Rare genetic variants underlie outlying levels of DNA methylation and gene-expression.

Testing the effect of rare variants on phenotypic variation is difficult due to the need for extremely large cohorts to identify associated variants given expected effect sizes. An alternative approach is to investigate the effect of rare genetic variants on DNA methylation (DNAm) as effect sizes are expected to be larger for molecular traits compared with complex traits. Here, we investigate DNAm in healthy ageing populations-the Lothian Birth Cohorts of 1921 and 1936-and identify both transient and stable outlying DNAm levels across the genome. We find an enrichment of rare genetic single nucleotide polymorphisms (SNPs) within 1 kb of DNAm sites in individuals with stable outlying DNAm, implying genetic control of this extreme variation. Using a family-based cohort, the Brisbane Systems Genetics Study, we observed increased sharing of DNAm outliers among more closely related individuals, consistent with these outliers being driven by rare genetic variation. We demonstrated that outlying DNAm levels have a functional consequence on gene expression levels, with extreme levels of DNAm being associated with gene expression levels toward the tails of the population distribution. This study demonstrates the role of rare SNPs in the phenotypic variation of DNAm and the effect of extreme levels of DNAm on gene expression.

A locus conferring tolerance to Theileria infection in African cattle.

East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Bos indicus (Boran) cattle demonstrating heritable tolerance to infection with T. parva (h2 = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on bovine chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained variant in a paralogue of the FAF1 gene in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africa's most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva.

Using machine learning to detect the differential usage of novel gene isoforms.

BACKGROUND: Differential isoform usage is an important driver of inter-individual phenotypic diversity and is linked to various diseases and traits. However, accurately detecting the differential usage of different gene transcripts between groups can be difficult, in particular in less well annotated genomes where the spectrum of transcript isoforms is largely unknown. RESULTS: We investigated whether machine learning approaches can detect differential isoform usage based purely on the distribution of reads across a gene region. We illustrate that gradient boosting and elastic net approaches can successfully identify large numbers of genes showing potential differential isoform usage between Europeans and Africans, that are enriched among relevant biological pathways and significantly overlap those identified by previous approaches. We demonstrate that diversity at the 3' and 5' ends of genes are primary drivers of these differences between populations. CONCLUSION: Machine learning methods can effectively detect differential isoform usage from read fraction data, and can provide novel insights into the biological differences between groups.

Assessment of genotyping array performance for genome-wide association studies and imputation in African cattle.

BACKGROUND: In cattle, genome-wide association studies (GWAS) have largely focused on European or Asian breeds, using genotyping arrays that were primarily designed for European cattle. Because there is growing interest in performing GWAS in African breeds, we have assessed the performance of 23 commercial bovine genotyping arrays for capturing the diversity across African breeds and performing imputation. We used 409 whole-genome sequences (WGS) spanning global cattle breeds, and a real cohort of 2481 individuals (including African breeds) that were genotyped with the Illumina high-density (HD) array and the GeneSeek bovine 50 k array. RESULTS: We found that commercially available arrays were not effective in capturing variants that segregate among African indicine animals. Only 6% of these variants in high linkage disequilibrium (LD) (r2 > 0.8) were on the best performing arrays, which contrasts with the 17% and 25% in African and European taurine cattle, respectively. However, imputation from available HD arrays can successfully capture most variants (accuracies up to 0.93), mainly when using a global, not continent-specific, reference panel, which partially reflects the unusually high levels of admixture on the continent. When considering functional variants, the GGPF250 array performed best for tagging WGS variants and imputation. Finally, we show that imputation from low-density arrays can perform almost as well as HD arrays, if a two-stage imputation approach is adopted, i.e. first imputing to HD and then to WGS, which can potentially reduce the costs of GWAS. CONCLUSIONS: Our results show that the choice of an array should be based on a balance between the objective of the study and the breed/population considered, with the HD and BOS1 arrays being the best choice for both taurine and indicine breeds when performing GWAS, and the GGPF250 being preferable for fine-mapping studies. Moreover, our results suggest that there is no advantage to using the indicus-specific arrays for indicus breeds, regardless of the objective. Finally, we show that using a reference panel that better represents global bovine diversity improves imputation accuracy, particularly for non-European taurine populations.

