CRELD1 modulates homeostasis of the immune system in mice and humans.

CRELD1 is a pivotal factor for heart development, the function of which is unknown in adult life. We here provide evidence that CRELD1 is an important gatekeeper of immune system homeostasis. Exploiting expression variance in large human cohorts contrasting individuals with the lowest and highest CRELD1 expression levels revealed strong phenotypic, functional and transcriptional differences, including reduced CD4+ T cell numbers. These findings were validated in T cell-specific Creld1-deficient mice. Loss of Creld1 was associated with simultaneous overactivation and increased apoptosis, resulting in a net loss of T cells with age. Creld1 was transcriptionally and functionally linked to Wnt signaling. Collectively, gene expression variance in large human cohorts combined with murine genetic models, transcriptomics and functional testing defines CRELD1 as an important modulator of immune homeostasis.

ABO genotype alters the gut microbiota by regulating GalNAc levels in pigs.

The composition of the intestinal microbiome varies considerably between individuals and is correlated with health1. Understanding the extent to which, and how, host genetics contributes to this variation is essential yet has proved to be difficult, as few associations have been replicated, particularly in humans2. Here we study the effect of host genotype on the composition of the intestinal microbiota in a large mosaic pig population. We show that, under conditions of exacerbated genetic diversity and environmental uniformity, microbiota composition and the abundance of specific taxa are heritable. We map a quantitative trait locus affecting the abundance of Erysipelotrichaceae species and show that it is caused by a 2.3 kb deletion in the gene encoding N-acetyl-galactosaminyl-transferase that underpins the ABO blood group in humans. We show that this deletion is a ≥3.5-million-year-old trans-species polymorphism under balancing selection. We demonstrate that it decreases the concentrations of N-acetyl-galactosamine in the gut, and thereby reduces the abundance of Erysipelotrichaceae that can import and catabolize N-acetyl-galactosamine. Our results provide very strong evidence for an effect of the host genotype on the abundance of specific bacteria in the intestine combined with insights into the molecular mechanisms that underpin this association. Our data pave the way towards identifying the same effect in rural human populations.

The rate of de novo structural variation is increased in in vitro-produced offspring and preferentially affects the paternal genome.

Assisted reproductive technologies (ARTs), including in vitro maturation and fertilization (IVF), are increasingly used in human and animal reproduction. Whether these technologies directly affect the rate of de novo mutation (DNM), and to what extent, has been a matter of debate. Here we take advantage of domestic cattle, characterized by complex pedigrees that are ideally suited to detect DNMs and by the systematic use of ART, to study the rate of de novo structural variation (dnSV) in this species and how it is impacted by IVF. By exploiting features of associated de novo point mutations (dnPMs) and dnSVs in clustered DNMs, we provide strong evidence that (1) IVF increases the rate of dnSV approximately fivefold, and (2) the corresponding mutations occur during the very early stages of embryonic development (one- and two-cell stage), yet primarily affect the paternal genome.

An organism-wide ATAC-seq peak catalog for the bovine and its use to identify regulatory variants.

We report the generation of an organism-wide catalog of 976,813 cis-acting regulatory elements for the bovine detected by the assay for transposase accessible chromatin using sequencing (ATAC-seq). We regroup these regulatory elements in 16 components by nonnegative matrix factorization. Correlation between the genome-wide density of peaks and transcription start sites, correlation between peak accessibility and expression of neighboring genes, and enrichment in transcription factor binding motifs support their regulatory potential. Using a previously established catalog of 12,736,643 variants, we show that the proportion of single-nucleotide polymorphisms mapping to ATAC-seq peaks is higher than expected and that this is owing to an approximately 1.3-fold higher mutation rate within peaks. Their site frequency spectrum indicates that variants in ATAC-seq peaks are subject to purifying selection. We generate eQTL data sets for liver and blood and show that variants that drive eQTL fall into liver- and blood-specific ATAC-seq peaks more often than expected by chance. We combine ATAC-seq and eQTL data to estimate that the proportion of regulatory variants mapping to ATAC-seq peaks is approximately one in three and that the proportion of variants mapping to ATAC-seq peaks that are regulatory is approximately one in 25. We discuss the implication of these findings on the utility of ATAC-seq information to improve the accuracy of genomic selection.

