WO2013079560A1

WO2013079560A1 - Genetic marker for polycystic ovary syndrome (pcos)

Info

Publication number: WO2013079560A1
Application number: PCT/EP2012/073881
Authority: WO
Inventors: Arseni Markov; Nadja BOGDANOVA MARKOV; Volker Gerke
Original assignee: Westfälische Wilhelms-Universität Münster
Priority date: 2011-11-29
Filing date: 2012-11-29
Publication date: 2013-06-06

Abstract

The present invention relates to a novel genetic marker for polycystic ovary syndrome (PCOS), and in particular to a method for diagnosing polycystic ovary syndrome (PCOS) or detecting a predisposition of a female subject to polycystic ovary syndrome (PCOS), comprising examining the human annexin A5 (ANXA5) promoter in a sample obtained from the female subject and to detect nucleotide exchanges therein. The present invention also relates to a nucleic acid sequence comprising a human annexin A5 (ANXA5) promoter, which promoter comprises specific nucleotide exchanges defined herein, for use in the methods disclosed herein. The present invention further relates to a kit for use in the diagnostic methods disclosed herein.

Description

Genetic marker for polycystic ovary syndrome (PCOS)

Annexin A5 shares the essential annexin family properties, but is a member that can be found extracellularly [1]. It can inhibit coagulation by various mechanisms [2, 3]. The regulation of ANXA5 gene expression in specific tissues is largely unknown. Annexin A5 is distributed abundantly and ubiquitously, mostly in kidney, liver and placenta [4]. The human ANXA5 gene generates several transcripts and has a complex promoter [5]. Annexin A5 is also known as placental anticoagulant protein. Lowered annexin A5 expression on placental trophoblast villi has been detected in the presence of antiphospholipid antibodies [6] and has also been confirmed immunohistochemically in patients with preeclampsia (PE) [7]. Polycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders. PCOS is a complex, heterogeneous disorder of uncertain etiology, but there is evidence that PCOS can be classified as a genetic disease [17]. Such evidence include familial clustering of cases, greater concordance in monozygotic compared with dizygotic twins and heritability of endocrine and metabolic features of PCOS. The genetic pattern can be inherited from either the father or the mother (or both), and can be passed along to the offspring. The allele appears to manifest itself at least partially via heightened androgen levels secreted by ovarian follicle theca cells from women with the allele. Unfortunately, any exact gene that is affected has not yet been identified [18]. PCOS is meanwhile thought to be the most frequent endocrine problem in women of reproductive age.

The principal features of patients suffering of PCOS are anovulation, resulting in irregular menstruation, amenorrhea, ovulation-related infertility, and polycystic ovaries; excessive amounts or effects of androgenic (masculinizing) hormones, resulting in acne and hirsutism; and insulin resistance, often associated with obesity, Type 2 diabetes, and high cholesterol levels. The symptoms and severity of the syndrome vary greatly among affected women.

The diagnosis of PCOS is to the present date still dependent on the interpretation of the syndrome itself. There are methods how ovarian cysts -but not PCOS as such- are diagnosed such as Pelvic Examination, Pelvic Ultrasound or Laparoscopy. During a Pelvic Examination an ovarian cyst may be discovered by palpating the ovaries. If a cyst is suspected, an ultrasound examination is usually the next step. Ultrasound examination is a procedure where sound waves are transmitted through the pelvic area imaging the ovaries. The image is analyzed to determine the nature of the cyst. Laparoscopy is a surgical procedure performed in order to observe the cyst. A thin, lighted telescope, called a laparoscope, is inserted through a small incision into the abdomen. Laparoscopy may be used for treatment as well as diagnosis. The disadvantage of the described diagnosis methods is the necessity of a surgical examination. Furthermore the outcome of this diagnosis gives only results with regard to one part of the syndrome. A further diagnosis method regarding a different part of the PCOS is the measurement of blood serum levels of androgens including androstenedione and testosterone, which may be elevated. The free testosterone level is thought to be a possible method as ~60% of PCOS patients show supranormal levels. However, as the altered male hormone level in the ovaries is only one possible part of the PCOS that might not necessarily be part of the syndrome also this diagnosis method remains unsatisfactory.

Summarizing, there are only poor developed methods for diagnosing single symptoms of the polycystic ovary syndrome (PCOS). Moreover, it is up to date not possible to detect a female^'s predisposition to develop PCOS as no genetic marker has been identified. The knowledge of such a predisposition would, however, be of great benefit regarding e.g. decisions on the treatment of PCOS.

Thus, the technical problem underlying the present invention is to provide means and methods for diagnosing a predisposition of females to develop the polycystic ovary syndrome (PCOS). The present invention addresses this need and provides methods for diagnosing the above mentioned predisposition in females.

