Genetics of one-carbon metabolism in sheep in relation to productivity, fertility and health

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

This project is focussed on the health, productivity and welfare of sheep. We will concentrate on aspects of metabolism that affect lifelong health and wellbeing. Specifically, we will study a key aspect of metabolism referred to as one-carbon (1C) metabolism. This is important because it affects many key processes in the cell, including DNA synthesis, DNA methylation and cell proliferation. It does this by delivering methyl groups, which are central to these biochemical reactions. Deficiencies in metabolites involved in these pathways, such as choline, methionine, folate and vitamin B12, have adverse effects on animal development and wellbeing. For example, deficiencies in vitamin B12 and/or folate can affect fertility and fetal development, and lead to poor growth, vascular disease and metabolic syndrome in adult animals and humans. 1C metabolism pathways are complex and are affected by many genes. We hypothesise that mutations in these genes will affect 1C metabolism and the vulnerability of animals to micronutrient deficiencies. In this study we will identify such mutations, determine their functional significance (i.e. what they do and how important they are), test their impact in animals fed different diets, and find ways to use this information to improve the welfare of farm animals.

Firstly, we will identify mutations (i.e. single nucleotide polymorphisms or SNPs) in around 40 genes directly affecting 1C metabolism, and in very closely related pathways involved in energy metabolism, cell proliferation, DNA synthesis and DNA methylation. To achieve this in sheep we will create our own sequence data. We estimate that we will identify 2000-4000 SNPs by these methods.

We will then conduct a large-scale study with liver samples collected from around 300 sheep slaughtered at local abattoirs. We will genotype each sample (to determine which SNPs are present), measure gene expression and conduct a comprehensive analysis of all metabolites involved in 1C metabolism. Interpretation of these data will inform us on SNP function and how these SNPs affect pathways involved in 1C metabolism. To achieve this we will collate the required information and, using our knowledge of these genomes combined with complex bioinformatic analyses, determine the subset of SNPs that have the greatest impact on 1C metabolism. We expect to identify an estimated 100 or so functionally significant SNPs in these genes. We will then construct a 'SNP chip'; i.e. a tool to genotype sheep simultaneously and cheaply for many SNPs.

This chip will be used to screen several flocks of sheep to identify 24 'Low-risk' and 24 'High-risk' weaned lambs, and 24 'Low-risk' and 24 'High-risk' breeding ewes. We will then monitor these animals in separate studies involving Control and Methyl-Deficient diets (i.e. two genotypes by two diets). For weaned lambs we will focus on effects on growth, animal health and liver metabolism, and carcass yields and composition. For breeding ewes we will also focus on animal health and liver metabolism, but extend studies to consider effects on chemical modifications to DNA (i.e. DNA methylation) in early (Day 16) male and female embryos.

This research will provide novel insights into nutrient x gene interactions for many components of 1C metabolism and how they affect (a) lamb production efficiency, health and welfare, and (b) early development of mammalian embryos influencing fertility and the long-term health and wellbeing of offspring. We will be able to use this information to help breed animals with better functioning 1C metabolism (leading to permanent improvements in welfare and productivity) and/or to improve animal diets. As 1C metabolism also influences human development and health our results will also have biomedical research benefits, and increase the utility of sheep as a model species for this type of research.

Technical Summary

One-carbon (1C) metabolism delivers methyl groups for use in a plethora of key cellular reactions including DNA synthesis and methylation. Dietary deficiencies in 1C-related metabolites impair lifelong health and wellbeing in humans and farm animals, and reduce farm-animal productivity. We hypothesise that SNPs in genes associated with 1C metabolism alter the sensitivity of animals to micronutrient deficiencies. This project will identify SNPs in 1C metabolism in sheep, determine their functional significance, and confirm their practical relevance in two selection-based nutritional studies involving weaned lambs and breeding ewes. By re-sequencing and data mining, we expect to identify between 2000-4000 SNPs in or near 40 genes encoding enzymes involved in 1C metabolism and closely related pathways related to energy metabolism, cell proliferation, DNA synthesis and DNA/histone methylation. A comprehensive analysis of SNPs, transcripts and all metabolites involved in 1C metabolism will follow using liver samples collected from ca. 300 sheep. Outputs from these experiments will be the source of data for a 'SNP selection' pipeline, to identify an estimated 100 or so functionally significant SNPs, associated with these genes, for chip construction. This chip will be used to screen several sheep flocks to identify 24 'Low-risk' and 24 'High-risk' breeding ewes, and similar groups of lambs, to participate in two separate prospective 2 x 2 factorial studies involving Control and Methyl-Deficient diets. Measureable endpoints will be growth, immune status, liver metabolism and carcass quality (for lambs), liver metabolism and DNA methylation (MBD-Seq) in Day 16 embryonic cells (for breeding ewes). This research will provide novel insights into nutrient x gene interactions for the many components of 1C metabolism with benefits for animal productivity, health and welfare, improving our understanding of how diet can influence long-term epigenetic programming via the germline.

