Melanocortin-4 receptor and proopiomelanocortin: Candidate genes for obesity in domestic shorthair cats.

Obesity is an escalating global health problem affecting both humans and companion animals. In cats it is associated with increased mortality and multiple diseases, including diabetes mellitus. Two genes coding for proteins known to play a critical role in energy homeostasis across species are the proopiomelanocortin (POMC) gene and the melanocortin-4 receptor (MC4R) gene. A missense variant in the coding sequence of the feline MC4R (MC4R:c.92C>T) has been reported to be associated with diabetes and overweight in domestic shorthair cats, and while variants in the POMC gene are known to cause obesity in humans and dogs, variants in POMC and their association with feline obesity and diabetes mellitus have not been investigated to date. The current study aimed to assess the association between the previously described MC4R variant and body condition score (BCS), as well as body fat content (%BF) in 89 non-diabetic domestic shorthair cats. Furthermore, we investigated the feline POMC gene as a potential candidate gene for obesity. Our results indicate that the MC4R:c.92C>T polymorphism is not associated with BCS or %BF in non-diabetic domestic shorthair cats. The mutation analysis of all POMC exons identified two missense variants, with a variant in exon 1 (c.28G>C; p.G10R) predicted to be damaging. The variant was subsequently assessed in all 89 cats, and cats heterozygous for the variant had a significantly increased body condition score (p = 0.03) compared with cats homozygous for the wild-type allele. Results from our study provide additional evidence that the previously described variant in MC4R is not associated with obesity in domestic shorthair cats. More importantly, we have identified a novel variant in the POMC gene, which might play a role in increased body condition score and body fat content in domestic shorthair cats.

Canine coat pigmentation genetics: a review.

Our understanding of canine coat colour genetics and the associated health implications is developing rapidly. To date, there are 15 genes with known roles in canine coat colour phenotypes. Many coat phenotypes result from complex and/or epistatic genetic interactions among variants within and between loci, some of which remain unidentified. Some genes involved in canine pigmentation have been linked to aural, visual and neurological impairments. Consequently, coat pigmentation in the domestic dog retains considerable ethical and economic interest. In this paper we discuss coat colour phenotypes in the domestic dog, the genes and variants responsible for these phenotypes and any proven coat colour-associated health effects.

Roan, ticked and clear coat patterns in the canine are associated with three haplotypes near usherin on CFA38.

White coat patterning is a feature of many dog breeds and is known to be coded primarily by the gene micropthalmia-associated transcription factor (MITF). This patterning in the coat can be modified by other factors to produce the attractive phenotypes termed 'ticked' and 'roan' that describe the presence of flecks of color that vary in distribution and intensity within otherwise 'clear' white markings. The appearance of the pigment in the white patterning caused by ticking and roaning intensifies in the weeks after birth. We applied genome-wide association to compare English Cocker Spaniels of roan phenotype (N = 34) with parti-color (non-roan) English Cocker Spaniels (N = 9) and identified an associated locus on CFA 38, CFA38:11 057 040 (Praw  = 8.9 × 10-10 , Pgenome  = 2.7 × 10-5 ). A local case-control association in English Springer Spaniels comparing 11 ticked and six clear dogs identified indicative association with a different haplotype, CFA38:11 122 467G>T (Praw  = 1.7 × 10-5 ) and CFA38:11 124 294A>C (Praw  = 1.7 × 10-5 ). We characterize three haplotypes in Spaniels according to their putative functional variant profiles at CFA38:11 111 286C>T (missense), CFA38:11 131 841-11 143 239DUP.insTTAA (using strongly linked marker CFA38:11 143 243C>T) and CFA38:11 156 425T>C (splice site). In Spaniels, the haplotypes work as an allelic series including alleles (t, recessive clear; T, dominant ticked/parti-color; and TR , incomplete dominant roan) to control the appearance of pigmented spots or flecks in otherwise white areas of the canine coat. In Spaniels the associated haplotypes are t (CCT), T (TCC) and TR (TTT) for SNP markers on CFA38 at 11 111 286C>T, 11 143 243C>T and 11 156 425T>C respectively. It is likely that other alleles exist in this series and together the haplotypes result in a complex range of patterning that is only visible when dogs have white patterning resulting from the epistatic gene Micropthalmia-associated transcription factor (the S-locus).

