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PubMed2006

Mutational analysis of human eIF4AIII identifies regions necessary for exon junction complex formation and nonsense-mediated mRNA decay.

The exon junction complex (EJC) is deposited on mRNAs by the process of pre-mRNA splicing and is a key effector of downstream mRNA metabolism. We previously demonstrated that human eIF4AIII, which is essential for nonsense-mediated mRNA decay (NMD), constitutes at least part of the RNA-binding platform anchoring other EJC components to the spliced mRNA. To determine the regions of eIF4AIII that are functionally important for EJC formation, for binding to other EJC components, and for NMD, we now report results of an extensive mutational analysis of human eIF4AIII. Using GFP-, GST- or Flag-fusions of eIF4AIII versions containing site-specific mutations or truncations, we analyzed subcellular localizations, protein-protein interactions, and EJC formation in vivo and in vitro. We also tested whether mutant proteins could rescue NMD inhibition resulting from RNAi depletion of endogenous eIF4AIII. Motifs Ia and VI, which are conserved among the eIF4A family of RNA helicases (DEAD-box proteins), are crucial for EJC formation and NMD, as is one eIF4AIII-specific region. An additional eIF4AIII-specific motif forms part of the binding site for MLN51, another EJC core component. Mutations in the canonical Walker A and B motifs that eliminate RNA-dependent ATP hydrolysis by eIF4AIII in vitro are of no detectable consequence for EJC formation and NMD activation. Implications of these findings are discussed in the context of other recent results and a new structural model for human eIF4AIII based on the known crystal structure of Saccharomyces cerevisiae eIF4AI.

Adenosine Triphosphatases↗

PubMed2006

In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a disease characterized by microvascular thrombosis, often associated with deficiency of the vonWillebrand factor (VWF) cleaving protease ADAMTS13. We investigated the spectrum of ADAMTS13 gene mutations in patients with TTP and congenital ADAMTS13 deficiency to establish the consequences on ADAMTS13 processing and activity. We describe five missense (V88M, G1239V, R1060W, R1123C and R1219W), 1 nonsense (W1016Stop) and 1 insertion (82_83insT) mutations. In two patients no mutation was identified despite undetectable protease activity. Expression in HEK293 mammalian cells (V88M, G1239V, R1123C and R1219W) documented that three missense mutants were not secreted, whereas theV88M was secreted at low levels and with reduced activity. We also provide evidence that impaired secretion of ADAMTS13 mutants observed in vitro translates into severely reduced ADAMTS13 antigen levels in patients in vivo. To evaluate whether the small amounts of mutant protease present in the circulation of patients had VWF cleaving activity, WT and mutant rADAMTS13 were stably expressed in Drosophila S2 cells under the influence of the Drosophila BiP protein signal sequence, which allows protein secretion. Drosophila expression system showed a 40-60% protease activity in the mutants. Several single nucleotide polymorphisms (SNPs) within exons and intron boundaries were found in patients, suggesting that the interplay of SNPs could at least in part account for ADAMTS13 functional abnormalities in patients without mutations. In conclusion, defective secretion and impaired activity of the mutants concur to determine an almost complete deficiency of ADAMTS13 activity in patients with a homozygous or two heterozygous ADAMTS13 mutations.

ADAM Proteins↗

PubMed2006

Efficient screening of the cystinuria-related C663T Slc3a1 nonsense mutation in Newfoundland dogs by denaturing high-performance liquid chromatography.

Cystinuria in Newfoundland dogs is a metabolic disease associated with a nonsense mutation in the exon 2 of the Slc3a1 gene. Similar to type I human cystinuria, heterozygote carriers are not affected by the disease and do not reveal differences in urinary concentration of dibasic amino acids when compared with normal dogs. However, through a recessive mode of inheritance, these dogs are able to transmit the disease to their offspring. Early detection of mutation carriers through cost-effective reliable methods is therefore essential for the implementation of breeding methods aimed at the eradication of the disease. Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid and efficient screening of nucleotide polymorphisms in polymerase chain reaction-amplified products. This technique was used for the identification of the C663T Slc3a1 mutation in Portuguese Newfoundland dogs. Polymerase chain reaction products amplified from a region containing the C663T locus were subjected to DHPLC analysis, and results were double checked by DNA sequencing. Results showed the presence of the mutation in 6 of the 22 dogs tested. Urine biochemical parameters correlated well with the number of mutated Slc3a1 copies, and homozygotes for the C663T mutation were the only dogs diagnosed with cystinuria. Sequence analysis confirmed the DHPLC results, demonstrating that the technique could be a reliable alternative to sequencing for the rapid and cost-effective identification of mutations in canine breeds.

