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.

scholarly journals Characterization of Loss-Of-Function KCNJ2 Mutations in Atypical Andersen Tawil Syndrome

2021 ◽  
Vol 12 ◽  
Author(s):  
Pauline Le Tanno ◽  
Mathilde Folacci ◽  
Jean Revilloud ◽  
Laurence Faivre ◽  
Gabriel Laurent ◽  
...  
Keyword(s):  
De Novo ◽  
Beta Blockers ◽  
Periodic Paralysis ◽  
Dominant Negative ◽  
Polymorphic Ventricular Tachycardia ◽  
Loss Of Function ◽  
Functional Studies ◽  
Dysmorphic Features ◽  
Therapeutic Decisions

Andersen-Tawil Syndrome (ATS) is a rare disease defined by the association of cardiac arrhythmias, periodic paralysis and dysmorphic features, and is caused by KCNJ2 loss-of-function mutations. However, when extracardiac symptoms are atypical or absent, the patient can be diagnosed with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), a rare arrhythmia at high risk of sudden death, mostly due to RYR2 mutations. The identification of KCNJ2 variants in CPVT suspicion is very rare but important because beta blockers, the cornerstone of CPVT therapy, could be less efficient. We report here the cases of two patients addressed for CPVT-like phenotypes. Genetic investigations led to the identification of p. Arg82Trp and p. Pro186Gln de novo variants in the KCNJ2 gene. Functional studies showed that both variants forms of Kir2.1 monomers act as dominant negative and drastically reduced the activity of the tetrameric channel. We characterize here a new pathogenic variant (p.Pro186Gln) of KCNJ2 gene and highlight the interest of accurate cardiologic evaluation and of attention to extracardiac signs to distinguish CPVT from atypical ATS, and guide therapeutic decisions. We also confirm that the KCNJ2 gene must be investigated during CPVT molecular analysis.

scholarly journals Loss–of–function variants in the KCNQ5 gene are associated with genetic generalized epilepsies

2021 ◽  
Author(s):  
Johanna Krueger ◽  
Julian Schubert ◽  
Josua Kegele ◽  
Audrey Labalme ◽  
Miaomiao Mao ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Surface Expression ◽  
Epileptic Encephalopathy ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
K Channel ◽  
Loss Of Function ◽  
Normal Surface ◽  
Missense Variants

Objective: De novo missense variants in KCNQ5, encoding the voltage–gated K+ channel KV7.5, have been described as a cause of developmental and epileptic encephalopathy (DEE) or intellectual disability (ID). We set out to identify disease–related KCNQ5 variants in genetic generalized epilepsy (GGE) and their underlying mechanisms. Methods: 1292 families with GGE were studied by next-generation sequencing. Whole–cell patch–clamp recordings, biotinylation and phospholipid overlay assays were performed in mammalian cells combined with docking and homology modeling. Results: We identified three deleterious heterozygous missense variants, one truncation and one splice site alteration in five independent families with GGE with predominant absence seizures, two variants were also associated with mild to moderate ID. All three missense variants displayed a strongly decreased current density indicating a loss–of–function (LOF). When mutant channels were co–expressed with wild–type (WT) KV7.5 or KV7.5 and KV7.3 channels, three variants also revealed a significant dominant–negative effect on WT channels. Other gating parameters were unchanged. Biotinylation assays indicated a normal surface expression of the variants. The p.Arg359Cys variant altered PI(4,5)P2–interaction, presumably in the non–conducting preopen–closed state. Interpretation: Our study indicates that specific deleterious KCNQ5 variants are associated with GGE, partially combined with mild to moderate ID. The disease mechanism is a LOF partially with dominant–negative effects through functional, rather than trafficking deficits. LOF of KV7.5 channels will reduce the M–current, likely resulting in increased excitability of KV7.5–expressing neurons. Further studies on a network level are necessary to understand which circuits are affected and how the variants induce generalized seizures.

