EP2046987A2

EP2046987A2 - Assay for diagnosing a predisposition to pathological bone conditions and anticipating response to treatment

Info

Publication number: EP2046987A2
Application number: EP07789345A
Authority: EP
Inventors: María Luisa VILLAHERMOSA JAEN; Ana I. Moraga
Original assignee: Genomica SA
Current assignee: Genomica SA
Priority date: 2006-07-24
Filing date: 2007-07-23
Publication date: 2009-04-15
Also published as: WO2008012576A3; WO2008012576A2; GB0614620D0

Abstract

Methods and kits are provided for detection of specific polymorphisms that predispose towards osteoporosis and/or are indicative of likely response to treatment. Particular genes analysed are i) calcitonin Receptor; Vitamin D Receptor; Estrogen Receptor; Collagen I A1; and/or ii) Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR genes. The two groups may be analysed in separate multiplex amplification reactions on a sample.

Description

Assay for diagnosing a predisposition to patholoqicai bone conditions and anticipating response to treatment-

Field of the invention

The present invention relates to an in vitro assay and method characterized by the simultaneous genotyping of Calcitonin Receptor and Collagen I Al genes. More specifically, in preferred embodiments the assay further allows genotyping of Vitamin D Receptor and Estrogen Receptor and, in still more preferred embodiments, the genotyping also of Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR genes in ciinicai samples.

In preferred embodiments, the present invention relates to genotyping of polymorphisms AIuI of the Calcitonin Receptor, Fokl and Bsml of the Vitamin D Receptor, Xbal and PvuII of the Estrogen Receptor and COLlAl SpI of Collagen I Al, and of Osteoprotegerin OPG 209 G>A, OPG 245 T>G and OPG163 A>G, LRP5 C171346A, C135242T and G138351A, CYP17 T(27)-C; CYP19 C(1558)-T and MTHFR C677T.

Further, the present invention relates to an assay and method comprising independent genotyping of the group of genes Calcitonin Receptor, Vitamin D Receptor, Estrogen Receptor and Collagen I Al genes; and of the group of genes Osteoprotegerin, LRP5, CYP 17, CYP19 and MTHFR. The invention also relates to diagnostic kits comprising above-mentioned methods and their use for diagnosing predisposition to osteoporosis and other bone pathological conditions, as well as to treatment responsiveness.

Background of the invention

Osteoporosis is a disorder in which loss of bone strength leads to fragility fractures (Raisz, 2005, J. Clin. Invest 115(12):3318-25). The fundamental pathogenetic mechanisms underlying this disorder include: (a) failure to achieve a skeleton of optimal strength during growth and development; (b) excessive bone resorption resulting in loss of bone mass and disruption of architecture; and (c) failure to replace lost bone due to defects in bone formation. Estrogen deficiency is known to play a critical role in the development of osteoporosis, while calcium and vitamin D deficiencies and secondary hyperparathyroidism also contribute. There are multiple mechanisms underlying the regulation of bone remodelling, and these involve not only the osteoblastic and osteoclastic cell lineages but also other marrow cells, in addition to the interaction of systemic hormones, local cytokines, growth factors, and transcription factors. Polymorphisms of a large number of genes have been associated with differences in bone mass and fragility (Raisz, 2005, 3 Clin Invest. 115(12):3318- 25).

Certain clinical trials have assessed that there is a relationship between the presence of certain genotypes of Calcitonin Receptor, Vitamin D Receptor, Estrogen Receptor, Collagen I Al, Osteoprotegerin (OPG), LRP5, Cytochrome P450cl7alpha (CYP17), Aromatase (CYP19) and Methylenetetrahydrofolate reductase (MTHFR) genes, and the predisposition to bone diseases such as osteoporosis, as well as to the effectiveness of treatment. Common allelic variants in the gene encoding the vitamin D receptor (VDR) have been used to predict differences in bone density, accounting for up to 75% of the total genetic effect on bone density in healthy individuals (Morrison et al., 1994, Nature 367(6460) :284-7). Further, the genotype associated with lower bone density was overrepresented in postmenopausal women with bone densities more than 2 standard deviations below values in young normal women. A relationship between VDR gene allelic variants and the effect of calcium intake on maintenance of bone mass has further been outlined (Ferrari et al., 1995, Lancet 345(8947):423-4).

To assess whether there is a relationship between the effectiveness of alendronate treatment in postmenopausal women with osteoporosis and Bsml

VDR genotypes, a prospective baseline-controlled clinical trial was carried out wherein sixty-eight Italian osteoporotic women were enrolled and treated with alendronate at a dose of 10 mg/day for 12 months. (Palomba et al., 2003, Clin

Endocrinol (Oxf)- 58(3);365-71). The conclusion of this analysis was that the different Bsml VDR genotypes modify the pharmacological response to alendronate treatment in postmenopausal women with osteoporosis. A relationship between Bsml VDR genotypes and response to raloxifene treatment has also been found in post-menopausal women with osteoporosis (Palomba et al., 2003, Hum Reprod. 18(1): 192-8).

A correlation has also been pointed at between another polymorphism of the VDR gene, the Fokl polymorphism, and the rates of bone mineral density (BMD) at the lumbar spine and of bone loss at the hip in a group of 100 postmenopausal Mexican-American Caucasian women (Gross et al., 1996, J Bone Miner Res. ll(12):1850-5).

Further analysis has found that COLlAl SpI alleles are associated with a modest reduction in BMD, and a significant increase in risk of osteoporotic fracture, particularly vertebral fracture (Mann & Ralston, 2003, Bone 32(6):711- 7).

A relationship has been detected between PvuII and Xbal restriction fragment length polymorphisms (RFLPs) of the estrogen receptor (ER) gene and BMD in postmenopausal women (Salmen et al., 2000, J Bone Miner Res. 15(2):315-21; Kobayashi et al., 1996, J Bone Miner Res. ll(3):306-ll).

A novel RFLP was discovered in a Japanese population at the CTR gene by AIu I restriction enzyme at the 1377th nucleotide, Analysis of the presence of this CTR gene RFLP in 307 postmenopausal Italian women, evidenced a relationship between the CTR gene genotype and lumbar BMD (Masi et a!., 1998, Biochem Biophys Res Commun. 245(2):622-6). Additional studies have suggested that the CTR gene polymorphism might influence the process of acquiring peak bone mass rather than the process of bone loss along aging (Braga et al., 2000, Calcif Tissue Int. 67(5):361-366).

The relationship between BMD and Osteoprotegerin (OPG) gene polymorphisms OPG 209 G>A, OPG 245 T>G and OPG163 A>G was analysed in Arko et al., 2002, J Clin Endocrinol Metab. 87(9):4080-4 and in Choi et al., 2005, Calcif Tissue Int. 77(3):152-9. An association between BMD levels and LRP5 polymorphisms corresponding to SNPs at nucleotides C171346A, C135242T and G138351A was studied in Koay et al., 2004, J Bone Miner Res. 19(10):1619-27.

In a study carried out with recent postmenopausal women, an association between the A2 allele of the cytochrome P450cl7aipha (CYP17) gene (CYP17 T(27)-C polymorphism) and reduced bone mass and bone size was detected in lean perlmenopausal women, whereas high BMI seems to protect against this negative association (Tofteng et al., 2004, Calcif Tissue Int. 75(2): 123-32).

Further, polymorphisms in genes involved in synthesis of sex steroids, such as polymorphism CYP19 C(1558)-T of the CYP19 gene encoding aromatase, are associated with the magnitude of bone gain in response to hormone replacement therapy (Tofteng et al., 2004, Calcif Tissue Int. 74(i):25-34).

Finally, a polymorphism in nucleotide 677 of the gene encoding methylenetetrahydrofolate reductase (MTHFR), MTHFR C677T polymorphism, has been associated with BMD both in Japanese and European postmenopausal women (Abrahamsen et al., 2003, J Bone Miner Res. 18(4):723-9).

Several attempts to co-detect two or more genes involved in bone mineral density and related disorders, are to be found in the state of the art. In general, the technical approach for this detection consists of individual PCR amplification reactions for each gene, followed by sequencing or restriction enzyme analysis.

Thus, Braga et al., 2002, Calcif Tissue Int. 70(6):457-62, displays a method for co-detection of Calcitonin Receptor, COLlAl and Vitamin D Receptor which consists of the individual gene amplification reactions, followed by restriction enzyme analysis. Also, Bandres et al., 2005, J Endocrinol Invest. 28(4):3I2-21, displays detection of COLlAl, Vitamin D Receptor, Estrogen Receptor and Calcitonin Receptor genes, also by means of DNA extraction, followed by individual PCR amplification and restriction enzyme analysis. This analysis has shown that CoIlAl shows correlation with the prevalence of osteoporotic fractures in a postmenopausal Spanish women cohort, that Calcitonin Receptor and Vitamin D Receptor show correlation with BMD, while Estrogen Receptor does not shown any such correlation in the same study. The assays displayed both in Braga et a!., 2002, and in Bandres et al., 2005, consist of individual gene amplification reactions, followed by restriction enzyme analysis.

