Identification and functional characterisation of a novel KCNJ2 mutation, Val302del, causing Andersen–Tawil syndrome

Loss-of-function mutations of the KCNJ2 gene encoding for the inward rectifier potassium channel subunit Kir2.1 cause Andersen–Tawil Syndrome (ATS), a rare genetic disorder characterised by periodic paralysis, ventricular arrhythmias, and dysmorphic features. Clinical manifestations of the disease appear to vary greatly with the nature of mutation, therefore, functional characterisation of ATS-causing mutations is of clinical importance. In this study, we describe the identification and functional analysis of a novel KCNJ2 mutation, Val302del, identified in a patient with ATS. Heterologously expressed wild type (WT) and Val302del mutant alleles showed similar subcellular distribution of the Kir2.1 protein with high intensity labelling from the membrane region, demonstrating normal membrane trafficking of the Val302del Kir2.1 variant. Cells transfected with the WT allele displayed a robust current with strong inward rectification, while no current above background was detected in cells expressing the Val302del Kir2.1 subunit. Co-transfection of CHO cells with the WT and the Val302del Kir2.1 revealed a dose-dependent inhibitory effect of the Val302del Kir2.1 mutant subunit on WT Kir2.1 currents. These observations indicate that the WT and the Val302del mutant subunits co-assemble in the cell membrane and that the mutation affects potassium conductivity and (or) gating of the WT/Val302del heteromeric Kir2.1 channels.

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The Multifaceted Roles of MicroRNAs in Cystic Fibrosis

Diagnostics ◽

10.3390/diagnostics10121102 ◽

2020 ◽

Vol 10 (12) ◽

pp. 1102

Author(s):

Fatima Domenica Elisa De Palma ◽

Valeria Raia ◽

Guido Kroemer ◽

Maria Chiara Maiuri

Keyword(s):

Cystic Fibrosis ◽

Respiratory Infections ◽

Current Knowledge ◽

Pancreatic Insufficiency ◽

Clinical Manifestations ◽

Predictive Biomarkers ◽

Loss Of Function ◽

Gene Encoding ◽

Multisystemic Disease

Cystic fibrosis (CF) is a lifelong disorder affecting 1 in 3500 live births worldwide. It is a monogenetic autosomal recessive disease caused by loss-of-function mutations in the gene encoding the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR), the impairment of which leads to ionic disequilibria in exocrine organs. This translates into a chronic multisystemic disease characterized by airway obstruction, respiratory infections, and pancreatic insufficiency as well as hepatobiliary and gastrointestinal dysfunction. Molecular characterization of the mutational heterogeneity of CFTR (affected by more than 2000 variants) improved the understanding and management of CF. However, these CFTR variants are linked to different clinical manifestations and phenotypes, and they affect response to treatments. Expanding evidence suggests that multisystemic disease affects CF pathology via impairing either CFTR or proteins regulated by CFTR. Thus, altering the expression of miRNAs in vivo could constitute an appealing strategy for developing new CF therapies. In this review, we will first describe the pathophysiology and clinical management of CF. Then, we will summarize the current knowledge on altered miRNAs in CF patients, with a focus on the miRNAs involved in the deregulation of CFTR and in the modulation of inflammation. We will highlight recent findings on the potential utility of measuring circulating miRNAs in CF as diagnostic, prognostic, and predictive biomarkers. Finally, we will provide an overview on potential miRNA-based therapeutic approaches.

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Influence of Ferroportin Disease Mutations on Manganese Accumulation and Toxicity (P24-060-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz044.p24-060-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Eun-kyung Choi ◽

Young-Ah Seo

Keyword(s):

