Studies in Drosophila help implicate a variant in the human MTSS2/MTSS1L gene in a a syndromic form of intellectual disability

Am J Hum Genet. 2022 Oct 6;109(10):1923-1931. doi: 10.1016/j.ajhg.2022.08.011

The recurrent de novo c.2011C>T missense variant in MTSS2 causes syndromic intellectual disability

Huang Y, Lemire G, Briere LC, Liu F, Wessels MW, Wang X, Osmond M, Kanca O, Lu S, High FA, Walker MA, Rodan LH; Undiagnosed Diseases Network; Care4Rare Canada Consortium, Kernohan KD, Sweetser DA, Boycott KM, Bellen HJ

Abstract:

MTSS2, also known as MTSS1L, binds to plasma membranes and modulates their bending. MTSS2 is highly expressed in the central nervous system (CNS) and appears to be involved in activity-dependent synaptic plasticity. Variants in MTSS2 have not yet been associated with a human phenotype in OMIM. Here we report five individuals with the same heterozygous de novo variant in MTSS2 (GenBank: NM_138383.2: c.2011C>T [p.Arg671Trp]) identified by exome sequencing. The individuals present with global developmental delay, mild intellectual disability, ophthalmological anomalies, microcephaly or relative microcephaly, and shared mild facial dysmorphisms. Immunoblots of fibroblasts from two affected individuals revealed that the variant does not significantly alter MTSS2 levels. We modeled the variant in Drosophila and showed that the fly ortholog missing-in-metastasis (mim) was widely expressed in most neurons and a subset of glia of the CNS. Loss of mim led to a reduction in lifespan, impaired locomotor behavior, and reduced synaptic transmission in adult flies. Expression of the human MTSS2 reference cDNA rescued the mim loss-of-function (LoF) phenotypes, whereas the c.2011C>T variant had decreased rescue ability compared to the reference, suggesting it is a partial LoF allele. However, elevated  expression of the variant, but not the reference MTSS2 cDNA, led to similar defects as observed by mim LoF, suggesting that the variant is toxic and may act as a dominant-negative allele when expressed in flies. In summary, our findings support that mim is important for appropriate neural function, and that the MTSS2 c.2011C>T variant causes a syndromic form of intellectual disability.

DOI: 10.1016/j.ajhg.2022.08.011
PMID: 36067766

Video Protocol: Fly models of heart function

J Vis Exp. 2022 Aug 25;(186). doi: 10.3791/63939.

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

Gracheva E, Wang F, Matt A, Liang H, Fishman M, Zhou C

Abstract:

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of light-sensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.

DOI: 10.3791/63939
PMID: 36094265

Fly model helps illuminate mechanisms underlying contribution of GEMIN4 in an inherited neurodevelopmental disorder

Am J Med Genet A. 2022 Oct;188(10):2932-2940. doi: 10.1002/ajmg.a.62894 

Further delineation of GEMIN4 related neurodevelopmental disorder with microcephaly, cataract, and renal abnormalities syndrome

Altassan R, Qudair A, Alokaili R, Alhasan K, Faqeih EA, Alhashem A, Alowain M, Alsayed M, Rahbeeni Z, Albadi L, Alkuraya FS, Anderson EN, Rajan D, Pandey UB

Abstract:

Pathogenic variants in GEMIN4 have recently been linked to an inherited autosomal recessive neurodevelopmental disorder characterized with microcephaly, cataracts, and renal abnormalities (NEDMCR syndrome). This report provides a retrospective review of 16 patients from 11 unrelated Saudi consanguineous families with GEMIN4 mutations. The cohort comprises 11 new and unpublished clinical details from five previously described patients. Only two missense, homozygous, pathogenic variants were found in all affected patients, suggesting a founder effect. All patients shared global developmental delay with variable ophthalmological, renal, and skeletal manifestations. In addition, we knocked down endogenous Drosophila GEMIN4 in neurons to further investigate the mechanism of the functional defects in affected patients. Our fly model findings demonstrated developmental defects and motor dysfunction suggesting that loss of GEMIN4 function is detrimental in vivo; likely similar to human patients. To date, this study presents the largest cohort of patients affected with GEMIN4 mutations. Considering that identifying GEMIN4 defects in patients presenting with neurodevelopmental delay and congenital cataract will help in early diagnosis, appropriate management and prevention plans that can be made for affected families.

