scholarly journals Expanding the genetic spectrum of TUBB1-related thrombocytopenia

2021 ◽  
Author(s):  
Verónica Palma-Barqueros ◽  
Loredana Bury ◽  
Shinji Kunishima ◽  
Maria L Lozano ◽  
Agustin Rodriguez Alen ◽  
...  
Keyword(s):  
Phenotypic Expression ◽  
Incomplete Penetrance ◽  
Peripheral Blood Cd34 ◽  
Pathogenic Variants ◽  
Clinical Investigations ◽  
Microtubular Network ◽  
Proplatelet Formation ◽  
Insight Into ◽  
Platelet Shape

β1-tubulin plays a major role in proplatelet formation and platelet shape maintenance, and pathogenic variants in TUBB1 lead to thrombocytopenia and platelet anisocytosis (TUBB1-RT). To date, the reported number of pedigrees with TUBB1-RT and of rare TUBB1 variants with experimental demonstration of pathogenicity is limited. Here, we report 9 unrelated families presenting with thrombocytopenia carrying six β1-tubulin variants: p.Cys12Leufs12*, p.Thr107Pro, p.Gln423*, p.Arg359Trp, p.Gly109Glu, and p.Gly269Asp, the last of which novel. Segregation studies showed incomplete penetrance of these variants for platelet traits. Indeed, most carriers showed macrothrombocytopenia, some only increased platelet size and a minority no abnormalities. Moreover, only homozygous carriers of the p.Gly109Glu variant, displayed macrothrombocytopenia, highlighting the importance of allele burden in the phenotypic expression of TUBB1-RT. The p.Arg359Trp, p.Gly269Asp and p.Gly109Glu variants deranged β1-tubulin incorporation into the microtubular marginal ring in platelets, while had negligible effect on platelet activation, secretion or spreading, suggesting that β1-tubulin is dispensable for these processes. Transfection of TUBB1 missense variants in CHO cells altered β1-tubulin incorporation into the microtubular network. In addition, TUBB1 variants markedly impaired proplatelet formation from peripheral blood CD34+ cell-derived megakaryocytes. Our study, using in vitro modeling, molecular characterization, and clinical investigations provides a deeper insight into the pathogenicity of rare TUBB1 variants. These novel data expand the genetic spectrum of TUBB1-RT and highlight a remarkable heterogeneity in its clinical presentation, indicating that allelic burden or combination with other genetic or environmental factors modulate the phenotypic impact of rare TUBB1 variants.

FC 014INFLUENCE OF GENETIC VARIATION IN SLC7A13/AGT1 IN HUMAN CYSTINURIA

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Ria Schönauer ◽  
Anna Seidel ◽  
Linda Pöschla ◽  
Elena Hantmann ◽  
Soumeya Bekri ◽  
...  
Keyword(s):  
Complex Formation ◽  
Proximal Tubules ◽  
Patient Specific ◽  
Incomplete Penetrance ◽  
Renal Tubules ◽  
Pathogenic Variants ◽  
Cystine Transporter ◽  
S3 Segment

