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  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • 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
• • 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
  • C12Q2600/172—Haplotypes

Definitions

• the invention relates generally to the fields of molecular biology, genetics, and medicine.
• the invention relates to genetic polymorphisms and protein expression in serum useful for assessing jaw osteonecrosis risks in humans receiving or prescribed bisphosphonates.
• Bisphosphonates are prescribed to alleviate bone pain, bone destruction and hypercalcemia in many cancer patients, or to reduce bone loss in osteoporotic individuals.
• the bisphosphonates are a class of drugs that inhibit the activities and functions of osteoclasts (bone resorbing cells) and perturb the differentiation of osteoblasts (bone forming cells),
• Bisphosphonates inhibit bone resorption and thus bone renewal by suppressing the recruitment and activity of osteoclasts thus shortening their life span.
• IV bisphosphonates are primarily used to treat bone erosion and hypercalcemia associated with bone metastasis from multiple myeloma and other malignancies.
• Oral bisphosphonates are also used to prevent bone loss and are prescribed for patients with osteoporosis. Painful exposure of bone in the jaws of patients receiving the bisphosphonates pamidronate (Aredia®; Novartis Pharmaceuticals) and zoledronate (Zometa®; Novartis Pharmaceuticals) was first reported by Marx in 2003 (J Oral Maxillofac Surg, 61 : 1 115- 1 1 17, 2003). Since then, several authors have reported additional cases and many dental professionals, particularly oral and maxillofacial surgeons have identified numerous unpublished cases.
• Bisphosphonatcs are commonly prescribed to stabilize bone loss caused by osteoporosis in millions of postmenopausal women.
• the strategy in the treatment of osteoporosis is to inhibit the resorption of trabecular bone by osteoclasts and hence preserve its density.
• oral bisphosphonates are prescribed and include etidronate (Didronel®; Procter and Gamble), risedronate (Actonel®; Procter and Gamble), tiludronate (Skelid®; Sanofi-Synthe Lab Inc), and alendronate (Fosamax® ; Merck).
• More potent bisphosphonates are delivered intravenously and are indicated to stabilize metastatic cancer (primarily breast and prostate) deposits in bone, and to treat the bone resorption defects of multiple myeloma and correct severe hypercalcemia. These are pamidronate and zoledronate. In addition to the drugs mentioned here, several other bisphosphonates are known that are either not commonly used in the United States or that remain experimental.
• the exposed bone in the jaws is the direct result of the action of these bisphosphonates on the daily remodeling and replenishment of bone.
• Osteoblasts and osteocytes live for only about 150 days. If, upon their death, the mineral matrix is not resorbed by osteoclasts, which release the cytokines of bone morphogenetic protein and insulin-like growth factors to induce new osteoblasts from the stem cell population, the osteon becomes acellular and necrotic.
• the small capillaries within the bone become involuted, and the bone becomes a-vascular.
• a spontaneous breakdown of the overlying mucosa, some form of injury, or an invasive surgery to the jaws usually causes this necrotic bone to become exposed which then fails to heal.
• BONJ cases to date are diagnosed in cancer patients with bone metastases. However, BONJ cases have been also reported after oral therapy for osteoporosis. Thus, a large proportion of the general population ⁇ e.g. , post menopausal women and cancer patients) may be at risk.
• Described herein are methods of identifying the genetic basis for a patient's predisposition to BONJ, methods and kits for identifying patients who are prone to develop BONJ following bisphosphonate administration, and tools for physicians to prescribe treatment protocols for BONJ patients based on the patients' genomes ("personal/tailored medicine") and serum protein expression.
• proteins whose expression in patients' biological fluids e.g. , serum, saliva
• known methods e.g. , Western blots, ELISA, etc.
• the methods described herein include identifying genes that contribute to the development of BONJ using a candidate gene approach.
• a case- control design study is used that involves studying the effects of genes (e.g., expression and particular SNP(s)) and serum protein expression in patients with and without BONJ.
• the method includes the steps of: (a) obtaining a sample from the subject; (b) analyzing the sample for the presence of at least one gene having an SNP that is a biomarker for BON J or a predisposition to BONJ, a protein encoded by the at least one gene, or at least one SNP that is a marker for BONJ or a predisposition to BONJ; and (c) correlating the presence of the gene, protein or SNP that is a marker for BONJ or a predisposition to BONJ in the sample with a predisposition to BONJ in the subject.
• the sample can include, for example, blood, serum, plasma or saliva.
• the at least one gene can be a gene set forth in Table 1 , Table 3, or Table 1 1
• the at least one SNP can be an SNP set forth in Table 1 , Table 3, or Table 1 1.
• the at least one gene can comprise, for example, COL 1 A 1 , RANK, MMP2, OPG, or OPN.
• the genes comprise two, three, four, or all five genes from among COLl Al, RANK, MMP2, OPG, and OPN.
• the at least one gene comprises OPG, OPN, or both.
• the at least one SNP can be, for example, rsl24581 17 (SEQ ID NO: l), rs243865 (SEQ ID NO:2), rsl 800012 (SEQ ID NO: 15). rs2073618 (SEQ ID NO:24), or rsl 1730582 (SEQ ID NO:26).
• the SNPs comprise two, three, four, or all five SNPs from among rsl 24581 17 (SEQ ID NO: l), rs243865 (SEQ ID NO:2), rsl 800012 (SEQ ID NO: 15), rs2073618 (SEQ ID NO:24), and rsl 1730582 (SEQ ID NO:26).
• the at least one SNP comprises rs2073618 (SEQ ID NO:24), rsl 1730582 (SEQ ID NO:26), or both.
• the step of analyzing the sample for the presence of at least one gene having an SNP that is a biomarker for BONJ or a predisposition to BONJ, a protein encoded by the at least one gene, or at least one SNP that is a marker for BONJ or a predisposition to BONJ can include use of a microarray to detect the presence of the at least one gene or at least one SNP.
• Also described herein is a method of preventing BONJ in a subject having a predisposition to BONJ and receiving bisphosphonate therapy.