The Burmese cat as a genetic model of type 2 diabetes in humans.

The recent extension of genetic tools to the domestic cat, together with the serendipitous consequences of selective breeding, have been essential to the study of the genetic diseases that affect them. Cats are increasingly presented for veterinary surveillance and share many of human's heritable diseases, allowing them to serve as natural models of these conditions. Feline diabetes mellitus is a common condition in domestic cats that bears close pathological and clinical resemblance to type 2 diabetes in humans, including pancreatic ß-cell dysfunction and peripheral insulin resistance. In Australia, New Zealand and Europe, diabetes mellitus is almost four times more common in cats of the Burmese breed than in other breeds. This geographically based breed predisposition parallels familial and population clustering of type 2 diabetes in humans. As a genetically isolated population, the Australian Burmese breed provides a spontaneous, naturally occurring genetic model of type 2 diabetes. Genetically isolated populations typically exhibit extended linkage disequilibrium and increased opportunity for deleterious variants to reach high frequencies over many generations due to genetic drift. Studying complex diseases in such populations allows for tighter control of confounding factors including environmental heterogeneity, allelic frequencies and population stratification. The homogeneous genetic background of Australian Burmese cats may provide a unique opportunity to either refine genetic signals previously associated with type 2 diabetes or identify new risk factors for this disease.

A dominant TRPV4 variant underlies osteochondrodysplasia in Scottish fold cats.

OBJECTIVE: Scottish fold cats, named for their unique ear shape, have a dominantly inherited osteochondrodysplasia involving malformation in the distal forelimbs, distal hindlimbs and tail, and progressive joint destruction. This study aimed to identify the gene and the underlying variant responsible for the osteochondrodysplasia. DESIGN: DNA samples from 44 Scottish fold and 54 control cats were genotyped using a feline DNA array and a case-control genome-wide association analysis conducted. The gene encoding a calcium permeable ion channel, transient receptor potential cation channel, subfamily V, member 4 (TRPV4) was identified as a candidate within the associated region and sequenced. Stably transfected HEK293 cells were used to compare wild-type and mutant TRPV4 expression, cell surface localisation and responses to activation with a synthetic agonist GSK1016709A, hypo-osmolarity, and protease-activated receptor 2 stimulation. RESULTS: The dominantly inherited folded ear and osteochondrodysplasia in Scottish fold cats is associated with a p.V342F substitution (c.1024G>T) in TRPV4. The change was not found in 648 unaffected cats. Functional analysis in HEK293 cells showed V342F mutant TRPV4 was poorly expressed at the cell surface compared to wild-type TRPV4 and as a consequence the maximum response to a synthetic agonist was reduced. Mutant TRPV4 channels had a higher basal activity and an increased response to hypotonic conditions. CONCLUSIONS: Access to a naturally-occurring TRPV4 mutation in the Scottish fold cat will allow further functional studies to identify how and why the mutations affect cartilage and bone development.

Two variants in the KIT gene as candidate causative mutations for a dominant white and a white spotting phenotype in the donkey.

White spotting phenotypes have been intensively studied in horses, and although similar phenotypes occur in the donkey, little is known about the molecular genetics underlying these patterns in donkeys. White spotting in donkeys can range from only a few white areas to almost complete depigmentation and is characterised by a loss of pigmentation usually progressing from a white spot in the hip area. Completely white-born donkeys are rare, and the phenotype is characterised by the complete absence of pigment resulting in pink skin and a white coat. A dominant mode of inheritance has been demonstrated for spotting in donkeys. Although the mode of inheritance for the completely white phenotype in donkeys is not clear, the phenotype shows similarities to dominant white in horses. As variants in the KIT gene are known to cause a range of white phenotypes in the horse, we investigated the KIT gene as a potential candidate gene for two phenotypes in the donkey, white spotting and white. A mutation analysis of all 21 KIT exons identified a missense variant in exon 4 (c.662A>C; p.Tyr221Ser) present only in a white-born donkey. A second variant affecting a splice donor site (c.1978+2T>A) was found exclusively in donkeys with white spotting. Both variants were absent in 24 solid-coloured controls. To the authors' knowledge, this is the first study investigating genetic mechanisms underlying white phenotypes in donkeys. Our results suggest that two independent KIT alleles are probably responsible for white spotting and white in donkeys.

