WO2000042216A2 - Genetic predisposition to abnormal calcification conditions - Google Patents
Genetic predisposition to abnormal calcification conditions Download PDFInfo
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- WO2000042216A2 WO2000042216A2 PCT/EP2000/000319 EP0000319W WO0042216A2 WO 2000042216 A2 WO2000042216 A2 WO 2000042216A2 EP 0000319 W EP0000319 W EP 0000319W WO 0042216 A2 WO0042216 A2 WO 0042216A2
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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
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- 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
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- This invention relates to a method for assessing predisposition to various conditions based upon polymorphisms in a bone sialoprotein gene, a matrix gla protein gene, an osteopontm gene and/or an osteoprotegerin
- the invention relates to a method of assessing an individual's predisposition to various pathological calcification conditions including osteoporosis and atherosclerosis by screening for these polymorphisms.
- the method of the present invention is especially useful in determining allelic variations m the human bone sialoprotein gene, the human matrix gla protein gene, the human osteopontm gene and/or the osteoprotegerin
- the invention also relates to bone sialoprotein (BSP) genes, matrix gla protein (MGP) genes, ostepeopontm (OPN) genes and OPG/OCIF genes containing the polymorphisms and to probes and primers therefor.
- BSP bone sialoprotein
- MGP matrix gla protein
- OPN ostepeopontm
- Osteoporosis is today one of the most common diseases in individuals over 60 years of age. In America alone it affects an estimated 25 million people with a 5:1 ratio of women to men. This corresponds to approximately 25-30% of people over 60 years of age. In Europe the percentage of people affected by this disease is approximately the same At present there is no cure for osteoporosis. However, hormone replacement therapy as well as treatment with bisphosphonates can halt or slow accelerated bone loss . Hence, the sooner such a bone loss can De diagnosed the better the impact of treatment. It would, accordingly, be of particular advantage to be able to identify individuals predisposed to osteoporosis as early as possible.
- interleukin- 1 receptor antagonist gene Two tandem repeat polymorphisms recently found the interleukin- 1 receptor antagonist gene have been associated with rate of bone loss (Keen et al . , 1998; WO9844150) .
- a polymorphism in the apolipoprote E gene has been reported to be correlated to low BMD m postmenopausal Japanese women (Shiraki et al . , 1997). The reason for focusing on this gene was a report stating that the level of vitamin K which activates osteocalcm through ⁇ - glutamyl carboxylase was related to the apolipoprotem E phenotype (Saupe et al . , 1993) .
- WO9705275 discloses use of analysing for an allelic variant in the retinoic acid receptor gene for the prediction of bone density.
- the vitamin D receptor gene and the collagen type I 1 gene polymorphisms only seem to be associated with BMD in a fraction of the cohorts examined (the other gene polymorphisms have only been tested on small, national cohorts and most of them are rather peripheral to bone metabolism) . Since the genetic influence on the development of osteoporosis is caused by the inadequate action of multiple genes, this comes as no surprise. Obviously, more genetic polymorphisms with an impact on bone formation/ resorption need to be identified to get a better genetic prediction power valid within a wider geographical area.
- the present invention now provides a method of assessing an individual's predisposition to a selected calcification condition status, which method comprises determining the genotype of the promoter of the bone sialoprotein gene, the promoter of the matrix gla protein gene, the promoter of the osteopontm gene, or the promoter of the OPG/OCIF gene or all four or any combination of two or more out of the four promoters .
- the calcification condition status for which a predisposition is assessed according to the invention may be having a high or a low peak bone mass (as a future, present or as a past state) or having a high rate or a low rate of bone loss (as a future, present or past state) .
- the invention may be used to assess a predispostion to osteoporosis.
- the present invention provides a method of assessing predisposition of an individual to any condition associated with allelic variation of a said promoter or any such combination thereof.
- the method of the invention typically comprises determining whether an individual is homozygous or heter- ozygous for a bone sialoprotein promoter (BSP) , a matrix gla protein promoter (MGP) , an osteopontm promoter (OPN) , or an OPG/OCIF promoter or all four promoters or combinations of two or more out of these four promoters and particular polymorphisms thereof.
- BSP bone sialoprotein promoter
- MGP matrix gla protein promoter
- OPN osteopontm promoter
- OPG/OCIF promoter all four promoters or combinations of two or more out of these four promoters and particular polymorphisms thereof.
- a DNA sequence of the human bone sialoprotein promoter is known and has been published by Kim, R.H. et al . m Matrix Biol. 14: 31-40 (1994). The sequence submitted to GenBank by this group with accession # L24756 is referred to hereafter as the wild type sequence or the published sequence.
- a DNA sequence of the human matrix gla protein promoter is known and has been published by Cancela, L. et al. in J. Biol. Chem. 265 (25): 15040-15048 (1990). The sequence submitted to GenBank by this group with accession
- # M55270 is referred to hereafter as the wild type sequence or the published sequence.
- a DNA sequence of the human osteopontm promoter is known and has been published by Hijiya et al . in Biochem J., 303: 255-262 (1994). The sequence submitted to GenBank by this group with accession
- OPG/OCIF promoter is known and has been published by Mo ⁇ naga et al . , Eur. J. Biochem. 254 (3) : 658-691 (1998).
