Divergent selection for natural antibodies in poultry in the presence of a major gene.

BACKGROUND: Natural antibodies (NAb) are antibodies that are present in a healthy individual without requiring previous exposure to an exogenous antigen. Selection for high NAb levels might contribute to improved general disease resistance. Our aim was to analyse the genetic background of NAb based on a divergent selection experiment in poultry, and in particular the effect of a polymorphism in the TLR1A gene. METHODS: The study population consisted of a base population from a commercial pure-bred elite white leghorn layer line and seven generations of birds from a High and Low selection line. Birds were selected for total KLH-binding NAb titer (IgTotal). An enzyme-linked immunosorbent assay was performed to determine NAb titers in blood plasma for IgTotal and the antibody isotypes IgM and IgG. NAb titers were available for 10,878 birds. Genotypes for a polymorphism in TLR1A were determined for chickens in generations 5, 6 and 7. The data were analysed using mixed linear animal models. RESULTS: The heritability estimate for IgM was 0.30 and higher than that for IgG and IgTotal (0.12). Maternal environmental effects explained 2 to 3% of the phenotypic variation in NAb. Selection for IgTotal resulted in a genetic difference between the High and Low line of 2.4 titer points (5.1 genetic standard deviation) in generation 7. For IgM, the selection response was asymmetrical and higher in the Low than the High line. The frequency of the TLR1A C allele was 0.45 in the base population and 0.66 and 0.04 in generation 7 of the High and Low line, respectively. The TLR1A polymorphism had large and significant effects on IgTotal and IgM. Estimated genotypic effects suggest full dominance of the TLR1A C allele. Significant TLR1A by generation interactions were detected for IgM and IgTotal. CONCLUSIONS: The effect of a polymorphism in the TLR1A gene on IgTotal and IgM NAb was confirmed. Furthermore, we provide experimental verification of changes in allele frequencies at a major gene with dominant gene action on a quantitative trait that is subjected to mass selection. TLR1A by generation interactions indicate sensitivity to environmental factors.

Comparison of Methods to Measure Methane for Use in Genetic Evaluation of Dairy Cattle.

Partners in Expert Working Group WG2 of the COST Action METHAGENE have used several methods for measuring methane output by individual dairy cattle under various environmental conditions. Methods included respiration chambers, the sulphur hexafluoride (SF6) tracer technique, breath sampling during milking or feeding, the GreenFeed system, and the laser methane detector. The aim of the current study was to review and compare the suitability of methods for large-scale measurements of methane output by individual animals, which may be combined with other databases for genetic evaluations. Accuracy, precision and correlation between methods were assessed. Accuracy and precision are important, but data from different sources can be weighted or adjusted when combined if they are suitably correlated with the 'true' value. All methods showed high correlations with respiration chambers. Comparisons among alternative methods generally had lower correlations than comparisons with respiration chambers, despite higher numbers of animals and in most cases simultaneous repeated measures per cow per method. Lower correlations could be due to increased variability and imprecision of alternative methods, or maybe different aspects of methane emission are captured using different methods. Results confirm that there is sufficient correlation between methods for measurements from all methods to be combined for international genetic studies and provide a much-needed framework for comparing genetic correlations between methods should these become available.

Distribution and Functionality of Copy Number Variation across European Cattle Populations.

Copy number variation (CNV), which is characterized by large-scale losses or gains of DNA fragments, contributes significantly to genetic and phenotypic variation. Assessing CNV across different European cattle populations might reveal genetic changes responsible for phenotypic differences, which have accumulated throughout the domestication history of cattle as consequences of evolutionary forces that act upon them. To explore pattern of CNVs across European cattle, we genotyped 149 individuals, that represent different European regions, using the Illumina Bovine HD Genotyping array. A total of 9,944 autosomal CNVs were identified in 149 samples using a Hidden Markov Model (HMM) as employed in PennCNV. Animals originating from several breeds of British Isles, and Balkan and Italian regions, on average, displayed higher abundance of CNV counts than Dutch or Alpine animals. A total of 923 CNV regions (CNVRs) were identified by aggregating CNVs overlapping in at least two animals. The hierarchical clustering of CNVRs indicated low differentiation and sharing of high-frequency CNVRs between European cattle populations. Various CNVRs identified in the present study overlapped with olfactory receptor genes and genes related to immune system. In addition, we also detected a CNV overlapping the Kit gene in English longhorn cattle which has previously been associated with color-sidedness. To conclude, we provide a comprehensive overview of CNV distribution in genome of European cattle. Our results indicate an important role of purifying selection and genomic drift in shaping CNV diversity that exists between different European cattle populations.

