Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

Bull, Katherine; Rimmer, Andrew; Siggs, Owen M.; Miosge, Lisa A.; Roots, Carla; Enders, Anselm; Bertram, Edward M.; Crockford, Tanya L.; Whittle, Belinda; Potter, Paul K.; Simon, Michelle; Mallon, Ann Marie; Brown, Steve; Beutler, Bruce; Goodnow, Christopher C.; Lunter, Gerton; Cornall, Richard J.

doi:10.1371/journal.pgen.1003219

Cited by 45 publications

(45 citation statements)

References 48 publications

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“…The Fnip1 hamel allele was generated on a pure C57BL/6J background by ENU mutagenesis as previously described (39) Sequencing. Genomic DNA from three C57BL/6J outcrossed hamel mutants was subjected to whole-genome sequencing as described (41). Briefly, 100-bp paired-end libraries were prepared and sequenced on a single lane of an Illumina HiSEq.…”

Section: Methodsmentioning

confidence: 99%

Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity

Siggs

Stockenhuber

Deobagkar-Lele

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Folliculin (FLCN) is a tumor-suppressor protein mutated in the BirtHogg-Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive lossof-function variant of Fnip1. Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51-like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.irt-Hogg-Dubé (BHD) syndrome is a hereditary condition caused by germ-line or somatic mutations of the folliculin (FLCN) tumor-suppressor gene (1) and characterized by cutaneous hamartomas, pulmonary cysts, and an increased risk of renal cancer (2).FLCN forms a complex with paralogous folliculin interacting proteins, folliculin-interacting protein (FNIP)1 and FNIP2, both of which bind the C terminus of FLCN with the potential to form homo-or heterotrimeric multimers (3-5). FLCN is a nonredundant component of the FLCN/FNIP1/FNIP2 complex, whereas FNIP1 and FNIP2 appear to be functionally redundant in several tissues. This distinction is illustrated by the fact that kidney-specific deletion of mouse Flcn or compound deletion of Fnip1 and Fnip2 is sufficient to cause renal tumors, and yet deletions of Fnip1 or Fnip2 alone are not (6). This redundancy is consistent with the equivalent expression of Fnip1 and Fnip2 mRNA in kidney tissue (6). In contrast, FNIP1 is nonredundant in other tissues, including bone marrow, myocardium, and skeletal muscle: all of which express relatively more Fnip1 than Fnip2 (6-9).FNIP1 and FNIP2 also bind to the α, β, and γ subunits of the heterotrimeric AMP-activated protein kinase (AMPK) complex (3, 4). A critical regulator of cellular metabolism, AMPK senses and is activated by increased concentrations of AMP and ADP in the energy-depleted cell and subsequently phosphorylates an array of regulatory targets to restore cellular energy status (10). The multifaceted roles of AMPK include growth suppression by inhibiting synthesis of cellular macromolecules, in particular, through phosphorylation of the TSC2 tumor suppressor and inhibition of the mammalian target of rapamycin complex (mTORC)1 signaling pathway (11). AMPK also promotes autophagy via multiple pathways including mTORC1...

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Section: Methodsmentioning

confidence: 99%

Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity

Siggs

Stockenhuber

Deobagkar-Lele

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…For ENUmutagenized mice, early experiments used massively parallel sequencing for mutation identification within a critical region defined by traditional or bulk segregation mapping using recombinant mapping populations produced by outcrossing the mutant to another inbred laboratory strain and backcrossing or intercrossing a second time (2)(3)(4). Later reports demonstrated mapping with the identified sequence variants themselves as markers, which eliminated the need for outcrossing and its potential for altering the mutant phenotype (5,6). However, whole genome/exome sequencing currently remains very costly to apply as the sole method for genotyping large collections of G3 mice.…”

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confidence: 99%

Real-time resolution of point mutations that cause phenovariance in mice

Wang

Zhan

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.N-ethyl-N-nitrosourea | genetic mapping | forward genetics | mutagenesis | massively parallel sequencing P henotypic variation in mice can be induced with N-ethyl-Nnitrosourea (ENU), which creates single base pair substitutions in germ line DNA. However, the positional cloning of ENU-induced mutations causative for phenotypes of interest has historically been a time-consuming process, beginning with generation of an outcrossed recombinant mapping population of phenotypically mutant and WT mice, genotyping individual mice at genetic markers across the genome to create a linkage map, and finally targeted sequencing to identify the causative mutation within the critical region. The advent of massively parallel sequencing techniques has given rise to more rapid "mapping-bysequencing" methods in which genome-wide marker genotyping and DNA sequencing are combined into a single step applied to either individual or pooled groups of organisms (1). For ENUmutagenized mice, early experiments used massively parallel sequencing for mutation identification within a critical region defined by traditional or bulk segregation mapping using recombinant mapping populations produced by outcrossing the mutant to another inbred laboratory strain and backcrossing or intercrossing a second time (2-4). Later reports demonstrated mapping with the identified sequence variants themselves as markers, which eliminated...

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“…Thus far, several proof-of-principle studies, each applying different approaches to successfully map a chemically induced mutation using WGS, have been documented in the literature (Sarin et al 2008Blumenstiel et al 2009;Zhang et al 2009;Earley and Jones 2011;Fairfield et al 2011;Andrews et al 2012;Bull et al 2013). In general, a subset of SNVs is first removed based on assay-specific criteria upon which some form of mapping is performed to reduce the number of candidate mutations.…”

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confidence: 99%

“…This method has allowed the successful mutation identification of a handful of mutants in flies, worms, zebrafish, and mice, but the number of complementation groups that were mapped per report is limited. It thus remains unclear how scalable this approach is or what its success rate is when one attempts to apply WGS to identify their mutant of interest Earley and Jones 2011;Andrews et al 2012;Leshchiner et al 2012;Bull et al 2013;Henke et al 2013). The drawback of combining meiotic mapping and WGS is that (1) recombination mapping requires several generations of back-crossing and is less straightforward when recessive lethal mutations are being mapped, and (2) in order to sequence multiple animals per genotype, animals are typically pooled and sequenced on one lane of the Illumina sequencer at a low coverage (43-53), which fails to identify many SNVs that are present in the genome.…”

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confidence: 99%

Large-scale identification of chemically induced mutations in Drosophila melanogaster

Haelterman

Jiang

et al. 2014

Genome Res.

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Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of~3500 to 35 single-nucleotide variants per chromosome. By combining WGS with a rough mapping method based on large duplications, we were able to map 274 (~70%) mutations. We show that these mutations are causative, using small 80-kb duplications that rescue lethality. Hence, our findings demonstrate that combining rough mapping with WGS dramatically expands the toolkit necessary for assigning function to genes.

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Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

Cited by 45 publications

References 48 publications

Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity

Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity

Real-time resolution of point mutations that cause phenovariance in mice

Large-scale identification of chemically induced mutations in Drosophila melanogaster

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