Genetic differentiation of mainland-island sheep of Greece: Implications for identifying candidate genes for long-term local adaptation.

In Greece, a number of local sheep breeds are raised in a wide range of ecological niches across the country. These breeds can be used for the identification of genetic variants that contribute to local adaptation. To this end, 50k genotypes of 90 local sheep from mainland Greece (Epirus, n = 35 and Peloponnesus, n = 55) were used, as well as 147 genotypes of sheep from insular Greece (Skyros, n = 21), Lemnos, n = 36 and Lesvos, n = 90). Principal components and phylogenetic analysis along with admixture and spatial point patterns analyses suggested genetic differentiation of 'mainland-island' populations. Genome scans for signatures of selection and genome-wide association analysis (GWAS) pointed to one highly differentiating marker on OAR4 (FST = 0.39, FLK = 21.93, FDR p-value = 0.10) that also displayed genome wide significance (FDR p-value = 0.002) during GWAS. A total number of 6 positional candidate genes (LOC106990429, ZNF804B, TEX47, STEAP4, SRI and ADAM22) were identified within 500 kb flanking regions around the significant marker. In addition, two QTLs related to fat tail deposition are reported in genomic regions 800 kb downstream the significant marker. Based on gene ontology analysis and literature evidence, the identified candidate genes possess biological functions relevant to local adaptation that worth further investigation.

Allele and genotype frequencies of the SOD1 gene polymorphism associated with canine degenerative myelopathy in Belgian Malinois dogs in Greece.

BACKGROUND AND AIM: Canine degenerative myelopathy (CDM) is an adult-onset fatal disorder associated with a point mutation of the superoxide dismutase 1 (SOD1) gene (SOD1:c.118G>A). This study aimed to determine the allele and genotype frequencies of this mutation in a group of Belgian Malinois dogs in Greece. MATERIALS AND METHODS: Samples (n=72) of whole blood were collected from 72 purebred dogs of the Hellenic Armed Forces; these samples were processed for DNA isolation, polymerase chain reaction, and digestion with the restriction endonuclease AcuI. Sample testing was conducted in compliance with ISO17025 accreditation requirements. RESULTS: The observed relative genotype frequencies were 71% for the homozygous (GG), 25% for the heterozygous (AG), and 4% for the homozygous mutant (AA) alleles. These frequencies were close to those expected, indicating no significant departure from Hardy-Weinberg equilibrium (HWE, p=0.395). The frequency of heterozygous animals indicates that a high risk of developing CDM in forthcoming generations exists in the tested population because mating among carriers would result in 25% AA progeny. The medical record of the group of study animals indicated selection against leishmaniosis, as applied throughout generations by owners and breeders. The potential association of this selection with the HWE status of the study population was discussed. CONCLUSION: The SOD1:c.118G>A mutation was common in the tested group of dogs; thus, they are suitable for a follow-up investigation on the development and progression of CDM. A case-control study on animals with evidence of sensitivity to infectious myelopathy could provide new insights into disease pathogenesis.

Detection of loci exhibiting pleiotropic effects on body weight and egg number in female broilers.

The objective of the present study was to discover the genetic variants, functional candidate genes, biological processes and molecular functions underlying the negative genetic correlation observed between body weight (BW) and egg number (EN) traits in female broilers. To this end, first a bivariate genome-wide association and second stepwise conditional-joint analyses were performed using 2586 female broilers and 240 k autosomal SNPs. The aforementioned analyses resulted in a total number of 49 independent cross-phenotype (CP) significant SNPs with 35 independent markers showing antagonistic action i.e., positive effects on one trait and negative effects on the other trait. A number of 33 independent CP SNPs were located within 26 and 14 protein coding and long non-coding RNA genes, respectively. Furthermore, 26 independent markers were situated within 44 reported QTLs, most of them related to growth traits. Investigation of the functional role of protein coding genes via pathway and gene ontology analyses highlighted four candidates (CPEB3, ACVR1, MAST2 and CACNA1H) as most plausible pleiotropic genes for the traits under study. Three candidates (CPEB3, MAST2 and CACNA1H) were associated with antagonistic pleiotropy, while ACVR1 with synergistic pleiotropic action. Current results provide a novel insight into the biological mechanism of the genetic trade-off between growth and reproduction, in broilers.

Clustering patterns mirror the geographical distribution and genetic history of Lemnos and Lesvos sheep populations.

