p values for the phenotypic selection and RIST experiments were determined using linear least squares statistical analyses. For each marker tested, individual mice were assigned a genotypic score of zero, one, or two based on gene dosage (the number of D2 alleles at that locus). Correlation coefficients (Pearson's r) were determined and are equivalent to regressing BMD on gene dosage for each marker. Only a subset (30%) of the B6D2F2 population was genotyped and therefore scored, but all mice from the 3rd generation of phenotypic selection and 2nd filial generation of RIST mice were genotyped and scored, as above. The theoretical rationale underlying the QTL analysis methods have been described previously (Plomin et al. , Behav. Genet. 21 :99-1 16, 1991 ; Gora Maslak et al, Psychopharm. Berl. 104:413-424, 1991). The B6D2F2 genotyping data were subjected to analysis using the Map Manager QT (beta version 28) computer program to determine the position of the peak logarithm of the likelihood for linkage (LOD) and 1 LOD support intervals. StatView® statistical software for the Macintosh was used to perform all other statistics. Results
Microsatellite markers, spaced at a mean interval of ~ 10 cM across the length of chromosome 2, were chosen for analysis (Table 2). A BMD-related QTL was verified on this chromosome. FIG. 1 illustrates the average BMD of animals with the three possible genotypes for the D2MU94 marker. The large difference between mice homozygous for the D2 allele versus those homozygous for the B6 allele is apparent, as is the intermediate BMD of heterozygous mice.
Table 2: Chromosome 2 QTL mapping results for B6D2F2 mice.
Figure imgf000022_0001
The distribution of whole body BMD for the B6D2F2 population (n = 994) was not different from normal (p > 0.9), thus implying polygenic control of peak BMD in mice. A total of 30% of the genetically segregating B6D2F2 population (15% from each tail of the frequency distribution) was genotyped. The average BMD for mice chosen from the upper and lower ends of this distribution was 70.0 ± 1.9 mg/cm2 (mean ± SD) and 60.5 ± 2.1 mg/cm2, respectively. This selective genotyping approach, first suggested by Lander and Botstein (Genetics 121 : 185- 199, 1989), required only 30% of the genotyping expense compared to genotyping the entire F2 population, yet retained 70% of the linkage information needed to detect and map QTLs. Microsatellite markers, numbers of mice with each genotype (D2/D2, D2/B6, and B6/B6) within the lower and upper ends of the BMD distribution that contributed to the analysis, and the significance values are given in Table 2. Map location estimates are from the Mouse Genome Database (Blake et al, Nucleic Acids Res. 27:95-98, 1999). If close linkage exists between the marker D2MU94 and a QTL for peak BMD, then allele frequencies for this marker should diverge in the expected direction in mice selected for BMD. After three generations of selective breeding, the mean peak BMD for low BMD mice was 61.1 ± 3.1 mg/cm2 and for high BMD mice 69.6 ± 2.8 mg/cm2 (p = 5.2xl0"12). The allelic frequencies for D2MU94 in the third generation of mice selectively bred for low BMD was 70% B6 and 30% D2, while in the high BMD mice the allelic frequency was 100% D2 (fixation).
The divergence of D2MU94 allele frequency in the two oppositely selected lines significantly exceeded that expected from random (genetic) drift and allele frequency estimation error (LOD = 2.1, df = 1). Thus, the short-term selected line data strongly supported the presence of a QTL influencing BMD at a chromosomal site near D2MH94. Interval mapping was performed on B6D2F2 data for the verified chromosome 2 QTL with Map Manager QT The LOD plot based on least squares regression methods (df = 2) is shown in FIG 2 The curve is fairly broad, with the 1-LOD confidence interval (LOD 5 6 - 6 6) ranging over a ~ 30 cM interval Within this chromosomal region bounded by D2Mιt94 and D2Mιtl66 reside a number of candidate genes (FIG 3B)
A higher resolution mapping approach, known as recombinant inbred segregation testing (RIST), was used to further characterize gene involvement in BMD (Darvasi, Nat Genet 18 19-24, 1998) This experimental strategy takes advantage of the enhanced mapping resolution present in mouse recombinant inbred (RI) strains (FIG 3A) RI strain BXD-8 (The Jackson Laboratory, Bar Harbor, ME) was selected for this experiment, because it possesses a recombination within the 1- LOD confidence interval of the QTL map location (FIG 3 A) A highly significant correlation between genotype (D2Mιt94) and BMD was found in the F2 mice derived from the D2 X BXD-8 cross (FIG 4A), while no correlation was observed in the BXD-8 X B6 cross (FIG 4A) Thus, the chromosomal region of interest was reduced to = 9 - 10 cM This eliminated all but one hypothetical candidate gene, Ext2
This region of the mouse genome is homologous to the centromeπc region of human chromosome 1 1 , a region that has been previously linked to BMD in both large kindred (Johnson et al , Am J Hum Genet 60 1326-1332, 1997, U S Patent No 5,691 , 153) and sib-pair QTL analyses (Koller et al , J Bone Miner Res 13 1903- 1908, 1998) None of these studies pinpointed the Ext2 gene as being involved in BMD