Genetic Disorder ◽

Functional Results ◽

Site Directed Mutagenesis ◽

Loss Of Function ◽

Gene Encoding ◽

Excess Iron ◽

Funding Sources ◽

Disease Mutations ◽

The Impact

Abstract Objectives Hemochromatosis is a frequent genetic disorder characterized by the accumulation of excess iron across tissues. Mutations in the FPN1 gene, encoding a cell-surface iron exporter ferroportin (Fpn), are responsible for hemochromatosis type 4, also known as ferroportin disease. Recently, Fpn has been implicated in the regulation of manganese (Mn), another essential nutrient required for numerous cellular enzymes. However, the roles of Fpn in Mn regulation remain ill defined, and the impact of disease mutations on cellular Mn levels is unknown. Thus, this study aimed to define the role of Fpn in Mn regulation and determine the functional consequences of ferroportin disease mutations in cellular Mn levels. Methods Thus far, over 50 mutations in Fpn have been identified in hemochromatosis type 4/ferroportin disease. To test whether these mutations alter cellular Mn metabolism, we constructed an expression vector encoding human Fpn with a C-terminal HA epitope tag and introduced nine clinically relevant mutations by site-directed mutagenesis. Based on previously reported in vitro functional results, we selected five ferroportin disease mutations from each of the two groups: five loss-of-function (LOF) mutations (G80S, R88G, D157G, D157Y, and V162Δ) and four gain-of-function (GOF) mutations (N144H, N144T, C326S, and and S338R). Results Here, we provide evidence that Fpn can export Mn from cells into extracellular space. Fpn appears to play protective roles in Mn-induced cellular toxicity and oxidative stress. Finally, disease mutations interfere with Fpn's role in controlling Mn levels as well as the stability of Fpn. Conclusions These results define the function of Fpn as an exporter of both iron and Mn and highlight the potential involvement of Mn dysregulation in ferroportin disease. Funding Sources National Institutes of Health (NIH) to Y.A.S. (K99/R00 ES024340).

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A Novel Nonsense Mutation in FGB (c.1421G>A; p.Trp474Ter) in the Beta Chain of Fibrinogen Causing Hypofibrinogenemia with Bleeding Phenotype

Biomedicines ◽

10.3390/biomedicines8120605 ◽

2020 ◽

Vol 8 (12) ◽

pp. 605

Author(s):

Tomas Simurda ◽

Rui Vilar ◽

Jana Zolkova ◽

Eliska Ceznerova ◽

Zuzana Kolkova ◽

...

Keyword(s):

Structural Changes ◽

Phenotypic Expression ◽

Clinical Manifestations ◽

Severe Bleeding ◽

Loss Of Function ◽

Protein Modelling ◽

Gene Encoding ◽

Heterozygous Variant ◽

Thrombotic Complications ◽

Congenital Hypofibrinogenemia

Congenital hypofibrinogenemia is a rare bleeding disorder characterized by a proportional decrease of functional and antigenic fibrinogen levels. Hypofibrinogenemia can be considered the phenotypic expression of heterozygous loss of function mutations occurring within one of the three fibrinogen genes (FGA, FGB, and FGG). Clinical manifestations are highly variable; most patients are usually asymptomatic, but may appear with mild to severe bleeding or thrombotic complications. We have sequenced all exons of the FGA, FGB, and FGG genes using the DNA isolated from the peripheral blood in two unrelated probands with mild hypofibrinogenemia. Coagulation screening, global hemostasis, and functional analysis tests were performed. Molecular modeling was used to predict the defect of synthesis and structural changes of the identified mutation. DNA sequencing revealed a novel heterozygous variant c.1421G>A in exon 8 of the FGB gene encoding a Bβ chain (p.Trp474Ter) in both patients. Clinical data from patients showed bleeding episodes. Protein modelling confirmed changes in the secondary structure of the molecule, with the loss of three β sheet arrangements. As expected by the low fibrinogen levels, turbidity analyses showed a reduced fibrin polymerisation and imaging difference in thickness fibrin fibers. We have to emphasize that our patients have a quantitative fibrinogen disorder; therefore, the reduced function is due to the reduced concentration of fibrinogen, since the Bβ chains carrying the mutation predicted to be retained inside the cell. The study of fibrinogen molecules using protein modelling may help us to understand causality and effect of novel genetic mutations.

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A Thyroid Hormone–Based Strategy for Correcting the Biochemical Abnormality in X-Linked Adrenoleukodystrophy

Endocrinology ◽

10.1210/en.2016-1842 ◽

2017 ◽

Vol 158 (5) ◽

pp. 1328-1338 ◽

Cited By ~ 20

Author(s):

Meredith D. Hartley ◽

Lisa L. Kirkemo ◽

Tapasree Banerji ◽

Thomas S. Scanlan

Keyword(s):

