Single nuclei RNA-seq reveals a medium spiny neuron glutamate excitotoxicity signature prior to the onset of neuronal death in an ovine Huntington's disease model.

Huntington's disease (HD) is a neurodegenerative genetic disorder caused by an expansion in the CAG repeat tract of the huntingtin (HTT) gene resulting in behavioural, cognitive, and motor defects. Current knowledge of disease pathogenesis remains incomplete, and no disease course-modifying interventions are in clinical use. We have previously reported the development and characterisation of the OVT73 transgenic sheep model of HD. The 73 polyglutamine repeat is somatically stable and therefore likely captures a prodromal phase of the disease with an absence of motor symptomatology even at 5-years of age and no detectable striatal cell loss. To better understand the disease-initiating events we have undertaken a single nuclei transcriptome study of the striatum of an extensively studied cohort of 5-year-old OVT73 HD sheep and age matched wild-type controls. We have identified transcriptional upregulation of genes encoding N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in medium spiny neurons, the cell type preferentially lost early in HD. Further, we observed an upregulation of astrocytic glutamate uptake transporters and medium spiny neuron GABAA receptors, which may maintain glutamate homeostasis. Taken together, these observations support the glutamate excitotoxicity hypothesis as an early neurodegeneration cascade-initiating process but the threshold of toxicity may be regulated by several protective mechanisms. Addressing this biochemical defect early may prevent neuronal loss and avoid the more complex secondary consequences precipitated by cell death.

ICARUS v3, a massively scalable web server for single-cell RNA-seq analysis of millions of cells.

MOTIVATION: In recent years, improvements in throughput of single-cell RNA-seq have resulted in a significant increase in the number of cells profiled. The generation of single-cell RNA-seq datasets comprising >1 million cells is becoming increasingly common, giving rise to demands for more efficient computational workflows. RESULTS: We present an update to our single-cell RNA-seq analysis web server application, ICARUS (available at https://launch.icarus-scrnaseq.cloud.edu.au) that allows effective analysis of large-scale single-cell RNA-seq datasets. ICARUS v3 utilizes the geometric cell sketching method to subsample cells from the overall dataset for dimensionality reduction and clustering that can be then projected to the large dataset. We then extend this functionality to select a representative subset of cells for downstream data analysis applications including differential expression analysis, gene co-expression network construction, gene regulatory network construction, trajectory analysis, cell-cell communication inference, and cell cluster associations to GWAS traits. We demonstrate analysis of single-cell RNA-seq datasets using ICARUS v3 of 1.3 million cells completed within the hour. AVAILABILITY AND IMPLEMENTATION: ICARUS is available at https://launch.icarus-scrnaseq.cloud.edu.au.

Challenges for gene editing in common variable immunodeficiency disorders: Current and future prospects.

The original CRISPR Cas9 gene editing system and subsequent innovations offers unprecedented opportunities to correct severe genetic defects including those causing Primary Immunodeficiencies (PIDs). Common Variable Immunodeficiency Disorders (CVID) are the most frequent symptomatic PID in adults and children. Unlike many other PIDs, patients meeting CVID criteria do not have a definable genetic defect and cannot be considered to have an inborn error of immunity (IEI). Patients with a CVID phenotype carrying a causative mutation are deemed to have a CVID-like disorder consequent to an IEI. Patients from consanguineous families often have highly penetrant early-onset autosomal recessive forms of CVID-like disorders. Individuals from non-consanguineous families may have autosomal dominant CVID-like disorders with variable penetrance and expressivity. This essay explores the potential clinical utility as well as the current limitations and risks of gene editing including collateral genotoxicity. In the immediate future the main application of this technology is likely to be the in vitro investigation of epigenetic and polygenic mechanisms, which are likely to underlie many cases of CVID and CVID-like disorders. In the longer-term, the CRISPR Cas9 system and other gene-based therapies could be utilized to treat CVID-like disorders, where the underlying IEI is known.

A common regulatory haplotype doubles lactoferrin concentration in milk.

