scholarly journals Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Daniel L Kober ◽  
Jennifer M Alexander-Brett ◽  
Celeste M Karch ◽  
Carlos Cruchaga ◽  
Marco Colonna ◽  
...  
Keyword(s):  
Disease Risk ◽  
Surface Expression ◽  
Patient Specific ◽  
Loss Of Function ◽  
Wild Type ◽  
Functional Surface ◽  
Functional Studies ◽  
Functional Differences ◽  
Risk Variants ◽  
Disease Mutations

Genetic variations in the myeloid immune receptor TREM2 are linked to several neurodegenerative diseases. To determine how TREM2 variants contribute to these diseases, we performed structural and functional studies of wild-type and variant proteins. Our 3.1 Å TREM2 crystal structure revealed that mutations found in Nasu-Hakola disease are buried whereas Alzheimer’s disease risk variants are found on the surface, suggesting that these mutations have distinct effects on TREM2 function. Biophysical and cellular methods indicate that Nasu-Hakola mutations impact protein stability and decrease folded TREM2 surface expression, whereas Alzheimer’s risk variants impact binding to a TREM2 ligand. Additionally, the Alzheimer’s risk variants appear to epitope map a functional surface on TREM2 that is unique within the larger TREM family. These findings provide a guide to structural and functional differences among genetic variants of TREM2, indicating that therapies targeting the TREM2 pathway should be tailored to these genetic and functional differences with patient-specific medicine approaches for neurodegenerative disorders.

scholarly journals Disease-associated GRIN protein truncating variants trigger NMDA receptor loss-of-function

2020 ◽  
Author(s):  
Ana Santos-Gómez ◽  
Federico Miguez-Cabello ◽  
Adrián García-Recio ◽  
Sílvia Locubiche ◽  
Roberto García-Díaz ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
De Novo ◽  
Surface Expression ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Neuronal Function ◽  
Clinical Phenotypes ◽  
Functional Studies ◽  
Therapeutic Decisions ◽  
A Subunit

Abstract De novo GRIN variants, encoding for the ionotropic glutamate NMDA receptor subunits, have been recently associated with GRIN-related disorders (GRDs), a group of rare paediatric encephalopathies. Current investigational and clinical efforts are focused to functionally stratify GRIN variants, towards precision therapies of this primary disturbance of glutamatergic transmission that affects neuronal function and brain. In the present study, we aimed to comprehensively delineate the functional outcomes and clinical phenotypes of GRIN protein truncating variants (PTVs) -accounting for ~ 20% of disease-associated GRIN variants- hypothetically provoking NMDAR hypofunctionality. To tackle this question, we created a comprehensive GRIN PTVs variants database compiling a cohort of 9 individuals harbouring GRIN PTVs, together with previously identified variants, to build-up an extensive GRIN PTVs repertoire composed of 293 unique variants. Genotype–phenotype correlation studies were conducted, followed by cell-based assays of selected paradigmatic GRIN PTVs, allowing their functional annotation. Genetic and clinical phenotypes metaanalysis revealed that heterozygous GRIN1, GRIN2C, GRIN2D, GRIN3A and GRIN3B PTVs are non-pathogenic. In contrast, heterozygous GRIN2A and GRIN2B PTVs are associated with specific neurological clinical phenotypes in a subunit- and domain-dependent manner. Mechanistically, cell-based assays showed that paradigmatic pathogenic GRIN2A and GRIN2B PTVs result on a decrease of NMDAR surface expression and NMDAR-mediated currents, ultimately leading to NMDAR functional haploinsufficiency.. Overall, these findings contribute to delineate GRIN PTVs genotype–phenotype association, and GRIN variants stratification. Functional studies showed that GRIN2A and GRIN2B pathogenic PTVs trigger NMDAR hypofunctionality, and thus accelerate therapeutic decisions for this neurodevelopmental condition.