Fatty Acids ◽

Thyroid Hormone ◽

Thyroid Hormone Receptor ◽

Genetic Disorder ◽

Saturated Fatty Acids ◽

Clinical Stage ◽

Loss Of Function ◽

Biochemical Abnormality ◽

Gene Encoding ◽

Cns Demyelination

Abstract X-linked adrenoleukodystrophy (X-ALD) is a rare, genetic disorder characterized by adrenal insufficiency and central nervous system (CNS) demyelination. All patients with X-ALD have the biochemical abnormality of elevated blood and tissue levels of very long chain fatty acids (VLCFAs), saturated fatty acids with 24 to 26 carbons. X-ALD results from loss of function mutations in the gene encoding the peroxisomal transporter ABCD1, which is responsible for uptake of VLCFAs into peroxisomes for degradation by oxidation. One proposed therapeutic strategy for genetic complementation of ABCD1 is pharmacologic upregulation of ABCD2, a gene encoding a homologous peroxisomal transporter. Here, we show that thyroid hormone or sobetirome, a clinical-stage selective thyroid hormone receptor agonist, increases cerebral Abcd2 and lowers VLCFAs in blood, peripheral organs, and brains of mice with defective Abcd1. These results support an approach to treating X-ALD that involves a thyromimetic agent that reactivates VLCFA disposal both in the periphery and the CNS.

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DictyosteliumLvsB Mutants Model the Lysosomal Defects Associated with Chediak-Higashi Syndrome

Molecular Biology of the Cell ◽

10.1091/mbc.01-09-0454 ◽

2002 ◽

Vol 13 (2) ◽

pp. 656-669 ◽

Cited By ~ 46

Author(s):

Edward Harris ◽

Ning Wang ◽

Wei-l Wu ◽

Alisha Weatherford ◽

Arturo De Lozanne ◽

...

Keyword(s):

Membrane Trafficking ◽

Genetic Disorder ◽

Fluid Phase ◽

Negative Regulator ◽

Cysteine Proteinases ◽

Gene Encoding ◽

Lysosomal Trafficking ◽

Lysosome Biogenesis ◽

Genes Encoding ◽

Chediak Higashi Syndrome

Chediak-Higashi syndrome is a genetic disorder caused by mutations in a gene encoding a protein named LYST in humans (“lysosomal trafficking regulator”) or Beige in mice. A prominent feature of this disease is the accumulation of enlarged lysosome-related granules in a variety of cells. The genome of Dictyostelium discoideumcontains six genes encoding proteins that are related to LYST/Beige in amino acid sequence, and disruption of one of these genes,lvsA (large volumesphere), results in profound defects in cytokinesis. To better understand the function of this family of proteins in membrane trafficking, we have analyzed mutants disrupted in lvsA, lvsB, lvsC, lvsD, lvsE, and lvsF. Of all these, onlylvsA and lvsB mutants displayed interesting phenotypes in our assays. lvsA-null cells exhibited defects in phagocytosis and contained abnormal looking contractile vacuole membranes. Loss of LvsB, theDictyostelium protein most similar to LYST/Beige, resulted in the formation of enlarged vesicles that by multiple criteria appeared to be acidic lysosomes. The rates of endocytosis, phagocytosis, and fluid phase exocytosis were normal inlvsB-null cells. Also, the rates of processing and the efficiency of targeting of lysosomal α-mannosidase were normal, although lvsB mutants inefficiently retained α-mannosidase, as well as two other lysosomal cysteine proteinases. Finally, results of pulse-chase experiments indicated that an increase in fusion rates accounted for the enlarged lysosomes inlvsB-null cells, suggesting that LvsB acts as a negative regulator of fusion. Our results support the notion that LvsB/LYST/Beige function in a similar manner to regulate lysosome biogenesis.

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The spectrum of mutations in the PCFT gene, coding for an intestinal folate transporter, that are the basis for hereditary folate malabsorption

Blood ◽

10.1182/blood-2007-02-077099 ◽

2007 ◽

Vol 110 (4) ◽

pp. 1147-1152 ◽

Cited By ~ 122

Author(s):

Rongbao Zhao ◽

Sang Hee Min ◽

Andong Qiu ◽

Antoinette Sakaris ◽

Gary L. Goldberg ◽

...

Keyword(s):

Membrane Trafficking ◽

Autosomal Recessive ◽

Autosomal Recessive Disorder ◽

Amino Acid Residues ◽

Loss Of Function ◽

Folate Transport ◽

Gene Encoding ◽

Gene Coding ◽

The Central Nervous System ◽

Racial Ethnic

Abstract Hereditary folate malabsorption (HFM) is a rare autosomal recessive disorder caused by impaired intestinal folate absorption and impaired folate transport into the central nervous system. Recent studies in 1 family revealed that the molecular basis for this disorder is a loss-of-function mutation in the PCFT gene encoding a proton-coupled folate transporter. The current study broadens the understanding of the spectrum of alterations in the PCFT gene associated with HFM in 5 additional patients. There was no racial, ethnic, or sex pattern. A total of 4 different homozygous mutations were detected in 4 patients; 2 heterozygous mutations were identified in the fifth patient. Mutations involved 4 of the 5 exons, all at highly conserved amino acid residues. A total of 4 of the mutated transporters resulted in a complete loss of transport function, primarily due to decreased protein stability and/or defects in membrane trafficking, while 2 of the mutated carriers manifested residual function. Folate transport at low pH was markedly impaired in transformed lymphocytes from 2 patients. These findings further substantiate the role that mutations in PCFT play in the pathogenesis of HFM and will make possible rapid diagnosis and treatment of this disorder in infants, and prenatal diagnosis in families that carry a mutated gene.

