Effect of BTD gene variants on in vitro biotinidase activity

AbstractThe goal of this study was to identify MSX1 gene variants in multiple Chinese families with nonsyndromic oligodontia and analyse the functional influence of these variants. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify the causal gene variants in five families with nonsyndromic oligodontia, and a series of bioinformatics databases were used for variant confirmation and functional prediction. Phenotypic characterization of the members of these families was described, and an in vitro analysis was performed for functional evaluation. Five novel MSX1 heterozygous variants were identified: three missense variants [c.662A>C (p.Q221P), c.670C>T (p.R224C), and c.809C>T (p.S270L)], one nonsense variant [c.364G>T (p.G122*)], and one frameshift variant [c.277delG (p.A93Rfs*67)]. Preliminary in vitro studies demonstrated that the subcellular localization of MSX1 was abnormal with the p.Q221P, p.R224C, p.G122*, and p.A93Rfs*67 variants compared to the wild type. Three variants (p.Q221P, p.G122*, and p.A93Rfs*67) were classified as pathogenic or likely pathogenic, while p.S270L and p.R224C were of uncertain significance in the current data. Moreover, we summarized and analysed the MSX1-related tooth agenesis positions and found that the type and variant locus were not related to the severity of tooth loss. Our results expand the variant spectrum of nonsyndromic oligodontia and provide valuable information for genetic counselling.

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The identification of a novel splicing mutation in the DMD gene of a Chinese family

10.22541/au.163308103.31867516/v1 ◽

2021 ◽

Author(s):

Wanlu Liu ◽

Xinwei Shi ◽

Yuqi Li ◽

Fuyuan Qiao ◽

Yuanyuan Wu

Keyword(s):

Duchenne Muscular Dystrophy ◽

Pathogenic Mutation ◽

Chinese Family ◽

Gene Variants ◽

Splicing Mutation ◽

Aberrant Splicing ◽

Disease Phenotypes ◽

Dmd Gene ◽

Minigene Expression

The Duchenne Muscular Dystrophy (DMD) gene variants are associated with the disease phenotypes. The pathogenic mutation, c.2293-1G>C, was detected in DMD gene in the proband and the fetus, which has not been reported in the literature.The minigene expression in vitro confirmed that c.2293-1G>C is responsible of aberrant splicing.

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OR31-06 Candidate Gene Variants in a Large Cohort of Women with Primary Ovarian Insufficiency

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1419 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Bushra Gorsi ◽

Mika Moriwaki ◽

Marvin B Moore ◽

Aleksandar Rajkovic ◽

Lawrence M Nelson ◽

...

Keyword(s):

Functional Model ◽

Primary Ovarian Insufficiency ◽

Composite Likelihood ◽

Large Cohort ◽

Gene Variants ◽

Loss Of Function ◽

Test Statistic ◽

Ovarian Insufficiency ◽

Total N

Abstract Primary ovarian insufficiency (POI) is highly heritable. The majority of cases have no known cause. We hypothesized that mutations in previously identified genes or genes from the same pathways are the cause of POI in a recessive or dominant manner. Subjects included 294 women diagnosed with POI (amenorrhea with an elevated FSH level). All had a 46XX karyotype, and normal FMR1 repeat number. Subjects were recruited in Boston (n=95), at the NIH and Washington University (n=98), and in Pittsburgh (n=98). Controls included subjects recruited for health in old age and disorders unrelated to reproduction or cancer, and subjects from the 1000 Genomes Project (total n=587). Variants were called using the Sentieon software package (https://www.sentieon.com). Case and control samples were stratified on ethnicity, relatedness and heterozygosity. Peddy and XPAT were used to calculate quality control metrics to detect outlier samples for removal from analysis to create a homogenous dataset. The number of cases (227) and controls (458) was adjusted for downstream analysis. XPAT imposed additional quality filters and removed variants. A second filter removed variants that did not pass a Gnomad filter of <0.001 allele frequency. VAAST was used to determine a composite likelihood ratio (CLR) as the test statistic to represent the aggregate burden of variants of affected individuals in each transcript relative to a set of 458 control genomes. The significance of each transcript’s VAAST CLR score was evaluated by 1 million permutations. We screened exomes for variants in previously identified genes causing POI in humans and those demonstrating infertility in a male or female mouse model. We also used the American College of Medical Genetics and Genomics standards for interpretation of pathogenicity of a variant, with priority on null variants in genes with probability of loss of function intolerance based on the observed vs. expected rate in gnomAD, in vivo or in vitro functional evidence of a damaging effect, significantly increased prevalence compared to controls, i.e. not found in any controls or in fewer than 10 in the gnomAD database if the subject had a matching race/ethnicity. Thirty-four subjects were removed for poor quality exomes and relatedness. Fifty-three subjects had at least one variant in a previously identified POI gene or one in which there was a previously identified functional model. Two subjects carried recessive variants and 30 carried at least one novel heterozygous candidate variant for follow up. Analysis of genetic causes of POI in this large cohort identified candidate causal gene variants in over half of the subjects. The data demonstrate that the genetic architecture is heterogeneous. Although recessive mutations have been identified in consanguineous families, the data suggest that a dominant or oligogenic pattern of inheritance may be important.

