Sequence characterization of the melanocortin 1 receptor (MC1R) gene in sheep with different coat colours and identification of the putative e allele at the ovine Extension locus

Sequence of the melanocortin 1 receptor (MC1R) gene (the Extension locus) was obtained from a panel of 73 animals belonging to 9 Italian sheep breeds or populations (Appenninica, Bergamasca, Comisana, Cornigliese-like, Delle Langhe, Massese, Merinizzata Italiana, Sarda and Valle del Belice) with different coat colours. Evaluation of the identified polymorphisms on this phenotype was reported with in silico predictions and comparative approaches within and across breeds and across species. Five novel single nucleotide polymorphisms (SNPs), organized in three haplotypes, were detected. Another haplotype, including the two missense mutations already described for the E^D allele, was identified in few Massese sheep. One SNP (c.199C > T) caused a predicted amino acid substitution (p.R67C) in a highly conserved position of the first intracellular loop of the MC1R protein. The same substitution causes recessive pheomelanism in other species. We propose that the p.67C allele represents the recessive e allele at the ovine Extension series that was, so far, not completely recognized in sheep by classical genetic studies. This polymorphism was analysed in a total of 388 sheep of the 9 investigated breeds. The p.67C allele was identified only in the Valle del Belice breed (allele frequency of 21.3% in 176 analysed animals of this breed) in which the presence of epistatic white-determining loci might mask, at least in part, its effects. Confirming the effect of this novel allele on coat colour will lead to new perspectives on the composition of specialized coloured sheep lines.     

Genotyping of the prion protein (PrP) gene in Red Maasai and Black Head Persian sheep in Tanzania

Susceptibility to scrapie in sheep is largely influenced by four polymorphic amino acid positions of the ovine PrP gene at codons 136, 141, 154 and 171. Genotyping of corresponding DNA polymorphism can be used as a basis for selection decisions. A total of 100 Red Maasai and 79 Black Herd Persian sheep, representative of the widely distributed breeds in Tanzania, were genotyped by real-time PCR. Genomic DNA was extracted from buccal epithelial cells. We report six genotypes derived from four different alleles, with the ARQ/ARQ genotype being more frequent (p = 0.0081) in the Red Maasai than in the Black Head Persian sheep. Our study also demonstrated higher allelic frequency (p = 0.00055) of the ARQ in the Red Maasai than in Black Head Persian sheep, while the AHQ allelic frequency was higher (p = 0.00086) in the Black Head Persian. All the animals were homozygous LL₁₄₁. The highly susceptible VRQ allele was not found in any of the sheep breeds examined in this study. Both breeds were genetically low-level resistant to scrapie (NSP3). Due to absence or very low frequency of the ARR allele in the two breeds, selection for genetic resistance to scrapie through an increase of the ARR allele does not seem very relevant. If new breeds of sheep are to be introduced for crossbreeding in Tanzania, care should be taken to avoid import of the VRQ allele. To our knowledge, this is the first study exploring the PrP genotypes in sub-Saharan African sheep and in the Red Maasai and Black Head Persian sheep breeds in particular. The data provide base-line information on genetic susceptibility of the two sheep breeds to scrapie, which may be useful in policy formulation for prevention and future research on prion diseases.     

Diversity of copy number variation in a worldwide population of sheep

Copy number variation (CNV) represents a major source of genomic variation. We investigated the diversity of CNV distribution using SNP array data collected from a comprehensive collection of geographically dispersed sheep breeds. We identified 24,558 putative CNVs, which can be merged into 619 CNV regions, spanning 197 Mb of total length and corresponding to ~ 6.9% of the sheep genome. Our results reveal a population differentiation in CNV between different geographical areas, including Africa, America, Asia, Southwestern Asia, Central Europe, Northern Europe and Southwestern Europe. We observed clear distinctions in CNV prevalence between diverse groups, possibly reflecting the population history of different sheep breeds. We sought to determine the gene content of CNV, and found several important CNV-overlapping genes (BTG3, PTGS1 and PSPH) which were involved in fetal muscle development, prostaglandin (PG) synthesis, and bone color. Our study generates a comprehensive CNV map, which may contribute to genome annotation in sheep.     