Shifted inferior frontal laterality in women with major depressive disorder is related to emotion-processing deficits.

BACKGROUND: Facial emotion perception (FEP) is a critical human skill for successful social interaction, and a substantial body of literature suggests that explicit FEP is disrupted in major depressive disorder (MDD). Prior research suggests that weakness in FEP may be an important phenomenon underlying patterns of emotion-processing challenges in MDD and the disproportionate frequency of MDD in women. Method Women with (n = 24) and without (n = 22) MDD, equivalent in age and education, completed a FEP task during functional magnetic resonance imaging. RESULTS: The MDD group exhibited greater extents of frontal, parietal and subcortical activation compared with the control group during FEP. Activation in the inferior frontal gyrus (IFG) appeared shifted from a left >right pattern observed in healthy women to a bilateral pattern in MDD women. The ratio of left to right suprathreshold IFG voxels in healthy controls was nearly 3:1, whereas in the MDD group, there was a greater percentage of suprathreshold IFG voxels bilaterally, with no leftward bias. In MDD, relatively greater activation in right IFG compared with left IFG (ratio score) was present and predicted FEP accuracy (r = 0.56, p < 0.004), with an inverse relationship observed between FEP and subgenual cingulate activation (r = - 0.46, p = 0.02). CONCLUSIONS: This study links, for the first time, disrupted IFG activation laterality and increased subgenual cingulate activation with deficient FEP in women with MDD, providing an avenue for imaging-to-assessment translational applications in MDD.

Overweight and impaired insulin sensitivity present in growing cats.

Obesity is a growing problem in pets as well as in humans. Overweight and obesity are linked to insulin sensitivity and subsequently in older cats, to an increased risk of developing diabetes mellitus. In the experimental cat population of the Institute of Animal Nutrition of the Vetsuisse Faculty, University of Zurich, an overweight phenotype in intact cats younger than 1 year became evident. The aims of the present study were to determine whether an association between insulin sensitivity and body condition score (BCS) or feline body mass index (FBMI) is already present during young adulthood in these cats and to test the hypothesis that the phenotype lean/overweight is significantly associated with monthly body weight during the growing period. Therefore, 41 kittens from the mentioned cat breeding colony were studied. They were weighed weekly and checked monthly (third to eighth month after birth) for BCS and FBMI. At the age of 8 months, they were classified into an overweight and lean phenotype based on BCS on a scale of 9 (median; maximum and minimum: overweight male (6.4; 6.8; 6.0); overweight female (6.1; 6.2; 6.0); lean male (5.4; 5.7; 5.0); lean female (5.2; 5.6; 5.0). A significant association between the phenotype and body weight was obvious during the growing period from the third to the 8 months (p = 0.0001). At month 8, body fat content was measured by dual energy X-ray absorptiometry and a glucose tolerance test to determine the insulin sensitivity index was performed. Insulin sensitivity was significantly associated with BCS (p = 0.0007) and body fat content (p < 0.0001) but not with sex (p = 0.61). Our data provide evidence that already in young intact cats; insulin insensitivity is significantly associated with BCS or a presumed phenotype lean/overweight.

[Clinical evaluation of the new coat colour macchiato in a male Franches-Montagnes horse]. / Klinische und genetische Befunde bei einem männlichen Freiberger mit der neuen Fellfarbe Macchiato.

