scholarly journals Interrogation of Carboxy-Terminus Localized GJA1 Variants Associated with Erythrokeratodermia Variabilis et Progressiva

2022 ◽  
Vol 23 (1) ◽  
pp. 486
Author(s):  
Sergiu A. Lucaciu ◽  
Qing Shao ◽  
Rhett Figliuzzi ◽  
Kevin Barr ◽  
Donglin Bai ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
De Novo ◽  
Gene Mutations ◽  
Brefeldin A ◽  
Underlying Mechanism ◽  
Epidermal Keratinocytes ◽  
Carboxy Terminus ◽  
Whole Exome ◽  
Uptake Studies ◽  
Inhibitor Of Protein Synthesis

Although inherited GJA1 (encoding Cx43) gene mutations most often lead to oculodentodigital dysplasia and related disorders, four variants have been linked to erythrokeratodermia variabilis et progressiva (EKVP), a skin disorder characterized by erythematous and hyperkeratotic lesions. While two autosomal-dominant EKVP-linked GJA1 mutations have been shown to lead to augmented hemichannels, the consequence(s) of keratinocytes harboring a de novo P283L variant alone or in combination with a de novo T290N variant remain unknown. Interestingly, these variants reside within or adjacent to a carboxy terminus polypeptide motif that has been shown to be important in regulating the internalization and degradation of Cx43. Cx43-rich rat epidermal keratinocytes (REKs) or Cx43-ablated REKs engineered to express fluorescent protein-tagged P283L and/or T290N variants formed prototypical gap junctions at cell–cell interfaces similar to wildtype Cx43. Dye coupling and dye uptake studies further revealed that each variant or a combination of both variants formed functional gap junction channels, with no evidence of augmented hemichannel function or induction of cell death. Tracking the fate of EKVP-associated variants in the presence of the protein secretion blocker brefeldin A, or an inhibitor of protein synthesis cycloheximide, revealed that P283L or the combination of P283L and T290N variants either significantly extended Cx43 residency on the cell surface of keratinocytes or delayed its degradation. However, caution is needed in concluding that this modest change in the Cx43 life cycle is sufficient to cause EKVP, or whether an additional underlying mechanism or another unidentified gene mutation is contributing to the pathogenesis found in patients. This question will be resolved if further patients are identified where whole exome sequencing reveals a Cx43 P283L variant alone or, in combination with a T290N variant, co-segregates with EKVP across several family generations.

scholarly journals Coxiella burnetii Expresses a Functional Δ24 Sterol Reductase

2010 ◽  
Vol 192 (23) ◽  
pp. 6154-6159 ◽  
Author(s):  
Stacey D. Gilk ◽  
Paul A. Beare ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Fluorescent Protein ◽  
De Novo ◽  
Cholesterol Metabolism ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Cholesterol Biosynthesis ◽  
Brefeldin A ◽  
Ergosterol Biosynthesis

ABSTRACT Coxiella burnetii, the etiological agent of human Q fever, occupies a unique niche inside the host cell, where it replicates in a modified acidic phagolysosome or parasitophorous vacuole (PV). The PV membrane is cholesterol-rich, and inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. The precise source(s) of PV membrane cholesterol is unknown, as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. C. burnetii lacks enzymes for de novo cholesterol biosynthesis; however, the organism encodes a eukaryote-like Δ24 sterol reductase homolog, CBU1206. Absent in other prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis. In the present study, we examined the functional activity of CBU1206. Amino acid alignments revealed the greatest sequence identity (51.7%) with a Δ24 sterol reductase from the soil amoeba Naegleria gruberi. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under the control of a galactose-inducible promoter. Erg4 is a yeast Δ24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-green fluorescent protein (GFP), a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum. Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during C. burnetii intracellular growth.

scholarly journals Case Report: Causative De novo Variants of KCNT2 for Developmental and Epileptic Encephalopathy

2021 ◽  
Vol 12 ◽  
Author(s):  
Pan Gong ◽  
Xianru Jiao ◽  
Dan Yu ◽  
Zhixian Yang
Keyword(s):  
De Novo ◽  
Gene Mutations ◽  
Epileptic Encephalopathy ◽  
Infantile Spasms ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Dysmorphic Features ◽  
Pathogenic Variants ◽  
Ohtahara Syndrome ◽  
Whole Exome