DOI: 10.1002/ajmg.a.62894
PMID: 35861185

Drosophila studies contribute to a study associating variants of PP2A/PPP2R4 with juvenile-onset parkinsonism

Brain. 2022 Sep 8:awac326. doi: 10.1093/brain/awac326 

PTPA variants and impaired PP2A activity in early-onset parkinsonism with
intellectual disability

Fevga C, Tesson C, Mascaro AC, Courtin T, van Coller R, Sakka S, Ferraro F, Farhat N, Bardien S, Damak M, Carr J, Ferrien M, Boumeester V, Hundscheid J, Grillenzoni N, Kessissoglou IA, Kuipers DJS, Quadri M; French and Mediterranean Parkinson disease Genetics Study Group; International Parkinsonism Genetics Network, Corvol JC, Mhiri C, Hassan BA, Breedveld GJ, Lesage S, Mandemakers W, Brice A, Bonifati V

Abstract:

The protein phosphatase 2A complex (PP2A), the major Ser/Thr phosphatase in the brain, is involved in a number of signaling pathways and functions, including the regulation of crucial proteins for neurodegeneration, such as alpha-synuclein, tau, and LRRK2. Here, we report the identification of variants in the PTPA/PPP2R4 gene, encoding a major PP2A activator, in two families with early-onset parkinsonism and intellectual disability. We carried out clinical studies and genetic analyses, including genome-wide linkage analysis, whole-exome sequencing, and Sanger sequencing of candidate variants. We next performed functional studies on the disease-associated variants in cultured cells and knock-down of ptpa in Drosophila melanogaster. We first identified a homozygous PTPA variant, c.893T > G (p.Met298Arg), in patients from a South African family with early-onset parkinsonism and intellectual disability. Screening of a large series of additional families yielded a second homozygous variant, c.512C > A (p.Ala171Asp), in a Libyan family with a similar phenotype. Both variants co-segregate with disease in the respective families. The affected subjects display juvenile-onset parkinsonism and intellectual disability. The motor symptoms were responsive to treatment with levodopa and deep brain stimulation of the subthalamic nucleus. In overexpression studies, both the PTPA p.Ala171Asp and p.Met298Arg variants were associated with decreased PTPA RNA stability and decreased PTPA protein levels; the p.Ala171Asp variant additionally displayed decreased PTPA protein stability. Crucially, expression of both variants was associated with decreased PP2A complex levels and impaired PP2A phosphatase activation. PTPA ortholog knock-down in Drosophila neurons induced a significant impairment of locomotion in the climbing test. This defect was age-dependent and fully reversed by L-DOPA treatment. We conclude that bi-allelic missense PTPA variants associated with impaired activation of the PP2A phosphatase cause autosomal recessive early-onset parkinsonism with intellectual disability. Our findings might also provide new insights for understanding the role of the PP2A complex in the pathogenesis of more common forms of neurodegeneration.

DOI: 10.1093/brain/awac326
PMID: 36073231

Drosophila studies contribute to identification of a novel disease-associated gene, ZMYND8

Genet Med. 2022 Sep;24(9):1952-1966. doi: 10.1016/j.gim.2022.06.001

De Novo ZMYND8 variants result in an autosomal dominant neurodevelopmental
disorder with cardiac malformations

Dias KR, Carlston CM, Blok LER, De Hayr L, Nawaz U, Evans CA, Bayrak-Toydemir P, Htun S, Zhu Y, Ma A, Lynch SA, Moorwood C, Stals K, Ellard S, Bainbridge MN, Friedman J, Pappas JG, Rabin R, Nowak CB, Douglas J, Wilson TE, Guillen Sacoto MJ, Mullegama SV, Palculict TB, Kirk EP, Pinner JR, Edwards M, Montanari F, Graziano C, Pippucci T, Dingmann B, Glass I, Mefford HC, Shimoji T, Suzuki T, Yamakawa K, Streff H, Schaaf CP, Slavotinek AM, Voineagu I, Carey JC, Buckley MF, Schenck A, Harvey RJ, Roscioli T