Amino Acid Transport Systems↗

PubMed2000

A common variant of the AMPD1 gene predicts improved cardiovascular survival in patients with coronary artery disease.

OBJECTIVE: We tested whether a common AMPD1 gene variant is associated with improved cardiovascular (CV) survival in patients with coronary artery disease (CAD). BACKGROUND: Reduced activity of adenosine monophosphate deaminase (AMPD) may increase production of adenosine, a cardioprotective agent. A common, nonsense, point variant of the AMPD1 gene (C34T) results in enzymatic inactivity and has been associated with prolonged survival in heart failure. METHODS: Blood was collected from 367 patients undergoing coronary angiography. Genotyping was done by polymerase chain reaction amplification and restriction enzyme digestion, resulting in allele-specific fragments. Coronary artery disease was defined as > or =70% stenosis of > or =1 coronary artery. Patients were followed prospectively for up to 4.8 years. Survival statistics compared hetero- (+/-) or homozygotic (-/-) carriers with noncarriers. RESULTS: Patients were 66 +/- 10 years old; 79% were men; 22.6% were heterozygous and 1.9% homozygous for the variant AMPD1(-) allele. During a mean of 3.5 +/- 1.0 years, 52 patients (14.2%) died, 37 (10.1%) of CV causes. Cardiovascular mortality was 4.4% (4/90) in AMPD1(-) allele carriers compared with 11.9% (33/277) in noncarriers (p = 0.046). In multiple variable regression analysis, only age (hazard ratio, 1.11/year, p < 0.001) and AMPD1(-) carriage (hazard ratio, 0.36, p = 0.053) were independent predictors of CV mortality. CONCLUSIONS: Carriage of a common variant of the AMPD1 gene was associated with improved CV survival in patients with angiographically documented CAD. The dysfunctional AMPD1(-) allele may lead to increased cardiac adenosine and increased cardioprotection during ischemic events. Adenosine monophosphate deaminase-1 genotyping should be further explored in CAD for prognostic, mechanistic and therapeutic insights.

AMP Deaminase↗

PubMed2026

Loss of function of the chromatin remodeling gene INO80D leads to neurogenic features of schizophrenia.

Schizophrenia has been linked to severely damaging de novo mutations in synaptic junction proteins, neurotransmitter receptors, transcription factors, and chromatin remodeling proteins. In a patient with schizophrenia in the absence of a family history of severe mental illness, we identified de novo nonsense mutation, INO80D p.Q568X, associated with both a truncated protein and partial nonsense-mediated decay. Three experiments were undertaken to evaluate the consequences of the mutation. 1) In neural stem cells (iNSCs) differentiated from WTC11 iPSCs, CRISPRi knockdown of INO80D led to downregulation of three subunits of the AMPA-glutamate receptor, of multiple genes mutant in schizophrenia, and of genes of synaptic function. 2) INO80D p.Q568X iNSCs and neurons differentiated from patient-derived induced pluripotent stem cells (iPSCs) had significantly lower expression of neurogenesis genes compared to patient-derived cells with the mutation corrected by CRISPR-Cas9 gene editing. Patient-derived INO80D p.Q568X neurons had significantly higher expression of cell division genes compared to lines with the mutation corrected, consistent with the possibility that some of these cells may be undergoing mitosis, which is not normal for neurons. 3) Finally, on microelectrode array (MEA) plates, WTC11-derived glutamatergic neurons with reduced expression of INO80D had more rapid firing rate and increased average network burst duration, both features of neurons derived from patients with neurodevelopmental disorders. Overall, these findings suggest that partial loss of INO80D function due to de novo mutation may have disrupted normal neurodevelopment and contributed to the schizophrenia of this patient.