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Fanconi anemia and homologous recombination gene variants are associated with functional DNA repair defects in vitro and poor outcome in patients with advanced head and neck squamous cell carcinoma

Oncotarget ◽

10.18632/oncotarget.24797 ◽

2018 ◽

Vol 9 (26) ◽

pp. 18198-18213 ◽

Cited By ~ 8

Author(s):

Caroline V.M. Verhagen ◽

David M. Vossen ◽

Kerstin Borgmann ◽

Floor Hageman ◽

Reidar Grénman ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Dna Repair ◽

Homologous Recombination ◽

Cell Carcinoma ◽

Fanconi Anemia ◽

Gene Variants ◽

Advanced Head ◽

Functional Dna ◽

Recombination Gene

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Genetic variants in the p53 pathway influence implantation and pregnancy maintenance in IVF treatments using donor oocytes

Journal of Assisted Reproduction and Genetics ◽

10.1007/s10815-021-02324-9 ◽

2021 ◽

Author(s):

Arturo R. Palomares ◽

Adrián Alberto Castillo-Domínguez ◽

Maximiliano Ruiz-Galdón ◽

Kenny A. Rodriguez-Wallberg ◽

Armando Reyes-Engel

Keyword(s):

Pregnancy Rates ◽

Gene Variants ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Vitro Fertilization ◽

Genotype Frequencies ◽

Nested Case Control ◽

Donor Oocytes

Abstract Purpose Single-nucleotide polymorphisms (SNPs) in the p53 pathways have shown to play a role in endometrial receptivity and implantation in infertile women undergoing in vitro fertilization (IVF). The present study aimed to assess the influence of these gene variants over pregnancy success through a receptivity model in recipients of egg donation treatments, when factors such as age and quality of the oocytes are standardized. Methods A nested case–control study was performed on 234 female patients undergoing their first fresh IVF treatment as recipients of donor oocytes. Genotyping of TP53 Arg72Pro (rs1042522), LIF (rs929271), MDM4 (rs1563828), and USP7 (rs1529916) SNPs in the recipients allowed comparison of allele and genotype frequencies and their association with the IVF treatment outcome. Results Grouped by genotypes, patients showed differences in IVF outcomes after the embryo transfer. Arg72Pro (rs1042522) gene variant was associated to changes in implantation and clinical pregnancy rates. The polymorphisms USP7 (rs1529916) and MDM4 (rs1563828) were associated to differential ongoing pregnancy rates and variable miscarriage events, respectively. Conclusions This study highlights the association between gene polymorphisms related to P53 function and their influence over IVF reproductive outcomes. Arg72Pro variant may influence early events, as lower implantation rates were found in homozygous for Pro72 allele. By contrast, MDM4 (rs1563828) and USP7 (rs1529916) gene variants were associated with the later maintenance of pregnancy.

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Identification and In Vitro Functional Verification of Two Novel Mutations of GHR Gene in the Chinese Children with Laron Syndrome

Frontiers in Endocrinology ◽

10.3389/fendo.2021.605736 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ran Li ◽

Fengying Gong ◽

Hui Pan ◽

Hanting Liang ◽

Hui Miao ◽

...