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Morphologic alteration in the muscles of patients with hypokalemic periodic paralysis or myopathy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158650 ◽

1990 ◽

Vol 48 (3) ◽

pp. 222-223

Author(s):

T. Shimizu ◽

Y. Muranaka ◽

I. Ohta ◽

N. Honda

Keyword(s):

Clinical Manifestations ◽

Quadriceps Muscle ◽

Honeycomb Structure ◽

Periodic Paralysis ◽

Ultrastructural Changes ◽

Hypokalemic Periodic Paralysis ◽

Electron Microscopic ◽

Tubular Aggregates ◽

Hypokalemic Myopathy ◽

Transmission Electron

There have been many reports on ultrastructural alterations in muscles of hypokalemic periodic paralysis (hpp) and hypokalemic myopathy(hm). It is stressed in those reports that tubular structures such as tubular aggregates are usually to be found in hpp as a characteristic feature, but not in hm. We analyzed the histological differences between hpp and hm, comparing their clinical manifestations and morphologic changes in muscles. Materials analyzed were biopsied muscles from 18 patients which showed muscular symptoms due to hypokalemia. The muscle specimens were obtained by means of biopsy from quadriceps muscle and fixed with 2% glutaraldehyde (pH 7.4) and analyzed by ordinary method and modified Golgimethod. The ultrathin section were examined in JEOL 200CX transmission electron microscopy.Electron microscopic examinations disclosed dilated t-system and terminal cistern of sarcoplasmic reticulum (SR)(Fig 1), and an unique structure like “sixad” was occasionally observed in some specimens (Fig 2). Tubular aggregates (Fig 3) and honeycomb structure (Fig 4) were also common characteristic structures in all cases. These ultrastructural changes were common in both the hypokalemic periodic paralysis and the hypokalemic myopathy, regardless of the time of biopsy or the duration of hypokalemia suffered.

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Association of Micro RNA and Postoperative Cognitive Dysfunction: A Review

Mini-Reviews in Medicinal Chemistry ◽

10.2174/1389557520666200621182717 ◽

2020 ◽

Vol 20 (17) ◽

pp. 1781-1790

Author(s):

Noor Anisah Abu Yazit ◽

Norsham Juliana ◽

Srijit Das ◽

Nur Islami Mohd Fahmi Teng ◽

Nadia Mohd Fahmy ◽

...

Keyword(s):

Cognitive Dysfunction ◽

Chromosomal Abnormalities ◽

Current Knowledge ◽

System Development ◽

Genetic Disorder ◽

Postoperative Cognitive Dysfunction ◽

Clinical Importance ◽

Nervous System Development ◽

Micro Rna ◽

A Genome

Postoperative Cognitive Dysfunction (POCD) refers to the condition of neurocognitive decline following surgery in a cognitive and sensory manner. There are several risk factors, which may be life-threatening for this condition. Neuropsychological assessment of this condition is very important. In the present review, we discuss the association of apolipoprotein epsilon 4 (APOE ε4) and few miRNAs with POCD, and highlight the clinical importance for prognosis, diagnosis and treatment of POCD. Microarray is a genome analysis that can be used to determine DNA abnormalities. This current technique is rapid, efficient and high-throughout. Microarray techniques are widely used to diagnose diseases, particularly in genetic disorder, chromosomal abnormalities, mutations, infectious diseases and disease-relevant biomarkers. MicroRNAs (miRNAs) are a class of non-coding RNAs that are widely found distributed in eukaryotes. Few miRNAs influence the nervous system development, and nerve damage repair. Microarray approach can be utilized to understand the miRNAs involved and their pathways in POCD development, unleashing their potential to be considered as a diagnostic marker for POCD. This paper summarizes and identifies the studies that use microarray based approaches for POCD analysis. Since the application of microarray in POCD is expanding, there is a need to review the current knowledge of this approach.

