Biallelic loss of function NEK3 mutations deacetylate α-tubulin and downregulate NUP205 that predispose individuals to cilia-related abnormal cardiac left–right patterning

AbstractDefective left–right (LR) organization involving abnormalities in cilia ultrastructure causes laterality disorders including situs inversus (SI) and heterotaxy (Htx) with the prevalence approximately 1/10,000 births. In this study, we describe two unrelated family trios with abnormal cardiac LR patterning. Through whole-exome sequencing (WES), we identified compound heterozygous mutations (c.805-1G >C; p. Ile269GlnfsTer8/c.1117dupA; p.Thr373AsnfsTer19) (c.29T>C; p.Ile10Thr/c.356A>G; p.His119Arg) of NEK3, encoding a NIMA (never in mitosis A)-related kinase, in two affected individuals, respectively. Protein levels of NEK3 were abrogated in Patient-1 with biallelic loss-of function (LoF) NEK3 mutations that causes premature stop codon. Subsequence transcriptome analysis revealed that NNMT (nicotinamide N-methyltransferase) and SIRT2 (sirtuin2) was upregulated by NEK3 knockdown in human retinal pigment epithelial (RPE) cells in vitro, which associates α-tubulin deacetylation by western blot and immunofluorescence. Transmission electron microscopy (TEM) analysis further identified defective ciliary ultrastructure in Patient-1. Furthermore, inner ring components of nuclear pore complex (NPC) including nucleoporin (NUP)205, NUP188, and NUP155 were significantly downregulated in NEK3-silenced cells. In conclusion, we identified biallelic mutations of NEK3 predispose individual to abnormal cardiac left–right patterning via SIRT2-mediated α-tubulin deacetylation and downregulation of inner ring nucleoporins. Our study suggested that NEK3 could be a candidate gene for human ciliopathies.

Download Full-text

Novel WEE2 compound heterozygous mutations identified in patients with fertilization failure or poor fertilization

Journal of Assisted Reproduction and Genetics ◽

10.1007/s10815-021-02285-z ◽

2021 ◽

Author(s):

Jiamin Jin ◽

Xiaomei Tong ◽

Yin-Li Zhang ◽

Weijie Yang ◽

Yerong Ma ◽

...

Keyword(s):

Hela Cells ◽

Fertilization Rate ◽

Chinese Patients ◽

Compound Heterozygous ◽

Fertilization Failure ◽

Loss Of Function ◽

Protein Levels ◽

Compound Heterozygous Mutations ◽

Poor Fertilization

Abstract Purpose To study associations between novel WEE2 mutations and patients with fertilization failure or poor fertilization. Methods Thirty-one Chinese patients who underwent treatment with assisted reproductive technology and suffered from repeated (at least two times) total fertilization failure (TFF) or a low fertilization rate were enrolled. Genomic DNA was extracted from patients for whole-exome sequencing. Suspicious mutations were validated by Sanger sequencing. WEE2 protein levels in oocytes from affected patients were examined by immunofluorescence. Disruptive effects of mutations on WEE2 protein stability, subcellular localization, and kinase function were analyzed through western blotting, immunofluorescence, and flow cytometry in HeLa cells. Results Three of thirty-one (9.6%) enrolled patients had six compound heterozygous mutations of the WEE2 gene, and three of them were reported here for the first time (c.115_116insT, c.756_758delTGA, and c.C1459T). Oocytes from affected patients showed decreased WEE2 immunofluorescence signals. In vitro experiments showed that the mutant WEE2 gene caused reduced WEE2 protein levels or cellular compartment translocation in HeLa cells, leading to decreased levels of the phosphorylated Cdc2 protein. Compared with the wild-type WEE2 protein, the mutant WEE2 proteins were also found to have different effects on the cell cycle. Conclusion Three novel compound heterozygous WEE2 variants were found in patients with pronucleus formation failure. This study provides new evidence that WEE2 mutations result in loss of function, which could result in fertilization failure.

Download Full-text

Loss of cysteine 584 impairs the storage and release, but not the synthesis of von Willebrand factor

Thrombosis and Haemostasis ◽

10.1160/th14-04-0391 ◽

2014 ◽

Vol 112 (12) ◽

pp. 1159-1166 ◽

Cited By ~ 1

Author(s):

Viviana Daidone ◽

Giovanni Barbon ◽

Elena Pontara ◽

Grazia Cattini ◽

Lisa Gallinaro ◽

...

