Biology subjects

Wangler, M. F.

Publications and source records attributed to Wangler, M. F..

9 recordsLinked to original sources

bioRxiv2026

NLGN3 autism variants have distinct functional impact on synapses and sleep behavior in Drosophila

Neuroligin-3 (NLGN3) was first identified as a risk gene associated with autism spectrum disorder (ASD). The initial variant, p.R451C, associating NLGN3 with ASD has been heavily investigated, yet little is known about the functional consequences of other NLGN3 variants. Furthermore, while most of the identified variants are present in males with maternally inherited variants from unaffected mothers, several de novo variants were observed in females, suggesting a possible functional difference between de novo and maternally inherited variants. To address the functional consequences of NLGN3 variants in vivo, we generated transgenic Drosophila models corresponding to one de novo variant (p.R175W) identified in one female proband, and two maternally inherited variants (p.R451C and p.R597W) identified in male probands. In Drosophila, loss of the fly homolog, Nlg3, altered sleep patterns, synaptic architecture, and vesicle dynamics, which were rescued by the expression of the human NLGN3Ref allele. When comparing the variants, the de novo p.R175W variant and the maternally inherited p.R451C variant altered synapse morphology and sleep patterns, with minimal effects on vesicle dynamics, and the p.R597W variant altered sleep and vesicle dynamics with minimal impact on synapse morphology. Using overexpression models, human NLGN3Ref altered sleep patterns and synaptic morphology. Moreover, the p.R175W variant exacerbated sleep phenotypes, and the p.R175W and p.R451C variants exacerbated synapse morphology phenotypes. Together, our findings suggest that de novo NLGN3 variants identified in females are likely gain-of-function, while maternally inherited variants have mixed loss-and gain-of-function effects. Moreover, the location of the variants may contribute to the distinct functional differences we observed. Some NLGN3 variants disrupt synaptic development, while other variants alter synaptic function, suggesting that NLGN3 variants have differential effects. These functional differences may provide insight into the heterogeneity of individuals with ASD. Author SummaryAutism spectrum disorder (ASD) is a common neurodevelopmental disorder. Mutations in the Neuroligin-3 (NLGN3) gene are associated with ASD but very few of these mutations have been characterized in animal models. Most of these mutations affect male individuals who maternally inherited their genetic mutation; however, more rarely female individuals may present with a genetic mutation that was not identified in either of the parents. Here, we utilized the fruit fly model to investigate how three different mutations, one mutation identified in a female and two mutations identified in males, affect the flys behavior and synapse development. We identified altered sleep patterns in some of our mutants which is consistent with sleep disturbances being highly comorbid with ASD. Additionally, we identified alterations in synapse development and function which is consistent with the role of NLGN3 in synapse formation and maturation. Together, our findings support that NLGN3 is important for regulating the synapse and mutations in this gene can alter its function. However, different mutations can have differential effects. This demonstrates the need to assess multiple variants simultaneously because each variant may have distinct functional significances.

genetics↗

bioRxiv2024

Distinguishing PEX gene variant severity for mild, severe, and atypical peroxisome biogenesis disorders in Drosophila