Additional documents of interest are EP 1 054 066 and US 6,566,064, which describe methods for anticipating sensitivity to a medicine for osteoporosis characterized by analysing genetic polymorphisms of a vitamin D receptor gene, an estrogen receptor gene, and an apolipoprotein E gene. Within these publications, proposed methods for measuring genetic polymorphisms are classified into three types based on their principle. That is, (1) a method in which a gene fragment containing the polymorphism site is isolated and the base sequence of the site is determined or the polymorphism site is directly detected by use of a specific probe or primer, (2) a method in which a difference in higher level structure of a gene fragment containing the polymorphism site is used to distinguish polymorphisms based on electrophoretic mobility, and (3) a method in which the possibility of cleavage at the polymorphism site with a restriction enzyme is used to distinguish polymorphisms based on electrophoretic mobility. As specific examples of (1), mention is made of, for example, a sequencing method, a sequence specific oligonucleotide probe (SSOP) method, a mutant allele-specific amplification (MASA) method, etc.

In particular, examples are provided wherein a VDR gene fragment, an ER gene fragment, and an ApoE gene fragment are separately PCR amplified using the genome DNA as a template and specific primer pairs for each gene fragment. Each amplification reaction mixture is then treated with restriction enzymes with specific cleavage sites at the polymorphism site, followed by agarose gel electrophoresis. Polymorphisms of the VDR, ER, and ApoE genes are discriminated based on the band patterns.

Alternatively, in further examples, polymorphism identification is carried out by incubation of the amplified products with polyT-added probes coated at separate sites on a single sheet of nylon membrane. Also, Tempfer et al., 2004, Fertil Steril, 82(1): 132-7, displays an analysis of some SNPs associated with risks and benefits of estrogen therapy and hormone therapy, wherein genomic regions containing the specific SNPs are amplified through multiplex PCR. These regions include CYP17, CYP19, estrogen receptor, MTHFR and Vitamin D Receptor, among others. After amplification through multiplex PCR, the PCR products have then to be purified, and subjected to primer extension combined with fluorophore incorporation. The fluorophore- labeled single strands are hybridized to an ailele-specific oiigo array bound to a glass support. Summary of the invention

Aspects of the invention allow the provision of a reliable and high through-put assay that allows the simultaneous detection of Calcitonin Receptor and Collagen I Al in a sample.

It is an aim of aspects of the present invention to provide a kit comprising reagents, protocols and specific probes, for performing above-mentioned assay on clinical samples.

It is also an aim of aspects of the present invention to provide a reliable and high through-put method for diagnosing individual predisposition to pathological bone conditions such as osteoporosis, and for anticipating response to treatment.

The solution is based on the multiplex amplification reaction of the Calcitonin Receptor and Collagen I Al genes followed by hybridisation of the amplified fragments with ailele-specific probes.

Accordingly, a first aspect of the present invention relates to an assay comprising genotyping Calcitonin Receptor and Collagen I Al in a sample, characterized in that a multiplex amplification reaction of the Calcitonin Receptor and Collagen I Al genes is performed on the sample.

In a preferred embodiment, the assay further comprises multiplex amplification of the Vitamin D Receptor and/or the Estrogen Receptor genes; preferably in the same multiplex amplification as that of Calcitonin Receptor and Collagen I Al genes.

In another preferred embodiment, the assay, further to the multiplex amplification reaction of the group of genes i), comprising Calcitonin Receptor and Collagen I Al, and optionally, Vitamin D Receptor and/or Estrogen Receptor, comprises an additional multiplex amplification reaction of one or more genes being selected from the group of genes ii) comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR.

More specifically, the genotypes that are determined correspond to polymorphisms AIuI of the Calcitonin Receptor, Fokl and Bsml of the Vitamin D Receptor, Xbal and PvuII of the Estrogen Receptor and COLlAl SpI of Collagen I Al, and of Osteoprotegerin OPG 209 G>A, OPG 245 T>G and OPG163 A>G, LRP5 C171346A, C135242T and G138351A, CYP17 T(27)-C; CYP19 C(1558)-T and MTHFR C677T. The Genbank accession numbers of the various genes are as follows. Calcitonin Receptor: NM_001742; Vitamin D Receptor: AY342401; Estrogen Receptor: AY425004; Collagen I Al: AF017178; Osteoprotegerin: AB008822; LRP5: NM_002335; CYP17: NM_000102; CYP19: NMJB1226; MTHFR: NM_005957. The polymorphisms are known to those of skill in the art; selected probes useful for detecting the polymorphisms are set out in the tables disclosed herein.

Second and third aspects of the present invention relate to a solid support comprising a plurality of specific probes for polymorphisms of Calcitonin Receptor, Vitamin D Receptor, Estrogen Receptor and Collagen I Al, Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR, as well as to a reaction vessel comprising the solid support.

A fourth aspect of the present invention relates to a diagnostic kit comprising above-mentioned assay and yet a fifth aspect relates to the use of the kit for diagnosing predisposition to pathological bone conditions, as well as for anticipating response to treatment. Additional aspects of the present invention relate to specific probes and amplification primers.

It is an object of certain aspects of the present invention to provide a highly reliable and high-throughput method for genotyping Calcitonin Receptor and Collagen I Ai in a sample, by way of a multiplex amplification reaction,

The present invention further provides a method for simultaneous genotyping of Calcitonin Receptor, Collagen I Al, as well as Vitamin D Receptor and/or Estrogen Receptor, also allowing the possibility to further genotype Osteoprotegerin, LRP5, CYPl 7, CYP 19 and MTHFR genes. The assay of the present invention makes possible above-mentioned genotyping without the drawbacks of the methods of the state of the art, wherein individual amplification followed by genotyping of each gene of interest is required.

The use of such the assay of the present invention allows the diagnosis of individual predisposition to osteoporosis and to other bone pathologies, as well as the anticipation of responsiveness to treatment.

The genotyping assay comprises: performing a nucleic acid amplification reaction on a sample, obtaining single stranded oligonucleotides from the amplification products, allowing single stranded oligonucleotides to hybridise with alleie-specific probes corresponding to the genes of interest, which are immobilised on a solid support, and detecting hybridised oligonucleotides.

The amplification reaction is preferably PCR, Single stranded oligonucleotides may be obtained by denaturing any double stranded oligonucleotides present, for example by heating, Allele-specific probes are preferably selected to specifically bind to the single-stranded oligonucleotides from amplification products under the same hybridisation conditions for ail probes.

Design of a multiplex PCR is not straightforward. It is unlikely that an arbitrary primer combination will be successful, and will avoid unwanted interactions between the primers, or between primers and non-target sequences. On the contrary, the following criteria have to be fulfilled: i) Primers have to share the same Melting Temperature, and ii) the hybridization temperature of the cycling profile of the Multiplex-PCR must suit the Meiting Temperature of the amplification primers.

Further, not any probe with ability to hybridize with the amplified fragment is useful for genotyping the genes of interest. As a matter of fact, the selected probe must hybridize with the region of the amplified fragment in which the polymorphism is located, being able to distinguish between the two genotypes of a polymorphism.

Additionally, an equilibrium between the length of the probe and the amount and distribution of G/C within it has to be contemplated. Should the probe length be too small with the purpose of providing a good discrimination between genotypes, it might not even hybridise to the target nucleic acid sequence. On the contrary, with probes longer than 30 nt, hybridization to the target nucleic acid sequence will probably be guaranteed, but discrimination between genotypes certainly will not.

The probes are conveniently 15 to 30 nt in length, more preferably 15 to 23 nt. In a more preferred embodiment, the probe length is 15 to 20 nt. And in the particular case of polymorphism COLlAl SpI of gene Collagen I Al, the most suitable probe length is 19 nt. All probes need not be the same length. Preferred probes are selected from the group comprising SEQ ID NO 11 to SEQ ID NO 29, SEQ ID NO 44 to SEQ ID NO 211 and SEQ ID NO 212-222. The probes may be duplicated on the solid support, to provide for multiple detection locations for redundancy.

One or more control sequences may also be immobilised to the solid support; for example, a probe which does not hybridise to the target sequence from any allele of the selected polymorphisms.

In preferred embodiments, the sample and the solid support are contained within a reaction vessel; for example, that described in US2005064469.

Brief description of the drawings Figure 1: Drawing showing an arrangement of probes on the surface of a microarray with 12 x 10 = 120 locations. Numbers correspond to the SEQ ID NO from the sequence listing. Single probes were fixed at four different locations for detection of 18 different genotypes (FF, ff, Ff; SS, ss, Ss; AA, aa, Aa; BB. Bb, Bb, XX, xx, Xx; PP, pp, Pp) -taking in mind the presence of two alleles which are present in each cell-, and amplification control of each gene. M = probes for location reference (Marker- 1 [GCA GTA TAA GAT TAT TGA TGC CGG AAC]; Marker-2 [GTC AAA ACC TGG GAT AGT AGT TTT ACC]).

Figure 2: Photograph of an array tube used in the present invention. The arrangement of probes corresponds to the one displayed in Figure 1.

