A Novel Variant in CMAH Is Associated with Blood Type AB in Ragdoll Cats

The enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase is associated with the production of sialic acids on cat red blood cells. The cat has one major blood group with three serotypes; the most common blood type A being dominant to type B. A third rare blood type is known as AB and has an unclear mode of inheritance. Cat blood type antigens are defined, with N-glycolylneuraminic acid being associated with type A and N-acetylneuraminic acid with type B. Blood type AB is serologically characterized by agglutination using typing reagents directed against both A and B epitopes. While a genetic characterization of blood type B has been achieved, the rare type AB serotype remains genetically uncharacterized. A genome-wide association study in Ragdoll cats (22 cases and 15 controls) detected a significant association between blood type AB and SNPs on cat chromosome B2, with the most highly associated SNP being at position 4,487,432 near the candidate gene cytidine monophospho-N-acetylneuraminic acid hydroxylase. A novel variant, c.364C>T, was identified that is highly associated with blood type AB in Ragdoll cats and, to a lesser degree, with type AB in random bred cats. The newly identified variant is probably linked with blood type AB in Ragdoll cats, and is associated with the expression of both antigens (N-glycolylneuraminic acid and N-acetylneuraminic acid) on the red blood cell membrane. Other variants, not identified by this work, are likely to be associated with blood type AB in other breeds of cat.


Introduction
Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) is associated with the production of the sialic acids present on cat erythrocytes [1].CMAH converts N-acetylneuraminic acid (NeuAc) to N-glycolylneuraminic acid (NeuGc) by substituting one of the hydrogen atoms in the methyl moiety with a carboxyl group [2,3].Feline CMAH was the first gene shown to control blood types in non-primate mammals [4].The gene is non-functional in humans [5,6], New World monkeys [7], birds and reptiles [8].The ability to synthesize NeuGc was lost in hominids ~3 million years ago when an Alu element disrupted exon 6 of CMAH [5], and ~30 million years ago in the New World monkeys, through an inversion of exons 4 to 13 [7].Loss of function of genes involved in membrane glycan synthesis is often an evolutionary defense mechanism to combat pathogen cell invasion [9,10], such as in the case of the malaria parasite Plasmodium falciparum, which prefers to bind to NeuGc [11,12].By inactivating CMAH, the NeuAc precursor accumulates and NeuGc is absent on the erythrocyte cell surface, thus reducing parasite infection in humans and promoting a positive selection during evolution, which results in the enrichment of "resistant" blood type antigens.
The cat has one major blood group with three serotypes; the most common being blood type A that is dominant to type B. A third rare blood type is known as AB [13], and from breeding experiments has an unclear mode of inheritance due to apparently different inheritance patterns in different breeds [14].Cat blood type antigens are defined, NeuGc is associated with type A and NeuAc with type B. Blood type AB is characterized by agglutination with antibodies against both NeuGc and NeuAc [1].In a previous study, Bighignoli et al. (2007) demonstrated that the genomic structure of cat CMAH was similar to the homologous gene in other species [4].The cat AB blood group system resulted from variant(s) in CMAH that prevented the conversion of NeuAc to NeuGc, hence type B to type A. Several variants, including SNPs upstream of the start codon, a deletion in the 5' UTR of exon 1, and three variants in the coding region were concordant with A and B blood types in 18 different breeds of cat.Moreover, in the same study, the cats that tested positive for both NeuGc and NeuAc (type AB) could not be genetically differentiated from blood type A cats [4], hence blood type AB remained genetically uncharacterized.Note that the numbering in the original Bighignoli  The frequencies of the different blood types vary among cat breeds and random bred populations with different origins.Several studies of cats from different continents confirmed blood type A to be the most common with a prevalence from 73% in Australia [13], 88.2% in China [15] to 95.3% in the United States West coast and 99.7% in United States Northeast [16].Blood type B varies from 0.3% to 33% [16][17][18][19] and type AB is the rarest (0.2% to 20%) [13,20,21].Blood type AB is common in Ragdolls with a frequency of 20% [22], Devon Rex (1.4%),British shorthair (1.6%) and particular random bred populations.In Japan, the proportion of blood type AB in the random bred population is 9.7% [23], 6.8% in Portugal [24], 5% in the UK [20], 2.3% in Brazil [25], 1.6% in Australia [26], and less than 0.2% in Canada [27] and the USA [14].Whilst founder effects can explain the variation in frequency between breeds, and are probably the cause of the high percentage of type AB in Ragdolls, the variation between random bred populations strongly suggests an evolutionary enrichment of one blood type over another.
Cats possess naturally occurring antibodies against the blood antigen they lack [13,28,29].Blood type B cats have strong anti-A alloantibodies [13,28] and blood type A cats have weak anti-B alloantibodies [28], while blood type AB cats have no alloantibodies since they express both antigens.Adverse reactions (such as vomiting, pyrexia, hemolysis, electrolyte disturbances, circulatory overload and urticarial) are observed when transfusing type A blood to a B recipient.A mild transfusion reaction can be observed if a type A recipient receives type B blood.However, if the type A cat has been previously exposed to type B blood, such as during pregnancy, serious transfusion reactions can occur, although these tend to be less severe and are less common than when type A blood is given to a type B cat. [16,29].Blood type AB cats may develop a hemolytic reaction after a type B blood transfusion (due to strong anti-A alloantibodies in the blood donor) [14], hence transfusing type AB or type A blood is recommended.The lack of a feline universal blood donor requires that all cats must be blood typed and crossmatched before any transfusion and/or transplant.The characterization of blood type AB, and subsequent development of a genetic test, will increase the precision of genetic blood typing and help breeding programs.Moreover, once the type AB genetic test is developed, a substantial screening of blood types can be easily conducted and will allow the prevalence in different breeds and populations to be determined, offering insights into natural selection in different environmental conditions and resistance to pathogens associated with certain blood types.This study presents the genetic investigation of feline blood type AB in Ragdoll cats and confirms the power of the feline SNP array in detecting a phenotype-locus association.A genomewide association study showed the blood type AB locus to be in close proximity to CMAH, which has previously been shown to control blood types A and B. Detailed investigation and screening of hundreds of cats supports the characterization of a novel feline blood type variant in Ragdoll cats.

