scholarly journals Growth Hormone (GH)-Releasing Hormone Increases the Expression of the Dominant-Negative GH Isoform in Cases of Isolated GH Deficiency due to GH Splice-Site Mutations

Endocrinology ◽  
2010 ◽  
Vol 151 (6) ◽  
pp. 2650-2658 ◽  
Author(s):  
Vibor Petkovic ◽  
Michela Godi ◽  
Didier Lochmatter ◽  
Andrée Eblé ◽  
Christa E. Flück ◽  
...  
Keyword(s):  
Splice Site ◽  
Gh Deficiency ◽  
Splice Site Mutation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Pituitary Cells ◽  
Site Mutation ◽  
Dominant Form ◽  
Negative Effect ◽  
Rat Pituitary Cells

An autosomal dominant form of isolated GH deficiency (IGHD II) can result from heterozygous splice site mutations that weaken recognition of exon 3 leading to aberrant splicing of GH-1 transcripts and production of a dominant-negative 17.5-kDa GH isoform. Previous studies suggested that the extent of missplicing varies with different mutations and the level of GH expression and/or secretion. To study this, wt-hGH and/or different hGH-splice site mutants (GH-IVS+2, GH-IVS+6, GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GC-GHRHR). Upon GHRH stimulation, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed reduced hGH secretion and intracellular GH content when compared with cells expressing only wt-hGH, confirming the dominant-negative effect of 17.5-kDa isoform on the secretion of 22-kDa GH. Furthermore, increased amount of 17.5-kDa isoform produced after GHRH stimulation in cells expressing GH-splice site mutants reduced production of endogenous rat GH, which was not observed after GHRH-induced increase in wt-hGH. In conclusion, our results support the hypothesis that after GHRH stimulation, the severity of IGHD II depends on the position of splice site mutation leading to the production of increasing amounts of 17.5-kDa protein, which reduces the storage and secretion of wt-GH in the most severely affected cases. Due to the absence of GH and IGF-I-negative feedback in IGHD II, a chronic up-regulation of GHRH would lead to an increased stimulatory drive to somatotrophs to produce more 17.5-kDa GH from the severest mutant alleles, thereby accelerating autodestruction of somatotrophs in a vicious cycle.

Decreased Expression In NF-κB Essential Modulator Due to a Novel Splice-Site Mutation Causes Ectodermal Dysplasia with Immunodeficiency

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1732-1732
Author(s):  
Shuhei Karakawa ◽  
Satoshi Okada ◽  
Miyuki Tsumura ◽  
Yoko Mizoguchi ◽  
Norioki Ohno ◽  
...  
Keyword(s):  
Splice Site ◽  
Transcriptional Activity ◽  
Ectodermal Dysplasia ◽  
Splice Site Mutation ◽  
The Other ◽  
Dominant Negative Effect ◽  
Site Mutation ◽  
Streptococcus Pneumonia ◽  
Cell Counts ◽  
Ifn Γ