Figure 3: Details corresponding to Example 5. Panel A: Multiplex PCR conditions. Panel B: Multiplex PCR cycling profile. Panel C Visualization of the amplification products by means of agarose gei electrophoresis (2,5%),

Figure 4: Details corresponding to Example 6. Panel A: Probes designed for hybridisation with genotypes S or s of gene Collagen I Al. Panel B: Results obtained in an array tube assay with the probes of Panel A and the seven samples: op42 ss, op90 ss, op61 ss, op87 SS, op55 Ss, op72 Ss, op58 Ss (Values in Arbitrary Units).

Figure 5: Details corresponding to Example 7. Panel A: Probes designed for hybridisation with the amplified fragment of gene Collagen I Al, in a region common to genotypes S and s. Panel B: Results obtained in an array tube assay with the probes of Panel A and the seven samples: op42 ss, op90 ss, op61 ss, op87 SS, op55 Ss, op72 Ss, op58 Ss (Values in Arbitrary Units).

Figure 6: Comparison of SEQ ID No 217 and SEQ ID No 15 in detecting Collagen I Al gene.

Figure 7: Comparison of SEQ ID No 14, 218, and 219 in detecting Estrogen Receptor gene.

Definitions.

The following terms have the indicated meanings in the specification unless expressly indicated to have a different meaning: - Amplification Primers: Nucleic acids or nucleic acid analogs that bind to and allow amplification of one or more target sequences.

- Multiplex amplification reaction: reaction that allows amplification of two or more nucleic acid sequences if present.

- Probes: Nucleic acid with ability to specifically bind a target nucleic acid sequence. This includes DNA, RNA, PNA, and so forth.

- Target sequences: Sequences to be amplified.

- Target-specific probes: Probes that hybridize specifically with the target sequences amplifiable in the amplification reactions.

- Array tube: A reaction vessel which has a shape and size typical of a laboratory reaction vessel (for example, a 1.5 ml Eppendorf tube) with a microarray arranged therein in which microarray based tests can be carried out.

- Array strip: A set of reaction vessels, usually 8, or multiples of 8, each with a microarray arranged therein, in which microarray based tests can be carried OUt.

Detailed description of the Snvention

The present invention relates to a method and a kit for the simultaneous genotyping of Calcitonin Receptor and Collagen I Al genes. More specifically, the assay further allows genotyping of Vitamin D Receptor and Estrogen Receptor and, in a more preferred embodiment, additionally the genotyping of Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR genes in clinical samples.

In preferred embodiments, the gene regions to be amplified comprise the polymorphisms: i) AIuI of the Calcitonin Receptor; Fokl and/or Bsml of the Vitamin D Receptor; Xbal and/or PvuII of the Estrogen Receptor; COLlAl SpI of Collagen I Al; ii) Osteoprotegerin OPG 209 G>A, OPG 245 T>G and/or OPG163 A>G; LRP5 C171346A, C135242T and/or G138351A; CYP17 T(27)-C; CYP19 C(1558)- T; MTHFR C677T. Preferably these two groups are amplified in two separate multiplex reactions. We have determined that multiplexes involving genes from both groups simultaneously are less effective. There may be two sequential multiplexes carried out on the same sample; or the sample may be split and separate multiplexes carried out on each portion of the sample.

In preferred embodiments of the present invention the assay for genotyping polymorphisms of interest comprises the following steps:

1. Amplification of sample DNA: DNA obtained from clinical samples is amplified, preferably by PCR. Although PCR is the preferred amplification method, amplification of target sequences in a sample may be accomplished by any other method known in the art (ligase chain reaction, transcription-based amplification system, strand displacement amplification, etc).

Preferably, the group of genes i) comprising Calcitonin Receptor, Vitamin D Receptor, Estrogen Receptor and Collagen I Al is amplified in a first reaction tube, while the group of genes ii) comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR is amplified in a second reaction tube. Amplification primers may preferably be selected from those displayed in Tables 1 and 3, respectively.

Nucleotides of the sequences of Tables 1 to 5 are designated as follows: G for Guanine, A for Adenine, T for Thymine, C for Cytosine, M for nucleotides A or C, and Y for nucleotides T or C. The nucleotides as used in the present invention may be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups which do not essentially alter their hybridization characteristics. 2. A label is introduced in the amplified DNA during its amplification to allow further detection, preferably a label that provides a signal that may be detected by colorimetric methods. In a preferred embodiment, at least one of the primers used is labelled at the 5' end with biotin. However, any other kind of label known in the art may be used (e. g. digoxigenin). Furthermore, labelling of amplified DNA may be alternatively achieved by adding modified nucleotides bearing a label (e. g. biotinylated or digoxigenin dUTP derivatives) in the amplification mixture. Radioactive labels may be used, or fluorophores, in certain embodiments.

3. Hybridization: amplified DNA from step (i) is denatured (e.g. by heat). Other ways to prepare single stranded DNA after amplification may be used as well; for example, chemical means. The single stranded DNA is then incubated with a plurality of target-specific probes provided on a solid support. At least one, but preferably more than one probe with ability to hybridise with each target sequence, are provided on the solid support. In certain embodiments of the invention, the solid support is not necessary, and the single stranded DNA may be incubated with target-specific probes provided in solution; however, it is preferred that a solid support be used.

In a preferred embodiment, the solid support is located within a reaction vessel suitable for containing the sample. In a preferred embodiment, the probes of the present invention are provided on a solid support located within an array tube. A single array tube or, alternatively, an independent array tube for each set of genes may be used. In another embodiment, the probes of the present invention are provided on a solid support located within an array strip. In a preferred embodiment, the array strip of the present invention has 8 wells. In another preferred embodiment, the solid support is a coated glass slide. In a preferred embodiment, the probes of the present invention contain an amino group and the support comprises epoxy groups; this promotes binding of the probes to the support.

Preferably, one or more probes contained in the array tube are selected from the probes shown in Tables 2, 4 and 5.

4. Detection: DNA hybrids may be detected by recognition of the labei by specific binding to a lϊgand or by immunodetection. In the preferred embodiment, biotin label is detected by specific binding to streptavidin conjugated with horse-radish-peroxidase (HRP) and the subsequent conversion of tetramethyibenzidine (TMB) to a blue pigment that precipitates in the location where the corresponding specific probe was bound. Other kind of conjugates well known in the art may also be suitable for purposes of the present invention (e. g. streptavidin-Au conjugate). Fluorescently labelled detection systems may instead be used, either indirectly or directiy labelled. Alternatively, other enzyme-based systems may be used.

5. Analysis and processing of the results: array tubes so processed can be read using simple optical devices, such as an optical microscope or ATROl and ATS readers manufactured by CLONDIAG chip technologies GmbH (Jena, Germany). A preferred device for processing array strips is ASP, manufactured by CLONDIAG chip technologies GmbH (Jena, Germany).

Table 1: Amplification Primers for Calcitonin Receptor, Vitamin D Receptor, Estrogen Receptor and Collagen I Al genes.

Table 2: Detection probes for polymorphisms AIuI of the Calcitonin Receptor, Fokl and Bsml of the Vitamin D Receptor, Xbal and PvuII of the Estrogen Receptor and COLlAl SpI of Collagen I Al.

Table 3: Amplification primers for Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR.

Table 4: Detection probes for polymorphisms Osteoprotegerin OPG 209 G>A, OPG 245 T>G and OPG163 A>G, LRP5 C171346A, C135242T and G138351A, CYP17 T(27)-C; CYP19 C(1558)-T and MTHFR C677T.

Table 5: Additional detection probes for polymorphisms of Collagen I Al, Estrogen Receptor and Vitamin D Receptor.

The solid support used in the present invention may comprise one or more allele-specific probes selected from nucleotide sequences from the sequence lists of Tables 2, 4 and 5. The probes may be selected only from table 2; only from table 4; only from table 5; or from any combination of tables 2, 4 and 5. Probe sequences are represented as single stranded DNA oligonucleotides from the 5' to the 3' end.

It is apparent to those skilled in the art that any of these probes can be used as such, or in their complementary form, or in their RNA form (wherein T is replaced by U), as long as this change of sequence does not affect dramatically its functionality, Such probes are all within the scope of the present invention. Also apparent to the person skilled in the art is that any probe of tables 2, 4 or 5, which has been prepared by adding or changing one or more nucleotides of its sequence without dramatically affecting to its functionality, is also within the scope of the present invention. Preferably, no more than 5, 4, 3, 2, 1 nucleotides are modified.

Specific probes for SNPs associated with osteoporosis were selected as follows: The design of the probes was made based on the sequence of each gene deposited in GenBank, for each amplified region. The design was performed using a conventional nucleic acid program, such as Oligo (6 version). Potential sequences of oligonucleotides to be used as specific probes were selected from these sequence regions, preferably having following features: G+C balanced in both sides of the SNP, size of the probe, preferably an approximate length between 15-30 nt, more preferably 15-23 nt; preferably with no secondary structures or strings of consecutive same nucleotide longer than 4; preferably with a G+C ratio between 45 and 60 % and a Tm as much similar among all selected probes as possible.

Each potential probe sequence selected as aforementioned was compared between all the amplified products using the BLAST program (B2B) from the NCBI webpage (Altschul et al. Nucleic Acid Res. 1997, 25: 3389-3402).