It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Th us, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein . Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. At least one includes for example, one, two, th ree, fou r, or five or even more. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. Th roughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and va riations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Noth ing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

In the present work we have surprisingly detected in a cohort of female PCOS patients that the genotype frequencies of ANXA5 gene promoter haplotypes in PCOS patients differs significantly from two different control groups, which allows us to conclude that at least the genotypes M1/M2 and N/M2, and presumably also M2/M2 allows us to diagnose PCOS and/or to detect a predisposition of the female to develop PCOS (see Figure 8). Since all these genotypes are inter alia characterized by the risk haplotype M2, one can from now on easily detect the predisposition of a female to develop PCOS and/or diagnose PCOS in a female subject (for example a subject suspected of having PCOS, or diagnosed of suffering from PCOS) by way of testing for the presence or absence of at least the risk haplotype M2. The presence thereby indicates an increased risk of developing PCOS and/or allows a reliable method to diagnose PCOS. The present study complied with the ethical guidelines of all the institutions involved. Informed consent was obtained from all subjects examined. It is likewise envisaged that informed consent is obtained from all subjects (or their legal guardian) which are to be examined in accordance with the methods of the present invention. As already disclosed in WO2006/053725 or [8] or PCT/EP2011/064721, the risk haplotype M2 can be detected in the human ANXA5 promoter, and is characterized by the following four nucleotide exchanges: (1) G to A at a position which corresponds to nucleotide 186 of SEQ ID No. 2, (2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2, (3) T to C at a position which corresponds to nucleotide 229 of SEQ ID No. 2, and (4) G to A at a position which corresponds to nucleotide 276 of SEQ ID No. 2.

The risk haplotype Ml which can be detected in the human ANXA5 promoter as well, is also disclosed in WO2006/053725 or [8] or or PCT/EP2011/064721, and is characterized by the following two nucleotide exchanges (2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2, and (3) T to C at a position which corresponds to nucleotide 229 of SEQ ID No. 2.

SEQ ID No. 2 as used herein and depicted in Figure 3 corresponds to SEQ ID No. 2 disclosed in WO2006/053725. Said SEQ ID No 2 depicts an ANXA5 promoter structure as disclosed in Carcedo (2001), Biochem. J. 356, 571-579). Means and methods to determine and/or to detect these risk haplotypes are well-known (see for example WO2006/053725 and [8]) and additionally disclosed in detail herein. The ANXA5 allelic mRNA quantification in samples can be conducted as follows. Specific ANXA5 mRNA amplification formats for the normal (N) and haplotype M2 (M2) alleles may utilize primers with G/A variation of the last nucleotide, corresponding to the last substitution of the M2 haplotype, 76G>A [8], shared by all detected transcripts of ANXA5 (Fig.la). An oligonucleotide bridging exons 1 and 2 may serve as a reverse primer. Allele specific amplification of this format can be verified at respective annealing temperatures by direct product visualization (Fig.lb) and melting curve analysis (Fig.lc). Such methods have also been disclosed in great detail in WO2006/053725, [8], or PCT/EP2011/064721, which are all incorporated herein by way of reference thereto.

The terms "polycystic ovarian syndrome, " "PCOS " "polycystic ovarian disease" or "Stein- Leventhal syndrome" are used interchangeably herein and refer to a disease affecting females. PCOS is generally characterized by characteristic changes in the appearance of the ovaries, for example on an ultrasound scan (see Figure 2 - derived from www.pcos-uk.org) . The ovaries are in particular polycystic, with many small follicles scattered under the surface of the ovary (often more than 10 or 15 in each ovary) and almost none in the middle of the ovary. These follicles are all small and immature, and generally do not exceed 10mm in size and rarely, if ever, grow to maturity and ovulate. In PCOS, these follicles remain immature, never growing to full development or ovulating to produce an egg capable of being fertilised. Women diagnosed with PCOS may exhibit additionally one or more of the following symptoms: anovulation (irregular or absent menstrual periods) , hyperandrogenism (elevated serum testosterone and/or androstenedione) , abnormal uterine bleeding, enlarged multifollicular ovaries, infertility, obesity, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, type-2 diabetes mellitus, excess facial hair growth, hair loss, and acne. In PCOS, the ovaries are generally bigger than average. The outer surface of the ovary has an abnormally large number of small follicles (the sac of fluid that grows around the egg under the influence of stimulating hormones from the brain).

The skilled person, e.g. a medical doctor, more particularly a gynaecologist is in a position to identify and verify those symptoms whose presence is, up to the then knowledge, associated with/ or characteristic for PCOS (in addition to the above mentioned characteristic changes in the appearance of the ovaries).

The present invention relates in essence to a method for diagnosing PCOS in a female subject or detecting a predisposition of a female subject to develop polycystic ovary syndrome (PCOS), comprising:

(a) examining the human annexin A5 (ANXA5) promoter in a sample obtained from the said female subject to detect the following nucleotide exchanges

(i)

(1) G to A at a position which corresponds to nucleotide 186 of SEQ ID No. 2,

(2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2

(3) T to C at a position which corresponds to nucleotide 229 of SEQ ID No. 2, and

(4) G to A at a position which corresponds to nucleotide 276 of SEQ ID No. 2;

and/or

(ii)

(2) A to C at a position which corresponds to nucleotide 203 of SEQ I D No. 2, and

(3) T to C at a position which corresponds to nucleotide 229 of SEQ I D No. 2,

(b) determining whether the nucleotide exchanges defined in (i) and/or (ii) are present, wherein the presence of the nucleotide exchanges defined in (i) and/or (ii) indicates a predisposition of said female subject to develop polycystic ovary syndrome (PCOS) or diagnoses PCOS in sa id female subject .

As mentioned hereinbefore, we have su rprisingly detected in a cohort of female PCOS patients that the genotype frequencies of ANXA5 gene promoter haplotypes in PCOS patients d iffers significantly from two d ifferent control groups, which allows us to conclude that at least the genotypes M1/M2 and N/M2, and presumably also M2/M2 allows us to d iagnose PCOS and/or to detect a predisposition of the female to develop PCOS (see Figure 8). Since all these genotypes a re inter alia characterized by the risk haplotype M2, one can from now on easily detect the predisposition of a female to develop PCOS and/or diagnose PCOS in a female subject (for example a subject suspected of having PCOS, or diagnosed of suffering from PCOS) by way of testing for the presence or absence of at least the risk haplotype M2. The presence thereby indicates an increased risk of developing PCOS and/or allows a reliable method to diagnose PCOS.

