scholarly journals Identification and Functional Characterization of IDS Gene Mutations Underlying Taiwanese Hunter Syndrome (Mucopolysaccharidosis Type II)

2019 ◽  
Vol 21 (1) ◽  
pp. 114 ◽  
Author(s):  
Hsiang-Yu Lin ◽  
Ru-Yi Tu ◽  
Schu-Rern Chern ◽  
Yun-Ting Lo ◽  
Sisca Fran ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Gene Mutations ◽  
Hunter Syndrome ◽  
Missense Mutations ◽  
Mps Ii ◽  
Genomic Studies ◽  
Normal Enzyme ◽  
Positive Correlations ◽  
Highly Correlated

Hunter syndrome (mucopolysaccharidosis II; MPS II) is caused by a defect of the iduronate-2-sulfatase (IDS) gene. Few studies have reported integrated mutation data of Taiwanese MPS II phenotypes. In this study, we summarized genotype and phenotype correlations of confirmed MPS II patients and asymptomatic MPS II infants in Taiwan. Regular polymerase chain reaction and DNA sequencing were used to identify genetic abnormalities of 191 cases, including 51 unrelated patients with confirmed MPS II and 140 asymptomatic infants. IDS activity was analyzed in individual novel IDS variants using in vitro expression studies. Nineteen novel mutations were identified, in which the percentages of IDS activity of the novel missense mutations c.137A>C, c.311A>T, c.454A>C, c.797C>G, c.817C>T, c.998C>T, c.1106C>G, c.1400C>T, c.1402C>T, and c.1403G>A were significantly decreased (p < 0.001), c.254C>T and c.1025A>G were moderately decreased (p < 0.01), and c.851C>T was slightly decreased (p < 0.05) comparing with normal enzyme activity. The activities of the other six missense mutations were reduced but were insignificant. The results of genomic studies and their phenotypes were highly correlated. A greater understanding of the positive correlations may help to prevent the irreversible manifestations of Hunter syndrome, particularly in infants suspected of having asymptomatic MPS II. In addition, urinary glycosaminoglycan assay is important to diagnose Hunter syndrome since gene mutations are not definitive (could be non-pathogenic).

A Novel Loss of Function Melanocortin-4-Receptor Mutation (MC4R-F313Sfs*29) in Morbid Obesity

2020 ◽  
Author(s):  
Elisabetta Trevellin ◽  
Marnie Granzotto ◽  
Cristina Host ◽  
Francesca Grisan ◽  
Diego De Stefani ◽  
...  
Keyword(s):  
Early Onset ◽  
Functional Characterization ◽  
Gene Mutations ◽  
Melanocortin Receptor ◽  
Hek293 Cells ◽  
Intracellular Camp ◽  
Loss Of Function ◽  
Mc4r Gene ◽  
Melanocortin 4 Receptor

Abstract Context Melanocortin receptor-4 (MC4R) gene mutations are associated with early-onset severe obesity, and the identification of potential pathological variants is crucial for the clinical management of patients with obesity. Objective To explore whether and how a novel heterozygous MC4R variant (MC4R-F313Sfs*29), identified in a young boy (body mass index [BMI] 38.8 kg/m2) during a mutation analysis conducted in a cohort of patients with obesity, plays a determinant pathophysiological role in the obesity development. Design Setting and Patients The genetic screening was carried out in a total of 209 unrelated patients with obesity (BMI ≥ 35 kg/m2). Structural and functional characterization of the F313Sfs*29-mutated MC4R was performed using computational approaches and in vitro, using HEK293 cells transfected with genetically encoded biosensors for cAMP and Ca2+. Results The F313Sfs*29 was the only variant identified. In vitro experiments showed that HEK293 cells transfected with the mutated form of MC4R did not increase intracellular cAMP or Ca2+ levels after stimulation with a specific agonist in comparison with HEK293 cells transfected with the wild type form of MC4R (∆R/R0 = -90% ± 8%; P &lt; 0.001). In silico modeling showed that the F313Sfs*29 mutation causes a major reorganization in the cytosolic domain of MC4R, thus reducing the affinity of the putative GalphaS binding site. Conclusions The newly discovered F313Sfs*29 variant of MC4R may be involved in the impairment of α-MSH-induced cAMP and Ca2+ signaling, blunting intracellular G protein-mediated signal transduction. This alteration might have led to the dysregulation of satiety signaling, resulting in hyperphagia and early onset of obesity.

scholarly journals Functional characterization of Polr3a hypomyelinating leukodystrophy mutations in the S. cerevisiae homolog, RPC160