Harnessing genomic information for livestock improvement.

The world demand for animal-based food products is anticipated to increase by 70% by 2050. Meeting this demand in a way that has a minimal impact on the environment will require the implementation of advanced technologies, and methods to improve the genetic quality of livestock are expected to play a large part. Over the past 10 years, genomic selection has been introduced in several major livestock species and has more than doubled genetic progress in some. However, additional improvements are required. Genomic information of increasing complexity (including genomic, epigenomic, transcriptomic and microbiome data), combined with technological advances for its cost-effective collection and use, will make a major contribution.

Codon-specific translation reprogramming promotes resistance to targeted therapy.

Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation2,3. Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U34) tRNA (U34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U34 enzymes. Mechanistically, U34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U34 enzymes and HIF1α. Together, these results demonstrate that U34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.

A 12 kb multi-allelic copy number variation encompassing a GC gene enhancer is associated with mastitis resistance in dairy cattle.

Clinical mastitis (CM) is an inflammatory disease occurring in the mammary glands of lactating cows. CM is under genetic control, and a prominent CM resistance QTL located on chromosome 6 was reported in various dairy cattle breeds. Nevertheless, the biological mechanism underpinning this QTL has been lacking. Herein, we mapped, fine-mapped, and discovered the putative causal variant underlying this CM resistance QTL in the Dutch dairy cattle population. We identified a ~12 kb multi-allelic copy number variant (CNV), that is in perfect linkage disequilibrium with a lead SNP, as a promising candidate variant. By implementing a fine-mapping and through expression QTL mapping, we showed that the group-specific component gene (GC), a gene encoding a vitamin D binding protein, is an excellent candidate causal gene for the QTL. The multiplicated alleles are associated with increased GC expression and low CM resistance. Ample evidence from functional genomics data supports the presence of an enhancer within this CNV, which would exert cis-regulatory effect on GC. We observed that strong positive selection swept the region near the CNV, and haplotypes associated with the multiplicated allele were strongly selected for. Moreover, the multiplicated allele showed pleiotropic effects for increased milk yield and reduced fertility, hinting that a shared underlying biology for these effects may revolve around the vitamin D pathway. These findings together suggest a putative causal variant of a CM resistance QTL, where a cis-regulatory element located within a CNV can alter gene expression and affect multiple economically important traits.

High-resolution structural variants catalogue in a large-scale whole genome sequenced bovine family cohort data.

BACKGROUND: Structural variants (SVs) are chromosomal segments that differ between genomes, such as deletions, duplications, insertions, inversions and translocations. The genomics revolution enabled the discovery of sub-microscopic SVs via array and whole-genome sequencing (WGS) data, paving the way to unravel the functional impact of SVs. Recent human expression QTL mapping studies demonstrated that SVs play a disproportionally large role in altering gene expression, underlining the importance of including SVs in genetic analyses. Therefore, this study aimed to generate and explore a high-quality bovine SV catalogue exploiting a unique cattle family cohort data (total 266 samples, forming 127 trios). RESULTS: We curated 13,731 SVs segregating in the population, consisting of 12,201 deletions, 1,509 duplications, and 21 multi-allelic CNVs (> 50-bp). Of these, we validated a subset of copy number variants (CNVs) utilising a direct genotyping approach in an independent cohort, indicating that at least 62% of the CNVs are true variants, segregating in the population. Among gene-disrupting SVs, we prioritised two likely high impact duplications, encompassing ORM1 and POPDC3 genes, respectively. Liver expression QTL mapping results revealed that these duplications are likely causing altered gene expression, confirming the functional importance of SVs. Although most of the accurately genotyped CNVs are tagged by single nucleotide polymorphisms (SNPs) ascertained in WGS data, most CNVs were not captured by individual SNPs obtained from a 50K genotyping array. CONCLUSION: We generated a high-quality SV catalogue exploiting unique whole genome sequenced bovine family cohort data. Two high impact duplications upregulating the ORM1 and POPDC3 are putative candidates for postpartum feed intake and hoof health traits, thus warranting further investigation. Generally, CNVs were in low LD with SNPs on the 50K array. Hence, it remains crucial to incorporate CNVs via means other than tagging SNPs, such as investigation of tagging haplotypes, direct imputation of CNVs, or direct genotyping as done in the current study. The SV catalogue and the custom genotyping array generated in the current study will serve as valuable resources accelerating utilisation of full spectrum of genetic variants in bovine genomes.

