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WO1996003524A1 - Procede de diagnostic - Google Patents

Procede de diagnostic Download PDF

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WO1996003524A1
WO1996003524A1 PCT/AU1995/000452 AU9500452W WO9603524A1 WO 1996003524 A1 WO1996003524 A1 WO 1996003524A1 AU 9500452 W AU9500452 W AU 9500452W WO 9603524 A1 WO9603524 A1 WO 9603524A1
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genotype
osteocalcin
bsm
taq
bmd
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PCT/AU1995/000452
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Nigel Alexander Morrison
John Allan Eisman
Paul James Kelly
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Garvan Institute Of Medical Research
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Publication of WO1996003524A1 publication Critical patent/WO1996003524A1/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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • 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

  • VDR vitamin D receptor
  • the invention relates to the use of diagnostic kits for the discrimination of subjects with different set points of physiological characters associated with the vitamin D endocrine 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 risk of osteoporotic bone disease.
  • the use of VDR gene haplotypes has utility defined below, beyond the current state of the art.
  • the present invention provides a method of assessing a number of genetically variant sites in the VDR gene, which when combined to form haplotypes results in the further discrimination of genetic subtypes with differring traits.
  • 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. Since osteoporosis has a significant genetic component, the possiblity exists for genetic prediction of susceptibity and an understanding of the underlying pathophysiology of disease. Prediction of those at risk of osteoporosis may reduce the incidence of this disease by focussing early attention at those in greatest need. Our aim was to define a genetic test that is capable of identifying those individuals at risk of osteoporosis and those protected from disease.
  • Osteoporosis is defined generally as low bone density associated with fracture however for genetic analysis, a bone density two standard deviations below young normal is a more useful criterion of osteoporosis since subsequent fracture is dependent on a traumatic event.
  • Several humoral markers, notably osteocalcin, have been widely used as indicators of bone turnover. Genetic effects on osteocalcin levels have been reported in twin studies with a correlation between high osteocalcin levels and lower bone mineral density (Kelly et al., 1989).
  • haplotypes of the vitamin D receptor gene are combined with serum osteocalcin in an analysis of age related changes in bone mineral density inferred from a cross sectional population study.
  • the use of haplotypes defines extreme high and low bone density groups, and combined with osteocalcin data reveals a sub group essentially resistant to osteoporosis.
  • the different cross sectional rates of bone loss may provide a genetic basis for explaining "fast” and “slow” bone losers (Christiansen) .
  • the present invention consists in a method of assessing an individuals predisposition to low or high bone density and/or risk of fracture comprising assessing the vitamin D receptor genotype of the individual by haplotype analysis.
  • the genotype is assessed by haplotype analysis by assessing the genotype for the polymorphic restriction endonuclease sites for Bsm-1, Apa-1 and Taq-1 in the combinations of Bsm-1 and Apa-1, or Taq-1 and Apa-1, or Bsm-1, Apa-1 and Taq-1.
  • the assessment includes the polymorphic restriction endonuclease sites Sph-1 and/or the poly adenosine sequence microsatellite in the 3 prime untranslated region of exon 9 of the vitamin D receptor gene.
  • the assessment further includes measuring the individuals serum osteocalcin level.
  • PCR polymerase chain reaction
  • restriction endonucleases as described below to detect polymorphic sites and the subsequent analysis of the genotypes by combining Bsm-1 and Apa-1 or Apa-1 and Taq-1 RFLP data points and most preferably by the combination of all three RFLP genotypes.
  • FIG. 1 Lumbar spine bone density as a function of VDR genotype in premenopausal females.
  • the Mean ⁇ SEM of lumbar spine BMD is plotted according to genotype as described by haplotypes see Figure 1. Significant differences are shown and the p values shown are derived from Student's t-test. The difference in mean BMD between homozygotes 1,1 and 2,2 exceeds one SD of the young normal population (0.145 gm/cm*-*).
  • Figure 4 Menopause related change of lumbar spine bone density in major genotypic grouping defined by haplotypes of the vitamin D receptor gene.