2020 ◽  
Author(s):  
Robyn D. Moir ◽  
Christian Lavados ◽  
JaeHoon Lee ◽  
Ian M. Willis
Keyword(s):  
Functional Characterization ◽  
Rna Polymerase Iii ◽  
State Level ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Wild Type ◽  
Functional Deficits ◽  
Pol Iii ◽  
Transcriptional Output

AbstractMutations in RNA polymerase III (Pol III) cause hypomeylinating leukodystrophy (HLD) and neurodegeneration in humans. POLR3A and POLR3B, the two largest Pol III subunits, together form the catalytic center and carry the majority of disease alleles. Disease-causing mutations include invariant and highly conserved residues that are predicted to negatively affect Pol III activity and decrease transcriptional output. A subset of HLD missense mutations in POLR3A cluster in the pore region that provides nucleotide access to the Pol III active site. These mutations were engineered at the corresponding positions in the Saccharomyces cerevisiae homolog, Rpc160, to evaluate their functional deficits. None of the mutations caused a growth or transcription phenotype in yeast. Each mutation was combined with a frequently occurring pore mutation, POLR3A G672E, which was also wild-type for growth and transcription. The double mutants showed a spectrum of phenotypes from wild-type to lethal, with only the least fit combinations showing an effect on Pol III transcription. In one slow-growing temperature-sensitive mutant the steady-state level of tRNAs was unaffected, however global tRNA synthesis was compromised, as was the synthesis of RPR1 and SNR52 RNAs. Affinity-purified mutant Pol III was broadly defective in both factor-independent and factor-dependent transcription in vitro across genes that represent the yeast Pol III transcriptome. Thus, the robustness of yeast to Pol III leukodystrophy mutations in RPC160 can be overcome by a combinatorial strategy.

scholarly journals The mutational spectrum of Hunter syndrome reveals correlation between biochemical and clinical profiles in Tunisian patients

2020 ◽  
Author(s):  
latifa chkioua ◽  
Oussama Grissa ◽  
Nadia Leban ◽  
Moez Gribaa ◽  
Hela Boudabous ◽  
...  
Keyword(s):  
Nonsense Mutation ◽  
Dermatan Sulfate ◽  
Splice Site Mutation ◽  
Hunter Syndrome ◽  
Molecular Testing ◽  
Missense Mutations ◽  
Lysosomal Storage ◽  
High Concentration ◽  
Site Mutation ◽  
Mps Ii

Abstract Background: Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is an X-linked recessive lysosomal storage disorder resulting from deficient activity of iduronate 2-sulfatase (IDS) and the progressive lysosomal accumulation of sulfated glycosaminoglycans (GAGs). Methods: A diagnosis of MPS II or Hunter syndrome was performed based on the following approach after a clinical and paraclinical suspicion. Two biochemical and molecular tests were carried out separately and according to the availability of the biological material. Results: All patients in this cohort presented the most common MPS II clinical features. Electrophoresis of GAGs on a cellulose acetate plate in the presence of a high concentration of heparane sulfate showed an abnormal dermatan sulfate band in the patients compared with that in a control case. Furthermore, leukocyte IDS activity ranged from 0.00 to 0.75 nmol/h/mg of leukocyte protein in patients.Five previously reported mutations were identified in this study patients: one splice site mutation, c.240+1G>A; two missense mutations, p.R88P and p.G94D; a large deletion of exon 1 to exon 7; and one nonsense mutation, p.Q396*. In addition, two novel alterations were identified in the MPS II patients: one frame shift mutation, p.D450Nfs*95 and one nonsense mutation, p.Q204*. Additionally, five known IDS polymorphisms were identified in the patients: c.419-16 delT, c.641C>T (p.T214M), c.438 C>T (p.T146T), c.709-87G>A, and c.1006+38T>C.Conclusions: The high level of urine GAGs and the deficiency of iduronate 2-sulfatase activity was associated with the phenotype expression of Hunter syndrome. Molecular testing was useful for the patients’ phenotypic classification and the detection of carriers.

scholarly journals SAMHD1Gene Mutations Are Associated with Cerebral Large-Artery Atherosclerosis

2015 ◽  
Vol 2015 ◽  
pp. 1-8
Author(s):  
Wei Li ◽  
Baozhong Xin ◽  
Junpeng Yan ◽  
Ying Wu ◽  
Bo Hu ◽  
...  
Keyword(s):  
Small Vessel Disease ◽  
Direct Sequencing ◽  
Gene Mutations ◽  
Splice Site Mutation ◽  
Large Artery ◽  
Missense Mutations ◽  
Site Mutation ◽  
Chinese Han ◽  
Functionally Impaired

