METHOD AND DEVICE FOR DETERMINING THE BLOOD GROUP OF A CAT IN THE AB BLOOD GROUP SYSTEM

Description

[0001] The invention relates to a method for the genotypic determination of the blood group of a cat in the AB blood group system according to the preamble of the independent claims.

[0002] The feline blood group system was first described in 1915. However, it took several more years until the AB blood group system, commonly used in cats today, was officially characterized in 1981. Thus, in cats ( Felidae Three different serological blood groups are distinguished: A, B, and C, with blood group A being by far the most common. In the currently predominant literature, blood group C is usually found under the misleading designation AB.

[0003] It is known from the prior art, for example, that the phenotypic difference between the three blood groups is due to a single antigen embedded in the cell membrane of erythrocytes. N-glycolylneuraminic acid (NeuGc), a sialic acid, is found on erythrocytes of cats with blood group A, whereas in cats with blood group B, a precursor of the antigen, the sialic acid N-acetylneuraminic acid (NeuAc), is expressed. The erythrocytes in the blood of cats with blood group C possess both forms of the molecules. These epitopes serve as antigens for blood group-specific antibodies of the respective other blood group. Thus, cats with blood group B possess antibodies against the N-glycolylneuraminic acid of cats with blood group A, and conversely, cats with blood group A possess antibodies against the N-acetylneuraminic acid of cats with blood group B.Cats with C-type erythrocytes do not possess either antibody, as both antigens are present on their erythrocytes and the presence of either antibody would lead to an autoimmune reaction.

[0004] The AB blood group system is not only conserved in the various breeds of domestic cats, but also in wildcats and other felids. Studies from 1999 showed that pumas ( Puma concolor ) about bobcat ( Lynx rufus ) to the cheetah ( Acinonyx jubatus ) all share the same blood group system. Cross-reaction studies have shown that the antibodies and antigens of the different felids are the same or at least very similar.

[0005] The necessity of blood typing in cats stems from the fact that the reaction of antigen and antibody is known to lead to hemolysis and / or hemagglutination, making the determination of the serological blood group indispensable before a blood transfusion. An immune reaction can occur after a blood transfusion containing an AB-incompatible antibody.

[0006] However, blood types must be considered not only during transfusions but also in breeding. Cats with blood type A have relatively weak alloantibodies against the antigens of blood type B, and the antibody titer is usually relatively low. Cats with blood type B, on the other hand, develop very strong antibodies in high concentrations. These can cause a high mortality rate in kittens during breeding, a condition known as neonatal isoerythrolysis. The antibodies against the A antigens are found in high concentrations in the mother's milk. If a mother cat with blood type B has kittens with blood type A or C, the kittens will ingest the antibodies through her milk, which in most cases leads to their death.

[0007] However, the genetic cause of blood group expression in cats remained unknown for a long time, until 2007 when various mutations in the gene were discovered. Cytidine monophosphate N-acetylneuraminic acid hydroxylase ( CMAH) was identified as a trigger. However, to date, there is no evidence that these genetic variants are actually responsible for the enzyme's activity. This gene encodes the enzyme cytidine monophosphate N-acetylneuraminic acid hydroxylase, an oxidoreductase that catalyzes the reaction of N-acetylneuraminic acid (NeuAc) to N-glycolylneuraminic acid (NeuGc). The enzyme possesses both a Rieske-type (2Fe-2S) cluster and a non-heme iron ion in its reactive site, and it is known from the prior art that mutations at these sites are the most likely cause of enzyme dysfunction. Such dysfunction appears to be associated with the development of blood group B. If the CMAH enzyme is so severely mutated that it can no longer perform its function, NeuAc is no longer converted to NeuGc, resulting in the blood group B phenotype.The dominance of the A allele for blood group A suggests that even a reduced enzyme concentration of functional CMAH is sufficient to convert almost all NeuAc to NeuGc and thus produce the blood group A phenotype.

[0008] It is therefore not surprising that numerous rapid tests for determining a cat's blood type are available. For example, in one rapid test, monoclonal antibodies against specific antigens are immobilized at designated sites on the erythrocytes of the blood groups on a test membrane. When the buffer-treated erythrocytes migrate along the membrane, they encounter the antibodies, triggering agglutination. A disadvantage of the aforementioned rapid phenotypic tests is their lack of specificity, resulting in sometimes contradictory results. This is because no definitive genetic cause for the phenotypic expression of blood groups has yet been identified. Furthermore, the currently available methods and devices do not allow for a definitive determination of a cat's blood type.

[0009] It is also known that forward and backward typing can be performed. Both forward and backward typing utilize the fact that the blood of B cats contains antibodies against epitopes on erythrocytes from A cats, which can trigger hemagglutination. When A erythrocytes are mixed with B serum / plasma, agglutination occurs, whereas the reverse process does not work, or only works to a very limited extent, due to the low antibody titers in A cats. Another disadvantage of this method is that, due to the low titer and limited effectiveness of anti-B antibodies in the serum of A cats, only weak agglutination of A serum with B erythrocytes may be expected. Furthermore, a definitive determination of blood group C in cats is not possible with this method.

[0010] The publication by GANDOLFI, B. et al. from PLoS One (2016) 11 (5):e0154973, pages 1-15 entitled "A Novel Variant in CMAH Is Associated with Blood Type AB in Ragdoll Cats" reveals a variety of different mutations or deletions in the feline CMAH gene for determining the blood type of a cat in the AB blood group system.