• the method includes the steps of: (a) obtaining a sample from the subject; (b) analyzing the sample for the presence of at least one gene having an SNP that is a biomarker for BONJ or a predisposition to BONJ; a protein encoded by the gene, or at least one SNP that is a biomarker for BONJ or a predisposition to BONJ; (c) correlating the presence of the at least one gene, protein, or at least one SNP that is a biomarker for BONJ or a predisposition to BONJ in the sample with a predisposition to BONJ in the subject; and (d) administering to the subject a bisphosphonate that is not associated with BONJ or that is less likely to cause BONJ than other bisphopsphonates, administering to the subject a bisphosphonate at
• the bisphosphonate may be administered to the subject by any route suitable for the particular formulation administered (e.g., intravenous, oral).
• the sample can include, for example, blood, serum, plasma or saliva.
• the at least one gene can be a gene set forth in Table 1 , Table 3, or Table 11
• the at least one SNP can be an SNP set forth in Table 1 , Table 3 , or Table 11.
• the at least one gene can comprise, for example, COLl Al, RANK, MMP2, OPG. or OPN.
• the genes comprise two, three, four, or all five genes from among COL1A1 , RANK, MMP2, OPG, and OPN.
• the at least one gene comprises OPG. OPN, or both.
• the at least one SNP can be, for example, rsl 24581 17 (SEQ ID NO: 1), rs243865 (SEQ ID NO:2), rsl 800012 (SEQ ID NO: 15), rs2073618 (SEQ ID NO:24), or rsl 1730582 (SEQ ID NO:26).
• the SNPs comprise two, three, four, or all five SNPs from among rs l 24581 1 7 (SEQ ID NO:l), rs243865 (SEQ ID NO:2), rs 1800012 (SEQ ID N0:15), rs2073618 (SEQ ID NO:24). and rsl 1730582 (SEQ ID NG:26).
• the at least one SNP comprises rs2073618 (SEQ ID NO:24). rsl 1730582 (SEQ ID NO:26), or both.
• the step of analyzing the sample for the presence of at least one gene having an SNP that is a biomarker for BONJ or a predisposition to BONJ, a protein encoded by the at least one gene, or at least one SNP that is a marker for BONJ or a predisposition to BONJ can include use of a microarray to detect the presence of the at least one gene or at least one SNP.
• kits for identifying patients who have a predisposition to BONJ following biphosphonate administration includes (a) a solid support having a plurality of nucleic acids adhered thereto, wherein at least one of the nucleic acids specifically hybridizes to a gene having an SNP that is a biomarker for BONJ or a predisposition to BONJ; (b) a detection reagent; and (c) instructions for use.
• the gene can be a gene set forth in Table 1, Table 3, or Table 1 1
• the SNP can be an SNP set forth in Table 1 , Table 3, or Table 1 1.
• the gene can comprise, for example, COL1 A1 , RANK, MMP2, OPG, or OPN.
• the genes comprise two, three, four, or all five genes from among COL1A1 , RANK, MMP2, OPG, and OPN.
• the at least one gene comprises OPG, OPN. or both.
• the at least one SNP can be, for example. rsl24581 17 (SEQ ID NO:l), rs243865 (SEQ ID NO:2), rsl 800012 (SEQ ID NO: 15), rs2073618 (SEQ ID NO:24), or rsl 1730582 (SEQ ID NO:26).
• the SNPs comprise two, three, four, or all five SNPs from among rsl24581 17 (SEQ ID NO: l), rs243865 (SEQ ID NO:2), rsl 800012 (SEQ ID NO: 15), rs2073618 (SEQ ID NO:24), and rsl 1730582 (SEQ ID NO:26).
• the at least one SNP comprises rs2073618 (SEQ ID NO:24), rsl 1730582 (SEQ ID NO:26), or both.
• a method for assessing a subject's risk of developing BONJ following bisphosphonate treatment includes a) obtaining a biological sample from the subject; b) detecting one or more BONJ-associated biomarkers in said sample, wherein the biomarkers are related to one or more genes set forth in Table 1 , Table 3, or Table 1 1 , or said biomarkers are related to one or more polypeptides encoded by said genes resulting in a biomarker data set; c) comparing the biomarker data set to biomarker data from healthy people and people having BONJ; and d) determining the subject's risk of developing BONJ.
• At least one biomarker can be an SNP residing in a gene set forth in Table 1 , Table 3, or Table l l, a BONJ- associated polymorphic site associated with one or more of the SNP markers set forth in Table 1 , Table 3 , or Table 1 1 , or an expression product of a gene set forth in Table 1 , Table 3 , or Table 11.
• At least one biomarker can be an SNP being in complete linkage disequilibrium with one or more of the SNP markers set forth in Table 1, Table 3, or Table 1 1.
• the method includes the steps of: (a) obtaining a sample from the subject; (b) analyzing the sample for expression of a protein involved in bone homeostasis; and (c) correlating expression of the protein with a predisposition to BONJ in the subject.
• the protein involved in bone homeostasis can be one of: PTH, insulin, TNF-a, leptin, OPN, OC, OPG and IL6.
• the sample can be analyzed for overexpression of the protein.
• the protein can be one that shares at least 90% amino acid sequence identity with a protein listed in Table 4.
• the aforementioned methods of the invention further comprise determining whether the subject has other genetic (e.g. , based on presence of gene, SNP, and/or protein) or non-genetic (e.g. , life style factors such as smoking history) predictive risk factors for developing BONJ.
• other genetic e.g. , based on presence of gene, SNP, and/or protein
• non-genetic e.g. , life style factors such as smoking history
• an SNP includes a plurality of SNPs (i.e., at least one SNP)
• a gene includes a plurality of genes (i.e., at least one gene), and so forth.
• nucleic acid means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid).
• a “purified” nucleic acid molecule is one that has been substantially separated or isolated away from other nucleic acid sequences in a cell or organism in which the nucleic acid naturally occurs (e.g. , 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants).
• the term includes, e.g., a recombinant nucleic acid molecule incorporated into a vector, aplasmid, a virus, or a genome of a prokaryote or eukaryote.
• the term “gene” is meant a nucleic acid molecule that codes for a particular protein, or in certain cases, a functional or structural RNA molecule.