Background. To investigate whether one or moreSAMHD1gene mutations are associated with cerebrovascular disease in the general population using a Chinese stroke cohort.Methods. Patients with a Chinese Han background (N=300) diagnosed with either cerebral large-artery atherosclerosis (LAA,n=100), cerebral small vessel disease (SVD,n=100), or other stroke-free neurological disorders (control,n=100) were recruited. Genomic DNA from the whole blood of each patient was isolated, and direct sequencing of theSAMHD1gene was performed. Both wild type and mutant SAMHD1 proteins identified from the patients were expressed inE. coliand purified; then their dNTPase activities and ability to form stable tetramers were analysedin vitro.Results. Three heterozygous mutations, including two missense mutations c.64C>T (P22S) and c.841G>A (p.E281K) and one splice site mutation c.696+2T>A, were identified in the LAA group with a prevalence of 3%. No mutations were found in the patients with SVD or the controls (p=0.05). The mutant SAMHD1 proteins were functionally impaired in terms of their catalytic activity as a dNTPase and ability to assemble stable tetramers.Conclusions. HeterozygousSAMHD1gene mutations might cause genetic predispositions that interact with other risk factors, resulting in increased vulnerability to stroke.

In vitro functional characterization of missense mutations in the LDLR gene

2012 ◽  
Vol 225 (1) ◽  
pp. 128-134 ◽  
Author(s):  
S. Silva ◽  
A.C. Alves ◽  
D. Patel ◽  
R. Malhó ◽  
A.K. Soutar ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Missense Mutations ◽  
Ldlr Gene

scholarly journals The mutational spectrum of Hunter syndrome reveals correlation between biochemical and clinical profiles in Tunisian patients

2020 ◽  
Author(s):  
latifa chkioua ◽  
Oussama Grissa ◽  
Nadia Leban ◽  
Moez Gribaa ◽  
Hela Boudabous ◽  
...  
Keyword(s):  
Nonsense Mutation ◽  
Dermatan Sulfate ◽  
Splice Site Mutation ◽  
Hunter Syndrome ◽  
Molecular Testing ◽  
Missense Mutations ◽  
Lysosomal Storage ◽  
High Concentration ◽  
Site Mutation ◽  
Mps Ii

Abstract Background: Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is an X-linked recessive lysosomal storage disorder resulting from deficient activity of iduronate 2-sulfatase (IDS) and the progressive lysosomal accumulation of sulfated glycosaminoglycans (GAGs). Methods: A diagnosis of MPS II or Hunter syndrome was performed based on the following approach after a clinical and paraclinical suspicion. Two biochemical and molecular tests were carried out separately and according to the availability of the biological material. Results: All patients in this cohort presented the most common MPS II clinical features. Electrophoresis of GAGs on a cellulose acetate plate in the presence of a high concentration of heparane sulfate showed an abnormal dermatan sulfate band in the patients compared with that in a control case. Furthermore, leukocyte IDS activity ranged from 0.00 to 0.75 nmol/h/mg of leukocyte protein in patients. Five previously reported mutations were identified in this study patients: one splice site mutation, c.240+1G>A; two missense mutations, p.R88P and p.G94D; a large deletion of exon 1 to exon 7; and one nonsense mutation, p.Q396*. In addition, two novel alterations were identified in the MPS II patients: one frame shift mutation, p.D450Nfs*95 and one nonsense mutation, p.Q204*. Additionally, five known IDS polymorphisms were identified in the patients: IVS3-16 (c.419-16 delT), p.T214M (c.641C>T), p.T146T (c.438 C>T), IVS5-87 (c.709-87G>A), and IVS7+38 (c.1006+38T>C). Conclusions: The high level of urine GAGs and the deficiency of iduronate 2-sulfatase activity was associated with the phenotype expression of Hunter syndrome. Molecular testing was useful for the patients’ phenotypic classification and the detection of carriers.

scholarly journals Functional characterization of SMARCA4 variants identified by targeted exome-sequencing of 131,668 cancer patients

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Tharu M. Fernando ◽  
Robert Piskol ◽  
Russell Bainer ◽  
Ethan S. Sokol ◽  
Sally E. Trabucco ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Functional Characterization ◽  
Large Sample Size ◽  
Helicase Domain ◽  
Missense Mutations ◽  
Attractive Therapeutic Target ◽  
Genomic Studies ◽  
Domain Functional ◽  
Tumor Types

Abstract Genomic studies performed in cancer patients and tumor-derived cell lines have identified a high frequency of alterations in components of the mammalian switch/sucrose non-fermentable (mSWI/SNF or BAF) chromatin remodeling complex, including its core catalytic subunit, SMARCA4. Cells exhibiting loss of SMARCA4 rely on its paralog, SMARCA2, making SMARCA2 an attractive therapeutic target. Here we report the genomic profiling of solid tumors from 131,668 cancer patients, identifying 9434 patients with one or more SMARCA4 gene alterations. Homozygous SMARCA4 mutations were highly prevalent in certain tumor types, notably non-small cell lung cancer (NSCLC), and associated with reduced survival. The large sample size revealed previously uncharacterized hotspot missense mutations within the SMARCA4 helicase domain. Functional characterization of these mutations demonstrated markedly reduced remodeling activity. Surprisingly, a few SMARCA4 missense variants partially or fully rescued paralog dependency, underscoring that careful selection criteria must be employed to identify patients with inactivating, homozygous SMARCA4 missense mutations who may benefit from SMARCA2-targeted therapy.