[0011] In the publication OMI, T et al. from PLoS One (2016) 11 (10):e0165000, page 1-17 entitled "Molecular Characterization of the Cytidine Monophospate-N-Acetylneuraminic Acid Hydroxylase (CMAH) Gene Associated with the Feline AB Blood Group System", a variety of different mutations in the feline CMAH gene are revealed, which are associated with blood groups A, B or C in different cats.

[0012] The publication by BIGHIGNOLI et al. from BMC GENETICS (2007) 8 (1) entitled "Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) mutations associated with the domestic cat AB blood group" and the publication by TASKER et al.: "Feline blood genotyping versus phenotyping, and detection of non-AB blood type incompatibilities in UK cats", JOURNAL OF SMALL ANIMAL PRACTICE, Vol. 55, No. 4, 1 April 2014 also reveal a variety of different mutations in the feline CMAH gene for determining the blood type of a cat in the AB blood group system.

[0013] Disclosure document US 2008 / 0220427A1 deals with different cat breeds, their frequencies, and their expression for blood groups A, B, and C. This publication also cites various mutations in the feline CMAH gene as the cause of blood groups A, B, and C.

[0014] There is therefore a great need for a method for the genotypic determination of a cat's blood type that ensures a rapid, reliable, simple, and sufficiently accurate determination and / or differentiation of the three blood groups known for felines within the AB blood group system. Furthermore, the method should be cost-effective, reliable, and take into account the specific characteristics of a particular subfamily and / or breed of feline. Another important aspect is that the method should be practically self-explanatory and the interpretation straightforward.The invention therefore aims to provide a method for the genotypic determination of the blood group of a cat in the AB blood group system in order to overcome the above-mentioned difficulties, improve diagnostics and to minimize the burden on the cat being examined and the costs incurred in carrying out the procedure.

[0015] This task is solved in a surprisingly simple but effective way by methods for genotypic determination of the blood group of a cat in the AB blood group system according to the doctrine of independent claims.

[0016] According to the invention, a method for the genotypic determination of the blood group of a cat in the AB blood group system is proposed, which comprises the following steps: a) Determining the presence or absence of at least three mutations in one or both alleles of the feline gene cytidine monophosphate N-acetylneuraminic acid hydroxylase in a biological sample from a cat, wherein the mutations are a G to T substitution at position 179, a T to A substitution at position 268, and a T deletion at position 1322; and b) Determining the blood group of the cat, wherein the presence of one of the mutations in one or both alleles and / or the absence of at least one of the mutations in both alleles is indicative of blood group A, or wherein the presence of at least one of the mutations in both alleles and / or the presence of at least two of the mutations in one or both alleles is indicative of blood group B.

[0017] According to the invention, a method for the genotypic determination of the blood group of a cat in the AB blood group system is further proposed, which comprises the following steps: a) Determining the presence or absence of four mutations in one or both alleles of the feline gene Cytidine monophosphate N-acetylneuraminic acid hydroxylase ( CMAH ) in a biological sample from the cat, wherein the mutations are a G to T substitution at position 179, a T to A substitution at position 268, a C to T substitution at position 364 and a T deletion at position 1322; and b) determining the blood group of the cat, wherein the presence of the mutation at position 364 in both alleles or the presence of the mutation at position 364 in one allele and at least one of the mutations selected from the group comprising positions 179, 268 and 1322 in one of the two alleles or in both alleles is indicative of blood group C.

[0018] The method according to the invention is based on the fundamental idea that for the reliable, rapid, simple, sufficiently accurate and genotypic determination of the blood group, and thus the differentiation of all three blood groups known in cats in the AB blood group system from one another, it is sufficient to detect the presence or absence of at least three mutations in one of the two alleles or in both alleles of the feline gene. Cytidine monophosphate N-acetylneuraminic acid hydroxylase ( CMAH ) in a biological sample from the cat. Within the scope of the invention, it has been recognized that for the genotypic determination of the cat's blood group in the AB blood group system, it is sufficient to determine the presence, i.e., detectability in one or both alleles, or absence, i.e., non-detectable presence in both alleles, of at least three mutations in one or both alleles of the CMAH-gene in the biological sample of the cat, wherein the mutations are a G to T substitution at position 179, a T to A substitution at position 268, and a T deletion at position 1322, or wherein the mutations are a G to T substitution at position 179, a T to A substitution at position 268, a C to T substitution at position 364, and a T deletion at position 1322. The presence or absence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more mutations in the gene is preferred. CMAH determined.

[0019] The term "Cytidine monophosphate N-acetylneuraminic acid hydroxylase ( CMAH )" concerns a gene that codes for the enzyme cytidine monophosphate N-acetylneuraminic acid hydroxylase, an oxidoreductase. This enzyme catalyzes the reaction of N-acetylneuraminic acid (NeuAc) to N-glycolylneuraminic acid (NeuGc). The gene CMAHThe protein consists of 16 exons, with two isoforms existing, both of which are necessary to ensure catalytic activity. The isoforms CMAH 1a and CMAH 1b arise from different splicing of the leader exons. In CMAH 1a, exon 1a is spliced ​​to exon 2, whereas in CMAH 1b, exon 1b is spliced ​​to exon 2. According to the invention, the numbering preferably begins at the adenosine of the start codon ATG, which is located in exon 1a, as shown in SEQ ID NO: 1 for the nucleic acid sequence and in SEQ ID NO: 2 for the corresponding amino acid sequence.

