Nonsense Mutation In Gene Research Articles

A homozygous nonsense mutation in the gene for Tmem79, a component for the lamellar granule secretory system, produces spontaneous eczema in an experimental model of atopic dermatitis

Flaky tail (ma/ma Flg(ft/ft)) mice have a frameshift mutation in the filaggrin (Flg(ft)) gene and are widely used as a model of human atopic dermatitis associated with FLG mutations. These mice possess another recessive hair mutation, matted (ma), and develop spontaneous dermatitis under specific pathogen-free conditions, whereas genetically engineered Flg(-/-) mice do not. We identified and characterized the gene responsible for the matted hair and dermatitis phenotype in flaky tail mice. We narrowed down the responsible region by backcrossing ma/ma mice with wild-type mice and identified the mutation using next-generation DNA sequencing. We attempted to rescue the matted phenotype by introducing the wild-type matted transgene. We characterized the responsible gene product by using whole-mount immunostaining of epidermal sheets. We demonstrated that ma, but not Flg(ft), was responsible for the dermatitis phenotype and corresponded to a Tmem79 gene nonsense mutation (c.840C>G, p.Y280*), which encoded a 5-transmembrane protein. Exogenous Tmem79 expression rescued the matted hair and dermatitis phenotype of Tmem79(ma/ma) mice. Tmem79 was mainly expressed in the trans-Golgi network in stratum granulosum cells in the epidermis in both mice and humans. The Tmem79(ma/ma) mutation impaired the lamellar granule secretory system, which resulted in altered stratum corneum formation and a subsequent spontaneous dermatitis phenotype. The Tmem79(ma/ma) mutation is responsible for the spontaneous dermatitis phenotype in matted mice, probably as a result of impaired lamellar granule secretory system and altered stratum corneum barrier function.

Exome Sequencing Identifies Patterns of Disruptive Mutations in Oropharyngeal Squamous Cell Carcinoma

Objectives: 1) Describe the frequency of disruptive gene mutations in current or never smoking patients. 2) Analyze coding regions to identify tumor specific nonsense mutations in genes related to tumor development and critical signaling pathways. Methods: Tumor and adjacent normal oropharyngeal mucosal samples were obtained from 8 patients undergoing surgical treatment of oropharyngeal squamous cell carcinoma (OPSCC) from 2010-2012 representing HPV positive current and never smoking groups. Next generation sequencing following exome enrichment was performed to identify various classes of variants from each sample. Comparative analysis was conducted using Genesifter from PerkinElmer. Results: A global analysis of all mutation types determined that tumor tissues from current smoking patients contain 20% more missense mutations than never smoking groups, resulting in an amino acid substitution. Current smoking patients have tumor specific nonsense mutations in coding regions enriched for tumor suppressor genes and displayed extensive evidence of identical mutation in tumor and adjacent normal tissue. Never smoking patients have a greater number of tumor specific mutations that enrich for genes related to immune system function. Conclusions: Gene mutations that result in truncated proteins are known to associate with cancer development. We have identified a panel of nonsense mutations in current smoking patients that represent tumor suppressor and cell cycle regulatory genes. This group also shows extensive evidence of field cancerization. Never smoking patients carry larger numbers of tumor specific mutations than enrich for genes related to immune system regulation. This may reflect the involvement of active HPV infection in this group.

Familial rhabdoid tumour 'avant la lettre '-from pathology review to exome sequencing and back again