Planned Impact

This BBSRC supported Industrial Partnership Award (with three Levy Boards: EBLEX, HCC and AgriSearch) will advance both fundamental and applied knowledge, with primary non-academic beneficiaries being livestock breeders (producers), companies involved in genetic improvement of sheep, the animal feed industry, veterinarians and the animal health-care pharmaceutical industry. However, we anticipate that the development of a validated software pipeline for associating SNPs with specific changes in metabolism will also be of value to human health-care professionals and the human health-care pharmaceutical industry.

Outputs from this programme (e.g. functional SNP chips, specific dietary advice) may require further industry-sponsored refinement. However, it is probable that specific SNPs in just a few genes will have the greatest influence on 1C metabolism and metabolic outcomes. As this becomes evident so this knowledge can be integrated into practical programmes quickly via the routes outlined below. It is likely that the initial impact of this study will be realised within 3-5 years of project completion, perhaps following further Levy-Board sponsored field-scale studies within the national flock. This is likely to involve a small number of monitor farms where, using Electronic Identification Systems (EID) and following specific pedigrees, we would validate the merits of our approach by (i) quantifying variant-allele frequencies for 1C-metabolism genes and (ii) relating these to measures of animal performance, health and wellbeing. We can also establish a selected resident population of ewes, with contrasting SNP profiles, for future industry-supported studies that could, for example, test the efficacy of pertinent trace-element supplementation strategies at key stages of the life-course (or annual production cycle). This would help identify, for susceptible animals, the most suitable stages of development to mitigate trace-element deficiencies; the efficacy of which would be related to the status of animals identified as being genetically tolerant to such deficiencies.

Differences in digestive metabolism between ruminants and non-ruminants are recognised and, as we have previously demonstrated, can be suitably accommodated when formulating diets. These studies reported similar metabolic responses and developmental effects on insulin resistance in both sheep and rat offspring. Furthermore, we have shown that ewes and women undergoing controlled ovarian stimulation during IVF elicit similar ovarian responses to folate status. Following the successful conclusion of the current proposal we would be able to conduct direct interventional studies (whole animal, cell, embryo culture) in sheep to further address the effects of various SNP combinations on metabolic, developmental and epigenetic responses to varied 1C-metabolite status and relate these observations to parallel human studies. In this regard, we can also make use of the validated software pipeline for associating 1C SNPs with specified changes in metabolism in human studies.

At project completion, and in collaboration with the Levy Boards, we will host seminars/workshops with breed societies from across the UK, to discuss trace-element deficiencies in sheep and the use of contemporary genomic tools to facilitate selection (e.g. via the National Sheep Association (NSA) or via the biennial 'Sheep Breeders Roundtable'). Project completion coincides with the BBSRC Animal Science Forum held in conjunction with the British Society of Animal Science (~500 delegates from industry, the veterinary profession, various public bodies and media). Nottingham also hosts a successful annual meeting (~120 delegates) directed specifically at the animal feed industry. Papers at this meeting are published in the acclaimed series 'Recent Advances in Animal Nutrition', Nottingham University Press. The PI and Co-I Bishop will coordinate these activities.
 
