US20050064453A1

US20050064453A1 - Diagnostics and therapeutics for early-onset menopause

Info

Publication number: US20050064453A1
Application number: US10/838,503
Authority: US
Inventors: Gordon Duff; Kenneth Kornman; Simon Dijk
Original assignee: Individual
Current assignee: Individual
Priority date: 1999-06-30
Filing date: 2004-05-03
Publication date: 2005-03-24
Also published as: US6730476B1; WO2001000880A3; WO2001000880A2; AU6067900A; IL147182A0; US20090163460A1; AU784224B2; EP1194590A2; CA2378221A1; JP2003508022A

Abstract

A method of predicting whether a subject is predisposed to developing early-onset menopause is provided. The method involves genotyping a patient at the IL-1 gene loci.

Description

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/632,657 filed Aug. 4, 2000, now U.S. Pat. No. 6,730,476, which is a continuation in part of U.S. Pat. No. 6,268,142, filed Jun. 30, 1999 the contents of which are incorporated herein by reference in their entireties.

1. BACKGROUND OF THE INVENTION

Menopause is defined as the permanent cessation of menstruation caused by failure of ovarian follicular development in the presence of elevated gonadotrophin levels. Comprehensive Gynecology (eds. Mishell et al 1997). A hallmark of menopause is the decrease in ovarian follicular estrogen synthesis, although this is only one aspect of the array of physiological events that accompanies the climacteric. Symptoms accompanying menopause vary from woman to woman, but usually include some component of vasomotor instability or hot flashes, often accompanied by psychological symptoms like mood swings and irritability. The loss of estrogen predisposes to coronary artery disease. Loss of estrogen further results in loss of overall bone mineral content caused by an increased resorption of bone without a correlative increase in bone formation. Unabated net loss of bone structural integrity following menopause can culminate in clinically significant osteoporosis. The severity of these climacteric symptoms can be substantially reduced with estrogen replacement therapy. These symptoms represent formidable challenges to the health care system. Recognizing that the current average life expectancy for a woman in the U.S. is 78 years, one can readily calculate that a substantial portion of a woman's lifespan will be post-menopausal. In 1990, for example, there were more than 50 million women in the U.S. over 50 years of age.
The mean age of physiological menopause in the U.S. is between 51 and 52 years of age, with a range between 45 and 55 years old distributed along a normal curve. Stanford et al., J. Chron. Dis 40:995, 1987. About 10% of women in the U.S. undergo menopause before age 46; 1% of U.S. women enter menopause before age 40. If a woman becomes menopausal before age 40, the condition is termed premature ovarian failure. A variety of factors have been identified that correlate weakly with age of menopause, including number of pregnancies, use of oral contraceptives, duration of lactation, age at menarche, age at last pregnancy, race, height, weight, education or occupational history. Cassou et al., Maturitas 26:165-74, 1997. Cigarette smoking, however, has been observed to decrease the age of onset of menopause by about two years. Menopause may also be therapeutically induced, either chemically, surgically or via radiation (e.g. to reduce the risk of developing breast cancer associated with exposure to estrogens).
Diagnosis of menopause may be made clinically by observing the absence of menses for a year. Most women experience progressive menstrual irregularity that presages menopause. The time between the onset of menstrual irregularity and menopause is called the perimenopause. The median age at onset of perimenopause is 47.5 years; its median length is 4 years. McKinlay et al., Maturitas 14:103, 1992. Prior to the onset of the perimenopause, the length of the menstrual cycle tends to decrease in length, due to the decreased duration of follicular functioning. Munster et al., Br. J. Obstet./Gynaecol. 99:422, 1992. About 10% of women do not enter a perimenopausal phase, rather continuing to have regular cycles until menses suddenly stop.
Hormonal changes often precede changes in menstrual patterns, and early diagnosis for menopause and perimenopause has traditionally involved the measurement of follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels. A significant increase in both FSH and LH levels occurs about 5 years before menopause, with the FSH increase more prominent than the LH increase. Levels of these gonadotrophins peak about one year postmenopausally for LH and two to three years after menopause for FSH. The incidence of ovulatory cycles, measured by elevated luteal phase progesterone levels, decreases from 60% during the 5-6 years before menopause to 5% in the 6 months before menopause. Estrogen levels fall as the number of ovulatory cycles decreases, particularly during the 6-12 months before menopause. FSH release, mainly controlled by inhibin, remains elevated even in the presence of post-menopausal hormone replacement. An initial fall in inhibin level is an early indication of diminishing ovarian function. FSH level rises accordingly, suggesting the woman's entry into the perimenopausal stage of reproductive function. Initial changes in FSH and inhibin are often transitory. It is therefore important to demonstrate a sustained increase in FSH. This value, combined with low estradiol levels, is used to diagnose the onset of permanent ovarian failure.
Diagnosis of menopause and perimenopause thus tends to be possible only after major changes in the endocrine system have already occurred. A younger woman without premonitory perimenopausal symptoms may have no reason to have her hormone levels measured; she may thus enter early onset menopause (EOM) without any warning. In such a patient, hormonal analysis may not yield a diagnosis until after EOM has taken place. Hormone assays in a younger patient may only be transiently abnormal prior to the onset of permanent ovarian failure. The younger patient destined for EOM is a particularly appropriate subject for early medical intervention (e.g. estrogen replacement therapy). Family planning needs to be considered if early loss of reproductive function is anticipated. Decisions about childbearing and preservation of fertilized ova should be undertaken at an early age in such a patient. Further medical advice is important about the value of hormone replacement therapy for a young woman who faces prolonged post-menopausal levels of estrogen. The relative contribution of estrogen replacement to bone strength and cardiovascular health must be balanced against the possibility of breast cancer development, in response to hormone replacement therapy (HRT).
A complex set of endocrine mechanisms regulates the female reproductive system. Understanding the interrelationship of these mechanisms provides the basis for discerning the factors involved in physiological menopause and EOM. No single organ secretes all the hormones responsible for these processes. The hypothalamus, the pituitary gland, and the ovaries, are primary organs, although adrenal and thyroid hormones also play roles. Feedback mechanisms enable the various hormones to affect the production of other hormones within the reproductive system.
The central nervous system controls reproductive hormone production through its release of gonadotropin-releasing hormone (GnRH). This hormone, produced by the hypothalamus, in turn affects gonadotropin secretion by the pituitary. GnRH secretion is responsive to levels of brain neurotransmitters, in particular the two catecholamines dopamine and norepinephrine. Opioids and prostaglandins in the hypothalamus have also been identified as regulators of GnRH release. The neurotransmitter serotonin has not been associated with GnRH release, but it does stimulate the release of prolactin by the hypothalamus. Other peptides have been identified in the brain that act as neurotransmitters. For example, the peptides activin and inhibin, members of TGF-beta superfamily, have been identified within the brain. These substances have opposite effects on pituitary gonadotropin secretion: inhibin diminishes FSH production but does not affect the release of LH; activin stimulates FSH but not LH.
GnRH, when it reaches the anterior lobe of the pituitary, stimulates the production of LH and FSH from the gonadotrophs in the pituitary gland. GnRH only acts to stimulate the production of the gonadotropic hormones. The periodic release of LH and FSH by the pituitary is responsive not only to GnRH but also to feedback systems involving the target organ of these hormones, the ovary. LH acts primarily on the thecal cells of the ovary to induce the synthesis of steroids, while FSH acts primarily on the granulosa cells of the ovary to stimulate the growth of the ovarian follicles. Both types of ovarian cells are thought to be involved in estrogen production. LH acts on the thecal cells to produce the androgens androstenedione and testosterone, which in turn are transported to the granulosa cells where they are aromatized to form the estrogens estrone and estradiol. Before puberty, FSH release is greater than LH. With the onset of the menstrual cycle, LH secretion is greater than FSH secretion. Increased levels of estradiol and inhibin during the years of menstruation act to inhibit FSH release. After menopause, FSH release again exceeds LH release.
Growth factors produced in the ovary provide means for regulating the hormonal behavior of this gland. Inhibin and activin in particular are related to FSH release. Inhibin is regulated positively by FSH levels. Inhibin preferentially affects FSH release over LH release. Levels of inhibin are observed to decrease dramatically during perimenopause, suggesting that this substance has a permissive role in the elevation of FSH before menopause. Activin is observed to stimulate FSH release. Other growth factors have been identified as having hormonal, autocrine, and paracrine effects within the ovary. Comprehensive Gynecology (eds. Mishell et al. 1997).
Cytokines are involved in the production of reproductive hormones via their activities within the brain and in the ovary. Interleukin-1 (IL-1) is a multifunctional cytokine implicated in a number of aspects of ovarian biology. IL-1 has been implicated in follicular development and atresia, ovulation, steroidogenesis and corpus luteum function. Terranova et al., Am. J. Reprod. Immunol. 37:50-63, 1997. IL-1, when found within the ovary, may be produced by immune and non-immune cells. Machelon et al., Hum. Reprod. 10:2198-03, 1995. IL-1 is involved in rescuing ovarian follicles from apoptosis. Kaipia et al., Annu. Rev. Physiol. 59:349-63, 1997. Conversely, the IL-1 receptor antagonist (IL-1RA) has been shown to block ovulation in vivo and in vitro. Tsafriri, Adv. Exp. Med. Biol. 377:121-40, 1995. Furthermore, IL-1 has been identified as a neurotransmitter, active in releasing norepinephrine, dopamine and serotonin, and affected by their intrahypothalamic levels. Tringali et al., Pharmacol. Res. 36:269-73, 1997. IL-1RA exerts a blocking effect by competitively inhibiting the binding of IL-1 to its receptors. Shintani et al., Mol. Neurobiol. 10:47-71, 1995. Through both central and end-organ mechanisms, cytokines and growth factors have been shown to be implicated in the regulation of reproductive endocrinology.
The age of physiological menopause is understood to have a genetic component. Cramer et al., Fertil. Steril. 64:740-45, 1995; Snieder et al., J. Clin. Endocrinol. and Metab. 83:1875-80, 1998. Because management of menopausal symptoms, prevention of post-menopausal health problems, and diagnosis of early post-menopausal illness form an important part of primary medical care, the ability to determine a genetic predisposition to EOM and to identify causative mutations would be valuable.

2. SUMMARY OF THE INVENTION

In one aspect, the present invention provides a novel method for identifying whether a woman is predisposed to developing early-onset menopause (EOM). In one embodiment, the method comprises determining whether an EOM associated allele is present in a nucleic acid sample obtained from a woman. In a preferred embodiment, the EOM associated allele is IL-1RN (+2018) allele 2 or an allele of the IL-1 (44112332) haplotype.
In certain embodiments, detection of an EOM associated allele may be accomplished directly, e.g. by analyzing the DNA, or indirectly, e.g. by analyzing the RNA or protein products of the DNA. Where the marker in question results in the translation of a mutant protein, the protein can be detected by any of a variety of protein detection methods. Such methods include immunodetection and biochemical tests, such as an activity assay, or size fractionation, where the protein has a change in apparent molecular weight either through truncation, elongation, altered folding or altered post-translational modifications.
In a preferred embodiment, the EOM associated allele can be detected by any of a variety of available techniques, including: 1) performing a hybridization reaction between a nucleic acid sample and a probe that is capable of hybridizing to the allele; 2) sequencing at least a portion of the allele; or 3) determining the electrophoretic mobility of the allele or fragments thereof (e.g., fragments generated by endonuclease digestion). The allele can optionally be subjected to an amplification step prior to performance of the detection step. Preferred amplification methods are selected from the group consisting of: the polymerase chain reaction (PCR), the ligase chain reaction (LCR), strand displacement amplification (SDA), cloning, and variations of the above (e.g. RT-PCR and allele specific amplification). Oligonucleotides necessary for amplification may be selected from anywhere in the IL-1 gene loci, either flanking the marker of interest (as required for PCR amplification) or directly overlapping the marker (as in allele-specific oligonuceotide hybridization). The DNA in the human IL-1 region has been mapped, and oligonucleotides for primers can easily be selected with a commercially available primer selection program. In a particularly preferred embodiment, the sample is hybridized with a set of primers, which hybridize 5′ and 3′ in a sense or antisense sequence to the EOM associated allele, and is subjected to a PCR amplification.
In another aspect, the invention features kits for performing the above-described assays. The kit can include nucleic acid sample collection means and a means for determining whether a subject carries an EOM associated allele. The kit may also comprise control samples, either negative or positive, or standards and/or an algorithmic device for assessing the results, and addition reagents and components, including DNA amplification reagents, DNA polymerases, nucleic acid purification reagents, restriction enzymes, buffers, a nucleic acid sampling device, deoxynucleotides, etc. Information obtained using the assays and kits described herein is useful for example for family planning and for treating or preventing the development of symptoms, which are associated with menopause (e.g. osteoporosis and coronary artery disease). In addition, the information can allow a more customized approach to delaying the onset of or treating the symptoms associated with EOM. For example, this information can enable a doctor to: 1) more effectively prescribe a drug that will address the molecular basis of EOM in the subject; and/or 2) better determine the appropriate dosage of a particular drug for the particular patient.
In another aspect, the invention provides in vitro and in vivo methods for identifying biomarkers that are useful in monitoring a subject's progress towards and through menopause. In preferred embodiments, such biomarkers vary depending on a subject's IL-1 genotype. In certain embodiments, such biomarkers may be identified by comparing said biomarkers in subjects with an EOM-associated genotype to those in subjects with a genotype not associated with EOM. In certain embodiments, biomarkers may be used to monitor a subject's progress towards and through menopause.
In another embodiment, the invention features transgenic non-human animals and their uses in identifying biomarkers that are useful in monitoring a subject's progress towards and through menopause. In yet another embodiment, the transgenic animals may be used to screen for EOM therapeutics. In a preferred variation, such animals may be used to identify agonists and antagonists of IL-1α and/or β activity or agonists and antagonists of IL-1RA activity.
In still another aspect, the invention provides in vitro and in vivo assays for screening test substances to identify EOM therapeutics. In one embodiment, the screening assay comprises contacting a cell or subject comprising an EOM associated IL-1 allele with a test substance. One or more biomarker is observed and changes in one or more biomarker from an EOM-associated phenotype to a non-EOM-associated phenotype indicates that the test substance is likely to be effective as an EOM therapeutic. In preferred embodiments, the one or more biomarker is an IL-1 bioactivity. In yet a further aspect, the invention features methods for treating or preventing the development of early onset menopause in a woman, by administering to the woman, a pharmaceutically effective amount of an EOM therapeutic of the invention.
Other features and advantages of the invention will be apparent from the following detailed description and claims.

3. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleic acid sequence for IL-1A (GEN X03833; SEQ ID No. 1).
FIG. 2 shows the nucleic acid sequence for IL-1B (GEN X04500; SEQ ID No. 2).
FIG. 3 shows the nucleic acid sequence for the secreted IL-1RN (GEN X64532; SEQ ID No. 3).
FIG. 4 shows the nucleic acid sequence for the intracellular IL-1RN (GEN X77090; SEQ ID No. 4).

4. DETAILED DESCRIPTION OF THE INVENTION

4.1 Definitions
For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below. In addition, these terms and phrases should be understood in relation to the specification as a whole.
The term “allele” refers to the different sequence variants found at different polymorphic regions. For example, IL-1RN (VNTR) has at least five different alleles. The sequence variants may be single or multiple base changes, including without limitation insertions, deletions, or substitutions, or may be a variable number of sequence repeats.
The term “allelic pattern” refers to the identity of an allele or alleles at one or more polymorphic regions. For example, an allelic pattern may consist of a single allele at a polymorphic site, as for IL-1RN (+2018) allele 1, which is an allelic pattern having at least one copy of IL-1RN allele 1 at position +2018 of the IL-1RN gene loci. Alternatively, an allelic pattern may consist of either a homozygous or heterozygous state at a single polymorphic site. For example, IL1-RN (VNTR) allele 2,2 is an allelic pattern in which there are two copies of the second allele at the VNTR marker of IL-1RN and that corresponds to the homozygous IL-RN (VNTR) allele 2 state. Alternatively, an allelic pattern may consist of the identity of alleles at more than one polymorphic site.
The term “antibody” as used herein is intended to refer to a binding agent including a whole antibody or a binding fragment thereof which is specifically reactive with an IL-1 B polypeptide. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be generated by treating an antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. The antibody of the present invention is further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for an IL-1 B polypeptide conferred by at least one CDR region of the antibody.
“Biological activity” or “bioactivity” or “activity” or “biological function”, which are used interchangeably, for the purposes herein means a function that is directly or indirectly performed by an IL-1 polypeptide (whether in its native or denatured conformation), or by any subsequence thereof. These terms are also intended to encompass properties of IL-1 proteins and genes, such as expression levels and post-translational modifications. Biological activities include binding to a target peptide, e.g., an IL-1 receptor. An IL-1 bioactivity can be modulated by directly affecting an IL-1 polypeptide. Alternatively, an IL-1 bioactivity can be modulated by modulating the level of an IL-1 polypeptide, such as by modulating expression of an IL-1 gene.
As used herein the term “bioactive fragment of an IL-1 polypeptide” refers to a fragment of a full-length IL-1 polypeptide, wherein the fragment specifically mimics or antagonizes the activity of a wild-type IL-1 polypeptide. The bioactive fragment preferably is a fragment capable of interacting with an interleukin receptor.
The term “an aberrant activity”, as applied to an activity of a polypeptide such as IL-1, refers to an activity which differs from the activity of the wild-type or native polypeptide or which differs from the activity of the polypeptide in a healthy subject. An activity of a polypeptide can be aberrant because it is stronger than the activity of its native counterpart. Alternatively, an activity can be aberrant because it is weaker or absent relative to the activity of its native counterpart. An aberrant activity can also be a change in an activity. For example an aberrant polypeptide can interact with a different target peptide. A cell can have an aberrant IL-1 activity due to overexpression or underexpression of an IL-1 locus gene encoding an IL-1 locus polypeptide.
The term “biomarker” refers to a phenotype of a subject or cells. Biomarkers encompass a broad range of intra- and extra-cellular events as well as whole organism physiological changes. Biomarkers may be any of these and are not necessarily involved in inflammatory responses. With respect to cells, biomarkers may be essentially any aspect of cell function, for example: levels or rate of production of signaling molecules, transcription factors, intermediate metabolites, cytokines, prostanoids, steroid hormones (eg. estrogen, progesterone, androstenedione or testosterone), gonadotropins (eg. LH and FSH), gene transcripts, post-translational modifications of proteins, gonadotropin releasing hormone (GnRH), catecholamines (eg. dopamine or norepinephrine), opioids, activin, inhibin, as well as IL-1 bioactivities. Biomarkers may include whole genome analysis of transcript levels or whole proteome analysis of protein levels and/or modifications. Additionally, biomarkers may be reporter genes. For example, an IL-1 promoter or an IL-1 promoter comprising an EOM-associated allele can be operationally linked to a reporter gene. In an alternative method, the promoter can be an IL-1-regulated promoter, such as IL-8. In this manner, the activity of the reporter gene is reflective of the activity of the promoter. Suitable reporter genes include GUS, LacZ, green fluorescent protein (GFP) (and variants thereof, such as Red Fluorescent Protein, Cyan Fluorescent Protein, Yellow Fluorescent Protein and Blue Fluorescent Protein), or essentially any other gene whose product is easily detected. Other preferred biomarkers include factors involved in immune and inflammatory responses, as well as factors involved in IL-1 production and signaling, as described below. In subjects, biomarkers can be, for example, any of the above as well as electrocardiogram parameters, pulmonary function, IL-6 activities, urine parameters or tissue parameters. “EOM associated biomarkers” are any of the above which are found to correlate with EOM, or which are preferentially found in subjects or cells comprising an EOM-associated allele.
“Cells”, “host cells” or “recombinant host cells” are terms used interchangeably herein to refer not only to the particular subject cell, but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact be identical to the parent cell, but are still included within the scope of the term as used herein.
A “chimera,” “mosaic,” “chimeric mammal” and the like, refers to a transgenic mammal with a knock-out or knock-in construct in at least some of its genome-containing cells.
The terms “control” or “control sample” refer to any sample appropriate to the detection technique employed. The control sample may contain the products of the allele detection technique employed or the material to be tested. Further, the controls may be positive or negative controls. By way of example, where the allele detection technique is PCR amplification, followed by size fractionation, the control sample may comprise DNA fragments of an appropriate size. Likewise, where the allele detection technique involves detection of a mutated protein, the control sample may comprise a sample of a mutant protein. However, it is preferred that the control sample comprises the material to be tested. For example, the controls may be a sample of genomic DNA or a cloned portion of the IL-1 gene cluster. However, where the sample to be tested is genomic DNA, the control sample is preferably a highly purified sample of genomic DNA.
The phrases “disruption of the gene” and “targeted disruption” or any similar phrase refers to the site specific interruption of a native DNA sequence so as to prevent expression of that gene in the cell as compared to the wild-type copy of the gene. The interruption may be caused by deletions, insertions or modifications to the gene, or any combination thereof.
The abbreviation “EOM” as used herein refers to “early-onset menopause.” The term “early-onset menopause” as used herein refers to a premature menopause, that is, onset of menopause before that time at which menopause normally occurs. The term “early-onset menopause” includes, but is not limited to, exemplary disorders such as premature ovarian failure. Premature ovarian failure refers to several disorders in which women under 40 years of age present with symptoms and signs of estrogen deficiency, and have elevated levels of the gonadotropins FSH and LH, along with low levels of estradiol. Ovarian failure may occur in association with autoimmune disorders. These disorders include but are not limited to thyroiditis, hypoparathyroidism, hypoadrenalism, diabetes mellitus, rheumatoid arthritis, myasthenia gravis, and pernicious anemia.
An “EOM associated allele” refers to an allele whose presence in a female indicates that the female is susceptible to developing early onset menopause. Examples of EOM associated alleles include allele 2 of the +2018 marker of IL-1RN (contains an Msp 1 site); allele 2 of the VNTR marker of IL-1RN (240 bp PCR product); allele 4 of the 222/223 marker of IL-1A (132 mobility units (mu) PCR product); allele 4 of the gz5/gz6 marker of IL-1A (91 mu PCR product); allele 1 of the −889 marker of IL-1A (contains an NcoI site); allele 1 of the +3954 marker of IL-1B (contains two TaqI sites); allele 2 of the −511 marker of IL-1B (contains a Bsu36I site); allele 3 of the gaat.p33330 marker (197 mu PCR product); and allele 3 of the Y31 marker (160 mu PCR product); allele 2 of the 1731 marker of the IL-1RN gene (A at position 1731); allele 2 of the 1812 marker of the IL-1RN gene (A at position 1812); allele 2 of the 1868 marker of the IL-1RN gene (G at position 1868); allele 2 of the 1887 marker of the IL-1RN gene (C at position 1887); allele 2 of the 8006 marker of the IL-1RN gene (contains an HpaII or MspI site), allele 2 of the 8061 marker of the IL-1RN gene (lacks an MwoI site) and allele 2 of the 9589 marker of the IL-1RN gene (contains an SspI site).
An “EOM causative functional mutation” refers to a mutation which causes or contributes to the development of early onset menopause in a woman. Preferred mutations occur within the IL-1 complex. Examples of EOM causative mutations include IL-1RN (+2018), which results in altered levels of IL-1 receptor antagonist.
An “EOM-associated phenotype” is a phenotype of subjects or cells that is associated with EOM or associated with an increased likelihood of developing EOM. An EOM-associated phenotype is also any phenotype found in a subject or cell having an EOM-associated allele, where such phenotype differs from that found in subjects or cells lacking an EOM-associated allele. Such phenotypes encompass essentially any characteristic of a biomarker. An EOM-associated phenotype may not be directly involved in EOM but may nonetheless serve as an indicator for EOM. A “non-EOM-associated phenotype” is a phenotype that is not associated with EOM or with an increased likelihood of developing EOM.
An “EOM therapeutic” refers to any agent that prevents or postpones the development or alleviates the symptoms of early onset menopause. An EOM therapeutic can be a polypeptide, peptidomimetic, nucleic acid, other inorganic or organic molecule, or a nutraceutical, preferably a “small molecule”. Preferably an EOM therapeutic can modulate at least one EOM-associated phenotype. For example, an EOM therapeutic may modulate an activity of an IL-1 polypeptide, e.g., interaction with an IL-1 receptor, by mimicking or potentiating (agonizing) or inhibiting (antagonizing) the effects of a naturally-occurring IL-1 polypeptide. An IL-1 agonist can be a wild-type IL-1 protein or derivative thereof having at least one bioactivity of the wild-type IL-1, e.g. receptor binding activity. An IL-1 agonist can also be a compound that upregulates expression of an IL-1 gene or which increases at least one bioactivity of an IL-1 protein. An agonist can also be a compound which increases the interaction of an IL-1 polypeptide with another molecule, e.g, an interleukin receptor. An IL-1 antagonist can be a compound which inhibits or decreases the interaction between an IL-1 protein and another molecule, e.g., a receptor, such as an IL-1 receptor. Accordingly, a preferred antagonist is a compound which inhibits or decreases binding to an IL-1 receptor and thereby blocks subsequent activation of the IL-1 receptor. An antagonist can also be a compound that downregulates expression of an IL-1 locus gene or which reduces the amount of an IL-1 protein present. The IL-1 antagonist can be a dominant negative form of an IL-1 polypeptide, e.g., a form of an IL-1 polypeptide which is capable of interacting with a target peptide, e.g., an IL-1 receptor, but which does not promote the activation of the IL-1 receptor. The IL-1 antagonist can also be a nucleic acid encoding a dominant negative form of an IL-1 polypeptide, an IL-1 antisense nucleic acid, or a ribozyme capable of interacting specifically with an IL-1 RNA. Yet other IL-1 antagonists are molecules which bind to an IL-1 polypeptide and inhibit its action. Such molecules include peptides, e.g., forms of IL-1 target peptides which do not have biological activity, and which inhibit binding by IL-1 to IL-1 receptors. Thus, such peptides will bind the active site of IL-1 and prevent it from interacting with target peptides, e.g., an IL-1 receptor. Yet other IL-1 antagonists include antibodies interacting specifically with an epitope of an IL-1 molecule, such that binding interferes with the biological function of the IL-1 locus polypeptide. In yet another preferred embodiment, the IL-1 antagonist is a small molecule, such as a molecule capable of inhibiting the interaction between an IL-1 polypeptide and a target IL-1 receptor. Alternatively, the small molecule can function as an antagonist by interacting with sites other than the IL-1 receptor binding site. An antagonist can be any class of molecule, including a nucleic acid, protein, carbohydrate, lipid or combination thereof, but for therapeutic purposes is preferably a small molecule.
“Genotyping” refers to the analysis of an individual's genomic DNA (or a nucleic acid corresponding thereto) to identify a particular disease causing or contributing mutation or polymorphism, directly or based on detection of a mutation or polymorphism (a marker) that is in linkage disequilibrium with the disease causing or contributing gene.
A “haplotype” refers to a set of alleles that are inherited together as a group (are in linkage disequilibrium). As used herein, haplotype is defined to include those haplotypes that occur at statistically significant levels (p_corr≦0.05). As used herein, the phrase “an IL-1 haplotype” refers to a haplotype in the IL-1 loci. At least two IL-1 proinflammatory haplotypes are known. The IL-1 (44112332) haplotype is associated with decreased IL-receptor antagonist activity, whereas the IL-1 (33441461) haplotype is associated with increased IL-1α and β agonist activity. The IL-1 (44112332) haplotype includes the following alleles: IL-1RN (+2018) allele 2; IL-1RN (VNTR) allele 2; IL-1A (222/223) allele 4; IL-1A (gz5/gz6) allele 4; IL-1A (−889) allele 1; IL-1B (+3954) allele 1; IL-1B (−511) allele 2; gaat.p33330 allele 3; Y31 allele 3; IL-1RN exon 1ic (1812) allele 2; IL-1RN exon 1ic (1868) allele 2; IL-1RN exon 1ic (1887) allele 2; Pic (1731) allele 2; IL-1A (+4845) allele 1; IL-1B (+6912) allele 1; and IL-1B (−31) allele 2.
“IL-1 gene cluster” and “IL-1 loci” as used herein include all the nucleic acid at or near the 2q13 region of chromosome 2, including at least the IL-1A, IL-1B and IL-1 RN genes and any other linked sequences. The terms “IL-1A”, “IL-1B”, and “IL-1RN” as used herein refer to the genes coding for IL-1α, IL-1β, and IL-1 receptor antagonist or IL-1ra, respectively. The DNA in this region has been mapped. Nicklin et al., Genomics 19:382-84, 1994; Nothwang H. G., et al., Genomics 41:370, 1997; Clark, et al., Nucl. Acids. Res. 14:7897-914, 1986, (erratum at Nucleic Acids Res. 15:868, 1987. The gene accession numbers (GEN) for IL-1A and IL-1B, are X03833 and X04500, respectively. In general, references to nucleotide positions for IL-1RN refer to the nucleotide sequence in GEN X64532, which is the secreted form of the protein, unless there is some indication, either expressly indicated or implied from the context, that the intracellular form, which has GEN X77090, is being referenced. The two forms of IL-1RA are encoded by a single gene by alternative use of two first exons. See generally Lennard et al., Crit. Rev. Immuno. 15:77-105, 1995.
“IL-1 functional mutation” refers to a mutation within the IL-1 gene cluster that results in an altered phenotype (i.e. affects the function of an IL-1 gene or protein). Examples include: IL-1A (+4845) allele 1, IL-1B (+3954) allele 2, IL-1B (+6912) allele 2 and IL-1RN (+2018) allele 2.