Objective:KCNT2 gene mutations had been described to cause developmental and epileptic encephalopathies (DEEs). In this study, we presented the detailed clinical features and genetic analysis of two unrelated patients carrying two de novo variants in KCNT2 and reviewed eight different cases available in publications.Methods: Likely pathogenic variants were identified by whole exome sequencing; clinical data of the patients were retrospectively collected and analyzed.Results: Our two unrelated patients were diagnosed with Ohtahara syndrome followed by infantile spasms (IS) and possibly the epilepsy of infancy with migrating focal seizures (EIMFS), respectively. They both manifested dysmorphic features with hirsute arms, thick hair, prominent eyebrows, long and thick eyelashes, a broad nasal tip, and short and smooth philtrum. In the eight patients reported previously, two was diagnosed with IS carrying a ‘change-of-function' mutation and a gain-of-function mutation, respectively, two with EIMFS-like carrying a gain-of-function mutation and a loss-of-function mutation, respectively, one with EIMFS carrying a loss-of-function mutation, three with DEE without functional analysis. Among them, two patients with gain-of-function mutations both exhibited dysmorphic features and presented epilepsy phenotype, which was similar to our patients.Conclusion: Overall, the most common phenotypes associated with KCNT2 mutation were IS and EIMFS. Epilepsy phenotype associated with gain- and loss-of-function mutations could overlap. Additional KCNT2 cases will help to make genotype-phenotype correlations clearer.

Whole Exon Deletion in the GFAP Gene Is a Novel Molecular Mechanism Causing Alexander Disease

2017 ◽  
Vol 49 (02) ◽  
pp. 118-122 ◽  
Author(s):  
Lydia Green ◽  
Ian Berry ◽  
Anne-Marie Childs ◽  
Helen McCullagh ◽  
Sandhya Jose ◽  
...  
Keyword(s):  
De Novo ◽  
Mutation Screening ◽  
Copy Number Variants ◽  
Gene Mutations ◽  
Fiber Formation ◽  
Alexander Disease ◽  
Missense Mutations ◽  
Gene Encoding ◽  
Whole Exome ◽  
Filament Network

AbstractAlexander disease (AD) is a leukodystrophy caused by heterozygous mutations in the gene encoding the glial fibrillary acidic protein (GFAP). Currently, de novo heterozygous missense mutations in the GFAP gene are identified in over 95% of patients with AD. However, patients with biopsy-proven AD have been reported in whom no GFAP mutation has been identified. We report identical twin boys presenting in infancy with seizures and developmental delay in whom MR appearances were suggestive of AD with the exception of an unusual, bilateral, arc of calcification at the frontal white–gray junction. Initial mutation screening of the GFAP gene did not identify a mutation. Whole exome sequencing in both brothers revealed a de novo heterozygous in-frame deletion of the whole of exon 5 of the GFAP gene. Mutations in the GFAP gene are thought to result in a toxic effect of mutant GFAP disrupting the formation of the normal intermediate filament network and resulting in Rosenthal fiber formation, which has hitherto not been linked to exonic scale copy number variants in GFAP. Further studies on mutation negative AD patients are warranted to determine whether a similar mechanism underlies their disease.

scholarly journals Grapevine Fanleaf Virus Replication Occurs on Endoplasmic Reticulum-Derived Membranes

2002 ◽  
Vol 76 (17) ◽  
pp. 8808-8819 ◽  
Author(s):  
C. Ritzenthaler ◽  
C. Laporte ◽  
F. Gaire ◽  
P. Dunoyer ◽  
C. Schmitt ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Virus Replication ◽  
Fluorescent Protein ◽  
De Novo ◽  
Rna Virus ◽  
Lipid Synthesis ◽  
Cell Movement ◽  
Brefeldin A ◽  
Cell Periphery ◽  
Green Fluorescent

ABSTRACT Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear “viral compartment.” We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles.

scholarly journals Comparison of the frequencies of ENU-induced point mutations in male germ cells and inherited germline mutations in their offspring

2021 ◽  
Vol 43 (1) ◽  
Author(s):  
Kenichi Masumura ◽  
Tomoko Ando ◽  
Naomi Toyoda-Hokaiwado ◽  
Akiko Ukai ◽  
Takehiko Nohmi ◽  
...  
Keyword(s):  
Germ Cells ◽  
De Novo ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Gene Mutations ◽  
Next Generation ◽  
Male Germ Cells ◽  
Gene Coding ◽  
Whole Exome ◽  
And Control