In April 2008 a Franches-Montagnes colt was born with an unusual coat colour phenotype which had never been observed in that population before. The foal showed extended white markings on body and legs, a white head and blue eyes. As both parents have an unremarkable bay coat colour phenotype, a de novo mutation was expected in the offspring and a candidate gene approach revealed a spontaneous mutation in the microphthalmia associated transcription factor gene (MITF). A detailed clinical examination in 2010 indicated an impaired hearing capacity. As in the American Paint Horse large white facial markings in combination with blue eyes are associated with deafness, the hearing capacity of the stallion was closer examined performing brainstem auditory-evoked responses (BAER). The BAER confirmed bilateral deafness in the Franches-Montagnes colt. It is assumed that the deafness is caused by a melanocyte deficiency caused by the MITF gene mutation. Unfortunately, due to castration of the horse, the causal association between the mutation in the MITF gene and clinical findings cannot be confirmed by experimental matings.

De-novo and genome-wide meta-analyses identify a risk haplotype for congenital sensorineural deafness in Dalmatian dogs.

Congenital sensorineural deafness (CSD) has been reported to affect up to 30% of Dalmatian dogs world-wide and while unilaterally deaf dogs can live a close to normal life, dogs suffering bilateral deafness are frequently euthanized. Extreme-white coat patterning as encoded by the gene Melanocyte Inducing Transcription Factor (MITF) has long been postulated as the major risk factor for CSD in the Dalmatian breed. While attempts to identify causative risk variants associated with CSD have been numerous, no genome-wide association study has positively identified MITF as a risk locus for either bilateral or unilateral deafness in the Dalmatian breed to date. In this study, we identified an association with CSD on CFA20 in the vicinity of MITF within Australian Dalmatian dogs. Although not genome-wide significant, the association signal was validated by reanalysing publicly available data and merging the wider data resource with the local data to improve statistical power. The merged data, representing three major global populations of Dalmatian dogs, enabled us to identify a single, well-defined genome-wide significant risk haplotype for CSD. The haplotype was formed by three genome-wide significant associated markers (BICF2G630233852T>C, BICF2G630233861T>C, BICF2G630233888G>A) on CFA20 with 62% of bilaterally deaf dogs homozygous for the risk haplotype (CCA), while 30% of bilaterally deaf and 45% of hearing dogs carried one copy of the risk haplotype. Animals homozygous or heterozygous for the low-risk haplotype were less likely to be unilaterally deaf. While the association between the risk haplotype and deafness is incomplete, animals homozygous for the risk haplotype were 10-times more likely to be bilaterally deaf. Although the underlying causative variants are yet to be discovered, results from this study can now assist with reducing deafness in Dalmatian dogs.

Genetic diversity in an indigenous horse breed: implications for mating strategies and the control of future inbreeding.

The Franches-Montagnes is an indigenous Swiss horse breed, with approximately 2500 foalings per year. The stud book is closed, and no introgression from other horse breeds was conducted since 1998. Since 2006, breeding values for 43 different traits (conformation, performance and coat colour) are estimated with a best linear unbiased prediction (BLUP) multiple trait animal model. In this study, we evaluated the genetic diversity for the breeding population, considering the years from 2003 to 2008. Only horses with at least one progeny during that time span were included. Results were obtained based on pedigree information as well as from molecular markers. A series of software packages were screened to combine best the best linear unbiased prediction (BLUP) methodology with optimal genetic contribution theory. We looked for stallions with highest breeding values and lowest average relationship to the dam population. Breeding with such stallions is expected to lead to a selection gain, while lowering the future increase in inbreeding within the breed.

Comparative human-horse sequence analysis of the CYP3A subfamily gene cluster.

