Identification of Polymorphisms in the Rabbit Growth Hormone Receptor (GHR) Gene and Association with Finishing Weight in a Commercial Meat Rabbit Line.

A shortcut to identify DNA markers associated with economic traits is to use a candidate gene approach that is still useful in livestock species in which molecular tools and resources are not advanced or not well developed. Mutations in the growth hormone receptor (GHR) gene associated with production traits have been already described in several livestock species. For this reason GHR could be an interesting candidate gene in the rabbit. In this study we re-sequenced all exons and non-coding regions of the rabbit GHR gene in a panel of 10 different rabbits and identified 10 single nucleotide polymorphisms (SNPs). One of them (g.63453192C>G or c.106C>G), located in exon 3 was a missense mutation (p.L36V) substituting an amino acid in a highly conserved position across all mammals. This mutation was genotyped in 297 performance tested rabbits of a meat male line and association analysis showed that the investigated SNP was associated with weight at 70 days (P < 0.05). The most frequent genotype (GG) was in animals with higher weight at this age, suggesting that the high directional selection pressure toward this trait since the constitution of the genotyped line might have contributed to shape allele frequencies at this polymorphic site.

Next generation semiconductor based-sequencing of a nutrigenetics target gene (GPR120) and association with growth rate in Italian Large White pigs.

The GPR120 gene (also known as FFAR4 or O3FAR1) encodes for a functional omega-3 fatty acid receptor/sensor that mediates potent insulin sensitizing effects by repressing macrophage-induced tissue inflammation. For its functional role, GPR120 could be considered a potential target gene in animal nutrigenetics. In this work we resequenced the porcine GPR120 gene by high throughput Ion Torrent semiconductor sequencing of amplified fragments obtained from 8 DNA pools derived, on the whole, from 153 pigs of different breeds/populations (two Italian Large White pools, Italian Duroc, Italian Landrace, Casertana, Pietrain, Meishan, and wild boars). Three single nucleotide polymorphisms (SNPs), two synonymous substitutions and one in the putative 3'-untranslated region (g.114765469C > T), were identified and their allele frequencies were estimated by sequencing reads count. The g.114765469C > T SNP was also genotyped by PCR-RFLP confirming estimated frequency in Italian Large White pools. Then, this SNP was analyzed in two Italian Large White cohorts using a selective genotyping approach based on extreme and divergent pigs for back fat thickness (BFT) estimated breeding value (EBV) and average daily gain (ADG) EBV. Significant differences of allele and genotype frequencies distribution was observed between the extreme ADG-EBV groups (P < 0.001) whereas this marker was not associated with BFT-EBV.

A missense mutation in the rabbit melanocortin 4 receptor (MC4R) gene is associated with finishing weight in a meat rabbit line.

In this study we resequenced 1729 bp of the rabbit melanocortin 4 receptor (MC4 R) gene in 31 rabbits from different breeds/lines and identified ten polymorphisms: one was an indel and 9 were single nucleotide polymorphisms (SNPs). The indel and 5 SNPs were in the 5'-flanking region, 3 were synonymous SNPs and one was a missense mutation (c.101G>A; p.G34D), located in a conserved position of the extracellular tail of the MC4 R protein. The missense mutation was analyzed in a panel of 74 rabbits of different breeds and in 516 performance tested rabbits of a commercial paternal line under selection for growth efficiency. Association analysis indicated that rabbits with the less frequent genotype in this population (DD) had a lighter weight at 70 postnatal days than animals with genotype GD (P < 0.10) and animals with genotype GG (P < 0.05). This is the third study on candidate genes, after those on GH1 and IGF2 that reported a marker associated with finishing weight. Therefore, it seems that a candidate gene approach in rabbit based on previous information accumulated in other livestock species could be useful to identify genes explaining a fraction of variability of performance traits with potential application on rabbit breeding and selection.