  • Figure 6 Illustrates the genetic effect on serum calcium, identified using a single RFLP (Bsm-1) (open circles premenopausal, closed circles postmenopausal).
  • Figure 7 Shows graphically the data presented in Table 20 illustrating haplotype derived genotypes and serum calcium in post menopausal women.
  • Figure 8 Shows the profound difference in bone density observed in male subjects across genotype using Taq-1.
  • Figure 9 Shows the analysis of bone density with VDR haplotypes in males .
  • DNA analysis Southern blot, PCR (polymerase chain reaction) and RFLP analysis using endonuclease digestion. 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 (10 mM Tris-HCl, pH7.4, 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 hour at 65 Celcius. DNA was extracted by repetitive phenol chloroform solvent extraction as described in Maniatis et al .
  • TE buffer lOmM Tris-HCl, ImM EDTA, pH 8.0
  • Other methods of DNA preparation are compatible with the PCR procedure.
  • the Bsm-1 and Apa-1 RFLPs (Morrison et al . , 1992) are in intron 8 while the Taq-1 RFLP is a synonymous isoleucine codon change (ATT to ATC, isoleucine codons) in the VDR coding sequence in exon 9 (Morrison et al . , 1994).
  • a 2.4 kilobasepair (kb) PCR fragment was generated spanning exons 7 to exon 9, in a 50 ⁇ l reaction using a Pharmacia GeneAtaq thermocycler.
  • This fragment originated in the end of intron 6 and terminated in exon 9, spanning intervening exons and introns and contains all the amino acid coding potential of exons 7 to 9.
  • Some subjects were genotyped using the 2.4 kb or an alternative 2.1 kb fragment spanning the RFLPs, prior to the use of shorter fragments .
  • Primer pairs derived from the sequence were tested for utility in fast capillary PCR using a Corbett Research FTS-1 thermocycler.
  • any oligonucleotide primer pair derived from the sequenced region or derived from immediately adjacent regions could be used for detection by amplification by PCR. Although a number of primer pairs were tested, four oligonucleotide primers were selected arbitrarily as standard primers to amplify regions of the VDR gene for these studies.
  • Detection of the Bsm-1 site was facilitated by amplifying a region spanning the site, with one primer originating in exon 7 (primer 1: 5'-CAACCAAGACTACAAGTACCGCGTCAGTGA-3' ) and the other in intron 8 (primer 2: 5 '-AACCAGCGGAAGAGGTCAAGGG-3' ) producing a 825 basepair (bp) fragment.
  • PCR reactions were 20 ⁇ l containing 200ng genomic DNA, 20pmol of each primer, 200 ⁇ M dNTPs, 50mM KC1, lOmM Tris (pH8.3), 1.5mM MgCl2 and 1U Tth DNA polymerase (Thermus thermophilus. Toyobo, Osaka, Japan) . Each sample was subjected to 30 amplification cycles as follows:
  • Total duration of amplification was 30 minutes. If product is insufficient this protocol can be extended another 10 cycles. Amplification regimes should be optimised for any particular thermal cycling device. Increased extension times may be necessary for standard tube machines and for 96 well microplate format machines.
  • the digested PCR products were separated on 1.2% (Bsm-1 and Apa-1), or 2.0% (Taq-1) agarose gels containing 0.5 ⁇ g/ml ethidium bromide, 0.09M Tris-borate and 0.002M
  • Heterozygotes for the Taq-1 RFLP exhibit fragments of 490 bp, 290 bp, 245 bp and 205 bp.
  • Apa-1 digestion of the 740 bp PCR product yields fragments of 220 bp and 520 bp for the presence of the site allele and in the case of the absence of the site allele the fragment is not digested.
  • Primer 1 and primer 4 can be combined in a standard slow PCR reaction (using a Pharmacia GeneAtaq cycler) to yield a 2.1 kb fragment suitable for genotyping all three RFLPs from the same fragment.
  • the smaller reaction volume and shorter time of fast capillary PCR proved more time effective for genotyping.
  • Sequence at the polymorphic Apa- 1 site ending in an adjacent invariant Pvu-2 site is; a allele GAGGfiGCCCAGCTG, in the A allele the underlined G is a T.