Mutations in the human Ext2 gene sequence are responsible for hereditary multiple exostoses (HME), a disease characterized by aberrant bone formation (exostoses) (Schmale et al , J Bone Joint Surg Am 76 986-992, 1994) Further experiments were carried out to identify variations (polymorphisms) between B6 and D2 Ext2 cDNA sequences Comparison with the known mouse Ext2 cDNA sequence (Stickens & Evans, Biochem Mol Med 61 16-21 , 1997) revealed two sequence variations between the B6 and D2 alleles One polymoφhism was found at nucleotide position 2055 (FIG 5A) This polymoφhism predicts an amino acid sequence variant at position 622 with threonine at this position in the B6 allele and alanine at this position in the D2 allele Secondary structure modeling predicts that the Ala-622 residue (D2 allele) lies in a region that corresponds to an α helix (Gamier et al , J Mol Biol 120 97-120, 1978) The Thr-622 residue (B6 allele) disrupts this helix region to a large extent
A second polymorphism was found to reside in the 3 '-untranslated region (3'-UTR) at nucleotide position 2707 The G for C substitution in the D2 allele interrupts a short (13 nucleotide) palindromic sequence (Clines et al , Genome Res 7 359-367, 1997) (FIG 5B) This 3'-UTR palindrome is conserved in both human and mouse genes (Stickens & Evans, Biochem Mol Med 61 16-21, 1997, Clines et al , Genome Res 7 359-367, 1997), despite the fact that the Ext2 3'UTR regions of these two species bear little overall resemblance to one another The function of the Ext2 gene product is currently unknown, but accumulating evidence suggests that it may participate in the synthesis of glycosoaminoglycans (GAGs) Ext2 not only possesses intrinsic glycosyltransferase activity (Lind et al , J Biol Chem 273 26265-26268, 1998) but also exhibits a strong physical interaction with a novel N-acetylgalactosaminyl-transferase (GalNac-T5) m vitro (Simmons et al , Hum Mol Genet 8 2155-2164, 1999) As such, Ext2 may comprise part of a larger multi-enzyme GAG synthesis complex Deleting a single histidine residue (H601) from the COOH-terminal region of Ext2 significantly reduced the Ext2-GalNAc-T5 interaction in vitro (Simmons et al , Hum Mol Genet 8 2155-2164, 1999) The Ext2 amino acid variation described in the present report resides in this same general region at position 622 Another member of the Ext gene family, designated tout-velu (ttv), exists in Drosoph a (Bellaiche et al ,
Nature 394 85-88, 1998) The ttv protein appears to be required for the diffusion of hedgehog (Hh) signals in Drosophda (The et al , Mol Cell 4 633-639, 1999) Considering its similarity to Ext proteins, ttv could be responsible for the synthesis of GAGs that specifically enhance Hh diffusion (The et al , Mol Cell 4 633-639, 1999, Toyoda et al , J Biol Chem 275 2269-2275, 2000) The mammalian homolog of Hh is Indian hedgehog (Ihh) (Iwamoto e/ -./ Crit Rev Oral Biol Med 10 477-486, 1999) Ihh is an important regulator of chondrocyte differentiation (Vortkamp et al , Science 273 613-622, 1996, St-Jacques et al , Genes Dev 13 2072-2086, 1999) Based on these observations, a plausible model for the effects of Ext proteins on bone growth in humans is that Ext's regulate chondrocyte function, and thus bone moφhogenesis, by directing the diffusion of Ihh
Example 2: Other Ext2 Polymorphisms
With the provision herein of the correlation between Ext2 gene polymorphisms and predisposition to bone mineral density and/or osteoporosis, the isolation and identification of additional Ext2 polymoφhisms is enabled Any conventional method for the identification of genetic polymorphisms in a population can be used to identify such additional polymoφhisms
For instance, selective breeding studies similar to those described herein are performed to isolate different polymoφhic variants of Ext2 Alternatively, existing populations (e g , mouse or human populations) are assessed for bone mineral density and/or osteoporosis conditions, and individuals within the population (particularly those with low BMD and/or osteoporosis) are genotyped as relates to an Ext2 sequence These Ext2 sequences are then compared to a reference
Ext2 sequence, such as the D2 high BMD allele described herein, 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 BMD and/or osteoporosis
Example 3: Clinical Uses of Ext2 Polymorphisms
To perform a diagnostic test for the presence or absence of a polymoφhism in an Ext2 sequence of an individual, a suitable genomic DNA-containing sample from a subject is obtained and the DNA extracted using conventional techniques Most typically, a blood sample, a buccal swab, a hair follicle preparation, or a nasal aspirate is used as a source of cells to provide the DNA sample The extracted DNA is then subjected to amplification, for example according to standard procedures. The allele of the single base-pair polymoφhism is determined by conventional methods including manual and automated fluorescent DNA sequencing, primer extension methods (Nikiforov, et al, Nucl Acids Res. 22:4167-4175, 1994), oligonucleotide ligation assay (OLA) (Nickerson et al, Proc. Nat Acad. Sci. USA 87:8923-8927, 1990), allele-specific PCR methods (Rust et al., Nucl. Acids Res. 6:3623-3629, 1993), RNase mismatch cleavage, single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), Taq-Man, oligonucleotide hybridization, and the like. Also, see the following U.S. Patents for descriptions of methods or applications of polymoφhism analysis to disease prediction and/or diagnosis: 4,666,828 (RFLP for Huntington's); 4,801,531 (prediction of atherosclerosis); 5,1 10,920 (HLA typing); 5,268,267 (prediction of small cell carcinoma); and 5,387,506 (prediction of dysautonomia).