- the sequence submitted to GenBank by this group with accession #AB008821 is referred to hereafter as the wild type sequence or the published sequence This terminology is not intended to imply that any of these published sequences is more prevalent m the population than variations thereof or that each or any of them is associated with the minimum risk of pathology.
- the method of the invention includes determining whether the individual being tested has a bone sialoprotein promoter, a matrix gla protein promoter, an osteopontm promoter, or an OPG/OCIF promoter or all four or combinations of two or more out of these four promoters which are identical with the published sequences (or are identical at selected regions of said sequences) or whether that individual has a bone sialoprotein promoter, a matrix gla protein promoter, an osteopontm promoter, or an OPG/OCIF promoter or all four or combinations of two or more out of these four which differ from the published sequences (or which differ at said selected locations), i.e. are polymorphisms of the published sequences, whether homozygous or heterozygous.
- the invention includes a method as described above m which one determines the sequence at location 1496 bp of the BSP, in particular whether the sequence at this location is A (published) or G, and/or at the location 1869 bp, and particular whether the sequence at said location is G (published) or A.
- the locations identified above are numbered from the start of the published sequence. In an alternative numbering scheme, these locations are -683 bp and -310 bp from the start of the transcribed sequence of the gene.
- these specific allelic variations are indicated using the terminology BSP-A1496G and BSP-G1869A.
- the invention includes such a method which said allelic variation is at MGP location 242 bp
- MGP-C242A (published) or A, referred to hereafter as MGP-C242A.
- the invention includes such a method in which said allelic variation is at OPN location 520 bp
- OPN-G520A (published) or A, referred to hereafter as OPN-G520A.
- the invention includes such a method in which said allelic variation is at OPN location 1825 bp (numbered from the start of the published sequence or -443 bp from the start of the transcribed sequence) and m particular whether the sequence at said location reads T
- OPN-T1825C (published) or C, referred to hereafter as OPN-T1825C.
- the invention includes a method as described above in which one determines the sequence at location 163 bp of the OPG/OCIF promoter, m particular whether the sequence at this location is A (published) or G.
- the location specified above is numbered from the start of the published sequence. In an alternative numbering scheme, this location is -943 bp from the start of the transcribed sequence of the gene.
- this specific allelic variation is indicated using the terminology OPG-A163G.
- the relevant determinations of gene promoter sequences can be carried out by generally known methods, which generally involve amplifying a relevant portion of the DNA of a said gene promoter of said individual .
- the sequence of said amplified portion may be determined by hybridisation assay or by restriction fragment length analysis.
- amplification may be conducted using a promoter chosen such that if a selected one of the published sequences or the variation of the published sequence is present, amplification will produce a new site at which the amplicon will be cut by a restriction enzyme. A different number of restriction fragments will thus be produced by enzyme treatment of the amplicon.
- the invention includes an oligonucleotide primer for use m amplification of a relevant portion of a said gene promoter.
- the invention includes such a promoter selected so as to produce a different restriction pattern depending on the presence or absence of a selected variation. Suitable promoters according to the invention are described in the examples hereafter.
- the invention includes a method of osteoporosis therapy comprising determining a predisposition as described above, and administering a medicament to the individual to prevent or treat osteoporosis or to delay the onset of osteoporosis if the individual is predisposed to low peak bone mass or to a high rate of loss of bone mass.
- Bone sialoprotein is a bone tissue specific 33-34 kDa nascent protein which is extensively modified post- translationally by glycosylation, phosphorylation and sulfation leading to a final MW of 57 kDa (Oldberg et al . , 1988; Ecarot-Charrier et al . , 1989; Fisher et al . , 1990; Zhang et ⁇ al . , 1990). Together with osteopontin, BSP is the most abundant non-collagenous protein in the bone matrix (Nagata et al . , 1991).
- Matrix gla protein is a small 79 ammo acid residues protein with molecular weight of appr. 14 kDa which contains five ⁇ -carboxyglutamic acid (gla) residues (Price & Williamson, 1985; Loeser & Wallm, 1992) .
- the gla residues are presumably products of a post-translational modification by the vitamin K dependent enzyme ⁇ - carboxylase .
- MGP strongly binds to hydroxyapatite m a gla dependent fashion (Dowd et al . , 1995). High levels of MGP are found m the extracellular matrix of bone, dentm and cartilage (Hale et al . , 1988).
- MGP is expressed in many tissues, with the highest levels of mRNA found in lung, heart, kidney and cartilage (Fraser & Price., 1988).
- a first indication of the function of MGP in bone came from experiments with rats treated with the ⁇ -carboxylase inhibitor warfarin. These animals showed excessive mineralization of the growth plate, indicating that one function of MGP in bone and cartilage could be inhibition of hydroxyapatite formation (Price et al . , 1982).
- Final proof for this function has come from a recent study on MGP knockout mice, which die within 2 months after birth as a result of arterial calcification leading to blood-vessel rupture.