“IL-1X (Z) allele Y” refers to a particular allelic form, designated Y, occurring at an IL-1 locus polymorphic site in gene X, wherein X is IL-1A, B, or RN or some other gene in the IL-1 gene loci, and positioned at or near nucleotide Z, wherein nucleotide Z is numbered relative to the major transcriptional start site, which is nucleotide+1, of the particular IL-1 gene X. As further used herein, the term “IL-1×allele (Z)” refers to all alleles of an IL-1 polymorphic site in gene X positioned at or near nucleotide Z. For example, the term “IL-1 RN (+2018) allele” refers to alternative forms of the IL-1 RN gene at marker +2018. “IL-1 RN (+2018) allele 1” refers to a form of the IL-1RN gene which contains a thymine (T) at position +2018 of the sense strand. Clay et al., Hum. Genet. 97:723-26, 1996. “IL-1RN (+2018) allele 2” refers to a form of the IL-1RN gene which contains a cytosine (C) at position +2018 of the plus strand. When a subject has two identical IL-1 RN alleles, the subject is said to be homozygous, or to have the homozygous state. When a subject has two different IL-1 RN alleles, the subject is said to be heterozygous, or to have the heterozygous state. The term “IL-1RN (+2018) allele 2,2” refers to the homozygous IL-1 RN (+2018) allele 2 state. Conversely, the term “IL-1RN (+2018) allele 1,1” refers to the homozygous IL-1 RN (+2018) allele 1 state. The term “IL-1RN (+2018) allele 1,2” refers to the heterozygous allele 1 and 2 state.
“IL-1 related” as used herein is meant to include all genes related to the human IL-1 locus genes on human chromosome 2 (2q 12-14). These include IL-1 genes of the human IL-1 gene cluster located at chromosome 2 (2q 13-14) which include: the IL-1A gene which encodes interleukin-1 a, the IL-1 B gene which encodes interleukin-1β, and the IL-1RN (or IL-1ra) gene which encodes the interleukin-1 receptor antagonist. Furthermore these IL-1 related genes include the type I and type II human IL-1 receptor genes located on human chromosome 2 (2q12) and their mouse homologs located on mouse chromosome 1 at position 19.5 cM. Interleukin-1 α, interleukin-1 β, and interleukin-1 RN are related in so much as they all bind to IL-1 type I receptors, however only interleukin-1α and interleukin-1β are agonist ligands which activate IL-1 type I receptors, while interleukin-1 RN is a naturally occurring antagonist ligand.
Where the term “IL-1” is used in reference to a gene product or polypeptide, it is meant to refer to all gene products encoded by the interleukin-1 locus on human chromosome 2 (2q 12-14) and their corresponding homologs from other species or functional variants thereof. The term IL-1 thus includes secreted polypeptides which promote an inflammatory response, such as IL-1α and IL-1β, as well as a secreted polypeptide which antagonizes inflammatory responses, such as IL-1 receptor antagonist and the IL-1 type II (decoy) receptor.
An “IL-1 receptor” or “IL-1 R” refers to various cell membrane bound protein receptors capable of binding to and/or transducing a signal from IL-1 locus-encoded ligand. The term applies to any of the proteins which are capable of binding interleukin-1 (IL-1) molecules and, in their native configuration as mammalian plasma membrane proteins, presumably play a role in transducing the signal provided by IL-1 to a cell. As used herein, the term includes analogs of native proteins with IL-1-binding or signal transducing activity. Examples include the human and murine IL-1 receptors described in U.S. Pat. No. 4,968,607. The term “IL-1 nucleic acid” refers to a nucleic acid encoding an IL-1 protein.
An “IL-1 polypeptide” and “IL-1 protein” are intended to encompass polypeptides comprising the amino acid sequence encoded by the IL-1 genomic DNA sequences shown in FIGS. 1, 2, and 3, or fragments thereof, and homologs thereof and include agonist and antagonist polypeptides.
“Increased risk” refers to a statistically higher frequency of occurrence of the disease or condition in an individual carrying a particular polymorphic allele in comparison to the frequency of occurrence of the disease or condition in a member of a population that does not carry the particular polymorphic allele.
The term “interact” as used herein is meant to include detectable relationships or associations (e.g. biochemical interactions) between molecules, such as interactions between protein-protein, protein-nucleic acid, nucleic acid-nucleic acid and protein-small molecule or nucleic acid-small molecule in nature.
The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule. For example, an isolated nucleic acid encoding one of the subject IL-1 polypeptides preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the IL-1 gene in genomic DNA, more preferably no more than 5 kb of such naturally occurring flanking sequences, and most preferably less than 1.5 kb of such naturally occurring flanking sequence. The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
A “knock-in” transgenic animal refers to an animal that has had a modified gene introduced into its genome and the modified gene can be of exogenous or endogenous origin. A “knock-out” transgenic animal refers to an animal in which there is partial or complete suppression of the expression of an endogenous gene (e.g, based on deletion of at least a portion of the gene, replacement of at least a portion of the gene with a second sequence, introduction of stop codons, the mutation of bases encoding critical amino acids, or the removal of an intron junction, etc.).
A “knock-out construct” refers to a nucleic acid sequence that can be used to decrease or suppress expression of a protein encoded by endogenous DNA sequences in a cell. In a simple example, the knock-out construct is comprised of a gene, such as the IL-1 RN gene, with a deletion in a critical portion of the gene so that active protein cannot be expressed therefrom. Alternatively, a number of termination codons can be added to the native gene to cause early termination of the protein or an intron junction can be inactivated. In a typical knock-out construct, some portion of the gene is replaced with a selectable marker (such as the neo gene) so that the gene can be represented as follows: IL-1 RN 5′/neo/IL-1 RN 3′, where IL-1 RN5′ and IL-1RN 3′, refer to genomic or cDNA sequences which are, respectively, upstream and downstream relative to a portion of the IL-1RN gene and where neo refers to a neomycin resistance gene. In another knock-out construct, a second selectable marker is added in a flanking position so that the gene can be represented as: IL-1RN/neo/IL-1RN/TK, where TK is a thymidine kinase gene which can be added to either the IL-1RN5′ or the IL-1RN3′ sequence of the preceding construct and which further can be selected against (i.e. is a negative selectable marker) in appropriate media. This two-marker construct allows the selection of homologous recombination events, which removes the flanking TK marker, from non-homologous recombination events which typically retain the TK sequences. The gene deletion and/or replacement can be from the exons, introns, especially intron junctions, and/or the regulatory regions such as promoters.
“Linkage disequilibrium” refers to co-inheritance of two alleles at frequencies greater than would be expected from the separate frequencies of occurrence of each allele in a given control population. The expected frequency of occurrence of two alleles that are inherited independently is the frequency of the first allele multiplied by the frequency of the second allele. As used herein, the term “linkage disequilibrium” also refers to linked sequences. Alleles that co-occur at expected frequencies are said to be in “linkage equilibrium” or “not linked.” When referring to allelic patterns that are comprised of more than one allele, a first allelic pattern is in linkage disequilibrium with a second allelic pattern if all the alleles that comprise the first allelic pattern are in linkage disequilibrium with at least one of the alleles of the second allelic pattern. An example of linkage disequilibrium is that which occurs between the alleles at the IL-1 RN (+2018) and IL-1RN (VNTR) polymorphic sites. The two alleles at IL-1RN (+2018) are >97% in linkage disequilibrium with the two most frequent alleles of IL-1RN (VNTR), which are allele 1 and allele 2.
The term “marker” refers to a sequence in the genome that is known to vary among individuals. For example, the IL-1RN gene has a marker that consists of a variable number of tandem repeats (VNTR). The different sequence variants at a given marker are called alleles, mutations or polymorphisms. For example, the VNTR marker has at least five different alleles, three of which are rare. Different alleles could have a single base change, including substitution, insertion or deletion, or could have a change that affects multiple bases, including substitutions, insertions, deletions, repeats, inversions and combinations thereof.
A “mutated gene” or “mutation” or “functional mutation” refers to an allelic form of a gene, which is capable of altering the phenotype of a subject having the mutated gene relative to a subject which does not have the mutated gene. The altered phenotype caused by a mutation can be corrected or compensated for by certain agents. If a subject must be homozygous for this mutation to have an altered phenotype, the mutation is said to be recessive. If one copy of the mutated gene is sufficient to alter the phenotype of the subject, the mutation is said to be dominant. If a subject has one copy of the mutated gene and has a phenotype that is intermediate between that of a homozygous and that of a heterozygous subject (for that gene), the mutation is said to be co-dominant.
A “non-human animal” of the invention includes mammals such as rodents, non-human primates, sheep, dogs, cows, goats, etc. Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse, though transgenic amphibians, such as members of the Xenopus genus, and transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation. The term “chimeric animal” is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant gene is expressed in some but not all cells of the animal. The term “tissue-specific chimeric animal” indicates that one of the recombinant IL-1 genes is present and/or expressed or disrupted in some tissues but not others. The term “non-human mammal” refers to any members of the class Mammalia, except for humans.
The term “nucleic acid” refers to polynucleotides or oligonucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). Nucleic acids may be full length genes or portions thereof that are useful, for example, as primers or as probes. The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs (e.g. peptide nucleic acids) and as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
“Nutraceuticals” are defined as substances comprising vitamins, minerals, proteins, amino acids, sugars, phytoestrogens, flavonoids, phenolics, anthocyanins, carotenoids, polymers of the above, and mixtures of the above.
The term “polymorphism” refers to the coexistence of more than one form of a gene or portion (e.g., allelic variant) thereof. A portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a “polymorphic region.” As used herein, the term “polymorphic region” includes, without limitation, a polymorphic site consisting of a single nucleotide, e.g., a single nucleotide polymorphism (SNP). A specific genetic sequence at a polymorphic region is an allele. A polymorphic region can be a single nucleotide, the identity of which differs in different alleles. A polymorphic region can also be more than one nucleotide long, and possibly significantly longer in length.
The term “propensity” as used herein in reference to a condition or disease state, as in “propensity” for a condition or disease, is used interchangeably with the expressions “susceptibility” or “predisposition” to a condition or disease. For example, the term “propensity” is used in reference to certain polymorphic alleles which are hereby discovered to be associated with a given condition or disease state. They are thus over-represented in individuals with a condition or disease as compared with healthy individuals. Therefore, the presence of such alleles indicates that an individual is at increased risk for the future development of a condition or disease, and the absence of such alleles indicates that the individual is not at increased risk for the condition or disease.
“Small molecule” as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be nucleic acids, peptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules.
As used herein, the term “specifically hybridizes” or “specifically detects” refers to the ability of a nucleic acid molecule to hybridize to at least approximately 6 consecutive nucleotides of a sample nucleic acid.
A “test substance” can comprise essentially any element, chemical compound (including a nucleic acid, protein, peptide, carbohydrate or lipid) or mixture thereof, including a nutraceutical or small molecule drug.
“Transcriptional regulatory sequence” is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
As used herein, the term “transgene” means a nucleic acid sequence (encoding, e.g., one of the IL-1 polypeptides, or an antisense transcript thereto) which has been introduced into a cell. A transgene could be partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can also be present in a cell in the form of an episome. A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
A “transgenic animal” refers to any animal, preferably a non-human mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In the typical transgenic animals described herein, the transgene causes cells to express a recombinant form of one of the IL-1 polypeptides, e.g. either agonistic or antagonistic forms. However, transgenic animals in which the recombinant IL-1 gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below. Moreover, “transgenic animal” also includes those recombinant animals in which gene disruption of one or more IL-1 genes is caused by human intervention, including both recombination and antisense techniques. The term is intended to include all progeny generations. Thus, the founder animal and all F1, F2, F3, and so on, progeny thereof are included.
The term “treating” as used herein is intended to encompass curing as well as ameliorating at least one symptom of a condition or disease.
The term “vector” refers to a nucleic acid molecule, which is capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids” which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification, “plasmid” and “vector” are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
The term “wild-type allele” refers to an allele of a gene which, when present in two copies in a subject results in a wild-type phenotype. There can be several different wild-type alleles of a specific gene, since certain nucleotide changes in a gene may not affect the phenotype of a subject having two copies of the gene with the nucleotide changes.
4.2 Predictive Medicine
4.2.1. IL-1 Linked Polymorphisms Associated with EOM
The invention is based, at least in part, on the identification of an allele (IL-1 RN (+2018) allele 2), that is associated with early onset menopause (EOM) in women. Because this allele is in linkage disequilibrium with other alleles in the IL-1 region, the detection of such other alleles can also indicate a predisposition to developing EOM in a woman. For example, IL-1RN (+2018) allele 2, also referred to as exon 2 (8006) (GenBank:X64532 at 8006) polymorphism, Clay et al., Hum. Genet. 97:723-26, 1996, is in linkage disequilibrium with IL-1 RN (VNTR) allele 2, which is a member of the IL-1 (44112332) human haplotype. Cox et al., Am. J. Human Genet. 62:1180-88, 1998; International Patent Application No. PCT/GB98/01481. Further, the following alleles of the IL-1 (44112332) proinflammatory haplotype are known to be in linkage disequilibrium with IL-1RN (+2018): allele 4 of the 222/223 marker of IL-1A (a dinucleotide repeat polymorphism (HUGO GDB: 190869); allele 4 of the gz5/gz6 marker of IL-1A (a trinucleotide repeat polymorphism (HUGO GDB: 177384; Zuliani et al., Am. J. Hum. Genet. 46:963-69, 1990); allele 1 of the −889 marker of IL-1 A (a single base variation marker-HUGO GDB: 210902; McDowell et al., Arthritis and Rheumatism 38:221-28, 1995); allele 1 of the +3954 marker of IL-1B (a single base C/T variation; di Giovine et al., Cytokine 7:606 (1995); Pociot et al. Eur J. Clin. Invest. 22:396-402, 1992); allele 2 of the −511 marker of IL-1B; allele 3 of the gaat.p33330 marker; allele 3 of the Y31 marker, IL-1A (+4825) allele 2, IL-1B (+6912) allele 1, and IL-1B (−31) allele 2.
Three other polymorphisms in an IL-1 RN alternative exon (Exon 1 ic, which produces an intracellular form of the gene product, GEN X77090) are in linkage disequilibrium with IL-1RN (+2018) allele 2. These include: the IL-1RN exon 1ic (1812) polymorphism (GenBank:X77090 at 1812); the IL-1RN exon 1ic (1868) polymorphism (GenBank:X77090 at 1868); and the IL-1RN exon 1ic (1887) polymorphism (GenBank:X77090 at 1887). Yet another polymorphism in the promoter for the alternatively spliced intracellular form of the gene, the Pic (1731) polymorphism (GenBank:X77090 at 1731), is also in linkage disequilibrium with IL-1RN (+2018) allele 2. The corresponding sequence alterations for each of these IL-1RN polymorphic loci is shown below.