Peroxisomal biogenesis disorders (PBD) are autosomal recessive disorders caused by loss-of-function mutations of one of the PEX genes responsible for peroxisomal formation. Impaired peroxisome assembly causes severe multisystemic failure with patient phenotypes ranging from epilepsy, liver disease, feeding issues, biochemical abnormalities, and neurodegeneration. Variants in the same PEX gene can produce wide differences in severity, ranging from individuals with death in the first year of life to adults with milder complications. To study this strong genotype-phenotype correlation, we selected specific human PEX gene mutations and utilized Drosophila as a model organism. We generated flies replacing the coding sequence of our Pex gene of interest with a KozakGAL4 (KZ) promoter trap sequence. These cassettes simultaneously knock-out of the Pex gene and knock-in a GAL4 driver, ideal for making "humanized" flies in which the human PEX gene can replace the fly loss. We assessed Pex2KZ and Pex16KZ lines in lifespan, bang sensitivity, and climbing assays and confirmed that these are strong loss-of-function alleles. In parallel, we generated human reference and variant UAS-cDNA lines of PEX2 and PEX16 variants in Drosophila. We observed nearly complete phenotypic rescue of Drosophila Pex2 and Pex16 loss when human PEX2Refor PEX16Ref, respectively, were expressed. We also provide evidence for an allele severity spectrum in PEX2 and PEX16 in which some missense alleles, such as PEX2C247R, are equally severe as early truncations, such as PEX2R119*. We also observed that alleles associated with mild PBD, such as PEX2E55K, show variability depending on the assay but do not fully rescue. Finally, alleles associated with atypical ataxia phenotypes, such as PEX16F332Del, can perform as well as PEX16Ref, depending on the assay. Altogether, these Drosophila lines effectively model the range of severity of peroxisomal biogenesis disorders.

genetics↗

bioRxiv2024

Genetic analysis of the X-linked Adrenoleukodystrophy ABCD1 gene in Drosophila uncovers a role in Peroxisomal dynamics

X-linked adrenoleukodystrophy (X-ALD) is a progressive neurodegenerative disorder caused by a loss-of-function (LOF) mutation in the ATP-binding cassette subfamily D member 1 (ABCD1) gene, leading to the accumulation of very long-chain fatty acids (VLCFAs). This disorder exhibits striking heterogeneity; some male patients develop an early childhood neuroinflammatory demyelination disorder, while other patients, including adult males and most affected female carriers, experience a chronic progressive myelopathy. Adrenocortical failure is observed in almost all male patients, with age of onset varying sometimes being the first diagnostic finding. The gene underlying this spectrum of disease encodes an ATP-binding cassette (ABC) transporter that localizes to peroxisomes and facilitates VLCFA transport. X-ALD is considered a single peroxisomal component defect and does not play a direct role in peroxisome assembly. Drosophila models of other peroxisomal genes have provided mechanistic insight into some of the neurodegenerative mechanisms with reduced lifespan, retinal degeneration, and VLCFA accumulation. Here, we perform a genetic analysis of the fly ABCD1 ortholog Abcd1 (CG2316). Knockdown or deficiency of Abcd1 leads to VLCFA accumulation, salivary gland defects, locomotor impairment and retinal lipid abnormalities. Interestingly, there is also evidence of reduced peroxisomal numbers. Flies overexpressing the human cDNA for ABCD1 display a wing crumpling phenotype characteristic of the pex2 loss-of-function. Surprisingly, overexpression of human ABCD1 appears to inhibit or overwhelm peroxisomal biogenesis to levels similar to null mutations in fly pex2, pex16 and pex3. Drosophila Abcd1 is therefore implicated in peroxisomal number, and overexpression of the human ABCD1 gene acts a potent inhibitor of peroxisomal biogenesis in flies.

genetics↗

bioRxiv2024

Drosophila Models Uncover Substrate Channeling Effects on Phospholipids and Sphingolipids in Peroxisomal Biogenesis Disorders