Here we provide compelling evidence that next-generation sequencing will revolutionize diagnostics. We reappraised a case from 1991, published in 1993, describing the unique occurrence of an ovarian immature teratoma arising in a young woman and a clonally distinct intracerebral immature teratoma developing in her daughter. We conducted whole-exome sequencing on constitutional DNA from the mother and her daughter and identified a previously unreported nonsense mutation (c.3533G>A; p.Trp1178*) in the chromatin remodelling gene, SMARCA4, that was present in both individuals and was subject to nonsense-mediated decay. Tumour analysis by Sanger sequencing revealed a somatic SMARCA4 mutation in both the mother (c.2438+1G>T) and her daughter (c.3229C>T; p.Arg1077*), which are predicted to be truncating. As immature teratomas are classified as germ cell tumours, we performed a comprehensive mutation survey of 106 apparently sporadic germ cell tumours, but did not find any other clearly deleterious SMARCA4 mutations. Recently, inactivating mutations in SMARCA4 have been found in two cases of rhabdoid tumour predisposition syndrome type 2. In the light of these findings, renewed efforts to locate previously unobtainable tumour samples were successfully undertaken. Histopathological and immunohistochemical re-analysis of the daughter's tumour revealed that it was indeed a rhabdoid tumour (atypical teratoid/rhabdoid tumour). In this context, the original pathology report of the mother's ovarian tumour was re-interpreted as describing a malignant rhabdoid tumour of the ovary. This report raises the question as to whether molecular genetic analysis should be included in tumour classification, alongside more traditional microscopy-based methods. The use of new sequencing technologies, particularly when applied to archived samples, will lead to many more 'molecular rediagnoses'. This is the earliest known case of rhabdoid tumour predisposition syndrome type 2 and the first described case with an autosomal dominant pattern of inheritance, only discovered through an exome sequencing project.

GW24-e2477 A novel mutation of FBN1 gene associated with Marfan’s syndrome in a Chinese family

ObjectivesTo describe a novel mutation in the fibrillin-1 (FBN1) gene in a Chinese family with autosomal dominant Marfan syndrome (MFS).MethodsIt has been reported that FBN1 mutations account for approximately 90%...

Left ventricular hypertrophy caused by a novel nonsense mutation in FHL1

Emery Dreifuss muscular dystrophy (EDMD) is a hereditary muscular disorder, characterized by contractures, progressive muscular wasting and cardiac involvement. The majority of EDMD patients harbor mutations in the lamin A/C (LMNA) and emerin (STA) genes. Emerging data implicate mutations in FHL1 (four and a half LIM protein 1) gene, located in chromosome Xq26, in EDMD pathogenesis. FHL1 is mainly expressed in striated and cardiac muscle, and plays an important role in sarcomeric protein synthesis, maintenance of cellular integrity, intracellular signaling and genetic transcription pathways. We report the identification of a novel nonsense mutation in FHL1 gene, associated with left ventricular hypertrophy and a family history of stroke and sudden cardiac death. The management implications of this diagnosis are also discussed.

A Nonsense Mutation in the Acid α-Glucosidase Gene Causes Pompe Disease in Finnish and Swedish Lapphunds

Pompe disease is a recessively inherited and often fatal disorder caused by the deficiency of acid α-glucosidase, an enzyme encoded by the GAA gene and needed to break down glycogen in lysosomes. This glycogen storage disease type II has been reported also in Swedish Lapphund dogs. Here we describe the genetic defect in canine Pompe disease and show that three related breeds from Scandinavia carry the same mutation. The affected dogs are homozygous for the GAA c.2237G>A mutation leading to a premature stop codon at amino acid position 746. The corresponding mutation has previously been reported in humans and causes infantile Pompe disease in combination with a second fully deleterious mutation. The affected dogs from both the Finnish as well as the Swedish breed mimic infantile-onset Pompe disease genetically, but also clinico-pathologically. Therefore this canine model provides a valuable tool for preclinical studies aimed at the development of gene therapy in Pompe disease.