- MGP deficient mice also display inappropriate calcification of the growth plate cartilage, where calcification has extended into the zone of proliferating chondrocytes rather than being restricted to the lower hypertrophic zone as observed in normal animals.
- the abnormal calcification led to a disorganization of chondrocyte columns, eventually resulting m short stature, osteopenia and fractures (Luo et al . , 1997).
- MGP functions to inhibit calcification in soft tissues and restrict mineralization within the growth plate cartilage to the lower hypertrophic zone - the latter possibly by inhibiting calcification in the underlying area of proliferating chondrocytes .
- Osteopontm is a phosphorylated and glycosylated protein of 44 kDa (Prince et al . , 1987). Together with BSP, OPN is the most abundant non-collagenous protein in the bone matrix (Nagata et al . , 1991), but it is, unlike BSP, also expressed in several other tissues (Denhardt & Guo 1993) . Osteopontm and BSP are clearly related: 1) they both have an RGD domain that mediates cell attachment via c v ⁇ 3 mtegrm class of cell surface receptor (Ross et al . , 1993; Wong et al . , 1996), and 2) they both have a high content of acidic ammo acids (OPN has poly-aspartic acid segments and BSP has poly-glutamic acid segments) and sialic acid (Franzen & Hemegard) .
- OPN has poly-aspartic acid segments and BSP has poly-gluta
- Phosphorylated osteopontm is a potent inhibitor of hydroxyapatite formation while the dephosphorylated form is far less potent (Hunter et al . , 1994).
- bone osteopontm is found at high concentrations in the lamma limitans that underlies bone lining cells and in reversal (cement) lines found at matrix-matrix interfaces where bone deposition has been preceded by a resorptive event (McKee & Nanci 1996; McKee et al . , 1993).
- osteopontm may act to seal off growing hydroxyapatite surfaces once active bone formation has ceased, as speculated by Hunter et al . , 1994.
- osteopontin knockout mice show normal development and bone structure but osteoclast formation is enhanced in vi tro (Rittling et al . , 1998) and osteoclast numbers are higher in epiphyseal regions in OPN -/- than in wild type mice (Yoshitake et al . , 1998).
- Atherosclerosis Another condition, which has been suggested to be promoted by matrix gla protein and osteopontin is atherosclerosis (Shanahan et al . , 1994; Sohma et al . , 1994) .
- WHHL Watanabe heritable hyperlipidemic
- Osteoprotegerin (OPG) /osteolastogenesis inhibitory factor (OCIF) was recently identified independently by two groups as a 380 amino acid residue long glycoprotein with a molecular weight of approximately 55 kD related to the tumor necrosis factor receptor (TNF-R) superfamily (Simonet et al., 1997: Yasuda et al . , 1997). Unlike the other TNF- R-like molecules this cytokine receptor lacks a trans- membrane domain (Simonet et al . , 1997). Accordingly, OPG/OCIF is a secreted protein appearing as a disulfide linked homodimer with a molecular weight of approximately 110 kD (Simonet et al . 1997).
- mice transgenic for rat OPG/OCIF were found to develop osteopetrosis, which indicated that OPG/OCIF either could function to increase osteoblast-mediated bone formation or to decrease osteoclast-mediated bone resorption (Simonet et al . , 1997) .
- OPG/OCIF was found to be a potent inhibitor of osteoclastogenesis (Simonet et al., 1997).
- OPG/OCIF knockout mice develop osteoporosis, emphasising that OPG/OFIC is an important regulator of postnatal bone mass (Bucay et al . , 1998) .
- OPG/OCIF is highly expressed in cartilage of developing bones, as well as m several major arteries, the gastrointestinal tract, and skin (Simonet et al . , 1997).
- OPG/OCIF expression is found in several tissues including heart, brain, lung and liver, which is in contrast to human tissue where OPG/OCIF expression is absent in brain and liver, highly expressed in kidney and detectable in various hematopoietic and immune organs (Simonet et al . , 1997) . No explanations have been offered on these observations, except that it could be due to true species-specific expression differences (Simonet et al . , 1997).
- the ligand for OPG/OCIF has also been identified independently by the same two groups, who identified
- OPG/OCIF The ligand, called OPG ligand (OPGL) (Lacey et al., 1998) or osteoclast differentiation factor (ODF )
- OPGL/ODF is a TNF-related cytokine, which binds to a hematopoietic progenitor cell committed to the osteoclast lineage and stimulates its differentiation into an osteoclast (Lacey et al . , 1998).
- OPGL/ODF also stimulates mature osteoclasts to resorb bone and recombinant OPGL/ODF injected subcutaneously stimulates bone resorption in mice (Lacey et al . , 1998).
- OPGL/ODF is produced either as a 45 kDa membrane bound protein or as a 31 kDa soluble, secreted C-terminal fragment (Lacey et al . , 1998) .