A genome wide association study for backfat thickness in Italian Large White pigs highlights new regions affecting fat deposition including neuronal genes.

BACKGROUND: Carcass fatness is an important trait in most pig breeding programs. Following market requests, breeding plans for fresh pork consumption are usually designed to reduce carcass fat content and increase lean meat deposition. However, the Italian pig industry is mainly devoted to the production of Protected Designation of Origin dry cured hams: pigs are slaughtered at around 160 kg of live weight and the breeding goal aims at maintaining fat coverage, measured as backfat thickness to avoid excessive desiccation of the hams. This objective has shaped the genetic pool of Italian heavy pig breeds for a few decades. In this study we applied a selective genotyping approach within a population of ~ 12,000 performance tested Italian Large White pigs. Within this population, we selectively genotyped 304 pigs with extreme and divergent backfat thickness estimated breeding value by the Illumina PorcineSNP60 BeadChip and performed a genome wide association study to identify loci associated to this trait. RESULTS: We identified 4 single nucleotide polymorphisms with P≤5.0E-07 and additional 119 ones with 5.0E-07

Association between polymorphisms in cathepsin and cystatin genes with meat production and carcass traits in Italian Duroc pigs: confirmation of the effects of a cathepsin L (CTSL) gene marker.

We genotyped single nucleotide polymorphisms (SNPs) in 5 cathepsin or cystatin genes (cathepsin F, CTSF; cathepsin L, CTSL; cathepsin S, CTSS; cathepsin Z, CTSZ; cystatin B, CSTB) in two groups of Italian Duroc pigs: the first group (n. 100) was chosen using a selective genotyping approach with extreme estimated breeding value (EBV) for visible intermuscular fat (VIF); the second group (n. 218) was made of performance-tested Duroc pigs not selected by any criteria. CTSL marker showed a tendency towards association (P<0.10) with VIF (first group) and back fat thickness (BFT) and average daily gain (ADG; second group). In the second group, the CTSL polymorphism was associated with weight of lean cuts (LC; P<0.05). Additive effects for all mentioned traits in the second group was significant (P<0.05). The results we obtained in the Italian Duroc pigs confirmed the results and the direction of the effects already reported for the Italian Large White breed.

The porcine TBC1D1 gene: mapping, SNP identification, and association study with meat, carcass and production traits in Italian heavy pigs.

TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1] is a Rab-GTPase-activating related protein implicated in regulating the trafficking of glucose transporter 4 (GLUT4 or SLC2A4) storage vesicles to the cell surface in response to insulin and AMPK-activating stimuli in skeletal muscle. Mutations in the human and mouse TBC1D1 genes confer risk of obesity or leanness. We identified five single nucleotide polymorphisms (SNPs) in the porcine TBC1D1 gene. One of them (FN677935:g.219G>A) was genotyped either by high resolution melting and PCR-RFLP analyses to study allele frequencies in a few pig breeds and evaluate association with meat production and carcass traits in five groups of sib-tested pigs of Italian Large White and Italian Duroc breeds. The g.219G>A SNP was associated (P < 0.05) with ham weight, back fat thickness and lean cuts content in Italian Large White and with visible intermuscular fat in Italian Duroc pigs.

Characterization of the rabbit agouti signaling protein (ASIP) gene: transcripts and phylogenetic analyses and identification of the causative mutation of the nonagouti black coat colour.

The agouti locus encodes the agouti signalling protein (ASIP) which is involved in determining the switch from eumelanin to pheomelanin synthesis in melanocytes. In the domestic rabbit (Oryctolagus cuniculus) early studies indicated three alleles at this locus: A, light-bellied agouti (wild type); a(t), black and tan; a, black nonagouti. We characterized the rabbit ASIP gene and identified the causative mutation (an insertion in exon 2) of the black nonagouti allele whose frequency was evaluated in 31 breeds. Phylogenetic analysis of ASIP sequences from Oryctolagus and 9 other species of the family Leporidae placed Oryctolagus as sister species to Pentalagus and Bunolagus. Transcription analysis in wild type agouti rabbits revealed the presence of two major transcripts with different 5'-untranslated regions having ventral or dorsal skin specific expression. ASIP gene transcripts were also detected in all examined rabbit tissues distinguishing the rabbit expression pattern from what was observed in wild type mice.

