WO2013109145A1

WO2013109145A1 - Annexin a5 snp

Info

Publication number: WO2013109145A1
Application number: PCT/NL2013/050024
Authority: WO
Inventors: Waander Laurens Van Heerde; Maria Catharina Henrica DE VISSER- VAN SOEST; Larissa Filaretovna HIDDINK-EMELIANOVA
Original assignee: Stichting Katholieke Universiteit
Priority date: 2012-01-18
Filing date: 2013-01-17
Publication date: 2013-07-25

Abstract

The present invention relates to an in vitro method for predicting a tendency to pregnancy loss. The method involves determining whether at least one point mutation at a defined position is present or absent in the ANXA5 gene promoter sequence. The invention also encompasses the ANXA5 gene promoter sequence comprising the at least one specific point mutation. Also provided is a kit for predicting a tendency to pregnancy loss.

Description

Title of the invention

ANNEXIN A5 SNP

Technical field

The present invention relates to an in vitro method for predicting a tendency to pregnancy loss. The method involves determining whether at least one point mutation at a defined position is present or absent in a specific gene promoter sequence. The invention also encompasses the gene promoter sequence comprising the at least one specific point mutation. Also provided is a kit for predicting a tendency to pregnancy loss.

Background of the invention

Pregnancy loss, and particularly recurrent pregnancy loss (two or more), can involve significant psychological distress to the couple seeking parenthood. While spontaneous pregnancy loss occurs in approximately 15% of clinically diagnosed pregnancies of reproductive aged women, recurrent pregnancy loss occurs in 1 -2% of this same population (Kutteh, WH. Recurrent pregnancy loss. In: Carr BR, Blackwell RE, Azziz R. (eds) Essential Reproductive Medicine, McGraw-Hill, NY, NY-2005, 34:585-592). Pregnancy loss can be defined as the spontaneous end of a pregnancy at a stage wherein the embryo, fetus, or infant is incapable of surviving. Viability at 24 weeks of pregnancy is about 50% of fetus/infants, but moderate or major neurological disability drop to 50% only by 26 weeks (Kaempf JW, Tomlinson M, Arduza C et al. (2006). Pediatrics 1 17 (1 ): 22-9).

Etiologies for pregnancy loss include genetic, anatomic, endocrine, infectious, and autoimmune causes (Holly B. Ford, MD,* Danny J. Schust, MD vol. 2 no. 2 2009 reviews in obstetrics & gynecology). Diagnosis of the chance of (recurrent) pregnancy loss relies on the evaluation of these etiologies.

Genetic etiologies include parental balanced structural chromosome rearrangement, such as Robertsonian translocations, or chromosomal inversions, insertions, and mosaicism (Holly B. Ford, MD,* Danny J. Schust, MD vol. 2 no. 2 2009 reviews in obstetrics & gynecology).

Another significant genetic factor are hypercoagulable disorders that promote thrombosis. These are collectively termed thrombophilia and include the factor V Leiden mutation, methylenetetrahydrofolatreductase (MTH-FR) genetic variants, and factor II (prothrombin) 20210G>A mutation (Rey E et al. Lancet 2003, vol 361 , 901 -908). Recently, several lines of evidence support that placental anticoagulant protein Annexin A5 plasma level may play a significant role in the maintenance of pregnancy (Rand JH, Arslan AA, Wu XX et al. Am.J.Obstet.Gynecol. 2006; 194(1 ): 182-188). Annexin A5 (ANXA5), a Ca²⁺- and phospholipid-binding protein of the annexin family, which is also known as annexin V, is a protein that is (highly) expressed by human trophoblasts, vascular endothelial cells, and other cell types that serve a barrier function between tissues and body fluids (Rand JH, Arslan AA, Wu XX et al. Am.J.Obstet.Gynecol. 2006; 194(1 ):182-188). The human Annexin A5 (ANXA5) gene produces several transcripts and has a complex promoter with intricate regulation (Carcedo. Biochem. J. 2000, vol 356, 571 -579). Annexin A5 has been described to have strong anticoagulant properties as well as anti-inflammatory and anti-apoptotic features, both in vitro and in vivo (Reutelingsperger CP, Kop JM, Hornstra G, Hemker HC. Eur.J. Biochem. 1988; 173(1 ):171 -178; Thiagarajan P, Benedict CR. Circulation 1997;96(7):2339-2347; Reutelingsperger CP, van Heerde WL. Cell Mol.Life Sci.

1997;53(6):527-532; Cederholm A, Frostegard J. Ann.N.Y.Acad.Sci. 2007; 1 10896-103; Ewing MM, de Vries MR, Nordzell M et al. Arterioscler.Thromb.Vasc.Biol. 201 1 ;31 (1 ):95- 101 ).

Annexin A5 is expressed densely on the apical surfaces of the syncytiotrophoblasts, which face the maternal circulation (Krikun G, Lockwood CJ, Wu XX, Zhou XD, Guller S, Calandri C, et al. Placenta 1994; 15:601 -12), which is an ideal anatomic position to promote blood fluidity in the uteroplacental circulation. The infusion of antibodies against annexin A5 into pregnant mice resulted in placental infarcts and pregnancy losses (Wang X, Campos B, Kaetzel MA, Dedman JR. Am J Obstet Gynecol 1999; 180:1008-16). A reduction of annexin A5 by antiphospholipid antibodies is associated with recurrent spontaneous pregnancy losses (Rand JH, Wu XX, Andree HA, Lockwood CJ, Guller S, Scher J, et al. N Engl J Med 1997;337: 154-60; Rand JH, Wu XX, Guller S, Gil J, Guha A, Scher J, et al. Am J Obstet Gynecol 1994; 171 :1566-72; Rand JH, Wu XX, Guller S, Scher J, Andree HAM, Lockwood CJ. Am J Obstet Gynecol 1997;177:918-23). Rand et al (2006) also showed significant association between reduced levels of Annexin A5 and pregnancy loss Rand JH, Arslan AA, Wu XX et al. Am.J.Obstet.Gynecol. 2006; 194(1 ): 182-188).