Abstract Background Gene mutations induced in germ cells may be transmitted to the next generation and cause adverse effects such as genetic diseases. Certain mutations may result in infertility or death in early development. Thus, the mutations may not be inheritable. However, the extent to which point mutations in male germ cells are transmitted to the next generation or eliminated during transmission is largely unknown. This study compared mutation frequencies (MFs) in sperm of N-ethyl-N-nitrosourea (ENU)-treated gpt delta mice and de novo MFs in the whole exome/genome of their offspring. Results Male gpt delta mice were treated with 10, 30, and 85 mg/kg of ENU (i.p., weekly × 2) and mated with untreated females to generate offspring. We previously reported a dose-dependent increase in de novo MFs in the offspring estimated by whole exome sequencing (WES) (Mutat. Res., 810, 30–39, 2016). In this study, gpt MFs in the sperm of ENU-treated mice were estimated, and the MFs per reporter gene were converted to MFs per base pair. The inherited de novo MFs in the offspring (9, 26 and 133 × 10− 8/bp for 10, 30, and 85 mg/kg ENU-treated groups, respectively) were comparable to those of the converted gpt MFs in the sperm of ENU-treated fathers (6, 16, and 69 × 10− 8/bp). It indicated that the gpt MFs in the ENU-treated father’s sperm were comparable to the inherited de novo MFs in the offspring as estimated by WES. In addition, de novo MFs in the offspring of 10 mg/kg ENU-treated and control fathers were estimated by whole genome sequencing (WGS), because WES was not sufficiently sensitive to detect low background MF. The de novo MF in the offspring of the ENU-treated fathers was 6 × 10− 8/bp and significantly higher than that of the control (2 × 10− 8/bp). There were no significant differences in de novo MFs between gene-coding and non-coding regions. WGS analysis was able to detect ENU-induced characteristic de novo base substitutions at a low dose group. Conclusions Despite a difference between exome/genome and exogenous reporter genes, the results indicated that ENU-induced point mutations in male germ cells could be transmitted to the next generation without severe selection.

scholarly journals Whole exome sequencing reveals a wide spectrum of ciliary gene mutations in nonsyndromic biliary atresia

2020 ◽  
Author(s):  
Wai-Yee Lam ◽  
Man-Ting So ◽  
Jacob Shujui Hsu ◽  
Patrick Ho-Yu Chung ◽  
Diem Ngoc Ngo ◽  
...  
Keyword(s):  
Biliary Atresia ◽  
Exome Sequencing ◽  
De Novo ◽  
Wide Spectrum ◽  
Gene Mutations ◽  
Compound Heterozygous ◽  
Genetic Contribution ◽  
Whole Exome ◽  
Compound Heterozygous Variants ◽  
End Stage

ABSTRACTBiliary atresia (BA) is the most common obstructive cholangiopathy in neonates, often progressing to end-stage cirrhosis. BA pathogenesis is believed to be multifactorial, but the genetic contribution remains poorly defined. We conducted exome sequencing on 89 nonsyndromic BA trios. In 31.5% of the patients, rare and deleterious de novo, homozygous recessive and/or compound heterozygous variants were detected in liver-expressed ciliary genes of diverse ciliary functions. Enrichment of deleterious mutations in liver-expressed ciliary geneset was significant compared to 148 control trios (OR 2.58, 95% CI 1.15-6.07). KIF3B, PCNT and TTC17 are essential for ciliogenesis. Reduced ciliary proteins expression were detected in the BA livers with KIF3B and TTC17 mutations. CRISPR/Cas9-engineered zebrafish knockouts of KIF3B, PCNT and TTC17 displayed reduced biliary flow. Our findings support a larger genetic contribution to nonsyndromic BA risk than expected. Ciliary gene mutations leading to cholangiocyte cilia malformation and dysfunction could be a key biological mechanism in BA pathogenesis.

scholarly journals Cell-specific basolateral membrane sorting of the human liver Na+-dependent bile acid cotransporter

2001 ◽  
Vol 280 (6) ◽  
pp. G1305-G1313 ◽  
Author(s):  
An-Qiang Sun ◽  
I'Kyori Swaby ◽  
Shuhua Xu ◽  
Frederick J. Suchy
Keyword(s):  
Low Temperature ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Brefeldin A ◽  
Temperature Shift ◽  
Surface Expression ◽  
Apical Surface ◽  
Uptake Studies ◽  
Green Fluorescent

The human Na+-taurocholate cotransporting polypeptide (Ntcp) is located exclusively on the basolateral membrane of hepatocyte, but the mechanisms underlying its membrane sorting domain have not been fully elucidated. In the present study, a green fluorescent protein-fused human NTCP (NTCP-GFP) was constructed using the polymerase chain reaction and was stably transfected into Madin-Darby canine kidney (MDCK) and Caco-2 cells. Taurocholate uptake studies and confocal microscopy demonstrated that the polarity of basolateral surface expression of NTCP-GFP was maintained in MDCK cells but was lost in Caco-2 cells. Nocodazole (33 μM), an agent that causes microtubular depolymerization, partially disrupted the basolateral localization of NTCP-GFP by increasing apical surface expression to 33.5% compared with untreated cells ( P < 0.05). Brefeldin A (BFA; 1–2 μM) disrupted the polarized basolateral localization of NTCP, but monensin (1.4 μM) had no affect on NTCP-GFP localization. In addition, low-temperature shift (20°C) did not affect the polarized basolateral surface sorting of NTCP-GFP and repolarization of this protein after BFA interruption. In summary, these data suggest that the polarized basolateral localization of human NTCP is cell specific and is mediated by a novel sorting pathway that is BFA sensitive and monensin and low-temperature shift insensitive. The process may also involve microtubule motors.