• the COL1A1 gene encodes the COL1A1 protein.
• protein or “polypeptide” are used synonymously to mean any peptide- linked chain of amino acids, regardless of length or post-translational modification, e.g. , glycosylation or phosphorylation.
• nucleic acid molecule or polypeptide When referring to a nucleic acid molecule or polypeptide, the term “native” refers to a naturally-occurring (e.g. , a WT) nucleic acid or polypeptide.
• osteopontin protein or "OPN” or “OPN polypeptide” is meant an expression product of an OPN gene such as a protein that shares at least 65% (but preferably 75. 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with native human osteopontin (OPN) protein and displays a functional activity of anative OPN protein.
• a "functional activity" of a protein is any activity associated with the physiological function of the protein.
• additional proteins described herein e.g. , parathyroid hormone (PTH), insulin, TNF-a, osteocalcin (OC), osteoprotegerin (OPG). etc.
• sequence identity means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. Sequence identity is present when a subunit position in both of the two sequences is occupied by the same nucleotide or amino acid, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then the molecules are identical at that position. For example, if 7 positions in a sequence 10 nucleotides in length are identical to the corresponding positions in a second 10-nucleotide sequence, then the two sequences have 70 %o sequence identity. Sequence identity is typically measured using sequence analysis software (e.g. , Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
• a “biomarker” in the context of the present invention refers to a SNP marker disclosed in Tables 1 , 3, or 1 1 , or to a polymorphism of a gene disclosed in Tables 1 , 3, or 1 1 , or at a locus closely linked thereto, or to an organic biomolecule which is related to a gene set forth in Tables 1 , 3, or 1 1 and which is differentially present in samples taken from subjects (patients) having BONJ compared to comparable samples taken from subjects who do not have BONJ.
• An “organic biomolecule” refers to an organic molecule of biological origin, e.g., steroids, amino acids, nucleotides, sugars, polypeptides, polynucleotides, complex carbohydrates or lipids.
• a biomarker is differentially present between two samples if the amount, structure, function or biological activity of the biomarker in one sample differs in a statistically significant way from the amount, structure, function or biological activity of the biomarker in the other sample.
• One or more genes, SNPs, or proteins that are biomarkers for BONJ or a predisposition to BONJ can be correlated with a predisposition to BONJ, such that their presence within a biological sample (such as blood, serum, plasma, or saliva) is indicative of a predisposition to BONJ.
• a haplotype refers to any combination of genetic markers ("alleles").
• a haplotype can include two or more alleles and the length of a genome region including a haplotype may vary from a few hundred bases up to hundreds of kilobases. As it is recognized by those skilled in the art, the same haplotype can be described differently by determining the haplotype defining alleles from different nucleic acid strands.
• the haplotypes described herein are differentially present in individuals with BONJ or having an increased risk of BONJ than in individuals without BONJ. Therefore, these haplotypes have diagnostic value for risk assessment, diagnosis and prognosis of BONJ or risk of BONJ in an individual.
• haplotypes can be accomplished by methods known in the art used for detecting nucleotides at polymorphic sites.
• the haplotypes described herein, e.g. having markers such as those shown in Tables 1 , 3, or 1 1 are found more frequently in individuals with BONJ or having an increased risk of BONJ than in individuals without BONJ. Therefore, these haplotypes have predictive value for detecting BONJ or a susceptibility (increased risk) to BONJ in an individual.
• a nucleotide position in a genome at which more than one sequence is possible in a population is referred to herein as a "polymorphic site” or "polymorphism".
• a polymorphic site is a single nucleotide in length, the site is referred to as a SNP.
• SNP single nucleotide in length
• polymorphic site may be several nucleotides in length due to insertions, deletions, conversions or translocations.
• Each version of the sequence with respect to the polymorphic site is referred to herein as an "allele" of the polymorphic site.
• the SNP allows for both an adenine allele and a thymine allele.
• the SNP markers having novel BONJ associations as described herein have official reference SNP (rs) ID identification tags assigned to each unique SNP by the National Center for Biotechnological Information (NCBI).
• rs ID has been linked to specific variable alleles present in a specific nucleotide position in the human genome, and the nucleotide position has been specified with the nucleotide sequences flanking each SNP.
• NBI National Center for Biotechnological Information
• an "allele” may refer to a nucleotide at a SNP position (wherein at least two alternative nucleotides arc present in the population at the SNP position, in accordance with the inherent definition of a SNP) or, for cSNPs, may refer to an amino acid residue that is encoded by the codon which contains the SNP position (where the alternative nucleotides that are present in the population at the SNP position form alternative codons that encode different amino acid residues).
• An “allele” may also be referred to herein as a "variant”.
• an amino acid residue that is encoded by a codon containing a particular SNP may simply be referred to as being encoded by the SNP.
• Probes or “primers” are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid molecules.
• base specific manner is meant that the two sequences must have a degree of nucleotide complementarity sufficient for the primer or probe to hybridize to its specific target. Accordingly, the primer or probe sequence is not required to be perfectly complementary to the sequence of the template. Non-complementary bases or modified bases can be interspersed into the primer or probe, provided that base substitutions do not inhibit hybridization.
• the nucleic acid template may also include "non-specific priming sequences" or “nonspecific sequences” to which the primer or probe has varying degrees of complementarity.
• Probes and primers may include modified bases as in polypeptide nucleic acids. Probes or primers typically include about 15 to 30 consecutive nucleotides and they may further include a detectable label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co- factor. Probes and primers to a SNP marker disclosed in Tables 1, 3, or 11 are either commercially available or easily designed using the flanking nucleotide sequences assigned to a SNP rs ID and standard probe and primer design tools. Primers and probes for SNP markers disclosed in Tables 1, 3, or 1 1 can be used in risk assessment as well as in molecular diagnostic methods and kits as described herein.
• arrays are used herein interchangeably to refer to an array of distinct polynucleotides affixed to a substrate, such as glass, plastic, paper, nylon or other type of membrane, filter, chip, or any other suitable solid support.
• the polynucleotides can be synthesized directly on the substrate, or synthesized separate from the substrate and then affixed to the substrate.