Biallelic Mutations in SLC1A2; an Additional Mode of Inheritance for SLC1A2-Related Epilepsy

2017 ◽  
Vol 49 (01) ◽  
pp. 059-062 ◽  
Author(s):  
Mirjana Gusic ◽  
Roman Günthner ◽  
Bader Alhaddad ◽  
Reka Kovacs-Nagy ◽  
Christine Makowski ◽  
...  
Keyword(s):  
De Novo ◽  
Epileptic Seizures ◽  
Epileptic Encephalopathy ◽  
Genetic Mechanism ◽  
Loss Of Function ◽  
De Novo Mutations ◽  
Functional Studies ◽  
Additional Mode ◽  
Biallelic Mutations ◽  
Mode Of Inheritance

AbstractRecently, heterozygous de novo mutations in SCL1A2 have been reported to underlie severe early-onset epileptic encephalopathy. In one male presenting with epileptic seizures and visual impairment, we identified a novel homozygous splicing variant in SCL1A2 (c.1421 + 1G > C) by using exome sequencing. Functional studies on cDNA level confirmed a consecutive loss of function. Our findings suggest that not only de novo mutations but also biallelic variants in SLC1A2 can cause epilepsy and that there is an additional autosomal recessive mode of inheritance. These findings also contribute to the understanding of the genetic mechanism of autosomal dominant SLC1A2-related epileptic encephalopathy as they exclude haploinsufficiency as exclusive genetic mechanism.

scholarly journals Identification of Cdca7 as a novel Notch transcriptional target involved in hematopoietic stem cell emergence

2014 ◽  
Vol 211 (12) ◽  
pp. 2411-2423 ◽  
Author(s):  
Jordi Guiu ◽  
Dylan J.M. Bergen ◽  
Emma De Pater ◽  
Abul B.M.M.K. Islam ◽  
Verónica Ayllón ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
De Novo ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Hematopoietic Differentiation ◽  
Loss Of Function ◽  
Promoter Regions ◽  
Hematopoietic Stem

Hematopoietic stem cell (HSC) specification occurs in the embryonic aorta and requires Notch activation; however, most of the Notch-regulated elements controlling de novo HSC generation are still unknown. Here, we identify putative direct Notch targets in the aorta-gonad-mesonephros (AGM) embryonic tissue by chromatin precipitation using antibodies against the Notch partner RBPj. By ChIP-on-chip analysis of the precipitated DNA, we identified 701 promoter regions that were candidates to be regulated by Notch in the AGM. One of the most enriched regions corresponded to the Cdca7 gene, which was subsequently confirmed to recruit the RBPj factor but also Notch1 in AGM cells. We found that during embryonic hematopoietic development, expression of Cdca7 is restricted to the hematopoietic clusters of the aorta, and it is strongly up-regulated in the hemogenic population during human embryonic stem cell hematopoietic differentiation in a Notch-dependent manner. Down-regulation of Cdca7 mRNA in cultured AGM cells significantly induces hematopoietic differentiation and loss of the progenitor population. Finally, using loss-of-function experiments in zebrafish, we demonstrate that CDCA7 contributes to HSC emergence in vivo during embryonic development. Thus, our study identifies Cdca7 as an evolutionary conserved Notch target involved in HSC emergence.

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 CNKSR2-related neurodevelopmental and epilepsy disorder: a cohort of 13 new families and literature review indicating a predominance of loss of function pathogenic variants

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Leigh Ann Higa ◽  
Jennifer Wardley ◽  
Christopher Wardley ◽  
Susan Singh ◽  
Timothy Foster ◽  
...  
Keyword(s):  
Developmental Delay ◽  
Sleep Disturbances ◽  
Right Hemisphere ◽  
De Novo ◽  
Autism Spectrum ◽  
Loss Of Function ◽  
Seizure Onset ◽  
Genomic Deletions ◽  
Functional Studies ◽  
Pathogenic Variants

Abstract Background Pathogenic variants in connector enhancer of kinase suppressor of Ras-2 (CNKSR2) located on the X chromosome (Xp22.12) lead to a disorder characterized by developmental delay and a characteristic seizure phenotype. To date, 20 affected males representing 13 different pathogenic variants have been published. Case presentation We identified an 8-year-old male with seizures, abnormal electroencephalogram (EEG) with epileptiform abnormalities in the right hemisphere, and developmental delay with notable loss of speech following seizure onset. Additional concerns include multiple nighttime awakenings, hyperactivity, and autism spectrum disorder. Genetic testing identified a de novo pathogenic nonsense variant in CNKSR2. Through an active family support group, an additional 12 males are described, each harboring a different CNKSR2 variant. The clinical presentation and natural history consistently show early developmental delay, sleep disturbances, and seizure onset in childhood that is initially intractable but later becomes better controlled. Virtually all of the pathogenic variants are predicted to be loss of function, including genomic deletions, nonsense variants, splice site mutations, and small insertions or deletions. Conclusions This expanded knowledge, combined with functional studies and work with animal models currently underway, will enable a better understanding and improved ability to care for individuals with CNKSR2-related neurodevelopmental and epilepsy disorder.