Humans↗

PubMed2005

A novel nonsense mutation in the SCN5A gene leads to Brugada syndrome and a silent gene mutation carrier state.

BACKGROUND: Brugada syndrome (BS) is an electrical cardiac disorder with a right bundle branch block and ST segment elevation in leads V1 to V3 on surface electrocardiograms (ECGs), and is a syndrome that may lead to sudden cardiac death. PURPOSE: The aim of the present study was to screen for mutations in the SCN5A gene in a family with BS, and to characterize the consequences of the mutation on channel function. RESULTS: A heterozygous nonsense SCN5A mutation (W822X) was identified in the index patient. The mutation was confirmed in the patient's asymptomatic 16-year-old brother and 48-year-old father. The mutation was absent in the index patient's sister and mother. The ECG of the index patient showed a BS type 2 ECG phenotype, which converted into a type 1 ECG phenotype in the presence of flecainide. The ECG of the patient's brother was not typical for BS, but ajmaline treatment unmasked a type 1 ECG phenotype. The ECG of the asymptomatic father was normal at baseline and in the presence of ajmaline. No Na+ currents could be measured in tsA201 cells transfected with W822X mutant channels. Heterozygote expression showed a nearly 50% reduction in Na+ current amplitude with no significant alterations of biophysical properties, indicating a loss of functional Na+ channels, obviously without any dominant-negative activity on wild type channels. CONCLUSIONS: The haploinsufficiency of the Nav1.5 protein is the plausible explanation for the clinical BS phenotype in this family. Because the heterozygous W822X mutation theoretically leads to channel expression at one-half of the normal level, the authors suggest that a modifier gene may influence or rescue the phenotype in the asymptomatic family members.

Adolescent↗

PubMed2002

Nonsense and missense mutations in the human hepatocyte nuclear factor-1 beta gene (TCF2) and their relation to type 2 diabetes in Japanese.

Mutations in transcription factors expressed in the pancreatic beta-cell are a major cause of maturity-onset diabetes of the young (MODY). They have also been found in patients diagnosed with type 1 and type 2 diabetes mellitus, which may highlight the difficulty in diagnosing these forms of diabetes or perhaps indicate a direct role in the development of multiple forms of diabetes. We have screened the hepatocyte nuclear factor-1 beta (HNF-1 beta/MODY5) gene for mutations in a group of 126 unrelated Japanese patients with type 2 diabetes and a family history of at least one first degree relative with diabetes. We identified one patient with a nonsense mutation (R276X) and another with a missense mutation (S465R). These mutations were present in the heterozygous state and were not found in 132 nondiabetic subjects (264 normal alleles). We identified a second patient with the S465R mutation on screening a second group of 272 randomly selected type 2 diabetic patients but not in another 122 nondiabetic subjects. Functional studies indicated that R276X-HNF-1 beta was inactive and S465R-HNF-1 beta exhibited a 22% reduction in activity compared with the wild-type protein. The S465R mutation may function in a dominant-negative manner. The subject with the R276X mutation had MODY5 misdiagnosed as common type 2 diabetes. He was diagnosed with diabetes at 13 yr of age and also had small kidneys with multiple bilateral renal cysts and decreased urinary concentrating ability. The two subjects with the S465R mutation had typical late-onset type 2 diabetes and no evidence of kidney disease. We have identified two novel mutations in human HNF-1 beta gene. The prevalence of MODY5 among our population of Japanese diabetes patients with a strong positive family of disease is 0.8%. The S465R mutation was found in 0.5% of our patients with common type 2 diabetes and thus may be a rare genetic risk factor contributing to the development of type 2 diabetes rather than MODY5.

Adolescent↗

PubMed2006

A novel loss of function mutation of PCSK9 gene in white subjects with low-plasma low-density lipoprotein cholesterol.