Examples of polymoφhisms associated with predisposition to osteoporosis and/or an increased likelihood of having low bone mineral density are the Ala622Thr and 3'UTR "stem-loop mutation" polymoφhisms of Ext2. The absence of these polymoφhisms indicates a relative resistance to osteoporosis and a relatively decreased likelihood of having low bone mineral density. In addition to these particular polymoφhisms, other alleles that may be associated with variable predisposition to osteoporosis or likelihood of having low bone mineral density can also be detected, and used in combination with the disclosed Ext2 polymoφhisms to predict the probability that a subject will tend to develop osteoporosis or be likely to display low bone mineral density. These other alleles include (without limitation) polymoφhisms found in the following genes: interleukin- 1 receptor antagonist (IL-l ra) (U.S. Patent No. 5,698,399); Col 1 alpha gene (U.S. Patent No. 5,922,542); and the human estrogen receptor gene (U.S. Patent No. 5,834,200).
The markers of the present disclosure can be utilized for the detection of, and differentiation of, individuals who are homozygous and heterozygous for the Ala622Thr or 3'UTR "stem-loop mutation" polymoφhisms. The value of identifying individuals who carry a low bone mineral density allele of Ext2 (i.e., individuals who are heterozygous or homozygous for the an allele that contains a low bone mineral density Ext2 polymoφhism, such as the G to C transversion at nucleotide position 2707) is that these individuals can then initiate or customize therapy (such as calcium supplementation, hormone therapy, and so forth) to reduce the occurrence of or reverse osteoporosis or improve bone mineral density, or undergo more aggressive treatment of the condition, and thereby beneficially alter its course.
Example 4: Polymorphism Gene Probes and Markers
Sequences surrounding and overlapping single base-pair polymoφhisms in the Ext2 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 the Ala622Thr or 3'UTR "stem-loop mutation" polymoφhism. As will be appreciated, probe sequences may be greater than about 12 or more oligonucleotides in length and possess sufficient complementarity to distinguish between the Alanine (at amino acid residue 622 in the D2 high BMD allele) and Threonine (in the B6 low BMD Ala622Thr polymoφhism), or between the G (in the D2 high BMD allele) and the C (in the B6 low BMD 3'UTR "stem-loop mutation" polymoφhism). Similarly, sequences surrounding and overlapping either of the specifically disclosed single base-pair polymoφhisms (or other polymoφhisms found in accordance with the present teachings), or longer sequences encompassing both specifically disclosed polymoφhisms, can be utilized in allele specific hybridization procedures. A similar approach can be adopted to detect other Ext2 polymoφhisms.
Sequence surrounding and overlapping a Ext2 polymoφhism, or any portion or subset thereof that allows one to identify the polymoφhism, are highly useful. Thus, another embodiment provides a genetic marker predictive of the Ala622Thr polymoφhism of Ext2, comprising a partial sequence of the human genome including at least about 10 contiguous nucleotide residues including "N" in the following nucleotide sequence: AACTGGGTAGATNCTCATATGAACTGTGAA (SEQ ID NO: 23), and sequences complementary therewith, wherein "N" represents A or a single base-pair polymoφhism of the G that is present at N in a human allele analogous to the murine D2 (high BMD) allele. One example polymoφhism is a G to A transition, but can also include a G to T transversion or G to C transversion.
Likewise, another specific embodiment is a genetic marker predictive of a 3'UTR "stem- loop mutation" polymoφhism of Ext2, comprising a partial sequence of the human genome including at least about 10 contiguous nucleotide residues in the following nucleotide sequence: CAGAGAAAAACAGAGGGTCTGTACTAGCCAT (SEQ ID NO: 24), and sequences complementary therewith. In particular, mutations in the 10th through 15th or 19lh through 23rd residues of this sequence, which destabilize the stem of the stem-loop structure (underlined), are of particular interest. For example, in the murine Ext2, it is a G to C transversion that results in the D2 (high BMD) allele. Thus, particular aspects include oligonucleotide molecules including at least about 10 contiguous nucleotide residues of SEQ ID NO: 24, with at least one residue changed in the sequence, in particular in the 10th through 15th residue or the 19th through 23rd residues of SEQ ID NO: 24.
Example 5: Detecting SNPs
The SNPs at nucleotide residue 2055 (the first position encoding amino acid residue 622) and/or nucleotide residue 2707 can be detected by a variety of techniques. These techniques include 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 new 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. Mol. 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 (Tm) that can be readily detected. A variety of other techniques can be used to detect the polymorphisms in DNA. Merely by way of example, see U.S. Patents No. 4,666,828; 4,801,531 ; 5,1 10,920; 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.
Example 6: Detection of Ext2 Nucleic Acid Level(s) Individuals carrying mutations in the Ext2 gene, or having amplifications or heterozygous or homozygous deletions of the Ext2 gene, may be detected at the DNA or RNA level with the use of a variety of techniques. The detection of point mutations was discussed above; in the following example, techniques are provided for detecting the level of Ext2 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 Ext2 encoding sequence, such as a genomic amplification of the Ext2 gene or an over- or under-abundance of a Ext2 mRNA. Suitable biological samples include samples containing genomic DNA or mRNA obtained from 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 Ext2 gene, a mutant Ext2 RNA, or an amplified or homozygously or heterozygously deleted Ext2 gene, may be performed by a number of methodologies.
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 Ext2 nucleic acids in samples of tissue. Probes generated from the encoding sequence of Ext2 (Ext2 probes or primers) can be used to investigate and measure genomic dosage of the Ext2 gene.