scholarly journals Factor XIII A is synthesized and expressed on the surface of U937 cells and alveolar macrophages

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 778-785
Author(s):  
RL Kradin ◽  
GW Lynch ◽  
JT Kurnick ◽  
M Erikson ◽  
RB Colvin ◽  
...  
Keyword(s):  
Cell Surface ◽  
Alveolar Macrophages ◽  
Factor Xiii ◽  
De Novo ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
U937 Cells ◽  
Serum Free ◽  
Dependent Activity ◽  
A Subunit

Factor XIII A subunit was detected in U937 cells and human alveolar macrophages by immunohistology and Western blotting. U937 cells synthesize factor XIII A subunit de novo under serum-free, platelet- free conditions, as indicated by 35S-methionine labeling and immunoprecipitation. Thrombin-dependent activity was demonstrated to account for 98% of the total transglutaminase activity in U937 cells (1.5 micrograms per 0.5 X 10(6) cells/mL). Intact U937 cells and alveolar macrophages and homogenates from these cells cross-linked fibrin to form gamma-gamma and alpha-polymers. Factor XIII A was detected on the surface of intact U937 cells and macrophages by flow cytometry and 125I-labeling and immunoprecipitation. Cell surface expression of factor XIII A was augmented in the presence of several soluble macrophage activators; however, no concurrent increase in its biosynthesis was observed. The presence and cell surface expression of factor XIII A subunit within macrophages suggest new pathways by which these cells may function in clotting and in the remodeling of the extracellular matrix during inflammation and wound healing.

scholarly journals Temporal order of RNase IIIb and loss-of-function mutations during development determines phenotype in pleuropulmonary blastoma / DICER1 syndrome: a unique variant of the two-hit tumor suppression model

F1000Research ◽  
2018 ◽  
Vol 4 ◽  
pp. 214 ◽  
Author(s):  
Mark Brenneman ◽  
Amanda Field ◽  
Jiandong Yang ◽  
Gretchen Williams ◽  
Leslie Doros ◽  
...  
Keyword(s):  
Temporal Order ◽  
De Novo ◽  
Lung Tumor ◽  
Malignant Tumors ◽  
Early Adulthood ◽  
Pleuropulmonary Blastoma ◽  
Neoplastic Disease ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Clinical Phenotypes

Pleuropulmonary blastoma (PPB) is the most frequent pediatric lung tumor and often the first indication of a pleiotropic cancer predisposition, DICER1 syndrome, comprising a range of other individually rare, benign and malignant tumors of childhood and early adulthood. The genetics of DICER1-associated tumorigenesis are unusual in that tumors typically bear neomorphic missense mutations at one of five specific “hotspot” codons within the RNase IIIb domain of DICER 1, combined with complete loss of function (LOF) in the other allele. We analyzed a cohort of 124 PPB children for predisposing DICER1 mutations and sought correlations with clinical phenotypes. Over 70% have inherited or de novo germline LOF mutations, most of which truncate the DICER1 open reading frame. We identified a minority of patients who have no germline mutation, but are instead mosaic for predisposing DICER1 mutations. Mosaicism for RNase IIIb domain hotspot mutations defines a special category of DICER1 syndrome patients, clinically distinguished from those with germline or mosaic LOF mutations by earlier onsets and numerous discrete foci of neoplastic disease involving multiple syndromic organ sites. A final category of PBB patients lack predisposing germline or mosaic mutations and have sporadic (rather than syndromic) disease limited to a single PPB tumor bearing tumor-specific RNase IIIb and LOF mutations. We propose that acquisition of a neomorphic RNase IIIb domain mutation is the rate limiting event in DICER1-associated tumorigenesis, and that distinct clinical phenotypes associated with mutational categories reflect the temporal order in which LOF and RNase IIIb domain mutations are acquired during development.