OBJECTIVES: The PCSK9 gene, encoding a pro-protein convertase involved in posttranslational degradation of low-density lipoprotein receptor, has emerged as a key regulator of plasma low-density lipoprotein cholesterol. In African-Americans two nonsense mutations resulting in loss of function of PCSK9 are associated with a 30% to 40% reduction of plasma low-density lipoprotein cholesterol. The aim of this study was to assess whether loss of function mutations of PCSK9 were a cause of familial hypobetalipoproteinemia and a determinant of low-plasma low-density lipoprotein cholesterol in whites. METHODS AND RESULTS: We sequenced PCSK9 gene in 18 familial hypobetalipoproteinemia subjects and in 102 hypocholesterolemic blood donors who were negative for APOB gene mutations known to cause familial hypobetalipoproteinemia. The PCSK9 gene variants found in these 2 groups were screened in 42 subjects in the lowest (<5th) percentile, 44 in the highest (>95th) percentile, and 100 with the average plasma cholesterol derived from general population. In one familial hypobetalipoproteinemia kindred and in 2 hypocholesterolemic blood donors we found a novel PCSK9 mutation in exon 1 (c.202delG) resulting in a truncated peptide (Ala68fsLeu82X). Two familial hypobetalipoproteinemia subjects and 4 hypocholesterolemic blood donors were carriers of the R46L substitution previously reported to be associated with reduced low-density lipoprotein cholesterol as well as other rare amino acid changes (T77I, V114A, A522T and P616L) not found in the other groups examined. CONCLUSIONS: We discovered a novel inactivating mutation as well as some rare nonconservative amino acid substitutions of PCSK9 in white hypocholesterolemic individuals.

Adult↗

PubMed2006

pho2, a phosphate overaccumulator, is caused by a nonsense mutation in a microRNA399 target gene.

We recently demonstrated that microRNA399 (miR399) controls inorganic phosphate (Pi) homeostasis by regulating the expression of UBC24 encoding a ubiquitin-conjugating E2 enzyme in Arabidopsis (Arabidopsis thaliana). Transgenic plants overexpressing miR399 accumulated excessive Pi in the shoots and displayed Pi toxic symptoms. In this study, we revealed that a previously identified Pi overaccumulator, pho2, is caused by a single nucleotide mutation resulting in early termination within the UBC24 gene. The level of full-length UBC24 mRNA was reduced and no UBC24 protein was detected in the pho2 mutant, whereas up-regulation of miR399 by Pi deficiency was not affected. Several characteristics of Pi toxicity in the pho2 mutant were similar to those in the miR399-overexpressing and UBC24 T-DNA knockout plants: both Pi uptake and translocation of Pi from roots to shoots increased and Pi remobilization within leaves was impaired. These phenotypes of the pho2 mutation could be rescued by introduction of a wild-type copy of UBC24. Kinetic analyses revealed that greater Pi uptake in the pho2 and miR399-overexpressing plants is due to increased Vmax. The transcript level of most PHT1 Pi transporter genes was not significantly altered, except PHT1;8 whose expression was enhanced in Pi-sufficient roots of pho2 and miR399-overexpressing compared with wild-type plants. In addition, changes in the expression of several organelle-specific Pi transporters were noticed, which may be associated with the redistribution of intracellular Pi under excess Pi. Furthermore, miR399 and UBC24 were colocalized in the vascular cylinder. This observation not only provides important insight into the interaction between miR399 and UBC24 mRNA, but also supports their systemic function in Pi translocation and remobilization.

Arabidopsis↗

PubMed2024

Functional analysis of a novel nonsense PPP1R12A variant in a Chinese family with infantile epilepsy.