scholarly journals Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1430 ◽  
Author(s):  
Marlène Deschuyter ◽  
Florian Pennarubia ◽  
Emilie Pinault ◽  
Sébastien Legardinier ◽  
Abderrahman Maftah
Keyword(s):  
Colorectal Cancer ◽  
Notch Signaling ◽  
Functional Characterization ◽  
Missense Mutations ◽  
Colorectal Tumors ◽  
Protein Targets ◽  
E Coli ◽  
Notch Receptors

Background: Protein O-fucosyltransferase 1 (POFUT1) overexpression, which is observed in many cancers such as colorectal cancer (CRC), leads to a NOTCH signaling dysregulation associated with the tumoral process. In rare CRC cases, with no POFUT1 overexpression, seven missense mutations were found in human POFUT1. Methods: Recombinant secreted forms of human WT POFUT1 and its seven mutated counterparts were produced and purified. Their O-fucosyltransferase activities were assayed in vitro using a chemo-enzymatic approach with azido-labeled GDP-fucose as a donor substrate and NOTCH1 EGF-LD26, produced in E. coli periplasm, as a relevant acceptor substrate. Targeted mass spectrometry (MS) was carried out to quantify the O-fucosyltransferase ability of all POFUT1 proteins. Findings: MS analyses showed a significantly higher O-fucosyltransferase activity of six POFUT1 variants (R43H, Y73C, T115A, I343V, D348N, and R364W) compared to WT POFUT1. Interpretation: This study provides insights on the possible involvement of these seven missense mutations in colorectal tumors. The hyperactive forms could lead to an increased O-fucosylation of POFUT1 protein targets such as NOTCH receptors in CRC patients, thereby leading to a NOTCH signaling dysregulation. It is the first demonstration of gain-of-function mutations for this crucial glycosyltransferase, modulating NOTCH activity, as well as that of other potential glycoproteins.

scholarly journals The mutational spectrum of Hunter syndrome reveals correlation between biochemical and clinical profiles in Tunisian patients

2019 ◽  
Author(s):  
latifa chkioua ◽  
Oussama Grissa ◽  
Nadia Leban ◽  
Moez Gribaa ◽  
Hela Boudabous ◽  
...  
Keyword(s):  
Nonsense Mutation ◽  
Dermatan Sulfate ◽  
Splice Site Mutation ◽  
Hunter Syndrome ◽  
Molecular Testing ◽  
Missense Mutations ◽  
Lysosomal Storage ◽  
High Concentration ◽  
Site Mutation ◽  
Mps Ii

Abstract Background: Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is an X-linked recessive lysosomal storage disorder resulting from deficient activity of iduronate 2-sulfatase ( IDS ) and the progressive lysosomal accumulation of sulfated glycosaminoglycans (GAGs). Methods: A diagnosis of MPS II or Hunter syndrome was performed based on the following approach after a clinical and paraclinical suspicion. Two biochemical and molecular tests were carried out separately and according to the availability of the biological material. Results: All patients in this cohort presented the most common MPS II clinical features. Electrophoresis of GAGs on a cellulose acetate plate in the presence of a high concentration of heparane sulfate showed an abnormal dermatan sulfate band in the patients compared with that in a control case. Furthermore, leukocyte IDS activity ranged from 0.00 to 0.75 nmol/h/mg of leukocyte protein in patients. Five previously reported mutations were identified in the study patients: one splice site mutation, c.240+1G>A; two missense mutations, p.R88P and p.G94D; a large deletion of exon 1 to exon 7; and one nonsense mutation, p.Q396*. In addition, two novel alterations were identified in the MPS II patients: one frame shift mutation, p.D450Nfs*95 and one nonsense mutation, p.Q204*. Additionally, five known IDS polymorphisms were identified in the patients: IVS3-16 (c.419-16 delT), p.T214M (c.641C>T), p.T146T (c.438 C>T), IVS5-87 (c.709-87G>A), and IVS7+38 (c.1006+38T>C). Conclusions: The high level of urine GAGs and the deficiency of iduronate 2-sulfatase activity was associated with the phenotype expression of Hunter syndrome. Molecular testing was useful for the patients’ phenotypic classification and the detection of carriers.

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