Abstract:

PURPOSE: ZMYND8 encodes a multidomain protein that serves as a central interactive hub for coordinating critical roles in transcription regulation, chromatin remodeling, regulation of super-enhancers, DNA damage response and tumor suppression. We delineate a novel neurocognitive disorder caused by variants in the ZMYND8 gene. 

METHODS: An international collaboration, exome sequencing, molecular modeling, yeast two-hybrid assays, analysis of available transcriptomic data and a knockdown Drosophila model were used to characterize the ZMYND8 variants. 

RESULTS: ZMYND8 variants were identified in 11 unrelated individuals; 10 occurred de novo and one suspected de novo; 2 were truncating, 9 were missense, of which one was recurrent. The disorder is characterized by intellectual disability with variable cardiovascular, ophthalmologic and minor skeletal anomalies. Missense variants in the PWWP domain of ZMYND8 abolish the interaction with Drebrin and missense variants in the MYND domain disrupt the interaction with GATAD2A. ZMYND8 is broadly expressed across cell types in all brain regions and shows highest expression in the early stages of brain development. Neuronal knockdown of the Drosophila ZMYND8 ortholog results in decreased habituation learning, consistent with a role in cognitive function. 

CONCLUSION: We present genomic and functional evidence for disruption of ZMYND8 as a novel etiology of syndromic intellectual disability.

DOI: 10.1016/j.gim.2022.06.001
PMID: 35916866

Assay in fly eye contributes to study related to ALS & FTD

J Biol Chem. 2022 Aug;298(8):102191. doi: 10.1016/j.jbc.2022.102191. 

Casein kinase 1δ/ε phosphorylates fused in sarcoma (FUS) and ameliorates FUS-mediated neurodegeneration

Kishino Y, Matsukawa K, Matsumoto T, Miyazaki R, Wakabayashi T, Nonaka T, Kametani F, Hasegawa M, Hashimoto T, Iwatsubo T

From the abstract:

Aberrant cytoplasmic accumulation of an RNA-binding protein, fused in sarcoma (FUS), characterizes the neuropathology of subtypes of ALS and frontotemporal lobar degeneration ... we show that casein kinase 1δ (CK1δ) phosphorylates FUS at 10 serine/threonine residues in vitro ... We also show that phosphorylation by CK1δ or CK1ε significantly increased the solubility of FUS in human embryonic kidney 293 cells. In transgenic Drosophila that overexpress wt or P525L ALS-mutant human FUS in the retina or in neurons, we found coexpression of human CK1δ or its Drosophila isologue Dco in the photoreceptor neurons significantly ameliorated the observed retinal degeneration, and neuronal coexpression of human CK1δ extended fly life span. .. our data suggest a novel regulatory mechanism of the assembly and toxicity of FUS through CK1δ/CK1ε-mediated phosphorylation, which could represent a potential therapeutic target in FUS proteinopathies.

DOI: 10.1016/j.jbc.2022.102191
PMCID: PMC9293781
PMID: 35753345

Multi-species study identifies the vacuolar H+ ATPase component ATP6V0C as a human disease-associated gene

Brain. 2022 Sep 8:awac330. doi: 10.1093/brain/awac330

ATP6V0C variants impair vacuolar V-ATPase causing a neurodevelopmental disorder
often associated with epilepsy