Thus, in a preferred embodiment of the methods of the present invention, said method comprises the step of examining the human annexin A5 (ANXA5) promoter in a sample obtained from a female subject to detect the following nucleotide exchanges:

(1) G to A at a position which corresponds to nucleotide 186 of SEQ ID No. 2,

(2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2

(4) G to A at a position which corresponds to nucleotide 276 of SEQ ID No. 2;

wherein the presence of the nucleotide exchanges (1), (2), (3), and (4) (= risk haplotype M2), preferably in a heterozygous genotype (M1/M2 or N/M2) indicates a predisposition of said female subject to develop polycystic ovary syndrome (PCOS) or diagnoses PCOS in said female subject. Beside the detection of a predisposition of a female subject to develop polycystic ovary syndrome (PCOS), the above described method allows for the preserving fertility of females before the age of 40 is reached since early diagnosis of polycystic ovary syndrome (PCOS) will allow an earlier treatment of polycystic ovary syndrome (PCOS). f

The method of the present invention might generally be applied for any female subject. The "female subject" defined herein is a mammal, preferably a human. Said female subject may be pregnant or a non-pregnant. The methods of the present invention therefore encompass situations wherein the female subject plans to test its predisposition to develop PCOS, e.g. prior to a desired pregnancy. This includes for example females which plan to become pregnant, either by natural procreation or by in vitro fertilization. It is also envisaged that the female subject of the present invention is tested in accordance with the methods of the present invention prior to, during or after the first ovulation. Alternatively, said female subject may be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or more years of age, a range of 9 to 40, 9 to 30 or 9 to 20 being preferred. It is envisaged that in the methods of the present invention, said sample obtained from the female subject is a blood sample, a tissue sample, or a cell sample, a blood sample being preferred. It will be understood that any biological sample will be suitable as long as the respective sample contains genetic material which allows the detection/diagnosis which are subject of the methods of the present invention. Such a sample may be obtained via biopsy such as needle biopsy, surgical biopsy, via any kind of smear technique, for example by use of a buccal swab, etc. or others. The skilled person is well aware of further means and methods enabling him or her to obtain a sample containing genetic material from a female subject. "Genetic material" includes all kinds of nucleic acid sequences which can be obtained from the samples of the present invention and allow for the detection of the risk-haplotype Ml and/or M2 by standard nucleic acid detection techniques.

As documented for example in WO 2006/053725, a genetic variant in the promoter region of ANXA5 (BamHI) was surprisingly found in the annexin A5 (ANXA5) gene among women who were examined for different hereditary thrombosis genetic defects because of repeated pregnancy loss. This variant consists of four nucleotide substitutions, which are inherited as a haplotype. These four changes are important for the activity of the annexin A5 promoter and result in a reduced gene expression. As defined herein, of particular relevance in this respect are four point mutations characterized as "-19 G to A", "1 A to C", "27 T to C" and "76 G to A", whereby "G" denotes guanine, "C" denotes cytosine, "A" denotes adenine and "T" thymine. The positions "-19", "1", "27" and "76" of the mutations/substitutions described herein relate to the numbering of the sequence as given in appended Figure 4, depicting the ANXA5 core promoter structure. The numbering in particular relates to the first transcription start point of the gene (tsp 1, as indicated "+1"). However, the corresponding substitutions/mutations are also defined in relation to specific sequences representing the ANXA5 promoter, and given in particular in SEQ ID NO: 2 (an ANXA5 promoter structure as disclosed in Carcedo (2001), Biochem. J. 356, 571-579). Said SEQ ID No 2 is depicted herein in Figure 3. SEQ ID No. 2 as depicted herein corresponds in addition to SEQ ID No. 2 as depicted in WO2006/053725, which is included herein in its entirety. Further sequences which may be used alternatively for the determination of the nucleotide positions in the human ANXA5 promoter have already been described in the cited WO2006/053725, and in particular on pages 3 and 4 of said document (which are incorporated herein by way of reference thereto). It cannot be excluded that the risk conferred by haplotypes M2 or Ml as defined herein, could be modified to some extent by other SNPs occurring in the promoter region of ANXA5 gene, as defined for example in Fig. 4 and 6 (SEQ ID No. 3). These additional SNP(s) could be identified in representative samples of these populations through nucleic acids sequencing of the ANXA5 promoter region, a technique well known to the skilled person and also described herein.

The method of the present invention might generally be applied for any human female subject. It is however preferred that the sample used for the inventive method described herein is of West-Eurasian, East-Eurasian, African, Native American or Australo-Melanesian origin, West- Eurasian being preferred, European being more preferred and West-European being most preferred. The present invention further relates to a method as described hereinbefore wherein one or more additional detection methods of detecting/diagnosing polycystic ovary syndrome (PCOS) or a predisposition thereof are carried out. Such methods are well known to the skilled person as exemplified for example by WO/2008/092164, EP1749891, WO/2003/089623, WO/2008/047086 or WO/2004/052390, to name some.