Abstract Background and Aims Cystinuria (CU) is an inherited renal disorder based on urinary wasting of dibasic amino acids, urinary precipitation, and consecutive cystine stone formation. It is caused by pathogenic variants in two distinct disease genes, SLC3A1 and SLC7A9, both of which encode subunits of a heterodimeric tubular amino acid transporter, rBAT/SLC3A1 and BAT1/SLC7A9, located at the apical membrane of proximal renal tubules. CU is marked by incomplete penetrance and substantial disease variability. Recently, a novel cystine transporter, consisting of the light chain AGT1/SLC7A13 and its heterodimeric partner rBAT/SLC3A1 has been identified in the S3 segment of murine proximal tubules. In this study, we aim at evaluating the role of AGT1 in cystinuric patients with or without mutations in either SLC3A1 or SLC7A9, analyzing the role of AGT1/SLC7A13 as novel disease gene or genetic modifier in CU. Method A multicenter European CU-cohort comprising 132 individuals was screened for pathogenic variants in SLC3A1, SLC7A9, and SLC7A13 using high-throughput multiplex PCR-based amplification and next-generation sequencing (MiSeq Illumina) followed by multiplex ligation-dependent probe amplification (MLPA) of SLC3A1 and SLC7A9. For functional in vitro studies, epitope-tagged human and murine rBAT and AGT1 proteins were transiently expressed in different cell systems. Heterodimer complex formation was analyzed by co-immunoprecipitation and western blot studies and membrane trafficking was evaluated by immunofluorescence microscopy. Results Genectic analysis of our CU-cohort did not reveal indiviuals with SLC7A13 variation only, however we found three patients harbouring heterozygous missense variants in addition to pathogenic or VUS variants in SLC3A1 or SLC7A9. To evaluate their influence on the generation of functional cystine transporters in vitro, different cell models were transiently transfected with plasmids expressing wildtype or mutant proteins. In line with previous reports, co-expression of AGT1 and rBAT wildtype allowed efficient complex formation as AGT1-induced maturation of rBAT was detected by increased mature N-glycosylation, co-immunoprecipitation and membrane insertion. Whereas AGT1 patient variants p.Met452Thr (SLC7A13 c.1355T>C) and p.Ile174Phe (SLC7A13 c.520A>T) behaved comparable to wildtype AGT1, variants p.Asn45Lys (SLC7A13 c.135C>G) and p.Leu270Phe (SLC7A13 c.808C>T) led to clearly reduced glycosylation patterns and trafficking deficits of rBAT wildtype protein. Next, the mutual influence of pathogenic variation in both, AGT1 and rBAT, will unravel the consequences of patient-specific molecular interactions on the functional expression of cystine transporter complexes. Conclusion Here, we report three CU-patients with variants in SLC7A13 combined with either SLC3A1 or SLC7A9. For two of these variants, in vitro functional analysis revealed pathogenic molecular mechanisms disturbing complex formation, maturation and trafficking of rBAT. We hypothesize that specific pathogenic variants in SLC7A13 interfere with efficient membrane localization of heterodimeric cystine transporters, which results in modulation of cystine transport in the S3 segment of proximal tubules in CU-patients.

scholarly journals New SHH and Known SIX3 Variants in a Series of Latin American Patients with Holoprosencephaly

2021 ◽  
pp. 1-15
Author(s):  
Viviane Freitas de Castro ◽  
Daniel Mattos ◽  
Flavia Martinez de Carvalho ◽  
Denise Pontes Cavalcanti ◽  
Milagros M. Duenas-Roque ◽  
...  
Keyword(s):  
Latin American ◽  
Sanger Sequencing ◽  
Phenotypic Variability ◽  
Phenotypic Expression ◽  
Driver Gene ◽  
Incomplete Penetrance ◽  
Phenotype Correlation ◽  
Pathogenic Variants ◽  
Facial Defects ◽  
Domain Conservation

Holoprosencephaly (HPE) is the failure of the embryonic forebrain to develop into 2 hemispheres promoting midline cerebral and facial defects. The wide phenotypic variability and causal heterogeneity make genetic counseling difficult. Heterozygous variants with incomplete penetrance and variable expressivity in the <i>SHH</i>, <i>SIX3</i>, <i>ZIC2</i>, and <i>TGIF1</i> genes explain ∼25% of the known causes of nonchromosomal HPE. We studied these 4 genes and clinically described 27 Latin American families presenting with nonchromosomal HPE. Three new <i>SHH</i> variants and a third known <i>SIX3</i> likely pathogenic variant found by Sanger sequencing explained 15% of our cases. Genotype-phenotype correlation in these 4 families and published families with identical or similar driver gene, mutated domain, conservation of residue in other species, and the type of variant explain the pathogenicity but not the phenotypic variability. Nine patients, including 2 with <i>SHH</i> pathogenic variants, presented benign variants of the <i>SHH</i>, <i>SIX3</i>, <i>ZIC2</i>, and <i>TGIF1</i> genes with potential alteration of splicing, a causal proposition in need of further studies. Finding more families with the same <i>SIX3</i> variant may allow further identification of genetic or environmental modifiers explaining its variable phenotypic expression.

scholarly journals Novel Pathogenic Variants in LHX3, LHX4 and GLI2 Identified in Pediatric Patients With Congenital Hypopituitarism: From Variant Calling To Variant Testing