  • the presence of the G can be detected by Ban-2, Aoc- 2, Pss-1, Pal-1, Hae-3, Cfr-3I, Asu-1, Sau-961, Eco-01091, Dra-2, and isoschizomers .
  • the presence of the T creates a polymorphism for Ban-1, and its isoschizomers.
  • Taq-1 polymorphism spanning invariant Hha- 1 to Hae-3 sites is: T allele GCGCTGATTGAGGCC, in the t allele the underlined T is a C.
  • This polymorphism can be also detected by Mbo-1, Sau-3A, Dpn-1 and their isoshizomers.
  • Genotypes were judged after electrophoresis by band sizes. Apa-1 and Taq-1 genotypes were assessed in different reactions of the same PCR product. Since Taq-1 can cut at 37°C it is possible to judge Apa-1 and Taq-1 genotypes by double digest of the product, although it was technically easier to digest separately. VDR gene haplotypes were given arbitrary numerical laboratory designations based on frequency. Statistics. Coassociation of genotype was tested using Chi-square and contingency table analysis. The Statview+graphics system by Abacus Inc. (Berkeley, California, USA) was run on a Macintosh IlSi. Results. Haplotype distributions in Caucasian subjects.
  • the + and - in the frequency refers to the presence and absence of the site alleles respectively.
  • RFLP genotypes are highly correlated. Contingency table analysis of RFLP genotypes detected by Bsm-1, Apa-1 and Taq-1. Bsm-1 versus Taq-1.
  • Taq-1 RFLPs were in high disequilibrium with Bsm-1 RFLPs giving coassociation of genotypes of about 98%. The relationship was such that presence of the Bsm-1 site was correlated with absence of the Taq-1 site.
  • Bsm-1 and Taq-1 were considered on a per chromosome basis (1432 in number) there were only 15 chromosomes representing recombination between Bsm-1 and Taq-1 or a 1.0% recombination frequency. In comparison, on a per chromosome basis there were 196 potential recombinants between Bsm-1 and Apa-1, or 13.7% recombination frequency.
  • Taq-1 RFLPs correlated with Apa-1 RFLPs in the same manner as descibed for Bsm-1 RFLPs: on a per chromosome basis there were 202 potential recombinants, giving a frequency of 14.1%. Further discrimination of genotype is possible using combined RFLP markers. If three dimorphic RFLPs are used, there are 2*3 or 8 possible haplotypes and subsequently 64 possible combinations of haplotypes, if the RFLPs are in equilibrium. Genotypes identified by RFLP can often appear the same but can be derived from different haplotype combinations. As a result there are 27 possible genotypic combinations of three dimorphic RFLPs (3*--).
  • Bsm-l/Apa-1 genotypes are listed in the first column with the last column giving the number of each Bsm-l/Apa-1 genotype observed. The last row shows the number of Taq-1 genotypes observed.
  • haplotypes can be inferred from the most frequent homozygous genotypes (Table 4) .
  • Table 4 Four most frequent haplotypes and frequencies in the test population as homozygote and heterozygote combinations.
  • genotypes are a minor proportion of the Caucasian sample, and may be the result of meiotic crossover, although it is possible they relate to minor ethnic admixture in Caucasians.
  • the order of markers is Bsm-1, Apa-1 and Taq-1 with the markers in close proximity ( lkb and 400bp between the RFLPs respectively).
  • Bsm-1 and Apa-1 have about 80% coassociation of genotype while Bsm-1 and Taq-1 (flanking the Apa-1 site) have a high 98% coassociation of genotype and low recombination rate ( ⁇ 2%).
  • Bsm-1 and Apa-1 have about 80% coassociation of genotype while Bsm-1 and Taq-1 (flanking the Apa-1 site) have a high 98% coassociation of genotype and low recombination rate ( ⁇ 2%).
  • 236 chromosomes of BB homozygotes only 13 were recombinants between the Bsm-1 B marker and Apa-1 A marker (5.5%).