Peroxisomal Biogenesis Disorders Zellweger Spectrum (PBD-ZSD) disorders are a group of autosomal recessive defects in peroxisome formation that produce a multi-systemic disease presenting at birth or in childhood. Well documented clinical biomarkers such as elevated very long chain fatty acids (VLCFA) are key biochemical diagnostic findings in these conditions. Additional, secondary biochemical alterations such as elevated very long chain lysophosphatidylcholines are allowing newborn screening for peroxisomal disease. In addition, a more widespread impact on metabolism and lipids is increasingly being documented by metabolomic and lipidomic studies. Here we utilize Drosophila models of pex2 and pex16 as well as human plasma from individuals with PEX1 mutations. We identify phospholipid abnormalities in Drosophila larvae and brain characterized by differences in the quantities of phosphatidylcholine (PC) and phosphatidylethanolamines (PE) with long chain lengths and reduced levels of intermediate chain lengths. For diacylglycerol (DAG) the precursor of PE and PC through the Kennedy pathway, the intermediate chain lengths are increased suggesting an imbalance between DAGs and PE and PC that suggests the two acyl chain pools are not in equilibrium. Altered acyl chain lengths are also observed in PE ceramides in the fly models. Interestingly, plasma from human subjects exhibit phospholipid alterations similar to the fly model. Moreover, human plasma shows reduced levels of sphingomyelin with 18 and 22 carbon lengths but normal levels of C24. Our results suggest that peroxisomal biogenesis defects alter shuttling of the acyl chains of multiple phospholipid and ceramide lipid classes, whereas DAG species with intermediate fatty acids are more abundant. These data suggest an imbalance between de novo synthesis of PC and PE through the Kennedy pathway and remodeling of existing PC and PE through the Lands cycle. This imbalance is likely due to overabundance of very long and long acyl chains in PBD and a subsequent imbalance due to substrate channeling effects. Given the fundamental role of phospholipid and sphingolipids in nervous system functions, these observations suggest PBD-ZSD are diseases characterized by widespread cell membrane lipid abnormalities.

genetics↗

bioRxiv2024

Loss of function in RBBP5 results in a syndromic neurodevelopmental disorder associated with microcephaly

PurposeEpigenetic dysregulation has been associated with many inherited disorders. RBBP5 encodes a core member of the protein complex that methylates histone 3 lysine-4 (H3K4) and has not been implicated in human disease. MethodsWe identify five unrelated individuals with de novo heterozygous pathogenic variants in RBBP5. Three truncating and two missense variants were identified in probands with neurodevelopmental symptoms including global developmental delay, intellectual disability, microcephaly, and short stature. Here, we investigate the pathogenicity of the variants through protein structural analysis and transgenic Drosophila models. ResultsBoth missense p.T232I and p.E296D variants affect evolutionarily conserved amino acids and are expected to interfere with the interface between RBBP5 and the histones. In Drosophila, ubiquitous overexpression of human RBBP5 is lethal in the larval developmental stage. Loss of Rbbp5 leads to a reduction in brain size, and the human reference, p.T232I, or p.E296D variant transgenes fail to rescue loss of Rbbp5. Expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both p.T232I and p.E296D variants are loss-of-function alleles. ConclusionDe novo heterozygous variants in RBBP5 are associated with a syndromic neurodevelopmental disorder. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/578086v1_ufig1.gif" ALT="Figure 1"> O_LINKSMALLFIG WIDTH=200 HEIGHT=24 SRC="FIGDIR/small/578086v1_ufig1a.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@11337daorg.highwire.dtl.DTLVardef@b7f4d8org.highwire.dtl.DTLVardef@12bc283org.highwire.dtl.DTLVardef@1ef4e09_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2022

De Novo Variants in MRTFB have gain of function activity in Drosophila and are associated with a novel neurodevelopmental phenotype with dysmorphic features.

Myocardin-Related Transcription Factor B (MRTFB) is an important transcriptional regulator which promotes the activity of an estimated 300 genes during different stages of development. Here we report two pediatric probands with de novo variants in MRTFB (R104G and A91P) and mild dysmorphic features, intellectual disability, global developmental delays, speech apraxia, and impulse control issues. As the MRTFB protein is highly conserved between vertebrate and invertebrate model organisms, we generated a humanized Drosophila model expressing the human MRTFB protein in the same spatial and temporal pattern as the fly gene. Expression of the human MRTFBR104G variant using a mrtf-T2A-GAL4 line proved to be embryonic lethal. Additional phenotypes were also identified by expressing the MRTFBR104G and MRTFBA91P variant in a subset of Drosophila tissues. Notably, expression within wing tissues resulted in an expansion of intervein tissue, wing vein thickening, shortening or loss of wing veins, and blistering. The MRTFBR104G and MRTFBA91P variants also display a decreased level of actin binding within critical RPEL domains, resulting in increased transcriptional activity and changes in the organization of the Actin cytoskeleton. These changes were not observed in flies expressing two additional candidate variants, MRTFBN95Sand MRTFBR109Q, highlighting that the location of the mutation within the 2nd RPEL domain is critical to the pathogenicity of the variant. These changes suggest that the MRTFBR104G and MRTFBA91P alleles we have identified affect the regulation of the protein and that these variants in MRTFB underly a novel neurodevelopmental disorder.