Al- Exon 2 Exon 1ic-1 Exon 1ic-2 Exon 1ic-3 Pic (1731

lele (+2018 of (1812 of GB: (1868 of GB: (1887 of GB: of GB:

No. IL-1RN) X77090) X77090 X77090) X77090)

1 T G A G G

2 C A G C A

Clay et al., Hum. Genet. 97:723-26, 1996. For each of these polymorphic loci, the allele 2 sequence variant has been determined to be in linkage disequilibrium with IL-1RN (+2018) allele 2.
In addition to the allelic patterns described above, one of skill in the art can readily identify other alleles (including polymorphisms and mutations) that are in linkage disequilibrium with IL-1RN (+2018) allele 2, and are thereby associated with EOM. For example, a nucleic acid sample from a first group of women without EOM can be collected, as well as DNA from a second group of women with EOM. The nucleic acid sample can then be compared to identify those alleles that are over-represented in the second group as compared with the first group, wherein such alleles are presumably associated with EOM. Alternatively, alleles that are in linkage disequilibrium with an EOM associated allele can be identified, for example, by genotyping a large population and performing statistical analyses to determine which alleles appear more commonly together than expected. Preferably the group is chosen to be comprised of genetically related individuals. Genetically related individuals include individuals from the same race, the same ethnic group, or even the same family. As the degree of genetic relatedness between a control group and a test group increases, so does the predictive value of polymorphic alleles which are ever more distantly linked to a disease-causing allele. This is because less evolutionary time has passed to allow polymorphisms which are linked along a chromosome in a founder population to redistribute through genetic cross-over events. Thus race-specific, ethnic-specific, and even family-specific diagnostic genotyping assays can be developed to allow for the detection of disease alleles which arose at ever more recent times in human evolution, e.g., after divergence of the major human races, after the separation of human populations into distinct ethnic groups, and even within the recent history of a particular family line.
Linkage disequilibrium between two polymorphic markers or between one polymorphic marker and a disease-causing mutation is a meta-stable state. Absent selective pressure or the sporadic linked reoccurrence of the underlying mutational events, the polymorphisms will eventually become disassociated by chromosomal recombination events and will thereby reach linkage equilibrium through the course of human evolution. Thus, the likelihood of finding a polymorphic allele in linkage disequilibrium with a disease or condition may increase with changes in at least two factors: decreasing physical distance between the polymorphic marker and the disease-causing mutation, and decreasing number of meiotic generations available for the dissociation of the linked pair. Consideration of the latter factor suggests that, the more closely related two individuals are, the more likely they will share a common parental chromosome or chromosomal region containing the linked polymorphisms and the less likely that this linked pair will have become unlinked through meiotic cross-over events occurring each generation. As a result, the more closely related two individuals are, the more likely it is that widely spaced polymorphisms may be co-inherited. Thus, for individuals related by common race, ethnicity or family, the reliability of ever more distantly spaced polymorphic loci can be relied upon as an indicator of inheritance of a linked disease-causing mutation. For example, menopause occurring before age 46 is 6 times more likely to occur in women with family histories of early menopause, especially in families where a first-degree relative entered menopause before age 40. Torgerson et al., Eur. J. Obstet. Gynec. Reprod. Biol. 74:63-66, 1997
Appropriate probes may be designed to hybridize to a specific region of the IL-1 locus, such as IL-1 A, IL-1B or IL-1RN. These genomic DNA sequences are shown in FIGS. 1-4, respectively, and further correspond to formal SEQ ID Nos. 1-4, respectively. Alternatively, these probes may incorporate other regions of the IL-1 genomic locus, including intergenic sequences. Indeed this region of human chromosome 2 spans some 400,000 base pairs and, assuming an average of one single nucleotide polymorphism every 1,000 base pairs, includes some 400 SNPs loci alone. Yet other polymorphisms available for use with the immediate invention are obtainable from various public sources. For example, the human genome database collects intragenic SNPs, is searchable by sequence and currently contains approximately 2,700 entries (http://hgbase.interactiva.de). Also available is a human polymorphism database maintained by the Massachusetts Institute of Technology (MIT SNP database (http://www.genome.wi.mit.edu/SNP/human/index.html)). From such sources SNPs as well as other human polymorphisms occurring in the region of the IL-1 locus on human chromosome 2, region q12-13 may be found.
For example, examination of this region of the human genome in any one of these databases reveals that the IL-1 locus genes are flanked by a centromere proximal polymorphic marker designated microsatellite marker AFM220ze3 at 127.4 cM (centiMorgans) (see GenBank Acc. No. Z17008) and a distal polymorphic marker designated microsatellite anchor marker AFM087xa1 at 127.9 cM (see GenBank Acc. No. Z16545). These human polymorphic loci are both CA dinucleotide repeat microsatellite polymorphisms, and, as such, show a high degree of heterozygosity in human populations. For example, one allele of AFM220ze3 generates a 211 bp PCR amplification product with a 5′ primer of the sequence TGTACCTAAGCCCACCCTTTAGAGC (SEQ ID No. 5) and a 3′ primer of the sequence TGGCCTCCAGAAACCTCCAA (SEQ ID No. 6). Furthermore, one allele of AFM087xa1 generates a 177 bp PCR amplification product with a 5′ primer of the sequence GCTGATATTCTGGTGGGAAA (SEQ ID No. 7) and a 3′ primer of the sequence GGCAAGAGCAAAACTCTGTC (SEQ ID No. 8). Equivalent primers corresponding to unique sequences occurring 5′ and 3′ to these human chromosome 2 CA dinucleotide repeat polymorphisms will be apparent to one of skill in the art. Reasonable equivalent primers include those which hybridize within about 1 kb of the designated primer, and which further are anywhere from about 17 bp to about 27 bp in length. A general guideline for designing primers for amplification of unique human chromosomal genomic sequences is that they possess a melting temperature of at least about 50° C., wherein an approximate melting temperature can be estimated using the formula T_melt=[2×(# of A or T)+4×(# of G or C)].
A number of other human polymorphic loci occur between these two CA dinucleotide repeat polymorphisms and provide additional targets for determination of an EOM prognostic allele in a family or other group of genetically related individuals. For example, the National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov/genemap/) lists a number of polymorphism markers in the region of the IL-1 locus and provides guidance in designing appropriate primers for amplification and analysis of these markers.
Accordingly, the nucleotide segments of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of human chromosome 2 q 12-13 or cDNAs from that region or to provide primers for amplification of DNA or cDNA from this region. The design of appropriate probes for this purpose requires consideration of a number of factors. For example, fragments having a length of between 10, 15, or 18 nucleotides to about 20, or to about 30 nucleotides, will find particular utility. Longer sequences, e.g., 40, 50, 80, 90, 100, even up to full length, are even more preferred for certain embodiments. Lengths of oligonucleotides of at least about 18 to 20 nucleotides are well accepted by those of skill in the art as sufficient to allow sufficiently specific hybridization so as to be useful as a molecular probe. Furthermore, depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids. For example, relatively low salt and/or high temperature conditions, such as provided by 0.02 M-0.15M NaCl at temperatures of about 50° C. to about 70° C. Such selective conditions may tolerate little, if any, mismatch between the probe and the template or target strand.
4.2.2 EOM Causative Functional Mutations
An EOM causative functional mutation occurring within an IL-1 gene (e.g. IL-1 A, IL-1 B or IL-1 RN) or a gene locus, which is linked thereto may alter, for example, the open reading frame or splicing pattern of the gene, thereby resulting in the formation of an inactive or hypoactive gene product. For example, a mutation which occurs in intron 6 of the IL-1A locus corresponds to a variable number of tandem repeat 46 bp sequences corresponding to from five to 18 repeat units (Bailly, et al. (1993) Eur. J. Immunol. 23: 1240-45). These repeat sequences contain three potential binding sites for transcriptional factors: an SP 1 site, a viral enhancer element, and a glucocorticoid-responsive element; therefore individuals carrying IL-1 A intron 6 VNTR alleles with large numbers of repeat units may be subject to altered transcriptional regulation of the IL-1A gene and consequent perturbations of inflammatory cytokine production. Indeed, there is evidence that increased repeat number at this polymorphic IL-1A locus leads to decreased IL-1α synthesis (Bailly et al. (1996) Mol Immunol 33: 999-1006).
Alternatively, a mutation can result in a hyperactive gene product. For example, allele 2 of the IL-1B (C at +6912) polymorphism occurs in the 3′ UTR (untranslated region) of the IL-1B mRNA and is associated with an approximately four-fold increase in the steady state levels of both IL-1B mRNA and IL-1B protein compared to those levels associated with allele 1 of the IL-1B gene (G at +6912). Further, an IL-1B (−511) mutation occurs near a promoter binding site for a negative glucocorticoid response element (Zhang et al. (1997) DNA Cell Biol 16: 145-52). This element potentiates a four-fold repression of IL-1 B expression by dexamethosone and a deletion of this negative response elements causes a 2.5-fold increase in IL-1B promoter activity. The IL-1 B (−511) polymorphism may thus directly affect cytokine production and inflammatory responses. These examples demonstrate that genetic variants occurring in the IL-1A or IL-1B gene can directly lead to the altered production or regulation of IL-1 cytokine activity.
4.2.3. Detection of Alleles
Many methods are available for detecting specific alleles at human polymorphic loci. The preferred method for detecting a specific polymorphic allele may depend, in part, upon the molecular nature of the polymorphism. For example, the preferred method of detection used for a single nucleotide polymorphism may differ from that employed for a VNTR polymorphism.
By way of general introduction, detection of specific alleles may be nucleic acid techniques based on hybridization, size, or sequence, such as restriction fragment length polymorphism (RFLP), nucleic acid sequencing, and allele specific oligonucleotide (ASO) hybridization. In one embodiment, the methods comprise detecting in a sample DNA obtained from a woman the existence of an allele associated with EOM. For example, a nucleic acid composition comprising a nucleic acid probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence to an allele associated with EOM can be used as follows: the nucleic acid in a sample is rendered accessible for hybridization, the probe is contacted with the nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected. Such technique can be used to detect alterations or allelic variants at either the genomic or mRNA level as well as to determine mRNA transcript levels, when appropriate.
In another exemplary embodiment, an allele associated with EOM at a VNTR polymorphism, such as IL-1RN (VNTR) allele 2, may be determined. For example, the number of tandem repeats of the IL-1RN (VNTR) polymorphic site may be determined by amplifying the nucleic acid to be analyzed, and determining the identity of the allele of that site by analyzing the size of said amplification product.
A preferred detection method is ASO hybridization using probes overlapping an allele associated with EOM and has about 5, 10, 20, 25, or 30 nucleotides around the mutation or polymorphic region. In a preferred embodiment of the invention, several probes capable of hybridizing specifically to other allelic variants involved in EOM are attached to a solid phase support, e.g., a “chip” (which can hold up to about 250,000 oligonucleotides). Oligonucleotides can be bound to a solid support by a variety of processes, including lithography. Mutation detection analysis using these chips comprising oligonucleotides, also termed “DNA probe arrays” is described e.g., in Cronin et al., Human Mutation 7:244, 1996. In one embodiment, a chip comprises all the allelic variants of at least one polymorphic region of a gene. The solid phase support is then contacted with a test nucleic acid and hybridization to the specific probes is detected. Accordingly, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridization experiment.
These techniques may also comprise the step of amplifying the nucleic acid before analysis. Amplification techniques are known to those of skill in the art and include, but are not limited to cloning, polymerase chain reaction (PCR), polymerase chain reaction of specific alleles (ASA), ligase chain reaction (LCR), nested polymerase chain reaction, self sustained sequence replication (Guatelli, J. C. et al., Proc. Natl. Acad. Sci. USA 87:1874-78, 1990), transcriptional amplification system (Kwoh, D. Y. et al., Proc. Natl. Acad. Sci. USA 86:1173-77, 1989), and Q-Beta Replicase (Lizardi, P. M. et al., Bio/Technology 6:1197, 1988).
Amplification products may be assayed in a variety of ways, including size analysis, restriction digestion followed by size analysis, detecting specific tagged oligonucleotide primers in the reaction products, ASO hybridization, allele specific 5′ exonuclease detection, sequencing, hybridization, and the like.
PCR based detection means can include multiplex amplification of a plurality of markers simultaneously. For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously. Alternatively, it is possible to amplify different markers with primers that have detectable labels that are different and thus can each be differentially detected. Of course, hybridization based detection means allow the differential detection of multiple PCR products in a sample. Other techniques are known in the art to allow multiplex analyses of a plurality of markers.
In a merely illustrative embodiment, the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to an allele associated with EOM, such as IL-1RN (+2018) allele 2, under conditions such that hybridization and amplification of the desired marker occurs, and (iv) detecting the amplification product. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
An allele associated with EOM can also be identified by alterations in restriction enzyme cleavage patterns through RFLP analysis. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis through size fractionization.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence a polymorphic site having at least one allele associated with EOM. Exemplary sequencing reactions include those based on techniques developed by Maxim and Gilbert (Proc. Natl. Acad. Sci. USA 74:560, 1977) or Sanger (Sanger et al., Proc. Nat. Acad. Sci. USA 74:5463, 1977). It is also contemplated that any of a variety of automated sequencing procedures may be utilized when performing the subject assays (Biotechniques 19:448, 1995), including sequencing by mass spectrometry (see, for example PCT publication WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-62, 1996; and Griffin et al., Appl. Biochem. Biotechnol. 38:147-59, 1993). It will be evident to one skilled in the art that, for certain embodiments, the occurrence of only one, two or three of the nucleic acid bases need be determined in the sequencing reaction. For instance, A-track or the like, e.g., where only one nucleic acid is detected, can be carried out.
In a further embodiment, protection from cleavage agents (such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine) can be used to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNA heteroduplexes (Myers et al., Science 230:1242, 1985). In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes formed by hybridizing (labelled) RNA or DNA containing the wild-type allele with the sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digest the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. (See, for example, Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397, 1988; Saleeba et al., Methods Enzymol. 217:286-95, 1992) In a preferred embodiment, the control DNA or RNA can have a detectable label.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes). For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al., Carcinogenesis 15:1657-62, 1994). According to an exemplary embodiment, a probe based on IL-1RN (+2018) allele 2 is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. (See, for example, U.S. Pat. No. 5,459,039.)
In other embodiments, alterations in electrophoretic mobility will be used to identify an allele associated with EOM. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al., Proc. Natl. Acad. Sci. USA 86:2766, 1989, see also Cotton, Mutat. Res. 285:125-44, 1993; and Hayashi, Genet. Anal. Tech. Appl. 9:73-79, 1992. Single-stranded DNA fragments of sample and control are denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes, such as primers with a detectable label. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al., Trends Genet. 7:5, 1991).
In yet another embodiment, the movement of an allele associated with EOM in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al., Nature 313:495, 1985). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner, Biophys. Chem. 265:12753, 1987).
Examples of other techniques for detecting alleles associated with EOM include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation or nucleotide difference (e.g., in allelic variants) is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al., Nature 324:163, 1986); Saiki et al., Proc. Natl. Acad. Sci. USA 86:6230, 1989). Such ASO hybridization techniques may be used to test one mutation or polymorphic region per reaction when oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations or polymorphic regions when the oligonucleotides are attached to the hybridizing membrane and hybridized with labelled target DNA.
Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation or polymorphic region of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al., Nucleic Acids Res. 17:2437-2448, 1989) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner, Tibtech 11:238, 1993. In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al., Mol. Cell Probes 6:1, 1992). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany, Proc. Natl. Acad. Sci USA 88:189, 1991). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
In another embodiment, identification of the allelic variant is carried out using an oligonucleotide ligation assay (OLA), as described, e.g., in U.S. Pat. No. 4,998,617 and in Landegren et al., Science 241:1077-80, 1988. The OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. One of the oligonucleotides is linked to a separation marker, e.g,. biotinylated, and the other has a detectable label. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand. Nickerson, D. A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson et al., Proc. Natl. Acad. Sci. USA 87:8923-27, 1990. In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
Several techniques based on this OLA method have been developed and can be used to detect alleles associated with EOM. For example, U.S. Pat. No. 5,593,826 discloses an OLA using an oligonucleotide having 3′-amino group and a 5′-phosphorylated oligonucleotide to form a conjugate having a phosphoramidate linkage. In another variation of OLA described in Tobe et al., Nucleic Acids Res. 24:3728, 1996, OLA combined with PCR permits typing of two alleles in a single microtiter well. By marking each of the allele-specific primers with a unique hapten, i.e. digoxigenin and fluorescein, each OLA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase. This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors.
Several methods have been developed to facilitate analysis of single nucleotide polymorphisms. In one embodiment, the single base polymorphism can be detected by using a specialized exonuclease-resistant nucleotide, as disclosed, e.g., in U.S. Pat. No. 4,656,127 (Mundy et al.). According to the method, a primer complementary to the allelic sequence immediately 3′ to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridized primer. Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymorphic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of extraneous sequence data.
In another embodiment of the invention, a solution-based method is used for determining the identity of the nucleotide of a polymorphic site. French Patent 2,650,840; PCT Appln. No. WO91/02087. As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3′ to a polymorphic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.
An alternative method, known as Genetic Bit Analysis or GBA™ is described by Goelet et al. in PCT Appln. No. 92/15712. The method of Goelet et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3′ to a polymorphic site. The labeled terminator that is incorporated is thus determined by, and complementary to, the nucleotide present in the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al., French Patent 2,650,840 and PCT Appln. No. WO91/02087, the method of Goelet et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.
Recently, several primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (Komher et al., Nucleic Acids Res. 17:7779-84, 1989; Sokolov, Nucleic Acids Res. 18:3671, 1990; Syvanen et al., Genomics 8:684-92, 1990; Kuppuswamy et al., Proc. Natl. Acad. Sci. USA 88:1143-47, 1991; Prezant et al., Hum. Mutat. 1:159-64, 1992; Ugozzoli et al., GATA 9:107-12, 1992; Nyren et al., Anal. Biochem. 208:171-75, 1993). These methods differ from GBA™ in that they all rely on the incorporation of labeled deoxynucleotides to discriminate between bases at a polymorphic site. In such a format, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms that occur in runs of the same nucleotide can result in signals that are proportional to the length of the run (Syvanen, et al., Amer. J Hum. Genet. 52:46-59, 1993).
For mutations that produce premature termination of protein translation, the protein truncation test (PTT) offers an efficient diagnostic approach (Roest et. al., Hum. Mol. Genet. 2:1719-21, 1993; van der Luijt et. al., Genomics 20:1-4, 1994). For PTT, RNA is initially isolated from available tissue and reverse-transcribed, and the segment of interest is amplified by PCR. The products of reverse transcription PCR are then used as a template for nested PCR amplification with a primer that contains an RNA polymerase promoter and a sequence for initiating eukaryotic translation. After amplification of the region of interest, the unique motifs incorporated into the primer permit sequential in vitro transcription and translation of the PCR products. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of translation products, the appearance of truncated polypeptides signals the presence of a mutation that causes premature termination of translation. In a variation of this technique, DNA (as opposed to RNA) is used as a PCR template when the target region of interest is derived from a single exon. In still another method known as Dynamic Allele Specific Hybridization (DASH), a target sequence is amplified by PCR in which one primer is biotinylated. The biotinylated product strand is bound to a streptavidin or avidin coated microtiter plate well, and the non-biotinylated strand is rinsed away with alkali. An oligonucleotide probe, specific for one allele, is hybridized to the target at low temperature. This forms a duplex DNA region that interacts with a double strand-specific intercalating dye. Upon excitation, the dye emits fluorescence proportional to the amount of double stranded DNA (probe-target duplex) present. The sample is then steadily heated while fluorescence is continually monitored. A rapid fall in fluorescence indicates the denaturing (or “melting”) temperature of the probe-target duplex. When performed under appropriate buffer and dye conditions, a single-base mismatch between the probe and the target results in a dramatic lowering of melting temperature (Tm) that can be easily detected (Howell, W. M. et al., (1999) Nature Biotechnology 17:)87-88.
Any cell type or tissue may be utilized in the diagnostics described herein. In a preferred embodiment the DNA sample is obtained from a bodily fluid, e.g, blood, obtained by known techniques (e.g. venipuncture) or saliva. Alternatively, nucleic acid tests can be performed on dry samples (e.g. hair or skin). When using RNA or protein, the cells or tissues that may be utilized must express at least one gene of the IL-1 loci.
Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, PCR in situ Hybridization: Protocols and Applications (Raven Press, NY, 1992)).
In addition to methods which focus primarily on the detection of one nucleic acid sequence, profiles may also be assessed in such detection schemes. Fingerprint profiles may be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT-PCR.
Another embodiment of the invention is directed to kits for detecting a propensity to EOM in a woman. This kit may contain one or more oligonucleotides, including 5′ and 3′ oligonucleotides that hybridize 5′ and 3′ to a polymorphic site having as allele associated with EOM, such as the +2018 marker, or detection oligonucleotides that hybridize directly to an allele associate with EOM. The kit may also contain one or more oligonucleotides capable of hybridizing near or at other alleles of the IL-1 gene cluster. PCR amplification oligonucleotides should hybridize between 25 and 2500 base pairs apart, preferably between about 100 and about 500 bases apart, in order to produce a PCR product of convenient size for subsequent analysis.

