scholarly journals Childhood Hypophosphatasia Due to a de Novo Missense Mutation in the Tissue-Nonspecific Alkaline Phosphatase Gene

2005 ◽  
Vol 90 (4) ◽  
pp. 2436-2439 ◽  
Author(s):  
A. Taillandier ◽  
S.-L. Sallinen ◽  
I. Brun-Heath ◽  
P. De Mazancourt ◽  
J.-L. Serre ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
De Novo ◽  
Serum Alkaline Phosphatase ◽  
Bone Alkaline Phosphatase ◽  
Site Directed Mutagenesis ◽  
De Novo Mutation ◽  
Missense Mutations ◽  
Anterior Fontanel ◽  
Skeletal Changes

Abstract Hypophosphatasia is an inherited disorder due to mutations in the bone alkaline phosphatase (ALPL) gene. We report here a patient with childhood hypophosphatasia diagnosed at 1.4 yr because of pectus excavatum, large anterior fontanel, rachitic skeletal changes, and low serum alkaline phosphatase. Sequencing of the ALPL gene produced evidence of two distinct missense mutations, E174K (c.571G>A), of maternal origin, and a de novo mutation, M45I (c.186G>C). The study of various microsatellite polymorphisms ruled out false paternity and therefore confirmed that M45I occurred de novo in the paternal germline or in the early development of the patient. Site-directed mutagenesis showed that M45I results in the absence of in vitro alkaline phosphatase activity, suggesting that the mutation is a severe allele. In conclusion, childhood hypophosphatasia in this patient is the result of compound heterozygosity for the moderate mutation E174K and a novel severe de novo mutation M45I.

scholarly journals X-linked hypophosphatemic rickets: Case report

2014 ◽  
Vol 142 (1-2) ◽  
pp. 75-78 ◽  
Author(s):  
Vladimir Radlovic ◽  
Zeljko Smoljanic ◽  
Nedeljko Radlovic ◽  
Zoran Lekovic ◽  
Dragana Ristic ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
De Novo ◽  
Serum Levels ◽  
High Serum ◽  
Hypophosphatemic Rickets ◽  
De Novo Mutation ◽  
Inherited Disease ◽  
X Ray ◽  
Phex Gene ◽  
Renal Tubular

Introduction. X-linked hypophosphatemic rickets (XLHR) is a dominant inherited disease caused by isolated renal phosphate wasting and impairment of vitamin D activation. We present a girl with X-linked hypophosphatemic rickets (XLHR) as a consequence of de novo mutation in the PHEX gene. Case Outline. A 2.2-year-old girl presented with prominent lower limb rachitic deformity, waddling gait and disproportionate short stature (79 cm, <P5; -1,85 SD). On the basis of hypophosphatemia, hyperphosphaturia, high serum level of alkaline phosphatase, normal calcemia, 25(OH)D and PTH, as well as characteristic clinical and X-ray findings, diagnosis of hypophosphatemic rickets (HR) was made. Normal calciuria and absence of other renal tubular disorders indicated HR as a consequence of isolated hyperphosphaturia. The treatment (phosphate 55 mg/kg and calcitriol 35 ng/kg per day), introduced 15 month ago, resulted in a stable normalization of alkaline phosphatase and phosphorus serum levels (with intact calcemia and calciuria), disappearance of X-ray signs of the active rickets and improvement of the child?s longitudinal growth (0.6 cm per month). Subsequently, by detection of already known mutation in the PHEX gene: c.1735G>A (p.G579R) (exon 17), XLHR was diagnosed. Analysis of the parental PHEX gene did not show the abnormality, which indicated that the child?s XLHR was caused by de novo mutation of this gene. Conclusion. Identification of genetic defects is exceptionally significant for diagnosis and differential diagnosis of hereditary HR.

scholarly journals Translationskopplung via Termination-Reinitiation in Archaea und Bacteria

2021 ◽  
Author(s):  
◽  
Madeleine Huber
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Stop Codon ◽  
Haloferax Volcanii ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Directed Mutagenesis ◽  
Ribosome Display ◽  
E Coli

Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt. Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.

scholarly journals In vivo, in vitro and in silico correlations of four de novo SCN1A missense mutations

PLoS ONE ◽  
2019 ◽  
Vol 14 (2) ◽  
pp. e0211901 ◽  
Author(s):  
Andreea Nissenkorn ◽  
Yael Almog ◽  
Inbar Adler ◽  
Mary Safrin ◽  
Marina Brusel ◽  
...  
Keyword(s):  
In Silico ◽  
De Novo ◽  
Missense Mutations

scholarly journals Case Report: A Novel de novo Mutation in DNM1L Presenting With Developmental Delay, Ataxia, and Peripheral Neuropathy

