Genetic variant of the annexin a5 gene

Abstract

The present invention relates to a nucleic acid molecule comprising an annexin A5 (ANXA5) gene regulation element which comprises at least one point mutation, whereby said at least one point mutation (substitution) is selected from the group consisting of (i) a point mutation G to A at a position which corresponds to nucleotide 186 of SEQ ID NO: 2; (ii) a point mutation A to C at a position which corresponds to nucleotide 203 of SEQ ID NO: 2; (iii) a point mutation T to C at a position which corresponds to nucleotide 229 of SEQ ID NO: 2; and (iv) a point mutation G to A at a position which corresponds to nucleotide 276 of SEQ ID NO: 2. Furthermore, the present invention provides for a vector comprising the nucleic acid molecule the invention and a host transformed with the vector. The invention also relates to specific uses, in particular diagnostic uses of the nucleic acid molecules described herein. Moreover, the invention relates to a method for haplotyping an ANXA5 gene regulation element in an individual comprising the steps of: (a) isolating a nucleic acid from a sample that has been removed from the individual; (b) determining the presence of the nucleotides present at positions 186, 203, 229 and 276 of the individual's copy of the ANXA5 gene regulation element, wherein the position numbers are determined by comparison to SEQ ID NO: 2; (c) assigning the individuals a particular haplotype by comparison of the nucleotides present at said positions to the nucleotides recited in the haplotypes as defined herein.

What is claimed is: 
     
         1 . A composition for analysis of the annexin A5 gene, the composition comprising forward and reverse oligomers, the composition configured to allow the polymerase chain reaction amplification of a DNA fragment comprising at least from nucleotide 1259 to nucleotide 1349 of SEQ ID NO: 1 wherein the forward oligomer and the reverse oligomer are either or both (a) covalently attached to a fluorescent label or (b) fixed to a solid phase, the solid phase comprising a material selected from the group consisting of plastic, silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver, gold, nitrocellulose, and nylon, the forward oligomer and reverse oligomer each having a length of at least 15 nucleotides, wherein the forward oligomer is capable of hybridizing to:
 a portion of SEQ ID NO: 1 and wherein the forward and reverse oligomers allow the polymerase chain reaction amplification of a DNA fragment comprising at least from nucleotide 1259 to nucleotide 1349 of SEQ ID NO: 1, wherein the forward or reverse oligomer has;   (i) a point mutation G to A at a position which corresponds to nucleotide 1259 of SEQ ID NO: 1;   (ii) a point mutation A to C at a position which corresponds to nucleotide 1276 of SEQ ID NO: 1;   (iii) a point mutation T to C at a position which corresponds to nucleotide 1302 of SEQ ID NO: 1; or   (iv) a point mutation G to A at a position which corresponds to nucleotide 1349 of SEQ ID NO: 1; or,   a portion of SEQ ID NO: 2 and wherein the forward and reverse oligomers allow the polymerase chain reaction amplification of a DNA fragment comprising at least from nucleotide 186 to nucleotide 276 of SEQ ID NO: 2, wherein the forward or reverse oligomer has;   (i) a point mutation G to A at a position which corresponds to nucleotide 186 of SEQ ID NO: 2;   (ii) a point mutation A to C at a position which corresponds to nucleotide 203 of SEQ ID NO: 2;   (iii) a point mutation T to C at a position which corresponds to nucleotide 229 of SEQ ID NO: 2; or   (iv) a point mutation G to A at a position which corresponds to nucleotide 276 of SEQ ID NO: 2; or,   
       a portion of SEQ ID NO: 3 and wherein the forward and reverse oligomers allow the polymerase chain reaction amplification of a DNA fragment comprising at least from nucleotide 243 to nucleotide 337 of SEQ ID NO: 3, wherein the forward or reverse oligomer has;
 (i) a point mutation G to A at a position which corresponds to nucleotide 243 of SEQ ID NO: 3; 
 (ii) a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO: 3; 
 (iii) a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO: 3; or 
 (iv) a point mutation G to A at a position which corresponds to nucleotide 337 of SEQ ID NO: 3; or 
 
       a portion of SEQ ID NO: 4 and wherein the forward and reverse oligomers allow the polymerase chain reaction amplification of a DNA fragment comprising at least from nucleotide 190 to nucleotide 284 of SEQ ID NO: 4, wherein the forward or reverse oligomer has;
 (i) a point mutation G to A at a position which corresponds to nucleotide 190 of SEQ ID NO: 4; 
 (ii) a point mutation A to C at a position which corresponds to nucleotide 209 of SEQ ID NO: 4; 
 (iii) a point mutation T to C at a position which corresponds to nucleotide 235 of SEQ ID NO: 4; or 
 (iv) a point mutation G to A at a position which corresponds to nucleotide 284 of SEQ ID NO: 4. 
 