BACKGROUND: Defects in PPP1R12A can lead to genitourinary and/or brain malformation syndrome (GUBS). GUBS is primarily characterized by neurological or genitourinary system abnormalities, but a few reported cases are associated with neonatal seizures. Here, we report a case of a female newborn with neonatal seizures caused by a novel variant in PPP1R12A, aiming to enhance the clinical and variant data of genetic factors related to epilepsy in early life. METHODS: Whole-exome and Sanger sequencing were used for familial variant assessment, and bioinformatics was employed to annotate the variant. A structural model of the mutant protein was simulated using molecular dynamics (MD), and the free binding energy between PPP1R12A and PPP1CB was analyzed. A mutant plasmid was constructed, and mutant protein expression was analyzed using western blotting (WB), and the interaction between the mutant and PPP1CB proteins using co-immunoprecipitation (Co-IP) experiments. RESULTS: The patient experienced tonic-clonic seizures on the second day after birth. Genetic testing revealed a heterozygous variant in PPP1R12A, NM_002480.3:c.2533 C > T (p.Arg845Ter). Both parents had the wild-type gene. MD suggested that loss of the C-terminal structure in the mutant protein altered its structural stability and increased the binding energy with PPP1CB, indicating unstable protein-protein interactions. On WB, a low-molecular-weight band was observed, indicating that the protein was truncated. Co-IP indicated that the mutant protein no longer interacted with PPP1CB, indicating an effect on the structural stability of the myosin phase complex. CONCLUSION: The PPP1R12A c.2533 C > T variant may explain the neonatal seizures in the present case. The findings of this study expand the spectrum of PPP1R12A variants and highlight the potential significance of truncated proteins in the pathogenesis of GUBS.

Female↗

PubMed2005

A homozygous nonsense mutation in SOX9 in the dominant disorder campomelic dysplasia: a case of mitotic gene conversion.

Campomelic dysplasia (CD; MIM 114290), an autosomal dominant skeletal malformation syndrome with XY sex reversal, is caused by heterozygous de novo mutations in and around the SOX9 gene on 17q. We report a patient with typical signs of CD, including sex reversal, who was, surprisingly, homozygous for the nonsense mutation Y440X. Since neither parent carried the Y440X mutation, possible mechanisms explaining the homozygous situation were a de novo mutation followed by uniparental isodisomy, somatic crossing over, or gene conversion. As the patient was heterozygous for six microsatellite markers flanking SOX9, uniparental isodisomy and somatic crossing over were excluded. Analysis of intragenic single-nucleotide polymorphisms suggested that the homozygous mutation arose by a mitotic gene conversion event involving exchange of at least 440 nucleotides and at most 2,208 nucleotides between a de novo mutant maternal allele and a wild-type paternal allele. Analysis of cloned alleles showed that homozygous mutant cells constituted about 80% of the leukocyte cell population of the patient, whereas about 20% were heterozygous mutant cells. Heterozygous Y440X mutations, previously described in three CD cases, have been identified in seven additional cases, thus constituting the most frequent recurrent mutations in SOX9. These patients frequently have a milder phenotype with longer survival, possibly because of the retention of some transactivation activity of the mutant protein on SOX9 target genes, as shown by cell transfection experiments. The fact that the patient survived for 3 months may thus be explained by homozygosity for a hypomorphic rather than a complete loss-of-function allele, in combination with somatic mosaicism. This is, to our knowledge, the first report of mitotic gene conversion of a wild-type allele by a de novo mutant allele in humans.

Abnormalities, Multiple↗

PubMed2006

Fibrinogen Guarenas, an abnormal fibrinogen with an Aalpha-chain truncation due to a nonsense mutation at Aalpha 467 Glu (GAA)-->stop (TAA).

Fibrinogen Guarenas is a dysfibrinogenemia with a nonsense mutation at G4731T that causes an Aalpha-chain truncation at Ser 466. This abnormal fibrinogen is associated with a bleeding diathesis, severe in the proposita and mild in one brother, even though the fibrinogen levels in plasma are normal. All other family members are asymptomatic. Fibrinogens from the proposita and one family member, the mother of the proposita, both heterozygous for the mutation, were studied. Turbidity curves of fibrin polymerization showed that the lateral association of protofibrils was impaired and the maximum rate of polymerization was slightly diminished. The binding of albumin to fibrinogen was increased compared to control due to the presence of a free sulfhydryl group because of the missing disulphide bridge between Aalpha-Cys 442-472 in the mutated molecules. The abnormal fibrinogen formed much less alpha-polymer, and gamma-dimer formation was delayed compared to the control. Plasminogen activation by t-PA in the presence of fibrin was decreased. When Guarenas clots were perfused with fibrinolytic enzymes, clot degradation was retarded. Clot structure studied by confocal 3D microscopy showed that the fibrin network was dense, made up of thin and highly branched fibers, which accounted for the decreased flow rates by buffer permeation and increased rigidity of the fibrin clots, measured using a torsion pendulum. It seems that the increased clot rigidity, decreased porosity, hypofibrinolysis and t-PA induced fibrinolysis, by itself are not necessarily associated with thrombotic disorders in dysfibrinogenemia.