Appropriate 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 al. (Nat. Genet. 20:207-21 1, 1998) ("High Resolution Analysis of DNA Copy Number Variation using Comparative Genomic Hybridization to Microarrays")
Determination of gene copy number in cells of a patient-derived sample using other techniques is known in the art For example, Ext2 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, inteφhase 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 fluorescence microscope By way of example, approximately 20 non- overlapping nuclei with intact moφhology 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% formamιde/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 WO9944063A2 and WO9944062A1
Overexpression of the Ext2 gene can also be detected by measuring the cellular level of Exf2-specιfic mRNA mRNA can be measured using techniques well known in the art, including for instance Northern analysis, RT-PCR and mRNA in situ hybridization
Example 7: Expression of Ext2 Polypeptides
The expression and purification of proteins, such as the Ext2 protein, can be performed using standard laboratory techniques After expression, purified Ext2 protein may be used for functional analyses, antibody production, diagnostics, and patient therapy Furthermore, the DNA sequence of the Ext2 cDNA can be manipulated in studies to understand the expression of the gene and the function of its product Mutant forms of the human Ext2 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 Ext2 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 Eschenchia coll (E cob) 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 Ext2 proteins may be used to prepare polyclonal and monoclonal antibodies against these proteins Thereafter, these antibodies may be used to purify proteins by lmmunoaffinity chromatography, in diagnostic assays to quantitate the levels of protein and to localize proteins in tissues and individual cells by lmmunofluorescence 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 πbosome-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 Set 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) and pET-3 (Studiar and Moffatt, J Mol Biol 189 1 13, 1986) Ext2 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 Ext2 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 Set USA 78 2072-2076, 1981), and introduced into cells, such as monkey COS-1 cells (Gluzman, Cell 23 175-182, 1981), to achieve transient or long-term expression The stable integration of the chimeπc gene construct may be maintained in mammalian cells by biochemical selection, such as neomycin (Southern and Berg, ./ Mol Appl Genet 1 327-341 , 1982) and mycophenohc 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 bacteπophage 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 Set 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 Mol 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 , Mol Cell Biol 1 486, 1981) or Epstein-Barr (Sugden et al , Mol 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 , Mol Cell Biol 7 2013, 1987), electroporation (Neumann et al , EMBO J 1 841 , 1982), hpofection (Feigner et al , Proc Natl Acad Sci USA 84 7413, 1987), DEAE dextran (McCuthan et al , J Natl Cancer Inst 41 351, 1968), microinjection (Mueller et al , Cell 15 579, 1978), protoplast fusion (Schafner, Proc Natl Acad Sci USA 11 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 1 235, 1985), adenoviruses (Ahmad et al , J Virol 57 267, 1986), or Heφes virus (Spaete et al , Cell 30 295, 1982) Ext2 encoding sequences can also be delivered to target cells in vitro via non-infectious systems, for instance posomes
These eukaryotic expression systems can be used for studies of Ext2 encoding nucleic acids and mutant forms of these molecules, the Ext2 protein and mutant forms of this protein Such uses include, for example, the identification of regulatory elements located in the 5' region of the Ext2 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 Ext2 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 puφose 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 Ext2 gene or cDNA sequences, for expression in a suitable host The Ext2 DNA is operatively linked in the vector to an expression control sequence in the recombinant DNA molecule so that the Ext2 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 £ coli, Pseudomonas, Bacillus subt is, 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 Ext2 DNA sequences, similar systems are employed to express and produce the mutant product In addition, fragments of the Ext2 protein can be expressed essentially as detailed above Such fragments include individual Ext2 protein domains or sub-domains, as well as shorter fragments such as peptides Ext2 protein fragments having therapeutic properties may be expressed in this manner also Example 8: Production of Ext2 Protein Specific Binding Agents
Monoclonal or polyclonal antibodies may be produced to either the normal Ext2 protein or mutai forms of this protein. Optimally, antibodies raised against these proteins or peptides would specifically dete the protein or peptide with which the antibodies are generated. That is, an antibody generated to the Exi protein or a fragment thereof would recognize and bind the Ext2 protein and would not substantially recogni; or bind to other proteins found in human cells.
The determination that an antibody specifically detects the Ext2 protein is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook 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 Ext2 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 Ext2 protein will, by this technique, be shown to bind to the Ext2 protein band (which will be localized at a given position on the gel determined by its molecular weight). Nonspecific 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-Ext2 protein binding. Substantially pure Ext2 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:
A. Monoclonal Antibody Production by Hybridoma Fusion
Monoclonal antibody to epitopes of the Ext2 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. En∑ymol. 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).
B. Polyclonal Antibody Production by Immunization Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein (Example 7), 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 el 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 M). Affinity 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).
C. Antibodies Raised against Synthetic Peptides
A third approach to raising antibodies against the Ext2 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 Ext2 protein or peptide. Polyclonal antibodies can be generated by injecting these peptides into, for instance, rabbits.