Generally any additional testing method can be carried out that is suitable to generate data that lead to a (potentially better) PCOS-diagnosis and/or predictability of the predisposition to be detected. Further suitable additional testing methods are known to the skilled person and include e.g. pelvic ultrasound examinations and/ or the measurement of blood serum levels of androgens or e.g. proteomic analysis of human omental adipose tissue as described in reference

[19] . -

The present invention further relates to a method as described hereinbefore, wherein the detection of the nucleotide exchanges defined in (i)

(1) G to A at a position which corresponds to nucleotide 186 of SEQ ID No. 2,

(2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2

(4) G to A at a position which corresponds to nucleotide 276 of SEQ ID No. 2;

or (ϋ)

(2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2, and

(3) T to C at a position which corresponds to nucleotide 229 of SEQ ID No. 2,

is carried out by nucleic acid detection techniques.

"Nucleic acid detection techniques" are well-known to the skilled person and include inter alia any kind of PCR-based techniques or any other suitable technique which allows the identification of the nucleotide exchanges which characterize the risk haplotype Ml and/or M2. Such methods are described herein (see the examples) and are also published for example in [8] or in WO2006/053725.

Said techniques may be selected from the non-limiting group consisting of hybridization techniques, nucleic acid sequencing, PCR, restriction fragment determination, single nucleotide polymorphism (SNPs)-determination, LCR (ligation chain reaction) or restriction fragment length polymorphism (RFLP)-determination, to name some. Corresponding examples and further details may be obtained from standard technical advise literature (like Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds.)). As documented in the examples of WO2006/053725, a further suitable method is the restriction fragment determination or the RFLP method, comprising the determination of a BamH I restriction site. As shown in WO2006/053725, the absence (BamHI ') or the presence (BamHI +) of a BamHI restriction site is determined, and is indicative for the absence or presence of a point mutation as defined herein. Details on this method are given in the appended examples of WO2006/053725. In one embodiment, a relevant DNA-stretch may be amplified from genomic DNA by PCR-technology. Potential primers to be employed comprise, but are not limited to, the primers as provided in SEQ ID NO: 22 (ANX5.P.F) and SEQ ID NO: 23 (ANX5.exl.R) of WO2006/053725. The person skilled in the art is readily in the position to deduce further primer pairs or primers to be employed in order to amplify relevant stretches of the herein defined annexin A5 (ANXA5) promoter or of its fragments. After the amplicon is obtained (see also experimental part) it can be digested (restriction digest) with the restriction enzyme BamHI (which can be obtained from various suppliers, inter alia: Roche Applied Science, Mannheim, Germany; MBI Fermentas, St. Leon-Rot, Germany; New England Biolabs, Frankfurt am Main, Germany. Again, details are given in the experimental part. After this digest, to be carried in accordance with methods well-known in the art (see inter alia Sambrook/Russel, 2001, (log.cit.)), further analysis of the BamHI /BamHI + restriction site can be carried out by known techniques, like gel analysis, e.g. agarose gel analysis.

A further technique which is particularly envisaged in the context of the present invention is the SNP detection technique established by IHG Pharmaco. Said technique is sufficiently explained in WO 2006/038037.

Thus, in a preferred embodiment, the detection of the nucleotide exchanges defined in (i) and/or (ii) is carried out by a method for genotyping a target gene sequence associated with an inherited genetic disorder, comprising:

(a) providing a population of induced heteroduplex generator (IHG) molecules corresponding to said target gene sequence, the IHG molecule being a synthetic DNA sequence including at least one nucleotide position which corresponds to a known polymorphic site in the genomic DNA sequence of the target gene sequence and at least one nucleotide substitution, deletion and/or insertion ("identifier") relative to the genomic sequence at a nucleotide position spaced by a distance of at least one base from the nucleotide position which corresponds to the known polymorphic site and wherein the nucleotide(s) between the nucleotide position which corresponds to the polymorphic site and the identifier are unchanged from the genomic sequence; wherein the at least one identifier comprises an insertion of one or more bases and the IHG molecule is selected to provide improved separation of the resolved bands by a method comprising comparing the separation obtained using the IHG molecule with the separation obtained using a corresponding IHG in which the identifier is not so spaced;

(b) providing a population of the target gene sequence;

(c) combining the respective populations of (a) and (b) under conditions suitable for heteroduplex formation, to obtain induced heteroduplexes between the target gene sequence and an IHG molecule corresponding to said target gene sequence;

(d) resolving the induced heteroduplexes into bands on a suitable support; and (e) analysing the resolved induced heteroduplexes to determine the genotype of the target gene sequence.

Said technique is, as mentioned before, disclosed in great detail in WO 2006/038037 which is disclosed herein in its entirety (including the respective definitions of the terms used in the embodiment above). It will be understood that said "target gene sequence" is the human ANXA5 promoter or a fragment thereof.

It will be understood that the methods of the present invention are carried out "in vitro" (which equates with "ex vivo"). It is thus envisaged that the methods disclosed herein do preferably not include the step of obtaining the respective sample. Rather, the sample is already obtained and is provided in suitable container, i.e. the sample is preferably contained in a container, which container (including the sample) is then employed in the methods of the present invention. The present invention also relates to an IHG which can be employed in the IHG method characterized above and explained in WO 2006/038037, i.e. an IHG which is capable of detecting the risk haplotype Ml or M2 by way of the above identified IHG method. The skilled person knows how to design such an IHG, as the document WO 2006/038037 (and thereby also the present specification) provides sufficient guidance in this regard.

In a further embodiment, said IHG is comprised in a buffer. It is preferred that said buffer allows for the formation of heteroduplexes in accordance with the IHG method explained hereinabove and as well as in WO 2006/038037.