SNP-based quantitative deconvolution of biological mixtures: application to the detection of cows with subclinical mastitis by whole-genome sequencing of tank milk.

Biological products of importance in food (e.g., milk) and medical (e.g., donor blood-derived products) sciences often correspond to mixtures of samples contributed by multiple individuals. Identifying which individuals contributed to the mixture and in what proportions may be of interest in several circumstances. We herein present a method that allows to do this by shallow whole-genome sequencing of the DNA in mixed samples from hundreds of donors. We show the efficacy of the approach for the detection of cows with subclinical mastitis by analysis of farms' tank mixtures containing milk from as many as 500 cows.

Sequenced-based GWAS for linear classification traits in Belgian Blue beef cattle reveals new coding variants in genes regulating body size in mammals.

BACKGROUND: Cohorts of individuals that have been genotyped and phenotyped for genomic selection programs offer the opportunity to better understand genetic variation associated with complex traits. Here, we performed an association study for traits related to body size and muscular development in intensively selected beef cattle. We leveraged multiple trait information to refine and interpret the significant associations. RESULTS: After a multiple-step genotype imputation to the sequence-level for 14,762 Belgian Blue beef (BBB) cows, we performed a genome-wide association study (GWAS) for 11 traits related to muscular development and body size. The 37 identified genome-wide significant quantitative trait loci (QTL) could be condensed in 11 unique QTL regions based on their position. Evidence for pleiotropic effects was found in most of these regions (e.g., correlated association signals, overlap between credible sets (CS) of candidate variants). Thus, we applied a multiple-trait approach to combine information from different traits to refine the CS. In several QTL regions, we identified strong candidate genes known to be related to growth and height in other species such as LCORL-NCAPG or CCND2. For some of these genes, relevant candidate variants were identified in the CS, including three new missense variants in EZH2, PAPPA2 and ADAM12, possibly two additional coding variants in LCORL, and candidate regulatory variants linked to CCND2 and ARMC12. Strikingly, four other QTL regions associated with dimension or muscular development traits were related to five (recessive) deleterious coding variants previously identified. CONCLUSIONS: Our study further supports that a set of common genes controls body size across mammalian species. In particular, we added new genes to the list of those associated with height in both humans and cattle. We also identified new strong candidate causal variants in some of these genes, strengthening the evidence of their causality. Several breed-specific recessive deleterious variants were identified in our QTL regions, probably as a result of the extreme selection for muscular development in BBB cattle.

Fine-mapping inflammatory bowel disease loci to single-variant resolution.

Inflammatory bowel diseases are chronic gastrointestinal inflammatory disorders that affect millions of people worldwide. Genome-wide association studies have identified 200 inflammatory bowel disease-associated loci, but few have been conclusively resolved to specific functional variants. Here we report fine-mapping of 94 inflammatory bowel disease loci using high-density genotyping in 67,852 individuals. We pinpoint 18 associations to a single causal variant with greater than 95% certainty, and an additional 27 associations to a single variant with greater than 50% certainty. These 45 variants are significantly enriched for protein-coding changes (n = 13), direct disruption of transcription-factor binding sites (n = 3), and tissue-specific epigenetic marks (n = 10), with the last category showing enrichment in specific immune cells among associations stronger in Crohn's disease and in gut mucosa among associations stronger in ulcerative colitis. The results of this study suggest that high-resolution fine-mapping in large samples can convert many discoveries from genome-wide association studies into statistically convincing causal variants, providing a powerful substrate for experimental elucidation of disease mechanisms.