2021 ◽  
Vol 9 ◽  
Author(s):  
Yanping Wei ◽  
Min Qian
Keyword(s):  
Peripheral Neuropathy ◽  
Developmental Delay ◽  
De Novo ◽  
Refractory Epilepsy ◽  
De Novo Mutation ◽  
Global Developmental Delay ◽  
Missense Mutations ◽  
Related Protein ◽  
Resonance Imaging ◽  
Genetic Results

DNM1L encodes dynamin-related protein 1 (Drp1), which is a member of the dynamin superfamily of GTPases and mediates mitochondrial and peroxisomal fission. In humans, several de novo heterozygous missense mutations in DNM1L have been reported, which were characterized by devastating courses with refractory epilepsy, myoclonus, and brain atrophy on MRI. We describe a 4.5-year-old male child harboring a novel de novo mutation in DNM1L presenting a phenotype of developmental delay, ataxia, and peripheral neuropathy. The clinical features, magnetic resonance imaging findings, and genetic results were summarized. Meanwhile, all the cases of DNM1L mutations reported were reviewed. DNM1L variants may need to be considered in phenotypes that include global developmental delay, peripheral neuropathy, and ataxia.

scholarly journals Identification of three candidate mutations causing type IIA von Willebrand disease using a rapid, nonradioactive, allele-specific hybridization method

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 830-836 ◽  
Author(s):  
A Inbal ◽  
T Englender ◽  
N Kornbrot ◽  
AM Randi ◽  
G Castaman ◽  
...  
Keyword(s):  
De Novo ◽  
Screening Method ◽  
Von Willebrand Disease ◽  
Rapid Screening ◽  
De Novo Mutation ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Hybridization Method ◽  
Allele Specific ◽  
Von Willebrand

Type IIA von Willebrand disease (vWD), the most common type II vWD variant, is characterized by decreased binding of von Willebrand factor (vWF) to platelet glycoprotein Ib (Gplb) and by a decrease in large and intermediate vWF multimers. Mutations reported to cause vWD type IIA are clustered within the A2 domain of vWF, which is encoded by exon 28. Genomic DNA from affected members of 12 unrelated families with type IIA vWD were screened for these mutations by a rapid, nonradioactive, allele-specific oligonucleotide (ASO) hybridization method. Oligonucleotides containing each of eight mutations were cross-linked onto a nylon membrane by UV irradiation. A fragment of vWF exon 28 was amplified from peripheral blood leukocyte DNA using biotinylated primers and hybridized to the immobilized oligonucleotides. Positive signals were detected with an avidin-alkaline phosphatase conjugate and chemiluminescent substrate. Thus, in a single hybridization reaction, a patient sample could be analyzed for a large number of mutations simultaneously. Polymerase chain reaction (PCR) products from four patients did not contain any of the tested mutations and therefore were sequenced. Three additional candidate missense mutations, two of them novel, were identified: Arg(834)-->Gln in one patient, Gly(846)-->Arg in one patient, and Val(902)-->Glu in three ostensibly unrelated patients. By ASO hybridization, the mutations were confirmed in the affected patients and excluded in unaffected relatives and 50 normal controls. In one family, the Val(902)-->Glu mutation was shown to be a de novo mutation. This rapid screening method is applicable to other subtypes of vWD for which mutations have been identified.

scholarly journals Leukoencephalopathia, demyelinating peripheral neuropathy and dural ectasia explained by a not formerly described de novo mutation in the SAMD9L gene, ends 27 years of investigations – A case report

2019 ◽  
Author(s):  
Sofia Thunström ◽  
Markus Axelsson
Keyword(s):  
Case Report ◽  
Peripheral Neuropathy ◽  
De Novo ◽  
Age Of Onset ◽  
De Novo Mutation ◽  
Missense Mutations ◽  
Dural Ectasia ◽  
Whole Exome ◽  
Demyelinating Peripheral Neuropathy ◽  
Multiple Cysts