The present invention also relates to a nucleic acid sequence comprising a human annexin A5 (ANXA5) promoter or a fragment thereof, which promoter or fragment thereof comprises the nucleotide exchanges defined in

(i)

(1) G to A at a position which corresponds to nucleotide 186 of SEQ ID No. 2,

(2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2

(4) G to A at a position which corresponds to nucleotide 276 of SEQ ID No. 2; or

(ϋ)

(3) T to C at a position which corresponds to nucleotide 229 of SEQ ID No. 2, for use in a method of the present invention.

Said nucleic acid sequence comprising a human annexin A5 (ANXA5) promoter is exemplified by SEQ ID No. 2, but the invention is in no way limited thereto. Further suitable nucleotide sequences have been disclosed in for example in WO2006/053725 (therein SEQ Id No 1, 3 or 4 - see for example the table on page 4, which is also depicted herein below).

SEQ ID NO: 1 thereby relates, in accordance with the definition in WO2006/053725 to an ANXA5 promoter structure as deposited under gene accession number U0181; NCBI) and as annotated as„human annexin V gene, 5'- untranslated region, exons 1 and 2). Two further annexin V- promoter regions/5 '- untranslated regions are defined herein (in accordance with WO2006/053725) as SEQ ID NOS: 3 and 4. The 5 '-untranslated region of an annexin V gene may, in context of this invention, be characterized as comprising two specific motifs "A" and "B" which are documented in Figure 4.

Also comprised in the definition of a nucleic acid sequence comprising a human annexin A5 (ANXA5) promoter of the present invention are nucleic acid molecules which are at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90% even more preferably 95% and most preferably at least 97% identical to the promoter sequences as shown in any one of SEQ ID NOS: 1 to 4, SEQ ID No. 2 being preferred. "Percent (%) nucleotide sequence identity" is defined as the percentage of nucleotide residues in a candidate sequence (sequence of interest) that are identical with the nucleotide residues in the ANXA5 nucleotide sequence shown in SEQ ID No:l, 2, 3 or 4; SEQ ID No. 2 being preferred, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publically available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. The degree of identity is preferably over the entire length with the nucleotide sequence of SEQ ID No:2. It will be understood, however, that also the respective nucleotide sequences having a certain degree of identity as disclosed hereinabove, still comprise the nucleotide exchanges defined in (i)

(1) G to A at a position which corresponds to nucleotide 186 of SEQ ID No. 2,

(2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2 (3) T to C at a position which corresponds to nucleotide 229 of SEQ ID No. 2, and

(4) G to A at a position which corresponds to nucleotide 276 of SEQ ID No. 2;

or

(ii)

(2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2, and (3) T to C at a position which corresponds to nucleotide 229 of SEQ ID No. 2.

The above defined nucleic acid sequence comprising a human annexin A5 (ANXA5) promoter are preferably capable to drive the transcription of the ANXA5 gene or another gene or nucleic acid sequence (e.g. a reporter gene) which is under the control of the respective nucleic acid sequence comprising a human annexin A5 (ANXA5) promoter.

The "fragments of the human annexin A5 (ANXA5) promoter", however, are not necessarily capable to drive the transcription of the ANXA5 gene or another gene or nucleic acid sequence (for example a reporter gene). These fragments have a length of at least 27 consecutive nucleotides in case of the risk haplotype Ml, or a length of at least 91 nucleotides in case of risk haplotype M2. The maximum length of these fragments is preferably less then 50, more preferably less then 35 nucleotides in regard to the risk haplotype Ml, and/or less then 120, more preferably less then 100 nucleotides in length as regards risk haplotype M2. Nucleic acid molecules which are at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90% even more preferably 95% and most preferably at least 97% identical to the fragments defined herein above, are also envisaged.

Examples of reporter genes are genes which encode luciferase, (green/red) fluorescent protein and variants thereof, like eGFP (enhanced green fluorescent protein), RFP (red fluorescent proteins, like DsRed or DsRed2), CFP (cyan fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), β-galactosidase, glucose oxidase, maltose binding protein or chloramphenicol acetyltransferase, to name some. In another aspect, the present invention relates to a nucleic acid sequence which hybridizes to a nucleic acid sequence comprising a human annexin A5 (ANXA5) promoter or a fragment thereof, which promoter or fragment thereof comprises the nucleotide exchanges defined in

(i)

(1) G to A at a position which corresponds to nucleotide 186 of SEQ ID No. 2,

(2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2

(4) G to A at a position which corresponds to nucleotide 276 of SEQ ID No. 2;

or

(ii)

(3) T to C at a position which corresponds to nucleotide 229 of SEQ ID No. 2,

for use in a method of the present invention. It will be understood that the respective hybridizing nucleic acid sequence, comprises the above identified nucleotide exchanges defined in (i) or (ii), as well.

The term "hybridize" as used herein refers to conventional hybridization conditions, preferably to hybridization conditions at which 5xSSPE, 1% SDS, IxDenhardts solution is used as a solution and/or hybridization temperatures are between 35°C and 70°C, preferably 65°C. After hybridization, washing is preferably carried out first with 2xSSC, 1% SDS and subsequently with 0.2xSSC at temperatures between 35°C and 70°C, preferably at 65°C (regarding the definition of SSPE, SSC and Denhardts solution see Sambrook, loc. cit.). Stringent hybridization conditions as for instance described in Sambrook, supra, are particularly preferred. Particularly preferred stringent hybridization conditions are for instance present if hybridization and washing occur at 65°C as indicated above. Non-stringent hybridization conditions, for instance with hybridization and washing carried out at 45°C, are less preferred.