D. Antibodies Raised by Injection of Ext2 Encoding Sequence
Antibodies may be raised against Ext2 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 Sci. Technol. 5:27-37, 1987) as described by Tang et al. (Nature 356: 152-154, 1992). Expression vectors suitable for this puφose may include those that express the Ext2 encoding sequence under the transcriptional control of either the human -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 Ext2 protein
For administration to human patients, antibodies, e g , Ext2 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)
Example 9: Protein-Based Diagnosis
An alternative method of diagnosing Ext2 gene amplification, deletion or mutation, as well as abnormal Ext2 expression, is to quantitate the level of Ext2 protein in the cells of an individual This diagnostic tool would be useful for detecting reduced levels of the Ext2 protein that result from, for example, mutations in the promoter regions of the Ext2 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 Ext2 gene Alternatively, duplications of a Ext2 encoding sequence may be detected as an increase in the expression level of Ext2 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 Ext2 gene
Localization and/or coordinated Ext2 expression (temporally or spatially) can also be examined using known techniques, such as isolation and comparison Ext2 from cell or tissue specific, or time specific, samples The determination of reduced or increased Ext2 protein levels, in comparison to such expression in a control cell (e , normal, as in taken from a subject not suffering from osteoporosis), would be an alternative or supplemental approach to the direct determination of Ext2 gene deletion, amplification or mutation status by the methods outlined above and equivalents
The availability of antibodies specific to the Ext2 protein will facilitate the detection and quantitation of cellular Ext2 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 Example 8
Any standard immunoassay format (e , ELISA, Western blot, or RIA assay) can be used to measure Ext2 polypeptide or protein levels, comparison is to wild-type (normal) Ext2 levels, and an alteration in Ext2 polypeptide may be indicative of an abnormal biological condition altered BMD and/or a predilection to development of osteoporosis Immunohistochemical techniques may also be utilized for Ext2 polypeptide or protein detection For example, a tissue sample may be obtained from a subject, and a section stained for the presence of Ext2 using a Ext2 specific binding agent (e g , antι-Ext2 antibody) and any standard detection system (e , one which includes a secondary antibody conjugated to horseradish peroxidase) General guidance regarding such techniques can be found in, e g , 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 puφoses of quantitating a Ext2 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 required 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 breast cells Quantitation of Ext2 protein can be achieved by immunoassay and compared to levels of the protein found in control cells (e , healthy, as in from a patient known not to have osteoporosis) A significant (e , 10% or greater) reduction in the amount of Ext2 protein in the cells of a subject compared to the amount of Ext2 protein found in normal human cells could be taken as an indication that the subject may have deletions or mutations in the Ext2 gene, whereas a significant (e , 10% or greater) increase would indicate that a duplication (amplification), or mutation that increases the stability of the Ext2 protein or mRNA, may have occurred Deletion, mutation and/or amplification of or within the Ext2 encoding sequence, and substantial under- or over-expression of Ext2 protein, may be indicative of altered BMD and/or a predilection to develop osteoporosis
Example 10: Differentiation of Individuals Homozygous versus Heterozygous for the Polymorphism(s)
As will be appreciated, the oligonucleotide ligation assay (OLA), as described at Nickerson et al (Proc Natl Acad Sci USA 87 8923-8927, 1990), allows the differentiation between individuals who are homozygous versus heterozygous for either the Ala622Thr or the 3'UTR polymorphisms This feature allows one to rapidly and easily determine whether an individual is homozygous for at least one B6-lιnked polymoφhism, which condition is linked to a relatively high predisposition to developing osteoporosis and/or an increased likelihood of having low bone mineral density Alternatively, OLA can be used to determine whether a subject is homozygous for either of these polymoφhisms
As an example of the OLA assay, when carried out in microtiter plates, one well is used for the determination of the presence of the Ext2 allele that contains a G at nucleotide position 2707 and a second well is used for the determination of the presence of the Ext2 allele that contains a C at nucleotide position 2707 Thus, the results for an individual who is heterozygous for the polymoφhism will show a signal in each of the G and C wells, and an individual who is homozygous for the 3'UTR "stem-loop mutation" polymoφhism will show a signal in only the C well
Example 11 : Suppression of Ext2 Expression
A reduction of Ext2 protein expression in a transgenic cell may be obtained by introducing into cells an antisense construct based on the Ext2 encoding sequence, including the human Ext2 cDNA or gene sequence (Accession number U62740 or AH00672, respectively) or flanking regions thereof For antisense suppression, a nucleotide sequence from an Ext2 encoding sequence, e g all or a portion of the Ext2 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 above (Example 7) The introduced sequence need not be the full length human Ext2 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 Ext2 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 Ext2 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 Ext2 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 Ext2 expression can also be achieved using ribozymes Ribozymes are synthetic RNA molecules that possess highly specific endoπbonuclease activity The production and use of ribozymes are disclosed in U S Patent No 4,987,071 to Cech and U S Patent No 5,543,508 to Haselhoff The inclusion of πbozyme 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 Finally, dominant negative mutant forms of Ext2 may be used to block endogenous Ext2 activity
Example 12: Ext2 Gene Therapy
Gene therapy approaches for combating alterations in BMD, or reducing the risk of developing osteoporosis, in subjects are now made possible by the present disclosure