  • the osteocalcin concentration in serum was estimated by radioimmunoassay using an in house polyclonal antisera raised against purified ovine osteocalcin. This assay has a normal range of 2 to 18 ng/ml. Serum samples were assayed once only for osteocalcin in the majority of cases. Where repeat osteocalcin data were available, the mean value was taken. Statistics. Analysis of variance (ANOVA) , multiple regression and stepwise multiple regression were used to compare parametric variables . Results pertaining to osteocalcin are presented based on In (1 + osteocalcin) osteocalcin values as previously described and on raw osteocalcin values. Natural logarithm transformed, In (1 + osteocalcin), in both parametric and non-parametric analysis (Kruskal-Wallis) gave similar results.
  • Serum calcium was measured by standard hospital automated blood chemistry procedures. Results.
  • Tt vs tt p 0.038_ Osteocalcin, VDR genotype and the menopause.
  • Osteocalcin levels are reported to rise at the menopause.
  • osteocalcin values were significantly different according to menopausal status. There was no evidence of interaction between menopausal status and genotype on osteocalcin. There were 175 premenopausal and 174 postmenopausal subjects that had serum osteocalcin data. The effect of menopause on osteocalcin (premenopausal 7.7 ⁇ 5.3 ; postmenopausal 11.1 ⁇ 7.7, ⁇ SD; p ⁇ 0.0001), indicated a rise of about 44% over the menopause with a large degree of value overlap. We found similar changes in osteocalcin values across the menopause between the various genotypes.
  • Bsm-1 genotypes the increases in osteocalcin over the menopause were virtually identical in each genotypic group ( BB, 52%; Bb, 40% and bb, 52%, not shown).
  • the Taq-1 genotype gave similar results.
  • the magnitude of the genetic effect (57.5% from bb to BB) was comparable to the magnitude of the total menopause effect (44%) .
  • the mean premenopausal osteocalcin of the genotype bb (6.0 ⁇ 4.3 SD) , rises after the menopause to a value (9.1 ⁇ 5.4 SD) similar to the premenopausal value for the genotype BB (9.5 ⁇ 5.2 SD) .
  • haplotype 3 is associated with a low osteocalcin phenotype in the presence of both haplotypes 1 and 2. It would be expected from this that haplotype 3,3 homozygotes would have a low osteocalcin phenotype.
  • Genotype n Mean SD SEM p vs BBAa p vs BbAa
  • Table 7 Mean osteocalcin values according to menopause and genotype using a four haplotype, nine genotype system with either Bsm-1 and Apa-1 or Apa-1 and Taq-1.
  • Genotype n Mean SD SEM n: Mean: SD SEM ⁇ %
  • Genotype n Mean SD SEM n: Mean: SD SEM ⁇ %
  • Table 7B Serum osteocalcin levels in the six frequent genotypic groupings and significance of differences, disregarding the menopause.
  • Genotype n: Mean SD SEM
  • Sig refers to the p value of Students' t-test between pre and postmenopausal groups.
  • Bone mineral density and haplotypes of the VDR gene Bone mineral density and haplotypes of the VDR gene. Bone density in premenopausal subjects was not significantly related to age. This enables a simple ANOVA analysis of the effect of genotype on bone density to be used to distinguish differences in genotypic groupings. The five most frequent genotypes were analysed, corresponding to haplotype combinations: 1,1; 1,2; 2,2; 1,3; 2,3 ( Figure 2 and Table 9 below) .
  • Table 9 Haplotype analysis of bone mineral density in premenopausal subjects.
  • the low bone density genotype (BB or tt) is not overly affected by using extra RFLP markers or haplotypes.
  • the increased genetic effect is realised by Apa-1 RFLPs splitting the high BMD group into two groups (haplotypes 1,1 and 1,3) one of which is revealed as an extra high BMD group (mean BMD of 1.275 gm/cm 2 ).
  • This group is haplotype 1,1, defined as genotype bbaaTT, but also detectable as genotype bbaa or aaTT, due to the high co-association of Bsm-1 and Taq-1 RFLPs.
  • Lumbar spine BMD 1.263 - 0.066 osteocalcin
  • Femoral neck BMD 0.95 - 0.043 osteocalcin.