Abstract 555: Functional Characterization Uncovered Novel Loss-of-function Mutations in ABCA1

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Xin Bi ◽  
James McParland ◽  
Jian Wang ◽  
Adam D McIntyre ◽  
Robert A Hegele ◽  
...  
Keyword(s):  
In Silico ◽  
Cholesterol Efflux ◽  
Functional Characterization ◽  
In Silico Analysis ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Wild Type ◽  
Functional Studies ◽  
Lipid Efflux

Objectives: ABCA1 encodes the membrane protein ATP-binding cassette transporter A1 (ABCA1), a pivotal player in nascent HDL formation via its ability to facilitate cholesterol and phospholipid efflux to apolipoprotein A-I (ApoA-I). ABCA1 variants are frequently found in subjects with primary hypoalphalipoproteinemia, however, their pathogenicity and causal link with the clinical phenotype are not always known. Methods: In silico analysis (Mutation Assessor, PANTHER, PolyPhen-2, PROVEAN, SIFT, and VEST) were performed to predict the functional consequences of ABCA1 missense variants found in our cohort of hypoalphalipoproteinemia. A subset of novel ABCA1 variants were generated in vitro through site-directed mutagenesis and their abilities in mediating lipid efflux to apoA-I were determined using standard methods. Results: A total of 32 mutations in ABCA1 were identified, among which 15 were classified as missense, 9 as nonsense or frameshift, 7 as intronic, and 1 as ”no-protein”. We selected 5 variants that were labeled as pathogenic or possibly pathogenic by in silico analysis to conduct functional studies. Two newly identified mutations in ABCA1, a nonsense mutation (p.E1005X) and a missense mutation (p.S2046R), resulted in complete loss of the canonical lipid efflux function of ABCA1 (2.5% and 1.8% of wild type cholesterol efflux level respectively). These results were concordant with the phenotypic characteristics of the carriers. Three additional mutations (p.G750W and p.R1341T and p.I1085F) resulted in only a partial loss of function (66-75% of wild type cholesterol efflux level). These results were somewhat discordant with the phenotype of the heterozygote carriers (HDL-C levels of 16, 14 and 38 mg/dl respectively), suggesting the presence of additional causal factors. Conclusions: These results support E1005X and S2046R as ABCA1 loss-of-function mutations and highlight the need to conduct functional studies on unknown variants to determine their pathogenicity.

scholarly journals In Vitro Analyses of Novel HCN4 Gene Mutations

2018 ◽  
Vol 49 (3) ◽  
pp. 1238-1248 ◽  
Author(s):  
Melina Möller ◽  
Nicole Silbernagel ◽  
Eva Wrobel ◽  
Birgit Stallmayer ◽  
Elsie Amedonu ◽  
...  
Keyword(s):  
Sinus Node ◽  
Membrane Surface ◽  
Functional Characterization ◽  
Surface Expression ◽  
Confocal Imaging ◽  
Xenopus Laevis Oocytes ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Wild Type ◽  
Adrenergic Stimulation

Background/Aims: The hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 contributes significantly to the generation of basic cardiac electrical activity in the sinus node and is a mediator of modulation by β–adrenergic stimulation. Heterologous expression of sick sinus syndrome (SSS) and bradycardia associated mutations within the human HCN4 gene results in altered channel function. The main aim was to describe the functional characterization of three (two novel and one known) missense mutations of HCN4 identified in families with SSS. Methods: Here, the two-electrode voltage clamp technique on Xenopus laevis oocytes and confocal imaging on transfected COS7 cells respectively, were used to analyze the functional effects of three HCN4 mutations; R378C, R550H, and E1193Q. Membrane surface expressions of wild type and the mutant channels were assessed by confocal microscopy, chemiluminescence assay, and Western blot in COS7 and HeLa cells. Results: The homomeric mutant channels R550H and E1193Q showed loss of function through increased rates of deactivation and distinctly reduced surface expression in all three homomeric mutant channels. HCN4 channels containing R550H and E1193Q mutant subunits only showed minor effects on the voltage dependence and rates of activation/deactivation. In contrast, homomeric R378C exerted a left-shifted activation curve and slowed activation kinetics. These effects were reduced in heteromeric co-expression of R378C with wild-type (WT) channels. Conclusion: Dysfunction of homomeric/heteromeric mutant HCN4-R378C, R550H, and E1193Q channels in the present study was primarily caused by loss of function due to decreased channel surface expression.