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A718-A719
Author(s):  
Sebastian Vishnopolska ◽  
María Florencia Mercogliano ◽  
Maria Andrea Camilletti ◽  
Amanda Helen Mortensen ◽  
Debora Giselle Braslavsky ◽  
...  
Keyword(s):  
Single Molecule ◽  
Pediatric Patients ◽  
Variant Calling ◽  
Luciferase Reporter ◽  
Incomplete Penetrance ◽  
Genetic Causes ◽  
Pathogenic Variants ◽  
Central Nervous System Defects ◽  
Congenital Hypopituitarism

Abstract Congenital hypopituitarism (CH), septo-optic dysplasia (SOD), and holoprosencephaly (HPE) constitute an important group of structural birth defects that cause significant morbidity and life-long consequences for quality of life and care. The genetic causes are highly overlapping. As such, these disorders can be considered as a spectrum of related disorders. Improved insight into genetic causes would be valuable for patients, families, and medical geneticists. Very few systematic genetic screens have been carried out for patients with CH. We implemented genetic screening using single-molecule molecular inversion probes sequencing to identify causative mutations in a set of 67 genes previously reported in CH patients and the spectrum encompassing SOD and HPE. We captured genomic DNA from 170 Argentinean pediatric patients with CH, and 54% of the patients in this cohort have craniofacial, ophthalmologic, and/or central nervous system defects. We found candidate pathogenic, likely pathogenic and variants uncertain significance (VUS) in 23% of the cases. In order to evaluate the functional consequences of VUS in LHX3, LHX4, and GLI2, we performed in-vitro functional assays to study the activity of the mutated proteins. To test LHX3/4 variants we co-transfected HEK293T cells with wild type (WT) or mutated LHX3/4 variant plasmids and luciferase reporter genes driven by the ɑGSU promoter or GH1 promoter and assayed for luciferase activity. For GLI2 functional analysis we used the cell line NIH/3T3-CG, stably transfected to express GFP under the presence of GLI2 activated form. Endogenous Gli2 was knocked out by CRISPR-Cas9 and clones were selected for absence of GFP expression upon activation of the sonic hedgehog pathway. We tested the ability of transfected WT or mutated GLI2 expression plasmids to restore GFP fluorescence. We concluded that variants LHX3:p.Pro187Ser LHX4:p.Arg84His, p.Gln100His and p.Trp204Leu and GLI2:p.1404Lfs impair activation of the reporter gene, while the LHX3:p.Leu220Met and GLI2:p.L761P have WT activity on their respective assays. Identification of disease-causing variants in CH is complicated by phenotypic variation, incomplete penetrance, and VUS. Functional testing of potentially pathogenic variants is critical to arrive at a definitive molecular diagnosis. A full catalogue of variant effects in known causative genes would be invaluable for clinicians in order to simplify the interpretation of novel variants and reduce the diagnostic odyssey that families often experience.

Functional assessment and phenotypic heterogeneity of SFTPA1 and SFTPA2 mutations in interstitial lung diseases and lung cancer

2020 ◽  
Vol 56 (6) ◽  
pp. 2002806 ◽  
Author(s):  
Marie Legendre ◽  
Afifaa Butt ◽  
Raphaël Borie ◽  
Marie-Pierre Debray ◽  
Diane Bouvry ◽  
...  
Keyword(s):  
Lung Transplantation ◽  
Lung Tissue ◽  
Lung Diseases ◽  
Ex Vivo ◽  
Phenotypic Expression ◽  
Surfactant Protein ◽  
Interstitial Lung Diseases ◽  
Incomplete Penetrance ◽  
Tissue Samples

IntroductionInterstitial lung diseases (ILDs) can be caused by mutations in the SFTPA1 and SFTPA2 genes, which encode the surfactant protein (SP) complex SP-A. Only 11 SFTPA1 or SFTPA2 mutations have so far been reported worldwide, of which five have been functionally assessed. In the framework of ILD molecular diagnosis, we identified 14 independent patients with pathogenic SFTPA1 or SFTPA2 mutations. The present study aimed to functionally assess the 11 different mutations identified and to accurately describe the disease phenotype of the patients and their affected relatives.MethodsThe consequences of the 11 SFTPA1 or SFTPA2 mutations were analysed both in vitro, by studying the production and secretion of the corresponding mutated proteins and ex vivo, by analysing SP-A expression in lung tissue samples. The associated disease phenotypes were documented.ResultsFor the 11 identified mutations, protein production was preserved but secretion was abolished. The expression pattern of lung SP-A available in six patients was altered and the family history reported ILD and/or lung adenocarcinoma in 13 out of 14 families (93%). Among the 28 SFTPA1 or SFTPA2 mutation carriers, the mean age at ILD onset was 45 years (range 0.6–65 years) and 48% underwent lung transplantation (mean age 51 years). Seven carriers were asymptomatic.DiscussionThis study, which expands the molecular and clinical spectrum of SP-A disorders, shows that pathogenic SFTPA1 or SFTPA2 mutations share similar consequences for SP-A secretion in cell models and in lung tissue immunostaining, whereas they are associated with a highly variable phenotypic expression of disease, ranging from severe forms requiring lung transplantation to incomplete penetrance.