Advances in basic and clinical immunology in 2012

Basic and clinical immunology articles published in the Journal in 2012 were mostly related to the expanding area of primary immunodeficiencies (PIDs). Novel forms of PID were identified by using whole-exome sequencing or after careful examination of flow cytometric data, as in the reports of lymphocyte-specific protein tyrosine kinase, CD27, and CD21 deficiencies. Absent IgG and IgA memory B cells were described in patients with hyper-IgE syndrome, which is consistent with defective antibody response and suggests a potential benefit of immunoglobulin replacement. Impaired production of antibodies to polysaccharide antigens by the human B-cell subset analog to murine B-1 cells was reported in a child with selective polysaccharide antibody deficiency. Increased production of inflammatory cytokines by monocyte-derived cells on Toll-like receptor activation was reported in patients with X-linked agammaglobulinemia, underscoring the important role of Bruton tyrosine kinase in modulation of inflammation. The mechanisms explaining susceptibility to yeast infections and development of chronic mucocutaneous candidiasis were extensively studied. Universal newborn screening for T-cell deficiencies is being implemented in several states, resulting in the diagnosis of a higher number of immunodeficient newborns than previously estimated. The use of laboratory testing to distinguish PIDs from HIV infection was clarified. In the management of PIDs, refinement of indication and strategies to hematopoietic stem cell transplantation resulted in improved outcomes. The use of anti-IL-6 mAbs showed promise as an alternative treatment in patients with Schnitzler syndrome.

Genetic analysis of structural proteins in the adsorption apparatus of bacteriophage epsilon 15

To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15 (E15) using genetic and biochemical methodology Hydroxylamine was used to create nonsense mutants of bacteriophage E15. The mutants were then screened for defects in their adsorption apparatus proteins, initially by measuring the concentrations of free tail spike proteins in lysates of cells that had been infected by the phage mutants under non-permissive growth conditions. Phage strains whose infected cell lysates contained above-average levels of free tail spike protein under non-permissive growth conditions were assumed to contain nonsense mutations in genes coding for adsorption apparatus proteins. These mutants were characterized by classical genetic mapping methods as well as automated sequencing of several of their genes. Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography were used to examine the protein compositions of the radioactive particles produced when the various mutants were grown on a non-permissive host cell in the presence of (35)S-methionine and co-purified along with E15wt phage on CsCl block gradients. Our results are consistent with gp4 forming the portal ring structure of E15. In addition, they show that proteins gp15 and gp17 likely comprise the central tube portion of the E15 adsorption apparatus, with gp17 being more distally positioned than gp15 and dependent upon both gp15 and gp16 for its attachment. Finally, our data indicates that tail spike proteins comprised of gp20 can assemble onto nascent virions that contain gp7, gp10, gp4 and packaged DNA, but which lack both gp15 and gp17, thereby forming particles that are of sufficient stability to survive CsCl buoyant density centrifugation. The portal ring (gp4) of E15 is bound to tail spikes (gp20) and the tail tube (gp15 and gp17); gp17's attachment requires both gp15 and gp16.

Treatment of a methylmalonyl-CoA mutase stopcodon mutation

There are limited treatment options for the metabolic disorder methylmalonic aciduria. The disorder can be caused by nonsense mutations within the methylmalonyl-CoA mutase gene, resulting in the production of a truncated protein with little or no catalytic activity. We used a genomic reporter assay and mouse primary cell lines which carry a stop-codon mutation in the human methylmalonyl-CoA mutase gene to test the effects of gentamicin and PTC124 for stop-codon read-through potential.Fibroblast cell lines were established from methylmalonic aciduria knockout-stop codon mice. Addition of gentamicin to the culture medium caused a 1.5- to 2-fold increase in mRNA expression of the human methylmalonyl-CoA mutase gene. Without treatment the cells contained 19% of the normal levels of methylmalonyl-CoA mutase enzyme activity which increased to 32% with treatment, suggesting a functional improvement. Treatment with PTC124 increased the amount of human methylmalonyl-CoA mutase gene mRNA by 1.6±0.3-fold and a trend suggesting increased enzyme activity.The genomic reporter assay, BAC_MMA∗EGFP, expresses enhanced green fluorescent protein when read-through of the stop codon occurs. Using flow cytometry, RT-real-time PCR and enzyme assay, read-through was measured. Treatment with PTC124 at 20μmol/L resulted in a significant increase in enhanced green fluorescent protein, a 2-fold increase in mRNA expression and a trend to a slight increase in enzyme activity.The clinical relevance of these effects may be tested in mouse models of MMA carrying nonsense mutations in the methylmalonyl-CoA mutase gene. Pharmacological approaches have the advantage of providing a broader effect on multiple tissues, which will benefit many different disorders with similar nonsense mutations.