- OPGL/ODF which is identical to two previously identified cytokines, TNF-related activation- induced cytokine (TRANCE) (Wong et al . , 1997) essential for T-cell activation and receptor activator of NF-kB ligand (RANKL) essential for dendritic cell activation (Anderson et al . , 1997) , is highly expressed in lymphoid tissues and trabecular bone (Lacey et al . , 1998: Yasuda et al . , 1998). Since OPG/OCIF binds to and inhibits the action of OPGL/ODF, these two proteins are seemingly important extra- cellular regulators of osteoclast development and, thus, eventually bone resorption.
- TRANCE TNF-related activation- induced cytokine
- RNKL NF-kB ligand
- OPG/OCIF knockout mice Apart from an osteoporotic phenotype OPG/OCIF knockout mice also displayed a marked calcification of the aorta and renal arteries by 2 months of age (Bucay et al . , 1998) . Thus, OPG/OCIF inhibits decalcification of the skeleton and at the same time inhibits calcification of certain blood vessels. A similar phenomenon has been observed previously in matrix gla protein (MGP) knockouts (Luo et al . , 1997). However, the arterial calcification is more disseminated and pronounced m the MGP knockouts, while the bone loss is more severe in the OPG/OCIF knockouts.
- MGP matrix gla protein
- the method of assessing an individual, predisposition to osteoporosis or other calcification condition related diseases described above may be combined with measurements of bone mass on a whole body or selected location basis. These include X-ray or ultrasound BMD measurements.
- the methods described herein may also or instead be combined with measurements of chemical bone resorption markers such as the CrosslapsTM measurement of C-telopeptide fragments of Type 1 collagen or measurements of N-telopeptide fragments of Type 1 collagen in body fluids such as serum or urine.
- Each of these types of measurement may be treated as a risk factor to be combined m a weighted manner with the one or more of the others (one of course being a genetic predisposition measurement according to this invention) .
- FIG. 1 panel A shows the location of the two polymorphisms, called BSP-A1496G and BPS-G1869A, in the bone sialoprotein gene promoter.
- Panel B shows the location of the polymorphism, called MGP-C242A, in the matrix gla protein gene promoter.
- Panel C shows the location of the polymorphisms, called OPN-G520A and OPN-T1825C, in the osteopont gene promoter.
- nucleotide sequences encompassing the four polymorphic sites for all three said promoters are shown with the nucleotide at the polymorphic position in bold and with the substituting nucleotide also in bold - positioned above the polymorphic site. All nucleotide numbering is relative to base pair number 1, which is the most 5' nucleotide of each of the promoter sequences as published in the GenBank nucleotide database.
- Figure 2 shows the time dependence of the mean BMCs measured at the distal arm when grouped into two genotypes for the BSP-A1496G polymorphic site.
- the BMCs were determined at four time points - 1977, 1979, 1989, and 1995 from each individual out of 133 in the 18 years study. Each point on the curves is the mean BMC for a given year and a given genotype with SEM error bars.
- the table below the chart lists the actual BMC values determined 1977, 1979, 1989, and 1995 for the two genotype groups as well as difference - in percent - between the two genotype groups for each of the four sampling years.
- Figure 3 shows the time dependence of the mean BMCs measured at the distal arm when grouped into two genotypes for the BSP-G1869A polymorphic site.
- the BMCs were determined at four time points - 1977, 1979, 1989, and 1995 from each individual out of 133 in the 18 years study. Each point on the curves is the mean BMC for a given year and a given genotype with SEM error bars.
- the table below the chart lists the actual BMC values determined in 1977, 1979, 1989, and 1995 for the two genotype groups as well as difference - in percent - between the two genotype groups for each of the four sampling years.
- Figure 4 shows time dependence of the mean BMCs measured at the distal arm when grouped into two genotypes for a combination of the BSP-A1496G and BSP-G1869A polymorphic sites.
- the upper curve represents the combination: BSP-A1496G heterozygous/homozygous polymorphic and BSP-G1869A wild type while the lower curve represents the combination: BSP-A1496G wild type and BSP-G1869A heterozygous/homozygous polymorphic.
- the BMCs were determined at four time points - 1977, 1979, 1989, and 1995 from each individual out of 133 in the 18 years study. Each point on the curves is the mean BMC for a given year and a given genotype with SEM error bars.
- FIG. 5 shows the time dependence of the mean BMCs measured at the distal arm when grouped into two genotypes for the MGP-C242A polymorphic site.
- the BMCs were determined at four time points - 1977, 1979, 1989, and 1995 from each individual out of 133 m the 18 years study. Each point on the curves is the mean BMC for a given year and a given genotype with SEM error bars.
- the table below the chart lists the actual BMC values determined in 1977, 1979, 1989, and 1995 for the two genotype groups as well as difference - in percent - between the two genotype groups for each of the four sampling years.
- Figure 6 shows the time dependence of the mean BMCs measured at the distal arm when grouped into two genotypes for the OPN-G520A polymorphic site.
- the BMCs were determined at four time points - 1977, 1979, 1989, and 1995 from each individual out of 133 in the 18 years study. Each point on the curves is the mean BMC for a given year and a given genotype with SEM error bars.
- the table below the chart lists the actual BMC values determined in 1977, 1979, 1989, and 1995 for the two genotype groups as well as difference - in percent - between the two genotype groups for each of the four sampling years.