BACKGROUND: Bovine lactoferrin (Lf) is an iron absorbing whey protein with antibacterial, antiviral, and antifungal activity. Lactoferrin is economically valuable and has an extremely variable concentration in milk, partly driven by environmental influences such as milking frequency, involution, or mastitis. A significant genetic influence has also been previously observed to regulate lactoferrin content in milk. Here, we conducted genetic mapping of lactoferrin protein concentration in conjunction with RNA-seq, ChIP-seq, and ATAC-seq data to pinpoint candidate causative variants that regulate lactoferrin concentrations in milk. RESULTS: We identified a highly-significant lactoferrin protein quantitative trait locus (pQTL), as well as a cis lactotransferrin (LTF) expression QTL (cis-eQTL) mapping to the LTF locus. Using ChIP-seq and ATAC-seq datasets representing lactating mammary tissue samples, we also report a number of regions where the openness of chromatin is under genetic influence. Several of these also show highly significant QTL with genetic signatures similar to those highlighted through pQTL and eQTL analysis. By performing correlation analysis between these QTL, we revealed an ATAC-seq peak in the putative promotor region of LTF, that highlights a set of 115 high-frequency variants that are potentially responsible for these effects. One of the 115 variants (rs110000337), which maps within the ATAC-seq peak, was predicted to alter binding sites of transcription factors known to be involved in lactation-related pathways. CONCLUSIONS: Here, we report a regulatory haplotype of 115 variants with conspicuously large impacts on milk lactoferrin concentration. These findings could enable the selection of animals for high-producing specialist herds.

ICARUS, an interactive web server for single cell RNA-seq analysis.

Here we present ICARUS, a web server to enable users without experience in R to undertake single cell RNA-seq analysis. The focal point of ICARUS is its intuitive tutorial-style user interface, designed to guide logical navigation through the multitude of pre-processing, analysis and visualization steps. ICARUS is easily accessible through a dedicated web server (https://launch.icarus-scrnaseq.cloud.edu.au/) and avoids installation of software on the user's computer. Notable features include the facility to apply quality control thresholds and adjust dimensionality reduction and cell clustering parameters. Data is visualized through 2D/3D UMAP and t-SNE plots and may be curated to remove potential confounders such as cell cycle heterogeneity. ICARUS offers flexible differential expression analysis with user-defined cell groups and gene set enrichment analysis to identify likely affected biological pathways. Eleven organisms including human, dog, mouse, rat, zebrafish, fruit fly, nematode, yeast, cattle, chicken and pig are currently supported. Visualization of multimodal data including those generated by CITE-seq and the 10X Genomics Multiome kit is included. ICARUS incorporates a function to save the current state of analysis avoiding computationally intensive steps during repeat analysis. The complete analysis of a typical single cell RNA-seq dataset by inexperienced users may be achieved in 1-2 h.

Soluble wild-type ACE2 molecules inhibit newer SARS-CoV-2 variants and are a potential antiviral strategy to mitigate disease severity in COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease of 2019 (COVID-19), has caused havoc around the world. While several COVID-19 vaccines and drugs have been authorized for use, these antiviral drugs remain beyond the reach of most low- and middle-income countries. Rapid viral evolution is reducing the efficacy of vaccines and monoclonal antibodies and contributing to the deaths of some fully vaccinated persons. Others with normal immunity may have chosen not to be vaccinated and remain at risk if they contract the infection. Vaccines may not protect some immunodeficient patients from SARS-CoV-2, who are also at increased risk of chronic COVID-19 infection, a dangerous stalemate between the virus and a suboptimal immune response. Intra-host viral evolution could rapidly lead to the selection and dominance of vaccine and monoclonal antibody-resistant clades of SARS-CoV-2. There is thus an urgent need to develop new treatments for COVID-19. The NZACE2-Patari project, comprising modified soluble angiotensin-converting enzyme 2 (ACE2) molecules, seeks to intercept and block SARS-CoV-2 infection of the respiratory mucosa. In vitro data presented here show that soluble wild-type ACE2 molecules retain the ability to effectively block the Spike (S) glycoprotein of SARS-CoV-2 variants including the ancestral Wuhan, delta (B.1.617.2) and omicron (B.1.1.529) strains. This therapeutic strategy may prove effective if implemented early during the nasal phase of the infection and may act synergistically with other antiviral drugs such as Paxlovid to further mitigate disease severity.

