WO2004055212A1

WO2004055212A1 - Genetic susceptibility

Info

Publication number: WO2004055212A1
Application number: PCT/GB2003/005400
Authority: WO
Inventors: Jonathan Mangion; Maxine Johanna Allen; Simon Thomas Bennet
Original assignee: Oxagen Limited
Priority date: 2002-12-13
Filing date: 2003-12-11
Publication date: 2004-07-01
Also published as: AU2003292404A1

Abstract

The present invention relates to the identification of a novel nucleotide substitution in the Msx2 interacting nuclear target (MINT) gene and the exploitation of this nucleotide substitution in the diagnosis of susceptibility to bone disorder, in particular low bone mineral density or fracture. Also provided are transgenic non-human animals comprising the polynucleotides of the present invention and methods and kits for diagnosing and/or determining susceptibility to bone disorder, in particular low bone mineral density or fracture.

Description

GENETIC SUSCEPTIBILITY

MINT is also known as SHARP (SMART/HDAC1 associated repressor protein). This gene encodes a hormone inducible trans criptional repressor. Repression of transcription by this gene product can occur through interactions with other repressors, by the recruitment of proteins involved in histone deacetylation, or through sequestration of transcriptional activators. The product of this gene contains a carboxy-terminal domain that permits binding to other corepressor proteins. This domain also permits interaction with members of the NuRD complex, a nucleosome remodelling protein complex that contains deacetylase activity. In addition, this repressor contains several RNA recognition motifs that confer binding to a steroid receptor RNA coactivator; this binding can modulate the activity of both liganded and nonliganded steroid receptors. The Msx2-interacting nuclear target (MINT) protein was first identified by a farwestern cloning strategy using an expression library derived from mouse brain.

Osteoporosis is a common disease characterized by reduced bone mineral density (BMD), deterioration of bone micro-architecture and increased risk of bone damage, such as fracture. It is a major public health problem which affects quality of life and increases costs to health care providers. In European populations, one in three women and one in twelve men over the age of fifty is at risk. The disease affects 25 million people in the USA, where the incidence of disease is 25% higher than it is in the UK, and a further 50 million people in Japan and Europe combined. It is estimated that by the middle of the next century the number of osteoporosis sufferers will double in the West, but may increase six-fold in Asia and South-America. Fracture is the most serious endpoint of osteoporosis, particularly fracture of the hip which affects up to 1.7 million people worldwide each year. It is estimated that by the year 2050, the number of hip fractures worldwide will increase to over 6 million, as life expectancy and age of the population increase.

Treatment of osteoporosis is unsatisfactory. In particular, once bone damage has occurred as a result of osteoporosis, there is little a physician can do other than let the bone heal. In the elderly, this may be a slow and painful process. Diagnosis of those at risk of developing osteoporosis allows more effective preventative measures. Strategies for the prevention of this disease include development of bone density in early adulthood, and minimisation of bone loss in later life. Changes in lifestyle, nutrition and hormonal factors have been shown to affect bone loss.

Osteoporosis can be considered a complex genetic trait with variants of several genes underlying the genetic determination of the variability of the phenotype. Low bone mineral density is an important risk factor for fractures, the clinically most relevant feature of osteoporosis .

In view of the polygenic control of bone mineral density and therefore disease states such as osteoporosis, it is apparent that the identification of additional genetic factors which contribute to susceptibility to bone damage will provide important new insights into such disease, and may enable preventative measures to be applied, and new diagnostic and or prognastic tests and treatments to be developed.

Testing for genetic susceptibility is an important enabling diagnostic tool. In many cases, the preferred test will be for more than one factor involved in a disease, e.g. osteoporosis. This provides a broader picture, with increased information, on the genetic susceptibility of an individual. Genetic susceptibility is the risk that an individual either suffers from, or will suffer from, a disease.

The present invention provides, amongst other aspects a method of determining an individual's susceptibility to bone disorder, in particular low bone mineral density or fracture.

In a first aspect, the present invention provides a method of determining susceptibility to bone disorder, in particular low bone mineral density or fracture, comprising determining the presence or absence of a polymorphism at position 121660 of the MINT gene. The MINT gene is detailed in the NT contig NT_002166. The full length of NTJ302166 was retrieved via the National Center Biotechnology Information (NCBI) website (http//:www.ncbi.nlm.gov) using the Entrez nucleotide search facility. This contig is composed of two overlapping full length BAG sequences, AL 334555 and AL 096775 to give a contiguous 163038bp sequence containing partial sequence of the human orthologue of the mouse MINT/SHARP gene. The single nucleotide polymorphism of the invention has been given a positional reference of 121660, as shown in Figure 1. RNA splicing utilizes conserved sequences at the junctions between exons and introns to direct the specific excision of introns and connections of exons to create the mRNA transcript. The MINT polymorphism mentioned is the 6^th base from the splice site and potentially may impact on RNA splicing and MINT protein production. The 5' end of the intron contains the donor splice site defined by a GT dinucleotide at the very 5' end of the intron.

