Name: in vivo functional study of disease-associated rare human variants using drosophila
Uploaded: 2019-08-20T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila at JoVE.com

We thank Jose Salazar, Julia Wang, and Dr. Karen Schulze for critical reading of the manuscript. We acknowledge Drs. Ning Liu and Xi Luo for the functional characterization of the TBX2 variants discussed here. Undiagnosed Diseases Network Model Organisms Screening Center was supported through the National Institutes of Health (NIH) Common Fund (U54 NS093793). H. T. C. was further supported by the NIH[CNCDP-K12 and NINDS (1K12 NS098482)], American Academy of Neurology (Neuroscience Research grant), Burroughs Wellcome Fund (Career Award for Medical Scientists), Child Neurology Society and Child Neurology Foundation (PERF Elterman grant), and the NIH Director&#8217;s Early Independence Award (DP5 OD026426). M. F. W. was further supported by Simons Foundation (SFARI Award: 368479). S. Y. was further supported by the NIH (R01 DC014932), the Simons Foundation (SFARI Award: 368479), the Alzheimer&#8217;s Association (New Investigator Research Grant: 15-364099), Naman Family Fund for Basic Research, and Caroline Wiess Law Fund for Research in Molecular Medicine. Confocal microscopy at BCM is supported in part by NIH Grant U54HD083092 to the Intellectual and Developmental Disabilities Research Center (IDDRC) Neurovisualization Core.

1. Gathering Human and MO Information to Assess: Likelihood of A Variant of Interest being Responsible for Disease Phenotypes and Feasibility of Functional Studies in Drosophila
<ol>
	<li>Perform extensive database and literature searches to determine whether the specific genes and variants of interest are good candidates to explain the phenotype of the patient of interest. Specifically, gather the following information.
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		<li>Assess if the gene of interest has been previously implicated...

Experimental studies using Drosophila melanogaster provide a robust assay system to assess the consequences of disease-associated human variants. This is due to the large body of knowledge and diverse genetic tools that have been generated by many researchers in the fly field over the past century89. Just like any other experimental system, however, it is important to acknowledge the caveats and limitations that exist.
Caveats Associated with Data Minin...