Mattison KA, Tossing G, Mulroe F, Simmons C, Butler KM, Schreiber A, Alsadah A, Neilson DE, Naess K, Wedell A, Wredenberg A, Sorlin A, McCann E, Burghel GJ, Menendez B, Hoganson GE, Botto LD, Filloux FM, Aledo-Serrano Á, Gil-Nagel A, Tatton-Brown K, Verbeek NE, van Hirtum-Das M, Breckpot J, Hammer TB, Møller RS, Whitney A, Douglas AGL, Kharbanda M, Brunetti-Pierri N, Morleo M, Nigro V, May HJ, Tao JX, Argili E, Sherr EH, Dobyns WB, Consortium GER, Baines RA, Warwicker J, Parker JA, Banka S, Campeau PM, Escayg A

Abstract:

The vacuolar H+-ATPase (V-ATPase) is an enzymatic complex that functions in an ATP-dependent manner to pump protons across membranes and acidify organelles, thereby creating the proton/pH gradient required for membrane trafficking by several different types of transporters. We describe heterozygous point variants in ATP6V0C, encoding the c-subunit in the membrane bound integral domain of the V-ATPase, in 27 patients with neurodevelopmental abnormalities with or without epilepsy. Corpus callosum hypoplasia and cardiac abnormalities were also present in some patients. In silico modeling suggested that the patient variants interfere with the interactions between the ATP6V0C and ATP6V0A subunits during ATP hydrolysis. Consistent with decreased V-ATPase activity, functional analyses conducted in Saccharomyces cerevisiae revealed reduced LysoSensor fluorescence and reduced growth in media containing varying concentrations of CaCl2. Knockdown of ATP6V0C in Drosophila resulted in increased duration of seizure-like behavior, and the expression of selected patient variants in Caenorhabditis elegans led to reduced growth, motor dysfunction, and reduced lifespan. In summary, this study establishes ATP6V0C as an important disease gene, describes the clinical features of the associated neurodevelopmental disorder, and provides insight into disease mechanisms.

DOI: 10.1093/brain/awac330
PMID: 36074901

Drosophila studies contribute to understanding impact of a variant of MTSS2 associated with intellectual disability

Am J Hum Genet. 2022 Aug 30:S0002-9297(22)00363-9. doi: 10.1016/j.ajhg.2022.08.011.

The recurrent de novo c.2011C>T missense variant in MTSS2 causes syndromic intellectual disability.

Huang Y, Lemire G, Briere LC, Liu F, Wessels MW, Wang X, Osmond M, Kanca O, Lu S, High FA, Walker MA, Rodan LH; Undiagnosed Diseases Network; Care4Rare Canada Consortium, Kernohan KD(, Sweetser DA, Boycott KM, Bellen HJ

Abstract: MTSS2, also known as MTSS1L, binds to plasma membranes and modulates their bending. MTSS2 is highly expressed in the central nervous system (CNS) and appears to be involved in activity-dependent synaptic plasticity. Variants in MTSS2 have not yet been associated with a human phenotype in OMIM. Here we report five individuals with the same heterozygous de novo variant in MTSS2 (GenBank: NM_138383.2: c.2011C>T [p.Arg671Trp]) identified by exome sequencing. The individuals present with global developmental delay, mild intellectual disability, ophthalmological anomalies, microcephaly or relative microcephaly, and shared mild facial dysmorphisms. Immunoblots of fibroblasts from two affected individuals revealed that the variant does not significantly alter MTSS2 levels. We modeled the variant in Drosophila and showed that the fly ortholog missing-in-metastasis (mim) was widely expressed in most neurons and a subset of glia of the CNS. Loss of mim led to a reduction in lifespan, impaired locomotor behavior, and reduced synaptic transmission in adult flies. Expression of the human MTSS2 reference cDNA rescued the mim loss-of-function (LoF) phenotypes, whereas the c.2011C>T variant had decreased rescue ability compared to the reference, suggesting it is a partial LoF allele. However, elevated expression of the variant, but not the reference MTSS2 cDNA, led to similar defects as observed by mim LoF, suggesting that the variant is toxic and may act as a dominant-negative allele when expressed in flies. In summary, our findings support that mim is important for appropriate neural function, and that the MTSS2 c.2011C>T variant causes a syndromic form of intellectual disability.