Natural cryptic variation in epigenetic modulation of an embryonic gene regulatory network.

Gene regulatory networks (GRNs) that direct animal embryogenesis must respond to varying environmental and physiological conditions to ensure robust construction of organ systems. While GRNs are evolutionarily modified by natural genomic variation, the roles of epigenetic processes in shaping plasticity of GRN architecture are not well understood. The endoderm GRN in Caenorhabditis elegans is initiated by the maternally supplied SKN-1/Nrf2 bZIP transcription factor; however, the requirement for SKN-1 in endoderm specification varies widely among distinct C. elegans wild isotypes, owing to rapid developmental system drift driven by accumulation of cryptic genetic variants. We report here that heritable epigenetic factors that are stimulated by transient developmental diapause also underlie cryptic variation in the requirement for SKN-1 in endoderm development. This epigenetic memory is inherited from the maternal germline, apparently through a nuclear, rather than cytoplasmic, signal, resulting in a parent-of-origin effect (POE), in which the phenotype of the progeny resembles that of the maternal founder. The occurrence and persistence of POE varies between different parental pairs, perduring for at least 10 generations in one pair. This long-perduring POE requires piwi-interacting RNA (piRNA) function and the germline nuclear RNA interference (RNAi) pathway, as well as MET-2 and SET-32, which direct histone H3K9 trimethylation and drive heritable epigenetic modification. Such nongenetic cryptic variation may provide a resource of additional phenotypic diversity through which adaptation may facilitate evolutionary changes and shape developmental regulatory systems.

From Pathogenesis to Therapeutics: A Review of 150 Years of Huntington's Disease Research.

Huntington's disease (HD) is a debilitating neurodegenerative genetic disorder caused by an expanded polyglutamine-coding (CAG) trinucleotide repeat in the huntingtin (HTT) gene. HD behaves as a highly penetrant dominant disorder likely acting through a toxic gain of function by the mutant huntingtin protein. Widespread cellular degeneration of the medium spiny neurons of the caudate nucleus and putamen are responsible for the onset of symptomology that encompasses motor, cognitive, and behavioural abnormalities. Over the past 150 years of HD research since George Huntington published his description, a plethora of pathogenic mechanisms have been proposed with key themes including excitotoxicity, dopaminergic imbalance, mitochondrial dysfunction, metabolic defects, disruption of proteostasis, transcriptional dysregulation, and neuroinflammation. Despite the identification and characterisation of the causative gene and mutation and significant advances in our understanding of the cellular pathology in recent years, a disease-modifying intervention has not yet been clinically approved. This review includes an overview of Huntington's disease, from its genetic aetiology to clinical presentation and its pathogenic manifestation. An updated view of molecular mechanisms and the latest therapeutic developments will also be discussed.

Novel PRMT7 mutation in a rare case of dysmorphism and intellectual disability.

Protein arginine N-methyltransferase 7 (PRMT7) encodes an arginine methyltransferase central to a number of fundamental biological processes, mutations in which result in an autosomal recessive developmental disorder characterized by short stature, brachydactyly, intellectual developmental disability and seizures (SBIDDS). To date, fewer than 15 patients with biallelic mutations in PRMT7 have been documented. Here we report brothers from a consanguineous Iraqi family presenting with a developmental disorder characterized by global developmental delay, shortened stature, facial dysmorphisms, brachydactyly, and kidney dysfunction. In both affected brothers, whole genome sequencing (WGS) identified a novel homozygous substitution in PRMT7 (ENST00000339507.5), c.1097 G > A (p.Cys366Tyr), considered to account for the majority of the phenotypic presentation. Rare compound heterozygous mutations in the dysplasia-associated perlecan-encoding HSPG2 gene (ENST00000374695.3) were also found (c.10721-2dupA, p.Ser71Asn and c.212 G > A), potentially accounting for the kidney dysfunction. In addition to expanding the known mutational spectrum of variably expressive PRMT7 mutations alongside potential digenic inheritance with HSPG2, this report underlines the diagnostic utility of a WGS-guided analysis in the detection of rare genetic disorders.