Genome-wide association study
The datasets supporting the results are included within additional supplementary files (including S1 and S2 Datasets).
The Ragdoll cases were selected by pedigree analysis to have minimal relationship to the controls, and 37 cats, including 22 cases (blood type AB) and 15 controls (blood type A, Ab or B), were submitted for SNP array genotyping (S1 Dataset, S2 Dataset).Testing for stratification revealed that four cats (one control and three cases) had a P^> 0.35, suggesting familiar relationships within the sample cohort.These samples were excluded from the genome-wide association study to reduce inflation of the results, leaving 19 cases and 14 controls for analysis.After the exclusion of the related samples, the genomic inflation (λ) was reduced from 1.72 to 1.50.Testing for stratification by multi-dimensional scaling (MDS) revealed that the cases and controls formed one main overlapping cluster, thus no additional samples were excluded (Fig 1A).
After initial quality control of the 62,897 SNPs on the array, 47,239 markers were included in the analysis; 15,557 were excluded for a minor allele frequency < 0.05 and 101 SNPs failed genotype frequency > 20% across all the samples.By analyzing 19 cases and 14 controls, a significant association was detected with SNPs on cat chromosome B2 (Fig 1B), and correcting P raw values for multiple hypotheses testing by permutation, two adjacent SNPs on chromosome B2 at positions 4,487,432 and 4,449,822 respectively (S1 Table ), were genome-wide significant (P genome = 0.00001 and P genome = 0.004) and remained significant after Bonferroni correction (1.8e -6 and 0.0097).The two SNPs are in linkage disequilibrium with the phenotype, the most common haplotype having a frequency of 79% in the cases and 10% in the controls, defining a short (~40 Kb) haplotype block.The haplotype block is in the intergenic region between two genes: the leucine-rich repeat-containing protein 16A isoform 2 (LRRC16A) located ~50 Kb upstream of the haplotype block and CMAH, which is located ~40 Kb downstream.Since CMAH has been shown to control feline blood types A and B, this gene was further examined for cDNA and genomic variants that might be responsible for blood type AB.The c.327A>C variant was unique to the blood type B random bred cat.Two of the three synonymous mutations (c.1158T>C, c.1269A>G) were found exclusively in the blood type A random bred (from reference sequence EF127684.1),while c.1398G>T was found in the type A and B random bred cats and was not present in the type AB phenotype cats (Table 1).