A composite six bp in-frame deletion in the melanocortin 1 receptor (MC1R) gene is associated with the Japanese brindling coat colour in rabbits (Oryctolagus cuniculus).

BACKGROUND: In the domestic rabbit (Oryctolagus cuniculus), classical genetic studies have identified five alleles at the Extension locus: ED (dominant black), ES (steel, weaker version of ED), E (wild type, normal extension of black), eJ(Japanese brindling, mosaic distribution of black and yellow) and e (non-extension of black, yellow/red with white belly). Sequencing almost the complete coding sequence (CDS) of the rabbit MC1R gene, we recently identified two in-frame deletions associated with dominant black (c.280_285del6; alleles ED or ES) and recessive red (c.304_333del30; allele e) coat colours. It remained to characterize the eJallele whose phenotypic effect is similar to the Orange and Sex-linked yellow loci of cat and Syrian hamster. RESULTS: We sequenced the whole CDS in 25 rabbits of different coat colours including 10 Japanese and 10 Rhinelander (tricolour) rabbits and identified another 6 bp-in frame deletion flanked by a G > A transition in 5' (c.[124G>A;125_130del6]) that was present in all animals with Japanese brindling coat colour and pattern. These mutations eliminate two amino acids in the first transmembrane domain and, in addition, cause an amino acid substitution at position 44 of the wild type sequence. Genotyping 371 rabbits of 31 breeds with different coat colour this allele (eJ) was present in homozygous state in Japanese, Rhinelander and Dutch tricolour rabbits only (except one albino rabbit). Rabbits with eJ/eJ genotype were non fixed at the non-agouti mutation we previously identified in the ASIP gene. Segregation in F1 and F2 families confirmed the order of dominance already determined by classical genetic experiments with a possible dose effect evident comparing eJ/eJ and eJ/e animals. MC1R mRNA was expressed in black hair skin regions only. CONCLUSIONS: The c.[124A;125_130del6] allele may be responsible for a MC1R variant determining eumelanin production in the black areas. However, the mechanism determining the presence of both red and black hairs in the same animal seems more complex. Expression analyses of the c.[124A;125_130del6] allele suggest that MC1R transcription may be regulated epigenetically in rabbits with the Japanese brindling phenotype. Further studies are needed to clarify this issue.

A single nucleotide polymorphism in the porcine cathepsin K (CTSK) gene is associated with back fat thickness and production traits in Italian Duroc pigs.

Cathepsin K (CTSK) was selected as a candidate gene for fat deposition in pigs because recently, in human and mouse, it was shown that this lysosomal proteinase is an obesity marker. A single nucleotide polymorphism (SNP) was identified in intron 4 of the porcine CTSK gene (g.15G>A; FM209043). Allele frequencies of this polymorphism were analysed in seven pig breeds. Radiation hybrid mapping confirmed the localization of CTSK to porcine chromosome 4, close to the FAT1 QTL region. Three populations of pigs (one Italian Large White and two Italian Duroc groups of pigs) were selected for association analysis. In the Italian Large White breed the g.15G>A SNP was not informative. Association analysis including all Italian Duroc pigs showed that the CTSK marker was associated with back fat thickness and lean cuts (P < 0.01), and average daily gain and feed:gain ratio (P < 0.05) estimated breeding values.

Confirmed association between a single nucleotide polymorphism in the FTO gene and obesity-related traits in heavy pigs.