- Figure 7 shows the time dependence of the mean BMCs measured at the distal arm when grouped into two genotypes for a combination of the MGP-C242A and OPN-G520A polymorphic sites.
- the BMCs were determined at four time points - 1977, 1979, 1989, and 1995 from each individual out of 133 in the 18 years study. Each point on the curves is the mean BMC for a given year and a given genotype with SEM error bars.
- the table below the chart lists the actual BMC values determined in 1977, 1979, 1989, and 1995 for the two genotype groups as well as difference - in percent - between the two genotype groups for each of the four sampling years.
- Figure 8 shows the time dependence of the mean BMCs measured at the distal arm when grouped into two genotypes for the OPN-T1825C polymorphic site.
- the BMCs were determined at four time points - 1977, 1979, 1989, and 1995 from each individual out of 133 m the 18 years study. Each point on the curves is the mean BMC for a given year and a given genotype with SEM error bars.
- the table below the chart lists the actual BMC values determined m 1977, 1979, 1989, and 1995 for the two genotype groups as well as difference - in percent - between the two genotype groups for each of the four sampling years.
- Figure 9 shows the time dependence of the mean BMCs measured at the distal arm when grouped into two genotypes for a combination of the BSP-G1869A and OPN-T1825C polymorphic sites.
- the BMCs were determined at four time points - 1977, 1979, 1989, and 1995 from each individual out of 133 in the 18 years study. Each point on the curves is the mean BMC for a given year and a given genotype with SEM error bars.
- the table below the chart lists the actual BMC values determined in 1977, 1979, 1989, and 1995 for the two genotype groups as well as difference - percent - between the two genotype groups for each of the four sampling years.
- Figure 10 shows the location of the OPG-A163G polymorphism m the OPG/OCIF gene promoter.
- the wild type sequence encompassing the polymorphic site for said promoter is shown with the nucleotide at the polymorphic position m bold and with the substituting nucleotide - also in bold - positioned above the polymorphic site. All nucleotide numbering is relative to base pair number 1, which is the 5' most nucleotide of the promoter sequence as published in the GenBank nucleotide database.
- Figure 11 shows the time dependence of the mean BMCs measured at the distal arm when grouped into two genotypes for the OPG-A163G polymorphic site.
- the BMCs were determined at four time points - 1977, 1979, 1989 and 1995 from each out of 133 individuals in the 18 years study. Each point on the curves is the mean BMC for a given year and a given genotype with SEM error bars .
- the table below the chart lists the actual BMC values determined in 1977, 1979, 1989 and 1995 for the two genotype groups as well as difference - in percent - between the two genotype groups for each of the four sampling years.
- Figure 12 shows time dependence of the mean BMCs measured at the distal arm when grouped into two genotypes for a combination of the BSP-A1496G and OPG-A163G polymorphic sites.
- the upper curve represents the combination: BSP-A1496G heterozygous/homozygous polymorphic and OPG-A163G wild type while the lower curve represents the combination: BSP-A1496G wild type and OPG-A163G heterozygous/homozygous polymorphic.
- the BMCs were determined at four time points - 1977, 1979, 1989 and 1995 from each out of 133 individuals in the 18 year study. Each point on the curves is the mean BMC for a given year and a given genotype with SEM error bars .
- the table below the chart lists the actual BMC values determined m 1977, 1979, 1989 and 1995 for the two genotype groups as well as difference - in percent - between the two genotype groups for each of the four sampling years.
- Figure 13 shows time dependence of the mean BMCs measured at the distal arm when grouped into two genotypes for a combination of the BSP-G1869A and OPG-A163G polymorphic sites.
- the upper curve represents the combination: BSP-A1496G homozygous polymorphic and OPG- A163G wild type while the lower curve represents the combination: BSP-G1869A wild type/heterozygous and OPG- A163G heterozygous/homozygous polymorphic
- the BMCs were determined at four time points - 1977, 1979, 1989 and 1995 from each out of 133 individuals m the 18 year study. Each point on the curves is the mean BMC for a given year and a given genotype with SEM error bars .
- the table below the chart lists the actual BMC values determined m 1977, 1979, 1989 and 1995 for the two genotype groups as well as difference - in percent - between the two genotype groups for each of the four sampling years.
- PCR polymerase chain reaction
- PCR techniques are well known in the art and it would be within the ambit of a person of ordinary skill in this art to identify primers for amplifying a suitable section of the BSP, MGP and OPN genes including the positions 1496bp and 1869bp in the BSP promoter, the position 242bp in the MGP promoter, and the positions 520bp and 1825bp in the osteopontin promoter.
- PCR techniques are described for example in patents US4683202 or EP0200362B1.