Elucidating the genetic variation and structure of Lemnos and Lesvos sheep is critical for maintaining local genetic diversity, ecosystem integrity and resilience of local food production of the two North Aegean islands. In the present study, we explored genetic diversity and differentiation as well as population structure of the Lemnos and Lesvos sheep. Furthermore, we sought to identify a small panel of markers with the highest discriminatory power to assign animals across islands. A total number of n = 424 (n = 307, Lemnos and n = 117, Lesvos) ewes, sampled from n = 24 herds dispersed at different geographic regions on the two islands, were genotyped with the 50K SNP array. Mean observed heterozygosity was higher (but not statistically significantly different) in Lesvos than in Lemnos population (0.384 vs. 0.377) while inbreeding levels were higher in Lemnos than Lesvos herds (0.065 vs. 0.031). Results of principal components along with that of admixture analysis and estimated genetic distances revealed genetic clusters corresponding to Lesvos and Lemnos origin and the existence of infrastructure within islands that were associated with geographical isolation and genetic history of the studied populations. In particular, genetic analyses highlighted three geographically isolated herds in Lemnos that are located at mountainous areas of the island and are characterized as representatives of the local sheep by historic data and reports. Admixture analysis also showed a shared genetic background between Lemnos and Lesvos sheep attributable to past gene flow. Little overall genetic differentiation was detected between the two island sheep populations, while 150 discriminatory SNPs could accurately assign animals to their origin. Present results are comparable with those reported in the worldwide sheep breeds, suggesting geography related genetic patterns across and within islands and the existence of the local Lemnos sheep.

Deciphering the mode of action and position of genetic variants impacting on egg number in broiler breeders.

BACKGROUND: Aim of the present study was first to identify genetic variants associated with egg number (EN) in female broilers, second to describe the mode of their gene action (additive and/or dominant) and third to provide a list with implicated candidate genes for the trait. A number of 2586 female broilers genotyped with the high density (~ 600 k) SNP array and with records on EN (mean = 132.4 eggs, SD = 29.8 eggs) were used. Data were analyzed with application of additive and dominant multi-locus mixed models. RESULTS: A number of 7 additive, 4 dominant and 6 additive plus dominant marker-trait significant associations were detected. A total number of 57 positional candidate genes were detected within 50 kb downstream and upstream flanking regions of the 17 significant markers. Functional enrichment analysis pinpointed two genes (BHLHE40 and CRTC1) to be involved in the 'entrainment of circadian clock by photoperiod' biological process. Gene prioritization analysis of the positional candidate genes identified 10 top ranked genes (GDF15, BHLHE40, JUND, GDF3, COMP, ITPR1, ELF3, ELL, CRLF1 and IFI30). Seven prioritized genes (GDF15, BHLHE40, JUND, GDF3, COMP, ELF3, CRTC1) have documented functional relevance to reproduction, while two more prioritized genes (ITPR1 and ELL) are reported to be related to egg quality in chickens. CONCLUSIONS: Present results have shown that detailed exploration of phenotype-marker associations can disclose the mode of action of genetic variants and help in identifying causative genes associated with reproductive traits in the species.

Discovery and characterization of functional modules associated with body weight in broilers.

Aim of the present study was to investigate whether body weight (BW) in broilers is associated with functional modular genes. To this end, first a GWAS for BW was conducted using 6,598 broilers and the high density SNP array. The next step was to search for positional candidate genes and QTLs within strong LD genomic regions around the significant SNPs. Using all positional candidate genes, a network was then constructed and community structure analysis was performed. Finally, functional enrichment analysis was applied to infer the functional relevance of modular genes. A total number of 645 positional candidate genes were identified in strong LD genomic regions around 11 genome-wide significant markers. 428 of the positional candidate genes were located within growth related QTLs. Community structure analysis detected 5 modules while functional enrichment analysis showed that 52 modular genes participated in developmental processes such as skeletal system development. An additional number of 14 modular genes (GABRG1, NGF, APOBEC2, STAT5B, STAT3, SMAD4, MED1, CACNB1, SLAIN2, LEMD2, ZC3H18, TMEM132D, FRYL and SGCB) were also identified as related to body weight. Taken together, current results suggested a total number of 66 genes as most plausible functional candidates for the trait examined.

Combined GWAS and 'guilt by association'-based prioritization analysis identifies functional candidate genes for body size in sheep.