Genomic Region Containing Toll-Like Receptor Genes Has a Major Impact on Total IgM Antibodies Including KLH-Binding IgM Natural Antibodies in Chickens.

Natural antibodies (NAb) are antigen binding antibodies present in individuals without a previous exposure to this antigen. Keyhole limpet hemocyanin (KLH)-binding NAb levels were previously associated with survival in chickens. This suggests that selective breeding for KLH-binding NAb may increase survival by means of improved general disease resistance. Genome-wide association studies (GWAS) were performed to identify genes underlying genetic variation in NAb levels. The studied population consisted of 1,628 adolescent layer chickens with observations for titers of KLH-binding NAb of the isotypes IgM, IgA, IgG, the total KLH-binding (IgT) NAb titers, total antibody concentrations of the isotypes IgM, IgA, IgG, and the total antibodies concentration in plasma. GWAS were performed using 57,636 single-nucleotide polymorphisms (SNP). One chromosomal region on chromosome 4 was associated with KLH-binding IgT NAb, and total IgM concentration, and especially with KLH-binding IgM NAb. The region of interest was fine mapped by imputing the region of the study population to whole genome sequence, and subsequently performing an association study using the imputed sequence variants. 16 candidate genes were identified, of which FAM114A1, Toll-like receptor 1 family member B (TLR1B), TLR1A, Krüppel-like factor 3 (KLF3) showed the strongest associations. SNP located in coding regions of the candidate genes were checked for predicted changes in protein functioning. One SNP (at 69,965,939 base pairs) received the maximum impact score from two independent prediction tools, which makes this SNP the most likely causal variant. This SNP is located in TLR1A, which suggests a fundamental role of TLR1A on regulation of IgM levels (i.e., KLH-binding IgM NAb, and total IgM concentration), or B cells biology, or both. This study contributes to increased understanding of (genetic) regulation of KLH-binding NAb levels, and total antibody concentrations.

Fine mapping of a quantitative trait locus for bovine milk fat composition on Bos taurus autosome 19.

A major quantitative trait locus (QTL) for milk fat content and fatty acids in both milk and adipose tissue has been detected on Bos taurus autosome 19 (BTA19) in several cattle breeds. The objective of this study was to refine the location of the QTL on BTA19 for bovine milk fat composition using a denser set of markers. Opportunities for fine mapping were provided by imputation from 50,000 genotyped single nucleotide polymorphisms (SNP) toward a high-density SNP panel with up to 777,000 SNP. The QTL region was narrowed down to a linkage disequilibrium block formed by 22 SNP covering 85,007 bp, from 51,303,322 to 51,388,329 bp on BTA19. This linkage disequilibrium block contained 2 genes: coiled-coil domain containing 57 (CCDC57) and fatty acid synthase (FASN). The gene CCDC57 is minimally characterized and has not been associated with bovine milk fat previously, but is expressed in the mammary gland. The gene FASN has been associated with bovine milk fat and fat in adipose tissue before. This gene is a likely candidate for the QTL on BTA19 because of its involvement in de novo fat synthesis. Future studies using sequence data of both CCDC57 and FASN, and eventually functional studies, will have to be pursued to assign the causal variant(s).

Genetic variation in vitamin B-12 content of bovine milk and its association with SNP along the bovine genome.