The present invention provides probes for specific detection of polymorphisms AIuI of the Calcitonin Receptor, Fokl and Bsml of the Vitamin D Receptor, Xbal and PvuII of the Estrogen Receptor and COLlAl SpI of Collagen I Al (Table 2; SEQ ID MO 11-29; Table 5; SEQ ID NO 212-222), and of Osteoprotegerin OPG 209 G>A, OPG 245 T>G and OPG163 A>G, LRP5 C171346A, C135242T and G138351A, CYP17 T(27)-C; CYP19 C(1558)-T and MTHFR C677T (Table 4, SEQ ID NO 44-211).

In addition, the solid support may comprise one or more probes for specific detection of controls such as PCR reaction control or adequacy of the DNA from the sample control. Furthermore, it may also comprise one or more labelled oligonucleotides (e.g. biotin modified oligonucleotides) for positive control of the detection reaction and for positioning reference so that all remaining probes can be located.

Oligonucleotide sequences, Marker-1 [GCA GTA TAA GAT TAT TGA TGC CGG AAC] and Marker-2 [GTC AAA ACC TGG GAT AGT AGT TTT ACC], have no significant homology to any of the amplified products of the present invention, When immobilized to the surface of the microarray, biotin modified oligonucleotides Marker-1 and Marker-2 serve as positive control of the PCR products detection reaction and as positioning reference so that all remaining probes can be located.

The probes of the present invention can be obtained by different methods, such as chemical synthesis (e. g. by the conventional phosphotriester method) or genetic engineering techniques, for example by molecular cloning of recombinant plasmids in which corresponding nucleotide sequences have been inserted and can be later obtained by digestion with nucleases.

In a preferred embodiment of the present invention, 5' amine-linked oligonucleotide probes are bound to the surface of a solid support in known distinct locations. Said probes may be immobilized individually or as mixtures to delineated locations on the solid support. Said probes or mixtures of probes may be immobilized in a single location of the solid support, preferably in two distinct locations of the solid support and more preferably in three distinct locations of the solid support. Figure 1 exemplifies a schematic representation of an arrangement of probes on the surface of the microarray. The numbers refer to the SEQ ID Nos as given in the above tables, while M represents markers, and O represents no probe.

In preferred embodiments of the present invention, the probes are provided on a solid support located within an array tube or within an array strip. The array tube and array strip of the present invention may comprise one or more probes selected from nucleotide sequences SEQ ID N⁰ 11 to SEQ ID N⁰ 29, SEQ ID N°44 to SEQ ID N⁰ 211, and SEQ ID N°212 to SEQ ID N⁰ 222. In addition, they may comprise one or more probes for specific detection of controls such as PCR reaction control or adequacy of the DNA from the sample control. Furthermore, they may also comprise one or more labelled oligonucleotides (e.g. biotin modified oligonucleotides) for positive control of the detection reaction and for positioning reference so that all remaining probes can be located.

After incubation of single stranded DNA with a plurality of target-specific probes, DNA hybrids may be detected by recognition of the label by specific binding to a ligand or by immunodetection.

In a preferred embodiment, biotin label is detected by specific binding to streptavidin conjugated with horse-radish-peroxidase (HRP) and the subsequent conversion of tetramethylbenzidine (TMB) to a blue pigment that precipitates in the concrete location where corresponding specific probe was bound. Other kind of conjugates well known in the art may also be suitable for purposes of the present invention (e. g. streptavidin-Au conjugate). Fluorescently labelled detection systems may instead be used, either indirectly or directly labelled. Alternatively, other enzyme-based systems may be used.

In a preferred embodiment, visualization of array tubes and array strips consists of the following steps; first, the image of the array is captured using an optical device, then, the image is analysed, and finally, a report containing an interpretation of the result is provided.

In the most preferred embodiment, the image is analysed by means of appropriate software. Any device suitable for this processing can be used. Preferred devices for processing array tubes are ATROl and ATS readers manufactured by CLONDIAG chip technologies GmbH (Jena, Germany). A preferred device for processing array strips is ASP, manufactured by CLONDIAG chip technologies GmbH (Jena, Germany). In a preferred embodiment, processing of the array strips can be automated in a specific workstation. In a more preferred embodiment such processing comprises all the washing and incubation steps of the method, starting from the PCR and ending with visualization of the result.

One process for preparing the array tube is disclosed in Patent Application No. US2005064469. In a preferred embodiment of the present invention, 5¹ amine- linked oligonucleotide probes are bound to the surface of a solid support in known distinct locations.

The present invention relates to an in vitro diagnostic kit for genotyping Calcitonin Receptor and Collagen I Al genes. In another embodiment such a kit might also allow genotyping of Vitamin D Receptor and/or Estrogen Receptor genes.

The present invention also relates to an in vitro diagnostic kit for genotyping Calcitonin Receptor and Collagen I Al, and optionally, Vitamin D Receptor and/or Estrogen Receptor, further allowing genotyping of one or more genes selected from the group of genes comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR.

The present invention also relates to an in vitro diagnostic kit for genotyping Calcitonin Receptor, Collagen I Al, Vitamin D Receptor and Estrogen Receptor genes and of Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR genes, in clinical samples.

Preferably, the mentioned kit comprises any or all of the following components: one or more amplification mix(es), including amplification buffer, dNTPs, primers, and control plasmid; wash buffer; detection reagents; array tube(s) including a solid support including allele-specific probes; reagents for obtaining and preparing a sample. The particular components will depend on the exact conditions under which the kit is intended to be used, although the skilled person will be able to determine suitable kit components and buffer compositions.

In a preferred embodiment the kit comprises: i) a nucleic acid amplification mix comprising two or more pairs of amplification primers, wherein at least one pair allows amplification of Calcitonin Receptor and at least another pair allows amplification of Collagen I Al, ii) a reaction vessel or set of reaction vessels containing a solid support that comprises target-specific probes, wherein at least one probe is specific for Calcitonin Receptor and at least another probe is specific for Collagen I Al, and iii) reagents for use in visualising hybridisation of nucleic acids to the probes of the solid support.

In another preferred embodiment the kit comprises:

i) a nucleic acid amplification mix comprising four or more pairs of amplification primers, wherein at least one pair allows amplification of

Calcitonin Receptor, at least another pair allows amplification of Collagen I Al, at least another pair allows amplification of Vitamin D Receptor and at least another pair allows amplification of Estrogen Receptor,

ii) a reaction vessel or set of reaction vessels containing a solid support that comprises target-specific probes, wherein at least one probe is specific for Calcitonin Receptor; at least another probe is specific for Collagen I

Al, at least another probe is specific for Vitamin D Receptor; and at least another probe is specific for Estrogen Receptor, and

iii) reagents for use in visualising hybridisation of nucleic acids to the probes of the solid support.

In yet another preferred embodiment the kit comprises: i) two nucleic acid amplification mixes, one of them comprising two or more pairs of amplification primers, wherein at least one pair allows amplification of Calcitonin Receptor and at least another pair allows amplification of Collagen I Al; the other amplification mix comprising two or more pairs of amplification primers that allow amplification of two or more of the genes of the group of genes comprising Osteoprotegerih, LRP5, CYP17, CYP19 and MTHFR, ii) a reaction vessel or set of reaction vessels containing a solid support that comprises target-specific probes, wherein at least one probe is specific for Calcitonin Receptor, at least another probe is specific for Collagen I Al, and at least two or more probes are specific for the genes of the group of genes comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR amplifyabie by the amplification mixture of i), iii) reagents for use in visualising hybridisation of nucleic acids to the probes of the solid support.

In yet another preferred embodiment the kit comprises:

i) two nucleic acid amplification mixes, one of them comprising two or more pairs of amplification primers, wherein at least one pair allows amplification of Calcitonin Receptor and at least another pair allows amplification of Collagen I Al; the other amplification mix comprising two or more pairs of amplification primers that allow amplification of two or more of the genes of the group of genes comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR, ii) two reaction vessels or sets of reaction vessels, one containing a solid support that comprises target-specific probes, wherein at least one probe is specific for Calcitonin Receptor, at least another probe is specific for Collagen I Al, the other containing a support comprising at least two or more probes specific for the genes of the group of genes comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR amplifyabie by the amplification mixture of i), iii) reagents for use in visualising hybridisation of nucleic acids to the probes of the solid support.

Examples The examples provided below merely illustrate the invention and in no way limit the scope of the accompanying claims.

EXAMPLE 1: preparation of array tubes

Array tubes of the present invention were manufactured at CLONDIAG chip Technologies GmbH (Jena, Germany) as follows. A standard reaction test tube from Eppendorf made of polypropylene and having a nominal receiving volume of 1,5 ml was modified by re-melting, so that, an opened recess for the microarray support with an adhesive edge was modelled into the tube.

