scholarly journals Three Mutations (p.Q36H, p.G418fsX482, and g.IVS19-2A>C) in the Dual Oxidase 2 Gene Responsible for Congenital Goiter and Iodide Organification Defect

2006 ◽  
Vol 52 (2) ◽  
pp. 182-191 ◽  
Author(s):  
Viviana Varela ◽  
Carina M Rivolta ◽  
Sebastián A Esperante ◽  
Laura Gruñeiro-Papendieck ◽  
Ana Chiesa ◽  
...  
Keyword(s):  
In Vitro Transcription ◽  
Sscp Analysis ◽  
Compound Heterozygous ◽  
Single Strand Conformational Polymorphism ◽  
Transcription Analysis ◽  
Unknown Compound ◽  
Dual Oxidase ◽  
Compound Heterozygous Mutations ◽  
Organification Defect

Abstract Background: Iodide organification defects are associated with mutations in the dual oxidase 2 (DUOX2) gene and are characterized by a positive perchlorate discharge test. These mutations produce a congenital goitrous hypothyroidism, usually transmitted in an autosomal recessive mode. Methods: We studied the complete coding sequence of the human DUOX2 gene by single-strand conformational polymorphism (SSCP) analysis of DNA from 17 unrelated patients with iodide organification defects. Samples showing an aberrant pattern were directly sequenced. All mutations were validated by SSCP analysis. Finally, the effect of a splicing mutation was studied by construction of minigenes. Results: Genomic DNA sequencing revealed 3 novel mutations [c.108G>C (p.Q36H), c.1253delG (p.G418fsX482), and g.IVS19-2A>C] and 1 previously reported mutation [c.2895-2898delGTTC (p.S965fsX994)] in 2 families with 1 (family 1) and 2 (family 2) affected members. This implies the inheritance of 2 compound heterozygous mutations, p.Q36H and p.S965fsX994 in family 1 and p.G418fsX482 and g.IVS19-2A>C in family 2. The c.1253delG mutation was associated with a c.1254C>A transversion. In vitro transcription analysis showed that exon 20 is skipped entirely when the g.IVS19-2A>C mutation is present. The wild-type glutamine residue at position 36 is strictly conserved. Conclusions: Two previously unknown compound heterozygous mutations in the DUOX2 gene, p.Q36H/p.S965fsX994 and p.G418fsX482/g.IVS19-2A>C, are responsible for iodide organification defects in 2 unrelated families. Identification of the molecular basis of this disorder might be helpful for understanding the pathophysiology of this congenital hypothyroidism.

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

scholarly journals Goitrous Congenital Hypothyroidism and Hearing Impairment Associated with Mutations in the TPO and SLC26A4/PDS Genes

2006 ◽  
Vol 91 (7) ◽  
pp. 2678-2681 ◽  
Author(s):  
Nicole Pfarr ◽  
Guntram Borck ◽  
Andrew Turk ◽  
Ulrike Napiontek ◽  
Annerose Keilmann ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Hearing Impairment ◽  
Congenital Hypothyroidism ◽  
Sensorineural Deafness ◽  
Primary Hypothyroidism ◽  
Thyroid Peroxidase ◽  
Compound Heterozygous ◽  
Sequence Variant ◽  
Organification Defect