     
     
         2 . The composition of  claim 1  wherein the forward oligomer comprises a nucleotide sequence that is complimentary to and hybridizes to: a nucleotide of SEQ ID NO: 1 that has
 (i) a point mutation G to A at a position which corresponds to nucleotide 1259 of SEQ ID NO: 1; 
 (ii) a point mutation A to C at a position which corresponds to nucleotide 1276 of SEQ ID NO: 1; 
 (iii) a point mutation T to C at a position which corresponds to nucleotide 1302 of SEQ ID NO: 1; or 
 (iv) a point mutation G to A at a position which corresponds to nucleotide 1349 of SEQ ID NO: 1; or, 
 
       a nucleotide of SEQ ID NO:2 that has
 (i) a point mutation G to A at a position which corresponds to nucleotide 186 of SEQ ID NO: 2; 
 (ii) a point mutation A to C at a position which corresponds to nucleotide 203 of SEQ ID NO: 2; 
 (iii) a point mutation T to C at a position which corresponds to nucleotide 229 of SEQ ID NO: 2; or 
 (iv) a point mutation G to A at a position which corresponds to nucleotide 276 of SEQ ID NO: 2; or, 
 
       a nucleotide of SEQ ID NO:3 that has
 (i) a point mutation G to A at a position which corresponds to nucleotide 243 of SEQ ID NO: 3; 
 (ii) a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO: 3; 
 (iii) a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO: 3; or 
 (iv) a point mutation G to A at a position which corresponds to nucleotide 337 of SEQ ID NO: 3; or, 
 
       a nucleotide of SEQ ID NO:4 that has
 (i) a point mutation G to A at a position which corresponds to nucleotide 190 of SEQ ID NO: 4; 
 (ii) a point mutation A to C at a position which corresponds to nucleotide 209 of SEQ ID NO: 4; 
 (iii) a point mutation T to C at a position which corresponds to nucleotide 235 of SEQ ID NO: 4; or 
 (iv) a point mutation G to A at a position which corresponds to nucleotide 284 of SEQ ID NO: 4. 
 
     
     
         3 . The composition of  claim 1 , wherein:
 (a) the forward oligomer has SEQ ID NO: 8 or SEQ ID NO: 9 and the reverse oligomer has SEQ ID NO: 10 or SEQ ID NO: 13;   (b) the forward oligomer has SEQ ID NO: 11, SEQ ID NO: 21, or SEQ ID NO: 12 and the reverse oligomer has SEQ ID NO: 13 or SEQ ID NO: 10;   (c) the forward oligomer has SEQ ID NO: 14 or SEQ ID NO: 15 and the reverse oligomer has SEQ ID NO: 16 or SEQ ID NO: 7; or   (d) the forward oligomer has SEQ ID NO: 5 or SEQ ID NO: 6 and the reverse oligomer has SEQ ID NO: 7 or SEQ ID NO: 16.   
     
     
         4 . A kit comprising the composition of  claim 1  for diagnosing or detecting a predisposition for or a tendency to pregnancy loss. 
     
     
         5 . The composition of  claim 1  wherein the forward oligomer is selected from the group consisting of:
 (i) a forward oligomer capable of hybridizing under stringent conditions to a portion of SEQ ID NO: 2 having a point mutation G to A at a position which corresponds to nucleotide 186 of SEQ ID NO: 2; 
 (ii) a forward oligomer capable of hybridizing under stringent conditions to a portion of SEQ ID NO: 2 having a point mutation A to C at a position which corresponds to nucleotide 203 of SEQ ID NO: 2; 
 (iii) a forward oligomer capable of hybridizing under stringent conditions to a portion of SEQ ID NO: 2 having a point mutation T to C at a position which corresponds to nucleotide 229 of SEQ ID NO: 2; and 
 iv) a forward oligomer capable of hybridizing under stringent conditions to a portion of SEQ ID NO: 2 having a point mutation G to A at a position which corresponds to nucleotide 276 of SEQ ID NO: 2. 
 
     
     
         6 . The composition of claim  25  wherein the forward oligomer is selected from the group consisting of:
 (i) a forward oligomer comprising SEQ ID NO: 9; 
 (ii) a forward oligomer comprising SEQ ID NO: 12; 
 (iii) a forward oligomer comprising SEQ ID NO: 15; and 
 (iv) a forward oligomer comprising SEQ ID NO: 6. 
 
     
     
         7 . The composition of  claim 1  consisting essentially of the forward and reverse oligomers. 
     