BACKGROUND: Body size in sheep is an important indicator of productivity, growth and health as well as of environmental adaptation. It is a composite quantitative trait that has been studied with high-throughput genomic methods, i.e. genome-wide association studies (GWAS) in various mammalian species. Several genomic markers have been associated with body size traits and genes have been identified as causative candidates in humans, dog and cattle. A limited number of related GWAS have been performed in various sheep breeds and have identified genomic regions and candidate genes that partly account for body size variability. Here, we conducted a GWAS in Frizarta dairy sheep with phenotypic data from 10 body size measurements and genotypic data (from Illumina ovineSNP50 BeadChip) for 459 ewes. RESULTS: The 10 body size measurements were subjected to principal component analysis and three independent principal components (PC) were constructed, interpretable as width, height and length dimensions, respectively. The GWAS performed for each PC identified 11 significant SNPs, at the chromosome level, one on each of the chromosomes 3, 8, 9, 10, 11, 12, 19, 20, 23 and two on chromosome 25. Nine out of the 11 SNPs were located on previously identified quantitative trait loci for sheep meat, production or reproduction. One hundred and ninety-seven positional candidate genes within a 1-Mb distance from each significant SNP were found. A guilt-by-association-based (GBA) prioritization analysis (PA) was performed to identify the most plausible functional candidate genes. GBA-based PA identified 39 genes that were significantly associated with gene networks relevant to body size traits. Prioritized genes were identified in the vicinity of all significant SNPs except for those on chromosomes 10 and 12. The top five ranking genes were TP53, BMPR1A, PIK3R5, RPL26 and PRKDC. CONCLUSIONS: The results of this GWAS provide evidence for 39 causative candidate genes across nine chromosomal regions for body size traits, some of which are novel and some are previously identified candidates from other studies (e.g. TP53, NTN1 and ZNF521). GBA-based PA has proved to be a useful tool to identify genes with increased biological relevance but it is subjected to certain limitations.

Sequencing analysis of the human glucocorticoid receptor (NR3C1) gene in multiple sclerosis patients.

Various specific human glucocorticoid receptor (NR3C1) gene polymorphisms have been described in multiple sclerosis (MS) patients and correlated with disease progression, susceptibility and aggressiveness. Herein, we investigated the presence of gene alterations in the entire coding region of the NR3C1 in MS patients of variable clinical status (CIS, RRMS and SPMS) and the association(s) of these alterations with severity of disease (EDSS), response to glucocorticoid (GC) treatment and clinical improvement. Sixty Caucasian Greek MS patients were included. Sequencing the coding sequences and intron-exon boundaries of the NR3C1 did not reveal the presence of mutation(s) in any of the MS patients. Three previously described polymorphisms were detected: p.N363S (rs6195), p.N766N (rs6196) and c.1469-16G>T (rs6188). None of the identified alleles/genotypes were found to be associated with the severity of disease, response to glucocorticoids and disease subtypes. Known polymorphism, such as ER22/23EK that has been previously detected in MS patients, was not detected. There is a considerable ethnicity-related variation in the frequency of the NR3C1 polymorphisms. Although a genetic basis of the glucocorticoid sensitivity exists in healthy population, in the presence of chronic inflammation and abundance of cytokines--such in MS patients--other factors appear to play a more important role in GC sensitivity.

Severe maternal undernutrition and post-weaning behavior of rabbits.

The objective of the present experiment was to investigate the implications of severe maternal undernutrition on the post-weaning behavior of rabbits. Thirty two does were randomly assigned to four groups: the control group (C) that was fed 100% of the recommended energy maintenance requirements throughout pregnancy and lactation and the U1, U2 and U3 groups that were fed 50% of the recommended energy maintenance requirements between the 6th and the 19th day of pregnancy, between the 20th and the 27th day of pregnancy and between the 3rd and the 10th day of lactation, respectively. At the age of 50 and 65 days, behavior of rabbits in cages was recorded and rabbits were further subjected to an open-field test; a paradigm used as an indicator of fear and emotional distress. Significant differences were found between the U2 and the other experimental groups for the duration of eating and drinking, and duration of locomotory and investigatory behaviors (P<0.05). At the age of 65 days, duration of comfort behaviors was also lower in the U2 compared to the other groups (P<0.05). On the other hand, duration of resting appeared to be the highest in the group of rabbits born from undernourished does between the 20th and the 27th day of pregnancy (P<0.05). Duration of resting was significantly increased, although frequency and duration of eating and drinking were significantly decreased at the age of 65 days (P<0.05). Male rabbits had greater values for locomotory and investigatory behaviors, and duration of eating and drinking compared to the females (P<0.01). A decreased activity of rabbits during the light period was observed (as expected), whereas an increase in duration of locomotory and investigatory behaviors was observed during the first four and the last 4h of the light and dark periods, respectively (P<0.05). Rabbits born from the U1 and U2 group of does displayed increased rates of latency to leave the start position compared to the other groups during the implementation of the open-field test (P<0.05). No other significant differences in the exhibition of the examined behaviors were found between the treatment groups, with the exception of 'moving forelegs' activity that had the lowest value in the U3 group of rabbits. It is concluded that severe maternal undernutrition (50% of the recommended maintenance requirements) especially between the 20th and the 27th day of pregnancy has significant effects on the exhibition of rabbits' post-weaning behavior, since the offspring from the U2 undernourished does were significantly less active compared to the other experimental groups.

Reduction in glucocorticoid receptors in renal biopsies of patients with lupus nephritis.