The method of the first aspect may be practised on any mammalian subject. Preferably, the mammalian subject will be a human, and most preferably an adult, preferably female.

The novel polymorphism at position 121660 of the MINT gene has been shown to be responsible for increased susceptibility to bone disorder, in particular low bone mineral density or fracture, low bone mineral density is an indication of susceptibility to bone damage and related conditions such as osteoporosis. In particular, the polymorphism of the present invention, either alone or in combination with other polymorphisms are useful in identifying individuals susceptible to or resistant to bone disorder, in particular low bone mineral density or fracture and in the prevention and/or treatment of this condition.

In this text, bone disorder has the same meaning as bone damage and bone disfunction. Bone disorder includes, in particular, low bone mineral density, osteoporosis and bone fracture. The bone fracture may be vertebral or non-vertebral.

The present invention is applicable to any disease in which low bone mineral density is a factor, such as bone damage, osteoporosis and osteoporotic fracture. Low bone mineral density has previously been defined statistically as two standard deviations below the aged-matched mean of bone mineral density for a given population. Bone damage may be defined as any form of damage resulting from low bone mineral density, and includes any form of structural damage, such as fractures, bones or chips, and degradation or deterioration of the bone other than normal wear and tear.

A polymorphism is typically defined as two or more alternative sequences, or alleles, of a gene in a population. A polymorphic site is the location in the gene at which divergence in sequence occurs. Examples of the ways in which polymorphisms are manifested include restriction fragment length polymorphisms, variable number of tandem repeats, hypervariable regions, minisatellites, di- or multi-nucleotide repeats, insertion elements and nucleotide deletions, additions or substitutions. The first identified allele is usually referred to as the reference allele, or the wild type. Additional alleles are usually designated alternative or variant alleles. Herein, the sequences of the first aspect are designated the reference sequences, and are not part of the invention. Nucleic acid sequences of the present invention which differ from these sequences at one or more of the positions indicated above may be referred to as variants of these sequences.

A single nucleotide polymorphism is a variation in sequence between alleles at a site occupied by a single nucleotide residue. Single nucleotide polymorphisms (SNP's) arise from the substitution, deletion or insertion of a nucleotide residue at a polymorphic site. Typically, this results in the site of the variant sequence being occupied by any base other than the reference base. For example, where the reference sequences contains a "T" base at a polymorphic site, a variant may contain a "C", "G" or "A" at that site.

The first aspect of the invention may comprise determining the presence of nucleotide base which is not C at position 121660. It may comprise determining the presence of nucleotide T at position 121660. The presence of the homozygous polymorphism at position 121660 in MINT is rare. Susceptibility to bone damage, in particular low bone mineral density or fracture occurs even when one of the alleles of an individual shows a polymorphism at position 121660. The polymorphism of the invention occurs in a non-coding region and as such may not affect protein sequence, but may exert phenotypic effects by influencing replication, transcription and/or translation. A polymorphism, may affect more than one phenotypic trait or may be related to a specific phenotype.

The method of the first aspect is carried out by methodology known to the person skilled in the art. Typically, the method involves contracting a sample of DNA, from an individual, with an agent which identifies the presence or absence of a polymorphism at position 121660 of the MINT gene. Such an agent may be a hybridizing nucleotide, an antibody, a PCR agent or another agent. Further descriptions of agents are set out below, and methodology is described in the Examples section of this text.

The method may include determining whether one or more particular alleles are present, or which combination of alleles (i.e. a haplotype) is present. The method may also include determining whether subjects are homozygous or heterozygous for the particular allele or haplotype. In a preferred embodiment, the method comprises determining which allele of the polymorphism of the invention is/are present.

Any method, including those known to persons skilled in the art, may be used to determine which allele(s) of the MINT gene are present. Preferably, the method comprises first removing a sample from a subject. More preferably, the method comprises obtaining an isolated sample of a polynucleotide or protein to determine therein which allele(s) of the MINT gene is/are present. Thus, the present invention relates to a non-invasive diagnostic method, the results of which provide an indication of susceptibility to bone damage, in particular low bone mineral density or fracture but do not lead to a diagnosis upon which an immediate medical intervention is required.

