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

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.

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

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Yuan Zhang ◽  
Weicheng Chen ◽  
Weijia Zeng ◽  
Zhouping Lu ◽  
Xiangyu Zhou
Keyword(s):  
Stop Codon ◽  
Retinal Pigment ◽  
Premature Stop Codon ◽  
Nuclear Pore ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Tem Analysis ◽  
Protein Levels ◽  
Biallelic Mutations

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.

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

BMC Cancer ◽  
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.

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

2016 ◽  
Vol 6 (1) ◽  
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.

P–199 A case report to suggest that there must be other mutations than PATL2 or TUBB8 to cause oocyte maturation arrest

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Molinari ◽  
M Yang ◽  
J Hu ◽  
L Zhang ◽  
D F Albertini ◽  
...  
Keyword(s):  
Case Report ◽  
Oocyte Maturation ◽  
Fertilization Rate ◽  
Functional Study ◽  
Embryo Morphology ◽  
Loss Of Function ◽  
Maturation Arrest ◽  
Polar Bodies

Abstract Study question What causes our patient’s repeated almost complete oocyte maturation arrest (OMA)? Summary answer Since we did not detect PATL2 and TUBB8 mutations, both known to cause OMA, this case was likely caused by mutations in HUS1 and ITGB3 What is known already OMA has been associated with loss-of-function in key genes, such as PATL2 and TUBB8. Such patients have, however, uniformly have been unable to conceive with IVF Study design, size, duration We here report the case of repeatedly presenting patient between 2009 until 2020 (age 30 at 1st and 41 at last visit). Participants/materials, setting, methods The couple underwent 7 IVF treatments under several ovarian stimulation protocols at different gonadotropin dosages and in different preparations to try to recruit mature eggs. She conceived in her 2nd IVF cycle in 2009 and delivered uneventfully in 2010. She then conceived spontaneously and delivered a healthy boy in 2014. The couple since then has been attempting another pregnancy. Remarkably, in all IVF cycles all eggs but one arrested at prophase. Main results and the role of chance The female demonstrates abnormally high ovarian reserve for age (AMH=5.9 ng/mL in 2019) (mean, 10.6 oocytes). In all cycles, all but one retrieved were immature. In vitro maturation rate for the GV oocytes was 28%. Resultant M2s, however, demonstrated morphological abnormalities, such as giant polar bodies. In vivo M2s, in contrast, were always morphologically unremarkable, and their fertilization rate was 85%. Embryo morphology deteriorated appreciatively with advancing age. Sanger sequencing for TUBB8 and PATL2 genes were unremarkable. Whole genome sequencing of her and her sister (who had no fertility problems) revealed mutations of genes belonging to the integrin family (ITGB3) and DNA repair checkpoint (HUS1), both of which could be determinants in the observed maturation arrest. Limitations, reasons for caution A functional study, coupled with imaging of the discarded material, will likely offer further information regarding the mechanisms leading to OMA in this female. Wider implications of the findings: This case report represents a new phenotype of female infertility, characterized by almost complete maturation arrest which, however, still offers opportunity for pregnancy. Further isolation of underlying mutation(s) may offer additional insights about checkpoints required for the transition of prophase to metaphase in human oocytes. Trial registration number NA

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

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.

scholarly journals Novel Compound Heterozygous Mutations in ZAP70 Leading to a SCID Phenotype with Normal Downstream In vitro Signaling

2020 ◽  
Author(s):  
Kelsey Kaman ◽  
Monique Abrams ◽  
Kerry Dobbs ◽  
Luigi D. Notarangelo ◽  
Ottavia M. Delmonte ◽  
...  
Keyword(s):  
Compound Heterozygous ◽  
Compound Heterozygous Mutations

CTSC compound heterozygous mutations in two Chinese patients with Papillon–Lefèvre syndrome

Oral Diseases ◽  
2019 ◽  
Vol 25 (5) ◽  
pp. 1394-1402 ◽  
Author(s):  
Yuelin Wu ◽  
Lei Zhao ◽  
Chunmei Xu ◽  
Yafei Wu
Keyword(s):  
Chinese Patients ◽  
Compound Heterozygous ◽  
Compound Heterozygous Mutations

scholarly journals TBC1D24-TLDc-related epilepsy exercise-induced dystonia: rescue by antioxidants in a disease model

