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.
	<ol>
		<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>
			<td style="width:189px;">BDSC</td>
			<td style="width:191px;">#24871</td>
			<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>
			<td height="25" style="height:25px;width:189px;">BDSC</td>
			<td style="width:191px;">#24872</td>
			<td style="width:385px;">Specific Reagent: VK37 (2nd chromosome) Injection line</td>
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			<td height="25" style="height:25px;width:705px;">Plasmid DNA</td>
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			<td height="25" style="height:25px;width:325px;">Cloning vector</td>
			<td style="width:189px;">Thermo Fisher</td>
			<td style="width:191px;">#12536-017</td>
			<td style="width:385px;">Specific Reagent: pDONR221</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 height="25" style="height:25px;width:705px;">Molecular biology kits and reagents</td>
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			<td style="width:189px;">Sigma-Aldrich</td>
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			<td style="width:385px;">Specific Reagent: Agarose (molecular biology grade)</td>
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			<td height="25" style="height:25px;width:325px;">Chemically Competent Cells (E. coli)</td>
			<td style="width:189px;">Thermo Fisher</td>
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			<td style="width:385px;">Specific Reagent: DH5&#945;</td>
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			<td height="25" style="height:25px;width:325px;">DNA Gel Extraction kit</td>
			<td style="width:189px;">Thermo Fisher</td>
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			<td style="width:385px;">Specific Reagent: PureLink Gel Extraction Kit</td>
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			<td height="25" style="height:25px;width:325px;">DNA Isolation and purification kit</td>
			<td style="width:189px;">Qiagen</td>
			<td style="width:191px;">#27104</td>
			<td style="width:385px;">Specific Reagent: QIAprep Spin Miniprep Kit</td>
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			<td height="25" style="height:25px;width:325px;">High Fidelity Polymerase</td>
			<td style="width:189px;">NEB</td>
			<td style="width:191px;">#M0491</td>
			<td style="width:385px;">Specific Reagent: Q5 Polymerase kit</td>
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			<td height="75" style="height:75px;width:325px;">Recombinase mediated cloning system</td>
			<td style="width:189px;">Thermo Fisher</td>
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			<td style="width:385px;">Specific Reagent: Gateway BP Clonase kit</td>
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			<td height="75" style="height:75px;width:325px;">Recombinase mediated cloning system</td>
			<td height="50" style="height:50px;width:189px;">Thermo Fisher</td>
			<td style="width:191px;">#11791100</td>
			<td style="width:385px;">Specific Reagent: Gateway LR Clonase II Enzyme kit</td>
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			<td height="50" style="height:50px;width:325px;">Site Directed Mutagenesis kit</td>
			<td style="width:189px;">Agilent</td>
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			<td style="width:385px;">Specific Reagent: Quick Change II Mutagenesis kit</td>
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			<td height="25" style="height:25px;width:705px;">Electroretinogram Rig related equipment</td>
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			<td height="50" style="height:50px;width:325px;">ERG Analysis</td>
			<td style="width:189px;">Molecular Devices</td>
			<td style="width:191px;">N/A</td>
			<td style="width:385px;">Specific Reagent: Axon pCLAMP 10 Data Software Package</td>
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			<td height="50" style="height:50px;width:325px;">ERG Data Collection</td>
			<td style="width:189px;">LabX</td>
			<td style="width:191px;">#R150358</td>
			<td style="width:385px;">Specific Reagent: ISO-DAM Isolated Biologic Amplifier</td>
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			<td height="25" style="height:25px;width:325px;">ERG Stimulator</td>
			<td style="width:189px;">Astro-Med</td>
			<td style="width:191px;">#S48</td>
			<td style="width:385px;">Specific Reagent: Square Pulse Stimulator</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 ...

