Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) mutations associated with the domestic cat AB blood group

Abstract

Background: The cat has one common blood group with two major serotypes, blood type A that is dominant to type B. A rare type AB may also be allelic and is suspected to be recessive to A and dominant to B. Cat blood type antigens are defined, N-glycolylneuraminic acid (NeuGc) is associated with type A and N-acetylneuraminic acid (NeuAc) with type B. The enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) determines the sugar bound to the red cell by converting NeuAc to NeuGc. Thus, mutations in CMAH may cause the A and B blood types.

Results: Genomic sequence of CMAH from eight cats and the cDNA of four cats representing all blood types were analyzed to identify causative mutations. DNA variants consistent with the blood types were genotyped in over 200 cats. Five SNPs and an indel formed haplotypes that were consistent with each blood type.

Conclusion: Mutations in type B cats likely disrupt the gene function of CMAH, leading to a predominance of NeuAc. Type AB concordant variants were not identified, however, cDNA species suggest an alternative allele that activates a downstream start site, leading to a CMAH protein that would be altered at the 5' region. The cat AB blood group system is proposed to be designated by three alleles, A > aab > b. The A and b CMAH alleles described herein can distinguish type A and type B cats without blood sample collections. CMAH represents the first blood group gene identified outside of non-human primates and humans.

Figure 1

Cat genomic sequence of CMAH including the 5'UTR to exon 1. Sequence generated from genomic DNA from cats of each blood type. Lower case letters represent the UTR. The six bold upper case letters are the last six nucleotides of exon 1 that are translated to the methionine start and a glycine prior to the splice with exon 2. ATG sites are underlined. The lower case in italics is the intronic 1 sequence that is the splice variant sequence found in the cDNA of Type B cats, spliced immediately upstream of the exon 2 sequence. Two GATA transcription factor sites are underlined. The mutations that are consistent with blood group phenotypes, A-217G and the T-371C, are presented in bold and underlined. One intron 1 variant and three 5' UTR mutations that are not consistent with blood types are also presented.

Figure 2

Pedigree of a British Shorthair cat family segregating for blood types and CMAH variants. Circles represent females, squares represent males, filled symbols indicate type B cats from hemagglutination assays. Symbols with a diagonal slash represent cats that were not available for the analyses. Numbers under the symbols represent the laboratory sample number. Genotypes for the CMAH variants are represented below the cat identification numbers, respectively, and are presented as haplotypes in the order; G-217A, T-371C, Δ-53, G139A, T265A.

Figure 3

Pedigree of a Birman cat family segregating for blood types and CMAH variants. Symbols, numbering and haplotype order are the same as described for Figure 2.

Figure 4

Schematic of putative cat CMAH protein deduced from mRNA. a. The ATG at -102 bp may be the true activation site in the cat, implying an extension to exon 1 exists in the cat, 5' to CMAH exon 1 that has been declared in mouse Cmah. Blood type A cats have at least one allele that creates the depicted fully functional protein. b. An 18 bp insertion is homozygous in the genomic sequence of type B cats. This insertion disrupts the reading frame created by the -102 bp ATG, causing a TGA stop codon. Approximately 50% of the mRNA of the type B cat had this insertion. c. Approximately 50% of the mRNA from the blood type B cats had the 3' portion of intron 1 spliced directly to exon 2, completely skipping exon 1, including the insertion. Two ATG sequences are present in this intronic leader but are not in frame. The first in-frame ATG occurs at bp 139 in exon 2 for cats with this mis-spliced mRNA species. The insertion and G139A mutation is concordant with the b allele. Cats that are blood type A but carry the b allele had all three species of mRNA. d. The mRNA from cats of the rare blood type AB did not extend to the -102 bp start and did not have the 18 bp insertion. The traditional ATG start at bp 1 would be the first in-frame start site. Approximately 50% of the mRNA from the blood type AB cat also had mRNA species as depicted in figure 5c but not the G139A mutation as found on the b allele.