Benchmarking phasing software with a whole-genome sequenced cattle pedigree.

BACKGROUND: Accurate haplotype reconstruction is required in many applications in quantitative and population genomics. Different phasing methods are available but their accuracy must be evaluated for samples with different properties (population structure, marker density, etc.). We herein took advantage of whole-genome sequence data available for a Holstein cattle pedigree containing 264 individuals, including 98 trios, to evaluate several population-based phasing methods. This data represents a typical example of a livestock population, with low effective population size, high levels of relatedness and long-range linkage disequilibrium. RESULTS: After stringent filtering of our sequence data, we evaluated several population-based phasing programs including one or more versions of AlphaPhase, ShapeIT, Beagle, Eagle and FImpute. To that end we used 98 individuals having both parents sequenced for validation. Their haplotypes reconstructed based on Mendelian segregation rules were considered the gold standard to assess the performance of population-based methods in two scenarios. In the first one, only these 98 individuals were phased, while in the second one, all the 264 sequenced individuals were phased simultaneously, ignoring the pedigree relationships. We assessed phasing accuracy based on switch error counts (SEC) and rates (SER), lengths of correctly phased haplotypes and the probability that there is no phasing error between a pair of SNPs as a function of their distance. For most evaluated metrics or scenarios, the best software was either ShapeIT4.1 or Beagle5.2, both methods resulting in particularly high phasing accuracies. For instance, ShapeIT4.1 achieved a median SEC of 50 per individual and a mean haplotype block length of 24.1 Mb (scenario 2). These statistics are remarkable since the methods were evaluated with a map of 8,400,000 SNPs, and this corresponds to only one switch error every 40,000 phased informative markers. When more relatives were included in the data (scenario 2), FImpute3.0 reconstructed extremely long segments without errors. CONCLUSIONS: We report extremely high phasing accuracies in a typical livestock sample. ShapeIT4.1 and Beagle5.2 proved to be the most accurate, particularly for phasing long segments and in the first scenario. Nevertheless, most tools achieved high accuracy at short distances and would be suitable for applications requiring only local haplotypes.

Genetic risk for insomnia is associated with objective sleep measures in young and healthy good sleepers.

Insomnia disorder (ID) is the second most common neuropsychiatric disorder. Its socioeconomic burden is enormous while diagnosis and treatment are difficult. A novel approach that reveals associations between insomnia genetic propensity and sleep phenotypes in youth may help understand the core of the disease isolated from comorbidities and pave the way for new treatments. We obtained quantitative nocturnal sleep electroencephalogram (EEG) features in 456 participants (18-31y, 49 women). Sleep EEG was recorded during a baseline night following at least 7 days of regular sleep times. We then assessed daytime sleep onset latency in a subsample of N = 359 men exposed to manipulations affecting sleep pressure. We sampled saliva or blood for polygenic risk score (PRS) determination. The PRS for ID was computed based on genome-wide common single nucleotide polymorphism assessments. Participants also completed a battery of behavioral and cognitive tests. The analyses revealed that the PRS for ID was negatively associated with cumulated EEG power in the delta (0.5-4 Hz) and theta (4-8 Hz) bands across rapid eye movement (REM) and non-REM sleep (p ≤ .0026; ß ≥ -0.13) controlling for age, sex and BMI. The PRS for ID was also negatively associated with daytime likelihood of falling asleep (ß = -0.19, p = .0009). Other explorations for associations with non-baseline-nights, cognitive measures, and mood did not yield significant results. These results propose that the need or the ability to fall asleep and to generate slow brain activity during sleep may constitute the core sleep-related risk factors for developing ID.

Defining the genetic control of human blood plasma N-glycome using genome-wide association study.

