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WO1997005275A1 - Procede de prediction de la densite osseuse - Google Patents

Procede de prediction de la densite osseuse Download PDF

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Publication number
WO1997005275A1
WO1997005275A1 PCT/AU1996/000474 AU9600474W WO9705275A1 WO 1997005275 A1 WO1997005275 A1 WO 1997005275A1 AU 9600474 W AU9600474 W AU 9600474W WO 9705275 A1 WO9705275 A1 WO 9705275A1
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WIPO (PCT)
Prior art keywords
rar
gene
vdr
bone density
mean
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PCT/AU1996/000474
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English (en)
Inventor
Nigel Alexander Morrison
John Allan Eisman
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Garvan Institute Of Medical Research
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Application filed by Garvan Institute Of Medical Research filed Critical Garvan Institute Of Medical Research
Priority to AU65103/96A priority Critical patent/AU6510396A/en
Publication of WO1997005275A1 publication Critical patent/WO1997005275A1/fr

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

Definitions

  • the present invention relates to a method of identifying allelic variations in trans-acting regulators as a means of identifying individuals at risk of suffering from an adverse pathophysiological condition.
  • the method of the present invention is particularly useful in assessing allelic variations in a retinoic acid receptor (RAR) gene(s) and thereby predicting predisposition to low or high bone density.
  • RAR retinoic acid receptor
  • the invention relates to the use of diagnostic kits for the discrimination of individuals with different set points of physiological character associated with the RAR regulatory system and to the discrimination of bone density traits associated with health and disease.
  • the invention also relates to the identification of individuals at low or high risk of osteoporosis.
  • any genetic variation in the TAR gene which leads to functionally different TAR gene variants can produce a coordinated change in the overall regulation of tl e cohort of downstream target genes.
  • Such a downstream effect can result in a change in the set point of a gross physiological variable, such as bone density, or any other physiological variable which, conceivably, is regulated downstream of the TAR.
  • genetic variation in the VDR gene was also related to different set points of the bone density trait and subsequently related to osteoporosis risk.
  • RAR-a and RXRs are derived from a family of related genes with similar function. These proteins form part of a complex web of cross-regulatory actions with other TAR, both of the steroid receptor family [VDR, T3R, ER and orphan receptors] as well as TAR of unrelated types such as the Jun/Fos family leucine zipper regulators.
  • RAR-a and related genes are therefore central regulators of diverse physiological processes. Variation in any member of the RAR-a family, if resulting in functional changes, should result in the alteration of subsequent physiological processes and be reflected in changed set points. If such variation could be identified it may serve as a useful genetic prognostic.
  • RAR-a is also an important co-factor in VDR gene responses, acting to regulate gene expression by the formation of heterodimers, although VDR and RAR-a can act as homodimers and can form complexes with other members of this and other TAR families as mentioned above.
  • Regulation of the osteocalcin gene by RA metabolites has been described by several authors, including Morrison, and direct association of RAR-a and its related protein X to the osteocalcin gene promoter has been described. Regulation of osteocalcin by RAR-a therefore provides the opportunity to detect functional genetic variation in the gene for RAR-a, and subsequently, members of the RAR-RXR family.
  • Osteoporosis is a debilitating bone disease that affects a high proportion of women and a lesser number of men. Due to the considerable public health problem associated with osteoporosis, efforts have been focussed at identifying diagnostic and predictive markers associated with the disease. Prediction of those at risk of osteoporosis may reduce the incidence of this disease by enabling early attention to those in greatest need. The aim of the present inventors has, therefore, been to define genetic tests that are capable of identifying those individuals at risk of osteoporosis and those protected from the disease.
  • the present invention consists in a method of assessing an individual's predisposition to low or high bone density, development of high or low bone turnover and/or responsiveness to therapy comprising analysing allelic variation in relation to a retinoic acid receptor (RAR) gene of the individual.