Abstract Background: Missense mutations in SAMD9L gene is associated with ataxia-pancytopenia syndrome (ATXPC), OMIM#159550. Common clinical features in these patients include neurological and hematological symptoms. The phenotype and age of onset is variable. Case Presentation: In this case report whole exome sequencing (WES) revealed a not previously reported de novo variant c.2686T>G, p.(Phe896Val) in SAMD9L in a patient with widespread findings of slow developing pathology in the peripheral and central nervous system. The clinical picture was dominated by neurological symptoms, unlike previously described cases, and in addition dural ectasias and multiple cysts in the brain was observed using magnetic resonance imaging. Conclusions: This case underscores the effect of variable expressivity, i.e. different mutations in the same gene can cause different phenotypes. Keywords: Leukoencephalopathia, demyelinating peripheral neuropathy, dural ectasia explained, de novo mutation, the SAMD9L gene.

scholarly journals Lowe syndrome identified in the offspring of an oocyte donor who was an unknown carrier of a de novo mutation: a case report and review of the literature

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
P. Tatsi ◽  
G. E. Papanikolaou ◽  
T. Chartomatsidou ◽  
I. Papoulidis ◽  
A. Athanasiadis ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Ethical Issues ◽  
De Novo ◽  
Dent Disease ◽  
De Novo Mutation ◽  
Lowe Syndrome ◽  
Genetic Constitution ◽  
Preimplantation Genetic Testing ◽  
Oculocerebrorenal Syndrome

Abstract Background Oculocerebrorenal syndrome of Lowe is an X-linked disorder with very low prevalence in the general population. The OCRL gene encodes the protein phosphatidylinositol 4,5-bisphosphate-5-phosphatase, a lipid phosphatase, located in the trans-Golgi network. Point mutations in the OCRL gene cause Lowe syndrome and Dent disease, which are characterized as a multisystemic disorder. The symptoms of Lowe syndrome are expressed primarily as dysfunction of the eyes, kidneys, and the central nervous system. Case presentation This report describes a case of a 31-year-old Georgian woman with a de novo pathogenic mutation causing oculocerebrorenal syndrome of Lowe, who was a volunteer in an oocyte donation program for in vitro fertilization purposes, and the outcome of the treatments of this particular donor’s oocyte receivers, describing the implications of the mutation for the children born as a result of the treatments. It raises important medical and ethical issues about the necessity of genetic testing of oocyte donors and the possibility of rare genetic disorders being inherited by the offspring of donors. Conclusion This particular case indicates the legal, medical, and emotional risks of utilizing donor oocytes from phenotypically healthy women, whose genetic constitution is unknown in terms of being silent carriers of rare diseases. In addition, all the necessary actions were followed; the further examinations that are required are mentioned. The donor and the offspring should be further tested. The remaining cryopreserved embryos should be destroyed or preimplantation genetic testing should be performed before they are utilized. Finally, all the people involved, the treated couples and the donor, alongside her family, should follow genetic and psychological counselling.

scholarly journals An Unusual Route for p-Aminobenzoate Biosynthesis in Chlamydia trachomatis Involves a Probable Self-Sacrificing Diiron Oxygenase

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Yamilet Macias-Orihuela ◽  
Thomas Cast ◽  
Ian Crawford ◽  
Kevin J. Brandecker ◽  
Jennifer J. Thiaville ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Active Site ◽  
De Novo ◽  
Sequence Similarity ◽  
Single Gene ◽  
The United States ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
Active Site Tyrosine

ABSTRACT Chlamydia trachomatis lacks the canonical genes required for the biosynthesis of p-aminobenzoate (pABA), a component of essential folate cofactors. Previous studies revealed a single gene from C. trachomatis, the CT610 gene, that rescues Escherichia coli ΔpabA, ΔpabB, and ΔpabC mutants, which are otherwise auxotrophic for pABA. CT610 shares low sequence similarity to nonheme diiron oxygenases, and the previously solved crystal structure revealed a diiron active site. Genetic studies ruled out several potential substrates for CT610-dependent pABA biosynthesis, including chorismate and other shikimate pathway intermediates, leaving the actual precursor(s) unknown. Here, we supplied isotopically labeled potential precursors to E. coli ΔpabA cells expressing CT610 and found that the aromatic portion of tyrosine was highly incorporated into pABA, indicating that tyrosine is a precursor for CT610-dependent pABA biosynthesis. Additionally, in vitro enzymatic experiments revealed that purified CT610 exhibits low pABA synthesis activity under aerobic conditions in the absence of tyrosine or other potential substrates, where only the addition of a reducing agent such as dithiothreitol appears to stimulate pABA production. Furthermore, site-directed mutagenesis studies revealed that two conserved active site tyrosine residues are essential for the pABA synthesis reaction in vitro. Thus, the current data are most consistent with CT610 being a unique self-sacrificing enzyme that utilizes its own active site tyrosine residue(s) for pABA biosynthesis in a reaction that requires O2 and a reduced diiron cofactor. IMPORTANCE Chlamydia trachomatis is the most reported sexually transmitted infection in the United States and the leading cause of infectious blindness worldwide. Unlike many other intracellular pathogens that have undergone reductive evolution, C. trachomatis is capable of de novo biosynthesis of the essential cofactor tetrahydrofolate using a noncanonical pathway. Here, we identify the biosynthetic precursor to the p-aminobenzoate (pABA) portion of folate in a process that requires the CT610 enzyme from C. trachomatis. We further provide evidence that CT610 is a self-sacrificing or “suicide” enzyme that uses its own amino acid residue(s) as the substrate for pABA synthesis. This work provides the foundation for future investigation of this chlamydial pABA synthase, which could lead to new therapeutic strategies for C. trachomatis infections.