Any biological sample comprising cells containing nucleic acid or protein is suitable for this purpose. Examples of suitable samples include whole blood, semen, saliva, tears, buccal, skin or hair. The sample and/or the nucleic acid may be immobilized on a support, such as a solid support.

In a preferred embodiment, the method is carried out using a polynucleotide. Any method for determining alleles in a polynucleotide may be used, including those known to persons skilled in the art. Preferably, the method may comprise the use of anti-sense polynucleotides, for example, as defined below. Such polynucleotides may be sequences which are able to distinguish between alleles of the MINT gene, by preferential binding, or sequences which hybridise under stringent conditions to a region either side of an allele to enable amplification of one or more of the polymorphisms.

Methods of this embodiment include those known to persons skilled in the art, for example direct probing, allele specific hybridisation, PCR methodology including Allele Specific Amplification (ASA), Allele Specific Hybridisation, single base extension, Genetic Bit Analysis and RFLP, or direct sequencing. The appropriate restriction enzyme, will, of course, be dependent upon the polymorphism and restriction site, and will include those known to persons skilled in the art. Analysis of the digested fragments may be performed using any method in the art, for example gel analysis, or southern blots.

Determination of an allele of a polymorphism using direct probing typically involves the use of anti-sense sequences, for example as described in the third aspect of the invention. These may be prepared synthetically or by nick translation. The anti-sense probes may be suitably labelled using, for example, a radiolabel, enzyme label, fluoro-label, biotin-avidin label for subsequent visualization in, for example, a southern blot procedure. A labelled probe may be reacted with a sample DNA or RNA, and the areas of the DNA or RNA which carry complimentary sequences will hybridise to the probe, and become labelled themselves. The labelled areas may then be visualized, for example by autoradiography.

The above described methods may require amplification of the DNA sample from the subject, and this can be done by techniques known in the art, such as PCR. Other suitable amplification methods include ligase chain reaction (LCR), transcription amplification, self sustained sequence replication and nucleic acid based sequence amplification (NASBA). The latter two methods both involve isothermal reactions based on isothermal transcription which produce both single stranded RNA and double stranded DNA as the amplification products, in a ratio of 30 or 100 to 1, respectively.

Where it is desirable to identify the presence of multiple single nucleotide polymorphisms, or haplotypes, in a sample from a subject, it may be preferable to use arrays. The array may contain a number of probes, each designed to identify one or more of the above single nucleotide polymorphisms of the invention.

A second aspect provides fragments of the MINT gene itself or sequences complimentary thereto. Such fragments are useful in the method according to the first aspect of the invention. They may be isolated or recombinant. The polynucleotide of this invention is preferably DNA, or may be RNA or other options.

By "isolated" is meant a polynucleotide sequence which has been purified to a level sufficient to allow allelic discrimination. For example, an isolated sequence will be substantially free of any other DNA or protein product. Such isolated sequences may be obtained by PCR amplification, cloning techniques, or synthesis on a synthesiser. By recombinant is meant polynucleotides which have been recombined by the hand of a man.

Preferred fragments are from at least 10 up to 100 nucleotides in length. More preferably, the fragments are from 10, 20, 30, 40, 50, 60, 70, 80 or 90 nucleotides in length. The fragments should comprise a sequence which includes position 121660 of the MINT gene.

According to the second aspect of the invention, the preferred fragments are complementary sequences of the MINT gene which hybridise a portion of the nucleic acid sequence as given in Figure 1 which includes position 121660. Such "anti-sense" sequences are useful as agents for the identification of individuals having or being susceptible to bone damage, in particular low bone mineral density or fracture.

The anti-sense sequences of the invention include those which hybridise to an allele (preferably the variant allele) of a polymorphism of the invention. These sequences are useful as probes. To be useful as a probe, the anti-sense sequence should bind preferentially one allele of one or more polymorphisms of the present invention and will, preferably, comprise the exact complement of one allele of one or more polymorphisms of the invention. Thus, for example, where the variant comprises an "A" residue at a polymorphic site, it is preferred that the anti-sense sequence will comprise a "T" residue. Such anti- sense sequences which are capable of specific hybridisation to detect a single base mis-match may be designed according to methods known in the art. Variation in the sequence of these anti-sense sequence is acceptable for the purposes of the present invention, provided that the ability of the anti-sense sequence to distinguish between alleles of a polymorphism is not compromised. Preferably, the antisense sequence will hybridize to the sequence of interest under stringent conditions which are defined below.