Vitamin B-12 (also called cobalamin) is essential for human health and current intake levels of vitamin B-12 are considered to be too low. Natural enrichment of the vitamin B-12 content in milk, an important dietary source of vitamin B-12, may help to increase vitamin B-12 intake. Natural enrichment of the milk vitamin B-12 content could be achieved through genetic selection, provided there is genetic variation between cows with respect to the vitamin B-12 content in their milk. A substantial amount of genetic variation in vitamin B-12 content was detected among raw milk samples of 544 first-lactation Dutch Holstein Friesian cows. The presence of genetic variation between animals in vitamin B-12 content in milk indicates that the genotype of the cow affects the amount of vitamin B-12 that ends up in her milk and, consequently, that the average milk vitamin B-12 content of the cow population can be increased by genetic selection. A genome-wide association study revealed significant association between 68 SNP and vitamin B-12 content in raw milk of 487 first-lactation Dutch Holstein Friesian cows. This knowledge facilitates genetic selection for milk vitamin B-12 content. It also contributes to the understanding of the biological mechanism responsible for the observed genetic variation in vitamin B-12 content in milk. None of the 68 significantly associated SNP were in or near known candidate genes involved in transport of vitamin B-12 through the gastrointestinal tract, uptake by ileum epithelial cells, export from ileal cells, transport through the blood, uptake from the blood, intracellular processing, or reabsorption by the kidneys. Probably, associations relate to genes involved in alternative pathways of well-studied processes or to genes involved in less well-studied processes such as ruminal production of vitamin B-12 or secretion of vitamin B-12 by the mammary gland.

Genomic regions associated with bovine milk fatty acids in both summer and winter milk samples.

BACKGROUND: In this study we perform a genome-wide association study (GWAS) for bovine milk fatty acids from summer milk samples. This study replicates a previous study where we performed a GWAS for bovine milk fatty acids based on winter milk samples from the same population. Fatty acids from summer and winter milk are genetically similar traits and we therefore compare the regions detected in summer milk to the regions previously detected in winter milk GWAS to discover regions that explain genetic variation in both summer and winter milk. RESULTS: The GWAS of summer milk samples resulted in 51 regions associated with one or more milk fatty acids. Results are in agreement with most associations that were previously detected in a GWAS of fatty acids from winter milk samples, including eight 'new' regions that were not considered in the individual studies. The high correlation between the -log10(P-values) and effects of SNPs that were found significant in both GWAS imply that the effects of the SNPs were similar on winter and summer milk fatty acids. CONCLUSIONS: The GWAS of fatty acids based on summer milk samples was in agreement with most of the associations detected in the GWAS of fatty acids based on winter milk samples. Associations that were in agreement between both GWAS are more likely to be involved in fatty acid synthesis compared to regions detected in only one GWAS and are therefore worthwhile to pursue in fine-mapping studies.

Genome-wide association of milk fatty acids in Dutch dairy cattle.

BACKGROUND: Identifying genomic regions, and preferably individual genes, responsible for genetic variation in milk fat composition of bovine milk will enhance the understanding of biological pathways involved in fatty acid synthesis and may point to opportunities for changing milk fat composition via selective breeding. An association study of 50,000 single nucleotide polymorphisms (SNPs) was performed for even-chain saturated fatty acids (C4:0-C18:0), even-chain monounsaturated fatty acids (C10:1-C18:1), and the polyunsaturated C18:2cis9,trans11 (CLA) to identify genomic regions associated with individual fatty acids in bovine milk. RESULTS: The two-step single SNP association analysis found a total of 54 regions on 29 chromosomes that were significantly associated with one or more fatty acids. Bos taurus autosomes (BTA) 14, 19, and 26 showed highly significant associations with seven to ten traits, explaining a relatively large percentage of the total additive genetic variation. Many additional regions were significantly associated with the fatty acids. Some of the regions harbor genes that are known to be involved in fat synthesis or were previously identified as underlying quantitative trait loci for fat yield or content, such as ABCG2 and PPARGC1A on BTA 6; ACSS2 on BTA 13; DGAT1 on BTA 14; ACLY, SREBF1, STAT5A, GH, and FASN on BTA 19; SCD1 on BTA26; and AGPAT6 on BTA 27. CONCLUSIONS: Medium chain and unsaturated fatty acids are strongly influenced by polymorphisms in DGAT1 and SCD1. Other regions also showed significant associations with the fatty acids studied. These additional regions explain a relatively small percentage of the total additive genetic variance, but they are relevant to the total genetic merit of an individual and in unraveling the genetic background of milk fat composition. Regions identified in this study can be fine mapped to find causal mutations. The results also create opportunities for changing milk fat composition through breeding by selecting individuals based on their genetic merit for milk fat composition.