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Genetic polymorphism for IFNγ +874T/A in patients with acute toxoplasmosis

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822012000600020 ◽

2012 ◽

Vol 45 (6) ◽

pp. 757-760 ◽

Cited By ~ 11

Author(s):

Elizabeth de Souza Neves ◽

André Luis Land Curi ◽

Maira Cavalcanti de Albuquerque ◽

Cassius Schnel Palhano-Silva ◽

Laura Berriel da Silva ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Case Control Study ◽

Severe Disease ◽

Clinical Manifestations ◽

Nucleotide Polymorphism ◽

Gene Encoding ◽

Single Nucleotide ◽

Duration Of Disease ◽

Acute Toxoplasmosis ◽

Control Study

INTRODUCTION: A single nucleotide polymorphism (SNP) in the gene encoding gamma interferon influences its production and is associated with severity of infectious diseases. This study aimed to evaluate the association of IFNγ+874T/A SNP with duration of disease, morbidity, and development of retinochoroiditis in acute toxoplasmosis. METHODS: A case-control study was conducted among 30 patients and 90 controls. RESULTS: Although statistical associations were not confirmed, A-allele was more common among retinochoroiditis cases and prolonged illness, while T-allele was more frequent in severe disease. CONCLUSIONS: Despite few cases, the results could indicate a relation between IFNγ+874T/A single nucleotide polymorphism and clinical manifestations of toxoplasmosis.

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Gain-of-function GABRB3 variants identified in vigabatrin-hypersensitive epileptic encephalopathies

Brain Communications ◽

10.1093/braincomms/fcaa162 ◽

2020 ◽

Vol 2 (2) ◽

Author(s):

Nathan L Absalom ◽

Vivian W Y Liao ◽

Kavitha Kothur ◽

Dinesh C Indurthi ◽

Bruce Bennetts ◽

...

Keyword(s):

De Novo ◽

Drug Effects ◽

Receptor Expression ◽

Aminobutyric Acid ◽

Loss Of Function ◽

Molecular Phenotype ◽

Gain Of Function ◽

Gene Encoding ◽

Epileptic Encephalopathies ◽

Receptor Phenotype

Abstract Variants in the GABRB3 gene encoding the β3-subunit of the γ-aminobutyric acid type A ( receptor are associated with various developmental and epileptic encephalopathies. Typically, these variants cause a loss-of-function molecular phenotype whereby γ-aminobutyric acid has reduced inhibitory effectiveness leading to seizures. Drugs that potentiate inhibitory GABAergic activity, such as nitrazepam, phenobarbital or vigabatrin, are expected to compensate for this and thereby reduce seizure frequency. However, vigabatrin, a drug that inhibits γ-aminobutyric acid transaminase to increase tonic γ-aminobutyric acid currents, has mixed success in treating seizures in patients with GABRB3 variants: some patients experience seizure cessation, but there is hypersensitivity in some patients associated with hypotonia, sedation and respiratory suppression. A GABRB3 variant that responds well to vigabatrin involves a truncation variant (p.Arg194*) resulting in a clear loss-of-function. We hypothesized that patients with a hypersensitive response to vigabatrin may exhibit a different γ-aminobutyric acid A receptor phenotype. To test this hypothesis, we evaluated the phenotype of de novo variants in GABRB3 (p.Glu77Lys and p.Thr287Ile) associated with patients who are clinically hypersensitive to vigabatrin. We introduced the GABRB3 p.Glu77Lys and p.Thr287Ile variants into a concatenated synaptic and extrasynaptic γ-aminobutyric acid A receptor construct, to resemble the γ-aminobutyric acid A receptor expression by a patient heterozygous for the GABRB3 variant. The mRNA of these constructs was injected into Xenopus oocytes and activation properties of each receptor measured by two-electrode voltage clamp electrophysiology. Results showed an atypical gain-of-function molecular phenotype in the GABRB3 p.Glu77Lys and p.Thr287Ile variants characterized by increased potency of γ-aminobutyric acid A without change to the estimated maximum open channel probability, deactivation kinetics or absolute currents. Modelling of the activation properties of the receptors indicated that either variant caused increased chloride flux in response to low concentrations of γ-aminobutyric acid that mediate tonic currents. We therefore propose that the hypersensitivity reaction to vigabatrin is a result of GABRB3 variants that exacerbate GABAergic tonic currents and caution is required when prescribing vigabatrin. In contrast, drug strategies increasing tonic currents in loss-of-function variants are likely to be a safe and effective therapy. This study demonstrates that functional genomics can explain beneficial and adverse anti-epileptic drug effects, and propose that vigabatrin should be considered in patients with clear loss-of-function GABRB3 variants.

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