OBJECTIVES: The first-line treatment for lupus nephritis is the administration of glucocorticoids (GC) that mediate their effects via the glucocorticoid receptor (GR). The aim of this study was to investigate the expression of GR protein in the cortical area of renal parenchyma of normal and diseased renal biopsies from treated and untreated patients. DESIGN AND METHODS: The immunohistochemical EnVision/HRP technique was performed on renal tissue to detect GR protein. Statistical analysis was performed by SAS (2001). RESULTS: The antigen was mainly detected in glomerular podocytes and in tubules. The number of GR-positive podocytes of the controls was significantly higher than in the untreated patients, which was accordingly higher than in patients who were under medication. CONCLUSIONS: The lower number of GR-positive cells in the diseased kidney compared to controls is possibly linked to tissue-specific GC resistance, whereas the decreased GR expression in podocytes of treated compared to untreated patients may be due to a down-regulation effect after GCs' administration.

Glucocorticoid receptor, nuclear factor kappaB, activator protein-1 and C-jun N-terminal kinase in systemic lupus erythematosus patients.

OBJECTIVE: Due to the crucial role of the glucocorticoid receptor (GR), nuclear factor kappaB (NFkappaB), activator protein-1 (AP-1) and c-jun N-terminal kinase (JNK) in regulating inflammatory mediators and immune responses, we investigated their potential role in systemic lupus erythematosus (SLE). PATIENTS AND METHODS: Whole cell and nuclear extracts from peripheral blood lymphocytes, isolated from 25 SLE patients and 25 controls, were immunoblotted using GR, p65/NFkappaB, c-fos and JNK1 antibodies. The electrophoretic mobility shift assay (EMSA) assessed GR, NFkappaB and AP-1-DNA binding in nuclear aliquots. Associations with the disease state and the doses of corticosteroids administered were studied. RESULTS: (i) SLE patients had lower GR-DNA binding (p < 0.001), NFkappaB-DNA binding (p < 0.001) and whole cell c-fos (p < 0.01) but higher nuclear NFkappaB (p < 0.01). (ii) SLE patients and controls had similar AP-1-DNA binding, nuclear c-fos, GR and JNK, whole cell GR, NFkappaB and JNK. (iii) No differences were detected between active and non-active SLE or high- and low-dose corticosteroid patients. (iv) In SLE, increases in GR-DNA binding were associated with increases in NFkappaB-DNA binding (p < 0.0001), and increases in nuclear JNK were associated with increases in AP-1-DNA binding (p < 0.01). (v) In controls, increases in GR-DNA binding were associated with increases in AP-1-DNA binding (p < 0.001). CONCLUSION: We suggest disturbed GR, NFkappaB, AP-1 and JNK signaling in SLE, characterized by a reduced GR- and NFkappaB-DNA binding, a significant association between GR-mediated and NFkappaB-driven pathways, and a significant correlation between nuclear JNK- and AP-1-driven pathways. These disturbances may contribute to abnormal cytokine production and the etiopathogenesis of SLE.

Molecular analysis of the estrogen receptor alpha gene in men with coronary artery disease: association with disease status.

BACKGROUND: The vasoprotective effects of estrogens are known to be mediated by their respective estrogen receptors (ER) alpha (ERalpha) and beta (ERbeta), which are present on the vascular wall. The amino-terminal part of the ERalpha appears to be important; genetic alterations in this region have been associated with arterial hypertension. This region has not been studied in atherosclerotic disease. In the present study, we examined the association between coronary artery disease (CAD) and alterations of the NH(2)-terminal part of ERalpha coding region. METHODS: Genomic DNA was isolated from 50 healthy men and 40 men with CAD confirmed by coronary angiography. The coding sequences of exons 1 and 2 were amplified by polymerase chain reaction (PCR) and analyzed by either denaturing gradient gel electrophoresis (DGGE) or single stranded conformational polymorphism (SSCP), or both, sequencing and restriction fragment length polymorphism (RFLP), as appropriate. In the same subjects, biochemical and vascular parameters were also determined by using the appropriate methodology. RESULTS: In exon 1, the codon 10 polymorphism was detected in both patients and healthy men either in heterozygous or homozygous form. The codon 87 polymorphism was detected mainly in homozygous form and only five individuals were heterozygotes. No mutations were found in exon 2. Statistical analysis of the allele distribution for either codon 10 or 87 between patients and healthy men showed no significant difference. In patients, the biochemical parameters were not statistically significantly different between ERalpha codon 10 genotypes or alleles. However, there was a clear effect of the TCT/TCT genotype and TCT allele on the vascular parameters whereas the right internal carotid artery (RICA) intima-media thickness was significantly associated with TCT/TCT genotype and TCT allele. CONCLUSIONS: We conclude that ERalpha genotypes play no role in the incidence of CAD disease, however, ERalpha codon 10 may be a genetic factor controlling some vessels' angiographic complications.