The term "comprises" includes in the context of the above identified nucleic acid sequences, that these sequences may contain further nucleotides or stretches of nucleotides which are not necessarily identical to the human ANXA5 promoter. Such stretches may resemble artificial hybridization sites for amplification primers (for example universal primers which can be used for the amplification of different nucleic acid sequences), restriction sites, tags, etc. Said further nucleotide nucleotides or stretches of nucleotides are preferably heterologous to the genetic sequences upstream and downstream of the human ANXA5 promoter. "Stretches" includes up to several kpb of consecutive nucleotides, however preferably not exceeding the typical length of a DNA vector, i.e. preferably not exceeding 5kbp.

All the above mentioned nucleic acid sequences can be used in the methods of the present invention.

The present invention further relates to the above identified nucleic acid sequences, which are under conditions suitable for the detection of said nucleic acid sequence. The present invention also relates to the above nucleotide sequences which are under conditions suitable for the detection of the human ANXA5 promoter, for example in a human sample described herein.

Provided that the detection is carried out by way of amplification methods such as PCR, the respective "suitable conditions" are adapted to the respective amplification method. "Suitable conditions" therefore typically denotes the presence of the nucleotide sequences described herein, in a buffer system, which buffer system contains as a minimum requirement all necessary elements which allow the detection of the respective nucleic acid as such, or which allow at least the very first step of that detection (which is typically a step of amplification, either via cloning in a host system or by way of a PCR technique, such as a real-time PCR as disclosed in the appended examples). Suitable buffer systems are well known to the skilled person. The skilled person is also well aware which components have to be included in order to amplify or clone a respective nucleic acid sequence.

It is particularly envisaged that the nucleic acid sequences of the present invention are contained in a PCR amplification buffer (preferably also including primers, nucleotides and/or the PCR-enzyme); or in a buffer which allows the formation of heteroduplexes in accordance with the methods disclosed herein, as well as in WO 2006/038037.

In another aspect, the present invention relates to a kit, preferably for use in a method of the present invention, said kit comprising:

(a) a package insert and/or an imprint indicating that said kit is to be employed in a method for diagnosing PCOS in a female subject or detecting a predisposition of a female subject to develop polycystic ovary syndrome (PCOS); and

(b) means to carry out said methods; and

(c) optionally one or more of the nucleic acid sequence(s) disclosed herein.

The term "package insert" is used to refer to instructions customarily included in commercial packages of diagnostic products, that contain information about the methods, usage, storage, handling, and/or warnings concerning the use of such diagnostic products. In the same way the term "imprints" is used to refer to instructions and/or information customarily imprinted on commercial packages of diagnostic products that contain information about the methods, usage, storage, handling, the contained materials and/or warnings concerning the use of such diagnostic products. The kit of the present invention may comprise one or more container(s), optionally with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic, and are preferably sterilized. The container holds a composition having an active agent or comprising a buffer which is effective for the detection of the risk haplotype Ml or M2. Further container may hold suitable amplification primers (for example PCR-primers) which allow the specific amplification of the human ANXA5 promoter or the fragments therefore which fragments have been defined herein elsewhere. It is also envisaged that containers are included which hold diverse buffers, for example amplification buffers, and/or buffers for the formation of heteroduplexes etc. The active agent in the composition is preferably an IHG, a positive control (for example an already isolated ANXA5 promoter or a fragment thereof), a negative control, a PCR-amplification enzyme such as the Taq enzyme etc. The kit may further comprise amplification primer pairs for the specific amplification of the human ANXA5 promoter (and in particular for the amplification of at least the fragment of the human ANXA5 promoter). The label on the container indicates that the composition is used for the detection of the risk haplotype Ml or M2 and/or for the amplification of the human ANXA5 promoter, and may also indicate directions for in vitro use, such as those described above.

In a further aspect, the present invention relates to the kit disclosed herein further comprising a nucleic acid sequence comprising a human annexin A5 (ANXA5) promoter or a fragment thereof, which promoter or fragment thereof comprises less than four of the following four nucleotide exchanges

(1) G to A at a position which corresponds to nucleotide 186 of SEQ ID No. 2,

(2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2

(4) G to A at a position which corresponds to nucleotide 276 of SEQ ID No. 2; The above embodiment discloses several possible positive controls of the human ANXA5 promoter (including fragments thereof) which comprise less then the four, i.e. three, two or just one or even none of the nucleotide exchanges defined herein.

The present invention also relates to the kits as disclosed herein, wherein said means comprises an IHG. Said IHG is capable of forming specific heteroduplexes with the intended target molecule which "target molecule" is in the context of the present invention the human ANXA5 promoter or a fragment thereof.

In an even further aspect, the present invention relates to the kit defined above, further comprising one or more primer pair(s) capable of amplifying at least a stretch of the human ANXA5 promoter, which stretch comprises at least one of the nucleotide exchanges (1) G to A at a position which corresponds to nucleotide 186 of SEQ ID No. 2, (2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2

(4) G to A at a position which corresponds to nucleotide 276 of SEQ ID No. 2. It is preferred that the stretches have a length of at least 20 consecutive nucleotides. Preferably said stretches cover nucleotide exchanges (2) and (3) defined herein, more preferably all four nucleotide exchanges (1), (2), (3), and (4), defined above.

The present invention also relates to the use of the nucleic acid sequences or fragments thereof, and/or the IHG(s), and/or the primer pair(s), disclosed herein for the manufacture of a kit or diagnostic composition, for use in a diagnostic method of the present invention.