For use in a kit, oligonucleotides may be any of a variety of natural and/or synthetic compositions such as synthetic oligonucleotides, restriction fragments, cDNAs, synthetic peptide nucleic acids (PNAs), and the like. The assay kit and method may also employ oligonucleotides having detectable labels to allow ease of identification in the assays. Examples of labels which may be employed include radio-labels, enzymes, fluorescent compounds, streptavidin, avidin, biotin, magnetic moieties, metal binding moieties, antigen or antibody moieties, and the like. Oligonucleotides useful in kits as well as other aspects of the present invention are selected from the group consisting of any oligonucleotides that overlap or are contained in any of the following sequences:


5′ CTC AGC AAC ACT CCT AT 3′	(SEQ ID No. 9)

5′ TCC TGG TCT GCA GGT AA 3′	(SEQ ID No. 10)

5′ CTA TCT GAG GAA CAA CCA ACT AGT AGC 3′	(SEQ ID No. 11)

5′ TAG GAC ATT GCA CCT AGG GTT TGT 3′	(SEQ ID No. 12)

5′ CTC AGG TGT CCT CGA AGA AAT CAA A 3′	(SEQ ID No. 13)

5′ GCT TTT TTG CTG TGA GTC CCG 3′	(SEQ ID No. 14)

5′ AAG CTT GTT CTA CCA CCT GAA CTA GGC 3′	(SEQ ID No. 15)

5′ TTA CAT ATG AGC CTT CCA TG 3′	(SEQ ID No. 16)

5′ TGG CAT TGA TCT GGT TCA TC 3′	(SEQ ID No. 17)

5′ GTT TAG GAA TCT TCC CAC TT 3′	(SEQ ID No. 18)

5′ ATG GTT TTA GAA ATC ATC AAG CCT AGG GCA 3′	(SEQ ID No. 19)

5′ AAT GAA AGG AGG GGA GGA TGA CAG AAA TGT 3′	(SEQ ID No. 20)

5′ TTACGCAGATAAGAACCAGTTTGG 3′	(SEQ ID No. 21)

5′ TTTCCTGGACGCTTGCTCACCAG 3′	(SEQ ID No. 22)

5′ ATGTATAGAATTCCATTCCTG 3′	(SEQ ID No. 23)

5′ TAAAATCAAGTGTTGATGTAG 3′	(SEQ ID No. 24)

5′ GGGATTACAGGCGTGAGCCACCGCG 3′	(SEQ ID No. 25)

5′ TTAGTATTGCTGGTAGTATTCATAT 3′	(SEQ ID No. 26)

5′ GAGGCGTGAGAATCTCAAGA 3′	(SEQ ID No. 27)

5′ GTGTCCTCAAGTGGATCTGG 3′	(SEQ ID No. 28)

5′ GGGCAACAGAGCAATGTTTCT 3′	(SEQ ID No. 29)

5′ CAGTGTGTCAGTGTACTGTT 3′	(SEQ ID No. 30)