Cytochrome P450 enzymes (CYP450s) represent a superfamily of haem-thiolate proteins. CYP450s are most abundant in the liver, a major site of drug metabolism, and play key roles in the metabolism of a variety of substrates, including drugs and environmental contaminants. Interaction of two or more different drugs with the same enzyme can account for adverse effects and failure of therapy. Human CYP3A4 metabolizes about 50% of all known drugs, but little is known about the orthologous CYP450s in horses. We report here the genomic organization of the equine CYP3A gene cluster as well as a comparative analysis with the human CYP3A gene cluster. The equine CYP450 genes of the 3A family are located on ECA 13 between 6.97-7.53 Mb, in a region syntenic to HSA 7 99.05-99.35 Mb. Seven potential, closely linked equine CYP3A genes were found, in contrast to only four genes in the human genome. RNA was isolated from an equine liver sample, and the approximately 1.5-kb coding sequence of six CYP3A genes could be amplified by RT-PCR. Sequencing of the RT-PCR products revealed numerous hitherto unknown single nucleotide polymorphisms (SNPs) in these six CYP3A genes, and one 6-bp deletion compared to the reference sequence (EquCab2.0). The presence of the variants was confirmed in a sample of genomic DNA from the same horse. In conclusion, orthologous genes for the CYP3A family exist in horses, but their number differs from those of the human CYP3A gene family. CYP450 genes of the same family show high homology within and between mammalian species, but can be highly polymorphic.

Molecular analysis of carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) as a candidate gene for bovine spongiform encephalopathy susceptibility.

Endogenous prion proteins (PrP) play the central role in the pathogenesis of transmissible spongiform encephalopathies. The carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) promotes the conversion of the cellular PrP(C) into the pathogenic PrP(d). Six sequence variants within the CHST8 gene were identified by comparative sequencing and genotyped for a sample of 623 animals comprising bovine spongiform encephalopathy (BSE)-affected and healthy control cows representing German Fleckvieh (German Simmental), German Holstein (Holstein-Friesian) and Brown Swiss. Significant differences in the allele, genotype and haplotype frequencies between BSE-affected and healthy cows indicate an association of sequence variant g.37254017G>T with the development of the disease in Brown Swiss cattle.

Seven novel KIT mutations in horses with white coat colour phenotypes.

White coat colour in horses is inherited as a monogenic autosomal dominant trait showing a variable expression of coat depigmentation. Mutations in the KIT gene have previously been shown to cause white coat colour phenotypes in pigs, mice and humans. We recently also demonstrated that four independent mutations in the equine KIT gene are responsible for the dominant white coat colour phenotype in various horse breeds. We have now analysed additional horse families segregating for white coat colour phenotypes and report seven new KIT mutations in independent Thoroughbred, Icelandic Horse, German Holstein, Quarter Horse and South German Draft Horse families. In four of the seven families, only one single white horse, presumably representing the founder for each of the four respective mutations, was available for genotyping. The newly reported mutations comprise two frameshift mutations (c.1126_1129delGAAC; c.2193delG), two missense mutations (c.856G>A; c.1789G>A) and three splice site mutations (c.338-1G>C; c.2222-1G>A; c.2684+1G>A). White phenotypes in horses show a remarkable allelic heterogeneity. In fact, a higher number of alleles are molecularly characterized at the equine KIT gene than for any other known gene in livestock species.

Genetic polymorphisms in toll-like receptors 1, 2, and 4 in feline upper respiratory tract aspergillosis.

Fungal species in the genus Aspergillus are environmental saprophytes that can act as opportunistic pathogens of the nasal cavity and paranasal sinuses in humans, cats and other species. Upper respiratory tract aspergillosis (URTA) presents as non-invasive and invasive forms with the latter occurring almost exclusively in immunocompromised hosts. However, in domestic cats, invasive URTA affects apparently immunocompetent patients. A defect in innate immunity has been proposed as a predisposing factor in invasive feline URTA. Single nucleotide polymorphisms (SNPs) in pattern recognition receptor genes have been implicated in the pathogenesis of aspergillosis in humans. The aims of this study were to identify non-synonymous SNPs in the coding regions of toll-like receptors involved in the immune response to Aspergillus spp. and to compare the frequency of these SNPs between affected and control cats. The coding and flanking regions of TLR1, TLR2 and TLR4 were sequenced in 14 cats with URTA and the sequences were compared with those in 20 control cats without aspergillosis. In total, 23 non-synonymous SNPs were identified in TLR1 (n = 11), TLR2 (n = 3) and TLR4 (n = 10). Differences in allelic frequency of non-synonymous SNPs between affected and controls were not identified either within breeds or overall or between non-invasive and invasive disease phenotypes. Although allelic frequency differed between cat breeds that are overrepresented for URTA and underrepresented breeds there was no association differences identified between affected cats and underrepresented breeds. The difference in allelic frequency of an INDEL point mutation identified in intron 1 of TLR4, between cats with non-invasive versus invasive aspergillosis approached significance (p = 0.054). While results from this study do not support a role for non-synonymous SNPs in the pathogenesis of feline URTA they do provide evidence that investigation for polymorphisms in non-coding regions of these genes and in other pattern recognition receptors are warranted.