• Microarrays can be prepared and used by a number of methods, including those described in U.S. Pat. No. 5,837,832 (Chee et al.), PCT application W095/1 1995 (Chee et al.), Lockhart, D. J. et al. (Nat. Biotech.
• such arrays can be produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.
• risk genes By the phrases “risk genes,” “risk genes for diseases,” and “disease loci” is meant genetic variants that confer an increased likelihood of developing disease.
• patient means a mammalian (e.g. , human) subject to be treated and/or to obtain a biological sample from.
• subject and “individual” are used interchangeably herein, and mean a mammalian (e.g. , human) subject to be treated and/or to obtain a biological sample from.
• FIG. 1 is a photograph of the mouth of a man suffering from painful, non-healing necrotic bone exposure in the mandible of a multiple myeloma patient treated with Zometa® (zoledronate, Novartis).
• SEQ ID NO: 1 is the nucleotide sequence assigned to SNP rs 124581 17 of the tumor necrosis factor receptor superfamily, member 1 1a, NFKB activator (TNFRSF11 A (RANK)) gene.
• SEQ ID NO:2 is the nucleotide sequence assigned to SNP rs243865 of the matrix metallopeptidase 2 (MMP2) gene.
• SEQ ID NO:3 is the nucleotide sequence assigned to SNP rs 1800211 of the collagen, type I, alpha 1 (COL1A1) gene.
• SEQ ID NO:4 is the nucleotide sequence assigned to SNP rs4147630 of the osteoclast associated receptor (OSCAR) gene.
• SEQ ID NO:5 is the nucleotide sequence assigned to SNP rs 10788796 of the cathepsin K
• SEQ ID NO:6 is the nucleotide sequence assigned to SNP rsl 800471 of the transforming growth factor, beta 1 (TGFB 1 ) gene.
• SEQ ID NO:7 is the nucleotide sequence assigned to SNP rsl 805034 of the RANK gene.
• SEQ ID NO: 8 is the nucleotide sequence assigned to SNP rs9562415 of the receptor activator of NFKB ligand (RANKL) gene.
• SEQ ID NO:9 is the nucleotide sequence assigned to SNP rs2069830 of the interleukin-6 (IL6) gene.
• SEQ ID NO: 10 is the nucleotide sequence assigned to SNP rs2228570 of the vitamin D receptor (VDR) gene.
• SEQ ID NO: l 1 is the nucleotide sequence assigned to SNP rs731236 of the VDR gene.
• SEQ ID NO: 12 is the nucleotide sequence assigned to SNP rsl 1498198 runt-related transcription factor 2 (RUNX2) gene.
• SEQ ID NO:13 is the nucleotide sequence assigned to SNP rsl934980 of the CYP2C8 gene.
• SEQ ID NO: 14 is the nucleotide sequence assigned to SNP rs l 93495 1 of the CYP2C8 gene.
• SEQ ID NO: 15 is the nucleotide sequence assigned to SNP rs l 800012 of the COL1A1 gene.
• SEQ ID NO: 16 is the amino acid sequence of parathyroid hormone (PTH).
• SEQ ID NO: 17 is the amino acid sequence of insulin.
• SEQ ID NO:l 8 is the amino acid sequence of TNF-alpha.
• SEQ ID NO: 19 is the amino acid sequence of leptin.
• SEQ ID NO:20 is the amino acid sequence of osteocalcin (OC).
• SEQ ID NO: 2 1 is the amino acid sequence of osteoprotegerin (OPG).
• SEQ ID NO:22 is the amino acid sequence of osteopontin (OPN).
• SEQ ID NO:23 is the amino acid sequence of interleukin-6 (IL6).
• SEQ ID NO: 24 is the nucleotide sequence assigned to SNP rs2073618 of the tumor necrosis factor receptor superfamily, member 1 lb (TNFRSF1 IB) gene (OPG).
• SEQ ID NO:25 is the nucleotide sequence assigned to SNP rs3102735 of the TNFRSF1 IB gene (OPG).
• SEQ ID NO:26 is the nucleotide sequence assigned to SNP rsl 1730582 of the secreted phosphoprotein 1 (SPP 1 ) gene (OPN).
• SEQ ID NO:27 is the nucleotide sequence assigned to SNP rs28357094 of the SPP1 gene (OPN).
• SEQ ID NO:28 is the nucleotide sequence assigned to SNP rs 1800629 of the tumor necrosis factor (TNF) gene.
• the invention relates to identifying risk factors for developing BONJ in a patient or subject (e.g., humans) receiving bisphosphonate treatment or potentially receiving bisphosphonate treatment. Described herein are methods of determining the pharmacogenetic, pharmacokinetic and cellular basis of BONJ. Also described herein are methods and kits for identifying the genetic basis for a patient's predisposition to BONJ. and methods of identifying patients who are prone to develop BONJ following bisphosphonate administration provide for the development of tools for physicians to prescribe treatment protocols for BONJ patients based on the patients' genomes ("personal/tailored medicine").
• a haplotype tagging SNP approach was used to analyze candidate genes involved in bone absorption and destruction and to examine the influence of genetic variants on the susceptibility of BONJ.
• Bone biomarkers of BONJ were examined using molecular cell techniques. The methods described herein can be used to identify differences in how patients are genetically predisposed to BONJ as well as genetic differences amongst patients that account for differences in how these patients clear bisphosphonates from their systems. Determining such genetic differences provides for improved monitoring of the drugs used to treat BONJ, improved prevention of BONJ, and optimized treatment of such individuals.
• the methods of the invention further comprise determining whether the subject has other genetic (e.g., based on presence of gene, SNP, and/or protein) or non-genetic (e.g., life style factors such as smoking history) predictive risk factors for developing BONJ.
• other genetic e.g., based on presence of gene, SNP, and/or protein
• non-genetic e.g., life style factors such as smoking history
• SNPs Single Nucleotide Polymorphisms
• SNPs are single base positions in DNA at which different alleles, or alternative nucleotides, exist in a population, and are the most common form of genetic variation in the genome.