Retroviruses have been considered a preferred vector for experiments in gene therapy, with a high efficiency of infection and stable integration and expression (Orkin et al , Prog Med Genet 1 130-142, 1988) The full-length Ext2 gene or cDNA can be cloned into a retroviral vector and driven from either its endogenous promoter or from the retroviral LTR (long terminal repeat) Other viral transfection systems may also be utilized for this type of approach, including adenovirus, adeno- associated virus (AAV) (McLaughlin et al J Virol 62 1963- 1973, 1988), Vaccinia virus (Moss et al , Annu Rev Immunol 5 305-324, 1987), Bovine Papilloma virus (Rasmussen et al , Methods Enzymol 139 642-654, 1987) or members of the heφesvirus group such as Epstein-Barr virus (Margolskee / α/ , Mol Cell Biol 8 2837-2847, 1988) Recent developments in gene therapy techniques include the use of RNA-DNA hybrid oligonucleotides, as described by Cole-Strauss, et al (Scιence 213 1386- 1389, 1996) This technique may allow for site-specific integration of cloned sequences, thereby permitting accurately targeted gene replacement In addition to delivery of an Ext2 encoding sequence to cells using viral vectors, it is possible to use non-infectious methods of delivery For instance, lipidic and hposome-mediated gene delivery has recently been used successfully for transfection with various genes (for reviews, see Templeton and Lasic, Mol Biotechnol 1 1 175- 180, 1999, Lee and Huang, Crit Rev Ther Drug Carrier Syst 14 173-206, and Cooper, Semm Oncol 23 172-187, 1996) For instance, cationic hposomes have been analyzed for their ability to transfect monocytic leukemia cells, and shown to be a viable alternative to using viral vectors (de Lima et al , Mol Membr Biol 16 103-109, 1999) Such cationic hposomes can also be targeted to specific cells through the inclusion of, for instance, monoclonal antibodies or other appropriate targeting ligands (Kao et al , Cancer Gene Ther 3 250- 256, 1996) To reduce the level of Ext2 expression, gene therapy can be carried out using antisense or other suppressive constructs, the construction of which is discussed above (Example 1 1)
Example 13: Kits
Kits are provided which contain the necessary reagents for determining the presence or absence of polymoφhιsm(s) in an Ext2-encodιng sequence, such as probes or primers specific for the Ext2 gene Such kits can be used with the methods described herein to determine whether a subject is predisposed to osteoporosis and/or low bone mineral density
The provided kits may also include written instructions The instructions can provide calibration curves or charts to compare with the determined (e g , experimentally measured) values Kits are also provided to determine elevated or depressed expression of mRNA (/ e , containing probes) or Ext2 protein (/ e , containing antibodies or other Ext2 -protein specific binding agents)
A. Kits for Amplification of Ext2 Sequences
The oligonucleotide probes and primers disclosed herein can be supplied in the form of a kit for use in detection of a predisposition to osteoporosis or low bone mineral density in a subject In such a kit, an appropriate amount of one or more of the oligonucleotide primers is provided in one or more containers The oligonucleotide primers may be provided suspended in an aqueous solution or as a freeze-dπed or lyophi zed powder, for instance The contaιner(s) in which the ohgonucleotιde(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microftige tubes, ampoules, or bottles In some applications, pairs of primers may be provided in pre-measured single use amounts in individual, typically disposable, tubes or equivalent containers With such an arrangement, the sample to be tested for the presence of an Ext2 polymoφhism can be added to the individual tubes and amplification carried out directly The amount of each oligonucleotide primer supplied in the kit can be any appropriate amount, depending for instance on the market to which the product is directed. For instance, if the kit is adapted for research or clinical use, the amount of each oligonucleotide primer provided would likely be an amount sufficient to prime several PCR amplification reactions. Those of ordinary skill in the art know the amount of oligonucleotide primer that is appropriate for use in a single amplification reaction. General guidelines may for instance be found in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990), Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989), and Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992).
A kit may include more than two primers, in order to facilitate the in vitro amplification of Ext2 sequences, for instance the Exl2 gene or the 5' or 3' flanking region thereof.
In some embodiments, kits may also include the reagents necessary to carry out nucleotide amplification reactions, including, for instance, DNA sample preparation reagents, appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesium chloride), and deoxyribonucleotides (dNTPs).
Kits may in addition include either labeled or unlabeled oligonucleotide probes for use in detection of Ext2 polymoφhism(s). In certain embodiments, these probes will be specific for a potential polymoφhism that may be present in the target amplified sequences. The appropriate sequences for such a probe will be any sequence that includes one or more of the identified polymoφhic sites, particularly nucleotide positions 2055 and 2707, such that the sequence the probe is complementary to a polymoφhic site and the surrounding Ext2 sequence. Oligonucleotides GTAGATGCTCATA (SEQ ID NO: 25) and ACAGAGGGTCTGT (SEQ ID NO: 26) exemplify such sequences, and an appropriate probe could comprise either (or both) of these sequences. It may also be advantageous to provide in the kit one or more control sequences for use in the amplification reactions. The design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art.
B. Kits for Detection of Ext2 mRNA Expression
Kits similar to those disclosed above for the detection of Ext2 polymoφhisms directly can be used to detect Ext2 mRNA expression, such as over- or under-expression. Such kits include an appropriate amount of one or more oligonucleotide primers for use in, for instance, reverse transcription PCR reactions, similarly to those provided above with art-obvious modifications for use with RNA amplification.
In some embodiments, kits for detection of altered expression of Ext2 mRNA may also include some or all of the reagents necessary to carry out RT-PCR in vitro amplification reactions, including, for instance, RNA sample preparation reagents (including e.g., an RNase inhibitor), appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesium chloride), and deoxyribonucleotides (dNTPs). Written instructions may also be included. Such kits may in addition include either labeled or unlabeled oligonucleotide probes for use in detection of the in vitro amplified target sequences. The appropriate sequences for such a probe will be any sequence that falls between the annealing sites of the two provided oligonucleotide primers, such that the sequence the probe is complementary to is amplified during the PCR reaction. In certain embodiments, these probes will be specific for a potential polymoφhism that may be present in the target amplified sequences, for instance specific for the B6 allele at the 3'UTR site (e.g., capable of detecting a C residue at position 2707 of the Ext2 sequence).
It may also be advantageous to provide in the kit one or more control sequences for use in the RT-PCR reactions. The design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art.
Alternatively, kits may be provided with the necessary reagents to carry out quantitative or semi-quantitative Northern analysis of Ext2 mRNA. Such kits include, for instance, at least one Ext2- specific oligonucleotide for use as a probe. This oligonucleotide may be labeled in any conventional way, including with a selected radioactive isotope, enzyme substrate, co-factor, ligand, chemiluminescent or fluorescent agent, hapten, or enzyme. In certain embodiments, such probes will be specific for a potential polymoφhism that may be present in the target amplified sequences, for instance specific for the B6 allele at the 3'UTR site (e.g., capable of detecting a C residue at position 2707 of the Ext2 sequence).