EP1819833 describes a method wherein a tendency to pregnancy loss is determined by examining the promoter region of the Annexin A5 gene for the presence of four specific point mutations. These four point mutations correspond to the M2 haplotype as described by Bogdanova (Bogdanova N, Horst J, Chlystun M et al. Hum. Mol. Genet. 2007; 16(5):573-578). There remains a need for alternative methodology for predicting a tendency to pregnancy loss, and possibly to overcome this tendency, particularly in women intending to become pregnant who are predisposed to such tendency. The present invention fulfils these and other needs. Detailed description of the invention

Definitions

Throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure in their entirety.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); March, Advanced Organic Chemistry Reactions,

Mechanisms and Structure 4th ed., J. Wiley & Sons (New York, N.Y. 1992); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2001 ) provide one skilled in the art with a general guide to many of the terms used in the present application. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

Methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al. ., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in

Enzymology, Academic Press, San Diego. As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, a method for isolating "a" DNA molecule, as used above, includes isolating a plurality of molecules (e.g. 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more molecules).

A "3' UTR" or "3' non-translated sequence" (also often referred to as 3' untranslated region, or 3'end) refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises for example a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal (such as e.g. AAUAAA or variants thereof). After termination of transcription, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the cytoplasm (where translation takes place).

A "5' UTR" or "leader sequence" or "5' untranslated region" is a region of the mRNA transcript, and the corresponding DNA, between the +1 position where mRNA transcription begins and the translation start codon of the coding region (usually AUG on the mRNA or ATG on the DNA). The 5'UTR usually contains sites important for translation, mRNA stability and/or turnover, and other regulatory elements. Aligning and alignment: With the term "aligning" and "alignment" is meant the comparison of two or more nucleotide sequence based on the presence of short or long stretches of identical or similar nucleotides. Several methods for alignment of nucleotide sequences are known in the art, as will be further explained below. "Identity" is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF

SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G.,

Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER; Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in GUIDE TO HUGE COMPUTERS, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1 ):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403).

As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a certain sequence it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference polypeptide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted and/or substituted with another nucleotide, and/or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5" or 3" terminal positions of the reference nucleotide sequence, or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

DNA amplification: the term DNA amplification will be typically used to denote the in vitro synthesis of double-stranded DNA molecules using PCR. It is noted that other amplification methods exist and they may be used in the present invention without departing from the gist.

The term "gene" means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3'non- translated sequence comprising e.g. transcription termination sites.

A nucleic acid according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably the nucleotides cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is herein incorporated by reference in its entirety for all purposes).

In general, the term primers refer to DNA strands which can prime the synthesis of DNA. DNA polymerase cannot synthesize DNA de novo without primers: it can only extend an existing DNA strand in a reaction in which the complementary strand is used as a template to direct the order of nucleotides to be assembled. We will refer to the synthetic

oligonucleotide molecules which are used in a polymerase chain reaction (PCR) as primers. As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Optionally the term "promoter" includes herein also the 5'UTR region (e.g. the promoter may herein include one or more parts upstream (5') of the translation initiation codon of a gene, as this region may have a role in regulating

transcription and/or translation.

The term sequencing refers to determining the order of nucleotides (base sequences) in a nucleic acid sample, e.g. DNA or RNA. Many techniques are available such as Sanger sequencing and high-throughput sequencing technologies (also known as next -generation sequencing technologies) such as the GS FLX platform offered by Roche Applied Science, and the Genome Analyzer from lllumina, both based on pyrosequencing.

Description of the invention The present invention relates to an (isolated) nucleic acid molecule comprising an Annexin A5 (ANXA5) gene promoter nucleotide sequence which sequence comprises the following point mutation:

i) a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 . It is further preferred that the nucleic acid molecule according to the invention has at least 80%, more preferably 90%, even more preferably 95%, most preferably 99%, or 99.5% identity with SEQ ID NO:1. The presence of an "T" at a position that corresponds to nucleotide 82 of SEQ ID NO: 1 is indicative for an increased level of plasma Annexin A5, in comparison the absence of a "T" at that position. The presence of either a "C" or a "T" at a position that corresponds to nucleotide 82 of SEQ ID NO:1 is therefore corresponding to an altered risk for a tendency to pregnancy loss, the altered risk being an increased risk or in case of a "T".

It will be understood by the skilled person that the term point mutation within the context of the current invention denotes the presence of a nucleotide in the material under study that is different from the nucleotide that is shown in SEQ ID NO:1 at the corresponding position. Based on the presence or absence of one or more of the point mutations described herein, a skilled person can determine whether the particular subject from wh ich the material has been obtained may have e.g. an increased or decreased plasma level of Annexin A5 and/or a increased tendency for pregnancy loss. The results may well be combined with other indications and/or measurements in the diagnosis of these cond itions.

In an embodiment according to the invention, the nucleic acid molecule comprising a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 further comprises at least one of the following point mutations:

i) a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 ;

ii) a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 .

As is witnessed from the Examples, although a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO: 1 is predictive for plasma Annexin A5 levels, and/or tendency for pregnancy loss, the further point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 and/or the further point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 also correlate with plasma levels of Annexin A5.