Mosaic IL6ST variant inducing constitutive GP130 cytokine receptor signaling as a cause of neonatal onset immunodeficiency with autoinflammation and dysmorphy

2021 ◽  
Author(s):  
Anna Materna-Kiryluk ◽  
Agnieszka Pollak ◽  
Karol Gawalski ◽  
Aleksandra Szczawinska-Poplonyk ◽  
Zuzanna Rydzynska ◽  
...  
Keyword(s):  
Lymphoblastoid Cell Line ◽  
Autosomal Recessive ◽  
De Novo ◽  
Buccal Swab ◽  
Loss Of Function ◽  
Urine Sediment ◽  
Functional Studies ◽  
Hyper Ige Syndrome ◽  
Neonatal Onset ◽  
Stat3 Phosphorylation

Abstract IL6ST encodes the GP130 protein which transduces the proinflammatory signaling of the IL6 cytokine family through JAK/STAT activation. Biallelic loss-of-function IL6ST variants cause autosomal recessive hyper-IgE syndrome or a variant of Stuve-Wiedemann syndrome. Somatic gain-of-function IL6ST mutations, in particular small monoallelic in-frame deletions of which the most prevalent is IL6ST Ser187_Tyr190del, are an established cause of inflammatory hepatocellular tumors (IHCA) but so far, no disease caused by such mutations present constitutively has been described. Herein, we report a pediatric proband with a novel syndrome of neonatal onset immunodeficiency with autoinflammation and dysmorphy associated with the IL6ST Tyr186_Tyr190del variant present constitutively. Tyr186_Tyr190del was found by exome sequencing and shown to be de novo (absent in proband’s parents and siblings) and mosaic (present in approx. 15–40% of cells depending on the tissue studied—blood, urine sediment, hair bulbs, buccal swab). Functional studies were performed in EBV-immortalized patient’s B-cell lymphoblastoid cell line, which carried the variant in approximately 95% of cells. Western blot showed that the patient’s cells exhibited constitutive hyperphosphorylation of Tyr705 in STAT3 indicative of IL6 independent activation of GP130. Interestingly, the STAT3 phosphorylation could be inhibited with ruxolitinib as well as tofacitinib, which are clinically approved JAK1 and JAK3 (to lesser extent JAK2 and JAK1) inhibitors, respectively. Given our results and the recent reports of ruxolitinib and tofacitinib use for the treatment of diseases caused by direct activation of STAT3 or STAT1 we speculate that these drugs may be effective in the treatment of our patient’s condition.

scholarly journals MIR172d Is Required for Floral Organ Identity and Number in Tomato

2021 ◽  
Vol 22 (9) ◽  
pp. 4659
Author(s):  
Wanping Lin ◽  
Suresh Kumar Gupta ◽  
Tzahi Arazi ◽  
Ben Spitzer-Rimon
Keyword(s):  
Transcription Factors ◽  
Transcriptional Repression ◽  
De Novo ◽  
Floral Organ ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Floral Organ Identity ◽  
Organ Identity ◽  
Unique Species ◽  
Dose Dependent

MicroRNA172 (miR172) functions as a central regulator of flowering time and flower development by post-transcriptional repression of APETALA2-LIKE transcription factors. In the model crop Solanum lycopersicum (tomato), the miR172 family is still poorly annotated and information about the functions of specific members is lacking. Here, de-novo prediction of tomato miR172 coding loci identified seven genes (SlMIR172a-g), that code for four unique species of miR172 (sly-miR172). During reproductive development, sly-miR172s are differentially expressed, with sly-miR172c and sly-miR172d being the most abundant members in developing flowers, and are predicted to guide the cleavage of eight APETALA2-LIKE transcription factors. By CRISPR-Cas9 co-targeting of SlMIR172c and SlMIR172d we have generated a battery of loss-of-function and hypomorphic mutants (slmir172c-dCR). The slmir172c-dCR plants developed normal shoot but their flowers displayed graded floral organ abnormalities. Whereas slmir172cCR loss-of-function caused only a slight greening of petals and stamens, hypomorphic and loss-of-function slmir172dCR alleles were associated with the conversion of petals and stamens to sepaloids, which were produced in excess. Interestingly, the degrees of floral organ identity alteration and proliferation were directly correlated with the reduction in sly-miR172d activity. These results suggest that sly-miR172d regulates in a dose-dependent manner floral organ identity and number, likely by negatively regulating its APETALA2-like targets.

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