Microarrays to be inserted into these tubes were produced by using a MicroGrid II Arrayer (BioRobotics, Cambridge, Great Britain). Probes consisting of 5' end amino-modified oligonucleotides having a sequence from the sequence list were deposited at defined sites on an epoxidized glass surface of a slide (slide size: 75 mm x 25 mm) and covalently immobilised. A single microarray included 12 x 10 = 120, or 12 x 11 = 132 concrete locations at which oligonucleotides could be deposited. These locations have a spacing of 0.2 mm, so that the DNA library included in each microarray covered an area of 2.4 mm x 2.4 mm and, in total, more than 100 identical DNA libraries could be produced in this way per slide. Depending on the type of experiment, either one single probe or a mixture of them could be deposited at each one of these locations. Usually, single probes were deposited at each location when specificity and sensitivity experiments for probes selection were carried out. Once the probes have been validated, mixtures of probes capable of hybridizing in separate regions of the amplified product of a specific polymorphism genotype associated with osteoporosis could be deposited in the same location when identification of polymorphism genotypes assays were performed. Figure 2 shows the localization of the probes within microarrays used for this invention. The schematic representation of the arrangement of probes corresponding to Figure 2 is displayed in Figure 1. Four replicates for each probe or mixture of probes were included in each microarray. Besides specific probes for SNP genotypes associated to osteoporosis and for detection of amplification control and adequacy of DNA control, microarrays included reference markers at several locations consisting of 5' end biotin modified oligonucleotides with no significant homology for any of the amplified sequences from this invention (Marker-1 [GCA GTA TAA GAT TAT TGA TGC CGG AAC] and Marker-2 [GTC AAA ACC TGG GAT AGT AGT TTT ACC]). These reference markers served both for verifying proper performance of the detection reaction and for optical orientation of the image by the reader so all remaining probes can be located and the data analyzed.

All oligonucleotides were deposited on the slide from a Ix QMT Spotting Solution I (Quantifoil Micro Tools GmbH, Jena, Germany). Total concentration of oligonucleotides in each spotting solution ranged from 2,5 mM for reference markers to 20 mM for specific probes. Oligonucleotides were then covalently linked to the epoxide groups on the glass surface by baking at 6O⁰C for 30 minutes followed by a multi-step washing process. Dried slides were cut into 3.15 mm x 3.15 mm glass pieces which, strictly speaking, are what we name microarrays. In the final step for array tubes manufacturing, these microarrays were then inserted into the aforementioned modified Eppendorf tubes and glued to the adhesive edge.

EXAMPLE 2: preparation of DNA samples

2.1. Clinical samples For the purpose of genotyping the genes of the present invention, it is first of all necessary to separate DNA from remaining biological material. Preparation of DNA procedures vary according to sample source. Specific examples are provided for preparation of DNA from blood samples:

A commercial kit (NucleoSpin® Tissue kit Catalogue No. 635966 from BD Biosciences Clontech, Palo Alto, CA, USA) designed for DNA isolation from samples from a variety of sources was used to process blood samples. In this case, protocol was performed following manufacturer specifications for isolation of genomic DNA from 200 μl of total blood: 25 μl of Proteinase K was added to

200 μl of total blood, 200 μl of Buffer B3 was added to the sampIe+Proteinase K. After a vigorous vortexing the sample was incubated at 7O⁰C for 30 minutes.

210 μl of Ethanoi 96% was added to the sample and it was vigorous vortexing. Sample material was transferred to a column and it was centrifuged at 11000 g for 1 minute. The column was washed with 500 μl of Buffer BW and it was centrifuged at 11000 g for 1 minute, the supernatant was discarded and the column was newly washed with 600 μl of Buffer B5 and it was centrifuged at HOOOg for 1 minute. The obtained supernatant was discarded and the column was newly centrifuged at HOOOg for 1 minute to dry the filter. The column was transferred to a 1.5 ml Eppendorf tube and 60 μl of Buffer BE (prewarmed at 7O⁰C) was added and incubated for 3 minutes at room temperature. The column was centrifuged at IiOOOg for 1 minute and supernatant was transferred to a clean and sterile tube. An Aliquot of 5 μl was subsequently used in the PCR reaction.

Whatever it was the type of clinical sample or the DNA preparation method, negative controls were run in parallel with each batch of samples.

EXAMPLE 3: PCR amplification

PCR amplification using primers described as SEQ ID N⁰I, 2, 3, 4, 5, 6, 7, 8, 9, and 10 was performed. Briefly, PCR amplification was carried out in a 50 μl final volume reaction containing 10 mM Tris-HCI pH 8.3, 50 mM KCI, 1 mM MgCI2, 0.2 μM each primer of SEQ ID NO I₁ 2, 3, 4, 5, 6, 7, 8, 9, and 10, 500 μM of dNTPs (dATP, dTTP, dGTP, dCTP), 5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA), and 5 μl of each clinical sample DNA from Example 2.1. All forward and reverse primers used in the PCR reaction were biotin modified at the 5' end so that any amplified DNA could be subsequently detected. The following concentrations of primers were also tested, giving the same result: SEQ ID NO 1, 0.09449 μM, SEQ ID NO 2, 0.10604 μM, SEQ ID NO 3, 0.09174 μM, SEQ ID NO 4, 0.10197 μM, SEQ ID NO 5, 0.13805 μM, SEQ ID NO 6, 0.13375 μM, SEQ ID NO 7, 0.14081 μM, SEQ ID NO 8, 0.04257 μM, SEQ ID NO 9, 0,11225 μM, and SEQ ID NO 10, 0.1449 μM.

Similarly, PCR amplification of sample DNAs was carried out in a 50 μl final volume reaction using 0.13-0.15 μM of each primer of SEQ ID NO 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42 and 43 in the presence of 500 μM of dNTPs

(dATP, dTTP, dGTP, dCTP), 2.25 μM Ci₂Mg, 5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA), and 5 μl of each clinical sample DNA from Example 2,1.

Negative controls constituted of 5 μl of blank samples from Example 2.1. or 5 μl of deionised water were processed in parallel with the sample DNA. The use of these kinds of negative controls serves to check that contamination does not occur at any point in sample handling or in PCR reaction setting up and all positive results represent true presence of DNA in the sample.

PCR reactions were run in a Mastercycler thermocycler (Eppendorf, Hamburg, Germany) programmed with the following cycling profile: one initial denaturing cycle at 95⁰C for 12 minutes, 40 cycles of 30 seconds at 95⁰C, 40 seconds at 55-56⁰C and 60 seconds at 72⁰C, and one final extension cycle at 6O⁰C for 30 minutes. After amplification, 5 μl of each reaction were used for subsequent detection with specific probes.

EXAMPLE 4: simultaneous identification of osteoporosis polymorphism genotypes using array tubes

Array tubes were pre-washed just before its use by addition of 300 μl of 0.5X PBS-Tween 20 buffer to each tube and inverting them several times. All liquid from inside each tube was removed using a Pasteur pipette connected with a vacuum system, Five microliters of amplification reactions from Example 3 were added to 95 μl of hybridization solution (250 mM sodium phosphate buffer, pH 7.2; SSC IX; SDS 4.5%; 10 mM EDTA, pH 8.0), This mixture was denatured by heating them to 95⁰C for 10 minutes and, immediately after, was applied to the array tube prepared in Example 1 prewarmed at 5O⁰C in a Thermomixer comfort (Eppendorf, Hamburg, Germany) for i or 2 minutes. Hybridization reaction was carried out in a Thermomixer comfort (Eppendorf, Hamburg, Germany) by incubating the array tubes at 5O⁰C for one hour and 30 minutes with shaking at 550 rpm. After incubation period, hybridization reaction was removed using a Pasteur pipette connected with a vacuum system and a washing step with 300 μl of 0,5X PBS-Tween 20 buffer was carried out. Hybridized DNA was detected by incubation in 100 μl of a 0.075 μg/ml PoIy- HRP Streptavidin (Pierce Biotechnology Inc., Rockford, IL, USA) solution at 3O⁰C for 15 minutes with shaking at 550 rpm. Then, all liquid from the array tube was quickly removed and two washing steps as that aforementioned were carried out. Colour developing reaction was performed in 100 μl of True BlueTM Peroxidase Substrate (KPL, Gaithersburg, MD, USA), which consists of a buffered solution containing 3,3',5,5'-tetramethylbenzidine (TMB) and H₂O₂, by incubation at 25⁰C for 10 minutes. The coloured precipitates so produced cause changes in the optical transmission at concrete locations of the microarray that can be read using an ATROl or an ATS reader manufactured by CLOfMDIAG chip technologies GmbH (Jena, Germany). Optionally, ATS reader may have specific software installed for automatic processing of the sample analysis result obtained with the array tube developed in the present invention.

EXAMPLE 5: Importance of the design of amplification primers on the result of the Multiplex PCR.

The five pairs of primers corresponding to SEQ ID N⁰ 1-10 were designed as amplification primers for fragments containing the polymorphisms AIuI of the Calcitonin Receptor (SEQ ID N⁰ 7 & 8), Fokl and Bsml of the Vitamin D Receptor (SEQ ID N⁰ 9 & 10 and 1 & 2, respetively), Xbal and PvuII of the Estrogen Receptor (SEQ ID N⁰ 3 & 4, which serve the purpose of amplification of a fragment containing both polymorphisms) and COLlAl SpI of Collagen I Al (SEQ ID N⁰ 5 & 6).