Increased ultra-rare variant load in an isolated Scottish population impacts exonic and regulatory regions.

Human population isolates provide a snapshot of the impact of historical demographic processes on population genetics. Such data facilitate studies of the functional impact of rare sequence variants on biomedical phenotypes, as strong genetic drift can result in higher frequencies of variants that are otherwise rare. We present the first whole genome sequencing (WGS) study of the VIKING cohort, a representative collection of samples from the isolated Shetland population in northern Scotland, and explore how its genetic characteristics compare to a mainland Scottish population. Our analyses reveal the strong contributions played by the founder effect and genetic drift in shaping genomic variation in the VIKING cohort. About one tenth of all high-quality variants discovered are unique to the VIKING cohort or are seen at frequencies at least ten fold higher than in more cosmopolitan control populations. Multiple lines of evidence also suggest relaxation of purifying selection during the evolutionary history of the Shetland isolate. We demonstrate enrichment of ultra-rare VIKING variants in exonic regions and for the first time we also show that ultra-rare variants are enriched within regulatory regions, particularly promoters, suggesting that gene expression patterns may diverge relatively rapidly in human isolates.

Shared regulatory sites are abundant in the human genome and shed light on genome evolution and disease pleiotropy.

Large-scale gene expression datasets are providing an increasing understanding of the location of cis-eQTLs in the human genome and their role in disease. However, little is currently known regarding the extent of regulatory site-sharing between genes. This is despite it having potentially wide-ranging implications, from the determination of the way in which genetic variants may shape multiple phenotypes to the understanding of the evolution of human gene order. By first identifying the location of non-redundant cis-eQTLs, we show that regulatory site-sharing is a relatively common phenomenon in the human genome, with over 10% of non-redundant regulatory variants linked to the expression of multiple nearby genes. We show that these shared, local regulatory sites are linked to high levels of chromatin looping between the regulatory sites and their associated genes. In addition, these co-regulated gene modules are found to be strongly conserved across mammalian species, suggesting that shared regulatory sites have played an important role in shaping human gene order. The association of these shared cis-eQTLs with multiple genes means they also appear to be unusually important in understanding the genetics of human phenotypes and pleiotropy, with shared regulatory sites more often linked to multiple human phenotypes than other regulatory variants. This study shows that regulatory site-sharing is likely an underappreciated aspect of gene regulation and has important implications for the understanding of various biological phenomena, including how the two and three dimensional structures of the genome have been shaped and the potential causes of disease pleiotropy outside coding regions.

Homozygous loss-of-function variants in European cosmopolitan and isolate populations.

Homozygous loss of function (HLOF) variants provide a valuable window on gene function in humans, as well as an inventory of the human genes that are not essential for survival and reproduction. All humans carry at least a few HLOF variants, but the exact number of inactivated genes that can be tolerated is currently unknown­as are the phenotypic effects of losing function for most human genes. Here, we make use of 1432 whole exome sequences from five European populations to expand the catalogue of known human HLOF mutations; after stringent filtering of variants in our dataset, we identify a total of 173 HLOF mutations, 76 (44%) of which have not been observed previously. We find that population isolates are particularly well suited to surveys of novel HLOF genes because individuals in such populations carry extensive runs of homozygosity, which we show are enriched for novel, rare HLOF variants. Further, we make use of extensive phenotypic data to show that most HLOFs, ascertained in population-based samples, appear to have little detectable effect on the phenotype. On the contrary, we document several genes directly implicated in disease that seem to tolerate HLOF variants. Overall HLOF genes are enriched for olfactory receptor function and are expressed in testes more often than expected, consistent with reduced purifying selection and incipient pseudogenisation.

hapbin: An Efficient Program for Performing Haplotype-Based Scans for Positive Selection in Large Genomic Datasets.