In a preferred embodiment the (isolated) nucleic acid molecule comprises all of the following point mutations: i) a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 ;

ii) a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 ; and

iii) a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 .

Also provided is a nucleic acid fragment of the nucleic acid molecule comprising an Annexin A5 (ANXA5) gene promoter comprising the point mutations C to T at a position which corresponds to nucleotide 82 of SEQ ID NO: 1 . Preferably said fragment is at least 1 0, 20, 50, or 100 nucleotides in size.

Preferably the fragment has at least 80%, more preferably 90%, even more preferably 95%, most preferably 99%, or 99.5% identity with the corresponding sequence comprised in SEQ ID NO:1 , over the entire length of said nucleic acid molecule. For example, the isolated nucleic acid according to the invention, and comprising a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 , may have a length of, e.g. 51 nucleotides, with the nucleotide that corresponds in the middle. Consequently, the nucleic acid according to the invention has 25 nucleotides on the 3'end and 25 nucleotides on the 5'end of said nucleotide. Identity in now determined relative to the corresponding positions in SEQ ID N0.1 .

For example, the nucleic acid molecule according to the invention may have a length that is between, and including, 10 - 10000 nucleotides, 20 - 8000 nucleotides, 50 -1000 nucleotides, or 100 - 250 nucleotides.

Also provided is a nucleic acid fragment of the nucleic acid molecule comprising an Annexin A5 (ANXA5) gene promoter comprising the point mutations C to T at a position which corresponds to nucleotide 82 of SEQ ID NO: 1 , further comprising at least one of the point mutations A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 or T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 .

In a preferred embodiment said fragment comprising a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 , further comprising at least one of the point mutations A to C at a position which corresponds to nucleotide 262 of SEQ ID NO: 1 or T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 has a length of at least 170, 180, 206, 250, 300, or 500 nucleotides. Preferably the fragment has at least 80%, more preferably 90%, even more preferably 95%, most preferably 99%, or 99.5% identity with the corresponding sequence comprised in SEQ ID NO:1 , over the entire length of said nucleic acid molecule.

The inventors have surprisingly found genetic variance in the Annexin A5 gene promoter in human individuals with increased Annexin A5 plasma levels.

Sequence analysis of a 496-bp promoter region of the Annexin A5 gene showed the presence of six common SNPs: SNP1 (g.-628C>T, rs62319820); SNP2 (g.-467G>A, rs1 12782763); SNP3 (g.-448A>C, rs28717001 ) and SNP4 (g.-422T>C, rs28651243); SNP5 (g.-373G>A, rs1 13588187); and SNP6 (g.-302T>G, rs1050606) .

Sequencing of exon 2 showed the presence of an SNP, indicated here as SNP7 (g.-1 C>T, rs1 1575945). Nucleotide numbering is from the ATG codon in accordance with current HGVS practice ((http://www.hgvs.org/mutnomen/); there is no nucleotide 0, nucleotide 1 is the A of the ATG-translation initiation codon, the nucleotide 5' of the ATG-translation initiation codon is -1 , the previous -2, etc). A schematic overview of the promoter and sequences is given in Figure 1 .

SNP1 corresponds with a point mutation C to T at a position that corresponds to nucleotide 82 of SEQ ID NO: 1 ;

SNP2 corresponds with a point mutation G to A at a position that corresponds to nucleotide 243 of SEQ ID NO: 1 ;

SNP3 corresponds with a point mutation A to C at a position that corresponds to nucleotide 262 of SEQ ID NO: 1 ;

SNP4 corresponds with a point mutation T to C at a position that corresponds to nucleotide 288 of SEQ ID NO: 1 ;

SNP5 corresponds with a point mutation G to A at a position that corresponds to nucleotide 337 of SEQ ID NO: 1 ;

SNP6 corresponds with a point mutation T to G at a position that corresponds to nucleotide 408 of SEQ ID NO: 1 ;

SNP7 corresponds with a point mutation C to T at a position that corresponds to nucleotide 709 of SEQ ID NO: 1.

Within the context of the current disclosure reference may be made to SNP1 , SNP2, SNP3, SNP4, SNP5, SNP6 and/or SNP7. This will be understood to reference to the corresponding positions, and depending on the context, corresponding single nucleotide polymorphism, as indicated herein, and/or either as shown in SEQ ID NO: 1 , or in Figure 1 , or within any stretch of (genomic) DNA comprising the corresponding nucleotide position, e.g. comprising the nucleotide that corresponds to rs62319820, rs1 12782763, rs28717001 , rs28651243, rs1 13588187, rs1050606, and/or rs1 1575945 (SNP ID's according to www.ncbi.nlm.nih. ov/projects/SNP/).

In the art, different sequences corresponding to the Annexin A5 gene, and in particular to the promoter region, have been published. Figure 1 shows a comparison between those different sequences. S1 corresponds with SEQ ID NO:4, S2 correspond with SEQ ID NO:5; S3 corresponds with SEQ ID NO:6, S4 corresponds with SEQ ID NO:7, S5 corresponds with SEQ ID NO:8, and S6 corresponds to SEQ ID NO 1 . SED ID NO:4- SEQ ID NO:8 were disclosed in EP1819833. The nucleic acid molecule defined herein as comprising (or consisting of) an Annexin A5 gene promoter nucleotide sequence is a promoter. Accordingly, most preferably, the nucleic acid molecule is capable of conferring the activity of the Annexin A5 gene, in particular in form of Annexin A5 gene expression. Said expression may be tested by methods known in the art, for example by operatively linking the nucleic acid molecule of the present invention to either a marker molecule to be expressed and/or to the coding sequence of annexin A5 and detecting whether said Annexin A5 or said marker molecule is expressed in, inter alia, an heterologous gene expression system.