  • RAR retinoic acid receptor
  • the RAR gene is the RAR-a gene.
  • the analysis comprises restriction fragment length polymorphism (RFLP) using endonuclease digestion.
  • a segment of the retinoic acid receptor (RAR) gene is amplified using polymerase chain reaction prior to endonuclease digestion.
  • the endonuclease is selected from the group of endonucleases which cleave at the nucleotide sequence CTGCAG. Examples of such endonucleases are Bbi I, Noc I, Pma I, Pst I, Xma II and Xor I. Most preferably, the endonuclease is Pst I.
  • the method further involves analysing allelic variation in relation to the vitamin D receptor gene and/or the estrogen receptor gene of the individual. Further information regarding the analysis of these genes may be found in International Patent Application No.s PCT/AU93/00394, PCT/AU95/00452 and PCT/AU96/00017. The disclosure of these applications is included herein by cross reference.
  • RAR-a genotypes are detected with the endonuclease Pst I.
  • Pst I RFLP may be in linkage disequilibrium with other sequence alteration, both known and unknown, that mediate this effect.
  • the present invention can be practised in a number of ways, but in particular:
  • RAR-a genotypes detected by any means, in combination with other genes, particularly the VDR gene and the estrogen receptor (ESR) gene, in determining risk of low bone density, differing rates of loss of bone density and subsequent osteoporosis risk; and 4. through the use of RAR-a genotypes, detected by any means, in combination with other genes, particularly the VDR gene and the (ESR) gene, in determining response to therapy directed at reducing osteoporosis risk.
  • Figure 1 provides a graphical representation of the RAR-a genotype effect across "all females".
  • Figure 4 provides a graphical representation of the relationship between RAR-a alleles and LS BMD when VDR genotype is controlled. This figure shows that the RAR-a genotype effect persists when the VDR genotype effect is controlled by taking only VDR heterozygotes into consideration.
  • Figure 5 provides a graphical representation of the relationship between RAR-a alleles and serum osteocalcin when the VDR genotype is constrained to heterozygotes.
  • Fisgure 6 provides a plot of LS BMD from "all female” subjects. This figure shows a scatter plot of lumbar spine bone densities of normal female subjects according to combined VDR and RAR-a genotypes showing an almost linear relationship between BMD and genetic factors.
  • Figure 7 provides a graph showing a plot of mean ⁇ SEM of LS BMD within each combined genotype group.
  • a sample of normal subjects was genotyped by standard Southern blot techniques for variants in the RAR-a gene by RFLP. These subjects had previously been genotyped for VDR variation and represented well characterised normal humans.
  • the study population was comprised of monozygotic (MZ) and dizygotic (DZ) twins, representing a total of 252 people with a mean age of 42 years. There were 24 male pairs and 103 female pairs composed of 78 premenopausal and 25 postmenopausal pairs.
  • the total number genotyped for RAR-a alleles consisted of 31 males, 47 postmenopausal females and 132 premenopausal females. All subjects were genotyped for VDR alleles except two MZ pairs (one male and one female).
  • twins who were selected only on the basis of being twins, comprised 71 MZ and 55 DZ twin pairs, including 7 male MZ pairs and 6 male DZ pairs. They were aged between 17 and 70 years; MZ 45 ⁇ 13 yrs and DZ 44 ⁇ 11 yrs, mean ⁇ SD. All female twin pairs were concordant for menopausal status and, if postmenopausal, for years since menopause.
  • Bone density was measured at the lumbar spine and proximal femur with a Lunar DP3 dual-photon absorptiometer (LUNAR Corporation, Madison, WI) as previously described (Pocock NA et al., 1987) or Lunar DEXA dual energy x-ray absorptiometry (Pocock NA et al., 1988).
  • LS BMD lower spine bone mineral density
  • sequence differences in genes can be detected by numerous methods including polymerase chain reaction (PCR), Southern blot, ligase chain reaction, allele specific hybridisation and by hybridisation in solution.
  • PCR polymerase chain reaction
  • Southern blot ligase chain reaction
  • allele specific hybridisation by hybridisation in solution.
  • the examples below use Southern blot with probes to identify polymorphic DNA sequences.
  • Blood was collected into heparin treated tubes and leukocytes separated by sedimentation through physiological saline solution in a clinical centrifuge. Purified leukocytes were lysed in leukocyte lysis buffer (lOmM Tris HCl, pH 7.4, physiological saline and 0.5% w/v sodium dodecyl sulphate).