Afibrinogenemia↗

PubMed2006

An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements.

A nonsense mutation c.4250T>A (p.Leu1417X) in the dystrophin gene of a patient with an intermediate phenotype of muscular dystrophy induces partial in-frame skipping of exon 31. On the basis of UV cross-linking assays and pull-down analysis, we present evidence that the skipping of this exon is because of the creation of an exonic splicing silencer, which acts as a highly specific binding site (UAGACA) for a known repressor protein, hnRNP A1. Recombinant hnRNP A1 represses exon inclusion both in vitro and in vivo upon transient transfection of C2C12 cells with Duchenne muscular dystrophy (DMD) minigenes carrying the c.4250T>A mutation. Furthermore, we identified a downstream splicing enhancer in the central region of exon 31. This region functions as a Tra2beta-dependent exonic splicing enhancer (ESE) in vitro when inserted into a heterologous splicing reporter, and deletion of the ESE showed that incorporation of exon 31 depends on the Tra2beta-dependent enhancer both in the wild-type and mutant context. We conclude that dystrophin exon 31 contains juxtaposed sequence motifs that collaborate to regulate exon usage. This is the first elucidation of the molecular mechanism leading to exon skipping in the dystrophin gene and allowing the occurrence of a milder phenotype than the expected DMD phenotype. The knowledge of which cis-acting sequence within an exon is important for its definition will be essential for the alternative gene therapy approaches based on modulation of splicing to bypass DMD-causing mutations in the endogenous dystrophin gene.

Amino Acid Motifs↗

PubMed2005

Characterization of a recurrent germ line mutation of the E-cadherin gene: implications for genetic testing and clinical management.

PURPOSE: To identify germ line CDH1 mutations in hereditary diffuse gastric cancer (HDGC) families and develop guidelines for management of at risk individuals. EXPERIMENTAL DESIGN: We ascertained 31 HDGC previously unreported families, including 10 isolated early-onset diffuse gastric cancer (DGC) cases. Screening for CDH1 germ line mutations was done by denaturing high-performance liquid chromatography and automated DNA sequencing. RESULTS: We identified eight inactivating and one missense CDH1 germ line mutation. The missense mutation conferred in vitro loss of protein function. Two families had the previously described 1003C>T nonsense mutation. Haplotype analysis revealed this to be a recurrent and not a founder mutation. Thirty-six percent (5 of 14) of the families with a documented DGC diagnosed before the age of 50 and other cases of gastric cancer carried CDH1 germ line mutations. Two of 10 isolated cases of DGC in individuals ages <35 years harbored CDH1 germ line mutations. One mutation positive family was ascertained through a family history of lobular breast cancer (LBC) and another through an individual with both DGC and LBC. Occult DGC was identified in five of six prophylactic gastrectomies done on asymptomatic, endoscopically negative 1003C>T mutation carriers. CONCLUSIONS: In addition to families with a strong history of early-onset DGC, CDH1 mutation screening should be offered to isolated cases of DGC in individuals ages <35 years and for families with multiple cases of LBC, with any history of DGC or unspecified GI malignancies. Prophylactic gastrectomy is potentially a lifesaving procedure and clinical breast screening is recommended for asymptomatic mutation carriers.

Adult↗

PubMed2002

Twist haploinsufficiency in Saethre-Chotzen syndrome induces calvarial osteoblast apoptosis due to increased TNFalpha expression and caspase-2 activation.