CMAH cDNA and genomic DNA comparison
The (*) indicates SNPs that were previously reported by

CMAH variant genotyping
The newly identified c.364C>T variant in exon 4 was genotyped in 280 cats with known blood types defined by serology and was found in 89 of 115 type AB cats with at least one allele coding for blood type A. The variant was only detected in Ragdoll and random bred cats.Four type AB Ragdoll and 13 type AB random bred cats were wild-type for c.364C>T.The variant was not associated with blood type AB in Devon rex (n = 8) and British shorthair (n = 1) cats and was never found in blood type A or B cats (n = 164) (Table 2).
Other CMAH variants were also investigated in the 280 cats: the 18 bp deletion within the CMAH 5'UTR, the exon 2 c.142G>A and the exon 2 c.139C>T (LVS cats only) (S2 Table ).In seven cats, the c.142G>A was associated with the blood type confirmed by serology while the deletion was discordant.For two cats, both variants were discordant and suggested a serology mistype.For one cat, the deletion was concordant with the blood type, suggesting the missense mutation (c.142G>A) as more likely causal for blood type B, although an error in blood serotyping cannot be excluded.
To determine the allele frequency of the c.364C>T variant, 786 cats from many breeds with unknown blood type were genotyped for the newly identified variant, as well as the 5' UTR deletion and the c.142G>A missense mutation.The c.364C>T variant was only detected in the Ragdoll, random bred and Cornish rex cats; the frequency of the T allele in these breeds was 8.8%, 10% and 2.4%, respectively (Table 3).Cats with the c.364C>T variant were genotyped as AB/AB, AB/A or AB/b.In the 786 cats tested, 53 (6.7%) showed a discrepancy between the 5' UTR deletion and the c.142G>A variant (S4 Table ).
Since the c.364C>T variant was not concordant in all blood type AB cats, genomic analysis of CMAH was conducted on two type AB Devon rex, one type AB British shorthair, one type AB random bred cat and one type A random bred cat that were wild-type for the c.364C>T variant.Twelve variants were detected: seven in the coding region, one in the 5' UTR and four in intronic regions.Five novel variants were identified, four located in introns (I3 c.307+-22A>G, I9 c.1112+29T>G, I9 c.1112+66G>A, I9 c.1113-70G>A) and one silent mutation in the coding region, (c.884G>A).Only the variant localized 70 bp upstream of exon 10 (I9 c.1113-70G>A), was concordant with blood type AB, but was not predicted to disrupt any conserved splice site regions and was not investigated in detail (S3 Table ).

Computer modeling of feline CMAH protein structure and c.142G>A (V48M)
Templates with known tertiary structure were identified for two regions of the CMAH protein sequence (from residues 21 to 94 and from 207 to 294) using PSI-BLAST [30] with an e-value threshold of 0.005.For the first region (residues 21 to 94), the top template is a Rieske iron-sulfur protein 2PQZ, which covers the first variant site of interest (c.142G>A,V48M).Iron-sulfur [Fe-S] clusters are ubiquitous and required to sustain central life processes.Fe-S clusters participate in electron transfer, substrate binding/activation, gene expression regulation and enzyme