• the SNP position (interchangeably referred to herein as SNP, SNP site, SNP locus, SNP marker, biomarker, or marker) is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations).
• An individual maybe homozygous or heterozygous for an allele at each SNP position.
• an SNP is referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP is an amino acid coding sequence.
• a SNP may arise from a substitution of one nucleotide for another at the polymorphic site. Substitutions can be transitions or trans versions. A transition is the replacement of one purine nucleotide by another purine nucleotide, or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine by a pyrimidine, or vice versa.
• a SNP may also be a single base insertion or deletion variant referred to as an "indel" (Weber et al., Am. J . Hum. Genet. 71 :854-62, 2002).
• references to SNPs and SNP genotypes include individual SNPs and/or haplotvpes, which are groups of SNPs that are generally inherited together. Haplotypes can have stronger correlations with diseases or other phenotypic effects compared with individual SNPs, and therefore may provide increased diagnostic accuracy in some cases.
• Causative SNPs are those SNPs that produce alterations in gene expression or in the expression, structure, and/or function of a gene product, and therefore are most predictive of a possible clinical phenotype.
• One such class includes SNPs falling within regions of genes encoding a polypeptide product, i.e. cSNPs.
• SNPs may result in an alteration of the amino acid sequence of the polypeptide product (i.e., non-synonymous codon changes) and give rise to the expression of a defective or other variant protein. Furthermore, in the case of nonsense mutations, a SNP may lead to premature termination of a polypeptide product. Such variant products can result in a pathological condition, e.g. genetic disease.
• causative SNPs do not necessarily occur in coding regions; causative SNPs can occur in, for example, any genetic region that can ultimately affect the expression, structure, and/or activity of the protein encoded by a nucleic acid.
• Such genetic regions include, for example, those involved in transcription, such as SNPs in transcription factor binding domains, SNPs in promoter regions, in areas involved in transcript processing, such as SNPs at intron-exon boundaries that may cause defective splicing, or SNPs in mRNA processing signal sequences such as polyadenylation signal regions.
• SNP SNP-associated neurotrophic factor
• the SNP identification information such as variable alleles and flanking nucleotide sequences assigned to a SNP will remain the same.
• the analysis of the nucleotides present in one or more SNPs set forth in Tables 1 , 3, and 11 of this invention in an individual's nucleic acid can be done by any method or technique capable of determining nucleotides present in a polymorphic site using the published sequence information to the rs IDs of the SNPs listed in Tables 1 , 3, and 1 1 .
• the nucleotides present in polymorphisms can be determined from either nucleic acid strand or from both strands.
• TagSNPs are loci that can serve as proxies for many other SNPs. The use of tagSNPs greatly improves the power of association studies as only a subset of loci needs to be genotyped while maintaining the same information and power as if one had genotyped a larger number of SNPs.
• polymorphic sites associated with the SNP markers listed in Tables 1 , 3, and 1 1 of this invention may be either equally useful as biomarkers or even more useful as causative variations explaining the observed BON.I- association of SNP markers and haplotypes as described herein.
• peptides and polypeptides encoded by genes listed in Tables 1 , 3, and 11 including polymorphic positions (e.g., SNPs) disclosed herein.
• the peptides and polypeptides are useful screening targets to identify drugs for treating BONJ.
• genes and SNPs that are thought to play a role in osteoclastogenesis, osteoclast differentiation, and bone resorption, bone mineral density (BMD); osteoclast-mediated bone resorptionin tissues of patients with BONJ; and aggressive periodontitis are analyzed.
• the genes listed in Table 1 are genes and SNPs that are thought to play a role in osteoclastogenesis, osteoclast differentiation, and bone resorption, bone mineral density (BMD); osteoclast-mediated bone resorption in tissues of patients with BONJ; and aggressive periodontitis, and were reported to be important and significant. These genes are described below.
• OSCAR Osteoclast Associated Receptor
• Cathepsin K is a lysosomal cysteine protease involved in bone remodeling and resorption. This protein, which is a member of the peptidase CI protein family, is predominantly expressed in osteoclasts. However, the encoded protein is also expressed in a significant fraction of human breast cancers, where it could contribute to tumor invasiveness. Mutations in this gene are the cause of pycnodysostosis, an autosomal recessive disease characterized by osteosclerosis and short stature (Saftig et al., Proc Natl Acad Sci USA 95 : 13453-13458, 1998; Haagerup et al., Eur J Hum Genet 8:431 -436, 2000).
• TGFB is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types.
• TGF B l which is encoded by TGFB 1 gene is the most abundant growth factor in human bone.
• TGF B l is produced by osteoblasts and inhibits osteoclast proliferation and activity. It also functions as a regulator of susceptibility to osteoporosis and has been shown to affect on both osteoblast and osteoclast function in vitro (Massague J and Chen YG.. Genes & Dev. 14:627-644, 2000; Langdahl et al.. Bone 32:297-310, 2003).
• CSF-1 Human macrophage-specific colony-stimulating factor (CSF-1) along with Receptor Activator of NF-KB Ligand (RANKL) are involved in activation and differentiation of monocyte subsets into osteoclasts (Rabello et al., Biochem Biophys Res Commun. 347:791 -796, 2006; Komano et al., Arthritis Res Ther. 8 :R 152, 2006).
• a recent study of Japanese population showed a positive association between aggressive periodontitis and three polymorphisms located in CSF1.
• Osteoclastogenesis in vivo is regulated by action of osteoblast/stromal cells that express membrane-bound, receptor activator of NF-kB ligand (RANKL).
• RANKL a member of TNF family
• RANKL is a cytokine which is essential for induction of osteoclastogenesis.
• Both osteoblasts and stromal cells produce this cytokine and the signal is transduced by specific receptor called RANK that is localized on the surface of osteoclast progenotors (Boyle et al., Nature, 423 :337-342, 2003).
• Both RANKL and CSF-1 are essential for stimulation and differentiating the monocytes into osteoclats ( Komano ct al..