  • Lysate was treated with proteinase K (Applied Biosciences, Palo Alto, USA) at 50 ⁇ g/ml for 2 hours at 65°C. DNA was extracted by repetitive phenol chloroform solvent extraction as described in Maniatis et al. (1982) and ethanol precipitated. DNA was redissolved in TE buffer (lOmM Tris ⁇ HCl, ImM EDTA, pH 8.0) and quantitated by absorbance at 260 nm.
  • TE buffer lOmM Tris ⁇ HCl, ImM EDTA, pH 8.0
  • the RAR-a gene probe was a 1.6kb Eco Rl fragment from a cDNA clone of the human RAR-a (Arveiler et al. 1988). Southern blotting was according to methods well known in the art and hybridisation of radioactive probe was under standard conditions at 65°C, and high stringency washes to 0.2% SSC and 0.1% SDS at 65°C prior to autoradiographic exposure.
  • the RFLP was a two allele Pst I polymorphism with bands at 3.0kb in the absence of the Pst I restriction site and two bands (2.6 and 0.4 kb) in the presence of the Pst I restriction site. Invariant bands are also present at high stringency at 5.7, 3.8, 1.5, 1.4, 0.9, 0.8 and 0.3 kb.
  • the allelic frequency was found to be: Presence of Pst I site: 0.81
  • Vitamin D receptor (VDR) genotype has a strong effect on bone density (Morrison et al Nature 1994).
  • VDR gene heterozygotes the most abundant VDR genotype group which have intermediate BMD
  • Bb Bsm-1 RFLP
  • the difference between the means of genotype groupings can be used as a estimate of the allele's power in regulating the trait of bone density.
  • the difference in the means was 0.100, which can be compared against the total variance of the total study group and of the VDR heterozygote group used in this comparison.
  • VDR Bsm-1 allele heterozygotes used to control for the VDR gene effect:
  • the mean ⁇ standard deviation of the heterozygote population is 1.174 ⁇ 0.131.
  • the mean difference of the RAR-a allele effect is 0.100 or 76% of the SD of the heterozygote controlled population.
  • VDR alleles give extreme means of 1.063 and 1.235 with a total mean difference of 0.172, exceeding one standard deviation of the young normal value.
  • RAR-a The genotypes of RAR-a were tested with the two strong variables years post menopause (YPM) and weight (Kg) in multiple regression against BMD in the female test population. The result was that RAR-a genotypes are significantly related to the bone mass trait. Regression Table:
  • Source DF Sum Squares: Mean Square: F-test: REGRESSION 3 0.926 0.309 17.465
  • LS BMD 0.954 + 0.003 KG -0.01 YPM -0.045 RAR-a + 0.08 VDR.
  • VDR and RAR-a are independent predictors of bone density, and the strength of the RAR-a system is about one half that of the VDR system.
  • BMD is highly significantly associated with the BMD trait and is of comparable strength to the other variables (standardised coefficient of ⁇ 0.199).
  • VDR and RAR-a alleles were cross coded to give a 9 genotype system as follows:
  • Contingency table analysis of RAR-a and VDR alleles in female subjects (VDR in columns and RAR-a in row). Coding was arranged to create an ascending series of combined genotypes.
  • the alleles of the RAR-a gene detected by RFLP can be used as markers defining functionally different alleles of the RAR-a gene, which are either themselves involved or are linked to genetic changes which are responsible for, functionally significant variation in physiological parameters of clinical, medical and prognostic use, reflected in the significant differences in the bone density and serum osteocalcin traits described herein.
  • RAR-a Due to the intimate molecular cross-talk between RAR-a and the VDR and the other protein members of the RAR-a system, it is likely that genetically different alleles of RAR-a and the VDR will have a functional impact on other apparently unrelated endocrine and physiological systems. For instance it is now known that the VDR can participate with other nuclear transactivators in regulation of target genes. RAR-a has the same property. The cross-regulatory participants are vitamin D, thyroid, retinoid, dioxin, peroxysomal proliferator regulator receptor molecules. It is therefore likely that genetic variation in RAR-a or VDR will result in regulatory disturbances in these other endocrine/metabolite/xenobiotic pathways. 7.
  • the present inventors have developed a method of predicting genetic risk of having,
  • RAR-a genotypes can be used to decipher relationships between important physiological variables regulated by VDR dependent processes as a result of the fact that permissive genotypes of RAR-a permit mathematical description of the relationship between clinical humoral variables, which on general examination do not have strong relationships.
  • a working example is the relationship between serum 1,25 dihydroxyvitamin D3 and serum osteocalcin which have a relationship governed by RAR-a genotype. 4 1 j RAR-a gene alleles have utility in predicting the diversity of response to therapy based on the vitamin D endocrine system.