Glycosylation is a common post-translational modification of proteins. Glycosylation is associated with a number of human diseases. Defining genetic factors altering glycosylation may provide a basis for novel approaches to diagnostic and pharmaceutical applications. Here we report a genome-wide association study of the human blood plasma N-glycome composition in up to 3811 people measured by Ultra Performance Liquid Chromatography (UPLC) technology. Starting with the 36 original traits measured by UPLC, we computed an additional 77 derived traits leading to a total of 113 glycan traits. We studied associations between these traits and genetic polymorphisms located on human autosomes. We discovered and replicated 12 loci. This allowed us to demonstrate an overlap in genetic control between total plasma protein and IgG glycosylation. The majority of revealed loci contained genes that encode enzymes directly involved in glycosylation (FUT3/FUT6, FUT8, B3GAT1, ST6GAL1, B4GALT1, ST3GAL4, MGAT3 and MGAT5) and a known regulator of plasma protein fucosylation (HNF1A). However, we also found loci that could possibly reflect other more complex aspects of glycosylation process. Functional genomic annotation suggested the role of several genes including DERL3, CHCHD10, TMEM121, IGH and IKZF1. The hypotheses we generated may serve as a starting point for further functional studies in this research area.

Genetic architecture of individual variation in recombination rate on the X chromosome in cattle.

Meiotic recombination is an essential biological process that ensures proper chromosome segregation and creates genetic diversity. Individual variation in global recombination rates has been shown to be heritable in several species, and variants significantly associated with this trait have been identified. Recombination on the sex chromosome has often been ignored in these studies although this trait may be particularly interesting as it may correspond to a biological process distinct from that on autosomes. For instance, recombination in males is restricted to the pseudo-autosomal region (PAR). We herein used a large cattle pedigree with more than 100,000 genotyped animals to improve the genetic map of the X chromosome and to study the genetic architecture of individual variation in recombination rate on the sex chromosome (XRR). The length of the genetic map was 46.4 and 121.2 cM in males and females, respectively, but the recombination rate in the PAR was six times higher in males. The heritability of CO counts on the X chromosome was comparable to that of autosomes in males (0.011) but larger than that of autosomes in females (0.024). XRR was highly correlated (0.76) with global recombination rate (GRR) in females, suggesting that both traits might be governed by shared variants. In agreement, a set of eleven previously identified variants associated with GRR had correlated effects on female XRR (0.86). In males, XRR and GRR appeared to be distinct traits, although more accurate CO counts on the PAR would be valuable to confirm these results.

Analysis of Genes Associated With Monogenic Primary Immunodeficiency Identifies Rare Variants in XIAP in Patients With Crohn's Disease.

BACKGROUND & AIMS: A few rare monogenic primary immunodeficiencies (PIDs) are characterized by chronic intestinal inflammation that resembles Crohn's disease (CD). We investigated whether 23 genes associated with 10 of these monogenic disorders contain common, low-frequency, or rare variants that increase risk for CD. METHODS: Common and low frequency variants in 1 Mb loci centered on the candidate genes were analyzed using meta-data corresponding to genotypes of approximately 17,000 patients with CD or without CD (controls) in Europe. The contribution of rare variants was assessed by high-throughput sequencing of 4750 individuals, including 660 early-onset and/or familial cases among the 2390 patients with CD. Variants were expressed from vectors in SW480 or HeLa cells and functions of their products were analyzed in immunofluorescence, luciferase, immunoprecipitation, and immunoblot assays. RESULTS: We reproduced the association of the interleukin 10 locus with CD (P = .007), although none of the significantly associated variants modified the coding sequence of interleukin 10. We found XIAP to be significantly enriched for rare coding mutations in patients with CD vs controls (P = .02). We identified 4 previously unreported missense variants associated with CD. Variants in XIAP cause the PID X-linked lymphoproliferative disease type 2, yet none of the carriers of these variants had all the clinical features of X-linked lymphoproliferative disease type 2. Identified XIAP variants S123N, R233Q, and P257A were associated with an impaired activation of NOD2 signaling after muramyl dipeptide stimulation. CONCLUSIONS: In a systematic analysis of variants in 23 PID-associated genes, we confirmed the association of variants in XIAP with CD. Further screenings for CD-associated variants and analyses of their functions could increase our understanding of the relationship between PID-associated genes and CD pathogenesis.