The criterion that was followed for the primers' design was that all of them shared the same Melting Temperature. Multiplex-PCRs were carried out on 5 μl of DNA extracted from samples 1, 2 or 3, under the conditions displayed on panel A of Figure 3. Three different cycling profiles were tested for each sample: The one of panel B of Figure 3, wherein the hybridization temperature is 55⁰C, as well as two profiles wherein the hybridization temperatures were 49.9⁰C and 53.6⁰C,

The result of these PCRs is displayed on Figure 3, panel C. Lanes 1, 4 and 7 correspond to Sample 1; Lanes 2, 5 and 8 correspond to Sample 2, and Lanes 3, 6 and 9 correspond to Sample 3. The lower amplification levels corresponding to Sample 2 may be due to the fact that the concentration of the DNA extracted from this sample was lower than the optimal value of 40 ng/μl. Differently, the concentration of the DNA extracted from the samples 1 and 3 was 40 ng/μl. Amplification fragments corresponding to Calcitonin Receptor as well as to polymorphism Fokl of the Vitamin D Receptor are 157 and 188 bp long, respectively. Their similar size is the reason why they appear as a single band.

As it can be observed of Figure 3, panel C, at hybridization temperatures of 49.9⁰C and 53,6⁰C, amplification of some of the fragments containing the polymorphisms of interest was obtained. However, at hybridization temperatures of 55⁰C an optimal amplification was obtained,

Therefore, not any random primer combination results in a successful Multiplex PCR. On the contrary, the following criteria have to be fulfilled: i) Primers have to share the same Melting Temperature, and ii) the hybridization temperature of the cycling profile of the Multipiex-PCR must suit the Melting Temperature of the amplification primers.

EXAMPLE 6: Analysis of probes designed for the detection of genotypes S or s corresponding to the polymorphism COLlAl SpI of gene Collagen I Al.

The three allelic variants of the polymorphism COLlAl SpI of gene Collagen I Al are:

Seven samples (op42 ss, op90 ss, opβl ss, op87 SS, op55 Ss, op72 Ss, op58 Ss), whose genotypes corresponding to polymorphism COLlAl SpI of gene Collagen I Al were known by DNA sequencing, were tested. Genotype of samples op42 ss, op90 ss, opβl ss is ss; Genotype of sample op87 SS is SS; Genotype of samples op55 Ss, op72 Ss, op58 Ss is Ss, Samples were subjected to DNA amplification in a Multiplex-PCR according to conditions described in example 5, the hybridization temperature being of 55⁰C, Amplification products were denatured and incubated with an array tube comprising the probes of Figure 4, Panel A, under the conditions already described in Example 4. The results obtained after visualization are displayed in Figure 4, Panel B (Values in Arbitrary Units).

In the particular case of polymorphism COLlAl SpI of gene Collagen I Al, the most suitable probe length was 19 nt. As it can be observed in Figure 4, Panel B, of all the tested probes COLT-S19-AR (SEQ ID N⁰ 27) detects genotype s in the samples op42 ss, op90 ss, opβl ss, op55 Ss, op72 Ss, op58 Ss with intensity values higher than 200 Arbitrary Units, while it provides an intensity value lower than 20 Arbitrary Units in sample op87 SS, homozygote for S.

Of the remaining probes tested, either absence of hybridization or of genotype discrimination was observed. EXAMPLE 7: Analysis of probes designed for the detection of the amplified fragment corresponding to polymorphism COLiAl SpI of gene Collagen I Al.

Probes were designed that might hybridise with the amplification fragment of the Multiplex-PCR corresponding to polymorphism COLlAl SpI of gene Collagen I Al, independently of the genotype that might be present. Thus, a requisite of such probes was that they had the ability to hybridise with a region of the amplification fragment wherein the polymorphism would not be contained.

Two of such probes are displayed in Figure 5, Panel A. Amplification fragments corresponding to samples of Example 6 (op42 ss, op90 ss, opβl ss, op87 SS, op55 Ss, op72 Ss, op58 Ss), obtained as already described in Example 6, were denatured and incubated with an array tube comprising the probes of Figure 5, Panel A, under the conditions already described in Example 4. The results obtained after visualization are displayed in Figure 5, Panel B (Values in Arbitrary Units). As it can be observed, the probe COLGE-S30-AR (SEQ ID N⁰ 217) gives a good intensity value, while COLl Al G does not.

EXAMPLE 8: Comparative Testing of Probes.

Additional experiments directed to the testing of probes of the present invention were carried out under the experimental conditions of Examples 6 and 7 (i.e. amplification conditions of Example 6 and hybridization conditions according to Example 4).

8.1 Probes corresponding to polymorphism COLlAl SpI of gene Collagen I AL

Probe of SEQ ID N⁰ 217 was again compared with another probe, probe of SEQ ID N⁰ 15, both of which were designed to hybridise with the amplification fragment of the Muitiplex-PCR corresponding to polymorphism COLlAl SpI of gene Collagen I Al independently of the genotype that might be present. As already stated, a requisite of such probes is that they have the ability to hybridise with a region of the amplification fragment wherein the polymorphism is not contained.

21 samples were tested in duplicate, under the experimental conditions specified above,

As can be observed in Figure 6, probe of SEQ ID N⁰ 217 provides a better hybridization than probe of SEQ ID N⁰ 15, in 14 out of the 21 samples.

Conclusion: Probe of SEQ ID N⁰ 217 provides better hybridization levels than probe of SEQ ID N⁰ 15 in the detection of gene CoIlAl.

Analysis of probes for the detection of genotype "s" corresponding to polymorphism COLlAl SpI of gene Collagen I AL

Probes that were tested were:

The same 21 samples as above were tested in duplicate, under the same experimental conditions.

Genotype distribution was as follows: SS: 8 samples; Ss: 7 samples; ss: 6 samples.

Results of this analysis show that probes of SEQ ID N⁰ 215 and of SEQ ID N⁰ 27 provide similar values in the detection of polymorphic allele "s" of COLlAl SpI, while probe of SEQ ID N⁰ 216 provides worse results.

Analysis of probes for the detection of genotype "S" corresponding to polymorphism COLlAl SpI of gene Collagen I Al.

Probes that were tested were:

None of the three probes hybridises with samples of genotype ss, thus providing a 100% discrimination of genotype ss. Nevertheless, none of these probes provides a particular good intensity when detecting allele "S", the intensity being lost in 7 samples out of 15.

Therefore, probes of SEQ ID N⁰ 212, 213 and 214 do not provide a good alternative for the detection of allele ^HS" of COLlAl SpI, they constitute an alternative for the discrimination of allele "s".

8.2 Probes corresponding to the amplification fragment of the Estrogen Receptor gene.

Within the amplification fragment of the Multiplex-PCR corresponding to the Estrogen Receptor gene, both polymorphisms Xbal and PvuII are contained. Probes of SEQ ID N⁰ 14, 218 and 219 were designed to hybridise with the amplification fragment of the Multipiex-PCR corresponding to the Estrogen Receptor gene. A requisite of such probes is that they have the ability to hybridise with a region of the amplification fragment wherein no polymorphism is contained.

15 samples were tested in duplicate, under the experimental conditions specified above. The results show that probes of SEQ ID N⁰ 218 and 219 provide high intensity values, while those of probe of SEQ ID N⁰ 14 are lower (see Figure 7). The results obtained with probe of SEQ ID N⁰ 14 are enclosed by circles, while those enclosed within the rectangles correspond to probes of SEQ ID N⁰ 218 and 219, in this order.

Analysis of probes for the detection of genotype "p" corresponding to polymorphism PvuII of the Estrogen Receptor gene.

The following two probes were tested:

15 samples were tested in duplicate, under the experimental conditions specified above. Genotype distribution was as follows: PP: 5 samples; Pp: 5 samples; pp: 5 samples.

The probe of SEQ ID N⁰ 220 improves the detection of genotype ^Λλpp" with respect to SEQ ID N⁰ 19, in 2 out of 5 samples. Further, the probe of SEQ ID N⁰ 220 improves the detection of genotype "Pp" in 5 out of 5 samples, when compared with the probe of SEQ ID N⁰ 19. Finally, detection of genotype "PP" of the final 5 samples remained the same both with probes of SEQ ID N⁰ 220 and 19.

According to the present results, probe of SEQ ID N⁰ 220 provides a better detection of allele "p" of polymorphism PvuII of the Estrogen Receptor gene, than probe of SEQ ID N⁰ 19.

8.3 Probes corresponding to polymorphism Fokl of gene Vitamin D Receptor.

Analysis of probes for the detection of genotype "f" corresponding to polymorphism Fokl of gene Vitamin D Receptor.

The following two probes were tested:

16 samples were tested in duplicate, under the experimental conditions specified above. Genotype distribution was as follows: FF: 7 samples; Ff: 7 samples; ff: 2 samples.

According to results of these experiments, both probes of SEQ ID N⁰ 16 and SEQ ID N⁰ 222 are able to discriminate the presence or absence of allele "f". Nevertheless, the probe of SEQ ID N⁰ 222 provides higher hybridization levels in 8 out of 9 samples.

Conclusion: Although both probes of SEQ ID N⁰ 16 and of SEQ ID N⁰ 222 are able to discriminate the presence or absence of allele "f" of polymorphism Fokl of gene Vitamin D Receptor, probe of SEQ ID N⁰ 222 provides higher hybridization levels than probe of SEQ ID N⁰ 16 in the detection of allele "f".