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Abstract

Procédé pour déterminer la prédisposition d'un individu à une densité osseuse basse ou élevée, à une capacité basse ou élevée de reconstitution de l'os et/ou de réaction à une thérapie, qui consiste à analyser la variation allèle par rapport à un gène de récepteur d'acide rétinoïque (RAR) chez l'individu. Dans un mode de réalisation préféré, ce procédé consiste à analyser la variation allèle du gène de RAR-a par l'intermédiaire du polymorphisme de la longueur d'un fragment de restriction (RFLP) au moyen d'une digestion par endonucléase Pst I.
PCT/AU1996/000474 1995-07-27 1996-07-29 Procede de prediction de la densite osseuse WO1997005275A1 (fr)

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AU65103/96A AU6510396A (en) 1995-07-27 1996-07-29 Method of predicting bone density

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AUPN4450A AUPN445095A0 (en) 1995-07-27 1995-07-27 Method of predicting bone density

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000038707A1 (fr) * 1998-12-24 2000-07-06 Garvan Institute Of Medical Research Methode de traitement de la perte osseuse
EP1537969A1 (fr) 1998-07-29 2005-06-08 Masonite Entry Door Corporation Porte avec une partie centrale creux
WO2009071661A1 (fr) * 2007-12-07 2009-06-11 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés de traitement et de diagnostic de maladies associées à la densité minérale osseuse

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4690093A (en) * 1992-07-31 1994-03-03 Garvan Institute Of Medical Research Assessment of trans-acting factors allelic variation
AU4427196A (en) * 1995-01-16 1996-08-07 Garvan Institute Of Medical Research Diagnostic method using estrogen receptor gene polymorphisms

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4690093A (en) * 1992-07-31 1994-03-03 Garvan Institute Of Medical Research Assessment of trans-acting factors allelic variation
AU4427196A (en) * 1995-01-16 1996-08-07 Garvan Institute Of Medical Research Diagnostic method using estrogen receptor gene polymorphisms

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEM J., (1995) 309, 721-724 SANESHIGE SHINGO et al., "Retinoic Acid Directly Stimulates Osteoclastic Bone Resorption and Gene Expression of Cathepsin K/OC-2". *
DEVELOPMENT, (1994) 120, 2723-2748 LOHNES D. et al., "Function of the Retinoic Acid Receptors (RARS) During Development". *
NATURE, (20 January 1994), Vol. 367, MORRISON N.A. et al., "Prediction of Bone Density from Vitamin D Receptor Alleles", pages 284-287. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1537969A1 (fr) 1998-07-29 2005-06-08 Masonite Entry Door Corporation Porte avec une partie centrale creux
WO2000038707A1 (fr) * 1998-12-24 2000-07-06 Garvan Institute Of Medical Research Methode de traitement de la perte osseuse
WO2009071661A1 (fr) * 2007-12-07 2009-06-11 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés de traitement et de diagnostic de maladies associées à la densité minérale osseuse
JP2011507491A (ja) * 2007-12-07 2011-03-10 アンセルム（アンスチチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル） 骨塩密度関連の疾患の治療および診断の方法
US9834820B2 (en) 2007-12-07 2017-12-05 Inserm (Institut National De La Sante Et De La Recherche Medicale) Methods for the treatment and diagnosis of bone mineral density related diseases

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