Refractory epilepsy and mitochondrial dysfunction due to GM3 synthase deficiency

We report two children, born from consanguineous parents, who presented with early-onset refractory epilepsy associated with psychomotor delay, failure to thrive, blindness and deafness. Polarographic and spectrophotometric analyses in fibroblasts and liver revealed a respiratory chain (RC) dysfunction. Surprisingly, we identified a homozygous nonsense mutation in the GM3 synthase gene by using exome sequencing. GM3 synthase catalyzes the formation of GM3 ganglioside from lactosylceramide, which is the first step in the synthesis of complex ganglioside species. Mass spectrometry analysis revealed that the complete absence of GM3 ganglioside and its biosynthetic derivatives was associated with an upregulation of the alternative globoside pathway in fibroblasts. The accumulation of Gb3 and Gb4 globosides likely has a role in RC dysfunction and in the decrease of mitochondrial membrane potential leading to apoptosis, which we observed in fibroblasts. We show for the first time that GM3 synthase deficiency, responsible for early-onset epilepsy syndrome, leads to a secondary RC dysfunction. Our study highlights the role of secondary mitochondrial disorders that can interfere with the diagnosis and the evolution of other metabolic diseases.

Detection of nonsense and frameshift mutations in BRCA1 gene using a new plasmid vector pPhoA-frame

The technique for the detection of frame shift and nonsense mutations in BRCA1 gene was suggested. The technique presumes the construction of recombinant plasmids where the tested DNA fragment placed in-frame with alkaline phosphatase gene of Escherichia coli (phoA). A plasmid pPhoA-frame was constructed for such analysis, the plasmid contains DNA fragment coding for alkaline phosphatase of E. coli. Synthetic DNA fragment with BglII, StuI, ApaI and SacII sites was inserted into the DNA fragment coding for alkaline phosphatase of E. coli between Ala218 and Gly219 codons to facilitate the cloning of BRCA1 gene fragments. Occurrence of the frame shift or nonsense mutation in the tested DNA fragment can be detected after transformation of E. coli by the recombinant plasmid containing the tested fragment. E. coli colonies with the newly constructed recombinant plasmids are plated out on the indicator agar. In the case of frame shift or nonsense mutation the colonies are not colored, DNA fragments without such mutations result in the formation of the blue colonies.

Genotype–phenotype correlations in 17 Chinese patients with autosomal recessive Alport syndrome

Autosomal recessive Alport syndrome (ARAS) results from mutations in the COL4A3 or COL4A4 gene. We analyzed the genotype and phenotype of 17 unrelated Chinese patients with ARAS. Clinical data were reviewed. All coding exons of COL4A3 and COL4A4 genes were PCR-amplified and sequenced from genomic DNA. We identified pathologic mutations in all patients, giving a mutation detection rate of 100%, with 82% in COL4A3 gene and 18% in COL4A4 gene. Sixteen novel mutations in COL4A3 gene and four novel mutations in COL4A4 gene were identified. Furthermore, a previously reported in-frame deletion mutation (40_63del24) in exon 1 of the COL4A3 gene was found in four patients in our study. A single 40_63del24 mutation in COL4A3 seems to result in mild or no renal manifestations, whereas the homozygous state of 40_63del24 in COL4A3 gene or compound heterozygous mutation of 40_63del24 plus another nonsense or frameshift mutation in COL4A3 gene seems to result severe ARAS with hearing loss. Half of the probands' parents had hematuria with or without mild proteinuria. Therefore, we recommend that ARAS be considered when a patient has a positive family history of hematuria, and screening for COL4A3 mutations firstly may be an efficient strategy for molecular diagnosis of ARAS.