Understanding how the genome is shaped by selective processes forms an integral part of modern biology. However, as genomic datasets continue to grow larger it is becoming increasingly difficult to apply traditional statistics for detecting signatures of selection to these cohorts. There is therefore a pressing need for the development of the next generation of computational and analytical tools for detecting signatures of selection in large genomic datasets. Here, we present hapbin, an efficient multithreaded implementation of extended haplotype homzygosity-based statistics for detecting selection, which is up to 3,400 times faster than the current fastest implementations of these algorithms.

Exome sequencing to detect rare variants associated with general cognitive ability: a pilot study.

Variation in human cognitive ability is of consequence to a large number of health and social outcomes and is substantially heritable. Genetic linkage, genome-wide association, and copy number variant studies have investigated the contribution of genetic variation to individual differences in normal cognitive ability, but little research has considered the role of rare genetic variants. Exome sequencing studies have already met with success in discovering novel trait-gene associations for other complex traits. Here, we use exome sequencing to investigate the effects of rare variants on general cognitive ability. Unrelated Scottish individuals were selected for high scores on a general component of intelligence (g). The frequency of rare genetic variants (in n = 146) was compared with those from Scottish controls (total n = 486) who scored in the lower to middle range of the g distribution or on a proxy measure of g. Biological pathway analysis highlighted enrichment of the mitochondrial inner membrane component and apical part of cell gene ontology terms. Global burden analysis showed a greater total number of rare variants carried by high g cases versus controls, which is inconsistent with a mutation load hypothesis whereby mutations negatively affect g. The general finding of greater non-synonymous (vs. synonymous) variant effects is in line with evolutionary hypotheses for g. Given that this first sequencing study of high g was small, promising results were found, suggesting that the study of rare variants in larger samples would be worthwhile.

Abundant pleiotropy in human complex diseases and traits.

We present a systematic review of pleiotropy among SNPs and genes reported to show genome-wide association with common complex diseases and traits. We find abundant evidence of pleiotropy; 233 (16.9%) genes and 77 (4.6%) SNPs show pleiotropic effects. SNP pleiotropic status was associated with gene location (p = 0.024; pleiotropic SNPs more often exonic [14.5% versus 4.9% for nonpleiotropic, trait-associated SNPs] and less often intergenic [15.8% versus 23.6%]), "predicted transcript consequence" (p = 0.001; pleiotropic SNPs more often predicted to be structurally deleterious [5% versus 0.4%] but not more often in regulatory sequences), and certain disease classes. We develop a method to calculate the likelihood that pleiotropic links between traits occurred more often than expected and demonstrate that this approach can identify etiological links that are already known (such as between fetal hemoglobin and malaria risk) and those that are not yet established (e.g., between plasma campesterol levels and gallstones risk; and between immunoglobulin A and juvenile idiopathic arthritis). Examples of pleiotropy will accumulate over time, but it is already clear that pleiotropy is a common property of genes and SNPs associated with disease traits, and this will have implications for identification of molecular targets for drug development, future genetic risk-profiling, and classification of diseases.

Widespread signatures of recent selection linked to nucleosome positioning in the human lineage.

In this study we investigated the strengths and modes of selection associated with nucleosome positioning in the human lineage through the comparison of interspecies and intraspecies rates of divergence. We identify significant evidence for both positive and negative selection linked to human nucleosome positioning for the first time, implicating a widespread and important role for DNA sequence in the location of well-positioned nucleosomes. Selection appears to be acting on particular base substitutions to maintain optimum GC compositions in core and linker regions, with, e.g., unexpectedly elevated rates of C→T substitutions during recent human evolution at linker regions 60-90 bp from the nucleosome dyad but significant depletion of the same substitutions within nucleosome core regions. These patterns are strikingly consistent with the known relationships between genomic sequence composition and nucleosome assembly. By stratifying nucleosomes according to the GC content of their genomic neighborhood, we also show that the strength and direction of selection detected is dictated by local GC content. Intriguingly these signatures of selection are not restricted to nucleosomes in close proximity to exons, suggesting the correct positioning of nucleosomes is not only important in and around coding regions. This analysis provides strong evidence that the genomic sequences associated with nucleosomes are not evolving neutrally, and suggests that underlying DNA sequence is an important factor in nucleosome positioning. Recent signatures of selection linked to genomic features as ubiquitous as the nucleosome have important implications for human genome evolution and disease.