Also comprised in the definition of an nucleic acid molecule defined herein as comprising (or consisting of) an Annexin A5 gene promoter nucleotide sequence are nucleic acid molecules which are 80%, more preferably 90%, even more preferably 95%, most preferably 99%, or 99.5% identical to the promoter sequences as shown in any one of SEQ ID NOS: 1 , 4-8, as discussed above. The Annexin A5 gene promoter nucleotide sequence is accordingly, a promoter that is highly homologous to the promoter sequences as defined in any one of SEQ ID NOS: 1 , 4 to 8, or as shown in Figure 1 , and which is capable of driving Annexin A5 expression in cells.

It will be understood by the skilled person that these sequences are also encompassed by the current invention and that the corresponding point mutations at positions that correspond to those described in view of SEQ ID NO:1 are also part of the current disclosure and invention. In other words, the skilled person is aware of the existence of different nucleotide sequences available for this part of the Annexin A5 gene. He understands that the position of the point mutations, in particular those referred to as SNP 1 - SNP7, in particular SNP1 , SNP 3 and SNP4, described in the context of SEQ ID NO:1 , has a corresponding position in these alternative nucleotide sequences, and that the particular point mutations described herein is also present in such alternative nucleotide sequences.

As defined herein below, of particular relevance with respect to the present disclosure are the point mutations characterized as SNP1 , SNP3, and SNP4 above, whereby "G" denotes guanine, "C" denotes cytosine, "A" denotes adenine and "T" thymine.

When comparing their presence and absence in human individuals these variant nucleotides are shown herein to be related to plasma levels of Annexin A5. Reduced plasma levels of Annexin A5 are related to the risk of pregnancy loss, and the variant nucleotides may thus be used to determine/diagnose a tendency to pregnancy loss. Analysis of the

presence/absence of the identified variations may provide for better understanding, diagnosis, and prediction of pregnancy loss.

As shown in the examples, a human individual at least carrying SNP1 (g.-628C>T) and/or SNP3 (g.-448A>CT), and/or SNP4 (.g-422T>C) e.g. an individual with haplotype H4 (see Table 1 ) has increased plasma levels of Annexin A5 if compared to non-carriers of said SNPs or said haplotype. Pregnant women not carrying one or all of these single-nucleotide polymorphisms in the Annexin A5 promotor may need closer medical care and supervision during their (subsequent) pregnancy. As shown in Table 3, each individual SNP of SNP1 , SNP3, and SNP4 appears associated with increased plasma levels of Annexin A5. Analysis of the presence of the identified variations provides for better understanding in disease development, and prediction of progression of atherosclerosis and cardiovascular disease, and is therefore helpful.

The haplotype H4 disclosed herein comprises the following

i. a point mutation C to T at a position that corresponds to nucleotide 82 of SEQ ID NO:1 ;

ii. a point mutation A to C at a position that corresponds to nucleotide

262 of SEQ ID NO:1 ;

iii. a point mutation T to C at a position that corresponds to nucleotide

288 of SEQ ID NO:1 ;

iv. a point mutation T to G at a position that corresponds to nucleotide

408 of SEQ ID NO:1 . Also provided is a method for haplotyping an individual, comprising the step of determining the presence (or absence) of at least one of the above-identified point-mutations, preferably all of the above-identified point mutations SNP1 , SNP3, SNP4, and SNP6. The disclosure further pertains to a vector comprising the nucleic acid molecule , or nucleic acid molecule fragment, according to the invention.

For example, there is provided for an (isolated) nucleic acid molecule according to the invention or fragment thereof, comprising the following substitutions:

i) a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 ;

a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 ; and/or

iii) a point mutation T to C at a position which corresponds to nucleotide

288 of SEQ ID NO:1

which (isolated) nucleic acid fragment may be comprised in a vector. A vector may be used to transfer the nucleic acid molecule of fragment thereof to a host cell.

Also documented herein, the presence of a "T" at a position which corresponds to nucleotide 82 of SEQ ID NO: 1 indicates an increased plasma level of Annexin A5 and a lowered risk/tendency to pregnancy loss, while the presence of a C at a position which corresponds to nucleotide 82 of SEQ ID NO:1 would indicates an increased risk/tendency to pregnancy loss. Comparably, the presence of a C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 and/or a C at a position which corresponds to nucleotide 288 of SEQ ID NO: 1 indicates a lowered risk/tendency to pregnancy loss, while the presence of an A at a position which corresponds to nucleotide 262 of SEQ ID NO:1 and/or the presence of a T at a position which corresponds to nucleotide 288 of SEQ ID NO:1 indicates an increased risk/tendency to pregnancy loss.

To assist detection of a nucleic acid molecule or fragment thereof, according to the invention, i.e. comprising the following point mutations:

ii) a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 ; and/or

iii) a point mutation T to C at a position which corresponds to nucleotide

288 of SEQ ID NO:1 an oligomer or set of oligomers may be used, said oligomer or set of oligomers having a length of at least 8, more preferably at least 12, even more preferably at least 15, most preferably at least 20 nucleotides, which specifically hybridize to the a nucleic acid molecule or nucleic acid molecule fragment according to the invention.

Also provided is a method for predicting tendency to pregnancy loss and/or increased plasma level of Annexin A5 of a subject, the method comprising examining the presence or absence of the point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 or to determine the presence or absence of the point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 , and to further determine the presence or absence of at least one point mutation selected from the group consisting of a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 and a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 . The analysis can for example be performed in an Annexin A5 (ANXA5) promoter sequence obtained from said subject or one or more fragments thereof.

