Maria Longeri

Highly effective SNP-based association mapping and management of recessive defects in livestock

The widespread use of elite sires by means of artificial insemination in livestock breeding leads to the frequent emergence of recessive genetic defects, which cause significant economic and animal welfare concerns. Here we show that the availability of genome-wide, high-density SNP panels, combined with the typical structure of livestock populations, markedly accelerates the positional identification of genes and mutations that cause inherited defects. We report the fine-scale mapping of five recessive disorders in cattle and the molecular basis for three of these: congenital muscular dystony (CMD) types 1 and 2 in Belgian Blue cattle and ichthyosis fetalis in Italian Chianina cattle. Identification of these causative mutations has an immediate translation into breeding practice, allowing marker assisted selection against the defects through avoidance of at-risk matings.

Extent of Linkage Disequilibrium in the Domestic Cat, Felis silvestris catus, and Its Breeds

Domestic cats have a unique breeding history and can be used as models for human hereditary and infectious diseases. In the current era of genome-wide association studies, insights regarding linkage disequilibrium (LD) are essential for efficient association studies. The objective of this study is to investigate the extent of LD in the domestic cat, Felis silvestris catus, particularly within its breeds. A custom illumina GoldenGate Assay consisting of 1536 single nucleotide polymorphisms (SNPs) equally divided over ten 1 Mb chromosomal regions was developed, and genotyped across 18 globally recognized cat breeds and two distinct random bred populations. The pair-wise LD descriptive measure (r 2) was calculated between the SNPs in each region and within each population independently. LD decay was estimated by determining the non-linear least-squares of all pair-wise estimates as a function of distance using established models. The point of 50% decay of r2 was used to compare the extent of LD between breeds. The longest extent of LD was observed in the Burmese breed, where the distance at which r2 ≈ 0.25 was ∼380 kb, comparable to several horse and dog breeds. The shortest extent of LD was found in the Siberian breed, with an r2 ≈ 0.25 at approximately 17 kb, comparable to random bred cats and human populations. A comprehensive haplotype analysis was also conducted. The haplotype structure of each region within each breed mirrored the LD estimates. The LD of cat breeds largely reflects the breeds’ population history and breeding strategies. Understanding LD in diverse populations will contribute to an efficient use of the newly developed SNP array for the cat in the design of genome-wide association studies, as well as to the interpretation of results for the fine mapping of disease and phenotypic traits.

An international parentage and identification panel for the domestic cat (Felis catus)

Seventeen commercial and research laboratories participated in two comparison tests under the auspices of the International Society for Animal Genetics to develop an internationally tested, microsatellite-based parentage and identification panel for the domestic cat (Felis catus). Genetic marker selection was based on the polymorphism information content and allele ranges from seven random-bred populations (n = 261) from the USA, Europe and Brazil and eight breeds (n = 200) from the USA. Nineteen microsatellite markers were included in the comparison test and genotyped across the samples. Based on robustness and efficiency, nine autosomal microsatellite markers were ultimately selected as a single multiplex ‘core’ panel for cat identification and parentage testing. Most markers contained dinucleotide repeats. In addition to the autosomal markers, the panel included two gender-specific markers, amelogenin and zinc-finger XY, which produced genotypes for both the X and Y chromosomes. This international cat parentage and identification panel has a power of exclusion comparable to panels used in other species, ranging from 90.08% to 99.79% across breeds and 99.47% to 99.87% in random-bred cat populations.

Comparison between ultrasound and genetic testing for the early diagnosis of polycystic kidney disease in Persian and Exotic Shorthair cats