Description Full genome sequences were obtained on 20 unrelated Texel ewes. The animals were sequenced in two pools, to a depth of 30X. These data were submitted to the University of Nottingham partner and passed initial quality control. Reads were mapped to the reference Sheep genome assembly (Oar_v3.1) and the discovery of single nucleotide polymorphisms (SNPs) in these genome sequences is now complete. A total of 22,894,749 SNPs were identified and examined for evidence that they were associated with 48,782 gene transcripts encoded in the genome. 13,824,954 SNPS are located in gene sequences or their regulatory regions and 97,797 represent putative missense mutations within a total of 25,280 transcripts. Prioritisation of genes containing variants was undertaken. All discovered SNPs have or will shortly be deposited into publicly accessible databases. Many of these SNPs represent evidence to support the presence of SNPs reported previoulsy by others. There are currently >60 million putative SNP loci recognised in the sheep genome. Functional annotation of SNP variants was undertaken. Amino acid changing SNPs in coding sequence (452 SNPs, 167 genes) were tested for effects on structure by prediction of crystal structure of all proteins. To assess the potential impact of non-coding variants, the degree of conservation (variation between mammalian species at each orthologous position with the sheep genome) was calculated.

Liver tissue samples were collected by the University of Nottingham partner from 270 Texel lambs for metabolomic, SNP and transcript analyses. A custom SNP genotyping array was designed to allow the genotyping of SNPs within or close to the genes of interest, i.e. genes of relevance to 1-carbon metabolism and epigenetic regulator genes that use SAM as a methyl donor. In designing these arrays sequence and sequence-variant information was derived from i) the current reference sheep genome (Oar_v3.1), ii) earlier re-sequencing of 24 Texel sheep undertaken within the current project, and iii) SNPs in the public databases (e.g. dbSNP). 5,628 identified single nucleotide polymorphisms (SNPs) were provided to Edinburgh Genomics for final validation. Of these 5,550 passed quality control for SNP design. 257 genes of interest were captured by the array design. These redesigned arrays were purchase in October 2016 by Edinburgh Genomics. The identified SNPs are dispersed between coding (1,086) and non-coding sequences (130 splice site 684 UTR and 3650 upstream).

The University of Nottingham partner developed new and refined methodologies using LC-MS/MS for simultaneous measurements of key one-carbon metabolites such as total and mono-glutamated folates, S-adenosylmethionine and S-adenosylhomocysteine.

DNA was prepared from the 270 liver samples supplied to the Edinburgh Genomics facility. The QC of the DNA indicated that the samples were acceptable, in that they contained a lot of high molecular weight DNA. This DNA was subsequently analysed on the custom array.

Quantitative estimates of transcripts for key 1C genes were completed and some incorporated into bioinformatics analyses to identify functional SNPs.

Genetic quantitative trait association analyses (by GenABEL in R) on metabolite concentrations were adjusted for substrates, co-factors and allosteric regulators. Following Bonferroni correction, and re-estimation in ASReml, a prioritised list of 26 SNPs in 15 metabolic genes and 21 SNPs in 12 epigenetic-regulator genes was generated, and these were consistent across covariate categories. A number of interesting variants were identified but others, which we had expected to observe, were absent. This could in part be due to the nutrient status of the animals at the point of slaughter. A retrospective analysis of liver cobalt levels (by ICP-MS) revealed that only around 10% of these animals, from which these samples were obtained, were deficient in this trace element. This is in keeping with prevalence figures established by AHVLA and SAC for the UK.

The most important association to emerge from this study, therefore, was that for CUBN. This gene encodes the protein cubilin, a multi-ligand hydrophobic protein which binds to intrinsic factor-cobalamin (Cbl-IF) complexes with high affinity facilitating B12 uptake from the gut. Variants of the CUBN gene have been shown in human studies to be associated with lower B12 levels due to decreased binding and transport of B12 in the ileum. This selective intestinal B12 malabsorption has been shown to be associated with megaloblastic anaemia. In the current study, when compared to wild-type genotypes, lambs with mutant genotypes for dbrs413573281 and dbrs413234073 polymorphisms in CUBN were found to have decreased liver propionate concentrations. B12 is an essential co-factor to methylmalonyl-CoA mutase (MUT; EC 5.4.99.2), involved in the metabolism of propionate in the liver, prior to gluconeogenesis. The reduction in B12 transport associated with variants of this gene could, therefore, lead to reduced B12 availability for this reaction, and alter the conversion of propionate to succinate in the liver. With this in mind, the genetic association of SNPs in CUBN with varying propionate concentrations are biologically plausible. However, both these SNPs were synonymous and so further research is required to gain a better understanding of their importance and possible mechanism of action.