Breed-specific variations in the coding region of toll-like receptor 4 in the domestic cat.

Specific point mutations in the human toll-like receptor 4 (TLR4) confer altered risk for diverse diseases including sepsis, aspergillosis and inflammatory bowel disease. Some of these TLR4 polymorphisms are racially specific. We hypothesised that feline TLR4 polymorphisms might underlie an observed increased risk to infectious and inflammatory diseases in some cat breeds. The aim of this study was to identify breed-specific variations in the coding region of feline TLR4 and to model the effect of mutations on protein structure and function in silico. The entire coding region of TLR4 was sequenced in 8 groups (7 pure-bred, 1 crossbred) of domestic cats (Felis catus) comprising 158 individuals. Twenty-two single nucleotide polymorphisms (SNPs) were identified in TLR4, with 16 located in the coding region (11 non-synonymous) and four in the 3'UTR. Comparison of breed specific allelic frequencies indicated that Burmese and British shorthairs most commonly differed from other breeds. In silico analyses to predict the impact of the 11 non-synonymous variants indicated a deleterious effect on protein structure for one SNP (c.869 G > A), which was not associated with a specific breed. Overall, findings from this study do not support a role of TLR4 dysfunction in breed-predispositions to infectious diseases in domestic cats in Australia.

An equine chromosome 3 inversion is associated with the tobiano spotting pattern in German horse breeds.

The tobiano white-spotting pattern is one of several known depigmentation phenotypes in horses and is desired by many horse breeders and owners. The tobiano spotting phenotype is inherited as an autosomal dominant trait. Horses that are heterozygous or homozygous for the tobiano allele (To) are phenotypically indistinguishable. A SNP associated with To had previously been identified in intron 13 of the equine KIT gene and was used for an indirect gene test. The test was useful in several horse breeds. However, genotyping this sequence variant in the Lewitzer horse breed revealed that 14% of horses with the tobiano pattern did not show the polymorphism in intron 13 and consequently the test was not useful to identify putative homozygotes for To within this breed. Speculations were raised that an independent mutation might cause the tobiano spotting pattern in this breed. Recently, the putative causative mutation for To was described as a large chromosomal inversion on equine chromosome 3. One of the inversion breakpoints is approximately 70 kb downstream of the KIT gene and probably disrupts a regulatory element of the KIT gene. We obtained genotypes for the intron 13 SNP and the chromosomal inversion for 204 tobiano spotted horses and 24 control animals of several breeds. The genotyping data confirmed that the chromosomal inversion was perfectly associated with the To allele in all investigated horses. Therefore, the new test is suitable to discriminate heterozygous To/+ and homozygous To/To horses in the investigated breeds.

Sequence analysis of the porcine IFNAR1 and IFNGR2 genes.

A porcine BAC clone harboring the tightly linked IFNAR1 and IFNGR2 genes was identified by comparative analysis of the publicly available porcine BAC end sequences. The complete 168,835 bp insert sequence of this clone was determined. Sequence comparisons of the genomic sequence with EST sequences from public databases were performed and allowed a detailed annotation of the IFNAR1 and IFNGR2 genes. The analyzed genes showed a conserved genomic organization with their known mammalian orthologs, however the sequence conservation of these genes across species was relatively low. In addition to the IFNAR1 and IFNGR2 genes, which were completely sequenced, the analyzed BAC clone also contained parts of an orphan gene encoding a putative transmembrane protein (TMEM50B). In contrast to the IFNAR1 and IFNGR2 genes the sequence conservation of the TMEM50B gene across different mammalian species was extremely high.