The influence of fruit and vegetable consumption and genetic variation on NAD(P)H:quinone oxidoreductase (NQO1) phenotype in an endoscopy-based population.

NAD(P)H:quinone oxidoreductase (NQO1) is an inducible detoxification enzyme relevant for colorectal cancer biochemoprevention. We evaluated the influence of recent fruit and vegetable (F&V) consumption and polymorphisms in NQO1 and transcription factor NFE2L2 on rectal NQO1 phenotype and also whether white blood cell (WBC) NQO1 activity reflects rectal activity. Among 94 sigmoidoscopy patients, we assessed F&V consumption by dietary record and determined the NQO1 c.609C > T and g.-718A > G and NFE2L2 g.-650C > A, g.-684G > A, and g.-686A > G polymorphisms. NQO1 mRNA level was measured in rectal biopsies and NQO1 activity in rectal biopsies and WBC. Consumption of F&V did not yield higher mRNA level or activity but rather appeared to have a repressive effect. Rectal activity was higher among NQO1 609CC-genotypes as compared to 609CT-genotypes (P < 0.0001; 609TT-genotypes were absent), whereas mRNA was higher among 609CT-genotypes (P < 0.001). mRNA and activity correlated among NQO1 609CC-genotypes (r = .50, P = 0.0001) but not among 609CT-genotypes (r = .14, P = 0.45). The NFE2L2-684A-allele was associated with higher mRNA levels (P = < 0.05). The other polymorphisms did not affect phenotype significantly. WBC and rectal activity did not correlate. In conclusion, genetic variation, especially the NQO1 609C > T polymorphism, is a more important predictor of rectal NQO1 phenotype than F&V consumption. WBC NQO1 activity is not a good surrogate for rectal activity.

NQO1 and NFE2L2 polymorphisms, fruit and vegetable intake and smoking and the risk of colorectal adenomas in an endoscopy-based population.

Both environment and genetics contribute to the pathogenesis and prevention of colorectal neoplasia. NAD(P)H: quinone oxidoreductase (NQO1) is a detoxification enzyme that is polymorphic and inducible. We investigated interactions between lifestyle factors and polymorphisms in NQO1 and its key regulatory transcription factor NFE2L2 in colorectal adenoma risk. The NQO1 c.609C>T and g.-718A>G and NFE2L2 g.-650C>A, g.-684G>A and g.-686A>G polymorphisms were determined among 740 Dutch adenoma cases and 698 endoscopy-based controls. Dietary intake was assessed by food frequency questionnaire, other lifestyle information by questionnaire. The NQO1 609CT genotype was associated with a higher adenoma risk (OR 1.27, 95% CI 1.00-1.62) compared with the 609CC genotype, whereas the 609TT genotype was not (OR 1.03, 95% CI 0.56-1.88). The higher risk with the NQO1 609CT-genotype was seen among smokers (OR 1.96, 95% CI 1.40-2.76), but not among nonsmokers (OR 0.91, 95% CI 0.62-1.35; interaction p = 0.030). Fruit and vegetable consumption did not protect smokers from adenomas and did not interact with the NQO1 609C>T polymorphism or the NFE2L2 polymorphisms. A higher adenoma risk seen with high fruit and vegetable consumption among NQO1 -718GG genotypes was absent among -718GA genotypes (interaction p = 0.071). Gene-gene interactions were observed between the NQO1 609C>T and NFE2L2 -686A>G polymorphisms (interaction p = 0.056) and between the NQO1 -718 G>A and NFE2L2 -650C>A polymorphisms (interaction p = 0.013). IN CONCLUSION: the NQO1 609CT genotype is associated with increased adenoma risk among smokers, which is not diminished by high fruit and vegetable consumption. The observed gene-gene interactions may point to a role for NFE2L2 polymorphisms in NQO1-related adenoma formation.

Glutathione S-transferase phenotypes in relation to genetic variation and fruit and vegetable consumption in an endoscopy-based population.