Identification of novel SNPs of ovine <Emphasis Type="Italic">PRL</Emphasis> gene and their association with milk production traits

Prolactin (PRL) is a lactogenic hormone that plays a significant role in milk production; its depletion in sheep provokes a severe reduction of milk secretion. Thus, PRL also could be used as a positional marker gene associated with milk production and composition traits. Therefore, the purpose of the study was to identify genotype frequencies of single nucleotide polymorphisms the intron 2 in ovine PRL gene and its possible association genotypes with milk traits in dairy sheep breeds. The genetic structures of ovine PRL gene were examined by PCR-RFLP and DNA sequencing methods in three sheep populations. Four hundred and fifty blood and milk samples were used in the study, which included 150 samples from each of Sakiz, Akkaraman and Awassi ewes respectively. As a result, PRL genotype AA showed a strong association with milk yields content, whereas the animals carrying BB genotype had a higher fat percentage value in the three sheep breeds. Haplotype analysis of the obtained sequences showed the presence of 12 haplotypes in the PRL intron 2 region. In the present study, we have reported for the first time 48 SNPs of the PRL gene for intron 2 in dairy sheep breeds. These preliminary results indicate that the identified SNPs lend themselves readily for further research regarding physiological impacts such as milk production and reproductive traits in other dairy sheep populations.     

Associations of polymorphisms in the promoter I of bovine acetyl‐CoA carboxylase‐α gene with beef fatty acid composition

The objectives of this study were to identify single nucleotide polymorphisms (SNPs) in the promoter I (PI) region of the bovine acetyl‐CoA carboxylase‐α (ACACA) gene and to evaluate the extent to which they were associated with lipid‐related traits. Eight novel SNPs were identified, which were AJ276223:g.2064T>A (SNP1), g.2155C>T (SNP2), g.2203G>T (SNP3), g.2268T>C (SNP4), g.2274G>A (SNP5), g.2340A>G (SNP6), g.2350T>C (SNP7) and g.2370A>G (SNP8). Complete linkage disequilibrium was observed among SNP1, 2, 4, 5, 6 and 8. Phenotypic data were collected from 573 cross‐bred steers with six sire breeds, including Hereford, Angus, Brangus, Beefmaster, Bonsmara and Romosinuano. The genotypes of SNP1/2/4/5/6/8 were significantly associated with adjusted backfat thickness. The genotypes of SNP3 were significantly associated with triacylglycerol (TAG) content and fatty acid composition of longissimus dorsi muscle (LM) in Brangus‐, Romosinuano‐ and Bonsmara‐sired cattle. Cattle with g.2203GG genotype had greater concentrations of TAG, total lipid, total saturated fatty acid and total monounsaturated fatty acid than did cattle with g.2203GT genotype. The genotypes of SNP7 were significantly associated with fatty acid composition of LM. Cattle with genotype g.2350TC had greater amounts of several fatty acids in LM than did cattle with genotype g.2350CC. Our results suggested that the SNPs in the PI region of ACACA gene are associated with variations in the fatty acid contents in LM.     

Preventive SNP–SNP interactions in the mitochondrial displacement loop (D-loop) from chronic dialysis patients

Chronic dialysis association study involving individual single nucleotide polymorphisms (SNPs) in the mitochondrial displacement loop (D-loop) has previously been reported. However, possible SNP–SNP interactions for SNPs in the D-loop which could be associated with a reduced risk for chronic dialysis were not investigated. The purpose of this study was to propose an effective algorithm to identify protective SNP–SNP interactions in the D-loop from chronic dialysis patients. We introduce ISGA that uses an initialization strategy for genetic algorithms (GA) to improve the computational analysis for protective SNP–SNP interactions. ISGA generates genotype patterns with combined SNPs (SNP barcodes) for chronic dialysis. Using our previously reported 77 SNPs in the D-loop, the algorithm-generated protective SNP barcodes for chronic dialysis were evaluated. ISGA provides the SNP barcodes with the maximum frequency differences of occurrence between the cases and controls. The identified SNP barcodes with the lowest odds ratio (OR) values were regarded as the best preventive SNP barcodes against chronic dialysis. The best ISGA-generated SNP barcodes (two to nine SNPs) are more closely associated with the prevention of chronic dialysis when more SNPs are chosen (OR = 0.64 to 0.32; 95% confidence interval = 0.882 to 0.198). The cumulative effects of SNP–SNP interactions were more dominant in ISGA rather than in GA without the initialization strategy. We provide a fast identification of chronic dialysis-associated protective SNP barcodes and demonstrate that the SNP–SNP interactions may have a cumulative effect on prediction for chronic dialysis.     