Multiple common susceptibility variants near BMP pathway loci GREM1, BMP4, and BMP2 explain part of the missing heritability of colorectal cancer.

Genome-wide association studies (GWAS) have identified 14 tagging single nucleotide polymorphisms (tagSNPs) that are associated with the risk of colorectal cancer (CRC), and several of these tagSNPs are near bone morphogenetic protein (BMP) pathway loci. The penalty of multiple testing implicit in GWAS increases the attraction of complementary approaches for disease gene discovery, including candidate gene- or pathway-based analyses. The strongest candidate loci for additional predisposition SNPs are arguably those already known both to have functional relevance and to be involved in disease risk. To investigate this proposition, we searched for novel CRC susceptibility variants close to the BMP pathway genes GREM1 (15q13.3), BMP4 (14q22.2), and BMP2 (20p12.3) using sample sets totalling 24,910 CRC cases and 26,275 controls. We identified new, independent CRC predisposition SNPs close to BMP4 (rs1957636, P = 3.93×10(-10)) and BMP2 (rs4813802, P = 4.65×10(-11)). Near GREM1, we found using fine-mapping that the previously-identified association between tagSNP rs4779584 and CRC actually resulted from two independent signals represented by rs16969681 (P = 5.33×10(-8)) and rs11632715 (P = 2.30×10(-10)). As low-penetrance predisposition variants become harder to identify-owing to small effect sizes and/or low risk allele frequencies-approaches based on informed candidate gene selection may become increasingly attractive. Our data emphasise that genetic fine-mapping studies can deconvolute associations that have arisen owing to independent correlation of a tagSNP with more than one functional SNP, thus explaining some of the apparently missing heritability of common diseases.

Genomic Reference Resource for African Cattle: Genome Sequences and High-Density Array Variants.

The diversity in genome resources is fundamental to designing genomic strategies for local breed improvement and utilisation. These resources also support gene discovery and enhance our understanding of the mechanisms of resilience with applications beyond local breeds. Here, we report the genome sequences of 555 cattle (208 of which comprise new data) and high-density (HD) array genotyping of 1,082 samples (537 new samples) from indigenous African cattle populations. The new sequences have an average genome coverage of ~30X, three times higher than the average (~10X) of the over 300 sequences already in the public domain. Following variant quality checks, we identified approximately 32.3 million sequence variants and 661,943 HD autosomal variants mapped to the Bos taurus reference genome (ARS-UCD1.2). The new datasets were generated as part of the Centre for Tropical Livestock Genetics and Health (CTLGH) Genomic Reference Resource for African Cattle (GRRFAC) initiative, which aspires to facilitate the generation of this livestock resource and hopes for its utilisation for complete indigenous breed characterisation and sustainable global livestock improvement.

Continent-wide genomic analysis of the African buffalo (Syncerus caffer).

The African buffalo (Syncerus caffer) is a wild bovid with a historical distribution across much of sub-Saharan Africa. Genomic analysis can provide insights into the evolutionary history of the species, and the key selective pressures shaping populations, including assessment of population level differentiation, population fragmentation, and population genetic structure. In this study we generated the highest quality de novo genome assembly (2.65 Gb, scaffold N50 69.17 Mb) of African buffalo to date, and sequenced a further 195 genomes from across the species distribution. Principal component and admixture analyses provided little support for the currently described four subspecies. Estimating Effective Migration Surfaces analysis suggested that geographical barriers have played a significant role in shaping gene flow and the population structure. Estimated effective population sizes indicated a substantial drop occurring in all populations 5-10,000 years ago, coinciding with the increase in human populations. Finally, signatures of selection were enriched for key genes associated with the immune response, suggesting infectious disease exert a substantial selective pressure upon the African buffalo. These findings have important implications for understanding bovid evolution, buffalo conservation and population management.