This protocol allows us to determine whether missense variants in human genes that are linked to diseases affect protein function. This is difficult to accomplish computationally even with state-of-the-art algorithms. This technique allows a rapid analysis of missense variants in human proteins using an in vivo system Drosophila melanogaster which is faster and more cost efficient than vertebrate model organisms.Demonstrating that a specific variant affects protein function directly contributes to the diagnosis of rare diseases and serves as a starting point for disease mechanism analysis and potential therapies. This protocol is particularly useful when studying variants linked to rare diseases, but it can be applied to more common diseases like autism and cancer. Identifying reproducible phenotypes in flies that reflect protein function can be challenging.The phenotypes may vary from gene to gene and can be subtle. Functional studies of variants in human proteins using Drosophila can be performed based on rescue experiments or overexpression studies. Before beginning the procedure, gather information about the human gene of interest and its model organism orthologs.To test variant function by overexpressing reference and variant human proteins in the fly visual system, generate transgenic flies that express reference or variant human cDNAs of interest under the control of the Gal4 upstream activating sequence or UAS system. To select a specific Gal4 line to express the human proteins in a specific fly tissue, cross three to five virgin females from the Gal4 line with three to five males from each of the UAS human cDNA transgenic lines in single vials or in duplicates. Transfer the crosses every two to three days to obtain as many animals eclosing from a single cross as possible and examine the eclosed animal under a dissection microscope to identify any differences between the reference and variant strains.If there is a visible defect, image the flies to document the phenotypes. To generate flies to test for functional defects in the visual system, cross virgin females from the rhodopsin 1 Gal4 line to males with reference or variant UAS human cDNA transgenes to express the human proteins of interest in the photoreceptors R1 to R6 in the fly retina. Once the flies begin to eclose, gather the progeny into fresh vials and return the vials to an incubator set to the appropriate experimental temperature for an additional three days to allow the visual system to mature.At the end of the incubation, immobilize the flies with an appropriate anesthesia method and gently glue one side of each fly onto a glass microscope slide. After setting up an electroretinogram rig, place a 1.2 millimeter glass capillary into a needle puller and break the capillary tube to obtain two sharp hollow tapered electrodes with less than 0.5 millimeter diameters. Fill the capillaries with 100 millimolar saline solution taking care to avoid air bubbles and slide the glass capillaries over the silver wire electrodes.Clamp the capillaries in place and acclimate the flies to complete darkness for at least 10 minutes at room temperature. At the end of the habituation, place the slide containing the flies onto the recording apparatus and move the micromanipulators carrying the reference and recording electrodes close to the fly of interest. Watching the tip of the electrode, carefully place the reference electrode into the thorax of the fly penetrating the cuticle and place the recording electrode on the surface of the eye.For successful electroretinogram recordings, take care to apply an appropriate amount of pressure to the recording electrode so that it causes a small dimple without penetrating the eye. When the electrodes have been placed, turn off the primary light for three minutes to re-acclimate the flies to the dark environment. At the end of the re-acclimation period, open and close the shutter once a second for 20 seconds recording the electroretinograms from at least 15 flies per slide per genotype per condition using the same electrodes.When the recordings from the reference and variant flies are complete, compare the electroretinogram recordings from the reference, variant and controls to assess for differences. Then evaluate the electroretinogram data for changes in on transients, depolarization, off transients and repolarization. In this representative experiment, overexpression-based studies were performed for TBX2 since the human reference TBX2 was unable to functionally replace the orthologous fly gene making rescue-based strategies impossible.When reference human TBX2 was overexpressed in the developing eye and parts of the brain using eyeless Gal4, the overexpression results in an approximately 85%lethality and a significant reduction in the eye size. In contrast, the variant transgene is less potent in causing lethality and induces a small eye phenotype using the same driver under identical experimental conditions suggesting that the variant affects protein function. In addition, when a reference human TBX2 is overexpressed in the photoreceptors using rhodopsin Gal4, the overexpression causes a significant alteration in the electroretinogram trace.This phenotype is milder when the variant protein is expressed providing further evidence that the variant of interest affects protein function in vivo. It is important to note that every gene is unique so there is no single experiment that can assess the impact of all disease associated variants. In addition to working with human cDNAs, one can also determine if the variant has functional consequences by introducing analogous variants into evolutionarily conserved residues of the orthologous fly gene.Using this method, we have contributed to a number of human disease gene discovery studies in collaboration with the Undiagnosed Disease Network and other groups.

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13.4K 조회수.  Baylor College of Medicine. 이 프로토콜은 우리가 질병에 연결되는 인간 적인 유전자에 있는 잘못된 감각 이체가 단백질 기능에 영향을 미치는지 결정하는 것을 허용합니다. 이는 최첨단 알고리즘에서도 계산적으로 수행하기가 어렵습니다. 이 기술은 척추 동물 모델 유기체보다 빠르고 비용 효율적인 생체 내 시스템 Drosophila 멜라노가스터를 사용하여 인간의 단백질에 있는 잘못된 감각 이체의 신속한 ...