[0020] The term "allele" is familiar to those skilled in the art and generally refers to any number of alternative forms of a gene or segments of a chromosome. In the AB blood group system, there are preferably three alleles that determine the blood groups of cats: A, a<ab, and b. For inheritance purposes, allele A is dominant over a<ab and b, and allele a<ab is dominant over b (A > a<ab > b). It is therefore clear to those skilled in the art that a heterozygous cat with only one A allele always has serological blood group A, whereas a heterozygous cat with one a<ab allele only has blood group C if it also possesses the b allele. A cat with serological blood group B must always be homozygous for the b allele.

[0021] The terms "homozygous" and "heterozygous" have the meanings traditionally associated with these terms in genetics and generally refer to the condition of having two identical (homozygous) or non-identical (heterozygous) alleles of a particular gene or segment of the chromosome.

[0022] The term "mutation" refers to a genetic alteration of the nucleic acid and / or amino acid sequence. Preferably, the mutation is a substitution, insertion, deletion, duplication, or inversion of at least one base in the nucleic acid sequence or the fragment thereof that is responsible for a CMAH encoded, as well as by at least one amino acid in the amino acid sequence.

[0023] It is understandable to a specialist that the mutation can be synonymous, i.e., it does not result in an amino acid substitution in the polypeptide encoded by the nucleic acid, or it can be non-synonymous, i.e., it results in an amino acid substitution and / or a frame shift in the polypeptide encoded by the nucleic acid. It is further understood that a non-synonymous mutation can lead to a partial or complete loss of function of the polypeptide encoded by the nucleic acid.

[0024] It is further known to those skilled in the art that, with regard to amino acid sequences, single substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that result in a single amino acid or a small percentage of amino acids in the encoded sequence constitute a "conservatively modified variant," in which the modification results in the substitution of one amino acid by a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the field. Such conservatively modified variants do not additionally exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0025] It is further known to a person skilled in the art that the term "conservatively modified variant" applies to amino acid and nucleic acid sequences. With regard to certain nucleic acid sequences, conservatively modified variants refer to those nucleic acids that code for identical or substantially identical amino acid sequences, or, if the nucleic acid does not code for an amino acid sequence, for substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for a protein. Thus, a "silent or synonymous variant" is a change in the nucleotide sequence that does not alter the encoded polypeptide. Every nucleic acid sequence herein that codes for a polypeptide also describes every possible silent, synonymous, and / or implicit variation of the nucleic acid.

[0026] Within the scope of the invention, it has been discovered that the three mutations for determining blood group A or blood group B in cats are a G to T substitution at position 179, a T to A substitution at position 268, and a T deletion at position 1322, and that the four mutations for determining blood group C in cats are a G to T substitution at position 179, a T to A substitution at position 268, a C to T substitution at position 364, and a T deletion at position 1322. A further preferred mutation is selected from the group comprising Delta-53, which is an 18 base pair insertion-deletion at position -53, i.e., in the CMAHExon 1b relates to a C to T substitution at position 139, a G to A substitution at position 142, a T to C substitution at position 305, an A to C substitution at position 327, a G to A substitution at position 376, a G to T substitution at position 383, an A to C substitution at position 593, an A to C substitution at position 868, an A to G substitution at position 898, an A deletion at position 933, and a G to A substitution at position 1603. According to the invention, the numbering preferably begins at the adenosine of the start codon ATG, which is located in exon 1a, as shown in SEQ ID NO: 1 for the nucleic acid sequence and in SEQ ID NO: 2 for the corresponding amino acid sequence. Further preference is given to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more mutations in the gene. CMAHThe presence or absence of mutations at positions 1322, 179 and 268 in one or both alleles of the gene is determined according to the invention. CMAH or the presence or absence of mutations at positions 1322, 179, 268 and 364 in one or both alleles of the gene CMAH determined.

[0027] It is conceivable that, in addition, the presence of a mutation selected from the group comprising positions Delta-53, 139, 142, 327, 364, and 1603 in one of the two alleles, and / or the absence of at least one mutation selected from the group comprising positions Delta-53, 139, 142, 327, 364, and 1603 in both alleles, is indicative of blood group A in the AB blood group system. Preferably, the presence of a mutation is heterozygous, meaning that it is detectable in only one allele. Furthermore, the absence of 2, 3, 4, 5, 6, 7, or 8 of the mutations, which are understandably not identical to the single heterozygous mutation present, in both alleles is indicative of blood group A.Preferred is the at least one mutation selected from the group comprising positions Delta-53, 139, 142, 327, 364 and 1603 that is homozygous in the wild type, meaning it is not detectable in any allele and is therefore absent.

[0028] Furthermore, it is conceivable that the presence of at least one mutation selected from the group comprising positions Delta-53, 139, 142, and 1603 in both alleles, and / or the presence of at least two mutations selected from the group comprising positions Delta-53, 139, 142, and 1603 in one of the two alleles, is indicative of blood group B in the AB blood group system. It is understood by a person skilled in the art that a homozygous mutation cannot simultaneously be heterozygous, and vice versa. The presence of 2, 3, 4, 5, 6, or 7 of the mutations in one of the two alleles, or in both alleles, is further preferred as indicative of blood group B. Even more preferred is the homozygous mutation, meaning that it is detectable in both alleles and therefore present.

[0029] Furthermore, it is conceivable that the presence of a mutation at position 327 and / or of at least one mutation selected from the group comprising positions 139, 142, and 1603 in one or both alleles is indicative of blood group C in the AB blood group system. Preferably, the presence of 3, 4, 5, 6, 7, or 8 of the mutations in one or both alleles is indicative of blood group C. Preferably, the mutation at position 327 is homozygous, meaning that it is detectable in both alleles and therefore present, and the at least one mutation selected from the group comprising positions 139, 142, and 1603 is heterozygous, meaning that it is detectable in one allele.