A Novel Nonsense Mutation of the EXT1 Gene in an Argentinian Patient with Multiple Hereditary Exostoses

Multiple hereditary exostoses (MHE), also known as multiple osteochondromatosis, is an autosomal-dominant O-linked glycosylation disorder recently classified as EXT1/EXT2-CDG in the congenital disorder of glycosylation (CDG) nomenclature1. MHE is characterized by the presence of multiple cartilage-capped tumors, called “osteochondromas,” which usually develop in the juxta-epiphyseal regions of the long bones. The prevalence of MHE is estimated at 1:50,000 in the general population2,3. The Online Mendelian Inheritance in Man (OMIM) database classified it as either 133700 or 133701, according to whether the mutations occurred in the EXT1 or the EXT2 gene. These genes are located at 8q24 and 11p11-11p12, respectively, and they encode the co-polymerases responsible for heparan sulfate biosynthesis. EXT1 and EXT2 are tumor suppressor genes of the EXT gene family. The EXT1 gene contains eleven exons with a coding region of 2238 base pairs (bp), and the EXT2 gene contains sixteen exons with a coding region of 2154 bp4-7. These genes encode two glycosyltransferases involved in heparan sulfate biosynthesis, exostosin-1 (EXT1) (EC2.4.1.224) and exostosin-2 (EXT2) (EC2.4.1.225), whose impairment leads to the formation of exostoses5,8-10. Inactivating mutations (nonsense, frameshift, and splice site mutations) in EXT1 and EXT2 genes represent the majority of mutations that cause MHE. An overview of the reported variants is provided by the online Multiple Osteochondroma Mutation Database11. The most important complication of MHE is the malignant transformation of osteochondroma to chondrosarcoma, which is estimated to occur in 0.5% to 5% of patients7. Chondrosarcomas arise de novo (primary) or as a result of a preexisting cartilage lesion (secondary). The biological aggressiveness of chondrosarcomas can be predicted by means of a histological grading system (grade I to grade III), based on three parameters: cellularity, degree of nuclear atypia, …

Rescue of Melanocortin 4 Receptor (MC4R) Nonsense Mutations by Aminoglycoside‐Mediated Read‐Through

Aminoglycoside-mediated read-through of stop codons was recently demonstrated for a variety of diseases in vitro and in vivo. About 30 percent of human genetic diseases are the consequence of nonsense mutations. Nonsense mutations in obesity-associated genes like the melanocortin 4 receptor (MC4R), expressed in the hypothalamus, show the impact of premature stop codons on energy homeostasis. Therefore, the MC4R could be a potential pharmaceutical target for obesity treatment and targeting MC4R stop mutations could serve as proof of principle for nonsense mutations in genes expressed in the brain. We investigated four naturally occurring nonsense mutations in the MC4R (W16X, Y35X, E61X, Q307X) located at different positions in the receptor for aminoglycoside-mediated functional rescue in vitro. We determined localization and amount of full-length protein before and after aminoglycoside treatment by fluorescence microscopy, cell surface and total enzyme linked immunosorbent assay (ELISA). Signal transduction properties were analyzed by cyclic adenosine monophosphate (cAMP) assays after transient transfection of MC4R wild type and mutant receptors into COS-7 cells. Functional rescue of stop mutations in the MC4R is dependent on: (i) triplet sequence of the stop codon, (ii) surrounding sequence, (iii) location within the receptor, (iv) applied aminoglycoside and ligand. Functional rescue was possible for W16X, Y35X (N-terminus), less successful for Q307X (C-terminus) and barely feasible for E61X (first transmembrane domain). Restoration of full-length proteins by PTC124 could not be confirmed. Future pharmaceutical applications must consider the potency of aminoglycosides to restore receptor function as well as the ability to pass the blood-brain barrier.

A blueprint for a mutationist theory of replicative strand asymmetries formation.