Saethre-Chotzen syndrome (SCS) is a human autosomal dominant disorder characterized by premature fusion of cranial sutures caused by mutations of the Twist gene encoding a basic helix-loop-helix (bHLH) transcription factor. We previously showed that Twist haploinsufficiency caused by a Y103X nonsense mutation in SCS alters both proliferation and osteoblast gene expression in human calvarial osteoblasts, indicating that Twist is an important regulator of osteoblast differentiation. Here we show that Twist haploinsufficiency alters osteoblast apoptosis in SCS. Analysis of terminal deoxynucleotidyl transferase-mediated nick-end labelling (TUNEL) demonstrated increased osteoblast and osteocyte apoptosis in coronal sutures from two SCS patients with nonsense mutations (Y103X and Q109X) that result in the synthesis of bHLH-truncated proteins, and one patient with a missense mutation in the basic domain (R118C) that abolishes Twist DNA binding. To assess the mechanisms involved, we studied osteoblast apoptosis in mutant (M-Tw) calvarial cells bearing the Y103X mutation resulting in decreased Twist mRNA and protein levels. M-Tw cells cultured in low serum conditions showed enhanced DNA fragmentation compared to normal (Nl) age-matched calvarial cells. Biochemical analysis showed increased activity of initiator caspases-2 and -8 and downstream effector caspases-3, -6 and -7 in mutant osteoblasts. Caspase-2 was upstream of caspase-8 and effector caspases-3, -6 and -7 because their activities were suppressed by a specific caspase-2 inhibitor. M-Tw osteoblasts also showed increased cytochrome c release from the mitochondria. However, the activity of the downstream effector caspase-9 was not increased due to overexpression of the antagonist protein Hsp70. Detection of differentially expressed genes using cDNA expression array revealed increased Bax and TNFalpha mRNA levels in M-Tw compared to Nl cells, a finding confirmed by RT-PCR and western blot analyses. Neutralization of TNFalpha overexpression using anti-TNFalpha or anti-TNF receptor 1 antibodies abolished the increased activity of caspase-2, caspase-8 and caspases-3, -6 and -7 in M-Tw osteoblasts. These studies provide novel evidence that Twist haploinsufficiency in SCS promotes osteoblast apoptosis by a TNFalpha-caspase-2-caspase-8-caspases-3, -6, -7 cascade, and uncover a molecular mechanism in which Twist plays an anti-apoptotic role in human calvarial osteoblasts.

Acrocephalosyndactylia↗

PubMed1999

Evolution of the genetic code: the nonsense, antisense, and antinonsense codes make no sense.

According to the molecular recognition theory, the complementarity of the sense and nonsense DNA strands is reflected in a complementarity of polypeptides and the corresponding nonsense polypeptides. A comparison of the sense and nonsense code matrices, and of the antisense and antinonsense code matrices, either by visual inspection or by comparing the corresponding hydrophobicity matrices (e.g. by simply adding them together), revealed no complementarity of these pairs of matrices in terms of possible attractive physical forces. Instead, it was evident that the codes divide the amino acids into two major groups: hydrophilic and hydrophobic, a division which is directly correlated with the folding property of proteins. A simple primordial genetic code distinguishing between these two types of amino acids would have been capable of generating three-dimensionally folded peptides, which could stabilize coding RNAs by forming ribonucleoprotein complexes. This evolutionary scheme is reflected in the present organisation of information processing and storage in essentially all organisms. RNAs are processed and translated into proteins by ribonucleoproteins, while other steps in information retrieval and processing, such as DNA replication, transcription, protein folding and posttranslational processing, are catalyzed by proteins. This shows that the evolution of DNA as an information storage medium was a secondary event, unrelated to the evolution of the genetic code. From the primordial hydrophilic/hydrophobic (f.ex. Leu/Arg) code, evolution proceeded by introduction of a catalytic amino acid (Ser). The further evolution of the code has mainly served to increase the number of functional hydrophilic amino acids, since there has not been a great advantage in increasing the number of structural, hydrophobic amino acids. At some stage during the evolution of the genetic code, double-stranded DNA was introduced as a maximally safe genetic copy of RNA. This required the action of highly specific enzymes, and was therefore preceded by the refinement of the genetic code. As a conclusion of this evolutionary scheme, it can be inferred that, in general only the sense strand encodes proteins.

Codon, Nonsense↗

PubMed2006

Homozygous nonsense mutation in the MCEE gene and siRNA suppression of methylmalonyl-CoA epimerase expression: a novel cause of mild methylmalonic aciduria.