Coding and noncoding variants in HFM1, MLH3, MSH4, MSH5, RNF212, and RNF212B affect recombination rate in cattle.

We herein study genetic recombination in three cattle populations from France, New Zealand, and the Netherlands. We identify 2,395,177 crossover (CO) events in 94,516 male gametes, and 579,996 CO events in 25,332 female gametes. The average number of COs was found to be larger in males (23.3) than in females (21.4). The heritability of global recombination rate (GRR) was estimated at 0.13 in males and 0.08 in females, with a genetic correlation of 0.66 indicating that shared variants are influencing GRR in both sexes. A genome-wide association study identified seven quantitative trait loci (QTL) for GRR. Fine-mapping following sequence-based imputation in 14,401 animals pinpointed likely causative coding (5) and noncoding (1) variants in genes known to be involved in meiotic recombination (HFM1, MSH4, RNF212, MLH3, MSH5) for 5/7 QTL, and noncoding variants (3) in RNF212B for 1/7 QTL. This suggests that this RNF212 paralog might also be involved in recombination. Most of the identified mutations had significant effects in both sexes, with three of them each accounting for ∼10% of the genetic variance in males.

NGS-based reverse genetic screen for common embryonic lethal mutations compromising fertility in livestock.

We herein report the result of a large-scale, next generation sequencing (NGS)-based screen for embryonic lethal (EL) mutations in Belgian beef and New Zealand dairy cattle. We estimated by simulation that cattle might carry, on average, ∼0.5 recessive EL mutations. We mined exome sequence data from >600 animals, and identified 1377 stop-gain, 3139 frame-shift, 1341 splice-site, 22,939 disruptive missense, 62,399 benign missense, and 92,163 synonymous variants. We show that cattle have a comparable load of loss-of-function (LoF) variants (defined as stop-gain, frame-shift, or splice-site variants) as humans despite having a more variable exome. We genotyped >40,000 animals for up to 296 LoF and 3483 disruptive missense, breed-specific variants. We identified candidate EL mutations based on the observation of a significant depletion in homozygotes. We estimated the proportion of EL mutations at 15% of tested LoF and 6% of tested disruptive missense variants. We confirmed the EL nature of nine candidate variants by genotyping 200 carrier × carrier trios, and demonstrating the absence of homozygous offspring. The nine identified EL mutations segregate at frequencies ranging from 1.2% to 6.6% in the studied populations and collectively account for the mortality of ∼0.6% of conceptuses. We show that EL mutations preferentially affect gene products fulfilling basic cellular functions. The resulting information will be useful to avoid at-risk matings, thereby improving fertility.

Reported analgesic and anaesthetic administration to non-human primates undergoing experimental surgical procedure: 2010-2015.

BACKGROUND: The use of non-human primates (NHPs) in research remains a major societal concern with public expectations that appropriate anaesthetics and analgesics are used to minimize any pain or distress caused to animals undergoing invasive procedures. A literature review was conducted to examine the reporting of anaesthesia and analgesia methods used in non-human primates undergoing surgical procedures, with recovery from anaesthesia. METHODS: A total of 397 papers from peer-review journals published between 2010 and 2015 were examined. RESULTS: Only 25.9% of papers reported the analgesic regimen used, with carprofen and buprenorphine the 2 most widely used agents. Reporting of the anaesthetic regimens was included in 49.9% of papers. Ketamine and isoflurane were the most frequently used anaesthetic agents. CONCLUSIONS: Anaesthetic and analgesic regimens administered to NHPs remain poorly reported. This lack of detailed descriptions of protocols does little to reassure the public or regulatory authorities that appropriate high standards of perioperative care are employed.