scholarly journals Archetypes of AML Defined Using Whole Exome Sequencing and Clinical Characteristics in a Diverse Group of Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 597-597
Author(s):  
Christopher B. Benton ◽  
Koichi Takahashi ◽  
Prithviraj Bose ◽  
Feng Wang ◽  
Hsiang-Chun Chen ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Research Funding ◽  
De Novo ◽  
Gene Mutations ◽  
Advisory Board ◽  
Driver Mutations ◽  
Whole Exome ◽  
Clinical Scenarios

Abstract Background: Acute myeloid leukemia (AML) comprises a heterogeneous collection of morphologically related diseases. The identification and study of somatic mutations in AML using next-generation sequencing has led to insights into the pathogenesis of AML. Constellations of specific mutations may correlate with clinical phenotype and reveal the most relevant cooperating molecular pathways. Understanding AML biological pathways will contribute to more personalized, directed therapies and improve outcomes. Most sequencing efforts have focused on select groups of AML patients or select gene panels. Characterization of mutations identified by whole exome sequencing for AML patients from a variety of clinical scenarios has not been the subject of one study. Methods: Whole exome sequencing (WES) was performed, in addition to complete clinical molecular diagnostics, on leukemia samples from a diverse group of 141 consecutive AML patients who underwent complete clinical evaluation at a single quaternary leukemia care institution (MD Anderson Cancer Center, MDACC). The cohort included adults of all ages, treatment histories (treated/untreated), and ontogenies (de novo/secondary/therapy-related). To facilitate the identification of driver mutations from WES data, an automatic pipeline was developed to filter and annotate raw variants, and then select non-polymorphism, protein-coding changing mutations. A relational mutation/clinical database was created to analyze gene mutations and gene categories in the context of well-annotated mutations and complete clinical indices including treatments and outcomes. Results: We characterized the landscape of driver mutations in an unselected group of AML patients, and classified mutations by 9 predefined functional categories of known aberrant pathways implicated in AML (Figure 1). The frequency and distribution of gene mutations and gene categories were determined and compared to TCGA AML whole exome sequencing mutation data, derived from a de novo, untreated AML cohort. Forty-six separate genes/fusions were mutated in the MDACC cohort, including 326 mutations. The MDACC cohort included 23 cases (16%) with a TP53 mutation. Overall, a mean of 2.3+/-1.4 mutations-of-interest were identified per patient. For the MDACC dataset, the likelihood of co-occurrence and mutual exclusivity was determined for each gene (mutated in greater than 2 cases, n=34) and each gene category (n=9) versus every other gene, gene category, as well as each individual annotated clinical characteristic (n=30). 156 statistically significant co-occurring pairs and 71 statistically significant mutually exclusive pairs were found. We analyzed hierarchically clustered, strength-of-association correlograms to identify likely mutational frameworks with similarly associated clinical/molecular characteristics. We identified patterns of predominant genetic alterations that defined 4 distinct AML archetypes: 1) Transcription factor [TRNSXN] (16% of cases), 2) Nucleolar/Methylation/Signaling [NMS] (40%), 3) Spliceosome [SPLICE] (21%), and 4) TP53-mutated [TP53] (16%). Ten cases had no identifiable mutation-of-interest, compared to 4 cases without mutation in TCGA data. The defining mutational characteristics of each AML archetype, and select associated characteristics are shown in Table 1. Conclusions: Analysis of whole exome sequencing data along with clinical information from all-comer AML patients identifies archetypal classes of the disease, each with particular tendencies for specific mutational, clinical, and outcome characteristics. Our approach demonstrates that genomic characterization of mutations in a variety of diverse clinical scenarios has the potential to identify representative mechanisms of AML development across the spectrum of disease presentations. Disclosures Daver: Otsuka: Consultancy, Honoraria; Sunesis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Karyopharm: Honoraria, Research Funding; Ariad: Research Funding; Kiromic: Research Funding; BMS: Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. DiNardo:Agios: Other: advisory board, Research Funding; Celgene: Research Funding; Novartis: Other: advisory board, Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding; Abbvie: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

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