In relation to the present invention, "stringent conditions" refers to the washing conditions used in a hybridisation protocol. In general, the washing conditions should be a combination of temperature and salt concentration so that the denaturation temperature is approximately 5 to 20°C below the calculated Tm of the nucleic acid under study. The Tm of a nucleic acid probe of 20 bases or less is calculated under standard conditions (1M NaCl) as [4(C x (G+C) + 2(C x (A+T)], according to Wallace rules for short oligonucleotides. For longer DNA fragments, the nearest neighbour method, which combines solid thermodynamics and experimental data may be used. The optimum salt and temperature conditions for hybridisation may be readily determined in preliminary experiments in which DNA samples immobilised on filters are hybridised to the probe of interest and then washed under conditions of different stringencies. While the conditions for PCR may differ from the standard conditions, the Tm may be used as a guide for the expected relative stability of the primers. For short primers of approximately 14 nucleotides, low annealing temperatures of around 44° C to 50°C are used. The temperature may be higher depending upon the base composition of the primer sequence used.

In a third aspect of the invention, the polynucleotides of the aforementioned aspects of the invention may be in the form of a vector, to enable the in vitro or in vivo expression of the polynucleotide sequence. The polynucleotides may be operably linked to one or more regulatory elements including a promoter; regions upstream or downstream of a promoter such as enhancers which regulate the activity of the promoter; an origin of replication; appropriate restriction sites to enable cloning of inserts adjacent to the polynucleotide sequence; markers, for example antibiotic resistance genes; ribosome binding sites: RNA splice sites and transcription termination regions; polymerisation sites; or any other element which may facilitate the cloning and/or expression of the polynucleotide sequence. Where two or more polynucleotides of the invention are introduced into the same vector, each may be controlled by its own regulatory sequences, or all sequences may be controlled by the same regulatory sequences. In the same manner, each sequence may comprise a 3' polyadenylation site. The vectors may be introduced into microbial, yeast or animal DNA, either chromosomal or mitochondrial, or may exist independently as plasmids. Examples of suitable vendors will be known to persons skilled in the art and include pBluescript II, LambdaZap, and pCMV-Script (Stratagene Cloning Systems, La Jolla (USA)).

Appropriate regulatory elements, in particular, promoters will usually depend upon the host cell into which the expression vector is to be inserted. Where microbial host cells are used, promoters such as the lactose promoter system, trytophan (Tip) promoter system, (-lactamase promoter system or phage lambda promoter system are suitable. Where yeast cells are used, preferred promoters include alcohol dehydrogenase I or glycolytic promoters. In mammalian host cells, preferred promoters are those derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma virus etc. Suitable promoters for use in various host cells would be readily apparent to a person skilled in the art. In a fourth aspect of the present invention, there is provided host cell comprising a polynucleotide according to any of the aforementioned aspects, for expression of the polynucleotide. The host cell may comprise an expression vector, or naked DNA encoding said polynucleotides. A wide variety of suitable host cells are available, both eukaryotic and prokaryotic. Examples include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, preferably immortalised, such as mouse, CHO, HeLa, myeloma or Jurkat cell lines, human and monkey cell lines and derivatives thereof. Such host cells are useful in drug screening systems to identify agents for use in diagnosis or treatment of individuals having, or being susceptible to bone damage, in particular low bone mineral density or fracture.

The method by which said polynucleotides are introduced into a host cell will usually depend upon the nature of both the vector/DNA and the target cell, and will include those known to a person skilled in the art. Suitable known methods include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook et al.

In a fifth aspect of the present invention, there are provided antibodies which react (optionally or specific for) with an antigen such as a polynucleotide of the second aspect. Antibodies can be made by the procedure set forth by standard procedures. Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells are then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen DNA clone libraries for cells secreting the antigen. Those positive clones can then be sequenced. Preferably, the antigen being detected and/or used to generate a particular antibody will comprise a polynucleotide or fragment according to the second aspect.

The detection of binding of the antibody to the antigen in a sample may be assisted by methods known in the art, such as the use of a secondary antibody which binds to the first antibody, or a ligand. Immunoassays including immunofluorescence assays (IFA) and enzyme linked immunosorbet assays (ELISA) and immunoblotting may be used to detect the presence of the antigen. For example, where ELISA is used, the method may comprise binding the antibody to a substrate, contacting the bound antibody with the sample containing the antigen, contacting the above with a second antibody bound to a detectable moiety (typically an enzyme such as horse radish peroxidase or alkaline phosphatase), contacting the above with a substrate for the enzyme, and finally observing the colour change which is indicative of the presence of the antigen in the sample.