scholarly journals Functional roles of aspartate residues of the proton-coupled folate transporter (PCFT-SLC46A1); a D156Y mutation causing hereditary folate malabsorption

Blood ◽  
2010 ◽  
Vol 116 (24) ◽  
pp. 5162-5169 ◽  
Author(s):  
Daniel Sanghoon Shin ◽  
Sang Hee Min ◽  
Laura Russell ◽  
Rongbao Zhao ◽  
Andras Fiser ◽  
...  
Keyword(s):  
Small Intestine ◽  
Critical Role ◽  
Surface Expression ◽  
Loss Of Function ◽  
Wild Type ◽  
Intracellular Loop ◽  
Folate Transport ◽  
Functional Roles ◽  
Proximal Small Intestine ◽  
Pakistani Origin

Abstract The proton-coupled folate transporter (PCFT; SLC46A1) mediates folate transport into enterocytes in the proximal small intestine; pcft loss-of-function mutations are the basis for hereditary folate malabsorption. The current study explored the roles of Asp residues in PCFT function. A novel, homozygous, loss-of-function mutation, D156Y, was identified in a child of Pakistani origin with hereditary folate malabsorption. Of the 6 other conserved Asp residues, only one, D109, is shown to be required for function. D156Y, along with a variety of other substitutions at this site (Trp, Phe, Val, Asn, or Lys), lacked function due to instability of the PCFT protein. Substantial function was preserved with Glu, Gly, and, to a lesser extent, with Ser, Thr, and Ala substitutions. This correlated with PCFT bio-tinylated at the cell surface. In contrast, all D109 mutants, including D109E, lacked function irrespective of pH (4.5, 5.5, and 7.4) or substrate concentration (0.5-100μM), despite surface expression comparable to wild-type PCFT. Hence, D156 plays a critical role in PCFT protein stability, and D109, located in the first intracellular loop between the second and third transmembrane domains, is absolutely required for PCFT function.

Heterozygous Mutation (Q459R) in the Calcium-Sensing Receptor Gene Causes Familial Hypocalciuric Hypercalcemia 1 (FHH1)

2019 ◽  
Vol 105 (4) ◽  
pp. e1322-e1330 ◽  
Author(s):  
Ida Marie Boisen ◽  
Iris Mos ◽  
Eva Merete Lerche-Black ◽  
Anders Juul ◽  
Hans Bräuner-Osborne ◽  
...  
Keyword(s):  
Receptor Gene ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Familial Hypocalciuric Hypercalcemia ◽  
Loss Of Function ◽  
Wild Type ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Calcium Sensing Receptor Gene

Abstract Context Several heterozygous loss-of-function mutations in the calcium-sensing receptor gene (CASR) leading to elevated ionized serum calcium and familial hypocalciuric hypercalcemia 1 (FHH1) have been characterized. Few mutations are not pathogenic, and previous studies suggested that the Q459R mutation does not result in an FHH1 phenotype. Objective We identified a family with a heterozygous CASR Q459R mutation and characterized their calcium homeostasis and the pathophysiological mechanisms of a homozygous and heterozygous Q459R mutation in vitro. Design The index patient and her family had clinical, biochemical, and genetic analyses performed. In vitro functional characterization of homozygous and heterozygous (Q459R) mutations was conducted by determining CaSR cell-surface expression and inositol monophosphate (IP1) signaling in transiently transfected human embryonic kidney 293A (HEK293A) cells. Results All 3 heterozygous carriers had mild asymptomatic hypercalcemia, hypocalciuria, and 2 had elevated serum parathyroid hormone (PTH). In vitro characterization in HEK293A cells revealed that CASR Q459R is a loss-of-function mutation with no impact on cell-surface expression. Cells with the homozygous Q459R genotype had significantly reduced calcium potency of IP1 signaling compared to wild type, whereas the heterozygous Q459R also had lower calcium potency albeit not significantly different from wild type. Conclusion A loss-of-function Q459R mutation in CASR in a family caused FHH1 characterized by elevated ionized calcium and PTH and low calcium excretion. The marked presence of CaSR at the membrane and inhibition of IP1 signaling in vitro suggest that calcimimetics may be functional in patients with this mutation, which seems to be a mild loss-of-function mutation associated with autosomal dominant transmission of FHH1.