Autosomal-dominant polycystic kidney disease (AD-PKD) is common in Persians and Persians-related breeds. The aims of this study were to evaluate the sensitivity and specificity of early ultrasound examination and to compare ultrasound and genetic testing for early diagnosis. Sixty-three Persians and seven Exotic Shorthairs were considered. All underwent ultrasonographic and genetic testing (polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) assay) between 2.5 and 3.5 months of age (10–14 weeks). With ultrasound, 41.4% showed renal cysts, while 37.1% were PKD positive by genetic testing and DNA sequencing. Six cats with at least one renal cyst were negative by genetic testing, while only one cat negative at ultrasound resulted positive at genetic test. DNA sequencing of three polycystic cats, negative by genetic test, revealed they were heterozygous for the mutation. Agreement was described by Cohens kappa that resulted 0.85, considering genetic test and DNA sequencing. Sensitivity and specificity of ultrasound were 96.2% and 91%, respectively. Sensitivity was higher and specificity lower than reported previously. The higher sensitivity could be due to improved technical capabilities of ultrasound machines and transducers. Other causes of PKD could explain the lower specificity. In conclusion, ultrasound resulted in a reliable diagnostic method for feline AD-PKD1 at early age and it should always be used with genetic testing, in order to reach a complete screening programme and eventually to identify other genetic mutations.

COLQ variant associated with Devon Rex and Sphynx feline hereditary myopathy

Summary Some Devon Rex and Sphynx cats have a variably progressive myopathy characterized by appendicular and axial muscle weakness, megaesophagus, pharyngeal weakness and fatigability with exercise. Muscle biopsies from affected cats demonstrated variable pathological changes ranging from dystrophic features to minimal abnormalities. Affected cats have exacerbation of weakness following anticholinesterase dosing, a clue that there is an underlying congenital myasthenic syndrome (CMS). A genome‐wide association study and whole‐genome sequencing suggested a causal variant for this entity was a c.1190G>A variant causing a cysteine to tyrosine substitution (p.Cys397Tyr) within the C‐terminal domain of collagen‐like tail subunit (single strand of homotrimer) of asymmetric acetylcholinesterase (COLQ). Alpha‐dystroglycan expression, which is associated with COLQ anchorage at the motor end‐plate, has been shown to be deficient in affected cats. Eighteen affected cats were identified by genotyping, including cats from the original clinical descriptions in 1993 and subsequent publications. Eight Devon Rex and one Sphynx not associated with the study were identified as carriers, suggesting an allele frequency of ~2.0% in Devon Rex. Over 350 tested cats from other breeds did not have the variant. Characteristic clinical features and variant presence in all affected cats suggest a model for COLQ CMS. The association between the COLQ variant and this CMS affords clinicians the opportunity to confirm diagnosis via genetic testing and permits owners and breeders to identify carriers in the population. Moreover, accurate diagnosis increases available therapeutic options for affected cats based on an understanding of the pathophysiology and experience from human CMS associated with COLQ variants.

Italian autochthonous chicken breeds conservation: evaluation of biodiversity in Valdarnese Bianca breed (Callus gallus domesticus).

Three fowl breeds, Valdarnese Bianca, a traditional white feathered breed from Tuscany, Golden Comet® a commercial hybrid and Livornese Bianca, a white leghorn type, were genotyped at eight microsatellite loci. A total of 74 alleles were detected with locus ADL181 recorded the lowest (six alleles) and locus ADL136 the highest (15 alleles) allele frequencies respectively. Heterozygosity values ranged from 0.210 (locus ADL210) to 0.742 (locus ADL176). The Wrights fixation index values were 0.089 (FST), 0.300 (FIs) and 0.363 (FIT). Factorial correspondence analysis and a dendrogram individual tree constructed using individual genetic distances showed genetic differentiation of the three breeds.

Persistent right aortic arch and associated axial skeletal malformations in cats

Persistent right aortic arch (PRAA) in cats is an uncommon vascular anomaly with clinical signs referable to oesophageal obstruction. To our knowledge no reports of axial skeletal malformations concomitant to PRAA have been reported in cats. The aim of this study is to depict a new clinical feature in cats affected by PRAA. In the study six cats with a diagnosis of vascular ring anomaly were enrolled. A complete physical examination, a neurological examination and a total body radiograph were performed on each animal. Four of the six cats showed contemporary PRAA and skeletal malformations. Additionally, for the first time, a genetic test was performed on one subject to detect DNA alterations in the homologous DiGeorge region of cat. The percentage of skeletal malformations reported in the normal population was compared with animals with PRAA and showed a higher frequency. Genetic testing failed to demonstrate a correlation between PRAA and DiGeorge genomic deletion. A review of veterinary and human diseases that presented both conditions was assessed. The few animals enrolled do not allow definitive conclusions. Further studies are required to corroborate the correlation between PRAA and axial skeletal malformations in cats.