In a sixth aspect of the present invention, there is provided a transgenic non- human animal comprising a polynucleotide according to the second aspect of the invention. Transgenic non-human animals are useful for the analysis of the single nucleotide polymorphisms and their phenotypic effect. Expression of a polynucleotide of the invention in a transgenic non-human animal is usually achieved by operably linking the polynucleotide to a promoter and/or enhancer sequence, preferably to produce a vector as previously described, and introducing this into an embryonic stem cell of a host animal by microinjection techniques. The transgene construct should then undergo homologous recombination with the endogenous gene of the host. Those embryonic stem cells comprising the desired polynucleotide sequence may be selected, usually by monitoring expression of a marker gene, and used to generate a non-human transgenic animal. Preferred host animals include mice and other rodents. In a preferred embodiment, the transgenic non-human animal may comprise an anti-sense nucleic acid sequence of the second aspect. The expression of an anti-sense sequence in a transgenic non-human animal may be useful in determining the effects of such sequences of high bone mineral density, or in neutralising deleterious effects of variant genes in an animal. Preferably, the host animal will be one, which is susceptible to bone damage, in particular low bone mineral density or fracture. The condition may be naturally occurring or artificially induced.

The seventh aspect of the invention also provides the use of the transgenic non-human animal of the sixth aspect, in screening for an agent for use in the determination of an individual having, or being susceptible to bone damage, in particular low bone mineral density or fracture.

In some preferred embodiments, for example where the susceptibility to bone damage, in particular low bone mineral density or fracture has been artificially induced, the transgenic non-human animal will be modulated to no longer express the corresponding endogenous MINT/SHARP gene. Such animals may be referred to as "knock out". In some cases, it may be appropriate to modulate the expression of the endogenous genes, or express the polynucleotides of the present invention, in specific tissues. This approach removes viability problems if the expression of a gene is abolished or induced in all tissues.

In a seventh aspect of the present invention there is provided a method of screening for an agent for use in the determination of individuals having, or being susceptible to, bone damage, in particular low bone mineral density or fracture, said method comprising contacting a putative agent with a polynucleotide according to the second aspect of the present invention, and monitoring the reaction therebetween. Potential agents are those which react differently with a variant of the invention and a reference allele. The reference allele may be the wild-type allele (i.e. without a polymorphism). It is envisaged that the present method may be carried out by contacting a putative agent with a host cell or transgenic non-human animal comprising a polynucleotide or protein according to the invention. Putative agents will include those known to persons skilled in the art, and include chemical or biological compounds, such as anti-sense polynucleotide sequences complementary to the coding sequences of the first aspect, or polyclonal or monoclonal antibodies which bind to a product such as a protein or protein fragment of the second aspect. The agents identified in the present method may be useful as agents which modulate MINT/SHARP binding, and therefore as a regulator of osteocalcin expression. They may also be useful in determining susceptibility to bone damage, in particular low bone mineral density or fracture, or in the diagnosis, prognosis or treatment of related conditions.

An eighth aspect of the invention provides primer sequences suitable for PCR reactions, for use in determining the presence or absence of a polymorphism at position 121660 of the MINT gene. Suitable sequences showed comprise at least 18 nucleotide bases and may be one or more sequences, selected from: any sequence which hybridizes to the MINT gene. The primer may be 100% complimentary to the MINT sequence or may be 80% or more complimentary. Preferably the sequence is from 18 to 25 bases in length.

In a further aspect, the present invention provides a kit for use in diagnosis of an individual having or being susceptible to bone damage, in particular low bone mineral density or fracture, the kit comprising an agent for determining the presence or absence of a polymorphism at position 121660 of the MINT gene. The agent may determine the presence of the nucleotide other than C, at position 121660. The agent of the kit may comprise polynucleotides, mostly preferably anti-sense sequences such as those of the second aspect, for use as probes or primers; sequences of the eighth aspect of the invention; antibodies which bind to alleles of the MINT polypeptides, such as those of the fifth aspect; or restriction enzymes for use in detecting the presence of a MINT polynucleotide. Preferably, the kit will also comprise means for detection of a reaction, such as nucleotide label detection means, labelled secondary antibodies or size detection means. In yet a further preferred embodiment, the agent may be fixed to a substrate, for example an array, as described in W095/11995. The kit further comprises means for indicating correlation between the genotype of a subject and susceptibility to high bone mineral density. Such means may be in the form of a chart or visual aid, which indicates that presence of one or more alleles of the MINT gene, is associated with susceptibility to bone damage, in particular low bone mineral density or fracture.