As a consequence of these findings, we therefore altered the design of the final 'proof-of-concept' study undertaken during 2017. Rather than having two studies (one lamb and one breeding ewe) involving 48 animals each, we elected to undertake a single study with 90 Texel lambs selected (from pedigree records) from several commercial flocks across the UK to ensure no common parents or grandparents. This meant that we had comprehensive representation of the genetic variance that exists within the Texel breed. We fed all animals a methyl-deficient (MD) diet, characterised by deficient levels of sulphur (methionine) and cobalt (vitamin B12), during the summer and early autumn periods of 2017. SNP analysis and genotyping were undertaken, and metabolic status and animal performance recorded. Furthermore, the University of Nottingham partner undertook a further, previously unscheduled, round of comprehensive metabolic profiling in liver samples collected from these deficient animals post mortem. We incorporated data from these 90 deficient animals into the larger database analysed earlier in order to identify a more comprehensive list of functional SNPs.
Exploitation Route We anticipate our results will lead to genetic tests that can be used by the sheep industry to breed animals with enhanced health characteristics.
Sectors Agriculture, Food and Drink

 
Title Custom sheep SNP chip for genes involved in one-carbon metabolism 
Description This DNA chip allows the simultaneous genotyping of ca. 4,000 independent single nucleotide polymorphism (SNP) loci in the sheep genome with an emphasis on SNP loci within or close to genes involved in one-carbon metabolism. This custom sheep SNP chip exploited knowledge of SNP loci in public databases (e.g. dbSNP, EVA) as well as new SNP variants discovered by re-sequencing DNA from Texel sheep. The bioinformatics analyses and SNP selection was undertaken by the University of Nottingham partner. The SNP chip was commissioned from Illumina Inc. The resulting custom sheep SNP chips were used by Edinburgh Genomics to genotype several hundred sheep DNA samples. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The use of the custom sheep SNP chip was essential for genotyping sheep in order to identify putative associations between genetic variation in the target genes and variation in one-carbon metabolites. 
 
Description Genetics of one-carbon metabolism 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution The Roslin Institute partners in this collaboration provide expertise in genome analysis in farmed animal species and expertise in molecular, quantitative and population genetic analyses.
Collaborator Contribution The University of Nottingham group bring expertise in reproductive biology, physiology, epigenetics and bioinformatics as well as the biological questions which the "Genetics of one-carbon metabolism in sheep in relation to productivity, fertility and health" project has been designed to address.
Impact This BBSRC-funded research project (2013-17) supported by grants to University of Nottingham (BB/K017810/1) and The Roslin Institute, University of Edinburgh (BB/K017993/1) is an outcome from this collaborative link that started three years earlier. Whilst this collaboration is multi-disciplinary, the disciplines involved are specialisms within biology: reproductive biology; physiology; genetics; genomics; bioinformatics.
Start Year 2010
 
Description A talk at an international meeting on one carbon metabolism and clinical outcomes 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 12th International conference on One carbon metabolism, B vitamins and homocysteine held in Reus, Catalonia in June 2019. Attended by around 300 delegates including fellow scientists, practitioners, health-care professionals and policy makers from across the world. Delivered a 30 minute talk and discussion on genetics, one carbon metabolism and epigenetic programming. Considerable debate around long-term health implications and some discussion around European policies towards fortification of flour-based foodstuffs with folic acid.
Year(s) Of Engagement Activity 2019
URL http://wwwa.fundacio.urv.cat/congressos/homocysteine-2019/
 
Description A talk at an international meeting on one carbon metabolism and clinical outcomes 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact International meeting of scientists, clinicians and post graduate students investigating the health outcomes of dietary/metabolic disturbances to one-carbon metabolism. Consierable debate and discussion afterwards
Year(s) Of Engagement Activity 2019
URL http://wwwa.fundacio.urv.cat/congressos/homocysteine-2019/
 