- A1496G and BSP-G1869A polymorphisms or 49°C (MGP-C242A polymorphism) or 46°C (OPN-G520A polymorphism) or 48°C
- BSP-A1496G polymorphism primer set Forward primer : 5 ' - GAA AAG ATA TAT ATA GAA GCC CAA G - 3 ' (SEQ ID No. 1) Reverse primer : 5 ' - TAA TAT CAT TTG ATG TTT CCT CCT G - 3 ' (SEQ ID No. 2)
- BSP-G1869A polymorphism primer set Forward primer: 5'- TTC TTT CGA CAT AGT GAA AAC ACG T - 3' (SEQ ID No. 3)
- Reverse primer 5'- CGT GGA TTC TCA CCA GAA AAC - 3' (SEQ ID No.4)
- MGP-C242A polymorphism primer set Forward primer : 5 ' - CAG TGA GAA AGC TCA TCA CTT GGT C - 3 ' (SEQ ID No. 5)
- Reverse primer 5'- ATT CTC CCA TCC ATC CAT CCA TGC A - 3' (SEQ ID No. . 6)
- OPN-G520A polymorphism primer set Forward primer : 5 ' - CGC TGG AAT TAA GAA AAT TGG TAG A - 3 ' (SEQ ID No. 7)
- Reverse primer 5'- GTT GTC AAT TTA GTG GAG GGA GAT C - 3' (SEQ ID No. 8)
- OPN-T1825C polymorphism primer set Forward primer : 5 ' - GAG TAG TAA AGG ACA GAG GCG AGC T - 3 ' (SEQ ID No. 9)
- Reverse primer 5' - CTA GCT TTT TCA TTT ACG GGA TGG G - 3' (SEQ ID No. 10)
- DNA fragments PCR amplified using the BSP-A1496G polymorphism primer set were restricted with Eco T14 I in a 20 ⁇ l reaction containing: lx buffer H (Amersham Pharmacia) , 4 units of Eco T14 I (Amersham Pharmacia) and 5 ⁇ l of the cycled PCR reaction.
- the reaction mixture was incubated at 37°C for 1 hour.
- Four ⁇ l 6xgel-loading buffer 0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol in water) were added to the 20 ⁇ l Eco T14 I digest and loaded on a 2.5% agarose gel.
- DNA fragments were then resolved by electrophoresis until the bromophenol blue marker had run 2/3 through the gel. If the DNA sample analyzed was homozygous for the wild type BSP sequence one band of 270 bp would be observed. If the DNA sample analyzed was heterozygous two bands of 270 bp and 245 bp would be observed. If the DNA sample was homozygous for the polymorphism one band of 245 bp would be observed. DNA fragments PCR amplified using the BSP-G1869A polymorphism primer set were restricted with Eco 72 I m a
- DNA fragments PCR amplified using the MGP-C242A polymorphism primer set were restricted with Eco T22 I in a 20 ⁇ l reaction containing: lx buffer H (Amersham Pharmacia) , 4 units of Eco T22 I (Amersham Pharmacia) and 5 ⁇ l of the cycled PCR reaction.
- the reaction mixture was incubated at 37°C for 1 hour.
- Four ⁇ l 6xgel- loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol in water) were added to the 20 ⁇ l Eco T22 I digest and loaded on a 2.5% agarose gel. DNA fragments were then resolved by electrophoresis until the bromophenol blue marker had run 2/3 through the gel.
- DNA fragments PCR amplified using the OPN-G520A polymorphism primer set were restricted with Bgl II in a 20 ⁇ l reaction containing: lx buffer H (Amersham Pharmacia), 4 units of Bgl II (Amersham Pharmacia) and 5 ⁇ l of the cycled
- PCR reaction The reaction mixture was incubated at 37°C for 1 hour.
- Four ⁇ l 6xgel-loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol in water) were added to the 20 ⁇ l Bgl II digest and loaded on a 2.5% agarose gel . DNA fragments were then resolved by electrophoresis until the bromophenol blue marker had run 2/3 through the gel. If the DNA sample analyzed was homozygous for the wild type OPN sequence one band of 278 bp would be observed. If the DNA sample analyzed was heterozygous two bands of 278 bp and 257 bp would be observed. If the DNA sample was homozygous for the polymorphism one band of 257 bp would be observed.
- DNA fragments PCR amplified using the OPN-T1825C polymorphism primer set were restricted with Sac I in a 20 ⁇ l reaction containing: lx buffer L (Amersham Pharmacia) , 4 units of Sac I (Amersham Pharmacia) and 5 ⁇ l of the cycled PCR reaction.
- the reaction mixture was incubated at 37°C for 1 hour.
- Four ⁇ l 6xgel-loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol in water) were added to the 20 ⁇ l Sac I digest and loaded on a 2.5% agarose gel . DNA fragments were then resolved by electrophoresis until the bromophenol blue marker had run 2/3 through the gel.
- DNA sample analyzed was homozygous for the wild type OPN sequence one band of 256 bp would be observed. If the DNA sample analyzed was heterozygous two bands of 256 bp and 235 bp would be observed. If the DNA sample was homozygous for the polymorphism one band of 235 bp would be observed.
- the BSP-A1496G, BSP-G1869A, MGP-C242A, OPN-G520A, and OPN-T1825C polymorphisms were coded as Xx, Yy, Zz, Bb, and Ss, respectively, where the uppercase letter signifies presence of the wild type base pair at the given polymorphic position and the lowercase letter signifies presence of the base pair different from the wild type base pair at the given polymorphic position.