8.4 Probes corresponding to polymorphism Bsml of gene Vitamin D Receptor.

Analysis of probes for the detection of genotype "b" corresponding to polymorphism Bsml of gene Vitamin D Receptor.

The following two probes were tested:

16 samples were tested in duplicate, under the experimentai conditions specified above. Genotype distribution was as follows: BB: 4 samples; Bb: 6 samples; bb: 6 samples.

The same results were obtained with both probes.

Conclusion: Both probes of SEQ ID N⁰ 17 and SEQ ID N⁰ 221 provide similar effects in the detection of genotype "b" corresponding to polymorphism Bsml of gene Vitamin D Receptor.

Claims

1. An assay comprising genotyping Caicitonin Receptor and Collagen I Al in a sample, characterized in that a multiplex amplification reaction of the Calcitonin Receptor and Collagen I Al genes is performed on the sample.

2. The assay of claim 1 wherein the multiplex amplification further comprises amplification of the Vitamin D Receptor and/or the Estrogen Receptor genes.

3. The assay according to claims 1 or 2, wherein, further to the multiplex amplification reaction of the group of genes i), comprising Calcitonin Receptor and Collagen I Al, and optionally, Vitamin D Receptor and/or Estrogen Receptor, an additional amplification reaction of one or more genes being selected from the group of genes ii) comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR, is carried out on the sample.

4. The assay according to claim 3, wherein the additional amplification reaction is a multiplex amplification reaction, and two or more genes from group ii) are amplified.

5. The assay of any preceding claim wherein the gene regions to be amplified comprise the polymorphisms: i) AIuI of the Calcitonin Receptor; COLlAl SpI of Collagen I Al.

6. The assay of claim 2, wherein the gene regions to be amplified comprise the polymorphisms: i) AIuI of the Calcitonin Receptor; Fokl and/or Bsml of the Vitamin D Receptor; Xbal and/or PvuII of the Estrogen Receptor; COLlAl SpI of Collagen I Al.

7. The assay of claim 3 or 4, wherein the gene regions to be amplified comprise the polymorphisms: i) A!ul of the Calcitonin Receptor; Fokl and/or Bsmϊ of the Vitamin D Receptor; Xbal and/or PvuII of the Estrogen Receptor; COLlAl SpI of Collagen I Al; ii) Osteoprotegerin OPG 209 G>A, OPG 245 T>G and/or OPG163 A>G;

LRP5 C171346A, C135242T and/or G138351A; CYP17 T(27)-C; CYP19 C(1558)- T; MTHFR C677T.

8. The assay of any preceding claim comprising:

performing a nucleic acid amplification reaction on a sample, wherein the selected genes from group i) are amplified in a first reaction; and/or the selected genes from group ii) are amplified in a second reaction; obtaining single stranded oligonucleotides from any amplification products;

allowing single stranded oligonucleotides to hybridise where possible with allele-specific probes corresponding to the genes of interest; and

detecting hybridised oligonucleotides.

9. The assay of any of claims 3, 4 or 7 comprising; performing a nucleic acid amplification reaction on a sample, wherein the selected genes from groups i) and ii) are amplified in the same reaction;

obtaining single stranded oligonucleotides from any amplification products; allowing single stranded oligonucleotides to hybridise where possible with a!le!e-specific probes corresponding to the genes of interest which are immobilised on a solid support, and detecting hybridised oligonucleotides.

10. The assay of claims 8 or 9 wherein the probes corresponding to the genes of interest are immobilised on a solid support, said solid support being located within a reaction vessel suitable for containing the sample.

11. The assay of any of claims 3, 4 or 7 comprising: performing a nucleic acid amplification reaction on a sample, wherein the selected genes from group i) are amplified in a first reaction; and the selected genes from group ii) are amplified in a second reaction; obtaining single stranded oligonucleotides from any amplification products; allowing single stranded oligonucleotides to hybridise where possible with aϋele-specific probes corresponding to the genes of interest which are immobilised on a solid support, the support being located within a reaction vessel suitable for containing the sample; and detecting hybridised oligonucleotides; wherein allele-specific probes for the selected genes from group 1) are immobilised on a first solid support, and allele-specific probes for the selected genes from group Ii) are immobilised on a second solid support, and said first and second solid supports are located within first and second reaction vessels.

12. The assay of claims 8 to 11 wherein the amplification reaction is

PCR.

13. The assay of claims 8 to 12 wherein one or more of the amplification primers for the selected genes from group i) are selected from the group comprising SEQ ID N⁰ 1 to SEQ ID N⁰ 10; and/or one or more of the amplification primers for the selected genes from group ii) are selected from the group comprising SEQ ID N⁰ 30 to SEQ ID N⁰ 43.

14. The assay of claim 13 wherein the amplification primers are biotin modified at the 5' end.

15. The assay of any of claims 8 to 14 comprising combining an amplification reaction mix with the sample to perform the amplification reaction.

16. The assay of any of claims 8 to 15 wherein single stranded oligonucleotides are obtained by denaturing any double stranded oligonucleotides present.

17. The assay of any of claims 8 to 16 wherein the solid support is fixed to the reaction vessel.

18. The assay of any of claims 8 to 17 wherein the solid support comprises a microarray.

19. The assay of any of claims 8 to 18 wherein the reaction vessel is an array tube.

20. The assay of any of claims 8 to 18 wherein the reaction vessel is an array strip.

21. The assay of any of claims 8 to 20 wherein allele-specific probes for polymorphisms of the selected genes are used; the polymorphisms being selected from i) AIuI of the Calcitonin Receptor, Fokl and Bsml of the Vitamin D

Receptor, Xbal and PvuII of the Estrogen Receptor and COLlAl SpI of Collagen I Al; and/or ii) Osteoprotegerin OPG 209 G>A, OPG 245 T>G and OPG163 A>G, LRP5 C171346A, C135242T and G138351A, CYP17 T(27)-C, CYP19 C(1558)-T and MTHFR C677T.

22. The assay of any of claims 8 to 21 wherein allele-specific probes corresponding to the genes of interest comprise DNA.

23. The assay of any of claims 8 to 22 wherein the probes are 15 to

30 nt in length.

24. The assay of any of claims 8 to 23 wherein the probes are 15 to 23 nt in length,

25. The assay of any of claims 8 to 24 wherein one or more of the probes are selected from i) the group comprising SEQ ID N⁰ 11 to SEQ ID N⁰ 29; and/or ii) the group comprising SEQ ID N°44 to SEQ ID N⁰ 211.

26. The assay of any of claims 8 to 25 wherein all of the probes are selected from i) the group comprising SEQ ID N⁰ 11 to SEQ ID N° 29; and/or ii) the group comprising SEQ ID N°44 to SEQ ID N⁰ 211.

27. The assay of any of claims 8 to 24 wherein one or more of the probes are selected from the group comprising SEQ ID N⁰ 212 to SEQ ID N⁰ 222.

28 The assay of any of claims 8 to 27, wherein allele-specific probes are selected to specifically bind to the single stranded oligonucleotides from amplification products under the same hybridisation conditions for all probes.

29 The assay of any of claims 8 to 28 wherein at least one probe is present on the solid support in at least two distinct locations.

30. The assay of any of claims 8 to 29 wherein all probes are present on the solid support in at least two distinct locations.

31. The assay of any preceding claim further comprising detecting one or more control sequences.

32. The assay of claim 31 wherein the control sequence comprises a probe immobilised to a solid support which does not hybridise to the target sequence from any allele selected from polymorphisms of i) the group comprising AIuI of the Calcitonin Receptor, Fokl and Bsml of the Vitamin D Receptor, Xbal and PvuII of the Estrogen Receptor and COLlAl SpI of Collagen I Al; and/or ii) the group comprising Osteoprotegerin OPG 209 G>A, OPG 245

T>G and OPG163 A>G, LRP5 C171346A, C135242T and G138351A, CYP17 T(27)-C, CYP19 C(1558)-T or MTHFR C677T.

33. The assay of any preceding claim comprising providing a reaction vessel including a solid support having two or more alleJe-specific probes immobilised thereon; the probes being for polymorphisms selected from i) the group comprising AIuI of the Calcitonin Receptor, Fokl and Bsmϊ of the Vitamin D Receptor, Xbal and PvuII of the Estrogen Receptor and COLlAl SpI of Collagen I Al; and/or ii) the group comprising Osteoprotegerin OPG 209 G>A, OPG 245 T>G and OPG163 A>G, LRP5 C171346A, C135242T and G138351A, CYP17 T(27)-C, CYP19 C(1558)-T or MTHFR C677T.

34. A solid support comprising two or more probes immobilised thereon, the probes being for genes selected from the group i) comprising Calcitonin Receptor and Collagen I Al.

35, The solid support of claim 34 further comprising probes for

Vitamin D Receptor and/or Estrogen Receptor.

36. The solid support of claim 34 or 35 further comprising one or more probes for genes selected from the group ii) Osteoprotegerin; LRP5; CYP17; CYP19; MTHFR.