One of skill in the art can readily determine additional useful oligonucleotide sequences based on the IL-1 gene sequences provided herein.
The kit may, optionally, also include DNA sampling means such as the AmpliCard™ (University of Sheffield, Sheffield, England S10 2JF; Tarlow, et al., J. of Invest. Dermatol. 103:387-389, 1994) and the like; DNA purification reagents such as Nucleon™ kits, lysis buffers, proteinase solutions and the like; PCR reagents, such as 10×reaction buffers, thermostable polymerase, dNTPs, and the like; and DNA detection means such as appropriate restriction enzymes, allele specific oligonucleotides, degenerate oligonucleotide primers for nested PCR, and the like.
4.3 EOM Therapeutics and Pharmacogenomics
4.3.1 Pharmacogenomics
The ability to rapidly genotype patients promises to fundamentally change the testing and development of therapeutic or disease-preventative substances. Currently, the effectiveness of a substance for treating or preventing a disease is assessed by testing it on a pool of patients. While many variables in the patient pool are controlled for, the effects of genetic variability are not typically tested. Consequently, a drug may be found to be statistically ineffective when examined in a genetically diverse pool of patients and yet be highly effective for a select group of patients with particular genetic characteristics. Unless patients are separated by genotype, many drugs with great promise for selected populations are likely to be rejected as useless for the population as a whole.
Knowledge of particular alleles associated with EOM, alone or in conjunction with information on other genetic defects contributing to EOM (the genetic profile of EOM) allows a customization of the therapy to the individual's genetic profile, the goal of “pharmacogenomics”. For example, as shown herein, women having an allele associated with EOM, such as IL-1RN (+2018) allele 2 are predisposed to EOM. Thus, comparison of a woman's IL-1 profile to the population profile for the disease, permits the selection or design of drugs that are expected to be safe and efficacious for a particular patient or patient population (i.e., a group of patients having the same genetic alteration).
The ability to target populations expected to show the highest clinical benefit, based on the IL-1 gene profile or the genetic profile of EOM, can enable: 1) the repositioning of marketed drugs with disappointing market results; 2) the rescue of drug candidates whose clinical development has been discontinued as a result of safety or efficacy limitations, which are patient subgroup-specific; and 3) an accelerated and less costly development for drug candidates and more optimal drug labeling (e.g. since measuring the effect of various doses of an agent on an EOM causative mutation is useful for optimizing effective dose).
4.3.2 IL-1 Production and Molecular Signaling Pathways
To better understand likely targets for therapeutic intervention and likely EOM biomarkers, it is necessary to understand general mechanisms for IL-1 signaling and production. IL-1 is part of a complex web of inter- and intra-cellular signaling events. Many proteins are involved in the inflammatory response and also in immune responses more generally. A partial list includes the interleukins, TNF, NF-kB, the immunoglobulins, clotting factors, lipoxygenase, as well as attendant receptors, antagonists and processing enzymes for the above.
The IL-1 polypeptides, IL-1 a and IL-1b, are abundantly produced by activated macrophages that have been stimulated with bacterial lipopolysaccharide (LPS), TNF, IL-1 itself, other macrophage-derived cytokines, or contact with CD4⁺ T cells. The IL-1 promoter contains several regulatory elements including a cAMP responsive element, an AP-1 binding site and an NF-kB binding site. Both and AP-1 (Jun and Fos) must be activated and translocated to the nucleus in order to regulate transcription. NF-kB is normally retained in the cytoplasm through binding with IkB. The NF-kB-IkB complex is disrupted by phosphorylation of IkB. IkB phosphorylation can be regulated by signaling from cell-surface receptors via activation of mitogen-activated protein kinase (MAP kinse) pathways and other kinase pathways. Jun and Fos are also substrates for regulatory kinases, such as JNK, in the case of Jun.
The IL-1 A and B transcripts are translated into pro-proteins by a process that may also be regulated by MAP kinase pathways. Inhibitors of MAP kinase phosphorylation such as trebufelone decrease translation of IL-1 transcripts. The IL-1 a and b precursor proteins require myristoylation for localization to the membrane and conversion to mature IL-1 by the Interleukin Converting Enzyme (ICE). Other extracellular proteases may also play a minor role in IL-1 maturation, including trypsin, elastase, chymotrypsin and mast cell chymase. ICE can be inhibited by several agents including the eICE isoform, antibodies to the ICE a, b and g isoforms, the cow pox-produced Crm-A protein and an endogenous tetrapeptide competitive inhibitor.
Mature IL-1a and IL-1b have similar activities and interact with the same receptors. The primary receptor for these factors is the type I IL-1 receptor. The active signaling complex consists of the IL-1 ligand, the type I receptor and the IL-1 receptor accessory protein. A type II receptor, as well as soluble forms of the type I and type II receptors appear to act as decoy receptors to compete for bioavailable IL-1. In addition, a natural inhibitor of IL-1 signaling, IL-1 receptor antagonist, is produced by monocytes. IL-1ra is also produced by hepatocytes and is a major component of the acute phase proteins produced in the liver and secreted into the circulation to regulate immune and inflammatory responses.
The IL-1 signaling complex activates several intracellular signal transduction pathways, including the activities of NF- and AP-1 described above. In signaling, IL-1 influences the activity of a host of factors including: PI-3 kinase, phospholipase A2, protein kinase C, the JNK pathway, 5-lipoxygenase, cyclooxygenase 2, p38 MAP kinase, p42/44 MAP kinase, p54 MAP kinase, Rac, Ras, TRAF-6, TRAF-2 and many others. IL-1 also affects expression of a large number of genes including: members of the IL-1 gene cluster, TNF, other interleukin genes (2, 3, 6, 8, 12, 2R, 3R and 5R), TGF-b, fibrinogen, matrix metalloprotease 1, collagen, elastase, leukemia inhibiting factor, IFN a, b, g, COX-2, inducible nitric oxide synthase, metallothioneins, and many more.
4.3.3 EOM Associated Biomarkers
In addition to having genetic tests for EOM, it would be desirable to have tests for monitoring a subject's progression towards EOM. In other words, certain biomarkers may be indicative of the timing of early onset of menopause. It would be desirable to be able to identify these biomarkers and monitor them to provide information about the onset of menopause. It is particularly desirable to find biomarkers that are tailored to the subject's genotype.
In a preferred embodiment, biomarkers likely to be associated with EOM can be identified by using subjects or cells comprising differing IL-1 genotypes. A set of biomarkers can be examined in a subject or cell having an EOM-associated allele, such as IL-1RN (+2018) allele 2 or another allele of the IL-1 (44112332) haplotype. The same set of biomarkers can be examined in another subject or cell not having an EOM-associated allele. Biomarkers that show a difference dependent upon the IL-1 genotype are likely to be useful for predicting the early onset of menopause. These differences constitute EOM-associated phenotypes.
The association between certain biomarkers and EOM can be further established by performing trials wherein certain biomarkers are measured in a population of subjects of various ages, some of which may have entered menopause. Optionally, multiple measurements may be done over time as subjects age. Preferably, the presence or absence of EOM-associated alleles is determined in the subjects. Standard statistical methods may be used to determine the correlation between certain biomarkers and the early onset of menopause.
Measurements of EOM-associated biomarkers may be used as an indicator of a subject's current risk of developing EOM or as an indicator of progression towards or through menopause.
With respect to cells, biomarkers may be essentially any aspect of cell function, for example: levels or rate of production of signaling molecules, transcription factors, intermediate metabolites, cytokines, prostanoids, steroid hormones (eg. estrogen, progesterone, androstenedione or testosterone), gonadotropins (eg. LH and FSH), gene transcripts, post-translational modifications of proteins, gonadotropin releasing hormone (GnRH), catecholamines (eg. dopamine or norepinephrine), opioids, activin, inhibin, as well as IL-1 bioactivities. Biomarkers may include whole genome analysis of transcript levels or whole proteome analysis of protein levels and/or modifications. Additionally, biomarkers may be reporter genes. For example, an IL-1 promoter or an IL-1 promoter comprising an EOM-associated allele can be operationally linked to a reporter gene. In an alternative method, the promoter can be an IL-1-regulated promoter, such as IL-8. In this manner, the activity of the reporter gene is reflective of the activity of the promoter. Suitable reporter genes include GUS, LacZ, green fluorescent protein (GFP) (and variants thereof, such as RFP, CFP, YFP and BFP), or essentially any other gene that is easily detected. In subjects, biomarkers can be, for example, any of the above as well as electrocardiogram parameters, pulmonary function, IL-6 activities, urine parameters or tissue parameters. Other preferred biomarkers include factors involved in immune and inflammatory responses, as well as factors involved in IL-1 production and signaling, as described above.
4.3.4 EOM Therapeutics
An EOM therapeutic can comprise any type of compound, including a protein, peptide, peptidomimetic, small molecule, nucleic acid, or nutraceutical. In preferred embodiments, an EOM therapeutic is a modulator of a factor involved in IL-1 production or signaling. In a particularly preferred embodiment, an EOM therapeutic is a modulator of IL-1 bioactivity (e.g. IL-1 α, IL-1β or an IL-1 receptor agonist or antagonist). Preferred agonists include nucleic acids (e.g. encoding an IL-1 protein or a gene that is up- or down-regulated by an IL-1 protein), protein (e.g. IL-1 proteins or a protein that is up- or down-regulated by an IL-1 protein) or a small molecule (e.g. that regulates expression of an IL-1 protein). Preferred antagonists, which can be identified, for example, using the assays described herein, include nucleic acids (e.g. single (antisense) or double stranded (triplex) DNA or PNA and ribozymes), protein (e.g. antibodies) and small molecules or nutraceuticals that act to suppress or inhibit IL-1 transcription and/or IL-1 activity.
4.3.5. In Vivo and Cell-based Screening Assays
Based on the identification of IL-1 mutations that cause or contribute to EOM, the invention further features in vivo and cell-based assays, e.g., for identifying EOM therapeutics. In one embodiment, a cell having an EOM-associated allele is contacted with a test compound and at least one biomarker is measured. If at least one biomarker changes such that the phenotype of the cell now more closely resembles that of a cell that does not have an EOM-associated allele, then the test substance is likely to be effective as an EOM therapeutic.
As an illustrative example, suppose that an IL-1 allele associated with EOM causes cells having that allele to overproduce an IL-1 polypeptide. Levels of the IL-1 polypeptide are used as a biomarker in this case. Treatment with a test substance causes the cells to produce the IL-1 polypeptide at a lower level, more closely resembling IL-1 polypeptide production in a cell that does not have an EOM-associated allele. Accordingly, the test substance is likely to be effective as an EOM therapeutic. In this manner, test substances with allele-specific effects may be identified. The specificity of the compound vis a vis the IL-1 signaling pathway can, if desired, be confirmed by various control analysis, e.g., measuring the expression of one or more control genes. In particular, this assay can be used to determine the efficacy of IL-1 antisense, ribozyme and triplex compounds.
In another variation a cell is contacted with a test compound and an IL-1 protein and the interaction between the test compound and the IL-1 receptor or between the IL-1 protein (preferably a tagged IL-1 protein) and the IL-1 receptor is detected, e.g., by using a microphysiometer (McConnell et al. (1992) Science 257:1906). An interaction between the IL-1 receptor and either the test compound or the IL-1 protein is detected by the microphysiometer as a change in the acidification of the medium. This assay system thus provides a means of identifying molecular antagonists which, for example, function by interfering with IL-1 protein-IL-1 receptor interactions, as well as molecular agonist which, for example, function by activating an IL-1 receptor.
Essentially any culturable cell type can be used for the cell-based assays. In particular, cells may be immune cells such as monocytes, macrophages or thymocytes, or other cell types such as fibroblasts or cells derived from female reproductive organs. Preferrably cells will express an IL-1 receptor.
In another variation, a subject having an EOM-associated allele is contacted with a test compound and at least one biomarker is measured. If at least one biomarker changes such that the phenotype of the cell now more closely resembles that of a cell that does not have an EOM-associated allele, then the test substance is likely to be effective as an EOM therapeutic. The subject may be a human or a transgenic non-human animal.
In preferred embodiments, cellular or in vivo assays are used to identify compounds which modulate expression of an IL-1 gene, modulate translation of an IL-1 mRNA, or which modulate the stability or activity of an IL-1 mRNA or protein. Accordingly, in one embodiment, a cell which is capable of producing IL-1 protein is incubated with a test compound and the amount of IL-1 protein produced in the cell medium is measured and compared to that produced from a cell which has not been contacted with the test compound. In another variation, an IL-1 bioactivity is measured and compared to the bioactivity measured in a cell which has not been contacted with a test compound. Additionally, the effects of test substances on different cells containing various IL-1 alleles may be compared.
4.3.6 Cell-free Assays
Cell-free assays can also be used to identify compounds which are capable of interacting with an IL-1 protein, to thereby modify the activity of the IL-1 protein. Such a compound can, e.g., modify the structure of an IL-1 protein thereby affecting its ability to bind to an IL-1 receptor. In a preferred embodiment, cell-free assays for identifying such compounds consist essentially in a reaction mixture containing an IL-1 protein and a test compound or a library of test compounds in the presence or absence of a binding partner. A test compound can be, e.g., a derivative of an IL-1 binding partner, e.g., a biologically inactive target peptide, or a small molecule.
Accordingly, one exemplary screening assay of the present invention includes the steps of contacting an IL-1 protein or functional fragment thereof with a test compound or library of test compounds and detecting the formation of complexes. For detection purposes, the molecule can be labeled with a specific marker and the test compound or library of test compounds labeled with a different marker. Interaction of a test compound with an IL-1 protein or fragment thereof can then be detected by determining the level of the two labels after an incubation step and a washing step. The presence of two labels after the washing step is indicative of an interaction.
An interaction between molecules can also be identified by using real-time BIA (Biomolecular Interaction Analysis, Pharmacia Biosensor AB) which detects surface plasmon resonance (SPR), an optical phenomenon. Detection depends on changes in the mass concentration of macromolecules at the biospecific interface, and does not require any labeling of interactants. In one embodiment, a library of test compounds can be immobilized on a sensor surface, e.g., which forms one wall of a micro-flow cell. A solution containing the IL-1β protein or functional fragment thereof is then flown continuously over the sensor surface. A change in the resonance angle as shown on a signal recording, indicates that an interaction has occurred. This technique is further described, e.g., in BIAtechnology Handbook by Pharmacia.
Another exemplary screening assay of the present invention includes the steps of (a) forming a reaction mixture including: (i) an IL-1 protein, (ii) an IL-1 receptor, and (iii) a test compound; and (b) detecting interaction of the IL-1 protein and IL-1 receptor. A statistically significant change (potentiation or inhibition) in the interaction of the IL-1 protein and IL-1 receptor in the presence of the test compound, relative to the interaction in the absence of the test compound, indicates a potential antagonist (inhibitor) of IL-1 bioactivity for the test compound.
The compounds of this assay can be contacted simultaneously. Alternatively, an IL-1 protein can first be contacted with a test compound for an appropriate amount of time, following which the IL-1β receptor is added to the reaction mixture. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison.
Complex formation between an IL-1 protein and IL-1 receptor may be detected by a variety of techniques. Modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled IL-1 protein or IL-1 receptors, by immunoassay, or by chromatographic detection.
Typically, it will be desirable to immobilize either IL-1 protein or the IL-1 receptor to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of IL-1 protein and IL-1 receptor can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase/IL-1β (GST/IL-1β) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the IL-1 receptor, e.g. an 35S-labeled IL-1 receptor, and the test compound, and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintilant), or in the supernatant after the complexes are subsequently dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of IL-1 protein or IL-1 receptor found in the bead fraction quantitated from the gel using standard electrophoretic techniques such as described in the appended examples. Other techniques for immobilizing proteins on matrices are also available for use in the subject assay. For instance, either IL-1 protein or IL-1 receptor can be immobilized utilizing conjugation of biotin and streptavidin.
4.3.7 Transgenic Animals
As described above, transgenic animals can be made for example, to assist in screening for EOM therapeutics. Transgenic animals of the invention can include non-human animals containing an IL-1 mutation, which is causative of EOM in humans, under the control of an appropriate IL-1 promoter or under the control of a heterologous promoter. Transgenic animals of the invention can also include an IL-1 gene expressed at such a level as to create an EOM phenotype. To compare the effects of different IL-1 alleles, transgenic animals may be generated with a variety of IL-1 alleles and differences in EOM phenotype can be identified. By testing different alleles and different expression levels, an animal with an EOM phenotype optimal for testing candidate drugs can be generated and identified.
The transgenic animals can also be animals containing a transgene, such as reporter gene, under the control of an IL-1 promoter or fragment thereof. These animals are useful, e.g., for identifying drugs that modulate production of an IL-1, such as by modulating gene expression. In certain variations, the IL-1 allele may be a promoter mutation. In this case it is particularly desirable to operationally fuse the altered promoter to a suitable reporter gene.
Methods for obtaining transgenic non-human animals are well known in the art. In preferred embodiments, the expression of the EOM causative mutation is restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, cis-acting sequences that control expression in the desired pattern. In the present invention, such mosaic expression of an IL-1 protein can be essential for many forms of lineage analysis and can additionally provide a means to assess the effects of, for example, expression level which might grossly alter development in small patches of tissue within an otherwise normal embryo. Toward this end, tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the IL-1 mutation in certain spatial patterns. Moreover, temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences. Genetic techniques, which allow for the expression of IL-1 mutation can be regulated via site-specific genetic manipulation in vivo, are known to those skilled in the art.
The transgenic animals of the present invention all include within a plurality of their cells an EOM causative mutation transgene of the present invention, which transgene alters the phenotype of the “host cell”. In an illustrative embodiment, either the cre/loxP recombinase system of bacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al. (1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCT publication WO 92/15694) can be used to generate in vivo site-specific genetic recombination systems. Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences. loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination. The orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats.
Accordingly, genetic recombination of the target sequence is dependent on expression of the Cre recombinase. Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element. Thus, the activation of expression of the EOM causative mutation transgene can be regulated via control of recombinase expression.
Use of the cre/loxP recombinase system to regulate expression of an EOM causative mutational transgene requires the construction of a transgenic animal containing transgenes encoding both the Cre recombinase and the subject protein. Animals containing both the Cre recombinase and the EOM causative mutation transgene can be provided through the construction of “double” transgenic animals. A convenient method for providing such animals is to mate two transgenic animals each containing a transgene.
Similar conditional transgenes can be provided using prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the transgene. Exemplary promoters and the corresponding trans-activating prokaryotic proteins are given in U.S. Pat. No. 4,833,080.
Moreover, expression of the conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the transactivating protein, e.g. a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner. By this method, the transgene could remain silent into adulthood until “turned on” by the introduction of the transactivator.
In an exemplary embodiment, the “transgenic non-human animals” of the invention are produced by introducing transgenes into the germline of the non-human animal. Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The specific line(s) of any animal used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness. In addition, the haplotype is a significant factor. For example, when transgenic mice are to be produced, strains such as C57BL/6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those with H-2b, H-2d or H-2q haplotypes such as C57BL/6 or DBA/1. The line(s) used to practice this invention may themselves be transgenics, and/or may be knockouts (i.e., obtained from animals which have one or more genes partially or completely suppressed).
In one embodiment, the transgene construct is introduced into a single stage embryo. The zygote is the best target for microinjection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1-2 pl of DNA solution. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). As a consequence, all cells of the transgenic animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.
Normally, fertilized embryos are incubated in suitable media until the pronuclei appear. At about this time, the nucleotide sequence comprising the transgene is introduced into the female or male pronucleus as described below. In some species such as mice, the male pronucleus is preferred. It is most preferred that the exogenous genetic material be added to the male DNA complement of the zygote prior to its being processed by the ovum nucleus or the zygote female pronucleus. It is thought that the ovum nucleus or female pronucleus release molecules which affect the male DNA complement, perhaps by replacing the protamines of the male DNA with histones, thereby facilitating the combination of the female and male DNA complements to form the diploid zygote.
Thus, it is preferred that the exogenous genetic material be added to the male complement of DNA or any other complement of DNA prior to its being affected by the female pronucleus. For example, the exogenous genetic material is added to the early male pronucleus, as soon as possible after the formation of the male pronucleus, which is when the male and female pronuclei are well separated and both are located close to the cell membrane. Alternatively, the exogenous genetic material could be added to the nucleus of the sperm after it has been induced to undergo decondensation. Sperm containing the exogenous genetic material can then be added to the ovum or the decondensed sperm could be added to the ovum with the transgene constructs being added as soon as possible thereafter.
Introduction of the transgene nucleotide sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection. Following introduction of the transgene nucleotide sequence into the embryo, the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention. One common method in to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.
For the purposes of this invention a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism. Generally, the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete or gametes. Thus, the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism. Generally, a euploid zygote is preferred. If an aneuploid zygote is obtained, then the number of chromosomes should not vary by more than one with respect to the euploid number of the organism from which either gamete originated.
In addition to similar biological considerations, physical ones also govern the amount (e.g., volume) of exogenous genetic material which can be added to the nucleus of the zygote or to the genetic material which forms a part of the zygote nucleus. If no genetic material is removed, then the amount of exogenous genetic material which can be added is limited by the amount which will be absorbed without being physically disruptive. Generally, the volume of exogenous genetic material inserted will not exceed about 10 picoliters. The physical effects of addition must not be so great as to physically destroy the viability of the zygote. The biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.
The number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. As regards the present invention, there will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences.
Any technique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, nuclear membrane or other existing cellular or genetic structures. The exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.
Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.
Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product. Typically, DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene. Alternatively, the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal. Where mating with a partner is to be performed, the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both. Alternatively, the partner may be a parental line. Where in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
The transgenic animals produced in accordance with the present invention will include exogenous genetic material. Further, in such embodiments the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.
Retroviral infection can also be used to introduce the transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985) PNAS 82:6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al. (1982) Nature 298:623-628). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322:445-448). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, R. (1988) Science 240:1468-1474.
4.4 Methods of Treatment
4.4.1. Effective Dose
The treatment of an individual with a particular therapeutic can be monitored by monitoring a biomarker known to be affected by the therapeutic. In particular, such biomarkers may include IL-1 protein (e.g. IL-1α, IL-1β, IL-1 RA), mRNA and/or IL-1 bioactivity. Depending on the level detected, the therapeutic regimen can then be maintained or adjusted (increased or decreased in dose). In a preferred embodiment, the effectiveness of treating a subject with an agent comprises the steps of: (i) obtaining a preadministration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an IL-1 protein, mRNA or bioactivity in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the IL-1 protein, mRNA or bioactivity in the post-administration sample; (v) comparing the level of expression or activity of the IL-1 protein, mRNA or bioactivity in the preadministration sample with the corresponding IL-1 protein, mRNA or bioactivity in the postadministration sample, respectively; and (vi) altering the administration of the agent to the subject accordingly.
Cells of a subject may also be obtained before and after administration of a therapeutic to detect the level of expression of genes other than IL-1, to verify that the therapeutic does not increase or decrease the expression of genes which could be deleterious. This can be done, e.g., by using the method of transcriptional profiling. Thus, mRNA from cells exposed in vivo to a therapeutic and mRNA from the same type of cells that were not exposed to the therapeutic could be reverse transcribed and hybridized to a chip containing DNA from numerous genes, to thereby compare the expression of genes in cells treated and not treated with the therapeutic.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the Ed50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissues in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
4.4.2. Formulation and Use
Pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, injection, inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
For such therapy, the compounds of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Other suitable delivery systems include microspheres which offer the possibility of local noninvasive delivery of drugs over an extended period of time. This technology utilizes microspheres of precapillary size which can be injected via a coronary catheter into any selected part of the e.g. heart or other organs without causing inflammation or ischemia. The administered therapeutic is slowly released from these microspheres and taken up by surrounding tissue cells (e.g. endothelial cells).
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing.
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application) are hereby expressly incorporated by reference.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques that are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, (2nd ed., Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds., 1984); U.S. Pat. No. 4,666,828; U.S. Pat. No. 5,192,659; U.S. Pat. No. 5,272,057; and U.S. Pat. No. 4,801,531.