Methylmalonyl-CoA epimerase (MCE) catalyzes the interconversion of D- and L-methylmalonyl-CoA in the pathway responsible for the degradation of branched chain amino acids, odd chain-length fatty acids, and other metabolites. Despite the occurrence of metabolic disorders in the enzymatic step occurring immediately upstream of MCE (propionyl-CoA carboxylase) and downstream of MCE (adenosylcobalamin-dependent methylmalonyl-CoA mutase), no disease-causing mutations have been described affecting MCE itself. A patient, formerly identified as belonging to the cblA complementation group of vitamin B12 disorders but lacking mutations in the affected gene, MMAA, was tested for mutations in the MCEE gene. The patient's fibroblasts had normal levels of adenosylcobalamin compared to controls, whereas other cblA cell lines typically had reduced levels of the cofactor. As well, this patient had a milder form of methylmalonic aciduria than usually observed in cblA patients. The patient was found to be homozygous for a c.139C>T (p.R47X) mutation in MCEE by sequence analysis that was confirmed by restriction digestion of PCR products. One sibling, also with mild methylmalonic aciduria, was homozygous for the mutation. Both parents and one other sibling were heterozygous. A nearby insertion polymorphism, c.41-160_161insT, heterozygous in both parents, showed the wild-type configuration on the mutant alleles. To assess the impact of isolated MCE deficiency in cultured cells, HeLa cells were transfected with a selectable vector containing MCEE-specific small interfering RNA (siRNA) to suppress gene expression. The reduced level of MCEE mRNA resulted in the reduction of [14C]-propionate incorporation into cellular macromolecules. However, siRNA only led to a small reduction in pathway activity, suggesting that previously postulated non-enzymatic conversion of D- to L-methylmalonyl-CoA may contribute to some flux through the pathway. We conclude that the patient's MCEE defect was responsible for the mild methylmalonic aciduria, confirming a partial requirement for the enzymatic activity in humans.

Cell Culture Techniques↗

PubMed2004

Striate palmoplantar keratoderma arising from desmoplakin and desmoglein 1 mutations is associated with contrasting perturbations of desmosomes and the keratin filament network.

BACKGROUND: Several hereditary human diseases are now known to be caused by distinct mutations in genes encoding various desmosome components. Although the effects of some of these mutant genes have been analysed by targeted disruption experiments in mouse models, little is known about the cell and tissue changes in affected human patients. OBJECTIVES: To investigate the effects of heterozygous nonsense mutations in desmoplakin (Dp) and desmoglein (Dsg) 1 which cause the autosomal dominant disorder striate palmoplantar keratoderma (SPPK), focusing on changes in desmosome structure and composition and the associated keratin intermediate filament (KIF) network in palm skin, and in cultured keratinocytes generated from the same site. METHODS: We analysed palm and nonpalm skin sections from four SPPK patients with Dp mutations and one patient with a Dsg1 mutation with respect to tissue and subcellular morphologies, and correlated the in vivo and in vitro findings. RESULTS: Using electron microscopy, we found abnormalities of desmosomes and cell-cell adhesion in the suprabasal layers in the epidermis from patients with both Dsg1- and Dp-associated SPPK. These changes were more advanced in skin from patients with Dp mutations. Both Dp and Dsg1 mutations were accompanied by significantly reduced numbers of desmosomes in the suprabasal layers, while decreased desmosome size was evident only in Dsg1-associated SPPK. Confocal microscopy analysis showed marked differences in the expression of keratins and of desmosome components, both between the two types of SPPK, and between SPPK and normal skin. The expression of keratins K5, K14 and K10 was reduced in Dsg1-associated SPPK skin, whereas perinuclear aggregation of keratin filaments was more evident in Dp-associated SPPK. In both types of SPPK upregulation of K16 was pronounced and involucrin labelling was abnormal. CONCLUSIONS: Mutations in Dp and Dsg1 genes causing SPPK may be associated with perturbations in epidermal differentiation accompanied by a marked disruption of several components of the epidermal scaffold including desmosomes and the KIF network.

Adult↗