scholarly journals Author response: Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms

2016 ◽  
Author(s):  
Daniel L Kober ◽  
Jennifer M Alexander-Brett ◽  
Celeste M Karch ◽  
Carlos Cruchaga ◽  
Marco Colonna ◽  
...  
Keyword(s):  
Neurodegenerative Disease ◽  
Author Response ◽  
Loss Of Function ◽  
Functional Surface ◽  
Disease Mutations

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals The NALCN Channel Regulator UNC-80 Functions in a Subset of Interneurons To Regulate Caenorhabditis elegans Reversal Behavior

2019 ◽  
Vol 10 (1) ◽  
pp. 199-210 ◽  
Author(s):  
Chuanman Zhou ◽  
Jintao Luo ◽  
Xiaohui He ◽  
Qian Zhou ◽  
Yunxia He ◽  
...  
Keyword(s):  
Plant Hormone ◽  
Neuronal Excitability ◽  
Resting Membrane Potential ◽  
Loss Of Function ◽  
Wild Type ◽  
C Elegans ◽  
Link Type ◽  
Complex Functions ◽  
And Function ◽  
Multiple Loss

NALCN (Na+leak channel, non-selective) is a conserved, voltage-insensitive cation channel that regulates resting membrane potential and neuronal excitability. UNC79 and UNC80 are key regulators of the channel function. However, the behavioral effects of the channel complex are not entirely clear and the neurons in which the channel functions remain to be identified. In a forward genetic screen for C. elegans mutants with defective avoidance response to the plant hormone methyl salicylate (MeSa), we isolated multiple loss-of-function mutations in unc-80 and unc-79. C. elegans NALCN mutants exhibited similarly defective MeSa avoidance. Interestingly, NALCN, unc-80 and unc-79 mutants all showed wild type-like responses to other attractive or repelling odorants, suggesting that NALCN does not broadly affect odor detection or related forward and reversal behaviors. To understand in which neurons the channel functions, we determined the identities of a subset of unc-80-expressing neurons. We found that unc-79 and unc-80 are expressed and function in overlapping neurons, which verified previous assumptions. Neuron-specific transgene rescue and knockdown experiments suggest that the command interneurons AVA and AVE and the anterior guidepost neuron AVG can play a sufficient role in mediating unc-80 regulation of the MeSa avoidance. Though primarily based on genetic analyses, our results further imply that MeSa might activate NALCN by direct or indirect actions. Altogether, we provide an initial look into the key neurons in which the NALCN channel complex functions and identify a novel function of the channel in regulating C. elegans reversal behavior through command interneurons.

scholarly journals Identification of a novel homozygous loss-of-function mutation in FUCA1 gene causing severe fucosidosis: A case report

2021 ◽  
Vol 49 (4) ◽  
pp. 030006052110059
Author(s):  
Xinwen Zhang ◽  
Shaozhi Zhao ◽  
Hongwei Liu ◽  
Xiaoyan Wang ◽  
Xiaolei Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Lysosomal Storage Disorder ◽  
Autosomal Recessive ◽  
Signs And Symptoms ◽  
Lysosomal Storage ◽  
Loss Of Function ◽  
Wild Type ◽  
Single Nucleotide ◽  
Whole Exome ◽  
Exon 1

Fucosidosis is a rare lysosomal storage disorder characterized by deficiency of α-L-fucosidase with an autosomal recessive mode of inheritance. Here, we describe a 4-year-old Chinese boy with signs and symptoms of fucosidosis but his parents were phenotypically normal. Whole exome sequencing (WES) identified a novel homozygous single nucleotide deletion (c.82delG) in the exon 1 of the FUCA1 gene. This mutation will lead to a frameshift which will result in the formation of a truncated FUCA1 protein (p.Val28Cysfs*105) of 132 amino acids approximately one-third the size of the wild type FUCA1 protein (466 amino acids). Both parents were carrying the mutation in a heterozygous state. This study expands the mutational spectrum of the FUCA1 gene associated with fucosidosis and emphasises the benefits of WES for accurate and timely clinical diagnosis of this rare disease.

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