Description ESHRE Campus Event - Embryo innovation: the legacy of the past and visions of the future 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact First of two talks at "Embryo innovation: the legacy of the past and visions of the future" ESHRE Campus symposium organised by the Special Interest Group Embryology
Bratislava, Slovakia. Aimed primarily at early career clinicians, nurses and other health-care professionals, the talk covered aspects of parental nutrition during the periconceptional period with emphasis on one carbon metabolism, and long-term epigenetic consequences for offspring health. Lot's of debate and discussion regarding implications on parental diet and fortification of flour with B vitamins.
Year(s) Of Engagement Activity 2019
URL https://www.eshre.eu/Eshre/Login.aspx?returnUrl=%2FSpecialty-groups%2FSpecial-Interest-Groups%2FEmbr...
 
Description ESHRE Campus Event - Embryo innovation: the legacy of the past and visions of the future - Second of two talks 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Second of two talks - this one focusing on safety of IVF in relation to child health. Heavy emphasis once again on one carbon metabolism, as it is affected by ART procedures, and embryo culture media composition. Much debate concerning implications for child health as human IVF labs now encountering problems observed in animal studies stretching back 20-30 years.
Year(s) Of Engagement Activity 2019
URL https://www.eshre.eu/Eshre/Login.aspx?returnUrl=%2FSpecialty-groups%2FSpecial-Interest-Groups%2FEmbr...
 
Description Presentation at Sheep Breeders Round Table meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Poster presentation and discussion with livestock producers (levy payers), feed industry representatives, veterinary practitioners regarding genetics of cobalt deficiency in sheep. Much discussion and debate. Willingness to participate in follow up studies.
Year(s) Of Engagement Activity 2017
URL https://www.nationalsheep.org.uk/sbrt/
 
Description Presentation of interim findings at Fertility 2018 - Liverpool - 959 delegates - research scientists, health-care professionals, students 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation of interim findings - considerable interest and prospect for future collaborative work with the University of Manchester and Innovis Ltd,
(http://www.innovis.org.uk/breeding-sheep/). Best Poster Prize awarded to PhD student (Amey Brassington) by the Society for Reproduction and Fertility: Title:- Genetics of one carbon metabolism: Explaining inter-individual and ethnic variation in epigenetic responses to peri-conceptional diet
Year(s) Of Engagement Activity 2018
URL http://edition.pagesuite-professional.co.uk/html5/reader/production/default.aspx?pubname=&pubid=417c...
 
Description Seminar at the University of Newcastle - Medical and Life Sciences 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Seminar to scientific colleagues and students in Medical and Life Sciences at the University of Newcastle
Year(s) Of Engagement Activity 2018
 
Description Symposium speaker on nutrition and early development 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Sponsored by the Society for Reproduction and Fertility this meeting aimed to increase awareness among fellow scientists, health-care professionals and students (both post graduate and undergraduate) of work and preliminary findings from this particular research award. Related data from two of my PhD students was also presented at this meeting. There were around 80 delegates at this meeting. A group from the University of Manchester have since been in contact regarding possible collaborative clinical studies involving human cohorts.
Year(s) Of Engagement Activity 2017
URL http://srf-reproduction.org/events/past-events/little-embryos-do-make-big-decisions/
 
Description Talk given as industry conference hosted by Trouw Nutrition 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Talk given at LifeStart calf conference in Birmingham - hosted by Trouw Nutrition (Nutreco). Focus on early life, diet and epigenetic programming of development, production performance and calf health.
Year(s) Of Engagement Activity 2018
 
Description Talks at Fertility 2020: an international meeting involving basic scientists, clinicians, nurses and health care professionals - first of two 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Over 1000 delegates attended this event in Edinburgh. Talk initiated questions and discussion - increased awareness of importance of parental diet and nutrition during the periconceptional period. Of interest to IVF clinics and health-care professionals more generally.
Year(s) Of Engagement Activity 2020
URL https://fertilityconference.org/
 
Description Talks at Fertility 2020: an international meeting involving basic scientists, clinicians, nurses and health care professionals - second of two 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Second on two talks - this one focused on efficiency and safety of ART with strong focus on aneuploidy. Generated much debate afterwards - safety of human ART and value of aneuploidy screening. Also - much interest from livestock breeding sector.
Year(s) Of Engagement Activity 2020
URL https://fertilityconference.org/