Keyword(s):

Von Willebrand Factor ◽

Stop Codon ◽

Premature Stop Codon ◽

Von Willebrand Disease ◽

Hek293 Cells ◽

Loss Of Function ◽

Von Willebrand ◽

Willebrand Factor

SummaryCysteines play a key part in von Willebrand factor (VWF) dimerisation and polymerisation, and their loss may severely affect VWF structure and function. We report on three patients with type 3 von Willebrand disease carrying the new c.1751G>T missense mutation that induces the substitution of cysteine 584 by phenylalanine (C584F), and the deletion of seven nucleotides in exon 7 (c.729_735del), producing a premature stop codon at position 454 (E244Lfs*211). VWF was almost undetectable in the patients’ plasma and platelets, while a single, poorly represented, oligomer emerged on plasma VWF multimer analysis. No post-DDAVP increase in VWF and factor VIII was observed. Expressing human recombinant C584F-VWF in HEK293T cells showed that C584F-VWF was synthesised and multimerised but not secreted – apart from the first oligomer, which was slightly represented in the conditioned medium, with a pattern similar to the patients’ plasma VWF. The in vitro expression of the E244Lfs*211–VWF revealed a defective synthesis of the mutated VWF, with a behavior typical of loss of function mutations. Cellular trafficking, investigated in HEK293 cells, indicated a normal C584F-VWF content in the endoplasmic reticulum and Golgi apparatus, confirming the synthesis and multimerisation of C584F-VWF. No pseudo-Weibel Palade bodies were demonstrable, however, suggesting that C584F mutation impairs the storage of C584F-VWF. These findings point to cysteine 584 having a role in the release of VWF and its targeting to pseudo-Weibel Palade bodies in vitro, as well as in its storage and release by endothelial cells in vivo.

Download Full-text

Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies

Physiological Genomics ◽

10.1152/physiolgenomics.00070.2004 ◽

2004 ◽

Vol 19 (1) ◽

pp. 74-83 ◽

Cited By ~ 69

Author(s):

María Isabel Niemeyer ◽

Yamil R. Yusef ◽

Isabel Cornejo ◽

Carlos A. Flores ◽

Francisco V. Sepúlveda ◽

...

Keyword(s):

Stop Codon ◽

Premature Stop Codon ◽

Atp Depletion ◽

Functional Evaluation ◽

Loss Of Function ◽

Gene Encoding ◽

Hek 293 ◽

Channel Expression ◽

Generalized Epilepsy

The ClC-2 Cl− channel has been postulated to play a role in the inhibitory GABA response in neurons or to participate in astrocyte-dependent extracellular electrolyte homeostasis. Three different mutations in the CLCN2 gene, encoding the voltage-dependent homodimeric ClC-2 channel, have been associated with idiopathic generalized epilepsy (IGE). We study their function in vitro by patch clamp and confocal microscopy in transiently transfected HEK-293 cells. A first mutation predicts a premature stop codon (M200fsX231). An altered splicing, due to an 11-bp deletion in intron 2 (IVS2-14del11), predicts exon 3 skipping (Δ74–117). A third is a missense mutation (G715E). M200fsX231 and Δ74–117 are nonfunctional and do not affect the function of the normal (wild type, WT) channel. Neither M200fsX231 nor Δ74–117 reach the plasma membrane. Concerning the IVS2-14del11 mutation, we find no difference in the proportion of exon-skipped to normally spliced mRNA using a minigene approach and, on this basis, predict no alteration in channel expression in affected individuals. G715E has voltage dependence and intracellular Cl− dependence indistinguishable from WT channels. ClC-2 channels are shown to be sensitive to intracellular replacement of ATP by AMP, which accelerates the opening and closing kinetics. This effect is diminished in the G715E mutant and not significant in WT+G715E coexpression. We do not know whether, in a situation of cellular ATP depletion, this might become pathological in individuals carrying the mutation. We postulate that loss of function mutation M200fsX231 of ClC-2 might contribute to the IGE phenotype through a haploinsufficiency mechanism.