Screening for phenotypic outliers identifies an unusually low concentration of a ß-lactoglobulin B protein isoform in bovine milk caused by a synonymous SNP.

BACKGROUND: Milk samples from 10,641 dairy cattle were screened by a mass spectrometry method for extreme concentrations of the A or B isoforms of the whey protein, ß-lactoglobulin (BLG), to identify causative genetic variation driving changes in BLG concentration. RESULTS: A cohort of cows, from a single sire family, was identified that produced milk containing a low concentration of the BLG B protein isoform. A genome-wide association study (GWAS) of BLG B protein isoform concentration in milk from AB heterozygous cows, detected a group of highly significant single nucleotide polymorphisms (SNPs) within or close to the BLG gene. Among these was a synonymous G/A variation at position + 78 bp in exon 1 of the BLG gene (chr11:103256256G > A). The effect of the A allele of this SNP (which we named B') on BLG expression was evaluated in a luciferase reporter assay in transfected CHO-K1 and MCF-7 cells. In both cell types, the presence of the B' allele in a plasmid containing the bovine BLG gene from -922 to + 898 bp (relative to the transcription initiation site) resulted in a 60% relative reduction in mRNA expression, compared to the plasmid containing the wild-type B sequence allele. Examination of a mammary RNAseq dataset (n = 391) identified 14 heterozygous carriers of the B' allele which were homozygous for the BLG B protein isoform (BB'). The level of expression of the BLG B' allele was 41.9 ± 1.0% of that of the wild-type BLG B allele. Milk samples from three cows, homozygous for the A allele at chr11:103,256,256 (B'B'), were analysed (HPLC) and showed BLG concentrations of 1.04, 1.26 and 1.83 g/L relative to a mean of 4.84 g/L in milk from 16 herd contemporaries of mixed (A and B) BLG genotypes. The mechanism by which B' downregulates milk BLG concentration remains to be determined. CONCLUSIONS: High-throughput screening and identification of outliers, enabled the discovery of a synonymous G > A mutation in exon 1 of the B allele of the BLG gene (B'), which reduced the milk concentration of ß-lactoglobulin B protein isoform, by more than 50%. Milk from cows carrying the B' allele is expected to have improved processing characteristics, particularly for cheese-making.

The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis.

BACKGROUND: Animal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour. RESULTS: No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across the edited and non-edited cloned calves revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, appeared to have a distinct mutation signature where de novo mutations were predominantly C > A mutations, and in cloned calves they were predominantly T > G mutations, deviating from the expected excess of C > T mutations. CONCLUSIONS: We found no detectable CRISPR-Cas9 associated off-target mutations in the gene-edited cells or calves derived from the gene-edited cell line. Comparison of de novo mutation in two gene-edited calves and three non-edited control calves did not reveal a higher mutation load in any one group, gene-edited or control, beyond those anticipated from spontaneous mutagenesis. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.

Widespread cis-regulation of RNA editing in a large mammal.

Post-transcriptional RNA editing may regulate transcript expression and diversity in cells, with potential impacts on various aspects of physiology and environmental adaptation. A small number of recent genome-wide studies in Drosophila, mouse, and human have shown that RNA editing can be genetically modulated, highlighting loci that quantitatively impact editing of transcripts. The potential gene expression and physiological consequences of these RNA-editing quantitative trait loci (edQTL), however, are almost entirely unknown. Here, we present analyses of RNA editing in a large domestic mammal (Bos taurus), where we use whole-genome and high-depth RNA sequencing to discover, characterize, and conduct genetic mapping studies of novel transcript edits. Using a discovery population of nine deeply sequenced cows, we identify 2413 edit sites in the mammary transcriptome, the majority of which are adenosine to inosine edits (98.6%). Most sites are predicted to reside in double-stranded secondary structures (85.1%), and quantification of the rates of editing in an additional 355 cows reveals editing is negatively correlated with gene expression in the majority of cases. Genetic analyses of RNA editing and gene expression highlight 152 cis-regulated edQTL, of which 15 appear to cosegregate with expression QTL effects. Trait association analyses in a separate population of 9989 lactating cows also shows 12 of the cis-edQTL coincide with at least one cosegregating lactation QTL. Together, these results enhance our understanding of RNA-editing dynamics in mammals, and suggest mechanistic links by which loci may impact phenotype through RNA editing mediated processes.