DOI: 10.1016/j.ajhg.2022.08.011
PMID: 36067766

Drosophila used to help understand the role of Filamin in cardiomyopathies

Biol Open. 2022 Sep 6:bio.059376. doi: 10.1242/bio.059376.

Drosophila CRISPR/Cas9 mutants as tools to analyse cardiac filamin function and pathogenicity of human FLNC variants.

Ader F, Russi M, Tixier-Cardoso L, Jullian E, Martin E, Richard P, Villard E, Monnier V

Abstract: Filamins are large proteins with actin binding properties. Mutations in FLNC, one of the three filamin genes in humans, have recently been implicated in dominant cardiomyopathies, but the underlying mechanisms are not well understood. Here, we aimed to use Drosophila melanogaster as a new in vivo model to study these diseases. First, we show that adult-specific cardiac RNAi-induced depletion of Drosophila Filamin (dFil) induced cardiac dilatation, impaired systolic function and sarcomeric alterations, highlighting its requirement for cardiac function and maintenance of sarcomere integrity in the adult stage. Next, we introduced in the cheerio gene, using CRISPR/Cas9 gene editing, three missense variants, previously identified in patients with hypertrophic cardiomyopathy. Flies carrying these variants did not exhibit cardiac defects or increased propensity to form filamin aggregates, arguing against their pathogenicity. Finally, we show that deletions of the C-term part of dFil carrying the last four Ig-like domains are dispensable for cardiac function. Collectively, these results highlight the relevance of this model to explore the cardiac function of filamins and increase our understanding of physio-pathological mechanisms involved in FLNC-related cardiomyopathies.

DOI: 10.1242/bio.059376
PMID: 36066120

Fly model of Tauopathies contributes to study related to pTau interaction with Hsp27

Elife. 2022 Sep 1;11:e79898. doi: 10.7554/eLife.79898.

Specific binding of Hsp27 and phosphorylated Tau mitigates abnormal Tau aggregation-induced pathology.

Zhang S, Zhu Y, Lu J, Liu Z, Lobato AG, Zeng W, Liu J, Qiang J, Zeng S, Zhang Y, Liu C, Liu J, He
Z, Zhai RG, Li D

Abstract:

Amyloid aggregation of phosphorylated Tau (pTau) into neurofibrillary tangles is closely associated with Alzheimer's disease (AD). Several molecular chaperones have been reported to bind Tau and impede its pathological aggregation. Recent findings of elevated levels of Hsp27 in the brains of patients with AD suggested its important role in pTau pathology. However, the molecular mechanism of Hsp27 in pTau aggregation remains poorly understood. Here, we show that Hsp27 partially co-localizes with pTau tangles in the brains of patients with AD. Notably, phosphorylation of Tau by microtubule affinity regulating kinase 2 (MARK2), dramatically enhances the binding affinity of Hsp27 to Tau. Moreover, Hsp27 efficiently prevents pTau fibrillation in vitro and mitigates neuropathology of pTau aggregation in a Drosophila tauopathy model. Further mechanistic study reveals that Hsp27 employs its N-terminal domain to directly interact with multiple phosphorylation sites of pTau for specific binding. Our work provides the structural basis for the specific recognition of Hsp27 to pathogenic pTau, and highlights the important role of Hsp27 in preventing abnormal aggregation and pathology of pTau in AD.

DOI: 10.7554/eLife.79898
PMID: 36048712

Drosophila used in a study related to spastic quadriplegic cerebral palsy

 Clin Genet. 2022 Aug 31. doi: 10.1111/cge.14220. Online ahead of print.

Hereditary spastic paraparesis (HSP) presenting as cerebral palsy due to ADD3 variant with mechanistic insight provided by a Drosophila γ-adducin model.

Sanchez Marco SB Buhl E, Firth R, Zhu B, Gainsborough M, Beleza-Meireles A, Moore S, Caswell R, Stals K, Ellard S, Kennedy C, Hodge JJL, Majumdar A
 

From the abstract:

INTRODUCTION: Cerebral palsy (CP) causes neurological disability in early childhood. Hypoxic-ischaemic injury plays a major role in its aetiology, nevertheless, genetic and epigenetic factors may contribute to the clinical presentation. Mutations in ADD3 (encoding γ-adducin) gene have been described in a monogenic form of spastic quadriplegic cerebral palsy (OMIM 601568).
 