A tenth aspect of the present invention provides a method for diagnosing and then acting on any diagnosis of a polymorphism in the MINT gene at position 121660 to prevent and/or treat bone damage, in particular low bone mineral density or fracture. The prevention and/or treatment may be by any means, including replacing the allele with the polymorphism. The replacement may be by addition of an allele, or part thereof, with a polynucleotide of the MINT gene which is not associated with bone damage, in particular low bone mineral density or fracture.

The preferred embodiments of each aspect apply to the other aspects of the invention, mutatis mutandis. The present invention will now be described by way of a non-limiting example, with reference to the figures in which:

Figure 1 shows the nucleotide numbers 120780 to 124560 of the MINT gene.

Example 1

Sequencing

In order to search for polymorphism in the human MINT gene responsible for susceptibility to bone damage all of the 15 exons encoding the 12 kb human MINT transcript were scanned by direct sequencing.

DNA samples from unrelated individuals were amplified by PCR using the primers and conditions in Table 1 and 2.

To identify the position and nature of the polymorphism, PCR products were sequenced initially using the Dynamic ET Terminator on Dynamic ET Terminator cycle sequencing was conducted following the sequencing kit protocol (Amersham, Cat. No. US80890).

Table 1. PCR Conditions

Table 2. Annealing temperatures and primer sequences fragment

Sequencing products were purified by gel filtration (plO gel) followed by ethanol precipitation. Dry pellets are resuspended on 2μl deionised formamide and then denatured at 96°C for 2.5 min, followed by snap cooling on ice. lμl of each reaction was loaded onto a 48cm sequencing gel and run for 10 hours on an automatic AB1 377 sequencer and the data collected using filter set A.

Table 3. Details of each SNP found by sequencing

Example 2

Biallelic polymorphism genotyping by Pyrosequencin .TM

A pair of oligonucleotides for amplication by PCR was designed on either side of the biallelic polymorphism to produce a product size between 50bp and 350bp. A sequencing oligonucleotide was designed to end within 30bp either 5' and 3' to the polymorphic site. All amplification oligonucleotides used to generate the complementary strand to the sequencing primer were labeled with a 5' - Biotin. (see Table 4).

Table 4. PSQ assay oligonucleotides and PCR annealing temperatures

The sample genotyped was amplified by PCR using the PCR amplification oligonucleotides. Each reaction used: 20ng DNA (dried down), 0.6 units of AmpliTaq Gold™ DNA polymerase, IX PCT Buffer II, 2.5mM MgCl₂, ImM dNTP, and lOpmol of each PCR oligonucleotide in a final volume of lOμl. The PCR cycling conditions used were: 95°C for 12 min, 45 cycles of: 94°C for 15 sec, T_A for 15 sec, 72°C for 30 sec, and 72°C for 5 min.

After amplification the DNA strand of each PCR template complementary to the sequencing primer was isolated, ready for pvrosequencing (PSQ). To do this, 1) 50μl of Dynabead solution (2mg/ml Dynabeads®, 5mM Tris-HCl, 1M NaCl, 0.5 mM EDTA, 0.05% Tween 20) was added to the PCR product and shaken at 65 °C for 15 min, 2) the template was transferred using magnets to 50μl of 0.5M MaOH for 1 min, 3) the template was transferred using magnets to lOOμl of IX Annealing buffer (20mM Tris-Acetate, 5mM MgAc₂) for 1 min, and 4) the template was transferred using magnets to 45 μl of IX Annealing buffer containing 15pmol of sequencing oligonucleotide (Table 4).

After template isolation, the sequencing oligonucleotide was annealed to the template by denaturing at 80°C for 2 min and then cooling to room temperature for 10 min. Each marker/sample combination was then sequenced/genotyped by pyrosequencing™ on a PSQ96™ (Pyrosequencing AB) (Figure 3). Genotype results were stored in the PSQ oracle® database ready for statistical analysis.

Example 3

MINT/SHARP gene polymorphism in determining genetic susceptibility.

We found the SNP, MIN04 (MINT x7 C300T), to be associated with bone mineral density at the lumbar spine (χ =7.11, p=0.008) in a set of families chosen because they included at least one individual with low bone mineral density, and thus increased risk of osteoporotic fracture.