EXAMPLES

Determining an Individual's Allelic Pattern (Genotyping)
1. IL-1RN (+2018); IL-1RN (VNTR); IL-1A (−889); IL-1B (+3954); IL-1B (−511); and IL-1A (+4845)
Sections 1.1 to 1.4 set forth general procedures that apply to Sections 1.5 to 1.10 unless otherwise indicated.
1.1 Preparation of DNA. Blood is taken by venepuncture and stored uncoagulated at −20° C. prior to DNA extraction. Ten milliliters of blood are added to 40 ml of hypotonic red blood cell (RBC) lysis solution (10 mM Tris, 0.32 Sucrose, 4 mM MgCl₂, 1% Triton X-100) and mixed by inversion for 4 minutes at room temperature (RT). Samples are then centrifuged at 1300 g for 15 minutes, the supernatant aspirated and discarded, and another 30 ml of RBC lysis solution added to the cell pellet. Following centrifugation, the pellet is resuspended in 2 ml white blood cell (WBC) lysis solution (0.4 M Tris, 60 mM EDTA, 0.15 M NaCl, 10% SDS) and transferred into a fresh 15 ml polypropylene tube. Sodium perchlorate is added at a final concentration of 1 M and the tubes are first inverted on a rotary mixer for 15 minutes at RT, then incubated at 65° C. for 25 minutes, being inverted periodically. After addition of 2 ml of chloroform (stored at−20° C.), samples are mixed for 10 minutes at room temperature and then centrifuged at 800 g for 3 minutes. At this stage a very clear distinction of phases can be obtained using 300 μl Nucleon Silica suspension (Scotlab, UK) and centrifugation at 1400 G for 5 minutes. The resulting aqueous upper layer is transferred to a fresh 15 ml polypropylene tube and cold ethanol (stored at−20° C.) is added to precipitate the DNA. This is spooled out on a glass hook and transferred to a 1.5 ml eppendorf tube containing 500 μl TE or sterile water. Following overnight resuspension in TE, genomic DNA yield is calculated by spectrophotometry at 260 nm. Aliquots of samples are diluted at 100 ug/ml, transferred to microtiter containers and stored at 4° C. Stocks are stored at −20° C. for future reference.
1.2 Polymerase Chain Reaction. Oligonucleotide primers designed to amplify the relevant region of the gene spanning the polymorphic site (as detailed below) are synthesised, resuspended in Tris-EDTA buffer (TE), and stored at −20° C. as stock solutions of 200 uM. Aliquots of working solutions (1:1 mixture of forward and reverse, 20 uM of each in water) are prepared in advance.

Typically PCR reaction mixtures are prepared as detailed below.



Stock
Concentration	Volume	Final Concentration

Sterile H20			29.5 μl
10 × PCR buffer	200 mM Tris-HCl	5.00 μl	20 mM Tris-HCl,
	(pH 8.4)
MgCI2	50 mM	1.75 μl	1.75 mM
dNTP	mix 10 mM of each	4.00 μl	0.2 mM of each
primer forward	20 μM	2.5 μl	1 μM
prime reverse	20 μM	2.5 μl	1 μM
Taq polymerase	5 U/μl	0.25 μl	1.25 units/50 μl
Detergent (eg W-1,	1%	2.5 μl	0.05%
Gibco)
Template	200 ng/μl	2.00 μl	2 ng/μl
Final Volume		50.00 μl

DNA template is dotted at the bottom of 0.2 ml tubes or microwells. The same volume of water or negative control DNA is also randomly tested. A master-mix (including all reagents except templates) is prepared and added to the wells or tubes, and samples are transferred to the thermocycler for PCR.
PCR can be performed in 0.5 ml tubes, 0.2 ml tubes or microwells, according to the thermocycler available. The reaction mixture is overlaid with mineral oil if a heated lid (to prevent evaporation) is not available.
1.3 Restriction Enzyme Digestion. A master mix of restriction enzyme buffer and enzyme is prepared and aliquotted in suitable volumes in fresh microwells. Digestion is carried out with an oil overlay or capped microtubes at the appropriate temperature for the enzyme on a dry block.
Restriction buffer dilutions are calculated on the whole reaction volume (i.e. ignoring salt concentrations of PCR buffer). Restriction enzymes are used 3-5 times in excess of the recommended concentration, to compensate for the unfavourable buffer conditions and to ensure complete digestion.
1.4 Electrophoresis. Polyacrylamide-gel electrophoresis (PAGE) of 20-40 μl PCR sample is carried out in Tris-Borate-EDTA buffer and at constant voltage. Depending on the size discrimination need, different PAGE conditions are used (9 to 12% acrylamide, 1.5 mm×200) and different DNA size marker (
X174-Hae III or
X 174-Hmf I). A 2% agarose horizontal gel can be used for genotyping the IL-1RN (VNTR) marker.
1.5 IL-1RN (+2018). The genotypes for the women in this Example were determined for the +2018 marker. PCR primers were designed (mismatched to the genomic sequence) to engineer two enzyme cutting sites on the two alleles to allow for RFLP analysis. The gene accession number is X64532. Oligonucleotide primers used were:

5′ CTATCTGAGGAACAACCAACTAGTAGC 3′ (SEQ ID No. 7)

5′ TAGGACATTGCACCTAGGGTTTGT 3′ (SEQ ID No. 8)
Cycling was performed at [96°, 1 min]; [94°, 1 min; 57°, 1 min; 70°, 2 min;]×35; [70°, 5 min]×1; 4° C. Each PCR reaction was divided in two 25 ul aliquots: to one was added 5 Units of Alu 1, to the other 5 Units of Msp 1, in addition to 3 ul of the specific 10×restriction buffer. Incubation is at 37° C. overnight. Electrophoresis was by PAGE 9%.
The two enzymes cut respectively the two different alleles. Alu 1 will produce 126 and 28 bp fragments for allele 1, while it does not digest allele 2 (154 bp). Msp 1 will produce 125 and 29 bp with allele 2, while allele 1 is uncut (154 bp). Hence the two reactions (separated side by side in PAGE) will give inverted patterns of digestion for homozygote women, and identical patterns in heterozygotes. Allelic frequencies are 0.74 and 0.26.
1.6 IL-1RN (VNTR). The IL1-RN (VNTR) marker may be genotyped in accordance with the following procedure. As indicated above, the two alleles of the IL1-RN (+2018) marker are >97% in linkage disequilibrium with the two most frequent alleles of IL-1RN (VNTR), which are allele 1 and allele 2. The gene accession number is X64532. The oligonucleotide primers used for PCR amplification are:

5′ CTCAGCAACACTCCTAT 3′ (SEQ ID No. 5)