• the COL1A1 gene is considered as an important functional candidate for the pathogenesis of osteoporosis because the type I collagen is the major protein of bone and mutation in this gene results in syndrome called osteogenesis imperfecta which is characterized by reduced BMD and increase bone fragility (Boyde et al., Calcif Tissue Int. 64: 185-190, 1999). Genetic variants may play important role in osteoporosis or osteoporotic fractures by affecting the metabolism of COL1 Al gene.
• Yamada Yamada (Yamada ct al., Hum Biol.
• IL-6 is a pleiotropic cytokine that plays a critical role in bone resorption.
• Ferrari et al. showed that a G>C polymorphism at -174 in the promoter region of IL-6 gene has a functional effect in vivo and affects gene transcription and results in decreasing of circulating levels of IL-6 (Ferrari et al., Arthritis Rheum. 44: 196-201, 2001). It was also shown that the haplotype of two allelic variants in the IL-6 promoter region -572 and - 174 G was associated with bone resorption markers (Ferrari et al., J Clin Endocrinol Metab. 88:255-259, 2003).
• VDR vitamin D receptor
• a steroid receptor acts as a transcriptional factor which responds to steroid vitamin D hormone.
• Vitamin D regulates bone cell differentiation, osteoblast differentiation, bone turnover and calcium homeostasis by interaction with vitamin D receptor (Haussler et al, J Bone Miner Res. 13;325-349, 1998).
• the VDR gene was one of the first genes that were studied in relation to osteoporosis.
• a large and comprehensive study on VDR and its relation to osteoporosis was conducted on 641 8 individuals by Fang (Fang et al., Am J Hum Genet. 77:807-823, 2005).
• the authors used the haplotype-tagging SNPs approach and analyzed 15 haplotype-tagging SNPs, and showed that haplotype alleles in the promoter and 3 ' UTR regions of VDR gene were associated with increased risk of osteoporotic fracture, and subjects who carried both risk alleles had significantly higher risk (48%) of developing fracture when compared with control individuals.
• the combination of risk haplotypes results in lower VDR luRNA expression level caused by decreased transcription and increased mRNA degradation (Fang et al., Am J Hum Genet. 77:807-823, 2005).
• Runt-related transcription factor 2 (RUNX2), also known as CBFA1 gene, encodes a protein that binds to osteoblast-specific cis-acting element and plays an essential role in the regulation of osteoblast differentiation and inducing osteoblast-specific transcripts, like the one encoding osteocalci (Ducy et al.. Cell 89:747-754, 1997; Schinke et al., J Biol Chem. 274:30182- 30189, 1999).
• RUNX2 polymorphisms withBMD.
• polymorphisms are located within the promoter or polyalanine and polyglutamine repeats of exon 1.
• polymorphisms in the promoter region affect RUNX2 transcription in a reporter assay, and are associated with BMD. It was also shown that polymorphisms in the RUNX2 gene that affect BMD are in linkage disequilibrium.
• Genomic DNA is isolated from whole blood using any suitable extraction method, e.g., the QIAamp DNA Blood kit from Qiagen (Valencia. CA). Isolated gDNA is stored at -20°C (e.g., in bar-coded 1.5 ml microcentrifuge tubes). Isolated gDNA is quantified by any suitable method, such as a spectrophotometric method using a 96 well plate reader.
• PCR-based genotyping methods are utilized. Selection of PCR primers and conditions is optimized through use of a suitable primer design software ⁇ e.g. , Oligo Primer Analysis Software Version 6).
• the genotyping platform used for genotyping is Taqman.
• the ABI Taqman Prism 7900 HT Sequence Detection System is a second generation, real-time quantitative PCR system with high-throughput capability that can use either 96 or 384-well microtiter plates and a Micro Fluidic Card. This system is also equipped with new software for large-scale genotyping of known SNPs that produces reliable and reproducible results at low cost.
• Applied Biosystems (Foster City. CA) has over 180,000 validated Taqman® SNP genotyping assays, with an additional 1 .6 million predesigned assays for non-coding SNPs. Custom assays for SNPs not in their assay library can also be developed.
• Pyrosequencing is a real-time DNA sequencing technique that involves hybridization of a sequencing primer to single-stranded, PCR-amplified DNA, along with various substrates and enzymes.
• the sequencing primers are designed by using special primer design software provided by Pyrosequencing.
• Upon incorporation of nucleotide(s) into a nucleic acid chain by DNA polymerase an equimolar quantity of inorganic pyrophosphate is released and subsequently converted to ATP by ATP sulfurylase.
• the ATP drives a luciferase reaction where luciferin molecule is oxidized to produce light.
• the light is captured on a charge coupled device camera and seen as a peak on a pyrogram.
• the pyrosequencing reaction generates sequence data of 20-30 bp and allows genotyping with > 99% accuracy and reproducibility.
• the PSQ ITS 96A system supports throughput of up to 3,000 genotypes per workday.
• genotyping is achieved by restriction fragment length polymorphism (RFLP) analysis.
• RFLP restriction fragment length polymorphism
• the invention provides a method for identifying a patient or subject (e.g., human) that is predisposed to or at risk of BONJ following bisphosphonate administration.
• a patient or subject e.g., human
• an individual who is at risk for BONJ is an individual in whom one or more BONJ- associated polymorphisms selected from Tables 1 , 3, or 1 1 are identified and/or expression (e.g., overexpression) of one or more of the proteins listed in Table 4 (or precursors, mature forms, or cleavage fragments thereof) is detected in their serum.
• polymorphisms associated with SNPs and haplotypes of Tables 1, 3, or 1 1 and/or serum expression of proteins listed in Table 4 may be used in risk assessment of BONJ .
• Patients who are candidates for biphosphonate treatment are screened for the presence of the specific gene or SNP or protein.
• Patients who test positive can be considered for an alternative treatment or otherwise complete a complete dental treatment prior to the biphosphonate therapy.
• a typical method of identifying a subject having a predisposition to BONJ following bisphosphonate treatment includes obtaining a sample from the patient; analyzing the sample for the presence of at least one gene having an SNP that is a biomarker for BONJ or a predisposition to BONJ, a protein encoded by the gene, or at least one SNP that is a biomarker for BONJ or a predisposition to BONJ; and correlating the presence of the at least one gene, protein, or at least one SNP that is a biomarker for BONJ or a predisposition to BONJ in the sample with a predisposition to BONJ in the subject.