We recently showed that a polymorphism in the fat mass and obesity associated (FTO) gene (AM931150: g.276T > G) is associated with fat deposition traits in pigs. To confirm this result, we genotyped this polymorphism in an Italian Duroc population made up by 313 performance tested pigs with known estimated breeding values (EBVs) for average daily gain, back fat thickness (BFT), feed:gain ratio, lean cuts (LC), and visible intermuscular fat (VIF, a measure of intermuscular fat in the hams). In addition, we genotyped 148 commercial heavy pigs for which several fat deposition traits and lean meat percentage were measured. The results of the association analyses confirmed the effect of the FTO mutation on obesity-related traits (VIF, BFT and LC) in the Italian Duroc pigs (P < 0.01) and in the commercial pigs (intramuscular fat content of different muscles, P < 0.05 or P < 0.10; lean meat content, P < 0.05; BFT, P < 0.05; intermuscular fat content in the hams, P < 0.05).

Differences of the porcine amelogenin X and Y chromosome genes (AMELX and AMELY) and their application for sex determination in pigs.

Molecular sexing in wild and domestic animals has becoming an important issue in several fields including reproduction. X and Y chromosome-specific sequence differences of the amelogenin genes (AMELX and AMELY) have been described in different mammalian species and used for sex determination. We studied the possibility to use sequence variability between the porcine AMELX and AMELY genes for sex determination in pigs. Sequence analysis of about 400 bp of intron 3 of the porcine amelogenin genes showed the presence of a 9-10 bp deletion in AMELY gene compared to AMELX sequences. Moreover, one single nucleotide polymorphism (SNP) was detected for the AMELY sequence. Four other SNPs and 1 bp insertion differentiated three AMELX haplotypes indicating an unexpected quite high nucleotide diversity for a chromosome X region. Two sex determination assays targeting the 9-10 bp difference between AMELX and AMELY were developed. Assessment of the accuracy of the amelogenin assays to correctly sex individuals was tested on 329 pigs belonging to different breeds/lines. All analysed animals were correctly sexed with the new designed amelogenin tests. No amplification was obtained in human, cattle, goat, sheep, and horse genomic DNA. These assays can be used for sex diagnosis of small amounts of genomic DNA (20 pg) obtained from different sources including embryo biopsies, hair, meat, and other biological specimens. Thus, apart from the application in the reproduction field, these tests can be useful in several other sectors including forensics, archaeozoology, meat production, and processing as well as for quality control in sample identification.

Effects of single nucleotide polymorphisms and haplotypes of the protein kinase AMP-activated non-catalytic subunit gamma 3 (PRKAG3) gene on production, meat quality and carcass traits in Italian Large White pigs.

PRKAG3 is a major gene for meat quality and production in pigs. In this study, we investigated two single nucleotide polymorphisms (SNPs) of the PRKAG3 promoter region (g.-995A>G and g.-311A>G) in 381 pigs of different breeds. Association between these SNPs and three major haplotypes, constructed including other three substitutions (c.89C>A, c.154A>G and c.595G>A) and different traits (meat quality parameters: pH2h, pHu, glycogen and lactate content, glycolytic potential and cathepsin B activity of Semimembranosus muscle; estimated breeding values and random residuals for growth performances and carcass traits) were analysed in 271 Italian Large White performance tested pigs. A significant effect (P=0.043) of the g.-995A>G SNP on cathepsin B activity and a suggestive effect (P=0.067) of the g.-311A>G SNP on pHu were found. The haplotype [g.-995G:g.-311G:c.89A:c.154G:c.595G] was associated with pHu (P=0.024), glycolytic potential (P=0.040) and cathepsin B activity (P=0.086). This study on haplotypes, including promoter region SNPs, contribute to explain the effect of this gene on meat quality traits in pigs.

Analysis of the 227 bp short interspersed nuclear element (SINE) insertion of the promoter of the myostatin (MSTN) gene in different horse breeds.