[0030] It is understandable to a person skilled in the art that the terms "nucleic acid," "gene," "cDNA," "mRNA," "nucleotide," "oligonucleotide," and "polynucleotide" are used interchangeably to refer to deoxyribonucleotides or ribonucleotides and polymers thereof, either single- or double-stranded. Thus, nucleic acids include, for example, but by no means exclusively, known nucleotide analogs or modified residues, whether synthetic, naturally occurring, or non-naturally occurring, which exhibit similar binding properties to nucleic acids and are metabolized in a similar manner. Examples include phosphorus thioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Furthermore, the term also includes conservatively modified versions of these, complementary sequences, and the explicitly stated sequence.In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0031] It is readily understood by a specialist that the terms "amino acid," "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. Thus, amino acids include, but are not limited to, naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified.Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R-groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure different from the general chemical structure of an amino acid, but function similarly to a naturally occurring amino acid.

[0032] The term "haplotype" refers to a set of closely related genetic markers present on a chromosome that tend to be inherited together (that is, are not easily separated, for example, by recombination). A haplotype can preferentially be characterized by a set of single nucleotide polymorphisms (SNPs) and / or mutations, as described elsewhere, that are present on a chromosome and are inherited together.

[0033] Within the scope of the invention, it has further been discovered that the blood group of the cat in the AB blood group system is determined based on the presence or absence of at least three mutations in one or both alleles, wherein the presence or absence is indicative of blood group A, B, or C. It has been discovered that the presence of one of the mutations at positions 1322, 179, and 268 in one or both alleles and / or the absence of at least one of the mutations in both alleles is indicative of blood group A, and that the presence of at least one of the aforementioned mutations in both alleles and / or the presence of at least two of the mutations in one or both alleles is indicative of blood group B.Furthermore, it has been recognized that the presence of the mutation at position 364 and / or of at least one of the mutations selected from the group comprising positions 179, 268 and 1322 in one or both alleles is indicative of blood group C.

[0034] A person skilled in the art knows how to determine the presence or absence of at least one mutation in a biological sample using techniques known from the prior art.

[0035] The term "blood group" generally refers to one of many groups / types into which an individual's blood can be categorized based on the presence or absence of specific antigens produced by the same gene on the surface of blood cells. Within the scope of the invention, the term "blood group" preferably refers to the AB blood group system, wherein three blood groups in the AB blood group system are known in cats, namely A, B, and C (AB).

[0036] The term "biological sample" refers to material from a cat containing a nucleic acid that codes for a mutated CMAH polypeptide. Such samples are familiar to those skilled in the art, such as cell- and nucleus-containing (DNA and / or RNA-containing) samples.

[0037] The term "method for genotypic determination of blood group" refers to a method for genotypically determining the blood group in the AB blood group system or for distinguishing between blood groups A, B, or C of a cat. The method according to the invention may include additional steps that are located after or between the explicitly listed essential steps a) to b). Preferably, the method is partially or fully automatable.

[0038] The term "genotypic determination of the blood group" of cats refers to the determination of the blood group in the AB blood group system, or the differentiation between the blood groups A, B, or C known for cats, based on the presence or absence of at least three mutations in one or both alleles of the feline gene. CMAHin the biological sample. Preferably, the genotype of the CMAH gene is determined, which correlates with the expression of blood group A, B, or C in the AB blood group system. It is understandable to a person skilled in the art that the determination is preferably carried out semi-quantitatively, quantitatively, directly, and / or indirectly using methods known from the prior art, such as an algorithm.

[0039] It is understood by a person skilled in the art that a genotypic determination cannot usually be 100% accurate. The term therefore refers to a statistically significant probability regarding the accuracy of the genotypic determination of blood type within the AB blood group system. Whether such a determination is statistically significant can be determined by a person skilled in the art without inventiveness using methods known in the field. Examples include statistical evaluation tools such as determining the confidence interval, the p-value, Student's t-test, Mann-Whitney U test, etc. The corresponding intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% accurate. The p-values ​​are preferably 0.1, 0.05, 0.01, 0.005, or 0.0001.Preferably, this determination of the cat's blood type within the scope of the present invention is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% accurate.

[0040] Using the methods according to the invention, it is therefore possible to significantly increase the accuracy of the genotypic determination of the cat's blood group, as well as to reliably distinguish between the three blood groups known for cats in the AB blood group system. In this way, it is possible to determine the blood group quickly and with only one gene analysis. CMAHThe aim is to obtain a reliable and sufficiently accurate genotypic blood type determination for the cat in question, thus completely eliminating additional costs for any repeat tests or lengthy laboratory analyses. This also means that the procedure is not only very gentle for the cat, but also spares the cat the need for repeated sample collection.

[0041] Advantageous further developments of the invention, which can be implemented individually or in combination, are presented in the dependent claims.

[0042] In a further development of the invention, it is conceivable that the determination includes: a) Amplifying a nucleic acid, wherein the nucleic acid is selected from the group comprising i) a nucleic acid sequence as shown in SEQ ID NO: 1; and ii) a nucleic acid sequence encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2; and iii) a nucleic acid sequence encoding a polypeptide that is at least 50% identical to a polypeptide encoded by the nucleic acid sequences from i) or ii), wherein the polypeptide is feline CMAH; and iv) a nucleic acid sequence for a fragment of a nucleic acid from i), ii) or iii), wherein the fragment encodes a polypeptide, the polypeptide being feline CMAH; and b) detecting the amplified nucleic acid sequence.