Discussion
Sialic acids are a family of sugars that share a nine carbon backbone and are typically found on cell surfaces and glycan molecules.About 40 sialic acids are documented with the most common being NeuAc, which is a substrate for the synthesis of NeuGc by CMAH.These two sialic acids are the major determinants of feline blood types A and B. A third, rare and genetically uncharacterized blood type (AB) also exists with differing frequencies amongst cat populations.
The A/B blood group system is clinically important in cats because mismatched breeding and transfusions can cause life threatening hemolytic reactions without prior sensitization [14].Therefore, a complete genetic characterization of all feline blood types is crucial so that genetic testing can accurately report an individual's blood type.
Recent advances in feline genomics, such as a new genome assembly [32] and the availability of a high density SNP array chip, have accelerated the discovery of several genetic diseases [33][34][35] and phenotypic traits [36,37] in the domestic cat.The Ragdoll is a relatively new breed with western origins (California, USA) [38,39] and characterized by point coloration and long hair [40,41].Ragdolls are not significantly related to any other breed.In a previous study, Alhaddad et al. (2013) [42] reported that a recently developed breed might have longer haplotype blocks, enabling genome-wide association studies with fewer individuals [36,37].Strong selection for fewer aesthetic traits and a breeding program dependent on very few founders results in high levels of LD and long haplotype blocks, characteristic of identity by descent traits.In this study, using only 33 individuals, an association of blood type AB with a variant on cat chromosome B2 in the Ragdoll breed was detected.Likely, during the Ragdoll breed development, a cat with the AB phenotype was extensively used in the breeding program, introducing the variant into the breed with a high frequency.
Due to the overall low prevalence of blood type AB, it was unclear whether CMAH was the locus associated with the AB blood type phenotype, or whether there were different loci involved, in fact, the possibility of cis-acting alleles was previously suggested [13].Griot-Wenk et al. [14] presented strong evidence that type AB was allelic, but the presence of a third allele could not explain blood type inheritance in British shorthair and Somali cats, additionally, the inheritance of blood type AB was never studied in Ragdoll cats, hence the need for an association study.The GWAS indicated that the locus influencing the rare blood type AB resided on cat chromosome B2, within one of two genes: LRRC16A and CMAH.Since LRRC16A is responsible for the inhibition of actin filament capping, which results in enhancing actin polymerization [43], CMAH remained the top candidate gene for the blood type AB phenotype, given that the gene is the major locus controlling blood types A and B in cats [4].Although Bighignoli et al. [7] included a type AB Siamese cat in their study they were not able to identify a causal variant for blood type AB.Furthermore, Ragdoll cats were not included in the previous study and their CMAH CDS was only determined in the current study.In this study, a novel variant in CMAH exon 4 (c.364C>T) that causes a proline to serine amino acid substitution at position 122 (p.122P>S) was identified.Proline is the only amino acid where the side chain is connected to the protein backbone twice and is often found in very tight turns in protein structures.The proline side chain is non-reactive and is rarely involved in a protein's active or binding sites, while serine is quite common in protein functional centers and is fairly reactive.
Prolines are considered important in protein structure, hence this substitution might be associated with an abnormal protein conformation, resulting in a partial or aberrant enzyme function.The newly identified variant is highly associated with blood type AB in Ragdoll cats, with only four of the 58 blood type AB Ragdoll cats being homozygous for the wild-type exon 4 allele.In these discordant cats, another variant at a second locus or at the same locus in the non-coding sequence may be the cause of their blood serotype or it could be due to a database or sampling error.Alternatively, their blood phenotype could be incorrect due to inaccurate serotyping or a change in blood type serology due to sickness.A previous study showed that humans absorb a portion of ingested NeuGc and incorporate a small amount into newly synthesized glycoconjugates [44,45].Julien et al. [46] and Mortezai et al. [47] demonstrated that the presence of NeuGc and NeuAc improves local inflammatory responses, hence causing a temporary variation in blood serology results.