The myostatin (MSTN) gene encodes a protein known to be a negative regulator of muscle mass in mammalian species. Different polymorphisms of the horse (Equus caballus) MSTN gene have been identified, including single nucleotide polymorphisms and a short interspersed nuclear element (SINE) insertion of 227 bp within the promoter of the gene. The SINE insertion has been associated with performance traits in Thoroughbred racehorses and it was proposed as a predictor of optimum racing distance. The aims of this study were to perform in silico analysis to identify putative gains or abrogation of transcription-factor binding sites (TFBSs) generated by the SINE allele of the promoter and to analyse the frequency of the SINE insertion in horses used for racing (gallop and trot) and other purposes. The SINE insertion was genotyped in 227 horses from 10 breeds belonging to different morphological types (brachimorphic, mesomorphic, meso-dolichomorphic and dolichomorphic). The presence of the insertion was confirmed in the Quarter Horse (SINE allele frequency of 0.81) and in the Thoroughbred (0.51), whereas the SINE allele did not segregate in any of the other analysed breeds. As the SINE MSTN gene polymorphism may be population or breed specific, it is not a useful marker for association studies in all breeds.

The KIT gene is associated with the english spotting coat color locus and congenital megacolon in Checkered Giant rabbits (Oryctolagus cuniculus).

The English spotting coat color locus in rabbits, also known as Dominant white spotting locus, is determined by an incompletely dominant allele (En). Rabbits homozygous for the recessive wild-type allele (en/en) are self-colored, heterozygous En/en rabbits are normally spotted, and homozygous En/En animals are almost completely white. Compared to vital en/en and En/en rabbits, En/En animals are subvital because of a dilated ("mega") cecum and ascending colon. In this study, we investigated the role of the KIT gene as a candidate for the English spotting locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of En/En rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (En/en) with nine Checkered Giant (En/en) and two en/en does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the KIT gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the English spotting coat color phenotype (θ=0.00 LOD  =75.56). KIT gene expression in cecum and colon specimens of En/En (pathological) rabbits was 5-10% of that of en/en (control) rabbits. En/En rabbits showed reduced and altered c-kit immunolabelled ICC compared to en/en controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (P<0.05) and substance P (P<0.01) immunoreactive neurons in En/En vs. en/en. Electron microscopy analysis showed neuronal and ICC abnormalities in En/En tissues. The En/En rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon.

[Secondary cardiovascular prevention after acute coronary syndrome in clinical practice]. / Prevenzione cardiovascolare secondaria dopo sindrome coronarica acuta nella pratica clinica.

Secondary prevention after acute coronary syndromes should be aimed at reducing the risk of further adverse cardiovascular events, thereby improving quality of life, and lengthening survival. Despite compelling evidence from large randomized controlled trials, secondary prevention is not fully implemented in most cases after hospitalization for acute coronary syndrome. The Lazio Region (Italy) has about 5.3 million inhabitants (9% of the entire Italian population). Every year about 11 000 patients are admitted for acute coronary syndrome in hospitals of the Lazio Region. Most of these patients receive state-of-the art acute medical and interventional care during hospitalization. However, observational data suggest that after discharge acute coronary syndrome patients are neither properly followed nor receive all evidence-based treatments. This consensus document has been developed by 11 Scientific Societies of Cardiovascular and Internal Medicine in order develop a sustainable and effective clinical approach for secondary cardiovascular prevention after acute coronary syndrome in the local scenario of the Lazio Region. An evidence-based simplified decalogue for secondary cardiovascular prevention is proposed as the cornerstone of clinical intervention, taking into account regional laws and relative shortage of resources. The following appropriate interventions should be consistently applied: smoking cessation, blood pressure control (blood pressure < 130/80 mmHg), optimal lipid management (LDL cholesterol < 80 mmHg), weight and diabetes management, promotion of physical activity and rehabilitation, correct use of antiplatelet agents, beta-blockers, renin-angiotensin-aldosterone system blockers.