Danny Arends

Feeding of Enterococcus faecium NCIMB 10415 Leads to Intestinal miRNA-423-5p-Induced Regulation of Immune-Relevant Genes

ABSTRACT Probiotics are widely used in human and animal health, but little is known about the mode of action of probiotics. One possible mechanism at the molecular level could be an influence on microRNAs (miRNAs) and the related immune-relevant target genes. Here, we analyzed differential expression of miRNA and potential target genes of ileal and jejunal lymphatic tissues from Enterococcus faecium NCIMB 10415-fed piglets versus untreated controls by using next-generation sequencing. We identified miR-423-5p as being greatly affected by the treatment group (2.32-fold; P = 0.014). Validation by reverse transcription-quantitative PCR (RT-qPCR) confirmed a significant upregulation of miR-423-5p (2.11-fold; P = 0.03) and, additionally, downregulation of the important immune-relevant immunoglobulin lambda light C region (IGLC) (0.61-fold; P = 0.03) and immunoglobulin kappa constant (IGKC) (0.69-fold; P = 0.04) target genes. Expression analysis of miR-423-5p and IGLC at different age points shows a clear anticorrelated relationship. Luciferase reporter assays with a HeLa cell line verified IGLC as a target of miR-423-5p. The results provided evidence for an effect of feeding of E. faecium on the expression of miR-423-5p and on the regulation of the IGLC gene through miR-423-5p. This might be a possible mode of action of E. faecium on immune cell regulation in the small intestine.

Whole genome population genetics analysis of Sudanese goats identifies regions harboring genes associated with major traits

BackgroundSudan is endowed with a variety of indigenous goat breeds which are used for meat and milk production and which are well adapted to the local environment. The aim of the present study was to determine the genetic diversity and relationship within and between the four main Sudanese breeds of Nubian, Desert, Taggar and Nilotic goats. Using the 50xa0K SNP chip, 24 animals of each breed were genotyped.ResultsMore than 96% of high quality SNPs were polymorphic with an average minor allele frequency of 0.3. In all breeds, no significant difference between observed (0.4) and expected (0.4) heterozygosity was found and the inbreeding coefficients (FIS) did not differ from zero. Fst coefficients for the genetic distance between breeds also did not significantly deviate from zero. In addition, the analysis of molecular variance revealed that 93% of the total variance in the examined population can be explained by differences among individuals, while only 7% result from differences between the breeds. These findings provide evidence for high genetic diversity and little inbreeding within breeds on one hand, and low diversity between breeds on the other hand. Further examinations using Nei’s genetic distance and STRUCTURE analysis clustered Taggar goats distinct from the other breeds. In a principal component (PC) analysis, PC1 could separate Taggar, Nilotic and a mix of Nubian and Desert goats into three groups. The SNPs that contributed strongly to PC1 showed high Fst values in Taggar goat versus the other goat breeds. PCA allowed us to identify target genomic regions which contain genes known to influence growth, development, bone formation and the immune system.ConclusionsThe information on the genetic variability and diversity in this study confirmed that Taggar goat is genetically different from the other goat breeds in Sudan. The SNPs identified by the first principal components show high Fst values in Taggar goat and allowed to identify candidate genes which can be used in the development of breed selection programs to improve local breeds and find genetic factors contributing to the adaptation to harsh environments.

Fine mapping of a distal chromosome 4 QTL affecting growth and muscle mass in a chicken advanced intercross line

In our previous research, QTL analysis in an F2 cross between the inbred New Hampshire (NHI) and White Leghorn (WL77) lines revealed a growth QTL in the distal part of chromosome 4. To physically reduce the chromosomal interval and the number of potential candidate genes, we performed fine mapping using individuals of generations F10 , F11 and F12 in an advanced intercross line that had been established from the initial F2 mapping population. Using nine single nucleotide polymorphism (SNP) markers within the QTL region for an association analysis with several growth traits from hatch to 20xa0weeks and body composition traits at 20xa0weeks, we could reduce the confidence interval from 26.9 to 3.4xa0Mb. Within the fine mapped region, markers rs14490774, rs314961352 and rs318175270 were in full linkage disequilibrium (Dxa0=xa01.0) and showed the strongest effect on growth and muscle mass (LODxa0≥xa04.00). This reduced region contains 30 genes, compared to 292 genes in the original region. Chicken 60xa0K and 600xa0K SNP chips combined with DNA sequencing of the parental lines were used to call mutations in the reduced region. In the narrowed-down region, 489 sequence variants were detected between NHI and WL77. The most deleterious variants are a missense variant in ADGRA3 (SIFTxa0=xa00.02) and a frameshift deletion in the functional unknown gene ENSGALG00000014401 in NHI chicken. In addition, five synonymous variants were discovered in genes PPARGC1A, ADGRA3, PACRGL, SLIT2 and FAM184B. In our study, the confidence interval and the number of potential genes could be reduced 8- and 10- fold respectively. Further research will focus on functional effects of mutant genes.