METHODS: We studied a sixteen-year-old male with spastic diplegia. ... clinical genetics assessment and Whole Exome Sequencing (WES) gave the diagnosis. We generated an animal model using Drosophila to study the effects of γ-adducin loss and gain of function.

RESULTS: ... Pan-neuronal over-expression or knock-down of the Drosophila ortholog of ADD3 called hts caused a reduction of life span and impaired locomotion thereby phenocopying aspects of the human disease.

CONCLUSION: Our animal experiments present a starting point to understand the biological processes underpinning the clinical phenotype and pathogenic mechanisms, to gain insights into potential future methods for treating or preventing ADD3 related spastic quadriplegic cerebral palsy.

PMID: 36046955

Review Article: Fly Research & Cancer Therapeutics

Front Genet. 2022 Aug 8;13:949241. doi: 10.3389/fgene.2022.949241. eCollection 2022.

Drosophila melanogaster: A platform for anticancer drug discovery and personalized therapies.

Munnik C, Xaba MP, Malindisa ST, Russell BL, Sooklal SA

Abstract:

Cancer is a complex disease whereby multiple genetic aberrations, epigenetic modifications, metabolic reprogramming, and the microenvironment contribute to the development of a tumor. In the traditional anticancer drug discovery pipeline, drug candidates are usually screened in vitro using two-dimensional or three-dimensional cell culture. However, these methods fail to accurately mimic the human disease state. This has led to the poor success rate of anticancer drugs in the preclinical stages since many drugs are abandoned due to inefficacy or toxicity when transitioned to whole-organism models. The common fruit fly, Drosophila melanogaster, has emerged as a beneficial system for modeling human cancers. Decades of fundamental research have shown the evolutionary conservation of key genes and signaling pathways between flies and humans. Moreover, Drosophila has a lower genetic redundancy in comparison to mammals. These factors, in addition to the advancement of genetic toolkits for manipulating gene expression, allow for the generation of complex Drosophila genotypes and phenotypes. Numerous studies have successfully created Drosophila models for colorectal, lung, thyroid, and brain cancers. These models were utilized in the high-throughput screening of FDA-approved drugs which led to the identification of several compounds capable of reducing proliferation and rescuing phenotypes. More noteworthy, Drosophila has also unlocked the potential for personalized therapies. Drosophila 'avatars' presenting the same mutations as a patient are used to screen multiple therapeutic agents targeting multiple pathways to find the most appropriate combination of drugs. The outcomes of these studies have translated to significant responses in patients with adenoid cystic carcinoma and metastatic colorectal cancers. Despite not being widely utilized, the concept of in vivo screening of drugs in Drosophila is making significant contributions to the current drug discovery pipeline. In this review, we discuss the application of Drosophila as a platform in anticancer drug discovery; with special focus on the cancer models that have been generated, drug libraries that have been screened and the status of personalized therapies. In addition, we elaborate on the biological and technical limitations of this system.

DOI: 10.3389/fgene.2022.949241
PMCID: PMC9393232
PMID: 36003330

Conflict of interest statement: BR is funded by the company Buboo (Pty) Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Fly study sheds light on function of MEGF8, which is associated with Carpenter Syndrome

J Neurosci. 2022 Aug 9:JN-RM-0442-22. doi: 10.1523/JNEUROSCI.0442-22.2022.

