Interpreting human genetic variation with in vivo zebrafish assays

Davis, Erica E.; Frangakis, Stephan; Katsanis, Nicholas

doi:10.1016/j.bbadis.2014.05.024

Cited by 69 publications

(48 citation statements)

References 96 publications

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“…Zebrafish has been increasingly used to validate candidate disease genes because they represent a model organism that is a good compromise between speed of testing and relative closeness to human biology (Davis et al 2014). For example, a recent, large-scale exome-sequencing project identified genes putatively causing developmental disorders.…”

Section: Discussionmentioning

confidence: 99%

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

et al. 2015

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The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis.Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloningfree single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F 1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/ Cas9 is sixfold more efficient than other techniques. We show that the majority of published "rules" for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5 ′ end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.

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Section: Discussionmentioning

confidence: 99%

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

et al. 2015

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“…Both our group and others have shown that in vivo complementation assays in zebrafish embryos are a sensitive and specific approach to test the pathogenicity of missense mutations implicated in human genetic disease (Zaghloul et al 2010; Wan et al 2012;Niederriter et al 2013;Davis et al 2014). To test the effect of p.K78E on RPL10 function, coinjection of rpl10 tb-MO with mRNA harboring p.K78E failed to rescue the morphant phenotype and resulted in significantly decreased forebrain cross-sectional area (43,390 mm 2 ) when compared to the WT rescue (60,203 mm 2 ; P , 0.0001; n = 30; repeated twice); body length was not affected (P = 0.34; n = 30; Figure 2, B and C, Table S3).…”

Section: Rpl10 Mutation Causes Microcephaly 727mentioning

confidence: 99%

A Novel Ribosomopathy Caused by Dysfunction of RPL10 Disrupts Neurodevelopment and Causes X-Linked Microcephaly in Humans

et al. 2014

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Neurodevelopmental defects in humans represent a clinically heterogeneous group of disorders. Here, we report the genetic and functional dissection of a multigenerational pedigree with an X-linked syndromic disorder hallmarked by microcephaly, growth retardation, and seizures. Using an X-linked intellectual disability (XLID) next-generation sequencing diagnostic panel, we identified a novel missense mutation in the gene encoding 60S ribosomal protein L10 (RPL10), a locus associated previously with autism spectrum disorders (ASD); the p.K78E change segregated with disease under an X-linked recessive paradigm while, consistent with causality, carrier females exhibited skewed X inactivation. To examine the functional consequences of the p.K78E change, we modeled RPL10 dysfunction in zebrafish. We show that endogenous rpl10 expression is augmented in anterior structures, and that suppression decreases head size in developing morphant embryos, concomitant with reduced bulk translation and increased apoptosis in the brain. Subsequently, using in vivo complementation, we demonstrate that p.K78E is a loss-of-function variant. Together, our findings suggest that a mutation within the conserved N-terminal end of RPL10, a protein in close proximity to the peptidyl transferase active site of the 60S ribosomal subunit, causes severe defects in brain formation and function. N EURODEVELOPMENTAL defects in humans represent a diagnostic challenge. Displaying marked phenotypic overlap, examples include autism spectrum disorders (ASD), intellectual disability (ID), microcephaly, and seizures; in some instances, common genetic defects can underscore each of these clinical entities. For example, mutations in the voltage-gated sodium channel Na v 1.2 encoded by SCN2A are associated with the manifestation of early infantile epilepsy (Sugawara et al. 2001). However, recent exome sequencing studies have also identified SCN2A mutations as rare contributors to disease in autism cohorts, thereby expanding the phenotypic spectrum underscored by Na v 1.2 channel dysfunction (Sanders et al. 2012). A gender bias of 1.3-1.4 males to 1 female with a neurodevelopmental disorder has complicated further our mechanistic understanding of such defects (Leonard and Wen 2002;Ellison et al. 2013). One obvious explanation for an unbalanced representation of the sexes among individuals with a structural or functional brain defect is an abundance of developmentally important genes on the X chromosome. To date, .100 genes have been associated with ASD, ID, microcephaly, or seizures primarily in hemizygous males and, to some extent, their carrier mothers (De Brouwer et al. 2007;Tarpey et al. 2009;Lubs et al. 2012).Here, we report the genetic dissection of a novel form of X-linked human genetic disease characterized by microcephaly, seizures, growth retardation, and hypotonia. Combined genetic, functional, and biochemical assays suggest that a missense mutation in RPL10, a component of the 60S large ribosomal subunit, can cause syndromic central nervo...

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“…Although computational methods are beginning to find acceptance as clinical diagnostic tools (Richards et al 2015), they have limited predictive power (Mathe et al 2006;Chan et al 2007;Cline and Karchin 2011;Thusberg et al 2011;Castellana and Mazza 2013;Frousios et al 2013;Gnad et al 2013). Experimental assessment of variant function in human cells is hampered by inefficient allele replacement technology and by the presence of paralogs with overlapping functions, making complementation testing in 'humanized' model organisms an attractive alternative (Marini et al 2008;Trevisson et al 2009;Mayfield et al 2012;Davis et al 2014;Laurent et al 2015). Complementation of mutant versions of model organism genes by cognate human genes is a classic method to identify human gene function (Lee and Nurse 1987;Levitan et al 1996;Osborn and Miller 2007;Keegan et al 2011).…”

mentioning

confidence: 99%

“…Complementation of mutant versions of model organism genes by cognate human genes is a classic method to identify human gene function (Lee and Nurse 1987;Levitan et al 1996;Osborn and Miller 2007;Keegan et al 2011). Such complementation relationships can then be exploited to assess the impact of amino acid changes in human proteins Cox 1994, 1995;Marini et al 2008;Trevisson et al 2009;Wei et al 2010;Mayfield et al 2012;Dimster-Denk et al 2013;Davis et al 2014). Like computational methods, complementation assays in experimentally tractable model organisms allow functional assessment of human variation at a scale that is commensurate with the size of the human population.…”

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confidence: 99%

An extended set of yeast-based functional assays accurately identifies human disease mutations

et al. 2016

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We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease-and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods.

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Interpreting human genetic variation with in vivo zebrafish assays

Cited by 69 publications

References 96 publications

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

A Novel Ribosomopathy Caused by Dysfunction of RPL10 Disrupts Neurodevelopment and Causes X-Linked Microcephaly in Humans

An extended set of yeast-based functional assays accurately identifies human disease mutations

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