The direction of cross affects obesity after puberty in male but not female offspring

BackgroundWe investigated parent-of-origin and allele-specific expression effects on obesity and hepatic gene expression in reciprocal crosses between the Berlin Fat Mouse Inbred line (BFMI) and C57Bl/6NCrl (B6N).ResultsWe found that F1-males with a BFMI mother developed 1.8 times more fat mass on a high fat diet at 10 weeks than F1-males of a BFMI father. The phenotype was detectable from six weeks on and was preserved after cross-fostering. RNA-seq data of liver provided evidence for higher biosynthesis and elongation of fatty acids (pu2009=u20090.00635) in obese male offspring of a BFMI mother versus lean offspring of a BFMI father. Furthermore, fatty acid degradation (pu2009=u20090.00198) and the peroxisome pathway were impaired (pu2009=u20090.00094). The circadian rhythm was affected as well (pu2009=u20090.00087). Among the highest up-regulated protein coding genes in obese males were Acot4 (1.82 fold, pu2009=u20090.022), Cyp4a10 (1.35 fold, pu2009=u20090.026) and Cyp4a14 (1.32 fold, pu2009=u20090.012), which hydroxylize fatty acids and which are known to be increased in liver steatosis. Obese males showed lower expression of the genetically imprinted and paternally expressed 3 (Peg3) gene (0.31 fold, pu2009=u20090.046) and higher expression of the androgen receptor (Ar) gene (2.38 fold, pu2009=u20090.068). Allelic imbalance was found for expression of ATP-binding cassette transporter gene Abca8b. Several of the differentially expressed genes contain estrogen response elements.ConclusionsParent-of-origin effects during gametogenesis and/or fetal development in an obese mother epigenetically modify the transcription of genes that lead to enhanced fatty acid synthesis and impair β-oxidation in the liver of male, but not female F1 offspring. Down-regulation of Peg3 could contribute to trigger this metabolic setting. At puberty, higher amounts of the androgen receptor and altered access to estrogen response elements in affected genes are likely responsible for male specific expression of genes that were epigenetically triggered. A suggestive lack of estrogen binding motifs was found for highly down-regulated genes in adult hepatocytes of obese F1 males (pu2009=u20090.074).

Milk protein polymorphisms and casein haplotypes in Butana cattle

Butana is a Bos indicus dairy cattle breed that is well adapted to the local environment of Sudan. The breed has been gradually declining in number due to breed substitution. Therefore, conservation and improvement strategies are required to maintain this breed. The aim of the present study was to assess genetic variation that is characteristic for Butana cattle in the milk protein genes CSN1S1, CSN2, CSN1S2, CSN3, LALBA, and LGB. In a first step, genomic DNA of five unrelated individuals was comparatively sequenced across all exon and flanking sequences. Ninety-three single nucleotide polymorphisms (SNPs) were identified in Butana cattle compared with the Bos taurus reference sequence at Ensembl. We confirmed the recently identified protein variants CSN2*J, CSN2*L, and LALBA*E. Fifty-two SNPs in non-coding regions are novel. Among the novel SNPs, five are located in promoter regions, three of them are in putative transcription factor binding sites (TFBSs) of the CSN1S2 promoter. Fifteen SNPs potentially affect miRNA target sites. In a second step, 50 unrelated Butana cattle were genotyped. This allowed deriving haplotypes for the casein gene cluster on BTA6. The most frequent haplotype was CSN1S1*C-CSN2*A2-CSN1S2*A-CSN3*A (C-A2-A-A, frequency 0.1546). Considering the newly identified CSN1S2 promoter variants, the most frequent haplotype was C-A2-TTC-A-A (0.1046), with TTC as the promoter variant. The information on protein and promoter variants can be used for the development of conservation and breeding strategies for this local breed.

An Integrated Systems Genetics and Omics Toolkit to Probe Gene Function

Identifying genetic and environmental factors that impact complex traits and common diseases is a high biomedical priority. Here, we developed, validated, and implemented a series of multi-layered systems approaches, including (expression-based) phenome-wide association, transcriptome-/proteome-wide association, and (reverse-) mediation analysis, in an open-access web server (systems-genetics.org) to expedite the systems dissection of gene function. We applied these approaches to multi-omics datasets from the BXD mouse genetic reference population, and identified and validated associations between genes and clinical and molecular phenotypes, including previously unreported links between Rpl26 and body weight, and Cpt1a and lipidxa0metabolism. Furthermore, through mediation and reverse-mediation analysis we established regulatory relations between genes, such as the co-regulation of BCKDHA and BCKDHB protein levels, and identified targets of transcription factors E2F6, ZFP277, and ZKSCAN1. Our multifaceted toolkit enabled the identification of gene-gene and gene-phenotype links that are robust and that translate well across populations and species, and can be universally applied to any populations with multi-omics datasets.