In the present review, we summarized current knowledge on replicative strand asymmetries in prokaryotic genomes. A cornerstone for the creation of a theory of their formation has been overviewed. According to our recent works, the probability of nonsense mutation caused by replication-associated mutational pressure is higher for genes from lagging strands than for genes from leading strands of both bacterial and archaeal genomes. Lower density of open reading frames in lagging strands can be explained by faster rates of nonsense mutations in genes situated on them. According to the asymmetries in nucleotide usage in fourfold and twofold degenerate sites, the direction of replication-associated mutational pressure for genes from lagging strands is usually the same as the direction of transcription-associated mutational pressure. It means that lagging strands should accumulate more 8-oxo-G, uracil and 5-formyl-uracil, respectively. In our opinion, consequences of cytosine deamination (C to T transitions) do not lead to the decrease of cytosine usage in genes from lagging strands because of the consequences of thymine oxidation (T to C transitions), while guanine oxidation (causing G to T transversions) makes the main contribution into the decrease of guanine usage in fourfold degenerate sites of genes from lagging strands. Nucleotide usage asymmetries and bias in density of coding regions can be found in archaeal genomes, although, the percent of “inversed” asymmetries is much higher for them than for bacterial genomes. “Homogenized” and “inversed” replicative strand asymmetries in archaeal genomes can be used as retrospective indexes for detection of OriC translocations and large inversions.

Editorial [Hot Topic: Replicating Strand Asymmetry in Bacterial and Eukaryotic Genomes (Guest Editor: Feng-Biao Guo)

It is my pleasure as a Guest Editor of Current Genomics to present you with a ‘hot topic issue’ on DNA replication. DNA replication adopts a set of asymmetric mechanisms. One of them is the division of leading and lagging strands. In 1991, the nucleotide composition bias between the two replicating strands was originally found in genomes of echinoderm and vertebrate mitochondria. In the following twenty years, more and more bacterial genomes are found to have much different nucleotide composition between the two replicating strands. More importantly, eukaryotes and even mammalians are found to have such strand bias (asymmetry) in recent years. Besides composition bias, there are also other types of biases between two replicating strands, such as gene orientation bias, gene function bias and substitution rate bias. This topic of replicating strand bias has attracted numerous researchers to perform abundant and in-depth researches. In my view, it will continue to be one of the hot topics of genomics. A better understanding of replicating strand asymmetry will greatly advance our knowledge about the mechanism of DNA replication. This theme issue aims mainly to summarize various types of biases between the two replicating strands in bacterial and eukaryotic genomes. Another aim is to try to elucidate the underlying mechanisms of various strand biases. Therefore, this theme issue includes papers reviewing types, extents, application and underlying mechanism of strand asymmetry both in bacterial and eukaryotic genomes. K. Arakawa and M. Tomita reviewed the measures of strand bias that have been proposed to date, including the ΔGC skew, the predictability score of linear discriminant analysis for gene orientation and so on. These methods measure the general composition bias, gene distribution bias and the composition bias of certain oligonucleotides, respectively. Although these measures were predominantly designed for and applied in analyzing replication-related mutational processes of prokaryotes, the authors also give research examples in eukaryotes. X. Xia summarized the diverse patterns of strand asymmetry among different taxonomic groups and made four suggestions. The survey was involved with bacterial, archaeal and mitochondrial genomes. The four suggestions concern the numbers of replication origins and replicating mechanism in certain taxa. Lin et al. reviewed composition bias between light and heavy strands of animal mitochondrial genomes. They discussed the influence of replication-associated mutation pressure on nucleotide and amino acid compositions as well as gene organization in these genomes. H. Seligmann reviewed coding constraints on replication-associated strand composition bias in mitochondrial genomes. He concludes that the bias is the most at 3rd and least at 2nd codon positions across mitochondrial genomes. There are not bias at synonymous positions forming off frame stops and in regions coding for putative overlapping genes. Also, he compared transcription and replication associated biases of nucleotide composition and mutation rate. The review from V.V. Khrustalev and E.V. Barkovsky focuses on their hypothesis on the formation of replicating strand asymmetries in prokaryotes. In this review, they summarized various proofs supporting the theory. The hypothesis is that the combined effects of cytosine deamination and guanine oxidation produce more nonsense mutations in genes from lagging strands than in genes from leading strands, and hence lead to the composition bias and gene density bias. All of the above papers reviewed mainly on computational researches. Because all types of strand asymmetries derive from the asymmetric mechanism of replication and/or transcription, we have also arrange one review concerning experiment works of DNA replication and transcription. In the paper by Lin and Pasero, mechanisms of the two processes are touched and the focus is put on interference between them. Overall, these reviews provide a fascinating survey of the types, extents, mechanisms and applications of replicating strand asymmetry. I hope that this issue will be useful for the researchers and students in the filed. Finally, I would like to thank all the reviewers for their valuable suggestions and comments, which have led to the improvement of review articles.