Download Full-text

TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽

10.1186/s12885-021-08562-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Chengwu Xiao ◽

Wei Zhang ◽

Meimian Hua ◽

Huan Chen ◽

Bin Yang ◽

...

Keyword(s):

Cell Lines ◽

Prognostic Indicator ◽

The Cancer Genome Atlas ◽

Mrna Levels ◽

Loss Of Function ◽

Protein Levels ◽

Kaplan Meier ◽

Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

Download Full-text

Cis-Segregation of c.1171C>T Stop Codon (p.R391*) in SERPINC1 Gene and c.1691G>A Transition (p.R506Q) in F5 Gene and Selected GWAS Multilocus Approach in Inherited Thrombophilia

Genes ◽

10.3390/genes12060934 ◽

2021 ◽

Vol 12 (6) ◽

pp. 934

Author(s):

Donato Gemmati ◽

Giovanna Longo ◽

Eugenia Franchini ◽

Juliana Araujo Silva ◽

Ines Gallo ◽

...

Keyword(s):

At Risk ◽

Stop Codon ◽

Association Studies ◽

Premature Stop Codon ◽

Large Family ◽

Genome Wide Association Studies ◽

Loss Of Function ◽

Gene Markers ◽

Inherited Thrombophilia ◽

Fv Leiden

Inherited thrombophilia (e.g., venous thromboembolism, VTE) is due to rare loss-of-function mutations in anticoagulant factors genes (i.e., SERPINC1, PROC, PROS1), common gain-of-function mutations in procoagulant factors genes (i.e., F5, F2), and acquired risk conditions. Genome Wide Association Studies (GWAS) recently recognized several genes associated with VTE though gene defects may unpredictably remain asymptomatic, so calculating the individual genetic predisposition is a challenging task. We investigated a large family with severe, recurrent, early-onset VTE in which two sisters experienced VTE during pregnancies characterized by a perinatal in-utero thrombosis in the newborn and a life-saving pregnancy-interruption because of massive VTE, respectively. A nonsense mutation (CGA > TGA) generating a premature stop-codon (c.1171C>T; p.R391*) in the exon 6 of SERPINC1 gene (1q25.1) causing Antithrombin (AT) deficiency and the common missense mutation (c.1691G>A; p.R506Q) in the exon 10 of F5 gene (1q24.2) (i.e., FV Leiden; rs6025) were coinherited in all the symptomatic members investigated suspecting a cis-segregation further confirmed by STR-linkage-analyses [i.e., SERPINC1 IVS5 (ATT)5–18, F5 IVS2 (AT)6–33 and F5 IVS11 (GT)12–16] and SERPINC1 intragenic variants (i.e., rs5878 and rs677). A multilocus investigation of blood-coagulation balance genes detected the coexistence of FV Leiden (rs6025) in trans with FV HR2-haplotype (p.H1299R; rs1800595) in the aborted fetus, and F11 rs2289252, F12 rs1801020, F13A1 rs5985, and KNG1 rs710446 in the newborn and other members. Common selected gene variants may strongly synergize with less common mutations tuning potential life-threatening conditions when combined with rare severest mutations. Merging classic and newly GWAS-identified gene markers in at risk families is mandatory for VTE risk estimation in the clinical practice, avoiding partial risk score evaluation in unrecognized at risk patients.

Download Full-text

Membrane blebbing as an assessment of functional rescue of dysferlin-deficient human myotubes via nonsense suppression

Journal of Applied Physiology ◽

10.1152/japplphysiol.01366.2009 ◽

2010 ◽

Vol 109 (3) ◽

pp. 901-905 ◽

Cited By ~ 30

Author(s):

Bingjing Wang ◽

Zhaohui Yang ◽

Becky K. Brisson ◽

Huisheng Feng ◽

Zhiqian Zhang ◽

...