     
         8 . The composition of  claim 1  further comprising a heat stable polymerase. 
     
     
         9 . A kit for analysis of the annexin A5 gene, the kit comprising a composition comprising a set of oligomers, the set of oligomers consisting essentially of two, three, or four oligomers either (a) covalently attached to a fluorescent label or (b) fixed to a solid phase, the solid phase comprising a material selected from the group consisting of plastic, silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver, gold, nitrocellulose, and nylon, wherein the oligomers are first, second, and optionally third and fourth oligomers, having lengths of at least 15 nucleotides capable of hybridizing under stringent conditions to at least two different portions of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4,
 wherein (a) the at least two different portions of SEQ ID NO: 1 are selected from the group consisting of sequences having point mutations (i)-(iv), and (b) the first and second, and optionally third and fourth oligomer, independently have point mutations selected from (i)-(iv) or a complimentary nucleotide thereof:   (i) a point mutation G to A at a position which corresponds to nucleotide 1259 of SEQ ID NO: 1;   (ii) a point mutation A to C at a position which corresponds to nucleotide 1276 of SEQ ID NO: 1;   (iii) a point mutation T to C at a position which corresponds to nucleotide 1302 of SEQ ID NO: 1; and   (iv) a point mutation G to A at a position which corresponds to nucleotide 1349 of SEQ ID NO: 1; or,   
       wherein (a) the at least two different portions of SEQ ID NO: 2 selected from the group consisting of sequences having point mutations (i)-(iv), and (b) the first and second, and optionally third and fourth oligomer, independently have point mutations selected from (i)-(iv) or a complimentary nucleotide thereof:
 (i) a point mutation G to A at a position which corresponds to nucleotide 186 of SEQ ID NO: 2; 
 (ii) a point mutation A to C at a position which corresponds to nucleotide 203 of SEQ ID NO: 2; 
 (iii) a point mutation T to C at a position which corresponds to nucleotide 229 of SEQ ID NO: 2; and 
 (iv) a point mutation G to A at a position which corresponds to nucleotide 276 of SEQ ID NO: 2; or, 
 
       wherein (a) the at least two different portions of SEQ ID NO: 3 selected from the group consisting of sequences having point mutations (i)-(iv), and (b) the first and second, and optionally third and fourth oligomer, independently have point mutations selected from (i)-(iv) or a complimentary nucleotide thereof:
 (i) a point mutation G to A at a position which corresponds to nucleotide 243 of SEQ ID NO: 3; 
 (ii) a point mutation A to C at a position which corresponds to nucleotide 262 of SEQ ID NO: 3; 
 (iii) a point mutation T to C at a position which corresponds to nucleotide 288 of SEQ ID NO: 3; and 
 (iv) a point mutation G to A at a position which corresponds to nucleotide 337 of SEQ ID NO: 3; or 
 
       wherein (a) the at least two different portions of SEQ ID NO: 4 selected from the group consisting of sequences having point mutations (i)-(iv), and (b) the first and second, and optionally third and fourth oligomer, independently have point mutations selected from (i)-(iv) or a complimentary nucleotide thereof:
 (i) a point mutation G to A at a position which corresponds to nucleotide 190 of SEQ ID NO: 4; 
 (ii) a point mutation A to C at a position which corresponds to nucleotide 209 of SEQ ID NO: 4; 
 (iii) a point mutation T to C at a position which corresponds to nucleotide 235 of SEQ ID NO: 4; and 
 (iv) a point mutation G to A at a position which corresponds to nucleotide 284 of SEQ ID NO: 4. 
 
     
     
         10 . The kit of  claim 9  wherein the set of oligomers consist essentially of first and second oligomers having lengths of at least 15 nucleotides capable of hybridizing under stringent conditions to:
 two different portions of SEQ ID NO: 1 having (ii) and (iii) point mutations; two different portions of SEQ ID NO: 2 having (ii) and (iii) point mutations; two different portions of SEQ ID NO: 3 having (ii) and (iii) point mutations; or two different portions of SEQ ID NO: 4 having (ii) and (iii) point mutations. 
 
     
     
         11 . The kit of  claim 9  wherein the set of oligomers consist essentially of first, second, third, and fourth oligomers having lengths of at least 15 nucleotides capable of hybridizing under stringent conditions to:
 four different portions of SEQ ID NO: 1 having (i), (ii), (iii), (iv) point mutations; four different portions of SEQ ID NO: 2 having (i), (ii), (iii), (iv) point mutations; four different portions of SEQ ID NO: 3 having (i), (ii), (iii), (iv) point mutations; or 
 four different portions of SEQ ID NO: 4 having (i), (ii), (iii), (iv) point mutations.