High glutathione S-transferase (GST) activity may contribute to colorectal cancer prevention. Functional polymorphisms are known in the GSTM1, GSTT1, GSTA1 and GSTP1 genes. The influence of these GST polymorphisms and recent fruit and vegetable consumption on GST levels and activity has not been investigated simultaneously in a human population. Also, it is not clear if blood GST activity reflects rectal GST activity. Therefore, we determined GST polymorphisms in 94 patients scheduled for sigmoidoscopy. Rectal GST isoenzyme levels (GSTM1, GSTM2, GSTT1, GSTA and GSTP1) were measured by quantitative western blotting, and rectal and white blood cell total GST activities were measured spectrophotometrically using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Vegetable and fruit consumption was assessed by dietary record. As expected, the GSTM1 and GSTT1 deletion polymorphisms, and the GSTA1 g.-69C-->T polymorphism significantly affected the respective isoenzyme levels. Also, rectal GST isoenzyme levels differed between those with and without recent consumption of Alliaceae, Cucurbitaceae, Apiaceae and citrus fruit. Rectal GST activity, however, was not clearly influenced by fruit and vegetable consumption. It was most significantly determined by the GSTP1 c.313A-->G polymorphism; compared with the 313AA genotypes, the 313AG and 313GG genotypes showed 36 and 67 nmol/min/mg protein (P < 0.001) lower GST activity, respectively. The correlation between rectal and white blood cell GST activities was low (r = 0.40, P < 0.001), and the relevance of the various genetic and dietary factors appeared to differ between the two tissues. In conclusion, this study indicates that the GST enzyme system is influenced by both GST polymorphisms and consumption of fruits and vegetables. The latter appeared more important for individual rectal GST isoenzyme levels than for total GST activity, which could affect detoxification of isoenzyme-specific substrates. The study results do no support the use of white blood cell GST activity as a surrogate measure for rectal GST activity.

Dietary intake of B-vitamins, polymorphisms in thymidylate synthase and serine hydroxymethyltransferase 1, and colorectal adenoma risk: a Dutch case-control study.

Thymidylate synthase and serine hydroxymethyltransferase are involved in folate metabolism. In a case-control study, including 768 cases and 709 controls, we investigated the associations between colorectal adenomas and TS tandem repeat and SHMT1 C1420T polymorphisms, and the interplay with B-vitamins. The polymorphisms were not associated with adenomas, but there was a borderline significant interaction between TS genotype and vitamin B6: the association between vitamin B6 and adenomas seemed positive in TS 3R/3R individuals, but inverse in TS 2R/2R individuals. This study does not provide evidence for a role of SHMT1 genotype in adenoma occurrence. Future research has to indicate whether the TS-B6 interplay is a real effect or a chance finding.

GSTP1 and GSTA1 polymorphisms interact with cruciferous vegetable intake in colorectal adenoma risk.

The possible interplay between cruciferous vegetable consumption, functional genetic variations in glutathione S-transferases (GST) M1, T1, P1, and A1, and colorectal adenomas, was investigated in a Dutch case-control study. The GSTM1 and GSTT1 deletion polymorphisms, and the single nucleotide polymorphisms in GSTP1 (A313G) and in GSTA1 (C-69T) were assessed among 746 cases who developed colorectal adenomas and 698 endoscopy-based controls without any type of colorectal polyps. High and low cruciferous vegetable consumption was defined based on a median split in the control group. High consumption was slightly positively associated with colorectal adenomas [odds ratio (OR) 1.15; 95% confidence interval, 0.92-1.44]. For GSTP1, a positive association with higher cruciferous vegetable intake was only apparent in individuals with the low-activity GSTP1 genotype (GG genotype, OR 1.94; 95% confidence interval, 1.02-3.69). This interaction was more pronounced in men, with higher age and with higher meat intake. The GSTA1 polymorphism may have a modifying role as well: the OR for higher intake compared with lower intake was 1.57 (0.93-2.65) for individuals homozygous for the low expression variant (TT genotype). This seemed to be stronger with younger age and higher red meat intake. Cruciferous vegetable consumption and the combined GSTA1 and GSTP1 genotypes showed a statistically significant interaction (P = 0.034). The GSTM1 and GSTT1 genotypes did not seem to modify the association between cruciferous vegetable intake and colorectal adenomas. In conclusion, GSTP1 and GSTA1 genotypes might modulate the association between cruciferous vegetable intake and colorectal adenomas.