Feline blood typing can be commercially performed using several methods including a card that uses monoclonal antibodies [48], a matrix gel column containing serum or lectin [49], a slide and tube assay that uses lectin and anti-A sera [50] and a commonly used typing kit based on immunochromatographic diffusion of red blood cells [51], which has recently been shown to outperform the card method with 100% specificity and sensitivity in anemic and non-anemic cats [52].Seth et al. (2011) [51] compared feline blood typing methods and showed that agreement between the methods varied from 99.4% to 91.4% when compared to the tube assay.Therefore, potentially the four discordant Ragdoll cats submitted as blood type AB, were actually type A or B [51].In the domestic cat population, the newly identified variant was found in 73% of blood type AB cats, and was absent in type AB cats from the British shorthair and Devon rex breeds.Direct sequencing of the CMAH CDS in Devon rex and British shorthair cats with blood type AB did not suggest any previously undetected missense or splice site disrupting variants, suggesting blood type AB is controlled by a different locus, or a variant in the same locus in a non-coding region, in these breeds.
Both NeuGc and NeuAc sialic acids appear to be present at low levels on the red cell surface of blood type AB cats [1].The presence of both antigens on type AB cats' erythrocytes can be explained by a reduction in CMAH activity.Several studies showed that the amount of NeuGc incorporated on the cell membrane is regulated by the level of hydroxylase activity [53,54], hence the newly identified c.364C>T variant may reduce CMAH activity.Tasker et al. [20] reported a previously unknown c.136C>T (numbered c.139C>T herein) variant associated with feline blood type B; potentially every novel variant that knocks-out CMAH activity could be associated with blood type B. By comparison, all variants that reduce CMAH activity may be associated with blood type AB, suggesting the possibility of more, as yet unidentified, variants associated with feline blood types and the need for further molecular investigation.A three-dimensional prediction of the effect of the c.142G>A (V48M) missense mutation associated with blood type B suggests that CMAH is still capable of binding the substrate (since the substitution does not disrupt the [Fe-S] domain) but, since NeuGc is not synthesized, the enzyme activity must be compromised by the variant [1].Unfortunately, no insights were gained from the protein structure prediction on the effects of the substitution associated with blood type AB, mainly due to the lack of a viable protein template for the region.
The AB serotype is allelic to A and B in cats, and the variant associated with type AB resides on the enzymatically active type A allelic background, and is likely associated with a reduction of CMAH activity and the presence of both sialic acids on the red cell surface.A three allele system is proposed: A > a AB > b, where a type A cat may have AA, Aa AB or Ab genotype.The type B cats are always homozygous bb.Possible genotypes / phenotypes (serotypes) would be AA (type A); Aa AB (type A); Ab (type A); a AB b (type AB); a AB a AB (type AB), and bb (type B).Whilst the majority of blood type AB cats in the study were a AB b or a AB a AB a significant number (n = 27) were genotype Aa AB , and would be predicted to be blood type A. This apparent discordancy could be explained by a second, yet to be identified, type AB variant on the A allele, and is not an unexpected finding since other breeds with blood type AB were shown not to have the c.364C>T variant.An in-depth molecular investigation of samples with discrepancies between genotype (c.142G>A, c.364C>T) and blood phenotype is needed in future studies to detect other alleles involved in feline blood types.