Abstract Context: Pendred syndrome (PS) and thyroid peroxidase (TPO) deficiency are autosomal-recessive disorders that result in thyroid dyshormonogenesis. They share congenital hypothyroidism, goiter, and an iodide organification defect as common features. Whereas the hallmark of PS is sensorineural deafness, other forms of congenital hypothyroidism may also lead to hearing impairment. Therefore, a definite diagnosis may be difficult and require molecular genetic analyses. Case Report: The propositus presented at birth with primary hypothyroidism and goiter. He also had congenital bilateral moderate hearing loss, and PS was suspected. Methods: We sequenced the SLC26A4/PDS and TPO genes in the propositus and tested familial segregation of mutations in all available family members who were phenotypically normal. The functional consequences of the identified pendrin mutation (p.R776C) were studied in vitro. Results: Sequencing of the SLC26A4/PDS gene revealed a single monoallelic missense mutation in the propositus (p.R776C). This mutation, which was inherited from his unaffected mother, has previously been identified in an individual with deafness and an enlarged vestibular aqueduct. Sequencing of the TPO gene revealed compound heterozygosity for a novel nonsense mutation (p.Q235X) and a known missense mutation (p.Y453D). The mutant pendrin (p.R776C) retained its ability to transport iodide in vitro. Conclusions: These results show that the propositus carries three sequence variants in two genes: a monoallelic SLC26A4/PDS sequence variant and compound heterozygous TPO mutations. Our study illustrates that if only a single heterozygous SLC26A4/PDS mutation is found in a patient with goiter and deafness, other genetic explanations should be considered.

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 (

scholarly journals Biallelic Inactivation of the Dual Oxidase Maturation Factor 2 (DUOXA2) Gene as a Novel Cause of Congenital Hypothyroidism

2008 ◽  
Vol 93 (2) ◽  
pp. 605-610 ◽  
Author(s):  
Ilaria Zamproni ◽  
Helmut Grasberger ◽  
Francesca Cortinovis ◽  
Maria Cristina Vigone ◽  
Giuseppe Chiumello ◽  
...  
Keyword(s):  
Congenital Hypothyroidism ◽  
Critical Role ◽  
Chinese Patient ◽  
Catalytic Core ◽  
Genetic Event ◽  
Hormone Synthesis ◽  
Maturation Factor ◽  
Dual Oxidase ◽  
Organification Defect

Abstract Context: Dual oxidase 2 (DUOX2) is the catalytic core of the H2O2 generator crucial for the iodination of thyroglobulin in thyroid hormone synthesis. DUOX2 deficiency produces congenital hypothyroidism (CH) in humans and mice. We recently cloned a novel gene, the product of which (dual oxidase maturation factor 2; DUOXA2) is required to express DUOX2 enzymatic activity. Objective: Our objective was to identify DUOXA2 mutations as a novel cause of CH due to dyshormonogenesis. Patients: Subjects included 11 CH patients with partial iodine organification defect but negative for other known genetic causes of partial iodine organification defect. Results: One Chinese patient born to nonconsanguineous parents was homozygous for a nonsense mutation (p.Y246X), producing a truncated DUOXA2 protein lacking transmembrane helix 5 and the C-terminal cytoplasmic domain. The mutant protein was inactive in reconstituting DUOX2 in vitro. Pedigree analysis demonstrated recessive inheritance, because heterozygous carriers had normal thyroid function including negative results in neonatal TSH screening. One heterozygous carrier of Y246X was identified in unrelated Chinese controls (n = 92) but not in Caucasian or Japanese controls, indicating that homozygosity for Y246X could be a frequent cause of CH in Chinese. Functional studies suggest that the DUOXA2 paralog (DUOXA1) can partially compensate DUOXA2 deficiency, consistent with the proband having a milder CH phenotype than patients with biallelic DUOX2 nonsense mutations. Conclusions: We report the first mutation in DUOXA2, identified in a patient with CH and dyshormonogenic goiter. Results of our studies provide evidence for the critical role of DUOXA2 in thyroid hormonogenesis. Biallelic DUOXA2 mutations are a novel genetic event in permanent CH.