Testes of DAZL null neonatal sheep lack prospermatogonia but maintain normal somatic cell morphology and marker expression.

Multiplying the germline would increase the number of offspring that can be produced from selected animals, accelerating genetic improvement for livestock breeding. This could be achieved by producing multiple chimaeric animals, each carrying a mix of donor and host germ cells in their gonads. However, such chimaeric germlines would produce offspring from both donor and host genotypes, limiting the rate of genetic improvement. To resolve this problem, we disrupted the RNA-binding protein DAZL and generated germ cell-deficient host animals. Using Cas9-mediated homology-directed repair (HDR), we introduced a DAZL loss-of-function mutation in male ovine fetal fibroblasts. Following manual single cell isolation, 4/48 (8.3%) of donor cell strains were homozygously HDR-edited. Sequence-validated strains were used as nuclear donors for somatic cell cloning to generate three lambs, which died at birth. All DAZL null male neonatal sheep lacked germ cells on histological sections and showed greatly reduced germ cell markers. Somatic cells within their testes were morphologically intact and expressed normal levels of lineage-specific markers, suggesting that the germ cell niche remained intact. This extends the DAZL mutant phenotype beyond mice into agriculturally relevant ruminants, providing a pathway for using absolute germline transmitters in rapid livestock improvement.

A Review of the Current Mammalian Models of Alzheimer's Disease and Challenges That Need to Be Overcome.

Alzheimer's disease (AD) is one of the looming health crises of the near future. Increasing lifespans and better medical treatment for other conditions mean that the prevalence of this disease is expected to triple by 2050. The impact of AD includes both the large toll on individuals and their families as well as a large financial cost to society. So far, we have no way to prevent, slow, or cure the disease. Current medications can only alleviate some of the symptoms temporarily. Many animal models of AD have been created, with the first transgenic mouse model in 1995. Mouse models have been beset by challenges, and no mouse model fully captures the symptomatology of AD without multiple genetic mutations and/or transgenes, some of which have never been implicated in human AD. Over 25 years later, many mouse models have been given an AD-like disease and then 'cured' in the lab, only for the treatments to fail in clinical trials. This review argues that small animal models are insufficient for modelling complex disorders such as AD. In order to find effective treatments for AD, we need to create large animal models with brains and lifespan that are closer to humans, and underlying genetics that already predispose them to AD-like phenotypes.

A new mechanism for a familiar mutation - bovine DGAT1 K232A modulates gene expression through multi-junction exon splice enhancement.

BACKGROUND: The DGAT1 gene encodes an enzyme responsible for catalysing the terminal reaction in mammary triglyceride synthesis, and underpins a well-known pleiotropic quantitative trait locus (QTL) with a large influence on milk composition phenotypes. Since first described over 15 years ago, a protein-coding variant K232A has been assumed as the causative variant underlying these effects, following in-vitro studies that demonstrated differing levels of triglyceride synthesis between the two protein isoforms. RESULTS: We used a large RNAseq dataset to re-examine the underlying mechanisms of this large milk production QTL, and hereby report novel expression-based functions of the chr14 g.1802265AA > GC variant that encodes the DGAT1 K232A substitution. Using expression QTL (eQTL) mapping, we demonstrate a highly-significant mammary eQTL for DGAT1, where the K232A mutation appears as one of the top associated variants for this effect. By conducting in vitro expression and splicing experiments in bovine mammary cell culture, we further show modulation of splicing efficiency by this mutation, likely through disruption of an exon splice enhancer as a consequence of the allele encoding the 232A variant. CONCLUSIONS: The relative contributions of the enzymatic and transcription-based mechanisms now attributed to K232A remain unclear; however, these results suggest that transcriptional impacts contribute to the diversity of lactation effects observed at the DGAT1 locus.