ADIPOQ gene polymorphisms and cancer risk: A meta-analysis

The results of studies investigating the association between ADIPOQ gene polymorphisms and risk of cancer have been inconsistent and often contradictory. The present meta-analysis was conducted in order to overcome the limitations of any individual study and to provide a more precise overall effect estimate. Relevant studies were identified by searching PubMed and Embase for articles published through May 2012. The strength of the relationship between the ADIPOQ gene and risk of cancer was assessed using odds ratios (ORs). Either a fixed-effects or a random-effects model was used to calculate the overall risk estimates. Fifteen studies were included and five SNPs were considered. A significant association was found between SNP rs2241766 and risk of cancer in the recessive genetic model (OR: 0.768, 95% CI: [0.626, 0.942], P = 0.011); a significant relationship was also found between SNP rs1501299 and risk of cancer in both an allele contrast (OR: 0.141, 95%CI: [0.113, 0.176], P < 0.001) and the dominant genetic model (OR: 0.904, 95%CI: [0.830, 0.985], P = 0.021); no association was found with the rs266729, rs822395, or rs822396 SNPs. Adjusted ORs were also considered, but no statistically significant association was found in homozygote contrasts for any of the five SNPs after adjustment. Our results suggest that two polymorphisms, SNP rs2241766 and SNP rs1501299, of the ADIPOQ gene may be associated with reduced risk of cancer. However, the overall strength of association is mild to moderate, and additional well-designed studies are needed to confirm the present conclusion.     

Assessing genetic diversity and differentiation in Portuguese coarse-wool sheep breeds with microsatellite markers

Population structure and genetic diversity in the Portuguese native breeds of sheep Algarvia (AL), Badana (BA), Galega Bragançana (GB), Galega Mirandesa (GM), Mondegueira (MO) and Churra da Terra Quente (TQ), as well as the exotic Assaf (AS), were analyzed by typing 25 microsatellite markers in 210 individuals. The markers used exhibited high levels of polymorphism, with means for total and effective number of alleles per locus of 13.0 and 4.2, respectively, and an expected heterozygosity of 0.72 across loci. The mean number of alleles per locus and expected heterozygosity were highest in GM and GB, and lowest in AS. Exclusive alleles were found in 10 of the 25 markers analysed, mostly in the AS breed. The proportion of loci which were not in Hardy–Weinberg equilibrium in each breed ranged between 0.12 (GB) and 0.40 (AL and GM), mostly due to a lower than expected number of heterozygotes in those loci. All breeds showed a significant deficit in heterozygosity, which was more pronounced in GM (F_IS = 0.113) and BA (F_IS = 0.103), suggesting that inbreeding might be a major concern in these breeds. The analysis of relationships among breeds, assessed by different methods, indicates that AS and AL are the more distanced breeds relative to the others, while the closest relationships were observed between TQ with MO and GM with GB. The estimated F_ST indicates that only 0.049 of the total genetic variability can be attributed to differences among breeds, and this ratio dropped to 0.029 when only the native breeds were considered. The analysis of individual distances based on allele-sharing indicates that only AS and AL had a tendency for animals of the same breed to cluster together, while for the other breeds there was overlapping among breeds. The results of this study confirm that native breeds of sheep represent an important reservoir of genetic diversity, even though the level of differentiation among closely located breeds tends to be rather small. For several of the breeds analyzed, the levels of inbreeding currently observed cause some apprehension, and recommend the establishment of appropriate conservation strategies, aimed at minimizing inbreeding to avoid further losses of genetic diversity.     