The in vitro neonatal rat spinal cord preparation: a new insight into mammalian locomotor mechanisms

2004 ◽  
Vol 190 (5) ◽  
pp. 343-357 ◽  
Author(s):  
F. Clarac ◽  
E. Pearlstein ◽  
J. F. Pflieger ◽  
L. Vinay
Keyword(s):  
Spinal Cord ◽  
Neonatal Rat ◽  
Rat Spinal Cord ◽  
Insight Into

scholarly journals An epilepsy-causing mutation leads to co-translational misfolding of the Kv7.2 channel

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Janire Urrutia ◽  
Alejandra Aguado ◽  
Carolina Gomis-Perez ◽  
Arantza Muguruza-Montero ◽  
Oscar R. Ballesteros ◽  
...  
Keyword(s):  
Structural Instability ◽  
Binding Assays ◽  
Iq Motif ◽  
Channel Function ◽  
Human Epilepsy ◽  
Pathogenic Variants ◽  
In Silico Studies ◽  
Nascent Chain

Abstract Background The amino acid sequence of proteins generally carries all the necessary information for acquisition of native conformations, but the vectorial nature of translation can additionally determine the folding outcome. Such consideration is particularly relevant in human diseases associated to inherited mutations leading to structural instability, aggregation, and degradation. Mutations in the KCNQ2 gene associated with human epilepsy have been suggested to cause misfolding of the encoded Kv7.2 channel. Although the effect on folding of mutations in some domains has been studied, little is known of the way pathogenic variants located in the calcium responsive domain (CRD) affect folding. Here, we explore how a Kv7.2 mutation (W344R) located in helix A of the CRD and associated with hereditary epilepsy interferes with channel function. Results We report that the epilepsy W344R mutation within the IQ motif of CRD decreases channel function, but contrary to other mutations at this site, it does not impair the interaction with Calmodulin (CaM) in vitro, as monitored by multiple in vitro binding assays. We find negligible impact of the mutation on the structure of the complex by molecular dynamic computations. In silico studies revealed two orientations of the side chain, which are differentially populated by WT and W344R variants. Binding to CaM is impaired when the mutated protein is produced in cellulo but not in vitro, suggesting that this mutation impedes proper folding during translation within the cell by forcing the nascent chain to follow a folding route that leads to a non-native configuration, and thereby generating non-functional ion channels that fail to traffic to proper neuronal compartments. Conclusions Our data suggest that the key pathogenic mechanism of Kv7.2 W344R mutation involves the failure to adopt a configuration that can be recognized by CaM in vivo but not in vitro.

scholarly journals Plant-Derived Extracellular Vesicles: Current Findings,Challenges, and Future Applications

Membranes ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 411
Author(s):  
Nader Kameli ◽  
Anya Dragojlovic-Kerkache ◽  
Paul Savelkoul ◽  
Frank R. Stassen
Keyword(s):  
Human Health ◽  
Extracellular Vesicles ◽  
Preliminary Results ◽  
In Vivo Experiments ◽  
Health And Disease ◽  
Conducting Research ◽  
Protocol Standardization ◽  
Insight Into

In recent years, plant-derived extracellular vesicles (PDEVs) have gained the interest of many experts in fields such as microbiology and immunology, and research in this field has exponentially increased. These nano-sized particles have provided researchers with a number of interesting findings, making their application in human health and disease very promising. Both in vitro and in vivo experiments have shown that PDEVs can exhibit a multitude of effects, suggesting that these vesicles may have many potential future applications, including therapeutics and nano-delivery of compounds. While the preliminary results are promising, there are still some challenges to face, such as a lack of protocol standardization, as well as knowledge gaps that need to be filled. This review aims to discuss various aspects of PDEV knowledge, including their preliminary findings, challenges, and future uses, giving insight into the complexity of conducting research in this field.