For example the method comprises examining a sequence obtained from said subject to determine the presence or absence of the following point mutations: i) a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 ;

iii) a point mutation T to C at a position which corresponds to nucleotide

288 of SEQ ID NO:1 .

Preferably, but not necessarily, all of said point mutations are determined. Alternatively, presence or absence of the whole H4 haplotype as documented above may be determined.

An increased tendency to pregnancy loss of subjects not carrying the point mutations is to be interpreted as increased as compared to subjects carrying the point mutations.

Determining the presence or absence of the point mutations in the Annexin A5 (ANXA5) gene promoter sequence, or fragment thereof, may be performed by nucleic acid techniques based on size or sequence, preferably chosen from the group consisting of hybridization techniques, nucleic acid sequencing, PCR, restriction fragment determination, single nucleotide polymorphism (SNPs)-determination, LCR (ligation chain reaction) and restriction fragment length polymorphism (RFLP)-determination, more preferably by nucleic acid sequencing. As pointed out above, the methods described herein are useful in the diagnosis of a tendency to pregnancy loss, or plasma levels of Annexin A5, and/or to determine a predisposition of an individual to pregnancy loss.

The examining of at least one point mutation as described herein, or of a haplotype as described, herein can be carried out by standard methods known in the art. For example, the detection of said at least one point mutation in said Annexin A5 gene, including gene regulatory elements like promoter regions, 5'UTR, 3'UTR, exons, introns, or the haplotyping may be carried out or determined by nucleic acid techniques based on size or sequence. Techniques suitable include, but are not limited to, nucleic acid sequencing techniques, hybridization techniques (e.g. using specific probes), PCR, single nucleotide polymorphism (SNPs)-determination, or restriction fragment length polymorphism (RFLP)-determination. Corresponding or additional techniques have been described in numerous handbooks, including Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001 ))./pct

The method according to the invention is preferably performed in vitro.

Although the method according to the invention can be performed with genetic material from any organism, the method is preferably performed on genetic material obtained from a mammal, preferably a human, more preferably a woman, a pregnant women or a woman intending to become pregnant. As will be understood by the skilled person, the method according to the invention can advantageously be performed on genetic material of women intending to become pregnant and who already have had (recurrent) pregnancy loss. The nucleotide sequence to be analyzed may be obtained from any part of the body, including a body fluid, preferably chosen from the group consisting of blood, serum, urine, amniotic fluid, vaginal secretion, or peripheral leukocytes.

The method disclosed herein can be performed on genomic DNA, preferably by amplifying a DNA-stretch of the Annexin A5 gene from genomic DNA by PCR and subsequent examination. For example, the following primers may be used:

forward 5' CCGAGCCCTGGACAGCTCCCCA-3' (SEQ ID NO:2)

and reverse 5'-GCCCCGCGACCACGCTCTCCTCT-3' (SEQ ID NO:3) However, a skilled person well understands that alternative techniques and/or primers may be employed in the analysis of one or more (different) DNA-stretch or stretches of the Annexin A5 gene, and either alone, or together comprise at least one mutations as disclosed herein ((SNP1 , SNP3, and/or SNP4); preferably the stretch or stretches alone, or together comprises at least SNP1 and/or SNP3, and/or SNP4). For example, a stretch may be provided comprising the nucleotide positions that corresponds with SNP1 , whereas in addition a stretch may be provide that comprises the nucleotide that corresponds with SNP3 and/or SNP4.

Preferably a stretch is at least about (± 5 nucleotides) 20, 40, 100, 200, 400, 1000, 1500 nucleotides long, depending on the technique for examining employed.

The present disclosure further provides a method for screening for molecules which are capable of interacting with a ANXA5 gene promoter nucleotide sequence not comprising the following point mutation(s) :

iii) a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 ,

the method comprising the steps of a) contacting said Annexin A5 (ANXA5) gene promoter nucleotide

sequence with a candidate molecule;

b) measuring a response to said contacting;

c) comparing said response with a standard response as measured in the absence of the candidate molecule.

Alternatively fragments as documented above may be used.

The skilled person knows how to perform such experiments. For example, the nucleic acid molecule may be in a vector operably linked to a gene encoding for a marker protein, a signal protein, a reporter gene, or a tag and modification in expression of e.g. the marker protein may be determined in the presence or absence of the candidate molecule. Non- limiting examples include fluorescent proteins, e.g. green fluorescent protein. A vector is a circular or linear nucleic acid molecule which can (autonomously) replicate in host cells into which they are introduced.

Alternatively, interacting of a compound, e.g. binding can be determined and compared to binding to a corresponding or identical nucleic acid but now comprising the said point mutations.

The above-described method may further comprise the step of

d) comparing said response to a response obtained with the candidate molecule and the Annexin A5 gene promoter nucleotide sequence comprising the following point mutation(s)

Also encompassed is the use of a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 or a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 , and at least one point mutation selected from the group consisting of a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 and a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO: 1 , a primer pair capable of amplifying stretches of the Annexin A5 (ANXA5) gene promoter sequence comprising said point mutation(s), an Annexin A5 (ANXA5) gene promoter nucleotide sequence having said point mutation(s), a fragment or set of fragments thereof, and described above, and/or the vector according to the invention, for predicting a tendency to pregnancy loss and/or reduced plasma level of Annexin A5 of a subject.