Drosophila Homolog of the Human Carpenter Syndrome Linked Gene, MEGF8, is Required for Synapse Development and Function

Chen S, Venkatesan A, Lin YQ, Xie J, Neely G, Banerjee S, Bhat MA

Abstract: Drosophila Multiple Epidermal Growth Factor-like Domains 8 (dMegf8) is a homolog of human MEGF8. MEGF8 encodes a multi-domain transmembrane protein which is highly conserved across species. In humans, MEGF8 mutations cause a rare genetic disorder called Carpenter syndrome, which is frequently associated with abnormal left-right patterning, cardiac defects and learning disabilities. MEGF8 is also associated with psychiatric disorders. Despite its clinical relevance MEGF8 remains poorly characterized, and though it is highly conserved, studies on animal models of Megf8 are also very limited. The presence of intellectual disabilities in Carpenter syndrome patients and association of MEGF8 with psychiatric disorders indicate that mutations in MEGF8 cause underlying defects in synaptic structure and functions. In this study, we investigated the role of Drosophila dMegf8 in glutamatergic synapses of the larval neuromuscular junctions (NMJ) in both males and females. We show that dMegf8 localizes to NMJ synapses and is required for proper synaptic growth. dMegf8 mutant larvae and adults show severe motor coordination deficits. At the NMJ, dMegf8 mutants show altered localization of pre- and post-synaptic proteins, defects in synaptic ultrastructure and neurotransmission. Interestingly, dMegf8 mutants have reduced levels of the type II BMP receptor Wishful thinking (Wit). dMegf8 displays genetic interactions with neurexin-1 (dnrx) and wit, and in association with Dnrx and Wit plays an essential role in synapse organization. Our studies provide insights into human MEGF8 functions and potentially into mechanisms that may underlie intellectual disabilities observed in Carpenter syndrome as well as MEGF8-related synaptic structural and/or functional deficits in psychiatric disorders.

Significance Statement: Carpenter Syndrome, known for over a century now, is a genetic disorder linked to mutations in Multiple Epidermal Growth Factor-like Domains 8 (MEGF8) gene and associated with intellectual disabilities among other symptoms. MEGF8 is also associated with psychiatric disorders. Despite the high genetic conservation and clinical relevance, the functions of MEGF8 remain largely uncharacterized. Patients with intellectual disabilities and psychiatric diseases often have an underlying defect in synaptic structure and function. This work defines the role of the fly homolog of human MEGF8, dMegf8, in glutamatergic synapse growth, organization and function and provide insights into potential functions of MEGF8 in human central synapses and synaptic mechanisms that may underlie psychiatric disorders and intellectual disabilities seen in Carpenter Syndrome.

Copyright © 2022 the authors.

DOI: 10.1523/JNEUROSCI.0442-22.2022
PMID: 35944997

Two new studies exemplify utility of Drosophila for human genetic disease-related studies

 (1)

Am J Hum Genet. 2022 Apr 7;109(4):571-586. doi: 10.1016/j.ajhg.2022.01.020. Epub
2022 Mar 2.

Loss-of-function variants in TIAM1 are associated with developmental delay,
intellectual disability, and seizures.

Lu et al.

TIAM Rac1-associated GEF 1 (TIAM1) regulates RAC1 signaling pathways that affect the control of neuronal morphogenesis and neurite outgrowth by modulating the actin cytoskeletal network. To date, TIAM1 has not been associated with a Mendelian disorder. Here, we describe five individuals with bi-allelic TIAM1 missense variants who have developmental delay, intellectual disability, speech delay, and seizures. Bioinformatic analyses demonstrate that these variants are rare and likely pathogenic. We found that the Drosophila ortholog of TIAM1, still life (sif), is expressed in larval and adult central nervous system (CNS) and is mainly expressed in a subset of neurons, but not in glia. Loss of sif reduces the survival rate, and the surviving adults exhibit climbing defects,are prone to severe seizures, and have a short lifespan. The TIAM1 reference (Ref) cDNA partially rescues the sif loss-of-function (LoF) phenotypes. We also assessed the function associated with three TIAM1 variants carried by two of the probands and compared them to the TIAM1 Ref cDNA function in vivo. TIAM1 p.Arg23Cys has reduced rescue ability when compared to TIAM1 Ref, suggesting that it is a partial LoF variant. In ectopic expression studies, both wild-type sif and TIAM1 Ref are toxic, whereas the three variants (p.Leu862Phe, p.Arg23Cys, and p.Gly328Val) show reduced toxicity, suggesting that they are partial LoF variants. In summary, we provide evidence that sif is important for appropriate neural function and that TIAM1 variants observed in the probands are disruptive, thus implicating loss of TIAM1 in neurological phenotypes in humans.

Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc.
All rights reserved.

DOI: 10.1016/j.ajhg.2022.01.020
PMID: 35240055 

Conflict of interest statement: Declaration of interests M.J.G.S. is a salaried employee of GeneDx Inc.

(2)

Am J Hum Genet. 2022 Apr 7;109(4):601-617. doi: 10.1016/j.ajhg.2022.03.002.

Germline variants in tumor suppressor FBXW7 lead to impaired ubiquitination and
a neurodevelopmental syndrome.

Stephenson et al.

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc.
All rights reserved.

DOI: 10.1016/j.ajhg.2022.03.002
PMID: 35395208

Conflict of interest statement: Declaration of interests I.E.S. has served on scientific advisory boards for UCB, Eisai, GlaxoSmithKline, BioMarin, Nutricia, Rogcon, Chiesi, Encoded Therapeutics, Xenon Pharmaceuticals, and Knopp Biosciences; has received speaker honoraria from GlaxoSmithKline, UCB, BioMarin, Biocodex, and Eisai; has received funding for travel from UCB, Biocodex, GlaxoSmithKline, Biomarin and Eisai; has served as an investigator for Zogenix, Zynerba, Ultragenyx, GW Pharma, UCB, Eisai, Anavex Life Sciences, Ovid Therapeutics, Epygenyx, Encoded Therapeutics and Marinus; and has consulted for Zynerba Pharmaceuticals, Atheneum Partners, Ovid Therapeutics, Care Beyond Diagnosis, Epilepsy Consortium and UCB. She may accrue future revenue on pending patent WO2009/086591; her patent for SCN1A testing is held by Bionomics and is licensed to various diagnostic companies; and she has a patent for a molecular diagnostic/therapeutic target for benign familial infantile epilepsy (BFIE) (PRRT2), WO/2013/059884. She receives and/or has received research support from the National Health and Medical Research Council of Australia, Medical Research Future Fund, Health Research Council of New Zealand, CURE, Australian Epilepsy Research Fund, and the National Institute of Neurological Disorders and Stroke of the National Institutes of Health. J.P. is co-chief scientific officer for Global Gene Corp. All other authors declare no competing interests.

Review: "Comprehensive survey" of model organism-based research on rare neurological diseases

Exp Neurobiol. 2022 Feb 28;31(1):1-16. doi: 10.5607/en22003.

Invertebrate Model Organisms as a Platform to Investigate Rare Human Neurological Diseases.

Lee JH

Abstract: 

 Patients suffering from rare human diseases often go through a painful journey for finding a definite molecular diagnosis prerequisite of appropriate cures. With a novel variant isolated from a single patient, determination of its pathogenicity to end such "diagnostic odyssey" requires multi-step processes involving experts in diverse areas of interest, including clinicians, bioinformaticians and research scientists. Recent efforts in building large-scale genomic databases and in silico prediction platforms have facilitated identification of potentially pathogenic variants causative of rare human diseases of a Mendelian basis. However, the functional significance of individual variants remains elusive in many cases, thus requiring incorporation of versatile and rapid model organism (MO)-based platforms for functional analyses. In this review, the current scope of rare disease research is briefly discussed. In addition, an overview of invertebrate MOs for their key features relevant to rare neurological diseases is provided, with the characteristics of two representative invertebrate MOs, Drosophila melanogaster and Caenorhabditis elegans, as well as the challenges against them. Finally, recently developed research networks integrating these MOs in collaborative research are portraited with an array of bioinformatical analyses embedded. A comprehensive survey of MO-based research activities provided in this review will help us to design a well structured analysis of candidate genes or potentially pathogenic variants
for their roles in rare neurological diseases in future.

DOI: 10.5607/en22003
PMID: 35256540