Further analysis showed that after standardisation for age and sex, the lumbar spine bone mineral density of individuals who were homozygous for the "C" allele at the MIN04 SNP was 0.45 standard deviation units higher, on average, than that in individuals who were heterozygous (genotype CT) at this locus. This shows that the MIN04 polymorphism in the MINT/SHARP gene can be used to identify individuals susceptible to low bone mineral density and thus with an increased risk of bone damage. Study subjects

The families used in this study were recruited by identification of an individual (known as the proband, or index case) who had a bone mineral density (BMD) in the lower 2.5% of the normal age and sex adjusted phenotypic range. First degree relatives of the proband, including siblings, parents, spouse and children, over the age of 20 years, were also invited to participate. When any of these first degree relatives had a BMD in the lower 10% of the phenotypic range, their first degree relatives (e.g. uncles, aunts, cousins of the proband) were also invited to participate. In this way, large, multi-generation familes were collected. The collection protocol was approved by the UK Multi-centre Research Ethics Committee, as well as the local medical ethics committee in Oxford, Cambridge, London, Southampton, Aberdeen and Glasgow in the UK, Rotterdam in the Netherlands and Aarhus in Denmark.

Measurements

Height and weight were measured at the initial examination. Bone mineral density values (in g/cm2), for femoral neck, trochanter, total over three hip sites and lumbar-spine vertebrae 2, 3 and 4, were determined by Dual Energy X-ray Absorptiometry (DXA) following standard procedures. Each study volunteer completed a questionnaire containing details of their medical history, dietary intake of milk, physical exercise, smoking, alcohol consumption and, for women, reproductive and gynaecological history, including age at menarch, age at menopause, use of contraceptive pill, pregnancies, breast feeding, use of hormone replacement therapy (HRT) and hysterectomy.

Bone mineral density phenotype

Raw BMD measurements, collected from 27 DXA machines, were standardized using calibration curves derived from data generated using a standard European Spine Phantom. Calibrated BMD values were then adjusted for age, sex, sex multiplied by age, height, weight, height multiplied by weight, and for the hospital at which the patient was measured.

Determination of MINT/SHARP genotypes Genomic DNA was extracted from peripheral venous blood samples according to standard procedures and the genotypes of the nine lp36 SNPs were determined using mismatch RFLP assays as described in Example 1.

Statistical Analysis The analysis of the association between the lp36 SNP genotypes and bone mineral density was performed in 2744 individuals (for whom lumbar-spine BMD measurements were available). These represented 561 families, which comprised 4464 individuals in total. BMD value was treated as a quantitative trait and association was tested using the quantitative transmission disequilibrium test (QTDT - Abecasis et al., Am. J. Hum. Gen, 68 1463-1474, 2001). We found a significant association between the MIN04 polymorphism and BMD at the lumbar spine (χ =7.11, p=0.008). Furthermore, the analysis showed that after standardisation for age and sex, the lumbar spine bone mineral density of individuals who were heterozygous (CT) at the MIN04 SNP was 0.45 standard deviation units lower, on average, than that in individuals who were homozygous (CC) at this locus.

Conclusions

These data show an association between the MIN04 (MINT x7 C300T) polymorphism, situated in the MINT/SHARP gene, and bone mineral density of the lumbar spine. We show that the MIN04 polymorphism or polymorphisms in linkage disequilibrium with the MIN04 SNP are associated with the determination of bone mineral density. Example 4

Study Subjects

The Rotterdam Study is a population-based cohort study of 7983 subjects aged 55 years or more, residing in the Ommoord district of the city of Rotterdam in the Netherlands. The study was designed to document the occurrence of disease in the elderly in relation to several potential determinants (Hofman et al Eur J Epidemiol 1991 7:403-422). A total of 10,275 persons, of whom 9161 (89%) were living independently, were invited to participate in the study in 1991. In those subjects living independently, the overall response rate was 77% for home interview and 71% for examination in a research centre, including measurement of anthropometric characteristics, BMD, and blood sampling. The Rotterdam Study was approved by the Medical Ethics Committee of the Erasmus University Medical School and written informed consent was obtained from each subject. The analysis of the association between MIN04, BMD and fractures was performed in a sample of men and women participating in the study.

The analysis of the association between MINT, fracture risk and BMD was done in a random sample of 1061 women and 925 men. In this subset, follow up data of non vertebral fracture were available for 60 men and 130 women and vertebral fracture data for 65 men and 81 women.

Measurements

At baseline height and weight were measured. BMD (in g/cm²) was measured at the femoral neck and lumbar spine by dual energy X-ray absorptiometry (Lunar DPX-L densitometer), as reported earlier (2). Body mass index (BMI) was computed as weight in kilograms divided by height in square meters (kg/m ). Age at menopause was assessed by questionnaire. Dietary intakes for calcium (mg/day) and vitamin D (mg/day) were assessed by food frequency questionnaire and adjusted for energy intake. Both at baseline, between 1990 and 1993 and at the follow-up visit, between 1997-1999, radiographs of the spine from the fourth thoracic to the fifth lumbar vertebrae were taken. All follow-up photos were analyzed for the presence of vertebral fractures by the McCloskey/Kanis method (3). The occurrence of non- vertebral fractures was recorded, confirmed and classified by a physician.