Keyword(s):

Skeletal Muscle ◽

Stop Codon ◽

Premature Stop Codon ◽

Nonsense Suppression ◽

Muscle Membrane ◽

Membrane Repair ◽

Vitro Assay ◽

Membrane Blebbing ◽

Human Myotubes

Mutations that result in the loss of the protein dysferlin result in defective muscle membrane repair and cause either a form of limb girdle muscular dystrophy (type 2B) or Miyoshi myopathy. Most patients are compound heterozygotes, often carrying one allele with a nonsense mutation. Using dysferlin-deficient mouse and human myocytes, we demonstrated that membrane blebbing in skeletal muscle myotubes in response to hypotonic shock requires dysferlin. Based on this, we developed an in vitro assay to assess rescue of dysferlin function in skeletal muscle myotubes. This blebbing assay may be useful for drug discovery/validation for dysferlin deficiency. With this assay, we demonstrate that the nonsense suppression drug, ataluren (PTC124), is able to induce read-through of the premature stop codon in a patient with a R1905X mutation in dysferlin and produce sufficient functional dysferlin (∼15% of normal levels) to rescue myotube membrane blebbing. Thus ataluren is a potential therapeutic for dysferlin-deficient patients harboring nonsense mutations.

Download Full-text

Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands

Scientific Reports ◽

10.1038/srep20331 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 23

Author(s):

Encarnación Medina-Carmona ◽

Rogelio J. Palomino-Morales ◽

Julian E. Fuchs ◽

Esperanza Padín-Gonzalez ◽

Noel Mesa-Torres ◽

...

Keyword(s):

Protein Dynamics ◽

Protein Function ◽

Conformational Dynamics ◽

Loss Of Function ◽

Quinone Oxidoreductase ◽

Protein Levels ◽

Terminal Domain ◽

Directional Preference

Abstract Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S and to develop new pharmacological therapies to rescue this function.

Download Full-text

Loss-of-function in IRF2BPL is associated with neurological phenotypes

10.1101/322495 ◽

2018 ◽

Cited By ~ 1

Author(s):

Paul C. Marcogliese ◽

Vandana Shashi ◽

Rebecca C. Spillmann ◽

Nicholas Stong ◽

Jill A. Rosenfeld ◽

...

Keyword(s):

Nervous System ◽

Population Genomics ◽

Stop Codon ◽

Ectopic Expression ◽

Premature Stop Codon ◽

Model Organism ◽

Global Developmental Delay ◽

Mendelian Disease ◽

Loss Of Function ◽

Missense Variants

AbstractThe Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) gene encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals affected with neurological symptoms who carry damaging heterozygous variants in IRF2BPL. Five cases carrying nonsense variants in IRF2BPL resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The bioinformatics signature for IRF2BPL based on population genomics is consistent with a gene that is intolerant to variation. We show that the IRF2BPL ortholog in the fruit fly, called pits (protein interacting with Ttk69 and Sin3A), is broadly expressed including the nervous system. Complete loss of pits is lethal early in development, whereas partial knock-down with RNA interference in neurons leads to neurodegeneration, revealing requirement for this gene in proper neuronal function and maintenance. The nonsense variants in IRF2BPL identified in patients behave as severe loss-of-function alleles in this model organism, while ectopic expression of the missense variants leads to a range of phenotypes. Taken together, IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

Download Full-text

Upregulation of SGOL2 Predicts Poor Prognosis and Facilitates Hepatocellular Carcinoma Progression via Dysregulating the Cell Cycle by Directly Activating MAD2

10.21203/rs.3.rs-560609/v1 ◽

2021 ◽

Author(s):