Nephrotoxicity Testing In Vitro: The Current Situation

1997 ◽  
Vol 25 (5) ◽  
pp. 497-503
Author(s):  
Jean-Paul Morin ◽  
Marc E. De Broe ◽  
Walter Pfaller ◽  
Gabriele Schmuck
Keyword(s):  
Proximal Tubule ◽  
Culture Media ◽  
Task Force ◽  
Primary Cultures ◽  
Phenotypic Expression ◽  
Transepithelial Transport ◽  
Specific Cell ◽  
Proximal Tubule Cells ◽  
Tubule Cells

An ECVAM task force on nephrotoxicity has been established to advise, in particular, on the follow-up to recommendations made in the ECVAM workshop report on nephrotoxicity testing in vitro. Since this workshop was held, in 1994, there have been several improvements in the techniques used. For example, the duration of renal slice viability, and the maintenance of functional activities in slices, have been improved by using dynamic incubation systems with higher oxygen tensions and more-appropriate cell culture media. Highly differentiated primary cultures of pig, human and rabbit proximal tubule cells have been established by using specific cell isolation procedures and/or selective culture media. To date, the most comparable phenotypic expression and transepithelial transport capacities to proximal tubules in vivo have been obtained with primary cultures of rabbit proximal tubule cells which are grown on bicompartmental supports; in this system, transepithelial substrate gradients are generated and the transepithelial transport of both organic anions and cations is highly active. This in vitro system has been selected by ECVAM for further evaluation and prevalidation. Industrial needs in the area of nephrotoxicity testing have been identified, and recommendations are made at the end of this report concerning possible future initiatives.

scholarly journals Bestrophinopathies: perspectives on clinical disease, Bestrophin-1 function and developing therapies

2021 ◽  
Vol 13 ◽  
pp. 251584142199719
Author(s):  
Simranjeet Singh Grewal ◽  
Joseph J. Smith ◽  
Amanda-Jayne F. Carr
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Induced Pluripotent Stem Cell ◽  
Underlying Disease ◽  
Incomplete Penetrance ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
High Acuity ◽  
Induced Pluripotent

Bestrophinopathies are a group of clinically distinct inherited retinal dystrophies that typically affect the macular region, an area synonymous with central high acuity vision. This spectrum of disorders is caused by mutations in bestrophin1 ( BEST1), a protein thought to act as a Ca2+-activated Cl- channel in the retinal pigment epithelium (RPE) of the eye. Although bestrophinopathies are rare, over 250 individual pathological mutations have been identified in the BEST1 gene, with many reported to have various clinical expressivity and incomplete penetrance. With no current clinical treatments available for patients with bestrophinopathies, understanding the role of BEST1 in cells and the pathological pathways underlying disease has become a priority. Induced pluripotent stem cell (iPSC) technology is helping to uncover disease mechanisms and develop treatments for RPE diseases, like bestrophinopathies. Here, we provide a comprehensive review of the pathophysiology of bestrophinopathies and highlight how patient-derived iPSC-RPE are being used to test new genomic therapies in vitro.

scholarly journals Platelet Shape Changes during Thrombus Formation: Role of Actin-Based Protrusions

2021 ◽  
Vol 41 (01) ◽  
pp. 014-021
Author(s):  
Markus Bender ◽  
Raghavendra Palankar
Keyword(s):  
Blood Loss ◽  
Actin Filaments ◽  
Thrombus Formation ◽  
Short Review ◽  
Critical Component ◽  
Clot Retraction ◽  
Shape Changes ◽  
Platelet Shape

AbstractPlatelet activation and aggregation are essential to limit blood loss at sites of vascular injury but may also lead to occlusion of diseased vessels. The platelet cytoskeleton is a critical component for proper hemostatic function. Platelets change their shape after activation and their contractile machinery mediates thrombus stabilization and clot retraction. In vitro studies have shown that platelets, which come into contact with proteins such as fibrinogen, spread and first form filopodia and then lamellipodia, the latter being plate-like protrusions with branched actin filaments. However, the role of platelet lamellipodia in hemostasis and thrombus formation has been unclear until recently. This short review will briefly summarize the recent findings on the contribution of the actin cytoskeleton and lamellipodial structures to platelet function.

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