The present invention further relates to a method for treating a female subject diagnosed of suffering from PCOS with the methods of the present invention, comprising treating said female subject with a medical treatment known to be or suspected of being able to alleviate and/or treat PCOS and/or the symptoms associated therewith. Such medical treatments are well-known to the skilled person and include, inter alia, those disclosed in WO/2003/061362 WO/2011/006040 or WO/2007/137066, to name same. It is however understood that the present invention is in no way limited to the aforementioned specific treatments which are merely cited for illustrating such PCOS-treatments.

In a further embodiment, the present invention relates to a pharmaceutical kit comprising a pharmaceutical PCOS-treatment and a leaflet and/or imprint indicating that the female subject to be treated is a subject of the present invention (i.e. a subject which has been diagnosed to carry the risk haplotyp M2 or a genotype comprising this haplotype (e.g. N/M2 or M1/M2)).

The above mentioned pharmaceutical kit may further comprise one or more components of the kits of the present invention. This disclosure may best be understood in conjunction with the accompanying drawings, incorporated herein by references. Furthermore, a better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration and are not intended as limiting.

The figures show:

Fig.l. (a) Scheme of the N and M2 ANXA5 allelic differences on the DNA and mRNA level and amplification strategy from cDNA. utr Exonl, non-translated Exon 1, tsp, transcription start point; *, nucleotide count start; 1., 2., 3., variant ANXA5 transcripts; forward primer

G anneals to all N ANXA5 transcripts, forward primer A anneals to all M2 ANXA5 transcripts, (b) N/M2 allele specific PCR does not generate product from N/N samples, as demonstrated by direct visualization on 1.5% agarose gel. Arrow points at the specific amplicon band of 131bp in the N/M2 control lane, asterisks below the N/N lanes denote non-utilized primers, (c) Melting curve analysis of N/M2 M2 allele-specific products.

Melting curves are homogenous at ca. 93° C, indicative of a single amplicon. Base line non-specific products appear only in water samples at ca. 82° C.

Fig.2 PCOS is generally characterized by characteristic changes in the appearance of the ovaries, for example on an ultrasound scan

Fig.3 Illustration of SEQ ID No. 2

Fig.4 Structure of the ANXA5 core promoter region. The region boundaries are marked with vertical bars and are numbered according to the position of the first transcription start point (tspl). Untranslated exon 1 is shaded in gray. Transcription factor consensi are in small print and abbreviations of corresponding transcription factors are italisized over the sequence rows. NotI and BamHI restriction sites are underlined and the sequence of the Z-DNA stretch in the promoter is in italics. Nucleotides marking transcription start points (tsp) are underlined. Regions important for promoter function (motifs A and B accordingly) occupy nucleotide positions 295 - 311 and 328 - 337. Nucleotides changed in the BamHI; haplotype are bolded and substituting nucleotides are indicated in bold capital letters over the matching respective positions. Fig.5 Illustration of SEQ ID No. 1 Fig.6 Illustration of SEQ ID No. 3

Fig.7 Illustration of SEQ ID No. 4

Fig.8 Genotype frequencies of ANXA5 gene promoter haplotypes in German PCOS patients and two different control groups.

Examples:

The following examples illustrate the invention. These exa mples shou ld not be construed as to limit the scope of th is invention. The examples are included for purposes of illustration and the present invention is limited only by the claims.

Expected: genotype frequency expected at Hardy-Weinberg equilibrium; a: genotype M1/M2 was observed in no patients, one Munster and five PopGen controls.

PCOS(Patients with polycystic ovary syndrome) patients are not in Hardy-Weinberg equilibrium (excess of heterozygotes).

HWE (Hardy-Weinberg equilibrium) was also not expected for this group.

'Immunochip analysis of 200000 genomic SNPs has confirmed isomorph genetic background of patient samples and PopGen controls.

Using the PopGen samples from Kiel as adequate controls for this group:

OR = 3.6, 95%CI (1.9 - 6.6), relative (population) risk of PCOS corresponds to equal 3.

Compared with the Munster supercontrols (having an equal genetic background as PopGen controls):

OR = 7.2, 95%CI (3.7 - 14.2), relative PCOS risk compared to normal fertile women corresponds to about 5 It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, a re within the scope of the appended claims.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, detailed Description, and Examples is hereby incorporated herein by reference.

References

[I] Gerke V, Creutz, CE, Moss SE. Annexins: linking Ca2+ signaling to membrane dynamics. Nat Rev Mol Cell Biol 2005; 6: 449-61.

[2] Thiagarajan P, Tait,JF. Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J Biol Chem 1990; 265: 17420-3. [3] Ravassa S, Bennaghmouch A, Kenis H, Lindhout T, Hackeng T, Narula J, Hofstra L,

Reutelingsperger C. Annexin A5 down-regulates surface expression of tissue factor: a novel mechanism of regulating the membrane receptor repertoir. J Biol Chem 2005; 280: 6028-35.

[4] Morgan RO, Bell DW, Testa JR, Fernandez MP. Genomic locations of ANX11 and ANX13 and the evolutionary genetics of human annexins. Genomics 1998; 48: 100-

[5] Carcedo MT, Iglesias JM, Bances P, Morgan RO, Fernandez MP. Functional analysis of the human annexin A5 gene promoter: a downstream DNA element and an upstream long terminal repeat regulate transcription. Biochem. J 2001; 356: 571-9.