Meta-analysis of heritability estimates and genome-wide association for tick-borne haemoparasites in African cattle.

The control of tick-borne haemoparasites in cattle largely relies on the use of acaricide drugs against the tick vectors, with some vaccination also being used against selected pathogens. These interventions can be difficult in Africa, where accessibility and cost of vaccines can be issues, and the increasing resistance of tick vectors to the widely used acaricides is a complication to disease control. A potential complementary control strategy could be the exploitation of any natural host genetic resistance to the pathogens. However, there are currently very few estimates of the extent of host resistance to tick-borne haemoparasites, and a significant contributing factor to this knowledge gap is likely to be the difficulty of collecting appropriate samples and data in the smallholder systems that predominate livestock production in low- and middle-income countries, particularly at scale. In this study, we have estimated the heritability for the presence/absence of several important haemoparasite species (including Anaplasma marginale, Babesia bigemina, Babesia bovis, and Ehrlichia ruminantium), as well as for relevant traits such as body weight and body condition score (BCS), in 1,694 cattle from four African countries (Burkina Faso, Ghana, Nigeria, and Tanzania). Heritability estimates within countries were mostly not significant, ranging from 0.05 to 0.84 across traits and countries, with standard errors between 0.07 and 0.91. However, the weighted mean of heritability estimates was moderate and significant for body weight and BCS (0.40 and 0.49, respectively), with significant heritabilities also observed for the presence of A. marginale (0.16) and E. ruminantium (0.19). In a meta-analysis of genome-wide association studies (GWAS) for these traits, two peaks were identified as reaching the suggestive significance threshold (p < 1.91 × 10-7 and p < 1.89 × 10-7, respectively): one on chromosome 24 for BCS and one on chromosome 8 for the E. ruminantium infection status. These findings indicate that there is likely to be a genetic basis that contributes to pathogen presence/absence for tick-borne haemoparasite species, which could potentially be exploited to improve cattle resistance in Africa to the economically important diseases caused by these pathogens.

Profiling the immune epigenome across global cattle breeds.

BACKGROUND: Understanding the variation between well and poorly adapted cattle breeds to local environments and pathogens is essential for breeding cattle with improved climate and disease-resistant phenotypes. Although considerable progress has been made towards identifying genetic differences between breeds, variation at the epigenetic and chromatin levels remains poorly characterized. Here, we generate, sequence and analyse over 150 libraries at base-pair resolution to explore the dynamics of DNA methylation and chromatin accessibility of the bovine immune system across three distinct cattle lineages. RESULTS: We find extensive epigenetic divergence between the taurine and indicine cattle breeds across immune cell types, which is linked to the levels of local DNA sequence divergence between the two cattle sub-species. The unique cell type profiles enable the deconvolution of complex cellular mixtures using digital cytometry approaches. Finally, we show distinct sub-categories of CpG islands based on their chromatin and methylation profiles that discriminate between classes of distal and gene proximal islands linked to discrete transcriptional states. CONCLUSIONS: Our study provides a comprehensive resource of DNA methylation, chromatin accessibility and RNA expression profiles of three diverse cattle populations. The findings have important implications, from understanding how genetic editing across breeds, and consequently regulatory backgrounds, may have distinct impacts to designing effective cattle epigenome-wide association studies in non-European breeds.

The conservation of human functional variants and their effects across livestock species.

Despite the clear potential of livestock models of human functional variants to provide important insights into the biological mechanisms driving human diseases and traits, their use to date has been limited. Generating such models via genome editing is costly and time consuming, and it is unclear which variants will have conserved effects across species. In this study we address these issues by studying naturally occurring livestock models of human functional variants. We show that orthologues of over 1.6 million human variants are already segregating in domesticated mammalian species, including several hundred previously directly linked to human traits and diseases. Models of variants linked to particular phenotypes, including metabolomic disorders and height, are preferentially shared across species, meaning studying the genetic basis of these phenotypes is particularly tractable in livestock. Using machine learning we demonstrate it is possible to identify human variants that are more likely to have an existing livestock orthologue, and, importantly, we show that the effects of functional variants are often conserved in livestock, acting on orthologous genes with the same direction of effect. Consequently, this work demonstrates the substantial potential of naturally occurring livestock carriers of orthologues of human functional variants to disentangle their functional impacts.