- Table 1 shows the genotype distribution of DNA samples from the 18 years study for all identified polymorphic sites: BSP-A1496G, BSP-G1869A, MGP-C242A, OPN-G520A, and OPN-T1825C.
- the left panel shows the actual number of samples categorized into three genotypes for the 3 identified polymorphic sites.
- the right panel displays the same analysis as the left except that the numbers represent the percent of total samples analyzed for each polymorphic site .
- OPN-G520A OPN-G520A 10.3 39.7 50.0 100.0
- the genotype distributions for the 5 polymorphisms are shown in table 1.
- the homozygous wild type genotype was the most abundant, followed by the heterozygous and homozygous polymorphic genotypes, with the homozygous polymorphic groups being quite small.
- the wild type genotype as defined by the BSP gene promoter sequence from GenBank, was rare, the heterozygous genotype was 10 times more frequent and the homozygous polymorphic genotype was twice as frequent as the heterozygous.
- the heterozygous genotype was the most abundant followed by the homozygous wild type and homozygous polymorphic genotypes.
- the homozygous polymorphic genotype was the most abundant followed by the heterozygous and the wild type homozygous genotypes.
- the genotype distribution of the OPN-T1825C polymorphism was, generally, the same as for the MGP-C242A polymorphism.
- Table 2 shows a compilation of a statistical analysis of the results obtained from screening of the DNA samples from the 18 years study for the presence of either of the 5 identified polymorphisms.
- Panel A The numbers represent the likelihood that the difference in mean BMD between groups of different genotype (homozygous wild type or heterozygous/homozygous polymorphic) are identical. The test includes BMC (bone mineral content) and BMD measured at the distal arm. Numbers parentheses represent the year of BMC/BMD measurement.
- Panel B The numbers represent the difference between the mean BMDs of two genotype groups (heterozygous/homozygous polymorphic group subtracted from the homozygous wild type group) m percent of the highest BMD for a given polymorphic site.
- Figures 2 and 3 show that this is, indeed, the case for the BSP-A1496G and BSP-G1869A polymorphic sites. Moreover, the two BSP promoter polymorphisms act in concert on peak bone mass to augment the mean BMD difference between genotypes even more than the isolated contribution of each polymorphism ( Figure 4) .
- the reaction was heated to 95°C for 9 minutes followed by 35 cycles of 95°C for 30 seconds, 46°C for 30 seconds and 72°C for 30 seconds - the latter incubation with a 5 second time extension per cycle. The reaction was finally incubated 7 minutes at 72 °C for completion of the extension reaction.
- the primer sequences for this PCR amplification were: OPG-A163G polymorphism primer set:
- Reverse primer 3 ' -AGT TAG AGC CAG AGA GAA TCT G-3' (SEQ ID No. 12)
- restriction enzyme analyses were performed as follows: DNA fragments PCR amplified using the OPG-C163A polymorphism primer set were restricted with Mfe I in a 20 ⁇ l reaction containing: 1 x NEBuffer 4 (New England Biolabs) , 4 units of Mfel (New England Biolabs) and 5 ⁇ l of the cycled PCR reaction. The reaction mixture was incubated at 37°C for 1 hour.
- OPG-A163G A previously unknown polymorphism, OPG-A163G, was identified by sequencing the promoter region from the human OPG/OCIF gene promoter, following a PCR amplification of 40 DNA samples from healthy women.
- the OPG-A163G polymorphism was coded as Mm, where the uppercase letter signifies presence of the wild type base pair at the given polymorphic position and the lowercase letter signifies presence of the base pair different from the wild type base pair at the given polymorphic position.
- the position of the polymorphism is depicted in Figure 10.
- the 133 DNA samples from the 18 year study were screened for the presence of the OPG-A163G polymorphism.
- the genotype could not be determined in 4 out of the 133 DNA samples.
- the impact of the identified polymorphic site on bone mass as represented by bone mineral content (BMC) and bone mineral density (BMD) measurements at the distal arm in 1977 and 1995, respectively, was analysed (Table 3) .
- OPG- A163G/BSP-A1496G and OPG-A163G/BSP-G1869A showed that those polymor-phisms certainly act in a co-operative fashion, in that the t-test p-values for the null -hypothesis (i.e. no difference between the genotype groups) dropped to statistically significant values and the percent difference in mean BMC/BMD values for two genotype groups increased (Table 3) .
- OPG-A163G polymorphism is a good site for predicting whether an individual is genetically predisposed for high or low BMC/BMD.
- Age, height and weight of the individuals involved in the 18 year study did not differ significantly between any of the genotype groups (data not shown) .
- the combination of the OPG-A163G and the BSP-A1496G polymorphisms show that they act in concert on peak bone mass to augment the mean BMD difference between genotypes even more than the isolated contribution of each polymorphism ( Figure 12) .
- this co-operation is completely additive (Table 4) , indicating that the two polymorphisms act on bone mass independently of one another.