scholarly journals Synthesis and secretion of alkaline phosphatase in vitro from first-trimester and term human placentas

1981 ◽  
Vol 194 (3) ◽  
pp. 857-866 ◽  
Author(s):  
H Galski ◽  
S E Fridovich ◽  
D Weinstein ◽  
N De Groot ◽  
S Segal ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Time Course ◽  
De Novo ◽  
Placental Tissue ◽  
First Trimester ◽  
Alkaline Phosphatases ◽  
Heat Stable ◽  
Alkaline Phosphatase Isoenzymes ◽  
Specific Activities

The synthesis and secretion of alkaline phosphatases in vitro by human placental tissue incubated in organ culture were studied. First-trimester placenta synthesizes and secretes two different alkaline phosphatase isoenzymes (heat-labile and heat-stable), whereas in term placenta nearly all the alkaline phosphatase synthesized and secreted is heat-stable. The specific activities of alkaline phosphatases in first-trimester and term placental tissue remain constant throughout the time course of incubation. In the media, specific activities increase with time. Hence, alkaline phosphatase synthesis seems to be the driving force for its own secretion. The rates of synthesis de novo and of alkaline phosphatases were measured. The specific radioactivities of the secreted alkaline phosphatases were higher than the corresponding specific radioactivities in the tissue throughout the entire incubation period. The intracellular distribution of the alkaline phosphatase isoenzymes was compared.

scholarly journals Identification of a druggable protein-protein interaction site between mutant p53 and its stabilizing chaperone DNAJA1

2020 ◽  
pp. jbc.RA120.014749
Author(s):  
Xin Tong ◽  
Dandan Xu ◽  
Rama K. Mishra ◽  
Ryan D Jones ◽  
Leyu Sun ◽  
...  
Keyword(s):  
Small Molecule ◽  
Tp53 Gene ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Dominant Negative Effect ◽  
Mutant P53 ◽  
Missense Mutations ◽  
Oncogenic Function

The TP53 gene is the most frequently mutated gene in human cancers, and the majority of TP53 mutations are missense mutations. As a result, these mutant p53 (mutp53) either directly lose wild-type p53 (wtp53) tumor suppressor function or exhibit a dominant negative effect over wtp53. In addition, some mutp53 have acquired new oncogenic function (gain of function). Therefore, targeting mutp53 for its degradation, may serve as a promising strategy for cancer prevention and therapy. Based on our previous finding that farnesylated DNAJA1 is a crucial chaperone in maintaining mutp53 stabilization, and by using an in silico approach, we built 3-D homology models of human DNAJA1 and mutp53R175H proteins, identified the interacting pocket in the DNAJA1-mutp53R175H complex, and found one critical druggable small molecule binding  site in the DNAJA1 glycine/phenylalanine rich region. We confirmed that the interacting pocket in the DNAJA1-mutp53R175H complex was crucial for stabilizing mutp53R175H using a site-directed mutagenesis approach. We further screened a drug-like library to identify a promising small molecule hit (GY1-22) against the interacting pocket in DNAJA1-mutp53R175H complex. The GY1-22 compound displayed an effective activity against DNAJA1-mutp53R175H complex. Treatment with GY1-22 significantly reduced mutp53 protein levels, enhanced Waf1p21 expression, suppressed cyclin D1 expression, and inhibited mutp53-driven pancreatic cancer growth both in vitro and in vivo. Together, our results indicate that the interacting pocket in the DNAJA1-mutp53R175H complex is critical for mutp53’s stability and oncogenic function, and DNAJA1 is a robust therapeutic target for developing the efficient small molecule inhibitors against oncogenic mutp53.

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