Brain ◽  
2019 ◽  
Vol 142 (8) ◽  
pp. 2319-2335 ◽  
Author(s):  
Kevin Lüthy ◽  
Davide Mei ◽  
Baptiste Fischer ◽  
Maurizio De Fusco ◽  
Jef Swerts ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Trafficking ◽  
Disease Model ◽  
Stress Sensitivity ◽  
Compound Heterozygous ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Rolandic Epilepsy ◽  
Compound Heterozygous Mutations ◽  
Exercise Induced

AbstractGenetic mutations in TBC1D24 have been associated with multiple phenotypes, with epilepsy being the main clinical manifestation. The TBC1D24 protein consists of the unique association of a Tre2/Bub2/Cdc16 (TBC) domain and a TBC/lysin motif domain/catalytic (TLDc) domain. More than 50 missense and loss-of-function mutations have been described and are spread over the entire protein. Through whole genome/exome sequencing we identified compound heterozygous mutations, R360H and G501R, within the TLDc domain, in an index family with a Rolandic epilepsy exercise-induced dystonia phenotype (http://omim.org/entry/608105). A 20-year long clinical follow-up revealed that epilepsy was self-limited in all three affected patients, but exercise-induced dystonia persisted into adulthood in two. Furthermore, we identified three additional sporadic paediatric patients with a remarkably similar phenotype, two of whom had compound heterozygous mutations consisting of an in-frame deletion I81_K84 and an A500V mutation, and the third carried T182M and G511R missense mutations, overall revealing that all six patients harbour a missense mutation in the subdomain of TLDc between residues 500 and 511. We solved the crystal structure of the conserved Drosophila TLDc domain. This allowed us to predict destabilizing effects of the G501R and G511R mutations and, to a lesser degree, of R360H and potentially A500V. Next, we characterized the functional consequences of a strong and a weak TLDc mutation (TBC1D24G501R and TBC1D24R360H) using Drosophila, where TBC1D24/Skywalker regulates synaptic vesicle trafficking. In a Drosophila model neuronally expressing human TBC1D24, we demonstrated that the TBC1D24G501R TLDc mutation causes activity-induced locomotion and synaptic vesicle trafficking defects, while TBC1D24R360H is benign. The neuronal phenotypes of the TBC1D24G501R mutation are consistent with exacerbated oxidative stress sensitivity, which is rescued by treating TBC1D24G501R mutant animals with antioxidants N-acetylcysteine amide or α-tocopherol as indicated by restored synaptic vesicle trafficking levels and sustained behavioural activity. Our data thus show that mutations in the TLDc domain of TBC1D24 cause Rolandic-type focal motor epilepsy and exercise-induced dystonia. The humanized TBC1D24G501R fly model exhibits sustained activity and vesicle transport defects. We propose that the TBC1D24/Sky TLDc domain is a reactive oxygen species sensor mediating synaptic vesicle trafficking rates that, when dysfunctional, causes a movement disorder in patients and flies. The TLDc and TBC domain mutations’ response to antioxidant treatment we observed in the animal model suggests a potential for combining antioxidant-based therapeutic approaches to TBC1D24-associated disorders with previously described lipid-altering strategies for TBC domain mutations.

Transient congenital hypothyroidism caused by compound heterozygous mutations affecting the NADPH-oxidase domain of DUOX2

2016 ◽  
Vol 29 (3) ◽  
Author(s):  
Atsuko Yoshizawa-Ogasawara ◽  
Kiyomi Abe ◽  
Sayaka Ogikubo ◽  
Satoshi Narumi ◽  
Tomonobu Hasegawa ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Congenital Hypothyroidism ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Nicotinamide Adenine ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Dual Oxidase ◽  
Compound Heterozygous Mutations ◽  
Transient Congenital Hypothyroidism

AbstractHere, we describe three cases of loss-of-function mutations in the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase (NOX) domain of dual oxidase 2 (

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