  • menopausal status coded as zero and one
  • No interaction between menopausal status and osteocalcin effect on lumbar spine BMD was found, demonstrating that these factors are independent variables.
  • Bone mineral density is affected by anthropomorphic factors such as height and weight and life variables such as age and years post menopause (YPM) .
  • Taq-1 Intercept 0.598
  • Table 15 Simple correlation coefficients between osteocalcin and lumbar spine BMD within genotypic groupings .
  • Haplotype systems Combined Bsm-1 Apa-1 groups.
  • haplotype 1,1 homozygotes the extreme high bone mineral density genotype
  • haplotype 1,1 homozygotes the extreme high bone mineral density genotype
  • osteocalcin was not a significant predictor of bone density at either the lumbar spine or femoral neck, in the other major genotypes, 2,2 and 1,2. In stepwise regression of these variables in the minor genotypes osteocalcin was not a significant predictor of bone density at either the lumbar spine or the femoral neck .
  • Table 17 Regression of multiple variables against BMD in homozygote haplotype 1,1 and 2,2; postmenopausal subjects .
  • a prevalent concept in osetoporosis research is the concept of a fracture threshold, which is a critical value of bone density at which heightened risk of disease is apparent.
  • Postmenopausal women were analysed for changes in bone density in relation to the fracture threshold (descibed above in the analysis section) using YPM as well as age.
  • Discriminating genetic groups using Bsm-1 RFLPs alone the results are similar to those reported previously (Morrison et al . Nature 1994) with intercepts with the fracture threshold for the genotypes of: 18.7, 32.8 and 33.7 YPM for the lumbar spine and 17.9, 24.2 and 23.7 YPM for the femoral neck for genotypes BB, Bb and bb, respectively.
  • haplotype 1,1 homozygotes resulted in further discrimination of the genetic effect, with an increase in the magnitude of the genetic effect and the difference in the fracture threshold intercept between genotypes (data presented below in Table 18).
  • haplotype 1,1 homozygotes the result was 40.4 YPM before the mean genotype LS BMD reached the critical fracture threshold. Since the mean menopausal age of all subjects was 48.4 years (this was not different between genotypes), this value translates to an age of 89.6 years before a person of this genotype will, on average, reach this critical fracture threshold.
  • the intercept value for haplotype 2,2 homozygotes was 18.3 years.
  • the extremes of the genotypic groupings therefore translate to an average 22.1 year difference in reaching the critical fracture threshold.
  • the 1,2 heterozygotes had an intercept value of 34.6 years, giving an 16.3 year difference with the low bone density haplotype 2,2 homozygote group (see Fig. 4).
  • Table 18 Regression analysis of bone density versus years post menopause in different genotypic groups.
  • the minor genotypes had intermediate phenotypes .
  • the fracture intercepts of 2,3 and 1,3 heterozygotes were 20.2 and 20.6 YPM, respectively making these genotypes comparable to the 2,2 homozygotes.
  • VDR genotype The effect of VDR genotype on serum calcium levels.
  • Serum calcium levels are different across genotypes. Serum calcium levels were measured on a subset of subjects and correlated with vitamin D receptor genetics (see Fig. 5). In premenopausal subjects genetic effects were not obvious (see Fig. 6). In postmenopausal females a highly significant effect of genotype on serum calcium was observed. In the subset of individuals in which serum calcium was measured the serum calcium result mirrored the findings with bone density, with low bone density groups having lower serum calcium (see Table 19 below) . The high bone density genotypes had higher serum calcium. A similar trend which was not statistically significant was observed when ionised calcium was examined.
  • Table 20 Different serum calcium levels in different genotypes as determined by haplotypes: comparing the 5 most frequent haplotype derived genotypes .
  • VDR haplotypes have utility in assessing the bone density of male subjects.
  • VDR genotype affects bone density in the total male study group.
  • haplotypes While the mean difference between genotypes is not increased in males by using haplotypes (0.157, 2,2 compared to 1,1) the use of haplotpe analysis demonstrates a difference between homozygote 2,2 and heterozygote 2,3 (mean difference 0.16).
  • a combination of haplotype and weight explained 19.1% of the variance in lumbar spine bone density and 8.9% of the variance in femoral neck BMD.