5′ TCCTGGTCTGCAGGTAA 3′ (SEQ ID No. 6)
Cycling is performed at [96°, 1 min]×1; [94°, 1 min; 60°, 1 min; 70°, 2 min]×35; [70°, 5 min]×1; 4° C. Electrophoresis is conducted in 2% agarose at 90V for 30 min.
The PCR product sizes are direct indication of number of repeats: the most frequent allele (allele 1) yields a 412 bp product. As the flanking regions extend for 66 bp, the remaining 344 bp imply four 86 bp repeats. Similarly, a 240 bp product indicates 2 repeats (allele 2), 326 is for 3 repeats (allele 3), 498 is 5 (allele 4), 584 is 6 (allele 6). Frequencies for the four most frequent alleles are 0.734, 0.241, 0.021 and 0.004.
1.7 IL-1A (−889). The IL-1A (−889) marker may be genotyped in accordance with the following procedure. McDowell et al., Arthritis Rheum. 38:221-28, 1995. One of the PCR primers has a base change to create an Nco I site when amplifying allele 1 (C at −889) to allow for RFLP analysis. The gene accession number is X03833. The oligonucleotide primers used for PCR amplification are:

(SEQ ID No. 11)

5′ AAG CTT GTT CTA CCA CCT GAA CTA GGC 3′

(SEQ ID No. 12)

5′ TTA CAT ATG AGC CTT CCA TG 3′

MgCl₂is used at 1 mM final concentration, and PCR primers are used at 0.8 μM. Cycling is performed at [96°, 1 min]×1; [94°, 1 min; 50°, 1 min; 72°, 2 min]×45; [72°, 5 min]×1; 4° C. To each PCR reaction is added 6 Units of Nco I in addition to 3 μl of the specific 10×restriction buffer. Incubation is at 37° overnight. Electrophoresis is conducted by 6% PAGE.
Nco I digest will produce fragments 83 and 16 bp in length, whereas the restriction enzyme does not cut allele 2. Correspondingly, heterozygotes will have three bands. Frequencies for the two alleles are 0.71 and 0.29.
1.8 IL-1A (+4845). The IL-1A (+4845) marker may be genotyped in accordance with the following procedure. The PCR primers create an Fnu 4H1 restriction site in allele 1 to allow for RFLP analysis. The gene accession number is X03833. The oligonucleotide primers used for PCR amplification are:

(SEQ ID No. 15)

5′ ATG GTT TTA GAA ATC ATC AAG CCT AGG GCA 3′

(SEQ ID No. 16)

5′ AAT GAA AGG AGG GGA GGA TGA CAG AAA TGT 3′
MgCl₂is used at 1 mM final concentration, and PCR primers are used at 0.8 μM. DMSO is added at 5% and DNA template is at 150ng/50 μl PCR. Cycling is performed at [95°, 1 min]×1; [94°, 1 min; 56°, 1 min; 72°, 2 min]×35; [72°, 5 min]×1; 4° C. To each PCR reaction is added 2.5 Units of Fnu 4H1 in addition to 2 μl of the specific 10×restriction buffer. Incubation is at 37° overnight. Electrophoresis is conducted by 9% PAGE.
Fnu 4H1 digest will produce a constant band of 76 bp (present regardless of the allele), and two further bands of 29 and 124 bp for allele 1, and a single further band of 153 bp for allele 2. Frequencies for the two alleles are 0.71 and 0.29.
1.9 IL-1B (−511). The IL-1B (−511) marker may be genotyped in accordance with the following procedure. The gene accession number is X04500. The oligonucleotide primers used for PCR amplification are:

5′ TGG CAT TGA TCT GGT TCA TC 3′ (SEQ ID No. 13)

5′ GTT TAG GAA TCT TCC CAC TT 3′ (SEQ ID No. 14)
MgCl₂is used at 2.5 mM final concentration, and PCR primers are used at 1 μM. Cycling is performed at [95°, 1 min]×1; [95°, 1 min; 53°, 1 min; 72°, 1 min]×35; [72°, 5 min]×1; 4° C. Each PCR reaction is divided into two aliquots: to one aliquot is added 3 Units of Ava I, to the other aliquot is added 3.7 Units of BSU 36I. To both aliquots is added 3 μl of the specific 10×restriction buffer. Incubation is at 37° overnight. Electrophoresis is conducted by 9% PAGE.
Each of the two restriction enzymes cuts one of the two alleles, which allows for RFLP analysis. Ava I will produce two fragments of 190 and 114 bp with allele 1, and it does not cut allele 2 (304 bp). BSU 36I will produce two fragments of 190 and 11 base pairs with allele 2, and it does not cut allele 1 (304 bp). Frequencies for the two alleles are 0.61 and 0.39.
1.10 IL-1B (+3954). The IL-1B (+3954) marker may be genotyped in accordance with the following procedure. The gene accession number is X04500. The oligonucleotide primers used for PCR amplification are:

(SEQ ID No. 9)

5′ CTC AGG TGT CCT CGA AGA AAT CAA A 3′

(SEQ ID No. 10)

5′ GCT TTT TTG CTG TGA GTC CCG 3′
MgCl₂is used at 2.5 mM final concentration, and DNA template at 150 ng/50 μl PCR. Cycling is performed at [95°, 2 min]×1; [95°, 1 min; 67.50, 1 min; 72°, 1 min]×35; [72°, 5 min]×1; 4° C. To each PCR reaction is added 10 Units of Taq I (Promega) in addition to 3 μl of the specific 10×restriction buffer. Incubation is at 65° overnight. Electrophoresis is conducted by 9% PAGE.
The restriction enzyme digest produces a constant band of 12 bp and either two further bands of 85 and 97 bp corresponding to allele 1, or a single band of 182 bp corresponding to allele 2. Frequencies for the two alleles are 0.82 and 0.18.
2. IL-1A (222/223); IL-1A (gz5/gz6); gaat.p33330; and Y31

Genotyping of these markers could proceed as described in Cox et al., Am. J. Human Genet. 62:1180-88, 1998. PCRs for these markers may be carried out by using fluorescently labeled forward primers (Cruachem) in a 10 μl reaction volume containing 50 mM KCL, 10 mM Tris-HCl, pH 9.0, 1.5 mM MgCl₂, 200 μM dNTPs, 25 ng of each primer, 50 ng DNA, 0.004% W-1 (Gibco-BRI), and 0.2 units Taq polymerase. The PCR conditions could be 94° for 1 min., 55° for 1 min., and 72° for 1 min. for 30 cycles. One unit PERFECT MATCH (Stratagene) would be added to gz5/gz6 PCRs. The primer sequences could be as follows: for IL-1A (222/223):


5′ ATGTATAGAATTCCATTCCTG 3′	(SEQ ID No. 19)

5′ TAAAATCAAGTGTTGATGTAG 3′	(SEQ ID No. 20)

For IL-1A (gz5/gz6):

5′ GGGATTACAGGCGTGAGCCACCGCG 3′	(SEQ ID No. 21)

5′ TTAGTATTGCTGGTAGTATTCATAT 3′	(SEQ ID No. 22)

For gaat.p33330:

5′ GAGGCGTGAGAATCTCAAGA 3′	(SEQ ID No. 23)

5′ GTGTCCTCAAGTGGATCTGG 3′	(SEQ ID No. 24)

For Y31:

5′ GGGCAACAGAGCAATGTTTCT 3′	(SEQ ID No. 25)

5′ CAGTGTGTCAGTGTACTGTT 3′	(SEQ ID No. 26)

A sample of PCR product could be examined by agarose-gel electrophoresis, and the remainder of the PCR products could be pooled according to the intensity of the ethidium-bromide staining. Two microliters of the pool could be analyzed on an automated sequencer, and allele sizes could be determined against the appropriate size standard.
3 IL-1RN exon 1ic (1812); IL-1RN exon 1ic (1868); IL-1RN exon 1ic (1887); Pic (1731)

Genotyping of these markers could proceed as described in Clay et al., Hum. Genet. 97:723-26, 1996. PCRs could be performed using 5 μg genomic DNA in a final reaction volume of 250 μl containing 250 pmol forward and reverse primers and 1.5 mM MgCl₂. The annealing temperature could be 57°. Primers for exon 1ic PCR and sequencing could be:

5′ TTACGCAGATAAGAACCAGTTTGG 3′ (SEQ ID No. 17)

5′ TTTCCTGGACGCTTGCTCACCAG 3′ (SEQ ID No. 18)

The resulting product would be 426 bp, and the forward primer could be biotinylated to allow for ready sequencing.
2. IL-1RN (+2018) allele 2 is associated with EOM
Applicants investigated the relationship between age of onset of menopause and specific polymorphism in the IL-1RN gene in two study populations, one in the UK and one in the USA.
The polymorphism at position (+2018) in the IL-1RN gene was amplified by PCR techniques and analyzed by the TaqMan® technique, using primers and probes described above (SEQ ID NOs.7 and 8).
The UK study population consisted of 140 postmenopausal Caucasian women (mean age 64.8 years, range 51.1 to 84.2). The mean age of onset of menopause was 49.3 years. Age of menopause was stratified by carriage of allele 2 of IL-1RN (+2018). Statistical analysis indicated that carriage of allele 2 of IL-1RN (+2018) resulted in an earlier onset of menopause by 1.03+/−0.47 (p=0.03) years per copy of allele 2.
The US study population was broken into two groups, those of Northern European descent and those of non-Northern European descent. To determine ancestry, subjects were asked the following question: “Which of the following best describes your ethnic origin or family's original nationality? Please mark no more than two.” A subject was considered of Northern European descent if the subject selected any one or two of the following ethnic origins: Canadian (except French Canadian), English, Irish, Scandinavian, Scottish or Welsh.
313 women of Northern European descent were examined. Age of menopause was again stratified by carriage of allele 2 of IL-1RN (+2018). Statistical analysis indicated that carriage of allele 2 of IL-1RN (+2018) resulted in an earlier onset of menopause by 0.96+/−0.42 (p=0.02) years per copy of allele 2.
226 women of non-Northern European descent were also analyzed. Age of menopause was stratified by carriage of allele 2 of IL-1RN (+2018). Statistical analysis indicated that carriage of allele 2 of IL-1RN (+2018) resulted in a later onset of menopause by 1.16+/−0.54 (p=0.03) years per copy of allele 2.
The ancestry of subjects was ascertained by asking subjects to indicate the most appropriate category of ancestry. Subjects were instructed to indicate their ancestry as Northern European if they felt that their ancestry could best be traced to England, Scotland, Wales, Ireland and/or Scandinavia.

Polymorphisms in the L-1 RN gene at position (+2018) and at other sites in disequilibrium with IL-1RN (+2018) are associated with timing and onset of menopause. The relationship may differ among different ethnic groups.

Summary Table: Onset of Menopause and IL-1RN (+2018)

allele 2

		US:	US:
		Northern	Non-Northern
	UK:	European	European
Study Group:	Caucasian	Ancestry	Ancestry

Number of Subjects	140	313	266
Change in onset of	−1.03 +/− 0.47	−0.96 +/− 0.42	+1.16 +/− 0.54
menopause per IL-	P = 0.03	P = 0.02	P = 0.03
1RN (+2018) allele 2

The specification and examples should be considered exemplary only with the true scope and spirit of the invention suggested by the following claims.

Claims

1. A kit for determining a woman's predisposition to early-onset menopause comprising a first primer that hybridizes 5′ or 3′ to a marker of an IL-1-related gene associated with early-onset menopause, wherein said kit is used to detect a predisposition to early-onset menopause.

2. The kit of claim 1, wherein said marker is selected from the group consisting of: IL-1RN (+2018); IL-1RN (VNTR); IL-1A (222/223); IL-1A (gz5/gz6); IL-1A (−889); IL-1B (+3954); IL-1B (−511); gaat.p33330; Y31; IL-1RN exon 1ic (1812); IL-1RN exon 1ic (1868); IL-1RN exon 1ic (1887); Pic (1731); IL-1A (+4845); IL-1B (+6912) allele 1; and IL-1B (−31) allele 2.

3. The kit as of claim 2, further comprising a second primer that hybridizes 3′ to said marker when said first primer hybridizes 5′ and hybridizes 5′ to said marker when said first primer hybridizes 3′.

4. The kit of claim 3, wherein said first primer and said second primer hybridize to a region that includes said marker, wherein said region is in the range of between about 50 and 1000 base pairs.

5. The kit of claim 3, wherein said primers are selected from the group consisting of:

(SEQ ID No. 5) 5′ CTC AGC AAC ACT CCT AT 3′; (SEQ ID No.6) 5′ TCC TGG TCT GCA GGT AA 3′; (SEQ ID No.7) 5′ CTA TCT GAG GAA CAA CCA ACT AGT AGC 3′; (SEQ ID No.8) 5′ TAG GAC ATT GCA CCT AGG GTT TGT 3′; (SEQ ID No.9) 5′ CTC AGG TGT CCT CGA AGA AAT CAA A 3′; (SEQ ID No.10) 5′ GCT TTT TTG CTG TGA GTC CCG 3′; (SEQ ID No.11) 5′ AAG CTT GTT CTA CCA CCT GAA CTA GGC 3′; (SEQ ID No.12) 5′ TTA CAT ATG AGC CTT CCA TG 3′; (SEQ ID No.13) 5′ TGG CAT TGA TCT GGT TCA TC 3′; (SEQ ID No.14) 5′ GTT TAG GAA TCT TCC CAC TT 3′; (SEQ ID No.15) 5′ ATG GTT TTA GAA ATC ATC AAG CCT AGG GCA 3′; (SEQ ID No.16) 5′ AAT GAA AGG AGG GGA GGA TGA CAG AAA TGT 3′; (SEQ ID No.17) 5′ TTACGCAGATAAGAACCAGTTTGG 3′; (SEQ ID No.18) 5′ TTTCCTGGACGCTTGCTCACCAG 3′; (SEQ ID No.19) 5′ ATGTATAGAATTCCATTCCTG 3′; (SEQ ID No.20) 5′ TAAAATCAAGTGTTGATGTAG 3′; (SEQ ID No.21) 5′ GGGATTACAGGCGTGAGCCACCGCG 3′; (SEQ ID No.22) 5′ TTAGTATTGCTGGTAGTATTCATAT 3′; (SEQ ID No.23) 5′ GAGGCGTGAGAATCTCAAGA 3′; (SEQ ID No.24) 5′ GTGTCCTCAAGTGGATCTGG 3′; (SEQ ID No.25) 5′ GGGCAACAGAGCAATGTTTCT 3′; and (SEQ ID No.26) 5′ CAGTGTGTCAGTGTACTGTT 3′.

6. The kit of claim 2, further comprising a DNA sampling means.

7. The kit of claim 2, further comprising a control.

8. The kit of claim 2, further comprising a DNA detection means.

9. The kit of claim 3, wherein said first primer and/or said second primer further comprises a detectable label.