Inconsistencies in histone acetylation patterns among different HD model systems and HD post-mortem brains.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in exon 1 of the huntingtin gene. Emerging evidence shows that additional epigenetic factors can modify disease phenotypes. Harnessing the ability of the epigenome to modify the disease for therapeutic purposes is therefore of interest. Epigenome modifiers, such as histone deacetylase inhibitors (HDACi), have improved pathology in a range of HD models. Yet in clinical trials, HDACi have failed to alleviate HD symptoms in patients. This study investigated potential reasons for the lack of translation of the therapeutic benefits of HDACi from lab to clinic. We analysed histone acetylation patterns of immuno-positive nuclei from brain sections and tissue microarrays from post-mortem human control and HD cases alongside several well-established HD models (OVT73 transgenic HD sheep, YAC128 mice, and an in vitro cell model expressing 97Q mutant huntingtin). Significant increases in histone H4 acetylation were observed in post-mortem HD cases, OVT73 transgenic HD sheep and in vitro models; these changes were absent in YAC128 mice. In addition, nuclear labelling for acetyl-histone H4 levels were inversely proportional to mutant huntingtin aggregate load in HD human cortex. Our data raise concerns regarding the utility of HDACi for the treatment of HD when regions of pathology exhibit already elevated histone acetylation patterns and emphasize the importance of searching for alternative epigenetic targets in future therapeutic strategies aiming to rescue HD phenotypes.

Brain urea increase is an early Huntington's disease pathogenic event observed in a prodromal transgenic sheep model and HD cases.

The neurodegenerative disorder Huntington's disease (HD) is typically characterized by extensive loss of striatal neurons and the midlife onset of debilitating and progressive chorea, dementia, and psychological disturbance. HD is caused by a CAG repeat expansion in the Huntingtin (HTT) gene, translating to an elongated glutamine tract in the huntingtin protein. The pathogenic mechanism resulting in cell dysfunction and death beyond the causative mutation is not well defined. To further delineate the early molecular events in HD, we performed RNA-sequencing (RNA-seq) on striatal tissue from a cohort of 5-y-old OVT73-line sheep expressing a human CAG-expansion HTT cDNA transgene. Our HD OVT73 sheep are a prodromal model and exhibit minimal pathology and no detectable neuronal loss. We identified significantly increased levels of the urea transporter SLC14A1 in the OVT73 striatum, along with other important osmotic regulators. Further investigation revealed elevated levels of the metabolite urea in the OVT73 striatum and cerebellum, consistent with our recently published observation of increased urea in postmortem human brain from HD cases. Extending that finding, we demonstrate that postmortem human brain urea levels are elevated in a larger cohort of HD cases, including those with low-level neuropathology (Vonsattel grade 0/1). This elevation indicates increased protein catabolism, possibly as an alternate energy source given the generalized metabolic defect in HD. Increased urea and ammonia levels due to dysregulation of the urea cycle are known to cause neurologic impairment. Taken together, our findings indicate that aberrant urea metabolism could be the primary biochemical disruption initiating neuropathogenesis in HD.

Multiple QTL underlie milk phenotypes at the CSF2RB locus.

BACKGROUND: Over many years, artificial selection has substantially improved milk production by cows. However, the genes that underlie milk production quantitative trait loci (QTL) remain relatively poorly characterised. Here, we investigate a previously reported QTL located at the CSF2RB locus on chromosome 5, for several milk production phenotypes, to better understand its underlying genetic and molecular causes. RESULTS: Using a population of 29,350 taurine dairy cows, we conducted association analyses for milk yield and composition traits, and identified highly significant QTL for milk yield, milk fat concentration, and milk protein concentration. Strikingly, protein concentration and milk yield appear to show co-located yet genetically distinct QTL. To attempt to understand the molecular mechanisms that might be mediating these effects, gene expression data were used to investigate eQTL for 11 genes in the broader interval. This analysis highlighted genetic impacts on CSF2RB and NCF4 expression that share similar association signatures to those observed for lactation QTL, strongly implicating one or both of these genes as responsible for these effects. Using the same gene expression dataset representing 357 lactating cows, we also identified 38 novel RNA editing sites in the 3' UTR of CSF2RB transcripts. The extent to which two of these sites were edited also appears to be genetically co-regulated with lactation QTL, highlighting a further layer of regulatory complexity that involves the CSF2RB gene. CONCLUSIONS: This locus presents a diversity of molecular and lactation QTL, likely representing multiple overlapping effects that, at a minimum, highlight the CSF2RB gene as having a causal role in these processes.