  • Haplotype data alone explained 8.1% and 5% of the variance in BMD for the lumbar spine and femoral neck, respectively.
  • the genotype of the VDR receptor gene predicts the risk of bone fracture.
  • the study plan was to conduct a prosepective epidemiological study of incident bone fracture events in the city of Dubbo New South Wales Australia. This study is described in Nguyen et al in the British Medical Journal Volume 307:1111-1115, 1993.
  • the utility of vitamin D receptor in predicting fracture was determined by genotyping a random sample of 269 females with mean age 70+7 years, selected from the Dubbo study. Bone density was measured at the femoral neck at recruitment into the study and the statistics of logistic regression were used to discriminate the relationship to subsequent fracture incidence. Fracture was assessed by radiology. Genotype was assessed using Taq-1 and Apa-1 RFLP sites. Results.
  • Osteocalcin is a serum marker derived from osteoblasts that is widely used as a marker of bone turnover (see Morrison et al 1992). Different mean serum levels of osteocalcin were previously reported in different genetic groups defined by simple RFLP markers in the vitamin D receptor gene, leading to the conclusion that the RFLPs are markers for functionally different vitamin D receptor gene alleles. The information supporting the utility of the invention has been increased and the present invention demonstrated to be an improvement on the state of the art. This has been achieved by investigating the relationships between osteocalcin, age and menopause related changes in bone density using analysis based on combined RFLPs and haplotypes. Strong linkage disequilibrium between the RFLPs provides the basis for haplotype analysis, which showed three major haplotypes in Caucasians.
  • haplotype analysis explains the relationship between the three RFLPs, since there are only three major haplotype forms seen in Caucasians (baT, BAt and bAT) .
  • the invention comprises, in a particular preferred embodiment, the use of certain oligonucleotide primers to amplify by PCR regions of the VDR gene containing polymorphic restriction endonuclease sites.
  • the genotypes thus derived are combined to increase the genetic information content.
  • the genetic information is combined with serum osteocalcin data to provide enhanced predictiveness of bone density within certain genetic groups.
  • the menopausal rise in osteocalcin seen in the genotypes characterised by low osteocalcin merely increased osteocalcin to the premenopausal values seen in the high osteocalcin haplotype 2,2 group.
  • the high osteocalcin groups had lower bone density in both premenopausal and postmenopausal years.
  • the inverse relationship between osteocalcin and bone mass was also seen.
  • osteocalcin was negatively associated with BMD.
  • haplotype 2 subjects were considered there was no relationship between osteocalcin and BMD.
  • haplotype 1,1 homozygotes postmenopausal BMD was strongly related to osteocalcin and not to YPM.
  • the invention permits the detection of different genetic groups in which bone turnover markers are differentially related to BMD.
  • genotype contributes to a substantial difference in premenopausal BMD it would be expected to contribute to postmenopausal BMD.
  • the present invention detects a difference in the inferred rate of change of BMD at the LS and FN ranging from 0.5% per annum (genotypes 1,1 and *1,2) up to 1.2% (genotype 2,2).
  • genotype 2,2 When this analysis was focussed within 20 YPM the difference was even greater, with 2.0% per annum for genotype 2,2 subjects and low rates and non ⁇ significant relationships (slopes not significantly different from zero) for the heterozygote 1,2 and homozygote 1,1.
  • the present invention detects a genetic group (haplotype 1,1 homozygotes) who, if female, will on average reach the fracture threshold at about 89 years of age. This result represents an effective genetic resistance to osteoporosis, since at least 50% will still be above the threshold at this age and bone fracture may not be their only health concern. Furthermore a preferred embodiment of the present invention, in which genotype data is combined with serum osteocalcin data, detects those individuals who have a propensity to different levels of bone density within this high bone density group.
  • serum osteocalcin is related to LS BMD in the postmenopausal years, so an embodiment of the present invention utilising a combination of serum osetocalin and genotype detects a smaller subgroup, referred to as "genetic resistance to osteoporosis", that has very little prospect of reaching the fracture threshold.