C. Kits For Detection of Ext2 Protein Expression Kits for the detection of Ext2 protein expression (such as over- or under-expression) are also encompassed. Such kits may include at least one target protein specific binding agent (e.g., a polyclonal or monoclonal antibody or antibody fragment that specifically recognizes the Ext2 protein) and may include at least one control (such as a determined amount of Ext2 protein, or a sample containing a determined amount of Ext2 protein). The Ext2-protein specific binding agent and control may be contained in separate containers.
The Ext2 protein expression detection kits may also include a means for detecting Ext2:binding agent complexes, for instance the agent may be detectably labeled. If the detectable agent is not labeled, it may be detected by second antibodies or protein A for example which may also be provided in some kits in one or more separate containers. Such techniques are well known. Additional components in specific kits may include instructions for carrying out the assay.
Instructions will allow the tester to determine whether Ext2 expression levels are elevated. Reaction vessels and auxiliary reagents such as chromogens, buffers, enzymes, etc. may also be included in the kits.
D. Kits for Detection of Homozygous versus Heterozygous Allelism Also provided are kits that allow differentiation between individuals who are homozygous versus heterozygous for either the Ala622Thr or the 3'UTR polymoφhisms of Ext2. Such kits provide the materials necessary to perform oligonucleotide ligation assays (OLA), as described at Nickerson et al. (Proc. Natl. Acad. Sci USA 87:8923-8927, 1990). In specific embodiments, these kits contain one or more microtiter plate assays, designed to detect polymoφhism(s) in the Ext2 sequence of a subject, as described herein.
Additional components in some of these kits may include instructions for carrying out the assay. Instructions will allow the tester to determine whether an Ext2 allele is homozygous or heterozygous. Reaction vessels and auxiliary reagents such as chromogens, buffers, enzymes, etc. may also be included in the kits.
It may also be advantageous to provide in the kit one or more control sequences for use in the OLA reactions. The design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art.
Example 14: Ext2 Knockout and Overexpression Transgenic Animals
Mutant organisms that under-express or over-express Ext2 protein are useful for research. Such mutants allow insight into the physiological and/or pathological role of Ext2 in a healthy and/or pathological organism. These mutants are "genetically engineered," 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-£x.2 promoter inserted upstream of a native Ext2 encoding sequence would be non-native. An extra copy of an Ext2 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 Ext2 or under-express Ext2, or that do not express Ext2 at all. Over-expression mutants are made by increasing the number of Ext2 genes in the organism, or by introducing an Ext2 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 Ext2 may be made by using an inducible or repressible promoter, or by deleting the Ext2 gene, or by destroying or limiting the function of the Ext2 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 Ext2 expression, as discussed above in Example 1 1.
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 Ext2 gene altered or functionally deleted, this refers to the Ext2 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, e.g., in the diploid mouse or human.
A mutant mouse over-expressing Ext2 may be made by constructing a plasmid having an Ext2 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, e.g., 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 (e.g., mouse) from which an Ext2 gene is deleted can be made by removing all or some of the coding regions of the Ext2 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). This disclosure provides specific polymoφhisms in a gene, Ext2, that are linked to differential bone mass density, and more particularly to predisposition to or the condition of osteoporosis. The disclosure further provides methods for identifying these polymoφhisms in a subject, and using them to determine or predict a subject's osteoporosis state. It will be apparent that the precise details of the methods described 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

We claim:
L A method of detecting a biological condition associated with an abnormal Ext2 nucleic acid or an abnormal Ext2 expression, comprising detecting the abnormal Ext2 nucleic acid or the abnormal Ext2 expression.
2. The method of claim 1 , wherein the abnormal Ext2 nucleic acid or abnormal Ext2 expression comprises an alteration in cellular level of Ext2 nucleic acid or Ext2 protein, in comparison to a normal level.
3. The method of claim 1 , wherein the biological condition is altered BMD, osteoporosis, or a tendency to develop osteoporosis.
4. The method of claim 1, wherein the abnormal Ext2 nucleic acid comprises an Ext2 polymoφhism.
5. The method of claim 1 , wherein the method is used for presymptomatic screening of an individual for osteoporosis.
6. The method of claim 1 , which is a method of predicting a predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject, comprising: determining whether the subject has a polymoφhism in a Ext2 sequence, wherein presence of the polymoφhism indicates the predisposition to osteoporosis or the increased likelihood of having low bone mineral density.
7. The method of claim 6, wherein the polymoφhism is Ala622Thr or 3'UTR stem- loop mutation.
8. The method of claim 7, wherein determining whether the subject has the polymoφhism comprises providing DNA from the subject, and assessing the DNA for the presence of either the Ala622Thr or 3'UTR stem-loop mutation polymorphism.
9 The method of claim 7, wherein determining whether the subject has the polymoφhism comprises providing DNA from the subject, and assessing the DNA for the presence of both the Ala622Thr and 3'UTR stem-loop mutation polymoφhisms.
10. The method of claim 8, further comprising determining whether the subject is homozygous or heterozygous for the polymoφhism.
1 1. The method of claim 7, further comprising determining whether the subject has one or more other alleles associated with predisposition to osteoporosis or increased likelihood of having low bone mineral density.