Further encompassed is also the use of the following point mutations

i) a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 ; alone or in combination with

ii) a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 ; and/or iii) a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 ,

or a primer pair capable of amplifying stretches of the Annexin A5 (ANXA5) gene promoter sequence comprising the above identified point mutation(s), an Annexin A5 (ANXA5) gene promoter nucleotide sequence having the above identified point mutation(s), a fragment or set of fragments of the nucleic acid molecule according to the invention comprising the above-identified point mutation(s), an oligomer having a length of at least 8, more preferably at least 12, even more preferably at least 15, most preferably at least 20 nucleotides, which specifically hybridize to the a nucleic acid molecule or nucleic acid molecule fragment comprising the above identified point mutation(s), and/or the vector comprising the nucleic acid molecule according to the invention or fragment thereof comprising the above-identified point mutation(s), for predicting a tendency to pregnancy loss and/or reduced plasma level of Annexin A5 of a subject, as compared to a subject carrying the above-identified point mutation(s).

Also provided is for the use of a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 , optionally combined with at least one point mutation selected from the group consisting of a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 and/or a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO: 1 , for predicting a tendency to pregnancy loss and/or reduced plasma level of Annexin A5 of a subject.

Lastly, provided is a kit for predicting a tendency to pregnancy loss (increased/reduced) and/or plasma level (reduced/increased) of Annexin A5 of a subject, the kit comprising means for determining presence or absence, in a sample obtained from a subject, of the point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 or a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 , and at least one point mutation selected from the group consisting of a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 and a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 , the kit preferably comprising a primer pair capable of amplifying stretches of the Annexin A5 (ANXA5) gene promoter sequence comprising a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 or a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 , and at least one point mutation selected from the group consisting of a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 and a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO: 1 . In particular there is provided for a kit for predicting a tendency to pregnancy loss and/or reduced plasma level of Annexin A5 of a subject, the kit comprising means for determining presence or absence, in a sample obtained from a subject, of the point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 .

In one embodiment, the above kit according to the invention comprises an oligonucleotide selectively hybridizing with the Annexin A5 (ANXA5) gene promoter sequence comprising a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 and not selectively hybridizing with the Annexin A5 (ANXA5) gene promoter sequence comprising a T at a position which corresponds to nucleotide 82 of SEQ ID NO: 1 .

In one embodiment, the kit according to the invention further or in addition comprises a primer pair capable of amplifying stretches of the Annexin A5 (ANXA5) gene promoter sequence comprising a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO: 1.

In one embodiment, the kit according to the invention further or in addition comprises means for determining presence or absence, in a sample obtained from a subject, of the point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 and/or a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 .

In one embodiment, the kit according to the invention is a kit wherein at least one primer in said primer pair is designed to selectively anneal to, and amplify, an Annexin A5 gene promoter sequence comprising a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 .

Legend to the figures

Figure 1 shows a comparison between different nucleotide sequences in the art for part of the Annexin A5 gene, including the promoter region of the Annexin A5. S1 corresponds with SEQ ID NO:4, S2 correspond with SEQ ID NO:5; S3 corresponds with SEQ ID NO:6, S4 corresponds with SEQ ID NO:7, S5 corresponds with SEQ ID NO:8, and S6 corresponds to SEQ ID NO 1 . SED ID NO:4- SEQ ID NO:8 were disclosed in EP1819833. Example Polymorphisms in the Annexin A5 gene promoter influence circulating Annexin A5 levels in healthy subjects.

Methods

Participants

The study population included 137 healthy individuals collected at the Radboud University Nijmegen Medical Center. In accordance with the Declaration of Helsinki, written informed consent was obtained from each participant. Genetic analysis

Genomic DNA was isolated from peripheral leukocytes. A 496-bp promoter region of the ANXA5 gene was amplified by polymerase chain reaction (PCR) using two oligonucleotide primers: forward 5'-CCGAGCCCTGGACAGCTCCCCA-3' (SEQ ID NO:2) and reverse 5'- GCCCCGCGACCACGCTCTCCTCT-3'.(SEQ ID NO:3) ¹⁷ PCR reactions were carried out in a final volume of 50 μΙ reaction mixture containing 5 μΙ 10x PCR Buffer (Invitrogen), 1 .5 mM MgCI₂, 5% DMSO (v/v), 1 M GC-RICH resolution solution (Roche Applied Science), 20 pM of each primer (forward and reverse), 0.2 mM of deoxynucleotide triphosphates, 100-150 ng genomic DNA and 1 .25 U Taq DNA polymerase (Invitrogen). Cycling conditions were: an initial denaturation step at 95°C for 3 minutes followed by denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds and elongation at 72°C for 30 seconds (30 cycles in total). Exon 2 with flanking regions was amplified as previously described (de Laat B, Derksen RH, Mackie IJ et al. Ann. Rheum. Dis. 2006;65(1 1 ):1468-1472). PCRs were performed in a GeneAmp^® PCR System 9700 Thermal Cycler (Applied Biosystems). PCR products were purified using MultiScreen PCR plates (Millipore). Purified amplicons were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and analyzed on the ABI 3730 PRISM DNA Analyzer (Applied Biosystems).

Measurement of plasma AnxA5 levels

Plasma samples obtained by centrifugation of citrated blood at 4200g for 10 minutes were stored at -80°C until use. Circulating ANXA5 levels were determined by a Zymutest ANXA5 ELISA (Hyphen Biomed) following the manufacturer's instructions.

Statistical analyses

Successful genotyping was possible in all 137 individuals (100%). Hardy-Weinberg equilibrium for each SNP was evaluated by a x² test. Haploview software was used to estimate the degree of linkage disequilibrium (r² values) between all SNP pairs and to determine haplotypes (Barrett JC, Fry B, Mailer J, Daly MJ. Bioinformatics. 2005;21 (2):263- 265). Since the haplotypes could easily be reconstructed, they were assigned manually to all individuals. Plasma ANXA5 levels were ¹ "log-transformed for normalization of their distribution. Log-transformed values of 131 subjects were used in the analyses, and differences between transformed values were tested by f-test. However, all values reported were reconverted to geometric means with the appropriate 95% confidence interval (CI). Two-sided P-values <.05 were considered statistically significant. All analyses were done using the SPSS version 16.0 software.