Qingqing Hu ◽

Xiaochu Hu ◽

Yalei Zhao ◽

Lingjian Zhang ◽

Ya Yang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

The Cancer Genome Atlas ◽

Loss Of Function ◽

Protein Levels ◽

Cancer Genome Atlas ◽

Centromeric Protein ◽

Hcc Cells

Abstract Background: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Shugoshin-like protein 2 (SGOL2) is a centromeric protein that ensures the correct and orderly process of mitosis by protecting and maintaining centripetal adhesions during meiosis and mitosis. However, the role of SGOL2 in cancer is not well understood. Methods: The mRNA and protein levels of SGOL2 and survival analysis were conducted in The Cancer Genome Atlas (TCGA) and further validated in 2 independent cohorts. Differential genes correlated with SGOL2 and mitotic arrest deficient 2 like 1 (MAD2) were obtained using LinkedOmics. Subsequently, loss-of-function and rescue assays were carried out in vitro and in vivo to assess the functions of SGOL2 in hepatic tumorigenisis. Findings: We found that SGOL2 was significantly overexpressed in HCC and predicted unfavorable overall survival in HCC patients. Next, we identified 47 differentially expressed genes positively correlated with both SGOL2 and MAD2 to be mainly involved in the cell cycle. In addition, SGOL2 downregulation suppressed the migration, invasion, proliferation, stemness and EMT of HCC cells and inhibited tumorigenesis in vivo. Furthermore, SGOL2 promoted tumor proliferation by activating MAD2-induced cell cycle dysregulation, which could be reversed by the MAD2 inhibitor M2I-1. We also proved that SGOL2 activated MAD2 by directly binding with MAD2. Conclusions: The results of this study showed that SGOL2 acts as an oncogene in HCC cells by directly activating MAD2 and then dysregulating the cell cycle, thereby providing a potential target for HCC patients in the future.

Download Full-text

91 DISRUPTION OF TET1 DURING PORCINE EMBRYOGENESIS USING CRISPR/Cas9 SYSTEM

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab91 ◽

2017 ◽

Vol 29 (1) ◽

pp. 153

Author(s):

K. Uh ◽

J. Ryu ◽

C. Ray ◽

K. Lee

Keyword(s):

Embryo Development ◽

Translation Initiation ◽

Stop Codon ◽

Premature Stop Codon ◽

Initiation Site ◽

Translation Initiation Site ◽

Epigenetic Marks ◽

Hydroxymethyl Cytosine ◽

Biallelic Mutations

Ten-eleven translocation (TET) enzymes catalyse oxidation of 5-methylcytosine to 5-hydroxymethyl cytosine. This TET-mediated conversion of 5-methylcytosine to 5-hydroxymethyl cytosine is implicated in initiating the DNA demethylation process, observed post-fertilization. Three members (TET1–3) of the TET family are differentially expressed during embryo development and appear to have different roles. Previous studies in mice suggest that TET1 is a key regulator in maintaining pluripotency in embryonic stem cells by managing epigenetic marks such as DNA methylation. This would imply that TET1 should be a regulator of epigenetic marks during embryo development, although this has not been demonstrated. Previously, we have cloned porcine TET1 from blastocysts (GenBank accession number KC137683) and demonstrated that the level of TET1 (mRNA and protein) was high in blastocysts. The protein level was greater in the inner cell mass compared with the trophectoderm. In this study, we generated TET1 knockout porcine embryos using CRISPR/Cas9 system to study the role of TET1 in controlling epigenetic marks during porcine embryo development. First, 2 sgRNA, immediately downstream of the presumable translation initiation site, were designed and synthesised; location of the sgRNA were nucleotide position at 2 to 21 bp and 23 to 42 bp, respectively (KC137683). Then, sgRNA (10 ng μL−1 each) and Cas9 mRNA (20 ng μL−1) were injected into the cytoplasm of IVF zygotes, and Day 7 blastocysts were genotyped. All embryos carried mutations on both alleles of TET1 (10/10), one homozygous and 9 biallelic mutations. However, immunocytochemistry analysis of other CRISPR/Cas9 injected embryos revealed that TET1 was not removed (10/10), indicating that the sgRNA may have not introduced a premature stop codon 3′ to the presumable translation initiation site. Therefore, 2 new sgRNA were designed to generate a premature stop codon at the 5′ side of a key functional domain, the 2-oxoglutarate-Fe(II)-dependent oxygenase domain (4690 to 5160 bp); the locations of the 2 sgRNA were 4450 to 4469 bp and 4501 to 4520 bp, respectively. Similarly, all of the embryos carried mutations in TET1 (7/7), 2 homozygous and 5 biallelic mutations. In addition, TET1 proteins were not detected in 11 of 16 blastocysts, confirmed by immunocytochemistry. In this study, we successfully generated embryos lacking TET1 by introducing designed CRISPR/Cas9 system during embryogenesis. Presence of TET1 from the first injection experiment suggests that the presumable translation initiation site is not accurate. Discrepancy between genotyping and immunocytochemistry results from the second injection experiment indicates that embryos possessing TET1 protein probably have mutations in triplets, thus no premature stop codon was synthesised. Further studies will focus on identifying the role of TET1 in maintaining pluripotency and epigenetic modification during pre-implantation stage using these embryos.

Download Full-text