Conclusion
This study identified a variant associated with the rare feline AB blood type in Ragdoll cats.A genome-wide association study suggested CMAH as a candidate gene and a newly identified c.364C>T missense mutation is probably linked with the feline AB blood type.Moreover, the variant is likely associated with a reduction of CMAH enzyme activity and the resultant expression of both antigens (NeuGc and NeuAc) on the red cell membrane.Since blood type AB cats from other breeds did not have the Ragdoll type AB variant, at least one other variant must exist that also results in a type AB phenotype.

Ethics statement
This study was approved by the Animal Care and Use Committee (ACUC) of the University of Davis (protocol # 16991) and the University of Missouri (protocol # 7808).Italian samples were collected with the informed consent of owners during routine wellness visits.Several samples were acquired by specialist in the field, such as a veterinarian, or voluntarily donated by owners and breeders with consent to use the specimens for research purposes.

Sample collection
European and USA cat owners and breeders voluntarily donated EDTA anti-coagulated whole blood or buccal swabs from blood type A, B and AB cats (n = 134).Additionally, 138 DNA samples from cats blood typed by hemagglutination and alloantibody assays [13] were also provided by several laboratory services:  2).
The Veterinary Genetic Laboratory (VGL) at the University of California, Davis, genotyped 786 samples with unknown blood serology/phenotypes, from several different breeds and random bred cats (S4 Table ).
DNA from 37 Ragdolls (22 type AB and 15 type A, Ab or B) were submitted to Geneseek Inc. (Lincoln, NE) for genotyping analysis on the array.DNA extraction and quality control was conducted as previously described [33].Supplementary files contain the genotyping data (S1 Dataset) and the location of each SNP typed on the array (S2 Dataset).Blood from four Ragdoll type AB, one random bred type AB and a random bred type B was also collected in PAXgene TM (Qiagen, Valencia, CA) tubes for RNA extraction.identity and the top three templates analyzed.Finally, using the 3-dimensional structure of the top ranked protein as a template in conjunction with the sequence alignment, a 3-dimensional structure for the wild-type CMAH protein was built, for the regions for which templates were found, using Modeller [60].The structural model of the wild-type protein was then used as a template to construct the structural model of the mutated protein.
et al. paper is mis-aligned by 3 bases compared to the position in the feline coding sequence (CDS).Positions are updated in the current work, hence the original c.139G>A variant is actually at position c.142 of the CMAH CDS.
Sequence data for CMAH cDNA was derived from six cats: four Ragdoll blood type AB, one random bred blood type AB, one random bred type B (serology B, genotype bb) and compared to the publicly available random bred type A (serology A, genotype AA) cDNA sequence (EF127684.1).Sequence analyses of the coding region identified nine cDNA variants, including five missense mutations.Four of the nine variants had previously been identified by Bighignoli et al. (2007), thus five CMAH variants (c.327A>C, c.364C>T, c.1158T>C, c.1269A>G, c.1398G>T) were novel to this study (Table 1, S1 Fig).Note that in Table 1, SNP positions within the CMAH CDS have been updated.Only the c.364C>T variant in exon 4, predicted in silico to change a nonpolar and hydrophobic amino acid (proline) to a polar and uncharged residue (serine) at codon 122, was present in the 5 type AB cats and absent in the type A and B cats.The c.364C>T variant was further investigated by genomic analysis in 280 cats with known blood type (see below).

Fig 1 .
Fig 1. Multidimensional scaling and Manhattan plot summarizing the GWAS for Ragdoll cats with blood type AB.a. MDS showing the distribution of the Ragdoll cases and controls included in the analyses.Significant overlap of cases and controls suggests minimal substructuring in the dataset.b.The plot represents the P raw (top) and P genome (bottom) values of the SNPs included in the GWAS case-control analysis.The black dashed line (bottom) suggests the SNPs with genome-wide significance after permutation.Two SNPs on chromosome B2 (positions 4,487,432 and 4,449,822, respectively) remained significantly associated with the AB blood type phenotype.doi:10.1371/journal.pone.0154973.g001 The exon 2 c.142G>A and c.139C>T variants were originally numbered c.139G>A and c.136C>T in Bighignoli et al. (2007) and Tasker et al. (2014), respectively.The 18 bp deletion and c.142G>A variant, previously associated with feline blood type B, were found on the feline B allele, confirming the findings from the Bighignoli et al. (2007) study.However, nine cats with known blood type A or B serology showed a discrepancy between the deletion and the c.142G>A variant.

activity [ 31 ]
. The template (S2 Fig) shows that the metal-ion binding site is spatially distant from the V48M variant.The other template covering the second region of the CMAH sequence (residues 207 to 294, S2 Fig) is part of the complex of a large ribosomal subunit from Deinococus radiodurans (3JQ4) with a zinc binding site.The 3-D protein structure surrounding the novel P122S variant could not be modeled due to the lack of an available 3-D template.

Table 1 .
Comparison of CMAH cDNA variants in cats with blood types A, B and AB.