Results and discussion

Sequence analysis of the ANXA5 gene promoter in 68 male (mean 41 .6±10.0 years, +SD) and 69 female (mean 41 .1 +9.9 years, ±SD) healthy subjects showed the presence of six common SNPs (with rs ID's according to NCBI dbSNP

(http://www.ncbi.nlm.nih.gov/projects/SNP/index.html):

SNP1 (g.-628C>T, rs62319820),

SNP2 (g.-467G>A, rs1 12782763),

SNP3 (g.-448A>C, rs28717001 ) and

SNP4 (g.-422T>C, rs28651243),

SNP5 (g.-373G>A, rs1 13588187),

SNP6 (g.-302T>G, rs1050606)

(see Table 1 ).

SNP2 corresponds with a point mutation G to A at a position that corresponds to nucleotide 243 of SEQ ID NO: 1 ; SNP3 corresponds with a point mutation A to C at a position that corresponds to nucleotide 262 of SEQ ID NO: 1 ;

SNP7 corresponds with a point mutation C to T at a position that corresponds to nucleotide 709 of SEQ ID NO: 1 .

All participants were also examined for the presence of g.-1 C>T SNP (SNP7, rs1 1575945) in exon 2. All seven polymorphisms were in Hardy-Weinberg equilibrium. SNP3 and SNP4 as well as SNP2, SNP5 and SNP7 were completely linked (1^=1 ). Because of the tight linkage, only four common haplotypes (H) (frequency >1 %) were present (Table 1 ) covering approximately 99% of the population .

Table 1. Polymorphisms and haplotypes of the ANXA5 gene in 284 subjects.

SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP7

-6280T -467G>A -448A>C -422T>C -373G>A -302T>G -1 C>T

rs rs623198 rs1 12782 rs287170 rs286512 rs1 13588 rs10506 rs1 15759

number 20 763 01 43 187 06 45

†

MAF* 0.09 0.1 1 0.19 0.19 0.1 1 0.49 0.10

Haplo

Haplo type type frequency

H1 C G A T G T C 0.51

H2 C G A T G G C 0.29

H3 C A C c A G T 0.099

H4 T G c c G G C 0.09

Nucleotide numbering from the ATG codon; According to http://www.ncbi.nlm.nih.gov/snp; MAF, minor allele frequency; SNP, single nucleotide polymorphism; minor alleles in bold and underlined. Bogdanova's haplotype N ⁷ (-467G/-448A/-422T/-373G) (Bogdanova N, Horst J, Chlystun M et al. Hum. Mol. Genet. 2007;16(5):573-578) was a part of two novel haplotypes (H1 and H2), which were discriminated by SNP6. Interestingly, one of the novel haplotypes, H2, was associated with decreased plasma ANXA5 levels (Table 2). Carriers of the H2 haplotype may therefore have increased risk/tendency to pregnancy loss.

Carriers of two H2 alleles (mean 0.52 ng/ml, 95% CI: 0.44-0.62) had much lower ANXA5 levels than carriers of one H2 allele (mean 0.85 ng/ml, 95% CI: 0.76-0.94) and non-H2 carriers (mean 1 .01 ng/ml, 95% CI: 0.91 -1.13).

Table 2. Association of ANXA5 haplotypes with plasma ANXA5 levels

Number (%) Geometric mean ANXA5 (95% CI),

Haplotype

n=131 ng/ml

Haplotype 1

H1 H1 31 (23.7) 0.94 (0.82-1 .07)

H1 Hx 71 (54.2) 0.93 (0.84-1 .02)

HxHx 29 (22.1 ) 0.77 (0.61 -0.96)

Haplotype 2

H2H2 10 (7.6) 0.52 (0.44-0.62)^*

H2Hx 56 (42.7) 0.85 (0.76-0.94)^*

HxHx 65 (49.6) 1.01 (0.91 -1 .13)

Haplotype 3

H3H3 1 (0.8) 0.60

H3Hx 27 (20.6) 0.82 (0.67-1 .02)

HxHx 103 (78.6) 0.92 (0.84-1 .00)

Haplotype 4

H4H4 1 (0.8) 1.56

H4Hx 21 (16.0) 1.40 (1 .14-1 .72)^*

HxHx 109 (83.2) 0.81 (0.76-0.88)

Hx indicates all haplotypes except for the one given; CI, confidence interval; ^*P<.05 as compared with non-carriers. Since H1 and H2 were only discriminated by SNP6, it is suggestive that the -302G allele is responsible for decreased ANXA5 levels. Individual SNP analysis also demonstrated that homozygous -302GG carriers had lower ANXA5 levels (mean 0.76 ng/ml) than TG heterozygotes (mean 0.92 ng/ml) and TT carriers (mean 0.94 ng/ml) (P>.05) (Table 3).