[0043] The term "amplification" generally refers to a biochemical, chemical, and / or enzymatic reaction by which an increase in the number of copies of a nucleic acid sequence from the biological sample is achieved. Preferably, the nucleic acid sequence is partially or completely isolated from the biological sample. More preferably, the nucleic acid sequence is not isolated from the biological sample, i.e., it is amplifiable from the biological sample. Such amplification reactions are known to a person skilled in the art, for example, but by no means exclusively, polymerase chain reaction (PCR), ligase chain reaction (LCR), multi-displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), and branched DNA assay (bDNA).A specialist is also familiar with the relevant components of the aforementioned techniques, such as primers.

[0044] The term "primer" refers to a nucleic acid sequence that shapes the synthesis of an isolated nucleic acid sequence in a corresponding amplification reaction. Typically, a primer comprises fewer than approximately 100 nucleotides and preferably fewer than approximately 30 nucleotides. Examples of primers range from approximately 5 to approximately 25 nucleotides.

[0045] The term "isolated nucleic acid" refers to a nucleic acid or polynucleotide that is essentially or mainly free of the components that normally accompany it or occur as it does in its native state. Preferably, the nucleic acid or polynucleotide is isolated from the biological sample of the cat. More preferably, this is achieved using techniques known from the prior art, such as, but by no means exclusively, polyacrylamide gel electrophoresis or high-performance liquid chromatography. For example, it is conceivable that an isolated nucleic acid is separated from a regulatory region and / or open reading frames, which it preferably contains. CMAH- gene ,flank and / or encode proteins other than CMAH. The term "isolated" also means that a nucleic acid or protein essentially produces a band in an electrophoretic gel. In particular, this means that the nucleic acid or protein is at least 85%, particularly preferably at least 95%, and most preferably at least 99% pure.

[0046] The invention includes nucleic acids relating to polymorphic variants, fragments, alleles, mutants and interspecies homologs of CMAH,as shown in SEQ ID NO:1 and / or SEQ ID NO:2. It is understood by a person skilled in the art that the nucleic acids comprise naturally occurring or recombinant molecules. The nucleic acids preferably have i) a nucleic acid sequence as shown in SEQ ID NO:1. More preferably, they have a nucleic acid sequence that exhibits an identity of at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more, preferably over a range of at least about 25, 100, 200, 500, 1000 or more consecutive nucleotides. Alternatively, and more preferably, they ii) have a nucleic acid sequence that encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO:2.Alternatively, preferably, these iii) have a nucleic acid sequence encoding a polypeptide exhibiting at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity, preferably over a range of at least about 25, 50, 100, 200, 500, 1000 or more consecutive amino acids to a polypeptide encoded by the nucleic acid sequences from i) or ii), wherein the polypeptide is the feline CMAH as shown in SEQ ID NO: 1 and in SEQ ID NO: 2.

[0047] A person skilled in the art is aware of a large number of techniques known from the prior art for comparing sequences, for example an isolated nucleic acid sequence with the nucleic acid sequence as shown in SEQ ID NO: 1 and / or an isolated nucleic acid sequence with the amino acid sequence as shown in SEQ ID NO: 2.

[0048] The term "comparison" refers to the partial or complete comparison of nucleic acids with each other, with the correspondingly encoded amino acids, and / or with a reference sequence known from the prior art. It is understood that a comparison as applied here refers to a comparison of relevant parameters. Within the scope of the invention, the comparison can be carried out manually and / or with computer assistance. For a computer-assisted comparison, all means known to a person skilled in the art are conceivable, such as a computer and / or a computer program, such as the BLAST and BLAST 2.0 algorithms.A computer program can additionally evaluate the result of the comparison, for example automatically assessing the amino acid encoded by the nucleic acid, as well as predicting the expected primary, secondary, tertiary and / or quaternary structure, and predicting areas such as active or inactive regions of the polypeptide.

[0049] The term "detection" generally refers to the analysis and / or detection of the nucleic acid sequence, in particular the presence or absence of at least one mutation in the nucleic acid sequence. A person skilled in the art is aware of numerous detection techniques for evaluating nucleic acids for the presence of a single base alteration, such as the aforementioned amplification reaction, a probe, and / or sequencing. This advantageously ensures that determining a cat's blood type within the AB blood group system is reliable, safe, fast, and easy.

[0050] In a further development of the invention, it is conceivable that the cat is a big cat, a small cat such as a wildcat, a domestic cat, a pedigree cat and / or a mixed breed.The preferred species, for example, but by no means exclusively a cat, is selected from the group comprising the family Felidae, the subfamilies Felinae, Pantherinae and Acinonychinae, the genus Old World wildcat, caracal, Asian golden cats, Acinonyx, true cats, pumas, Iberian cats, lynxes, Otocolobus, Leptailurus, old-world cats, Neofelis, true big cats and Pardofelis, the species domestic cat, Canaani cat, cheetah, lynx, ocelot, lion, tiger, jaguar, leopard, snow leopard, clouded leopard, Sunda clouded leopard, caracal, grey cat, jungle cat, sand cat, black-footed cat, wildcat, African golden cat, marbled cat, Borneo golden cat, Asian golden cat, Pampas cat, small-spotted cat, Chilean forest cat, Andean cat, tiger cat, long-tailed cat, serval, Canadian lynx, Eurasian lynx, Iberian lynx. Bobcat, Pallas's cat, Bengal cat, flat-headed cat, rusty-spotted cat, fishing cat, puma, jaguarundi, pedigree cat and / or a mix thereof.Within the scope of the invention, it is further preferred that the pedigree cat is, for example, but by no means exclusively, a pedigree cat selected from the group comprising Ragdoll, British Shorthair, British Longhair, Siberian cat and the closely related pointed variant Neva Masquerade, Maine Coon, Sacred Birman, Scottish Fold, Turkish Angora, Persian and Abyssinian.