Genome-wide association analysis reveals QTL and candidate mutations involved in white spotting in cattle.

BACKGROUND: White spotting of the coat is a characteristic trait of various domestic species including cattle and other mammals. It is a hallmark of Holstein-Friesian cattle, and several previous studies have detected genetic loci with major effects for white spotting in animals with Holstein-Friesian ancestry. Here, our aim was to better understand the underlying genetic and molecular mechanisms of white spotting, by conducting the largest mapping study for this trait in cattle, to date. RESULTS: Using imputed whole-genome sequence data, we conducted a genome-wide association analysis in 2973 mixed-breed cows and bulls. Highly significant quantitative trait loci (QTL) were found on chromosomes 6 and 22, highlighting the well-established coat color genes KIT and MITF as likely responsible for these effects. These results are in broad agreement with previous studies, although we also report a third significant QTL on chromosome 2 that appears to be novel. This signal maps immediately adjacent to the PAX3 gene, which encodes a known transcription factor that controls MITF expression and is the causal locus for white spotting in horses. More detailed examination of these loci revealed a candidate causal mutation in PAX3 (p.Thr424Met), and another candidate mutation (rs209784468) within a conserved element in intron 2 of MITF transcripts expressed in the skin. These analyses also revealed a mechanistic ambiguity at the chromosome 6 locus, where highly dispersed association signals suggested multiple or multiallelic QTL involving KIT and/or other genes in this region. CONCLUSIONS: Our findings extend those of previous studies that reported KIT as a likely causal gene for white spotting, and report novel associations between candidate causal mutations in both the MITF and PAX3 genes. The sizes of the effects of these QTL are substantial, and could be used to select animals with darker, or conversely whiter, coats depending on the desired characteristics.

Evaluation of the performance of copy number variant prediction tools for the detection of deletions from whole genome sequencing data.

BACKGROUND: Whole genome sequencing (WGS) has increased in popularity and decreased in cost over the past decade, rendering this approach as a viable and sensitive method for variant detection. In addition to its utility for single nucleotide variant detection, WGS data has the potential to detect Copy Number Variants (CNV) to fine resolution. Many CNV detection software packages have been developed exploiting four main types of data: read pair, split read, read depth, and assembly based methods. The aim of this study was to evaluate the efficiency of each of these main approaches in detecting germline deletions. METHODS: WGS data and high confidence deletion calls for the individual NA12878 from the Genome in a Bottle consortium were the benchmark dataset. The performance of BreakDancer, CNVnator, Delly, FermiKit, and Pindel was assessed by comparing the accuracy and sensitivity of each software package in detecting deletions exceeding 1 kb. RESULTS: There was considerable variability in the outputs of the different WGS CNV detection programs. The best performance was seen from BreakDancer and Delly, with 92.6% and 96.7% sensitivity, respectively and 34.5% and 68.5% false discovery rate (FDR), respectively. In comparison, Pindel, CNVnator, and FermiKit were less effective with sensitivities of 69.1%, 66.0%, and 15.8%, respectively and FDR of 91.3%, 69.0%, and 31.7%, respectively. Concordance across software packages was poor, with only 27 of the total 612 benchmark deletions identified by all five methodologies. CONCLUSIONS: The WGS based CNV detection tools evaluated show disparate performance in identifying deletions ≥1 kb, particularly those utilising different input data characteristics. Software that exploits read pair based data had the highest sensitivity, namely BreakDancer and Delly. BreakDancer also had the second lowest false discovery rate. Therefore, in this analysis read pair methods (BreakDancer in particular) were the best performing approaches for the identification of deletions ≥1 kb, balancing accuracy and sensitivity. There is potential for improvement in the detection algorithms, particularly for reducing FDR. This analysis has validated the utility of WGS based CNV detection software to reliably identify deletions, and these findings will be of use when choosing appropriate software for deletion detection, in both research and diagnostic medicine.