Genetic diversity of Nubian ibex in comparison to other ibex and domesticated goat species

Capra nubiana is a wild ibex species that is in danger of extinction. This study aimed at assessing the genetic diversity and population structure of Nubian ibex (Capra nubiana, nu2009=u20098) in comparison to Alpine ibex (Capra ibex, nu2009=u20098), Bezoar ibex (Capra aegagrus, nu2009=u20094), and domesticated Taggar goats (Capra aegagrus hircus, nu2009=u200924). All animals were genotyped with the 50K goat SNP chip. Since commercial SNP chips are not designed for wild species, data analysis was done in two ways: (1) using all callable SNPs (33,698) and (2) with a reduced set of SNPs segregating within three out of four populations (662). Using these two sets of SNPs, the observed heterozygosity in Nubian ibex ranged from 0.02 to 0.44, in Alpine ibex from 0.01 to 0.38, and in Bezoar ibex from 0.13 to 0.38, when analyzing 33,698 or 662 SNPs, respectively. In domesticated Taggar goats, the values for the observed heterozygosity using all 33,698 callable SNPs and the reduced set of 662 SNPs were similar (0.40–0.41). Pairwise FST values for the differentiation between species ranged from 0.17–0.35 (Bezoar ibex vs. Taggar goats) to 0.47–0.91 (Bezoar vs. Alpine ibex), and was 0.33–0.90 between Bezoar and Nubian ibex, respectively, to the two sets of SNPs. The analysis of molecular variance among all animals revealed that 74–78% can be explained by differences between species, while the residual 22–26% result from differences among individuals, respectively. Cluster analysis of Nei’s genetic distance allowed to detected two distinct clusters comprising Nubian and Alpine ibex on one hand and Taggar goats and Bezoar ibex on the other hand, and clear separation of all four breeds. Principal component (PC) analysis confirmed and further refined the clusters. SNPs that contributed most to PC1 allowed us to identify genomic regions accounting for the distances between species. These regions contain known milk protein genes. The identification of milk protein genes as contributors to the differentiation between species provides insights into the domestication of wild Capra breeds.

Genome-wide association study of body morphological traits in Sudanese goats

Long-term selection of goats for a certain production system and/or different environmental conditions will be reflected in the body morphology of the animals under selection. To investigate the variation contributing to different morphological traits and to identify genomic regions that are associated with body morphological traits in Sudanese goats, we genotyped 96 females belonging to four Sudanese goat breeds with the SNP52 BeadChip. After quality control of the data, the genome-wide association study was performed using 95 goats and 24xa0027 informative single nucleotide polymorphisms (SNPs). Bicoastal diameter was significantly associated (LODxa0=xa06.32) with snp10185-scaffold1365-620922 on chromosome 2. The minor allele has an additive effect, increasing the bicoastal diameter by 2.6xa0cm. A second significant association was found between body length and snp56482-scaffold89-467312 on chromosome 3 (LODxa0=xa05.65). The minor allele is associated with increased body length. Additionally, five regions were suggestive for cannon bone, head width, rump length and withers height (LODxa0>xa05). Only one gene (CNTNAP5) is located within the 1-Mb region surrounding the significant SNP for bicoastal diameter on chromosome 2. The body length QTL on chromosome 3 harbors 49 genes. Further research is required to validate the observed associations and to prioritize candidate genes.

Reducing the interval of a growth QTL on chromosome 4 in laying hens

In our previous research, we identified a QTL with an interval of 3.4xa0Mb for growth on chicken chromosome (GGA) 4 in an advanced intercross population of an initial cross between the New Hampshire inbred line (NHI) and the White Leghorn inbred line (WL77). In the current study, an association analysis was performed in a population of purebred white layers (WLA) with White Leghorn origin. Genotypic data of 130 SNPs within the previously identified 3.4-Mb region were obtained using a 60K SNP chip. In total, 24 significant SNPs (LOD ≥ 4.44) on GGA4 were detected for daily weigh gain from 8 to 14xa0weeks and two SNPs (LOD ≥ 4.80) for body weight at 14xa0weeks. The QTL interval was reduced by 1.9xa0Mb to an interval of 1.5xa0Mb (74.6-76.1xa0Mb) that harbors 15 genes. Furthermore, to identify additional loci for chicken growth, a genome-wide association study (GWAS) was carried out in a WLA population. The GWAS identified an additional QTL on GGA6 for body weight at six weeks (19.8-21.2xa0Mb). Our findings showed that by using a WLA population we were able to further reduce the QTL confidence interval previously detected using a NHIxa0×xa0WL77 advanced intercross population.

Systems Genetics of Obesity

Obesity is a complex trait, determined by many genes and influenced by environmental factors. Mapping genomic loci contributing to obesity helps to identify gene variants responsible for differences in the phenotype. However, measuring fat content alone is often not sufficient to identify the underlying gene or genes. Besides in-depth phenotyping, well-designed genetic populations and the combined analysis of data of different origins are necessary to detect one of several genetic determinants. Structured mouse populations and linking information from different experiments help to simplify the complexity in the search for direct genetic effects or factors that are hidden in the genome. In this chapter we present an example of how the physicochemical characterization of adipose tissue in BXD recombinant inbred lines contributes to enlighten the obese phenotype of mice. We describe the search for gene(s) contributing to collagen content in adipose tissue of BXD strains using the GeneNetwork platform.