[0051] It is also conceivable that the biological sample could be a bodily fluid, such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph, various external secretions of the respiratory tract, intestines, and urogenital tract, tears, saliva, white blood cells, myelomas, and the like; a biological fluid, such as a cell extract and / or cell culture supernatant; a tissue and / or cell sample, such as a skin and / or fur sample; a tissue section, such as a biopsy and / or autopsy sample; and / or a frozen section. These samples are well-known in the field.

[0052] It is assumed that the definitions and / or explanations of the above-mentioned terms apply to all aspects described below in this description, unless otherwise stated.

[0053] It is also known to a person skilled in the art that the term "antibody" refers to a polypeptide comprising a scaffold region of an immunoglobulin gene or fragments thereof, which specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the genes of the constant region (kappa, lambda, alpha, gamma, delta, epsilon, and mu) as well as the genes of the variable region of the immunoglobulin. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The antibody is preferably a monoclonal, polyclonal, or chimeric antibody. Even more preferably, the antibody is directed against an immunogen that specifically binds a polypeptide derived from a CMAH- gene (as shown in SEQ ID NO: 1), a cDNA or a fragment thereof, including a mutated CMAH -gene, cDNA or a fragment thereof encoding polypeptides (as shown in SEQ ID NO: 2).

[0054] Further details, features, and advantages of the invention will become apparent from the following description of the preferred embodiments in conjunction with the dependent claims. The respective features can be implemented individually or in combination with one another. The invention is not limited to the embodiments shown. The embodiments are depicted schematically in the figures. Identical reference numerals in the individual figures denote identical or functionally equivalent elements, or elements that correspond to one another with respect to their function.

[0055] In detail: Fig. 1 a comparison of the nucleic acid sequence of the feline CMAH, as shown in SEQ ID NO: 1, with the corresponding encoded amino acid sequence as shown in SEQ ID NO: 2.

[0056] In Fig. 1 The corresponding sequences (SEQ ID NO: 1 and 2) used within the scope of the present invention for the identification and naming of the SNPs and mutations are shown side by side. These begin with the base adenine of the start codon, which is located in exon 1a of the CMAH is located, as this is also the starting point for identification using current technology, thus making a comparison of SNPs and mutations easier.

[0057] The following exemplary embodiments serve only to illustrate the invention. They are not intended to limit the subject matter of the patent claims in any way. Examples: Example 1: DNA / RNA isolation from cat blood

[0058] DNA isolation from the test animals is performed fully automatically by the MagNA Pure 96 (Roche Molecular Systems) using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche). For this, 200 µl of the respective sample mixture (usually EDTA blood) are pipetted into the designated tray. This yields approximately 100 µl of DNA / RNA solution. Example 2: Sequencing

[0059] The search for new genetic variants that could be associated with blood group differentiation requires the sequencing of all 16 exons of the CMAH- Genes . Prior to sequencing, these exons must be amplified using PCR. Due to the relatively long introns between the exons, separate primer pairs are required for each exon, with the exception of exons 5 and 6. The oligonucleotides listed in Table 1 below are used as primers for the analysis of the biological sample. Table 1: Oligonucleotides used Primer (5'UTR → 3'UTR) sequence SEQ ID NO: Exon 1 Forward 3 Exon 1 Reverse 4 Exon 2 Forward 5 Exon 2 Reverse 6 Exon 3 Forward 7 Exon 3 Reverse 8 Exon 4 Forward 9 Exon 4 Reverse 10 Exon 5-6 Forward 11 Exon 5-6 Reverse 12 Exon 7 Forward 13 Exon 7 Reverse 14 Exon 8 Forward 15 Exon 8 Reverse 16 Exon 9 Forward 17 Exon 9 Reverse 18 Exon 10 Forward 19 Exon 10 Reverse 20 Exon 11 Forward 21 Exon 11 Reverse 22 Exon 12 Forward 23 Exon 12 Reverse 24 Exon 13 Forward 25 Exon 13 Reverse 26 Exon 14 / 15 3'-UTR Forward 27 Exon 14 / 15 3'-UTR Reverse 28 Exon 1a Forward 29 Exon 1a Reverse 30 5'-UTR Forward 31 5'-UTR Reverse 32 Forward Primer T268A-BG5F (Genotyping Assay 268) CAC AAA GCA CAA CTG GAG GTT 33 Reverse Primer T268A-BG5R (Genotyping Assay 268) CAA CTA GTT CGT CTT GAC AGA AGC T 34 Reporter probe T268A-BG5V1 (VIC-marked) CAC CAT GAA ATA CGT CAA T 35 Reporter probe T268A-BG5M1 (FAM-labeled) ACC ATG AAA AAC GTC AAT 36 Forward Primer G142A-BG4F (Genotyping Assay) GCA AAG ATT TCA TTC TGT ACA AGA GCA A 37 Reverse Primer G142A-BG4R (Genotyping Assay) CTT GAT GCT TGC ACA CGT TCT T 38 Reporter Probe G142A-BG4V2 (VIC) CCC TCA CGC GAT TC 39 Reporter probe G142A-BG4M2 (FAM) CCC TCA TGC GAT TC 40 Forward Primer CMAH_F (Genotyping Assay) GGT GCA ACG GAA TCA GTA GTG A 41 Reverse Primer CMAH_R (Genotyping Assay) GCC TCT CCC TGG GAA TTC TG 42 Reporter probe CMAH_wt (YAK-BBQ) YAK-CAG CTT CGG TTG CTC GTT TGC TC-BBQ 43 Reporter probe CMAH_mut (FAM-BBQ) FAM-ACG GTG GTT GCT CGT TTG CTC C-BBQ 44