Heterozygosity analysis of Bionda Piemontese and Bianca di Saluzzo chicken breeds by microsatellites markers: a preliminary study

Abstract Conservation of genetic variability is one of the main goals in animal production science and the analysis of breeds genetic asset can supply objective basis for effective conservation programs and selection strategies. Bionda Piemontese(PIB) and Bianca di Saluzzo (SAB) chicken breeds originated in Piemonte region. Breeds conservation programmes started in 1999 in Verzuolo (CN) aiming to preserve the breeds and to improve their diffusion being particularly adapted to freerange rearing systems thanks to their resistance. PIB and SAB are both suggested for traditional recipes and production (e.g. Morozzo capon) and are Slow Food presidia. A total of 76 birds were analysed: PIB (n=36), SAB (n=40). Genomic DNA was extracted from blood samples. All birds were genotyped at eight microsatellite loci. Each marker was subjected to PCRand the products were separated by electrophoresis in 4.2% denaturing polyacrylamide gels on ABI Prism 377 DNA Sequencerequipped with Genescan and Genotyper software. The results of this preliminary study highlight the genetic differences occurring between PIB and SAB populations

Mitochondrial DNA control region variation in Sanfratellano horse and two other Sicilian autochthonous breeds

Abstract Mitochondrial D-loop hypervariable region was analysed in 20 Sanfratellano and two other Sicilian autochthonous horse breeds (20 Sicilian Oriental Purebred and 20 Sicilian Indigenous) in order to investigate matrilineal genetic diversity. A total of 20 different haplotypes were identified sequencing a fragment of 397 bp; overall, haplotypes showed 31 polymorphic sites (7.8%). High diversity was detected in Sanfratellano (11 haplotypes) and Sicilian Indigenous (13 haplotypes), whereas only one haplotype was found in Sicilian Oriental Purebred. Sanfratellano sequences were compared with those belonging to the other Sicilian autochthonous horses and 118 sequences selected from the GenBank database in order to calculate the statistics of molecular diversity. Six haplotypes were exclusive of Sanfratellano which shares haplotype C, D, H, and O with the Sicilian Indigenous and haplotype U with the Sicilian Oriental Purebred; not significant differentiation was found between Sanfratellano and Sicilian Indigenous. BLAST search showed Sicilian haplotypes overlap with the database sequences but for three. Phylogenetic analysis did not show monophyletic group for Sanfratellano samples or the other breeds included in this analysis.

Early-onset progressive retinal atrophy associated with an IQCB1 variant in African black-footed cats (Felis nigripes)

African black-footed cats (Felis nigripes) are endangered wild felids. One male and full-sibling female African black-footed cat developed vision deficits and mydriasis as early as 3 months of age. The diagnosis of early-onset progressive retinal atrophy (PRA) was supported by reduced direct and consensual pupillary light reflexes, phenotypic presence of retinal degeneration, and a non-recordable electroretinogram with negligible amplitudes in both eyes. Whole genome sequencing, conducted on two unaffected parents and one affected offspring was compared to a variant database from 51 domestic cats and a Pallas cat, revealed 50 candidate variants that segregated concordantly with the PRA phenotype. Testing in additional affected cats confirmed that cats homozygous for a 2 base pair (bp) deletion within IQ calmodulin-binding motif-containing protein-1 (IQCB1), the gene that encodes for nephrocystin-5 (NPHP5), had vision loss. The variant segregated concordantly in other related individuals within the pedigree supporting the identification of a recessively inherited early-onset feline PRA. Analysis of the black-footed cat studbook suggests additional captive cats are at risk. Genetic testing for IQCB1 and avoidance of matings between carriers should be added to the species survival plan for captive management.