Screening for Booroola (FecB) and Galway (FecXG) mutations in Indian sheep

This study reports the status of the Booroola (FecB) and Galway (FecX^G) mutations in Indian sheep breeds. The Kendrapada sheep (n = 46) was genotyped for the presence of FecB and FecX^G mutations, while the Garole (n = 34), Malpura (n = 30), and Decanni sheep (n = 15) for the FecX^G mutation. The FecB and FecX^G genotyping was carried out by forced restriction fragment length polymorphism PCR technique. In the present study, FecB mutation was discovered in the Kendrapada sheep of Orissa, which is now the second prolific sheep of India after the Garole. Out of 46 individuals of Kendrapada sheep, 26 were homozygous (BB), 15 heterozygous (B+) and 5 non-carriers (++) for the FecB mutation. The frequency of the FecB allele in this sample was about 0.73. Results indicated that the frequency of the FecB mutation is high, but the gene is not fixed in the population as reported in Garole sheep. None of sheep breeds carried the FecX^G mutation. The discovery of the FecB mutation in Kendrapada sheep will facilitate the use of FecB allele in improving the prolificacy of non-prolific sheep breeds of India.     

A missense mutation (c.184C > T) in ovine CLN6 causes neuronal ceroid lipofuscinosis in Merino sheep whereas affected South Hampshire sheep have reduced levels of CLN6 mRNA

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of fatal recessively inherited neurodegenerative diseases of humans and animals characterised by common clinical signs and pathology. These include blindness, ataxia, dementia, behavioural changes, seizures, brain and retinal atrophy and accumulation of fluorescent lysosome derived organelles in most cells. A number of different variants have been suggested and seven different causative genes identified in humans (CLN1, CLN2, CLN3, CLN5, CLN6, CLN8 and CTSD). Animal models have played a central role in the investigation of this group of diseases and are extremely valuable for developing a better understanding of the disease mechanisms and possible therapeutic approaches. Ovine models include flocks of affected New Zealand South Hampshires and Borderdales and Australian Merinos. The ovine CLN6 gene has been sequenced in a representative selection of these sheep. These investigations unveiled the mutation responsible for the disease in Merino sheep (c.184C > T; p.Arg62Cys) and three common ovine allelic variants (c.56A > G, c.822G > A and c.933_934insCT). Linkage analysis established that CLN6 is the gene most likely to cause NCL in affected South Hampshire sheep, which do not have the c.184C > T mutation but show reduced expression of CLN6 mRNA in a range of tissues as determined by real-time PCR. Lack of linkage precludes CLN6 as a candidate for NCL in Borderdale sheep.     

Genetic analysis of Greek sheep breeds using microsatellite markers for setting conservation priorities

Ten Greek sheep breeds were analysed at 28 microsatellite markers in order to estimate their genetic diversity and differentiation. This study aims to provide information on the genetic structure of the breeds analysed and the ancestral populations, and give indications and proposals for the conservation strategies. The breeds included were the local sheep breeds raised in different regions of Greece. In total, 310 animals were sampled. Non-biased average expected heterozygosity ranged from 0.68 ± 0.134 (Skopelos breed) to 0.76 ± 0.103 (Karagouniko breed) with an average of 0.74, while the average observed heterozygosity ranged from 0.626 ± 0.132 (Skopelos) to 0.74 ± 0.135 (Kefallenias). Estimates of inbreeding coefficient (Fis) were significant for all breeds studied, except for Kefallenias and Lesvos breeds (P < 0.05). The results of the phylogenetic relationships are in accordance with the geographical location of the breeds, the history of the origin of the breeds and the breeding practices. The phylogenetic tree showed three groupings according to the bootstrapping values. Correspondence analysis showed the isolation of the Skopelos breed and the grouping of Sfakia and Anogeiano breeds in a separate cluster.     

Exonuclease 1 (EXO1) gene variation and melanoma risk

ObjectiveDNA repair pathway genes play an important role in maintaining genomic integrity and protecting against cancer development. This study aimed to identify novel SNPs in the DNA repair-related genes associated with melanoma risk from a genome-wide association study (GWAS).MethodsA total of 8422 SNPs from the 165 DNA repair-related genes were extracted from a GWAS of melanoma risk, including 494 cases and 5628 controls from the Nurses’ Health Study (NHS) and the Health Professionals Follow-up Study (HPFS). We further replicated the top SNPs in a GWAS of melanoma risk from the MD Anderson Cancer Center (1804 cases and 1026 controls).ResultsA total of 3 SNPs with P value <0.001 were selected for in silico replication. One SNP was replicated: rs3902093 [A] in EXO1 promoter region (P_discovery = 6.6 × 10^?4, P_replication = 0.039, P_joint = 2.5 × 10^?4; OR_joint = 0.80, 95% CI: 0.71, 0.90). This SNP was associated with the expression of the EXO1; carriers of the A allele showed lower expression (P = 0.002).ConclusionOur study found that a promoter region SNP in the editing and processing nucleases gene EXO1 was associated with decreased expression of EXO1 and decreased melanoma risk. Further studies are warranted to validate this association and to investigate the potential mechanisms.     