Patients with rare diseases often undergo an arduous journey referred to as the &#34;diagnostic odyssey&#34;&#160;to obtain an accurate diagnosis1. Most rare diseases are thought to have a strong genetic origin, making genetic/genomic analyses critical elements of the clinical workup. In addition to candidate gene panel sequencing and copy number variation analysis based on chromosomal microarrays, whole-exome (WES) and whole-genome sequencing (WGS) technologies have become increasingly valuable tools over the past decade2,3. Currently, the diagnostic rate for identifying a known pathogenic variant in WES and WGS is ~25% (higher in pediatric cases)4,5. For most cases that remain undiagnosed after clinical WES/WGS, a common issue is that there are many candidate genes and variants. Next-generation sequencing often identifies novel or ultra-rare variants in many genes, and interpreting whether these variants contribute to disease phenotypes is challenging. For example, although most nonsense or frameshift mutations in genes are thought to be loss-of-function (LOF) alleles due to nonsense-mediated decay of the encoded transcript, truncating mutations found in the last exons escape this process and may function as benign or gain-of-function (GOF) alleles6.
Moreover, predicting the effects of a missense allele is a daunting task, since it can result in a number of different genetic scenarios as first described by Herman Muller in the 1930s (i.e., amorph, hypomorph, hypermorph, antimorph, neomorph, or isomorph)7. Numerous in silico programs and methodologies have been developed to predict the pathogenicity of missense variants based on evolutionary conservation, type of amino acid change, position within a functional domain, allele frequency in the general population, and other parameters8. However, these programs are not a comprehensive solution to solving the complicated problem of variant interpretation. Interestingly, a recent study demonstrated that five broadly used variant pathogenicity prediction algorithms (Polyphen9, SIFT10, CADD11, PROVEAN12, Mutation Taster) agree on pathogenicity ~80% of the time8. Notably, even when all algorithms agree, they return an incorrect prediction of pathogenicity up to 11% of the time. This not only leads to flawed clinical interpretation but also may dissuade researchers from following up on new variants by falsely listing them as benign. One way to complement the current limitation of in silico modeling is to provide experimental data that demonstrates the effect of variant function in vitro, ex vivo (e.g., cultured cells, organoids), or in vivo.
In vivo functional studies of rare disease associated variants in MO have unique strengths13 and have been adopted by many rare disease research initiatives around the world, including the Undiagnosed Diseases Network (UDN) in the United States and Rare Diseases Models &#38; Mechanisms (RDMM) Networks in Canada, Japan, Europe, and Australia14. In addition to these coordinated efforts to integrate MO researchers into the workflow of rare disease diagnosis and mechanistic studies at a national scale, a number of individual collaborative studies between clinical and MO researchers have led to the discovery and characterization of many new human disease-causing genes and variants82,83,84.
In the UDN, a centralized Model Organisms Screening Center (MOSC) receives submissions of candidate genes and variants with a description of the patient&#8217;s condition and assesses whether the variant is likely to be pathogenic using informatics tools and in vivo experiments. In Phase I (2015-2018) of the UDN, the MOSC comprised of a Drosophila Core [Baylor College of Medicine (BCM)] and Zebrafish Core (University of Oregon) that worked collaboratively to assess cases. Using informatics analysis and a number of different experimental strategies in Drosophila and zebrafish, the MOSC has so far contributed to the diagnosis of 132 patients, identification of 31 new syndromes55, discovery of several new human disease genes (e.g., EBF315, ATP5F1D16, TBX217, IRF2BPL18, COG419, WDR3720) and phenotypic expansion of known disease genes (e.g., CACNA1A21, ACOX122).
In addition to projects within the UDN, MOSC Drosophila Core researchers have contributed to new disease gene discoveries in collaboration with the Centers for Mendelian Genomics and other initiatives (e.g., ANKLE223, TM2D324, NRD125, OGDHL25, ATAD3A26, ARIH127, MARK328, DNMBP29) using the same set of informatics and genetic strategies developed for the UDN. Given the significance of MO studies on rare disease diagnosis, the MOSC was expanded to include a C. elegans Core and second Zebrafish core (both at Washington University at St. Louis) for Phase II (2018-2022) of the UDN.
This manuscript describes an in vivo functional study protocol that is actively used in the UDN MOSC Drosophila Core to determine if missense variants have functional consequences on the protein of interest using transgenic flies that express human proteins. The goal of this protocol is to help MO researchers work collaboratively with clinical research groups to provide experimental evidence that a candidate variant in a gene of interest has functional consequences, thus facilitating clinical diagnosis. This protocol is most useful in a scenario in which a Drosophila researcher is approached by a clinical investigator who has a rare disease patient with a specific candidate variant in a gene of interest.
This protocol can be broken down into three elements: (1) gathering information to assess the likelihood of the variant of interest being responsible for the patient phenotype and the feasibility of a functional study in Drosophila, (2) gathering existing genetic tools and establishing new ones, and (3) performing functional studies in vivo. The third element can further be subdivided into two sub-elements based on how the function of a variant of interest can be assessed (rescue experiment or overexpression-based strategies). It is important to note that this protocol can be adapted and optimized to many scenarios outside of rare monogenic disease research (e.g., common diseases, gene-environment interactions, and pharmacological/genetic screens to identify therapeutic targets). The ability to determine the functionality and pathogenicity of variants will not only benefit the patient of interest by providing accurate molecular diagnosis but will also have broader impacts on both translational and basic scientific research.

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			<td height="21" style="height:22px;width:705px;">Drosophila Stocks for UAS-human cDNA transgenesis</td>
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			<td height="54" style="height:54px;width:325px;">Injection strains for transgenesis (D. melanogaster)</td>
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			<td style="width:385px;">Specific Reagent: VK33 (3rd chromosome) Injection line</td>
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			<td height="54" style="height:54px;width:325px;">Injection strains for transgenesis (D. melanogaster)</td>
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			<td style="width:385px;">Specific Reagent: VK37 (2nd chromosome) Injection line</td>
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			<td height="25" style="height:25px;width:325px;">Drosophila transgenesis vector</td>
			<td style="width:380px;">Gift from Drs. Johannes Bischof and Konrad Basler (Bischof et al., 2013 PNAS)</td>
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			<td style="width:385px;">Specific Reagent: pGW-HA.attB</td>
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			<td style="width:385px;">Specific Reagent: Agarose (molecular biology grade)</td>
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			<td style="width:385px;">Specific Reagent: QIAprep Spin Miniprep Kit</td>
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in vivo functional study of disease-associated rare human variants using drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

Functional Study of de novo Missense Variant in EBF3 Linked to Neurodevelopmental Phenotypes 
In a 7 year-old male with neurodevelopmental phenotypes including hypotonia, ataxia, global developmental delay, and expressive speech disorder, physicians and human geneticists at the National Institutes of Health Undiagnosed Diseases Project (UDP) identified a de novo missense variant (p.R163Q) in EBF3 (Early B-Cell Factor 3)15, a gene that encodes a COE ...

Watch this Scientific Journal Video about In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila at JoVE.com

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

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