Whole-Genome Sequence Data Suggest Environmental Adaptation of Ethiopian Sheep Populations.

Great progress has been made over recent years in the identification of selection signatures in the genomes of livestock species. This work has primarily been carried out in commercial breeds for which the dominant selection pressures are associated with artificial selection. As agriculture and food security are likely to be strongly affected by climate change, a better understanding of environment-imposed selection on agricultural species is warranted. Ethiopia is an ideal setting to investigate environmental adaptation in livestock due to its wide variation in geo-climatic characteristics and the extensive genetic and phenotypic variation of its livestock. Here, we identified over three million single nucleotide variants across 12 Ethiopian sheep populations and applied landscape genomics approaches to investigate the association between these variants and environmental variables. Our results suggest that environmental adaptation for precipitation-related variables is stronger than that related to altitude or temperature, consistent with large-scale meta-analyses of selection pressure across species. The set of genes showing association with environmental variables was enriched for genes highly expressed in human blood and nerve tissues. There was also evidence of enrichment for genes associated with high-altitude adaptation although no strong association was identified with hypoxia-inducible-factor (HIF) genes. One of the strongest altitude-related signals was for a collagen gene, consistent with previous studies of high-altitude adaptation. Several altitude-associated genes also showed evidence of adaptation with temperature, suggesting a relationship between responses to these environmental factors. These results provide a foundation to investigate further the effects of climatic variables on small ruminant populations.

Clinical Evaluation of Corridor Disease in Bos indicus (Boran) Cattle Naturally Infected With Buffalo-Derived Theileria parva.

Corridor disease (CD) is a fatal condition of cattle caused by buffalo-derived Theileria parva. Unlike the related condition, East Coast fever, which results from infection with cattle-derived T. parva, CD has not been extensively studied. We describe in detail the clinical and laboratory findings in cattle naturally infected with buffalo-derived T. parva. Forty-six cattle were exposed to buffalo-derived T. parva under field conditions at the Ol Pejeta Conservancy, Kenya, between 2013 and 2018. The first signs of disease observed in all animals were nasal discharge (mean day of onset was 9 days post-exposure), enlarged lymph nodes (10 days post-exposure), and pyrexia (13.7 days post-exposure). Coughing and labored breathing were observed in more than 50% of animals (14 days post-exposure). Less commonly observed signs, corneal edema (22%) and diarrhea (11%), were observed later in the disease progression (19 days post-exposure). All infections were considered clinically severe, and 42 animals succumbed to infection. The mean time to death across all studies was 18.4 days. The mean time from onset of clinical signs to death was 9 days and from pyrexia to death was 4.8 days, indicating a relatively short duration of clinical illness. There were significant relationships between days to death and the days to first temperature (chi2 = 4.00, p = 0.046), and days to peak temperature (chi2 = 25.81, p = 0.001), animals with earlier onset pyrexia died sooner. These clinical indicators may be useful for assessing the severity of disease in the future. All infections were confirmed by the presence of macroschizonts in lymph node biopsies (mean time to parasitosis was 11 days). Piroplasms were detected in the blood of two animals (4%) and 20 (43%) animals seroconverted. In this study, we demonstrate the successful approach to an experimental field study for CD in cattle. We also describe the clinical progression of CD in naturally infected cattle, including the onset and severity of clinical signs and pathology. Laboratory diagnoses based on examination of blood samples are unreliable, and alternatives may not be available to cattle keepers. The rapid development of CD requires recognition of the clinical signs, which may be useful for early diagnosis of the disease and effective intervention for affected animals.