Macro CK type 1 as a major component of serum creatine kinase activity in pregnant sheep

Electrophoretic separation of creatine kinase (ATP: creatine phosphotransferase, E.C. 2.7.3.2) isoenzymes in agarose gel was made in the serum of 93 sheep of three Slovenian domestic breeds (Solcavska, Pramenka and Bovska breeds). 44 sheep were pregnant between 112 and 135 days at the time of the blood collection. The median value of serum total CK activity for all animals investigated was 82 U/l. After direct immunoinhibition of CK with anti-M-CK monoclonal antibodies the total CK activity remained the same (88 U/l, P = 0.354). There were significant differences among breeds in CK activity for the Solcavska (101 U/l), Bovska (89 U/l) and Pramenka breeds (73 U/l), respectively (Kruskal–Wallis one way analysis of variance, P < 0.01), and between pregnant (105 U/l) and non-pregnant animals (76 U/l), irrespective of the breed (Mann–Whitney rank sum test, P < 0.05). According to electrophoresis, all non-pregnant sheep had exclusively free CK-BB serum bands activity. In all pregnant sheep coupled dimeric BB variant appeared as macro-CK type 1 in the range between 80% and 100% of total CK activity. The present study confirms the existence of an elimination mechanism for CK from the plasma abundance free CK-BB enzyme.     

Disentangling Two QTL on Porcine Chromosome 12 for Backfat Fatty Acid Composition

A previous study allowed the identification of two QTL regions at positions 11–34 cM (QTL1) and 68–76 cM (QTL2) on porcine chromosome SSC12 affecting several backfat fatty acids in an Iberian x Landrace F2 intercross. In the current study, different approaches were performed in order to better delimit the quoted QTL regions and analyze candidate genes. A new chromosome scan, using 81 SNPs selected from the Porcine 60KBeadChip and six previously genotyped microsatellites have refined the QTL positions. Three new functional candidate genes (ACOX1, ACLY, and SREBF1) have been characterized. Moreover, two putative promoters of porcine ACACA gene have also been investigated. New isoforms and 24 SNPs were detected in the four candidate genes, 19 of which were genotyped in the population. ACOX1 and ACLY SNPs failed to explain the effects of QTL1 on palmitic and gadoleic fatty acids. QTL2, affecting palmitoleic, stearic, and vaccenic fatty acids, maps close to the ACACA gene location. The most significant associations have been detected between one intronic (g.53840T > C) and one synonymous (c.5634T > C) ACACA SNPs and these fatty acids. Complementary analyses including ACACA gene expression quantification and association studies in other porcine genetic types do not support the expected causal effect of ACACA SNPs.     

Genetic diversity and population structure of Sicilian sheep breeds using microsatellite markers