Keyword(s):

Signal Transduction ◽

Subcellular Distribution ◽

Hepg2 Cells ◽

Functional Verification ◽

Gene Variants ◽

Laron Syndrome ◽

Novel Mutations ◽

Protein Levels ◽

Receptor Signal

PurposeLaron syndrome (LS) is a severe growth disorder caused by GHR gene mutation or post-receptor pathways defect. The clinical features of these patients collected in our present study were summarized, GHR gene variants were investigated and further in vitro functional verification was carried out.MethodsFour patients with LS were collected, their clinical characteristics were summarized, genomic DNA was extracted, and GHR gene was amplified and sequenced. GHR wild type (GHR-WT) and mutant GHR expression plasmids were constructed, and transiently transfected into HepG2 cells and HEK293T cells to observe the subcellular distribution of the GHR protein by immunofluorescence and to determine the expression of GHR and its post-receptor signaling pathway changes by Western blotting.ResultsAll of the four patients were male, and the median height was -4.72 SDS. Four GHR gene variants including c.587A>C (p.Y196S), c.766C>T (p.Q256*), c.808A>G (p.I270V) and c.1707-1710del (p.E570Afs*30) were identified, and the latter two were novel mutations. The results of mutant GHR plasmids transfection experiments and immunofluorescence assay showed that the subcellular distribution of GHR-Q256* and GHR-E570Afs*30 mutant proteins in HepG2 and HEK293T cells presented with a unique ring-like pattern, gathering around the nucleus, while GHR-Y196S mutant protein was evenly distributed on HepG2 cell membrane similar to GHR-WT. The GHR protein levels of HepG2 cells transiently transfected with GHR-Y196S, GHR-Q256* and GHR-E570Afs*30 were all significantly lower when compared with cells transfected with GHR-WT (P<0.05). Further mutant GHR post-receptor signal transduction investigation demonstrated that GH induced phosphorylated STAT5 levels of HepG2 cells transfected with three mutant plasmids were all significantly decreased in comparison with that of GHR-WT (P<0.05).ConclusionsTwo novel GHR gene mutations (I270V and E570Afs*30) were found in our patients with LS. GHR mutations influenced the subcellular distribution and GHR protein levels, then led to the impaired post-receptor signal transduction, suggesting that the GHR mutations contributed to the pathological condition of LS patients.

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Frequency of biotinidase gene variants and incidence of biotinidase deficiency in the Newborn Screening Program in Minas Gerais, Brazil

Journal of Medical Screening ◽

10.1177/0969141319892298 ◽

2019 ◽

Vol 27 (3) ◽

pp. 115-120

Author(s):

Nara de Oliveira Carvalho ◽

José Nélio Januário ◽

Gilsimary Lessa Pereira Felix ◽

Daniela Magalhães Nolasco ◽

Roberto Vagner Puglia Ladeira ◽

...

Keyword(s):

Newborn Screening ◽

Screening Program ◽

Critical Role ◽

Minas Gerais ◽

Incidence Rates ◽

Biotinidase Deficiency ◽

Gene Variants ◽

Newborn Screening Program ◽

Different Populations ◽

Biotinidase Activity

Objective The prevalence of biotinidase deficiency and the frequency of biotinidase gene variants in Brazil are not documented. We aimed to determine the incidence of partial and profound biotinidase deficiency in the state of Minas Gerais, Brazil, and to calculate the frequency of biotinidase gene variants in the newborn screening program of Minas Gerais. Methods Neonates (1,168,385) were screened from May 2013 to June 2018. Those detected with abnormal biotinidase activity based on semi-quantitative assays underwent confirmatory serum tests. The biotinidase gene was sequenced in all confirmed cases. Results The combined incidence of partial and profound biotinidase deficiency was estimated at 1:13,909 live births (95% confidence limit 1:11,235–1:17,217), much higher than the incidence rates reported in other populations worldwide. The most frequent biotinidase gene variants were p.D444H (allele frequency, 0.016), haplotype c.1330G>C;c.511G>A (p.D444H;A171T), p.D543E, c.310-15delT (intronic), p.V199M, and p.H485Q. Together these accounted for 74.6% of the alleles analysed. Conclusion Newborn screening for biotinidase deficiency, which revealed a higher incidence in Minas Gerais, is feasible and plays a critical role in the early identification of affected neonates and prevention of symptoms and irreversible sequelae. Biotinidase gene sequencing is a useful tool to confirm the diagnosis, and also provides valuable information about genetic variability among different populations.