• any appropriate sample can be obtained, e.g., blood, serum, plasma, saliva, etc.
• SNPs rs 124581 17 (SEQ ID NO: l ) and rs243865 (SEQ ID NO:2) were shown to be present at a higher rate in patients having BONJ than in patients without BONJ.
• the subject's sample is analyzed for the presence of SNPs rsl 24581 17 (SEQ ID NO: 1 ) and rs243865 (SEQ ID NO:2).
• the at least one SNP can be an SNP set forth in Table 1, Table 3, or Table 1 1 .
• the at least one SNP can be rsl24581 1 7 (SEQ ID NO: 1), rs243865 (SEQ ID NO:2), rs l 800012 (SEQ ID NO: 15). rs2073618 (SEQ ID NO:24), or rsl 1730582 (SEQ ID NO:26).
• the SNPs comprise two, three, four, or all five SNPs from among rsl 24581 1 7 (SEQ ID NO: l), rs243865 (SEQ ID NO:2), rsl 800012 (SEQ ID NO: 15), rs2073618 (SEQ ID NO:24), and rsl 1730582 (SEQ ID NO:26).
• the at least one SNP comprises rs2073618 (SEQ ID NO:24), rs l 1 730582 (SEQ ID NO:26), or both.
• the subject's sample can be analyzed for the presence of such SNPs using any suitable method, including use of a microarray.
• Microarrays or Chips usually contain thousands up to a million or a little over one million SNPs, which are also used for genotyping.
• a subject's sample can be analyzed for the presence of a gene in which a particular SNP resides, or a protein encoded by a gene in which a particular SNP resides.
• the at least one gene can be one or more genes set forth in Table 1 , Table 3 , or Table 11.
• the at least one gene can comprise, for example, COL1A1 , RANK, MMP2, OPG. or OPN.
• the genes comprise two, three, four, or all five genes among COL1A1 , RANK, MMP2, OPG, and OPN.
• the at least one gene comprises OPG, OPN, or both.
• Some biphosphonates are prone to less side effects like BONJ.
• Aredia® is less likely to cause BONJ compared to Zometa®.
• patients who express the specific genes associated with BONJ are treated with drugs that are less likely to cause BONJ.
• Zometa® would not be used, its frequency of use would be reduced, and/or its dosage would be reduced.
• kits for identifying patients who are prone to develop BONJ following biphosphonate administration include reagents, materials and protocols for assessing one or more biomarkers (e.g., nucleic acids, proteins), and instructions and optionally software for comparing the biomarker data from a subject to biomarker data from healthy and diseased people to make risk assessment, a diagnosis or a prognosis of BONJ.
• biomarkers e.g., nucleic acids, proteins
• Useful reagents and materials for kits include, but are not limited to PCR primers, hybridization probes and primers as described herein (e.g., labeled probes or primers), allele-specific oligonucleotides, reagents for genotyping SNP markers, reagents for detection of labeled molecules, restriction enzymes (e.g.
• DNA polymerases DNA polymerases
• RNA polymerases DNA ligases
• marker enzymes microarrays
• antibodies which bind to altered or to non-altered (native) BONJ risk gene encoded polypeptides means for amplification of nucleic acids fragments from one or more BONJ- risk genes selected from Tables 1 , 3, or 1 1 means for analyzing the nucleic acid sequence of one or more BONJ risk genes or fragments thereof, or means for analyzing the sequence of one or more amino acid residues of a BONJ risk gene-encoded polypeptide, etc.
• a typical kit for identifying patients who are prone to developing BONJ following biphosphonate administration includes a solid support having a plurality of nucleic acids adhered thereto (e.g., a nucleic acid array), wherein at least one (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10) of the nucleic acids specifically hybridizes to a gene having an SNP that causes BONJ or a predisposition to BONJ; a detection reagent; and instructions for use.
• the solid support will have two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) nucleic acids adhered thereto that specifically hybridize to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) genes having SNPs that are markers for a predisposition to BONJ.
• a kit includes a blood test and/or saliva test for the expression of specific genes and SNPs that are associated with BONJ.
• a kit for diagnosing or predicting susceptibility to BONJ includes primers and reagents for detecting the nucleotides present in one or more SNP markers selected from Tables 1 , 3, or 1 1 in an individual's nucleic acid. Information obtained from use of kits as described herein can be used to optimize treatment of individuals having BONJ or suspected of having BONJ.
• kits for detecting the presence of one or more proteins (e.g., proteins listed in Table 4, or precursors, mature forms, or cleavage fragments thereof) whose expression in serum can be used to assess an individual's risk of developing BONJ.
• a kit can include the reagents and instructions necessary for carrying out a Western blot, for example, or an ELIS A.
• Genomic DNA was isolated from lymphocytes in whole blood using a commercially available kit (Qiagen DNA Blood Isolation Kit, Qiagen, Valencia, CA). The isolated DNA samples were quantified by spectrophotometry and agarose gel electrophoresis methods and standardized to 20 ng/ul.
• TaqMan genotyping assay probes [(C _74771 74_30 for SNP [A/C], dbSNP ID (rsl 800012) (SEQ ID NO: 15), COL1A1 gene chromosome 17, and C _31393804 for SNP [A/G], dbSNP ID (rsl24581 17) (SEQ ID NO: l), TNFRSF11A gene chromosome 18] were purchased from Applied Biosystems, Foster City, USA.
• the probes are fluorescent dyes (VIC and FAM) labeled primers designed by ABI (Applied Biosystems) for genotyping.
• Genomic DNA was isolated from lymphocytes of blood samples from 50 subjects, 6 cases of BONJ and 45 controls (patients without BONJ) and were genotyped for 4 single nucleotide polymorphisms (SNPs): dbSNP ID (rsl934980 (SEQ ID NO: 13), and rsl934951 (SEQ ID NO: 14)) from the CYP2C8 gene; dbSNP ID (rsl 800012) (SEQ ID NO: 15) from the COL1A1 gene, and dbSNP ID (rsl 24581 17) (SEQ ID NO:l), from the TNFRSFl 1 A gene.
