Unlocking the bottleneck in forward genetics using whole-genome sequencing and identity by descent to isolate causative mutations

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.The High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics is funded by Wellcome Trust grant reference 090532/Z/09/Z and MRC Hub grant G0900747 91070. This study was supported by Wellcome Trust Strategic Award 082030 (CCG), Wellcome Trust Studentship 094446/Z/10/Z (KRB), the Oxford NIHR Biomedical Research Centre, and the MRC Human Immunology Unit (RJC). AJR and GL were supported by Wellcome Trust grant 090532/Z/ 09/Z, CCG and AE by a Major initiative Award from the Clive and Vera Ramaciotti Foundation, and AE by an NHMRC Career Development Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The International Mouse Phenotyping Consortium (IMPC): a functional catalogue of the mammalian genome that informs conservation.

The International Mouse Phenotyping Consortium (IMPC) is building a catalogue of mammalian gene function by producing and phenotyping a knockout mouse line for every protein-coding gene. To date, the IMPC has generated and characterised 5186 mutant lines. One-third of the lines have been found to be non-viable and over 300 new mouse models of human disease have been identified thus far. While current bioinformatics efforts are focused on translating results to better understand human disease processes, IMPC data also aids understanding genetic function and processes in other species. Here we show, using gorilla genomic data, how genes essential to development in mice can be used to help assess the potentially deleterious impact of gene variants in other species. This type of analyses could be used to select optimal breeders in endangered species to maintain or increase fitness and avoid variants associated to impaired-health phenotypes or loss-of-function mutations in genes of critical importance. We also show, using selected examples from various mammal species, how IMPC data can aid in the identification of candidate genes for studying a condition of interest, deliver information about the mechanisms involved, or support predictions for the function of genes that may play a role in adaptation. With genotyping costs decreasing and the continued improvements of bioinformatics tools, the analyses we demonstrate can be routinely applied

Erratum: Author Correction: Identification of genes required for eye development by high-throughput screening of mouse knockouts.

[This corrects the article DOI: 10.1038/s42003-018-0226-0.]

Finding and sharing: new approaches to registries of databases and services for the biomedical sciences

The recent explosion of biological data and the concomitant proliferation of distributed databases make it challenging for biologists and bioinformaticians to discover the best data resources for their needs, and the most efficient way to access and use them. Despite a rapid acceleration in uptake of syntactic and semantic standards for interoperability, it is still difficult for users to find which databases support the standards and interfaces that they need. To solve these problems, several groups are developing registries of databases that capture key metadata describing the biological scope, utility, accessibility, ease-of-use and existence of web services allowing interoperability between resources. Here, we describe some of these initiatives including a novel formalism, the Database Description Framework, for describing database operations and functionality and encouraging good database practise. We expect such approaches will result in improved discovery, uptake and utilization of data resources. Database URL: http://www.casimir.org.uk/casimir_dd

Mendelian gene identification through mouse embryo viability screening.

BACKGROUND: The diagnostic rate of Mendelian disorders in sequencing studies continues to increase, along with the pace of novel disease gene discovery. However, variant interpretation in novel genes not currently associated with disease is particularly challenging and strategies combining gene functional evidence with approaches that evaluate the phenotypic similarities between patients and model organisms have proven successful. A full spectrum of intolerance to loss-of-function variation has been previously described, providing evidence that gene essentiality should not be considered as a simple and fixed binary property. METHODS: Here we further dissected this spectrum by assessing the embryonic stage at which homozygous loss-of-function results in lethality in mice from the International Mouse Phenotyping Consortium, classifying the set of lethal genes into one of three windows of lethality: early, mid, or late gestation lethal. We studied the correlation between these windows of lethality and various gene features including expression across development, paralogy and constraint metrics together with human disease phenotypes. We explored a gene similarity approach for novel gene discovery and investigated unsolved cases from the 100,000 Genomes Project. RESULTS: We found that genes in the early gestation lethal category have distinct characteristics and are enriched for genes linked with recessive forms of inherited metabolic disease. We identified several genes sharing multiple features with known biallelic forms of inborn errors of the metabolism and found signs of enrichment of biallelic predicted pathogenic variants among early gestation lethal genes in patients recruited under this disease category. We highlight two novel gene candidates with phenotypic overlap between the patients and the mouse knockouts. CONCLUSIONS: Information on the developmental period at which embryonic lethality occurs in the knockout mouse may be used for novel disease gene discovery that helps to prioritise variants in unsolved rare disease cases

Comprehensive ECG reference intervals in C57BL/6N substrains provide a generalizable guide for cardiac electrophysiology studies in mice.

Reference ranges provide a powerful tool for diagnostic decision-making in clinical medicine and are enormously valuable for understanding normality in pre-clinical scientific research that uses in vivo models. As yet, there are no published reference ranges for electrocardiography (ECG) in the laboratory mouse. The first mouse-specific reference ranges for the assessment of electrical conduction are reported herein generated from an ECG dataset of unprecedented scale. International Mouse Phenotyping Consortium data from over 26,000 conscious or anesthetized C57BL/6N wildtype control mice were stratified by sex and age to develop robust ECG reference ranges. Interesting findings include that heart rate and key elements from the ECG waveform (RR-, PR-, ST-, QT-interval, QT corrected, and QRS complex) demonstrate minimal sexual dimorphism. As expected, anesthesia induces a decrease in heart rate and was shown for both inhalation (isoflurane) and injectable (tribromoethanol) anesthesia. In the absence of pharmacological, environmental, or genetic challenges, we did not observe major age-related ECG changes in C57BL/6N-inbred mice as the differences in the reference ranges of 12-week-old compared to 62-week-old mice were negligible. The generalizability of the C57BL/6N substrain reference ranges was demonstrated by comparison with ECG data from a wide range of non-IMPC studies. The close overlap in data from a wide range of mouse strains suggests that the C57BL/6N-based reference ranges can be used as a robust and comprehensive indicator of normality. We report a unique ECG reference resource of fundamental importance for any experimental study of cardiac function in mice