Molecular Characterization of the Cytidine Monophosphate-N-Acetylneuraminic Acid Hydroxylase (CMAH) Gene Associated with the Feline AB Blood Group System

Abstract

Cat's AB blood group system (blood types A, B, and AB) is of major importance in feline transfusion medicine. Type A and type B antigens are Neu5Gc and Neu5Ac, respectively, and the enzyme CMAH participating in the synthesis of Neu5Gc from Neu5Ac is associated with this cat blood group system. Rare type AB erythrocytes express both Neu5Gc and Neu5Ac. Cat serum contains naturally occurring antibodies against antigens occurring in the other blood types. To understand the molecular genetic basis of this blood group system, we investigated the distribution of AB blood group antigens, CMAH gene structure, mutation, diplotypes, and haplotypes of the cat CMAH genes. Blood-typing revealed that 734 of the cats analyzed type A (95.1%), 38 cats were type B (4.9%), and none were type AB. A family of three Ragdoll cats including two type AB cats and one type A was also used in this study. CMAH sequence analyses showed that the CMAH protein was generated from two mRNA isoforms differing in exon 1. Analyses of the nucleotide sequences of the 16 exons including the coding region of CMAH examined in the 34 type B cats and in the family of type AB cats carried the CMAH variants, and revealed multiple novel diplotypes comprising several polymorphisms. Haplotype inference, which was focused on non-synonymous SNPs revealed that eight haplotypes carried one to four mutations in CMAH, and all cats with type B (n = 34) and AB (n = 2) blood carried two alleles derived from the mutated CMAH gene. These results suggested that double haploids selected from multiple recessive alleles in the cat CMAH loci were highly associated with the expression of the Neu5Ac on erythrocyte membrane in types B and AB of the feline AB blood group system.

Fig 1. Structure of cat’s CMAH gene.

(A) Genomic structure of cat’s CMAH gene obtained from the comparative analysis of mRNA sequences and whole genome shotgun sequence (Accession No.NC_018727.2, chromosome B). Exon 1a is located upstream of exon 1b. CMAH 1a mRNA corresponds to the mRNA sequences deposited in Genbank under Accession No. EF127684.1 from blood type A cat, while CMAH 1b mRNA corresponds to first presorted cat CMAH mRNA from blood type A cat [13]. □: Exon(UTR), ■: Exon(CDS), ▬: Intron. (B) Nucleotide sequences of the 5’ region of two mRNA isoforms, which have different exons (exon 1a and exon 1b) with start codon ATG and are considered leader exons Both exons were spliced to exon 2 to produce the full length cat CMAH mRNA isoforms 1a (CMAH 1a) and 1b (CMAH 1b).

Fig 2. Schematic representation of the DNA polymorphisms detected in the CMAH gene in cat.

□: Exon(UTR) ■: Exon(CDS) ▬: Intron: _ SNP. The position of identified DNA polymorphism was numbered from nucleotide A of the initiator methionine ATG codon revealed in exon 1a.

Fig 3. Relationships among cat’ CMAH gene haplotypes.

The relationships among haplotypes were based on the haplotype networks obtained in TCS software [51]. Each circle is proportional to the haplotype frequency. Each small white circle represents an intermediate non-sampled or non-existent haplotype. Each line within the CMAH network represents a mutation.

Fig 4. Conceptual scheme of the molecular basis of the feline AB blood group system.

□: CMAH mutated alleles (recessive), ■: CMAH intact allele (dominant), color boxes: haplotypes revealed in types B and AB within cats expressing Neu5Ac. These results suggested that double haploids selected from multiple recessive alleles in cat’s CMAH loci were highly associated with the determination of blood types B and AB in the feline AB blood group systems.