Table 3. Association of individual SNPs with plasma ANXA5 levels

Geometric mean ANXA5

Analysis Genotype Number

CI), (ng/ml)

-6280T CC 109 0.81 (0.75-0.87)

CT 21 1.41 (1 .14-1 .72)^*

TT 1 1.56

-4670A GG 103 0.91 (0.84-0.99)

GA 27 0.82 (0.67-1 .02)

AA 1 0.6

-448A>C AA 84 0.84 (0.77-0.92)

AC 41 0.91 (0.79-1 .05)

CC 6 1.57 (0.73-3.38)

-422T>C TT 84 0.84 (0.77-0.92)

TC 41 0.91 (0.79-1 .05)

CC 6 1.57 (0.73-3.38)

-3730A GG 103 0.91 (0.84-0.99)

GA 27 0.82 (0.67-1 .02)

AA 1 0.6

-302T>G TT 31 0.94 (0.82-1 .07)

TG 72 0.92 (0.84-1 .02)

GG 28 0.76 (0.60-0.96)

-10T CC 103 0.91 (0.84-0.99)

CT 27 0.82 (0.67-1 .02)

TT 1 0.6

CI means confidence interval; ^*P=.002 as compared with the wild-type.

The important result was the association of haplotype H4 with increased levels of ANXA5. H4 heterozygotes had significantly higher circulating ANXA5 levels than non-H4 carriers (mean 1 .40 ng/ml, 95% CI: 1 .14-1.72 versus mean 0.81 ng/ml, 95% CI: 0.76-0.88, respectively). In one individual who was homozygous for H4 individual , plasma ANXA5 was also increased (1 .56 ng/ml). Haplotype H4 is an extension of the M1 haplotype (G-C-C-G) (Bogdanova N, Horst J, Chlystun M et al. Hum.Mol.Genet. 2007;16(5):573-578). The possession of the minor T-allele of SNP1 rs62319820, which also individually showed association with circulating ANXA5 levels (Table 3) and was unique for haplotype H4, appeared to be a major contributor to higher ANXA5 levels. Our results contradict findings of Bogdanova et al (Bogdanova N, Horst J, Chlystun M et al. Hum.Mol.Genet. 2007;16(5):573- 578), who demonstrated reduced ANXA5 promoter activity for the M1 haplotype. The absence of the rs62319820T-allele (SNP1 ) in their luciferase reporter constructs could be one of the reasons for this contradiction. Haplotype H3 was the third major haplotype in our population. Circulating ANXA5 levels in H3 heterozygotes (mean 0.82 ng/ml, 95% CI: 0.67- 1 .02) were slightly lower as compared with non-H3 carriers (mean 0.92 ng/ml, 95% CI: 0.84- 1 .00). Low plasma ANXA5 (0.60 ng/ml) was observed in one H3 homozygous individual. Haplotype H3 is an extension of the M2 haplotype (A-C-C-A) with the minor T-allele of SNP7 rs1 1575945.

In conclusion, we reported that polymorphic variations within the 5'-untranslated region of the ANXA5 gene may determine the variability of plasma ANXA5 levels in healthy subjects. In particular, we found point mutations -448C, -422C, and/or -628T to be associated with increased plasma levels of ANXA5, which in turn is negatively associated with an increased risk / tendency to pregnancy loss. These point mutations correspond to 262A>C, 288T>C, and/or 82C>T as set forth in SEQ ID NO:1 .

Claims

1 . A nucleic acid molecule comprising an Annexin A5 (ANXA5) gene promoter

nucleotide sequence which sequence comprises the following point mutation:

i) a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 .

Nucleic acid molecule according to claim 1 , further comprising at least one of the following point mutations:

A nucleic acid fragment of the nucleic acid molecule according to claim 1 , comprising the point mutations C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 , preferably wherein said fragment is at least 10, 20, 50, or 100 nucleotides in size.

A fragment according to claim 3, further comprising at least one of the following point mutations:

i) a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO: 1 ;

5. A vector comprising the nucleic acid molecule or nucleic acid molecule fragment according to any one of the claims 1 - 4.

6. A method for predicting tendency to pregnancy loss and/or increased plasma level of Annexin A5 of a subject, the method comprising examining the presence or absence of the point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 or the method comprising examining the presence or absence of the point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 , and further examining the presence or absence of at least one point mutation selected from the group consisting of a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO: 1 and a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 .

7. Method according to claim 6 wherein all (three) point mutations of claim 1 and claim 2 are determined.

8. Method according to claim 6 - 7, wherein the subject is a mammal, preferably a

human, more preferably a woman, a pregnant women or a woman intending to become pregnant, most preferably a woman intending to become pregnant and who had recurrent pregnancy loss.

9. The method according to claim 6 -8, wherein the Annexin A5 (ANXA5) promoter sequence is isolated from body fluid, preferably chosen from the group consisting of blood, serum, urine, amniotic fluid, vaginal secretion, and peripheral leukocytes.

10. Use of a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 , optionally combined with at least one point mutation selected from the group consisting of a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO:1 and/or a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 , for predicting a tendency to pregnancy loss and/or reduced plasma level of Annexin A5 of a subject.

1 1 . Kit for predicting a tendency to pregnancy loss and/or reduced plasma level of

Annexin A5 of a subject, the kit comprising means for determining presence or absence, in a sample obtained from a subject, of the point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 .

12. Kit according to claim 1 1 comprising an oligonucleotide selectively hybridizing with the Annexin A5 (ANXA5) gene promoter sequence comprising a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 and not selectively hybridizing with the Annexin A5 (ANXA5) gene promoter sequence comprising a T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 .

13. Kit according to any one of claims 1 1 or 12 comprising a primer pair capable of

amplifying stretches of the Annexin A5 (ANXA5) gene promoter sequence comprising a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 .

14. Kit according to any one of claims 1 1 - 13, said kit further comprising means for determining presence or absence, in a sample obtained from a subject, of the point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO: 1 and/or a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO:1 .

15. Kit according to any one of claims 13 - 14, wherein at least one primer in said primer pair is designed to selectively anneal to, and amplify, an Annexin A5 gene promoter sequence comprising a point mutation C to T at a position which corresponds to nucleotide 82 of SEQ ID NO:1 .