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An 11-nt sequence polymorphism at the 3′UTR of human SFTPA1 and SFTPA2 gene variants differentially affect gene expression levels and miRNA regulation in cell culture

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00313.2013 ◽

2014 ◽

Vol 307 (1) ◽

pp. L106-L119 ◽

Cited By ~ 10

Author(s):

Patricia Silveyra ◽

Susan L. DiAngelo ◽

Joanna Floros

Keyword(s):

Gene Expression ◽

Chinese Hamster Ovary Cells ◽

Regulation Of Gene Expression ◽

Chinese Hamster ◽

Normal Lung ◽

Gene Variants ◽

Translation Efficiency ◽

Mrna Levels ◽

Mirna Regulation

Surfactant protein A (SP-A) plays a vital role in maintaining normal lung function and in host defense. Two genes encode SP-A in humans (SFTPA1, SFTPA2), and several gene variants have been identified for these. We have previously shown that sequence elements of SFTPA1 and SFTPA2 3′ untranslated regions (UTRs) differentially affect translation efficiency in vitro. Polymorphisms at the 3′UTRs of mRNA variants may account for differential binding of miRNAs, a class of small noncoding RNAs that regulate gene expression. In this work, we generated 3′UTR reporter constructs of the SFTPA1 and SFTPA2 variants most frequently found in the population, as well as mutants of a previously described 11-nt indel element (refSNP rs368700152). Reporter constructs were transfected in NCI-H441 cells in the presence or absence of miRNA mimics, and reporter gene expression was analyzed. We found that human miRNA mir-767 negatively affected expression of constructs containing SFTPA1 and SFTPA2 variants, whereas mir-4507 affected only constructs with 3′UTRs of SFTPA1 variants 6A, 6A3, and 6A4 (not containing the 11-nt element). Three miRNAs (mir-183, mir-449b, and mir-612) inhibited expression of recombinants of SFTPA2 variants and the SFTPA1 variant 6A2, all containing the 11-nt element. Similar results were obtained for SP-A expression when these miRNAs were transfected in Chinese hamster ovary cells expressing SFTPA1 or SFTPA2 variants or in NCI-H441 cells (genotype 1A5/1A5-6A4/6A4). Moreover, transfection with a specific antagomir (antagomir-183) reversed the effects of mir-183 on SP-A mRNA levels. Our results indicate that sequence variability at the 3′UTR of SP-A variants differentially affects miRNA regulation of gene expression.

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Functional variants in the sucrase–isomaltase gene associate with increased risk of irritable bowel syndrome

Gut ◽

10.1136/gutjnl-2016-312456 ◽

2016 ◽

Vol 67 (2) ◽

pp. 263-270 ◽

Cited By ~ 46

Author(s):

Maria Henström ◽

Lena Diekmann ◽

Ferdinando Bonfiglio ◽

Fatemeh Hadizadeh ◽

Eva-Maria Kuech ◽

...

Keyword(s):

Enzymatic Activity ◽

Population Based ◽

Stool Frequency ◽

Gene Variants ◽

Faecal Microbiota ◽

Familial Cases ◽

Functional Variants ◽

Allele Dosage ◽

Increased Risk

ObjectiveIBS is a common gut disorder of uncertain pathogenesis. Among other factors, genetics and certain foods are proposed to contribute. Congenital sucrase–isomaltase deficiency (CSID) is a rare genetic form of disaccharide malabsorption characterised by diarrhoea, abdominal pain and bloating, which are features common to IBS. We tested sucrase–isomaltase (SI) gene variants for their potential relevance in IBS.DesignWe sequenced SI exons in seven familial cases, and screened four CSID mutations (p.Val557Gly, p.Gly1073Asp, p.Arg1124Ter and p.Phe1745Cys) and a common SI coding polymorphism (p.Val15Phe) in a multicentre cohort of 1887 cases and controls. We studied the effect of the 15Val to 15Phe substitution on SI function in vitro. We analysed p.Val15Phe genotype in relation to IBS status, stool frequency and faecal microbiota composition in 250 individuals from the general population.ResultsCSID mutations were more common in patients than asymptomatic controls (p=0.074; OR=1.84) and Exome Aggregation Consortium reference sequenced individuals (p=0.020; OR=1.57). 15Phe was detected in 6/7 sequenced familial cases, and increased IBS risk in case–control and population-based cohorts, with best evidence for diarrhoea phenotypes (combined p=0.00012; OR=1.36). In the population-based sample, 15Phe allele dosage correlated with stool frequency (p=0.026) and Parabacteroides faecal microbiota abundance (p=0.0024). The SI protein with 15Phe exhibited 35% reduced enzymatic activity in vitro compared with 15Val (p<0.05).ConclusionsSI gene variants coding for disaccharidases with defective or reduced enzymatic activity predispose to IBS. This may help the identification of individuals at risk, and contribute to personalising treatment options in a subset of patients.

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