genetics↗

bioRxiv2021

ModelMatcher: A scientist-centric online platform to facilitate collaborations between stakeholders of rare and undiagnosed disease research

Next-generation sequencing is a prevalent diagnostic tool for undiagnosed diseases, and has played a significant role in rare disease gene discovery. While this technology resolves some cases, others are given a list of possibly damaging genetic variants necessitating functional studies. Productive collaborations between scientists, clinicians, and patients can help resolve such medical mysteries, and provide insights into in vivo function of human genes. Furthermore, facilitating interactions between scientists and research funders, including non-profit organizations or commercial entities, can dramatically reduce the time to translate discoveries from bench to bedside. Several systems designed to connect clinicians and researchers with a shared gene of interest have been successful. However, these platforms exclude some stakeholders based on their role or geography. Here we describe ModelMatcher, a global online matchmaking tool designed to facilitate cross-disciplinary collaborations, especially between scientists and other stakeholders of rare and undiagnosed disease research. ModelMatcher is integrated into the Rare Diseases Models and Mechanisms Network and Matchmaker Exchange, allowing users to identify potential collaborators in other registries. This living database decreases the time from when a scientist or clinician is making discoveries regarding their genes of interest, to when they identify collaborators and sponsors to facilitate translational and therapeutic research.

genetics↗

bioRxiv2019

Biases in arginine codon usage correlate with genetic disease risk

PurposeThe persistence of hypermutable CGN (CGG, CGA, CGC, CGU) arginine codons at high frequency suggests the possibility of negative selective pressure at these sites and that arginine codon usage could be a predictive indicator of human disease genes.\n\nMethodsWe analyzed arginine codons (CGN, AGG, AGA) from all canonical Ensembl protein coding gene transcripts before comparing the frequency of CGN codons between genes with and without human disease associations and with gnomAD constraint metrics.\n\nResultsThe frequency of CGN codons among a genes total arginine codon count was higher in genes linked to syndromic autism spectrum disorder (ASD) compared to genes not associated with ASD. A comparison of genes annotated as dominant or recessive with control genes not matching either classification revealed a progressive increase in CGN codon frequency. Moreover, CGN frequency was positively correlated with a genes probability of loss-of-function intolerance (pLI) score and negatively correlated with observed-over-expected ratios for both loss of function and missense mutations.\n\nConclusionOur findings indicate that genes utilizing CGN arginine codons rather than AGG or AGA are more likely to underlie single gene disorders, particularly for dominant phenotypes, and thus constitute candidate genes for the study of human genetic disease.

genetics↗

bioRxiv2018

Loss-of-function in IRF2BPL is associated with neurological phenotypes

The Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) gene encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals affected with neurological symptoms who carry damaging heterozygous variants in IRF2BPL. Five cases carrying nonsense variants in IRF2BPL resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The bioinformatics signature for IRF2BPL based on population genomics is consistent with a gene that is intolerant to variation. We show that the IRF2BPL ortholog in the fruit fly, called pits (protein interacting with Ttk69 and Sin3A), is broadly expressed including the nervous system. Complete loss of pits is lethal early in development, whereas partial knock-down with RNA interference in neurons leads to neurodegeneration, revealing requirement for this gene in proper neuronal function and maintenance. The nonsense variants in IRF2BPL identified in patients behave as severe loss-of-function alleles in this model organism, while ectopic expression of the missense variants leads to a range of phenotypes. Taken together, IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

genetics↗