Abstract Abstract 1732 <Introduction> X-linked ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the nuclear factor κB (NF-κB) essential modulator (NEMO) gene, also called IKBKG, and these mutations impair but do not abolish NF-κB signaling, resulting in distinct clinical and immunologic phenotypes. We presented the patient with XL-EDA-ID showing a novel splice-site mutation. In this report we precisely study the involvement of this mutation in the molecular pathogenesis of XL-EDA-ID. <Patient> The patient is 12 years old Japanese boy. He had conical-shaped teeth and hypodontia. He had suffered from recurrent bacterial infections and these pathogenic bacteria were mostly streptococcus pneumonia. The laboratory examination revealed that the number of white blood cell counts, the classification of lymphocytes, and the serum immunoglobulin levels were within normal range. However the specific antibodies against measles and streptococcus pneumonia were negative in spite of these infections. <Results> We identified a novel hemizygous mutation, 769-1 G>C, at the splicing acceptor site of exon 7 in the IKBKG gene. In order to clarify the effect of this mutation on mRNA, we performed a cloning analysis. Although various abnormal spliced NEMO (mutant NEMO) mRNAs were observed, a small amount of wild-type NEMO (WT NEMO) mRNA was also identified. The rate of WT and mutant was variable on the time of blood collection. Further we performed FACS and immunoblot analysis in order to evaluate the effect of this mutation at protein level. Two major bands which were presumed to be derived from WT and one mutant were detected in immunoblots using EBV transfected B cells, however the expression levels of these bands were markedly decreased compared to those of the healthy controls. The NEMO protein expression was confirmed to be decreased in various lineages of leukocytes. We generated WT and two representative mutant NEMO constructs and measured their NF-κB transcriptional activity using reporter assay. One mutant NEMO abolished NF-κB transcriptional activity, the other showed weak activity. However, neither of mutant NENO showed a dominant-negative effect against WT NEMO activity. CD14+ cells from the patient produced a lower level of TNF-α in response to IFN-γ stimulation, on the other hand CD3+ cells produced very little IFN-γ in response to IL-12. CD4+ T-cell proliferation was impaired in response to measles and mumps, but not rubella. <Conclusion> The hemizygous 769-1 G>C mutation was shown to cause the decreased expression of WT NEMO protein, resulting in the decrease in NF-κB activation and the development of XL-EDA-ID. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Novel GALNT3 Mutation in a Pseudoautosomal Dominant Form of Tumoral Calcinosis: Evidence That the Disorder Is Autosomal Recessive

2005 ◽  
Vol 90 (4) ◽  
pp. 2420-2423 ◽  
Author(s):  
Shoji Ichikawa ◽  
Kenneth W. Lyles ◽  
Michael J. Econs
Keyword(s):  
Splice Site ◽  
Autosomal Recessive ◽  
Splice Site Mutation ◽  
Tumoral Calcinosis ◽  
Proper Function ◽  
Single Mutation ◽  
Recessive Trait ◽  
Site Mutation ◽  
Dominant Form ◽  
Biallelic Mutations

Abstract Familial tumoral calcinosis is a rare metabolic disorder, characterized by ectopic calcification and hyperphosphatemia. Recently biallelic mutations in the GalNAc transferase 3 (GALNT3) gene were identified in two families with tumoral calcinosis. In the present study, we performed mutation analysis of the GALNT3 gene in a multigenerational family, which was originally described to have an autosomal dominant form of tumoral calcinosis. We identified a novel splice site mutation in intron 1 (IVS1–2a→t), likely leading to skipping of exon 2. The proband was a compound heterozygote for the splice site mutation and the previously reported nonsense mutation (484C→T; R162X). His affected maternal great uncle was homozygous for the splice site mutation. Biallelic mutations found in two generations demonstrated that the family had pseudoautosomal dominant inheritance, confirming that tumoral calcinosis is in fact an autosomal recessive trait. However, genetic and biochemical findings suggest that carriers of a single mutation may also manifest subtle biochemical abnormalities. Furthermore, coexpression of GALNT3 and fibroblast growth factor 23 (FGF23), a key regulator of phosphate homeostasis, in certain tissues suggests that O-glycosylation of FGF23 by GALNT3 may be necessary for proper function of FGF23.

scholarly journals Multiple splicing defects caused by hERG splice site mutation 2592+1G>A associated with long QT syndrome

2011 ◽  
Vol 300 (1) ◽  
pp. H312-H318 ◽  
Author(s):  
Matthew R. Stump ◽  
Qiuming Gong ◽  
Zhengfeng Zhou
Keyword(s):  
Splice Site ◽  
Long Qt Syndrome ◽  
Splice Site Mutation ◽  
Dominant Negative ◽  
Wild Type ◽  
Site Mutation ◽  
Long Qt ◽  
Herg Current ◽  
Qt Syndrome ◽  
Splicing Defects

Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene ( hERG). Cryptic splice site activation in hERG has recently been identified as a novel pathogenic mechanism of LQT2. In this report, we characterize a hERG splice site mutation, 2592+1G>A, which occurs at the 5′ splice site of intron 10. Reverse transcription-PCR analyses using hERG minigenes transfected into human embryonic kidney-293 cells and HL-1 cardiomyocytes revealed that the 2592+1G>A mutation disrupted normal splicing and caused multiple splicing defects: the activation of cryptic splice sites within exon 10 and intron 10 and complete intron 10 retention. We performed functional and biochemical analyses of the major splice product, hERGΔ24, in which 24 amino acids within the cyclic nucleotide binding domain of the hERG channel COOH-terminus is deleted. Patch-clamp experiments revealed that the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that mutant channels did not traffic to the cell surface. Coexpression of wild-type hERG and hERGΔ24 resulted in significant dominant-negative suppression of hERG current via the intracellular retention of the wild-type channels. Our results demonstrate that 2592+1G>A causes multiple splicing defects, consistent with the pathogenic mechanisms of long QT syndrome.

Emery-Dreifuss Muscular Dystrophy Type 1 in a 15-Year-Old Patient Due to a Novel Putative Splice-Site Mutation

2017 ◽  
Vol 48 (S 01) ◽  
pp. S1-S45
Author(s):  
O. Schwartz ◽  
J. Althaus ◽  
B. Fiedler ◽  
K. Heß ◽  
W. Paulus ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Splice Site ◽  
Splice Site Mutation ◽  
Site Mutation

A rare cause of fever of unknown origin: hypohidrotic ectodermal dysplasia with a splice site mutation

2018 ◽  
Vol 70 (5) ◽  
Author(s):  
Melahat M. Oguz ◽  
Meltem Akcaboy ◽  
Asuman Gurkan ◽  
Esma Altinel Acoglu ◽  
Pelin Zorlu ◽  
...  
Keyword(s):  
Splice Site ◽  
Fever Of Unknown Origin ◽  
Ectodermal Dysplasia ◽  
Splice Site Mutation ◽  
Unknown Origin ◽  
Hypohidrotic Ectodermal Dysplasia ◽  
Site Mutation

scholarly journals A Dominant Negative Effect of eth-1r, a Mutant Allele of the Neurospora crassa S-Adenosylmethionine Synthetase-Encoding Gene Conferring Resistance to the Methionine Toxic Analogue Ethionine

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1455-1462
Author(s):  
José L Barra ◽  
Mario R Mautino ◽  
Alberto L Rosa
Keyword(s):  
Neurospora Crassa ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Resistant Phenotype ◽  
Negative Effect ◽  
Encoding Gene ◽  
Adenosylmethionine Synthetase ◽  
Adomet Synthetase ◽  
Identical Gene

eth-1r a thermosensitive allele of the Neurospora crassa S-adenosylmethionine (AdoMet) synthetase gene that confers ethionine resistance, has been cloned and sequenced. Replacement of an aspartic amino acid residue (D48 → N48), perfectly conserved in prokaryotic, fungal and higher eukaryotic AdoMet synthetases, was found responsible for both thermosensitivity and ethionine resistance conferred by eth-1r. Gene fusion constructs, designed to overexpress eth-1r in vivo, render transformant cells resistant to ethionine. Dominance of ethionine resistance was further demonstrated in eth-1  +/eth-1r partial diploids carrying identical gene doses of both alleles. Heterozygous eth-1  +/eth-1r cells have, at the same time, both the thermotolerance conferred by eth-1  + and the ethionine-resistant phenotype conferred by eth-1r. AdoMet levels and AdoMet synthetase activities were dramatically decreased in heterozygous eth-1  +/eth-1r cells. We propose that this negative effect exerted by eth-1r results from the in vivo formation of heteromeric eth-1  +/eth-1r AdoMet synthetase molecules.

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