[0060] PCR was performed using the FastStart PCR Master Kit (Roche) according to the manufacturer's instructions, which already includes a suitable reaction buffer, a Taq polymerase, and the necessary dNTPs. A 50 µl batch was prepared for each exon according to the manufacturer's instructions. Based on a previous gradient PCR, a thermocycler program was performed to amplify the 16 exons at standardized amplification temperatures under standard conditions.

[0061] The PCR product is detected by gel electrophoresis. For this purpose, 8 µl of the sample are pipetted into wells of a 4% agarose gel containing ethidium bromide (Invitrogen, Thermo Fisher Scientific), which are filled with 12 µl of water. The gel is electrophoreticized for 20 minutes in the E-Base electrophoresis system (Invitrogen) and then photographed under UV light. 15 µl samples of the PCR product are then sequenced or further processed as follows.

[0062] The PCR product must first be purified. For this, the Qiagen MinElute PCR Purification Kit (250) is used. The PCR product to be purified, previously mixed with 250 µl of binding buffer, is added to the chilled columns. The column is centrifuged at 13,000 rpm for one minute, and the flow is discarded. 600 µl of washing buffer is added to the column, and the centrifugation step is repeated. After discarding the flow, a dry centrifugation step is performed. The column is then placed in a fresh reaction vessel and filled with 30 µl of elution buffer. After one minute of centrifugation at 8,000 rpm, the purified PCR product is in the flow of the column.

[0063] The purified PCR product is used as a template for a standard PCR. The Roche BigDye Terminator v1.1 Cycle Sequencing Kit is used according to the manufacturer's instructions. This mixture is then processed with a thermocycler program at standardized amplification temperatures under standardized conditions, according to the manufacturer.

[0064] The PCR product is diluted 1:1 with water, and the spin columns of the Qiagen DyeEx 2.0 Spin Kit (250) are prepared by dry centrifuging them for 5 minutes at 2800 rpm and placing them on fresh reaction vessels. The diluted sample is then added to the column and centrifuged for 5 minutes at 2800 rpm. The flow will yield the desired samples, which are then transferred to the Genetic Analyzer for capillary electrophoresis. Example 3: SNP genotyping assay

[0065] The genotyping assay is based on two different reporter probes for each SNP being analyzed, each hybridized to a fluorophore and a quencher. The probe consists of an oligonucleotide complementary to a region surrounding the SNP under investigation. One probe binds specifically to either the wild-type or the mutant allele located between the two primers for amplification. If the probe is intact, the quencher suppresses the fluorescence of the dye due to the relative proximity of the two substances. The exonuclease activity of DNA polymerase detaches the fluorophore from the probe once it has bound to the template strand. With the fluorophore now separated from the quencher, its fluorescence is no longer suppressed and can be detected.

[0066] The Fast Start Essential MM kit (Roche) is used for the assay according to the manufacturer's instructions. The primers for amplification are prepared in a primer mix at a final concentration of 0.02 pM and the probes at a final concentration of 0.6 pM. Different PCR assays are used for the SNP assays of 142 and 268 and for Delta-53. modus operandi selected. The samples are incubated in the Light Cycler (Roche) using a standard program and analyzed for fluorescence.

[0067] Fluorescence is measured on the two fluorescence channels of the fluorophores VIC (absorption 538, emission 554) and FAM (absorption 494, emission 518) and plotted against each other. As expected, the investigations revealed three distinct groups. One group shows only the signal of the wild-type probe (VIC), another only that of the mutated probe (FAM), and the third group exhibits both signals. Example 4: Sequence analysis in cats of blood group B in different breed cats

[0068] The gene CMAH It consists of 16 exons, with two isoforms of the enzyme existing, both of which are necessary to ensure catalytic activity. The isoforms CMAH 1a and CMAH 1b arises from different splicing of the leader exons. CMAH In 1a, exon 1a is split to exon 2. CMAH 1b accordingly refers to exon 1b. The sequencing performed within the scope of the invention comprises the 16 exons described, namely exons 2 to 15 and the two possible starter exons 1a and 1b. The latter contain the 5' UTR of the mRNA, and exon 15 forms the 3' UTR. The identification of the SNPs and mutations, or rather their naming, begins with the adenine base of the start codon, which is located in exon 1a, since this is also the starting point for identification in the current state of the art, thus simplifying the comparison of the SNPs.