• SNPs single nucleotide polymorphisms
• VAMC Administration Medical Center
• BONJ bisphosphonate
• the median length of treatment with bisphosphonate (BP) before the diagnosis of BONJ was 28 months.
• BONJ involved the mandible in 21 patients, maxilla in 4 and both in 2 patients. The most frequent presentation was pain, swelling, and exposed bone (FIG. 1 ).
• the BONJ incidence differed among the 3 centers where these patients were treated.
• the outpatient bone marrow transplant clinic where all patients had myeloma, the total incidence was 13%; while it was 4% for pamidronate only.
• the incidence in the VAMC was 4.2 % while the incidence in the Cancer Center clinic at UF was 1.7 %. In the latter two groups, patients had mostly solid tumors.
• BPs were collected. Whether or not they had documented BONJ was noted. Sixty seven myeloma patients who had been treated with IV BPs were genotyped, of whom 8 had BONJ, using 5 SNPs; the results are shown in Table 2. Patients with BONJ were compared to those without, and a trend toward a higher odds ratio in BONJ patients was observed with 2 of the SNPs: TNFRSF1 1 A (rs 124581 1 7) (SEQ ID NO: l) and MMP2 (rs243865) (SEQ ID NO:2). Carriers of both SNPs had a 50% event rate, while those subjects who did not carry either SNP had an event rate of 7.9%. The odds ratio (95% confidence interval) was 1 1.6 (1.34 - 100.8).
• MMP2 matrix metalloproteina.se 2
• the purpose of the present study was to investigate whether genetic polymorphisms in several genes including CYP2C8, COL1 Al , RANK, OPN. MMP2, OPG and TNF are associated with the risk of developing BONJ in a group of multiple myeloma (MM) patients treated with monthly IV BP.
• MM myeloma
• BONJ was identified by known acceptable criteria and demographic and clinical data on all patients were collected.
• Peripheral blood was used to genotype 10 single nucleotide polymorphisms (SNPs) on 7 genes considered associated with drug metabolism or bone metabolism.
• SNPs single nucleotide polymorphisms
• the adjusted odds ratio was 1 1.2, 95% confidence interval of 1 .8 - 69.9 and P value of 0.0097.
• Smoking, type of BP treatment and combined genotype score of COLA1 , RANK, MMP2, OPG and OPN were significantly associated with BON J in MM patients undergoing IV BP therapy.
• CYP2C8 (rs 1934980 and rs 1934951 ) (SEQ ID NOs:13 and 14. respectively);
• OPN (rsl 1730582 and rs28357094) (SEQ ID NOs:26 and 27, respectively);
• OPG (rs2073618 and rs3102735) (SEQ ID NOs:24 and 25. respectively);
• TNF (rsl 800629) (SEQ ID NO:28).
• Genomic DNA was extracted from lymphocytes in whole blood using a commercially available kit (Qiagen DNA Blood Isolation Kit, Qiagen, Valencia, CA). Polymorphisms were genotyped by Taqman ® genotyping method or pyro sequencing (Langaee et al., Mutat Res, 65(3):369-76. 2007). The Applied Biosystems 7900 HT SNP genotyping platform was used for the Taqman ® assay. The PCR primers and probes for COL1A1 SNP G/T rsl 800012 (ID
• MMP2 SNP C/T rs243865 (ID C_3225943 10) were purchased from Applied Biosystems (Applied Biosystems, Foster City, U SA). Reactions of 5 ⁇ L each in 384-well plate were prepared and the assays were performed and analyzed according to the manufacture's recommendations. Pyrosequencing (Langaee et al., Mutat Res, 65(3):369-76, 2007) (Biotage, Uppsala, Sweden) was performed on the rest of the SNPs using a PSQ HS96A SNP reagent kit (Biotage AB, Uppsala, Sweden) (primers available upon request). Genotype accuracy was verified by genotyping 5-10% randomly selected duplicate samples for each SNP.
• Baseline characteristics were compared by student's t-test or chi-square tests as appropriate. Allele frequencies deviations from Hardy- Weinberg Equilibrium (HWE) were assessed using chi-square tests with one degree of freedom. A dominant mode of inheritance was used in single SNP association analyses. The event rates were compared between carriers and non-carriers using chi-square tests. Logistic regression was used to assess the odds ratio for BONJ, using wild-type homozygous as the reference group.
• HWE Hardy- Weinberg Equilibrium
• BONJ group Total of 78 MM patients including the 12 BONJ patients (15.3%) were enrolled and their characteristics are shown in Table 9. Two groups of patients are compared: BONJ group versus all the other patients receiving IV BP. The median age of the BONJ group and the BP group was similar. Fifty percent of BONJ patients were females, although the majority of the 78 patients were males (61.5%). Most of the BONJ patients were of white origin compared to the BP group. None of the BONJ patients were African Americans. Two out of 3 Hispanic patients in this study were diagnosed with BONJ. However, none of these differences were statistically significant.
• the median time to developing BONJ was 28 months, but that was not different from the median length of BP treatment for the whole group. However, the finding confirms prior published data that BONJ usually develops after more than 2 year BP therapy.
• SNPs for 7 genes have been genotyped in this group.
• the minor allele frequencies and univariate odds ratios for BONJ are shown in Table 11.
• Patients with BONJ were compared to those without, and a trend toward a higher odds ratio in BONJ patients was found with 5 of the SNPs: COL1A1 (rsl800012) (SEQ ID NO: 15); RANK (rsl 24581 17) (SEQ ID NO:l); MMP2 (rs243865) (SEQ ID NO:2); OPG (rs2073618) (SEQ ID NO:24); and OPN (rsl 1730582) (SEQ ID ON:26).
• ABSB1 multi drug resistance gene 1
• This study shows an association between the combined genotype score of COLA 1. RANK, MMP2, OPG and OPN SNPs and the risk of developing ONJ in MM patients receiving IV BP treatment. This finding can be used to modify treatment regimens by identifying patients with these variants with the aim to decrease the incidence of ONJ .

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