  • the present invention also detects the status of haplotype 1,2 heterozygotes as having a projected time of reaching the fracture threshold of 34.6 YPM (around 83 years), as a result of partial genetic dominance of haplotype 1 on the rate of postmenopausal bone loss.
  • the present invention detects a group most at risk of osteoporosis (the haplotype 2,2 genotype), who have lower premenopausal BMD and faster bone loss in the postmenopausal years.
  • This group reaches the fracture threshold at about 66 years of age, only 18 years after the menopause with many subjects being below the threshold earlier. The relationship between fracture and genotype is clearly defined.
  • the present invention provides a means of predicting different serum calcium set point levels in postmenopausal females and defines a link between serum calcium, and therefore presumably intestinal calcium uptake, such that low bone density genotypes are characterised by low serum calcium. Therefore the invention further relates to the potential for selection of subjects for calcium therapy.
  • Kerner SA Scott RA, Pike JW. Proc Natl Acad Sci (USA) 86: 4455-4459 (1989)

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Abstract

Procédé permettant de déterminer la prédisposition d'un individu à développer une faible ou une forte densité osseuse et/ou le risque de fractures, consistant à déterminer le génotype du récepteur de vitamine D de cet individu par analyse de l'haplotype. De préférence, la détermination du génotype dudit individu par analyse de l'haplotype consiste à déterminer le génotype pour les sites d'endonucléase de restriction polymorphe pour Bsm-1, Apa-1 et Taq-1 dans les combinaisons de Bsm-1 et Apa-1, ou Taq-1 et Apa-1, ou Bsm-1 et Taq-1, ou Bsm-1, Apa-1 et Taq-1.
PCT/AU1995/000452 1994-07-25 1995-07-25 Procede de diagnostic WO1996003524A1 (fr)

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WO2000015836A3 (fr) * 1998-09-15 2000-06-08 Signalgene Inc Combinaison de marqueurs au niveau des genes recepteurs des oestrogenes et de la vitamine d, ou equivalents de ces derniers, pour le pronostic d'une reponse au traitement de l'osteoporose
WO2000038707A1 (fr) * 1998-12-24 2000-07-06 Garvan Institute Of Medical Research Methode de traitement de la perte osseuse
WO2002059358A3 (fr) * 2001-01-25 2003-10-23 Adnagen Ag Trousse de diagnostic, procede et jeu ordonne de microechantillons pour determiner la predisposition a l'osteoporose

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AU3323293A (en) * 1991-12-12 1993-07-19 Horus Therapeutics, Inc. Method for diagnosis osteopenia and determining its severity

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AU3323293A (en) * 1991-12-12 1993-07-19 Horus Therapeutics, Inc. Method for diagnosis osteopenia and determining its severity

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JOURNAL OF BONE AND MINERAL RESEARCH, Volume 9, Number 12, 1994, UCHIDA M. et al., "Activation of the Human Osteocalcin Gene by 24R, 25 - Dihydroxyvitamin D3 Occurs Through the Vitamin D Receptor and the Vitamin D-Responsive Element", pages 1981-1987. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 86, June 1989, KERNER S.A. et al., "Sequence Elements in the Human Osteocalcin Gene Confer Basal Activation and Inducible Response to Hormonal Vitamin D3", pages 4455-4459. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 89, August 1992, MORRISON N.A. et al., "Contribution of Transacting Factor Alleles to Normal Physiological Variability: Vitamin D Receptor Gene Polymorphisms and Circulating Osteocalcin", pages 6665-6669. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000015836A3 (fr) * 1998-09-15 2000-06-08 Signalgene Inc Combinaison de marqueurs au niveau des genes recepteurs des oestrogenes et de la vitamine d, ou equivalents de ces derniers, pour le pronostic d'une reponse au traitement de l'osteoporose
WO2000038707A1 (fr) * 1998-12-24 2000-07-06 Garvan Institute Of Medical Research Methode de traitement de la perte osseuse
WO2002059358A3 (fr) * 2001-01-25 2003-10-23 Adnagen Ag Trousse de diagnostic, procede et jeu ordonne de microechantillons pour determiner la predisposition a l'osteoporose

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