Statistical analysis

Differences in mean age at baseline between the study group and the Rotterdam Study were evaluated by means of analysis of variance (ANOVA). All other differences in baseline characteristics were compared by analysis of covariance (ANCOVA) testing with age to adjust for possible confounding effects. We grouped subjects by genotype either containing at least one risk allele or containing no risk alleles.

Odds ratios (ORs) with 95% confidence intervals (95% CI) were calculated by (multiple) logistic regression analyses to estimate the relative risk of fractures at baseline by genotypes of the risk allele, with no copies of the risk allele as the reference group. First, we calculated crude odds ratios, and, secondly, we adjusted for potentially confounding factors (age, BMI, BMD, and age at menopause). We used SPSS version 9.0 (SPSS Inc., Chicago, USA) for all our analyses. Results

The allele frequency of the rare T was 2.8%, similar to that seen in FAMOS. An association to non- vertebral fracture was observed in men (p = 0.02 OR 3.0[1.3-6.8]).

Odds ratio (95% Cl)** : without BMD-adjustment Odds ratio (95% Cl)* : with BMD-adjustment

Claims

1. A method of determining susceptibility to bone damage, comprising determining the presence or absence of a polymorphism at position 121660 of at least one allele of the MINT gene in an individual.

2. A method, as claimed in claim 1, wherein the polymorphism is the presence of nucleotide base T at position 121660.

3. A method, as claimed in claim 1 or claim 2, wherein the bone damage is low bone mineral density or fracture.

4. An isolated or recombinant polynucleotide comprising from at least 10 to 100 consecutive nucleotide bases of the MINT sequence, which sequence comprises a nucleotide at position 121660.

5. A polynucleotide, as claimed in claim 4 wherein the nucleotide at position 121660 is not the nucleotide C.

6. A polynucleotide, as claimed in claim 5, wherein the nucleotide at position 121660 is the nucleotide T.

7. A vector comprising a polynucleotide according to any one of claims 4 to 6.

8. A host cell comprising a polynucleotide or vector according to any one of claims 4 to 7.

9. An antibody or antibody fragment which preferentially binds to a sequence, as claimed in any one of claims 4 to 6.

10. A transgenic non-human animal comprising a polynucleotide sequence,

> vector or host cell according to any one of claims 4 to 8.

11. A method, as claimed in claim 1, 2 or 3, which comprises the use of a polynucleotide, antibody or antibody fragment, as claimed in any one of claims 3 to 8.

12. Use of a transgenic non-human animal according to claim 10 in screening for an agent for use in the determination of an individual having, or being susceptible to bone damage.

13. Use, as claimed in claim 12, wherein the bone damage is low bone mineral density or fracture.

14. A method of screening for an agent for use in the determination of an individual having, or being susceptible to bone damage, said method comprising contacting a putative agent with a polynucleotide, as claimed in any one of claims 4 to 6 and monitoring the reaction there between.

15. A method, as claimed in claim 14, wherein the bone damage is low bone mineral density or fracture.

16. A polynucleotide which comprises a nucleic acid sequence of at least 18 bases which can be used to amplify, by PCR, a portion of the MINT gene which comprises position 121660.

17. A kit for use in diagnosis of an individual having, or being susceptible to bone damage, said kit comprising an agent for determining the presence or absence of a polymorphism of at least one allele at position 121660 of the MINT gene.

18. A kit, as claimed in claim 17, wherein the bone damage is low bone mineral density or fracture.

19. A kit according to claim 17, wherein the agent comprises a polynucleotide according to any one of claims 4 to 6, an antibody according to claim 9, a restriction enzymes for digestion of a polynucleotide according to any one of claims 4 to 6 or a primer polynucleotide as claimed in claim 16.

20. A kit according to claim 17 or claim 19, wherein the agent is identified according to a method of claim 14.

21. A method for diagnosing and preventing and/or treating bone damage, the method comprising

1) determining the presence of a polymorphism at position 121660 of at least one allele of the MINT gene in an individual; and

2) administering to the individual an agent which prevents and/or treats low bone mineral density.

22. A method as claimed in claim 21, wherein the bone damage is low bone mineral density or fracture.