12. The method of claim 1 1, wherein the one or more other alleles is one or more of: a polymoφhism at position 1245 of a Col 1 alpha gene; a polymoφhism of an interleukin- 1 receptor antagonist gene; or a polymoφhism in a first intron of a human estrogen receptor gene.
13. The method of claim 8, wherein the assessing step is performed by a process which comprises subjecting the DNA or RNA to amplification using oligonucleotide primers flanking the polymoφhism.
14. The method of claim 13, wherein the assessing step further comprises an oligonucleotide ligation assay.
15. A method of predicting predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject, comprising: obtaining a test sample of DNA containing a Ext2 sequence of the subject; and determining whether the subject has a polymoφhism in the Ext2 sequence, wherein the presence of the polymoφhism indicates the predisposition to osteoporosis or the increased likelihood of having low bone mineral density in a subject.
16. The method of claim 15, wherein determining whether the subject has the polymoφhism comprises using restriction digestion, probe hybridization, nucleic acid amplification, or nucleotide sequencing.
17. The method of claim 16, wherein determining whether the subject has the polymoφhism comprises using nucleic acid amplification.
18. The method of claim 17, wherein determining whether the subject has the polymoφhism comprises using polymerase chain reaction nucleic acid amplification.
19. The method of claim 16, wherein determining whether the subject has the polymoφhism comprises performing probe hybridization with a nucleic acid probe to the Ext2 polymoφhism in the test sample.
20. The method of claim 16, wherein determining whether the subject has the polymoφhism comprises detecting the polymorphism by hybridization of an allele-specific oligonucleotide with the Ext2 B6 allele.
21. The method of claim 15, wherein the polymoφhism is Ala622Thr or 3'UTR stem- loop mutation.
22. A method of predicting predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject, comprising: obtaining from the subject a test sample of DNA comprising an Ext2 sequence; contacting the test sample with at least one nucleic acid probe for an Ext2 sequence polymoφhism that is associated with increased predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject to form a hybridization sample; maintaining the hybridization sample under conditions sufficient for specific hybridization of the Ext2 sequence with the nucleic acid probe; and detecting whether there is specific hybridization of the Ext2 sequence with the nucleic acid probe, wherein specific hybridization of the Ext2 sequence with the nucleic acid probe indicates increased predisposition to osteoporosis or an increased likelihood of having low bone mineral density in the subject.
23. The method of claim 22, wherein the polymoφhism is Ala622Thr or 3'UTR stem- loop mutation.
24. The method of claim 23, wherein the probe is present on a substrate.
25. The method of claim 24, wherein the substrate is a nucleotide array.
26. A kit for use in diagnosing an increased predisposition to osteoporosis or an increased likelihood of having low bone mineral density in a subject, comprising a probe that specifically hybridizes to an Ext2 sequence polymoφhism that is associated with the increased predisposition to osteoporosis or an increased likelihood of having low bone mineral density.
27. The kit of claim 26, wherein the probe specifically hybridizes to an Ext2 Ala622Thr or 3'UTR stem-loop mutation polymoφhism.
28. A nucleic acid probe that specifically hybridizes to a human Ext2 polymoφhism.
29. The nucleic acid probe according to claim 28, wherein the probe hybridizes to an Ext2 Ala622Thr or 3'UTR stem-loop mutation polymoφhism.
30. A method of osteoporosis therapy comprising: screening an individual for a genetic predisposition to osteoporosis; and if such a predisposition is identified, treating that individual to prevent or reduce osteoporosis or to delay the onset of osteoporosis, wherein predisposition to osteoporosis is correlated with a polymoφhism in a Ext2 sequence.
31. The method according to claim 30 comprising treating the individual by hormone replacement therapy.
PCT/US2001/012447 2000-04-17 2001-04-17 Ext2 as a predictive marker for osteoporosis WO2001078575A2 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004055212A1 (en) * 2002-12-13 2004-07-01 Oxagen Limited Genetic susceptibility
WO2006008342A1 (en) * 2004-07-16 2006-01-26 Oy Jurilab Ltd Method for detecting the risk of and for treatment of type 2 diabetes
CN116287280A (en) * 2023-05-25 2023-06-23 北京大学第三医院（北京大学第三临床医学院） EXT1 or EXT2 diagnostic kit based on single cell transcriptome mRNA level

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* Cited by examiner, † Cited by third party
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US5698399A (en) * 1996-04-05 1997-12-16 Duff; Gordon W. Detecting genetic predisposition for osteoporosis
CN1180709A (en) * 1996-10-21 1998-05-06 湖南医科大学 Neopolynucleotide and polypeptide gene of genetic multiple exogenetic bone wart family
GB9706359D0 (en) * 1997-03-27 1997-05-14 Gemini International Holdings Polymorphisms of an il-1 receptor antagonist gene

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004055212A1 (en) * 2002-12-13 2004-07-01 Oxagen Limited Genetic susceptibility
WO2006008342A1 (en) * 2004-07-16 2006-01-26 Oy Jurilab Ltd Method for detecting the risk of and for treatment of type 2 diabetes
JP2008506379A (en) * 2004-07-16 2008-03-06 オイ ユリラブ アェルテーデー Method for detecting and treating the risk of type 2 diabetes
CN116287280A (en) * 2023-05-25 2023-06-23 北京大学第三医院（北京大学第三临床医学院） EXT1 or EXT2 diagnostic kit based on single cell transcriptome mRNA level
CN116287280B (en) * 2023-05-25 2023-08-15 北京大学第三医院（北京大学第三临床医学院） EXT1 or EXT2 diagnostic kit based on single cell transcriptome mRNA level

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