- the numbers for the BSP-A1496G and BSP-G1869A polymorphisms in Table 4 are from the results presented in Figures 2 and 3.
- the combination of the OPG-A163G and BSP-G1869A polymorphisms indicates a positive cooperation ( Figure 13) , which is almost additive (Table 4) .
- Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell , 93:165-76.
- Kalachikov S Cayani E, Bartlett FS 3 rd , Frankel W ⁇ , Lee
- TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun ⁇ - terminal kinase in T cells. J Bio . Chem, 272:25190-4.
- Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA, 95:3597-602.
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AU31492/00A AU3149200A (en) | 1999-01-18 | 2000-01-17 | Genetic predisposition |
EP00909076A EP1144686A2 (en) | 1999-01-18 | 2000-01-17 | Genetic predisposition to abnormal calcification conditions |
JP2000593773A JP2005517379A (en) | 1999-01-18 | 2000-01-17 | Genetic predisposition |
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GBGB9901037.3A GB9901037D0 (en) | 1999-01-18 | 1999-01-18 | Genetic predisposition |
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GBGB9912585.8A GB9912585D0 (en) | 1999-05-28 | 1999-05-28 | Genetic predisposition |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001023562A2 (en) * | 1999-09-27 | 2001-04-05 | Eli Lilly And Company | Osteoprotegerin regulatory region |
US10073101B2 (en) | 2007-03-30 | 2018-09-11 | Chu Sainte-Justine | Methods for the prevention or treatment of scoliosis |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0705842A2 (en) * | 1994-10-06 | 1996-04-10 | Hoechst Aktiengesellschaft | Regulated genes by stimulation of chondrocytes with 1L-1beta |
WO1997003555A1 (en) * | 1995-07-17 | 1997-02-06 | Hoechst Pharmaceuticals & Chemicals K.K. | Transgenic animal model of osteopenia |
EP0784093A1 (en) * | 1995-12-22 | 1997-07-16 | Amgen Inc. | Osteoprotegerin |
-
2000
- 2000-01-17 WO PCT/EP2000/000319 patent/WO2000042216A2/en not_active Application Discontinuation
- 2000-01-17 EP EP00909076A patent/EP1144686A2/en not_active Withdrawn
- 2000-01-17 AU AU31492/00A patent/AU3149200A/en not_active Abandoned
- 2000-01-17 JP JP2000593773A patent/JP2005517379A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0705842A2 (en) * | 1994-10-06 | 1996-04-10 | Hoechst Aktiengesellschaft | Regulated genes by stimulation of chondrocytes with 1L-1beta |
WO1997003555A1 (en) * | 1995-07-17 | 1997-02-06 | Hoechst Pharmaceuticals & Chemicals K.K. | Transgenic animal model of osteopenia |
EP0784093A1 (en) * | 1995-12-22 | 1997-07-16 | Amgen Inc. | Osteoprotegerin |
Non-Patent Citations (5)
Title |
---|
BRANDSTROM, H. (1) ET AL: "Polymorphism in the promoter region of the human gene for osteoprotegerin: Correlation with bone mineral density." JOURNAL OF BONE AND MINERAL RESEARCH, (SEPT., 1999) VOL. 14, NO. SUPPL. 1 PP. S334. MEETING INFO.: TWENTY-FIRST ANNUAL MEETING OF THE AMERICAN SOCIETY FOR BONE AND MINERAL RESEARCH ST. LOUIS, MISSOURI, USA SEPTEMBER 30-OCTOBER 4 1999 AMERICAN SOCIETY, XP000915449 * |
BUCAY N ET AL: "osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification" GENES AND DEVELOPMENT,US,COLD SPRING HARBOR LABORATORY PRESS, NEW YORK, vol. 12, no. 9, 1 May 1998 (1998-05-01), pages 1260-1268, XP002090118 ISSN: 0890-9369 cited in the application * |
KIM R H ET AL: "Identification of a vitamin D3-response element that overlaps a unique inverted TATA box in the rat bone sialoprotein gene." BIOCHEMICAL JOURNAL, (1996 AUG 15) 318 ( PT 1) 219-26. , XP002142619 * |
LUO: "Spontaneous calcification of arteries and cartilage in mice lacking GLA protein" NATURE, vol. 386, 6 March 1996 (1996-03-06), pages 78-81, XP002142620 cited in the application * |
MUNROE P B ET AL: "Mutations in the gene encoding the human matrix Gla protein cause Keutel syndrome." NATURE GENETICS, (1999 JAN) 21 (1) 142-4. , XP000929109 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001023562A2 (en) * | 1999-09-27 | 2001-04-05 | Eli Lilly And Company | Osteoprotegerin regulatory region |
WO2001023562A3 (en) * | 1999-09-27 | 2002-01-24 | Lilly Co Eli | Osteoprotegerin regulatory region |
US10073101B2 (en) | 2007-03-30 | 2018-09-11 | Chu Sainte-Justine | Methods for the prevention or treatment of scoliosis |
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EP1144686A2 (en) | 2001-10-17 |
JP2005517379A (en) | 2005-06-16 |
WO2000042216A3 (en) | 2000-11-02 |
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