[0069] The sequencing results of the 71 test specimens revealed 17 new mutations within the exons of the feline CMAH gene, in addition to the 19 variants already known from the state of the art. Eight of these mutations (103G>A, 141C>T, 213A>G, 501G>A, 636G>A, 1392T>C, 1452T>C, and 1649G>A) are synonymous mutations that do not result in an amino acid substitution. Two further mutations are classified as deletions (933delA and 1322delT), both of which shorten the gene by one base and cause a frameshift of the subsequent codons. The remaining seven new mutations (305T>C, 376G>A, 383G>T, 593A>C, 868A>C and 898A>G) are non-synonymous mutations, which consequently result in an amino acid substitution (see Table 2). Table 2: Newly discovered mutations in the CMAH gene of pedigree cats Position of the point mutation Effect on the amino acid sequence 103G>A No 141C>T No 213A>G No 501G>A No 636G>A No 1392T>C No 1452T>C No 1649G>A No 933delA Frame shift 1322delT Frame shift 305T>C Lysine > Proline 376G>A Serine > Leucine 383G>T Arginine > Isoleucine 593A>C Histidine > Proline 868A>C Threonine > Proline 898A>G Lysine > Glutamic acid Example 5: Fully sequenced cats sorted by breed and blood type

[0070] Within the scope of the present invention, the CMAH gene of a total of 71 cats was successfully sequenced. As can be seen from Table 3, the range of animals includes subjects of the Ragdoll (27 specimens), British Shorthair (6 specimens), British Longhair (1 specimen), Siberian cat and the closely related pointed variant Neva Masquerade (2 and 3 specimens), Maine Coon (4 specimens), Sacred Birman (5 specimens), Scottish Fold (3 specimens), Turkish Angora (8 specimens), Persian (1 specimen), Abyssinian (2 specimens), and European Shorthair (3 specimens). In addition, six samples of cats of unknown breed, which are referred to as mixed breeds, were also examined. Table 3: Fully sequenced cats sorted by breed and blood type race Number of test subjects Blood groups Ragdoll 27 1 A; 14 B; 12 C British Shorthair 6 1 A; 4 B; 1 C British Longhair 1 1 B Siberian cat 2 1 A; 1 C Neva Masquerade 3 1 A; 2 B Maine Coon 4 2A; 2B Sacred Burma 5 1 A; 4 B Scottish Fold cat 3 1 A; 2 B Turkish Angora 8 4A; 4B Persian 1 1 A Abyssinian 2 1 A; 1 B Mixed breeds 6 6 C European Shorthair 3 3 B

[0071] The sequences are compared using the Basic Local Alignment and Search Tool (BLAST) with the sequence of a cat as a reference genome from the NCBI to identify possible mutations or SNPs that could be the cause of the AB blood group system. The reference cat is an Abyssinian cat with blood group A, provided by the International Cat Genome Sequencing Consortium. Example 6: Sequencing results of different pedigree cats

[0072] Table 4 below, which is composed of Tables 4a and 4b, summarizes the sequencing results obtained within the scope of the present invention for the various purebred cats and the mixed-breed cats examined. The letters C, T, A, and G describe the corresponding nucleobases detected in the sequencing. The letters N and P stand for absent (N) and present (P), respectively, while x indicates that the sequencing was unsuccessful. Table 4: Presentation of sequencing results of different pedigree cats

[0073] Example 7: Results of the SNP assays

[0074] Table 5 below summarizes the diplotype results obtained in accordance with the present invention using SNP assays for the mutations 179G>T, 268T>A, and the deletion of 1322delT. Additionally, the mutations Delta-53 and 142G>A are examined in each cat. The letters N and b denote the corresponding alleles, with N representing allele A (wild-type) and b representing allele b. Results marked with an "x" indicate that the SNP assay was unsuccessful, while results marked with a "-" indicate that no further assays were performed on the respective cat.

Claims

1. A method for identifying the blood group of a cat in the AB blood group system, the method comprising the following steps: a) determining the presence or absence of three mutations in one of the two alleles or in both alleles of feline gene Cytidine Monophosphate-N-Acetylneuraminic Acid Hydroxylase (CMAH) in a biological sample, the mutations consisting in a G to T substitution at position 179, a T to A substitution at position 268, and a T deletion at position 1322; and b) identifying the blood group of the cat, the presence of one of the mutations in one of the two alleles and / or the absence of at least one of the mutations in both alleles being indicative of blood group A, or the presence of at least one of the mutations in both alleles and / or the absence of at least two of the mutations in one of the two alleles being indicative of blood group B.

2. A method for identifying the blood group of a cat in the AB blood group system, the method comprising the following steps: a) determining the presence or absence of four mutations in one of the two alleles or in both alleles of feline gene Cytidine Monophosphate-N-Acetylneuraminic Acid Hydroxylase (CMAH) in a biological sample, the mutations consisting in a G to T substitution at position 179, a T to A substitution at position 268, a C to T substitution at position 364, and a T deletion at position 1322; and b) identifying the blood group of the cat, the presence of the mutation at position 364 in both alleles or of the mutation at position 364 in one allele and of at least one of the mutations selected from the group comprising positions 179, 268, and 1322 in one of the two alleles or in both alleles being indicative of blood group C.

3. The method according to any one of the preceding claims, the determining step comprising: a) amplifying a nucleic acid, the nucleic acid being selected from the group comprising i) a nucleic acid sequence as shown in SEQ ID NO: 1; and ii) a nucleic acid sequence encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2; and iii) a nucleic acid sequence encoding a polypeptide that is at least 50 % identical to a polypeptide encoded by the nucleic acid sequences of i) or ii), the polypeptide being feline CMAH; and iv) a nucleic acid sequence for a fragment of a nucleic acid of i), ii) or iii), the fragment encoding a polypeptide, the polypeptide being feline CMAH; and b) detecting the amplified nucleic acid sequence.

4. The method according to any one of the preceding claims, wherein the cat is a big cat, a small cat, a domestic cat, a pedigree cat, and / or a mixed breed.

5. The method according to any one of the preceding claims, wherein the biological sample is a body fluid, a biological fluid, tissue, a cell sample, a tissue section, and / or a frozen section.