Genetic diversity studies in domestic animals aim at evaluating genetic variation within and across breeds mainly for conservation purposes. In Sicily, dairy sheep production represents an important resource for hilly and mountain areas economy. Their milk is used for the production of traditional raw milk cheeses, sometimes protected designation of origin (PDO) cheeses. In some cases, the quality of these products is linked to a specific breed, i.e. mono-breed labelled cheeses and it is therefore important to be able to distinguish the milk of a breed from that of others, in order to guarantee both the consumer and the breed itself. In order to investigate the genetic structure and to perform an assignment test, a total of 331 individuals (Barbaresca, BAR n = 57, Comisana, COM n = 65, Pinzirita, PIN n = 75, Sarda, SAR n = 64, and Valle del Belice, VDB n = 70) were analysed using a panel of 20 microsatellite markers. A total of 259 alleles were observed with average polymorphic information content equal to 0.76, showing that the microsatellites panel used was highly informative. Estimates of observed heterozygosity ranged from 0.65 in the BAR breed to 0.75 in the COM breed. The low value of genetic differentiation among breeds (F_st = 0.049) may indicate that these breeds are little differentiated probably due to common history and breeding practices. The low F_is and F_it values indicated low level of inbreeding within and among breeds. The unrooted neighbor-joining dendrogram obtained from the Reynold's genetic distances, and factorial correspondence analysis revealed a separation between BAR and the other sheep breeds. Recent migration rates were estimated, showing that four out of the five breeds have not received a significant proportion of migrants. Only for the PIN breed a recent introgression rate from the VDB breed (7.2%) was observed. The Bayesian assignment test showed that BAR and SAR breeds had a more definite genetic structure (proportion of assignment of 92% and 86.6%, respectively), whereas the lowest assignment value was found in the PIN breed (67.1%). Our results indicated high genetic variability, low inbreeding and low genetic differentiation, except for BAR breed, and were in accordance with geographical location, history, and breeding practices. The low robustness of the assignment test makes it unfeasible for traceability purposes, due to the high level of admixture, in particular for COM, PIN and VDB.     

Isoelectric focusing reveals additional casein variants in German sheep breeds

Isoelectric focusing (IEF) was applied for screening milk protein variants in milk samples from altogether 1078 sheep of different breeds, in detail Black Faced Mutton sheep (SKF; n = 57), East Friesian Milk sheep (OMS; n = 254), Gray Horned Heath (GGH; n = 190), Merinoland sheep (MLS; n = 363), Merino Mutton sheep (MMS; n = 88), and Rhön sheep (RHO; n = 126). Besides the known genetic variants of α_s1-casein (CSN1S1) (A, C, D), α_s2-casein (CSN1S2) (A, B), and β-lactoglobulin (LGB) (A, B, C) additional variants could be demonstrated in CSN1S1 (H, I) and CSN1S2 (C, D) and their genetic control confirmed by segregation analyses. CSN1S1*H corresponds to a previously mentioned phenotype “X” occurring in OMS, whereas CSN1S1*I was identified for the first time in GGH. CSN1S2*C appeared in OMS, GGH, MLS, and RHO in low frequencies and CSN1S2*D in MLS. Within LGB all three alleles occurred in Merino breeds while α-lactalbumin (LAA) and κ-CN (CSN3) were monomorph at protein level. The haplotype CSN1S1*C–CSN1S2*A was predominant in five out of six breeds with frequencies between 0.325 and 0.919.     

Novel gene variants predict serum levels of the cytokines IL-18 and IL-1ra in older adults

Activation of inflammatory pathways measured by serum inflammatory markers such as interleukin-18 (IL-18) and interleukin-1 receptor antagonist (IL-1ra) is strongly associated with the progression of chronic disease states in older adults. Given that these serum cytokine levels are in part a heritable trait, genetic variation may predict increased serum levels. Using the Cardiovascular Health Study and InCHIANTI cohorts, a genome-wide association study was performed to identify genetic variants that influence IL-18 and IL-1ra serum levels among older adults. Multiple linear regression models characterized the association between each SNP and log-transformed cytokine values. Tests for multiple independent signals within statistically significant loci were performed using haplotype analysis and regression models conditional on lead SNP in each region. Multiple SNPs were associated with these cytokines with genome-wide significance, including SNPs in the IL-18-BCO gene region of chromosome 2 for IL-18 (top SNP rs2250417, P = 1.9 × 10^–32) and in the IL-1 gene family region of chromosome 2 for IL-1ra (rs6743376, P = 2.3 × 10^–26). Haplotype tests and conditional linear regression models showed evidence of multiple independent signals in these regions. Serum IL-18 levels were also associated with a region on chromosome 2 containing the NLRC4 gene (rs12989936, P = 2.7 × 10^–19). These data characterize multiple robust genetic signals that influence IL-18 and IL-1ra cytokine production. In particular, the signal for serum IL-18 located on chromosome two is novel and potentially important in inflammasome triggered chronic activation of inflammation in older adults. Replication in independent cohorts is an important next step, as well as molecular studies to better understand the role of NLRC4.