37. The solid support of claim 36 wherein the probes are allele- specific probes for two or more polymorphisms selected from the groups comprising i) A!ul of the Calcitonin Receptor; Fokl and/or Bsml of the Vitamin D

Receptor; Xbal and/or PvuII of the Estrogen Receptor; COLlAl SpI of Collagen I Al; and/or ii) Osteoprotegerin OPG 209 G>A, OPG 245 T>G and/or OPG163 A>G; LRP5 C171346A, C135242T and/or G138351A; CYP17 T(27)-C; CYP19 C(1558)- T; MTHFR C677T.

38. The solid support of claims 34 to 37 further comprising one or more probes which hybridise to any amplification product of one or more control sequences.

39. The support of claims 34 to 38 fixed to a reaction vessel.

40. The support of claims 34 to 39 comprising a microarray.

41. The support of claims 34 to 40 wherein one or more of the probes are selected from I) the group comprising SEQ ID N⁰ 11 to SEQ ID N⁰ 29, ii) the group comprising SEQ ID N°44 to SEQ ID N⁰ 211, and iii) the group comprising SEQ ID N°212 to SEQ ID N⁰ 222.

42. The support of claims 34 to 41 wherein the probes comprise DNA.

43. The support of any of claims 34 to 42 wherein the probes are 15 to 30 nt in length.

44. The support of any of claims 34 to 43 wherein the probes are 15 to 23 nt in length.

45. The support of any of claims 34 to 44 wherein ali of the probes are selected from i) the group comprising SEQ ID N⁰ 11 to SEQ ID N⁰ 29; and/or ii) the group comprising SEQ ID N°44 to SEQ ID N⁰ 211,

46. The support of any of claims 34 to 45 wherein one or more of the probes are selected from the group comprising SEQ ID N⁰ 212 to SEQ ID N⁰ 222.

47. The support of any of claims 34 to 46 wherein allele-specific probes are selected to specifically bind to the single stranded oligonucleotides from amplification products under the same hybridisation conditions for all probes.

48. The support of any of claims 34 to 47 wherein at least one probe is present on the solid support in at least two distinct locations.

49. The support of any of claims 34 to 48 wherein all probes are present on the solid support in at least two distinct locations.

50. The support of any of claims 34 to 49 wherein the solid support further comprises at least one probe which does not hybridise to the target sequence from any allele selected from polymorphisms of

i) the group comprising AIuI of the Calcitonin Receptor, Fokl and Bsml of the Vitamin D Receptor, Xbal and PvuII of the Estrogen Receptor and COLlAl SpI of Collagen I Al; and/or

ii) the group comprising Osteo protege rin OPG 209 G>A, OPG 245 T>G and OPG163 A>G, LRP5 C171346A, C135242T and G138351A, CYP17 T(27)-C, CYP19 C(1558)-T or MTHFR C677T.

51. The support of any of claims 34 to 50 being an array tube.

52. The support of claims 34 to 51 being an array strip.

53. A reaction vesse! or set of reaction vessels for performing an assay for genotyping two or more genes in a sample, the genes being selected from the groups comprising i) Calcitonin Receptor; Vitamin D Receptor; Estrogen Receptor; Collagen I Al; and/or ii) Osteoprotegerin; LRP5; CYP 17; CYP 19; MTHFR, the vessel or vessels comprising a solid support according to any one of claims 34 to 52.

54. A kit for genotyping Calcitonin Receptor and Collagen I Al in a sample, characterized in that a multiplex amplification reaction of the Calcitonin Receptor and Collagen I Al genes is performed on the sample.

55. The kit of claim 54 further comprising amplification of the Vitamin D Receptor and/or the Estrogen Receptor genes within the same multiplex amplification reaction.

56. The kit of claims 54 or 55, wherein, further to the multiplex amplification reaction of the group of genes i), comprising Calcitonin Receptor and Collagen I Ai, and optionally, Vitamin D Receptor and/or Estrogen Receptor, an additional multiplex amplification reaction of two or more genes being selected from the group of genes ii) comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR, is carried out on the sample.

57. The kit of claim 56 wherein the gene regions to be amplified comprise the polymorphisms: i) AIuI of the Calcitonin Receptor; Fokl and/or Bsml of the Vitamin D Receptor; Xbal and/or PvuII of the Estrogen Receptor; COLlAl SpI of Collagen I Al; ii) Osteoprotegerin OPG 209 G>A, OPG 245 T>G and/or OPG163 A>G; LRP5 C171346A, C135242T and/or G138351A; CYP17 T(27)-C; CYP19 C(1558)-T; MTHFR C677T.

58. The kit of claims 54 to 57 wherein genotyping comprises an assay according to claims 1 to 33.

59.The kit of any of claims 54 to 58 comprising the solid support of claims 34 to 52.

60. The kit of any of claims 54 to 59 comprising the reaction vessel or set of reaction vessels of claim 53.

61. A kit for genotyping Calcitonin Receptor and Collagen I Al, said kit comprising: i) A nucleic acid amplification mix comprising two or more pairs of amplification primers, wherein at least one pair allows amplification of Calcitonin Receptor and at least another pair allows amplification of Collagen I Al, ii) a reaction vessel containing a solid support that comprises target- specific probes, wherein at least one probe is specific for Calcitonin Receptor and at least another probe Is specific for Collagen I Al, and iii) reagents for use in visualising hybridisation of nucleic acids to the probes of the solid support.

62. A kit for genotyping Calcitonin Receptor, Collagen I Al, Vitamin D Receptor and Estrogen Receptor, said kit comprising:

ii) a reaction vessel or set of reaction vessels containing a solid support that comprises target-specific probes, wherein at least one probe is specific for Calcitonin Receptor; at least another probe is specific for Collagen

I Al, at least another probe is specific for Vitamin D Receptor; and at least another probe is specific for Estrogen Receptor, and iii) reagents for use in visualising hybridisation of nucleic acids to the probes of the solid support.

63. A kit comprising: i)two or more nucleic acid amplification mixes, a first mix comprising two or more pairs of amplification primers, wherein at least one pair allows amplification of Calcitonin Receptor and at least another pair allows amplification of Collagen I Al; a second amplification mix comprising two or more pairs of amplification primers that allow amplification of two or more of the genes of the group of genes comprising Osteoprotegerin, LRP5, CYPl 7, CYP19 and MTHFR, ii) a reaction vessel or set of reaction vessels containing a solid support that comprises target-specific probes, wherein at least one probe is specific for Calcitonin Receptor, at least another probe is specific for Collagen I Al, and at least two or more probes are specific for the genes of the group of genes comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR amplifiable by the amplification mixture of i), iii) reagents for use in visualising hybridisation of nucleic acids to the probes of the solid support.

64. A kit comprising: i)two or more nucleic acid amplification mixes, a first mix comprising two or more pairs of amplification primers, wherein at least one pair allows amplification of Calcitonin Receptor and at least another pair allows amplification of Collagen I Al; a second amplification mix comprising two or more pairs of amplification primers that allow amplification of two or more of the genes of the group of genes comprising Osteoprotegerin, LRP5, CYP 17, CYP19 and MTHFR, ii) two reaction vessels or sets of reaction vessels, one containing a solid support that comprises target-specific probes, wherein at least one probe is specific for Calcitonin Receptor, at least another probe is specific for Collagen I Al, the other containing a support comprising at least two or more probes specific for the genes of the group of genes comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR amplifiable by the amplification mixture of i), iii) reagents for use in visualising hybridisation of nucleic acids to the probes of the solid support.

65. The kit of any of claims 61 to 64 wherein at least one nucleic acid of the pair of amplification primers that correspond to each target sequence of selected genes, is labelled.

66. The kit of any of claims 61 to 65 wherein the amplification mix comprises labelled dNTPs.

67. The kit of any of claims 61 to 66 wherein the reaction vessel is an array tube.

68. The kit of any of claims 61 to 66 wherein the set of reaction vessels is an array strip.

69. Use of the assay of claims 1 to 33 or the kit of claims 54 to 68 for diagnosing a predisposition to a pathological bone condition.

70. The use of claim 69 wherein the pathological bone condition is selected from the group comprising osteoporosis, metabolic bone diseases, raquitism, pediatric pathologies and bone diseases after organ transplant.

71. The use of claim 70 wherein osteoporosis is postmenopausic osteoporosis.

72. Use of the assay of claims 1 to 33 or the kit of claims 54 to 68 for anticipating response to a treatment.

73. A primer selected from the group of SEQ ID N⁰ 1 to SEQ ID N⁰

10; and SEQ ID N⁰ 30 to SEQ ID N⁰ 43.

74. A probe for genotyping a gene selected from the groups comprising

i) Calcitonin Receptor; Vitamin D Receptor; Estrogen Receptor; Collagen I Al; and/or ii) Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR genes; wherein the probe is selected from I) the group comprising SEQ ID N⁰ II to SEQ ID N⁰ 29 and/or ii) the group comprising SEQ ID N°44 to SEQ ID N⁰ 211.

75. A probe for genotyping a gene selected from the group comprising Vitamin D Receptor; Estrogen Receptor; Collagen I Al, wherein the probe is selected from the group comprising SEQ ID N⁰ 212 to SEQ ID N⁰ 222.