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 Genome-wide effects onEscherichia colitranscription from ppGpp binding to its two sites on RNA polymerase

2019 ◽  
Vol 116 (17) ◽  
pp. 8310-8319 ◽  
Author(s):  
Patricia Sanchez-Vazquez ◽  
Colin N. Dewey ◽  
Nicole Kitten ◽  
Wilma Ross ◽  
Richard L. Gourse
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Binding Sites ◽  
Promoter Sequence ◽  
In Vitro Transcription ◽  
Transcriptional Responses ◽  
Transcription Analysis ◽  
Genome Wide

The second messenger nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability. ppGpp binds to two sites on RNA polymerase (RNAP) inEscherichia coli, but it has also been reported to bind to many other proteins. To determine the role of the RNAP binding sites in the genome-wide effects of ppGpp on transcription, we used RNA-seq to analyze transcripts produced in response to elevated ppGpp levels in strains with/without the ppGpp binding sites on RNAP. We examined RNAs rapidly after ppGpp production without an accompanying nutrient starvation. This procedure enriched for direct effects of ppGpp on RNAP rather than for indirect effects on transcription resulting from starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of ppGpp induction depended on ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp.

In vitro transcription analysis of DNA alkylation by nitrogen mustard

Biochemistry ◽  
1991 ◽  
Vol 30 (32) ◽  
pp. 8036-8040 ◽  
Author(s):  
Peter J. Gray ◽  
Carleen Cullinane ◽  
Don R. Phillips
Keyword(s):  
Nitrogen Mustard ◽  
In Vitro Transcription ◽  
Dna Alkylation ◽  
Transcription Analysis

In vitro transcription analysis of DNA adducts induced by cyanomorpholinoadriamycin

Biochemistry ◽  
1992 ◽  
Vol 31 (40) ◽  
pp. 9513-9519 ◽  
Author(s):  
Carleen Cullinane ◽  
Don R. Phillips
Keyword(s):  
Dna Adducts ◽  
In Vitro Transcription ◽  
Transcription Analysis

scholarly journals Roles of SigB and SigF in the Mycobacterium tuberculosis Sigma Factor Network

2007 ◽  
Vol 190 (2) ◽  
pp. 699-707 ◽  
Author(s):  
Jong-Hee Lee ◽  
Petros C. Karakousis ◽  
William R. Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Ribosomal Proteins ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription Analysis ◽  
Recombinant Strains ◽  
Regulatory Cascade

ABSTRACTTo characterize the roles of SigB and SigF in sigma factor regulation inMycobacterium tuberculosis, we used chemically inducible recombinant strains to conditionally overexpresssigBandsigF.Using whole genomic microarray analysis and quantitative reverse transcription-PCR, we investigated the resulting global transcriptional changes aftersigBinduction, and we specifically tested the relative expression of other sigma factor genes after knock-in expression ofsigBandsigF. Overexpression ofsigBresulted in significant upregulation of genes encoding several early culture filtrate antigens (ESAT-6-like proteins), ribosomal proteins, PE-PGRS proteins, the keto-acyl synthase, KasA, and the regulatory proteins WhiB2 and IdeR. Of note, the induction ofsigBdid not alter the expression of other sigma factor genes, indicating that SigB is likely to serve as an end regulator for at least one branch of theM. tuberculosissigma factor regulatory cascade. Analysis of the 5′-untranslated region (UTR) of SigB-dependent transcripts revealed a putative consensus sequence of NGTGG-N14-18-NNGNNG. This sequence appeared upstream of bothsigB(Rv2710) and the gene following it,ideR(Rv2711), and in vitro transcription analysis with recombinant SigB-reconstituted RNA polymerase confirmed SigB-dependent transcription from each of these promoters. Knock-in expression ofsigFrevealed that only thesigCgene was significantly upregulated 6 and 12 h aftersigFinduction. The previously identified SigF promoter consensus sequence AGTTTG-N15-GGGTTT was identified in the 5′ UTR of thesigCgene, and SigF-dependent in vitro transcription of the promoter upstream ofsigCwas confirmed by using recombinant SigF-reconstituted RNA polymerase. These two knock-in recombinant strains were tested in a macrophage model of infection which showed that overexpression ofsigBandsigFresulted in reduced rates ofM. tuberculosisintracellular growth. These results define the SigB promoter consensus recognition sequence and members of the SigB regulon. Moreover, the data suggest that, in addition to serving as an end regulator in a sigma factor cascade, SigB may auto-amplify its own expression under certain conditions.

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