[6] Rand JH, Wu XX, Guller S, Gil J, Guha A., Scher J, Lockwood Ci. Reduction of annexin-V (placental anticoagulant protein-l) on placental villi of women with antiphospholipid antibodies and spontaneous abortion. Am J Obstet Gynecol 1994; 171: 1566-72. [7] Shu F, Sugimura M, Kanayama N, Kobayashi H, Kobayashi T, Terao T. Immunohistochemical study of annexin V expression in placentae of preeclampsia. Gynecol Obstet Invest 2000; 49: 17- 23.

[8] Bogdanova N, Horst J, Chlystun M, Croucher PJ, Nebel A, Bohring A, Todorova A, Schreiber S, Gerke V, Krawczak M, Markoff A. A common haplotype of the annexin A5 (ANXA5) gene promoter is associated with pregnancy loss. Hum Mol Genet 2007; 16: 573-78.

[9] Chinni E, Tiscia GL, Colaizzo D, Vergura P, Margaglione M, Grandone E. Annexin V expression in human placenta is influenced by the carriership of the common haplotype M2. Fertil Steril 2009; 91: 940-2.

[10] Tiscia G, Colaizzo D, Chinni E, Pisanelli D, Scianname N, Favuzzi G, Margaglione M, Grandone E. Haplotype M2 in the annexin A5 (ANXA5) gene and the occurrence of 171 obstetric complications. Thromb Haemost 2009; 102: 309-13.

[II] Chinni E, Colaizzo D, Margaglione M, Rubini C, D'Ambrosio RL, Giuliani F, Di Vagno G, Grandone E. Correlation between factors involved in the local haemostasis and angiogenesis in full term human placenta. Thromb Res 2008; 122: 376-82. [12] Meller M, Vadachkoria S, Luthy DA, Williams MA. Evaluation of housekeeping genes in placental comparative expression studies. Placenta 2005; 26: 601-77 [13] Bieche I, Onody P, Tozlu S, Driouch K, Vidaud M, Lidereau 180 R. Prognostic value of ERBB family mRNA expression in breast carcinomas. IntJ Cancer 2003; 106: 758- 65.

[14] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402-8

[15] Markoff A, Bogdanova N, Knop M, Riiffer C, Kenis H, Lux P, Reutelingsperger C, Todorov V, Dworniczak B, Horst J, Gerke V. Annexin A5 interacts with polycystin-1 and interferes with the polycystin-1 stimulated recruitment of E-cadherin into adherens junctions. J Mol Biol 2007; 369: 954-66.

[16] Sifakis S, Soufla G, Koukoura O, Soulitzis N, Koutroulakis D, Maiz N, Konstantinidou A, Melissari E, Spandidos DA. Decreased annexin A5 mRNA placental expression in pregnancies complicated by fetal growth restriction. Thromb Res 2010; 125: 326-31.

[17] Legro RS, Strauss JF. Molecular progress in infertility: polycystic ovary syndrome. Fertil Steril. 2002 Sep;78(3):569-76.

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[19] Human Reproduction 2008 Vol.23, No.3 pp. 651-661,

Claims

1. A method for diagnosing polycystic ovary syndrome (PCOS) in a female subject or for detecting a predisposition of a female subject to develop PCOS, said method comprising: (a) examining the human annexin A5 (ANXA5) promoter in a sample obtained from said female subject to detect the following nucleotide exchanges

(i)

(1) G to A at a position which corresponds to nucleotide 186 of SEQ ID No. 2,

(2) A to C at a position which corresponds to nucleotide 203 of SEQ ID No. 2

(4) G to A at a position which corresponds to nucleotide 276 of SEQ ID No. 2;

and/or

(ii)

(3) T to C at a position which corresponds to nucleotide 229 of SEQ ID No. 2,

(b) determining whether the nucleotide exchanges defined in (i) and/or (ii) are present, wherein the presence of the nucleotide exchanges defined in (i) and/or (ii) indicates a predisposition of said female subject to develop polycystic ovary syndrome (PCOS) or diagnoses PCOS in said female subject .

2. The method of claim 1, wherein the presence of the nucleotide exchanges depicted in l(a)(i), preferably in a heterozygous genotype (M1/M2 or N/M2), indicates a predisposition of said female subject to develop polycystic ovary syndrome (PCOS) or diagnoses PCOS in said female subject.

3. The method of any one of claims 1 or 2, wherein said sample is a blood sample, a tissue sample, or a cell sample. 4. The method of any one of the preceding claims, wherein the detection of the nucleotide exchanges defined in (i) and/or (ii), is carried out by nucleic acid detection techniques. The method of claim 4, wherein the detection of the nucleotide exchanges defined in (i) and/or (ii) is carried out by a method for genotyping a target gene sequence associated with an inherited genetic disorder, comprising:

(b) providing a population of the target gene sequence;

(d) resolving the induced heteroduplexes into bands on a suitable support; and

(e) analysing the resolved induced heteroduplexes to determine the genotype of the target gene sequence.

A nucleic acid sequence comprising a human annexin A5 (ANXA5) promoter, which promoter comprises the nucleotide exchanges defined in (i) or (ii) of the preceding claims for use in a method of any one of claims 1 to 5.

7. The nucleic acid sequence of claim 6, which is under conditions suitable for the amplification of said nucleic acid sequence.

8. The nucleic acid sequence of claim 7, wherein said amplification is carried out by way of PCR.

9. Kit, preferably for use in a diagnostic method of any one of the preceding claims, said kit comprising:

(b) means to carry out said methods; and

(c) optionally one or more of the nucleic acid sequence(s) disclosed herein.