A Novel Nonsense Mutation of F13A1 Gene Causing Inherited Factor XIII Deficiency in a Chinese Han Family

Abstract 4661 Introduction:Coagulation factor XIII (FXIII) is a protransglutaminase that has a major role in the final stage of blood coagulation process by forming cross-links between γ-glutamyl and ε-lysine residues of fibrin chains. The plasma FXIII (pFXIII) circulates in plasma as a heterotetramer (FXIII-A2B2) consisting of two catalytic A subunits (FXIII-A2) and two carrier B subunits (FXIII-B2). Inherited FXIII deficiency is a rare autosomal recessive disease with lifelong bleeding. Most cases of FXIII deficiency are heterogeneous due to mutations in the F13A gene. Currently, more than 100 mutations have been reported. Aim:To identify the genetic defect of inherited coagulation factor XIII (FXIII) deficiency in a Chinese Han family. Methods and Results:A 13 year-old patient complained of poor wound healing after operation and had a history of an excessive bleeding from the umbilical cord stump after her birth. The routine laboratory tests are normal. Her bleeding time is more than 15 minutes and fibrin clot was solubilized very quickly in 5mol/L urea, and became insoluble when normal plasma was mixed with her plasma in vitro. Her plasma FXIII activity was zero with the amine incorporation assay and plasma FXIII antigen was also near zero by one-step sandwich ELISA method, the plasma FXIIIA antigen was zero using an indirect competitive ELISA assay. The plasma FXIIIA antigen, FXIII activity and antigen were assayed in all available family members. The testing results of patient's grandfather and maternal grandmother were within normal range. But the other pedigree members' results were lower in different level compare with normal ranges. All members of her family had normal coagulation test.All the exons of F13A gene as well as F13B gene and their flanking regions were amplified by PCR for direct sequencing to identify the mutations in the proband. Direct DNA sequencing of all purified amplification products from the patient's F13A gene demonstrated a homozygous nonsense mutation in exon8 (C to A transversion at nucleotide 98531 which caused Cys327X). And the patient didn't have the Val34Leu polymorphism. In the pedigree except of the proband, the Cys327X mutation was found in the heterozygous state in all investigated members except for her grandfather and maternal grandmother. A family study revealed that the mutation was inherited from both parents.The identified mutation was validated by PCR-RFLP technique in the family members and healthy people. Restriction enzyme analysis of amplified exon 8 DNA fragment confirmed that the patient was homozygous for this mutation. Then the quantitative RT-PCR method was used for studying the mRNA expression level of mutant FXIIIA. And the results indicated that F13A mRNA transcripts in heterozygous mutant were reduced by 25% when compared to transcripts in wild-type one, while the homozygous mutant level of F13A mRNA transcript was nearly zero relative to the normal transcript. Conclusion:We have identified a novel Cys327X nonsense mutation in human FXIIIA gene which we have not found in the FXIII database (www.f13-database.de) or in previous publications.And the identified nonsense mutation is causative for severe factor XIII deficiency with a bleeding disorder. Further, in vitro expression studies of the factor XIII mutation is required to confirm their pathological mechanism. Disclosures:No relevant conflicts of interest to declare.

Post-transcriptionally Regulated Expression System in Human Xenogeneic Transplantation Models

Cells have developed a mechanism to discriminate between premature termination codons (PTCs) and normal stop codons during translation, sparking vigorous research to develop drugs promoting readthrough at PTCs to treat genetic disorders caused by PTCs. It was posed that this concept could also be applied to regulated gene therapy protocols by incorporating a PTC into a therapeutic gene, so active protein would only be made after administration of a readthrough agent. The strengths of the system are highlighted here by results demonstrating: (i) background expression levels were reduced to 0.01% to 0.0005% of wild type in unselected mass populations of cells depending upon the specific stop codon utilized and its position within the gene; (ii) expression levels responded well to multiple "On" and "Off" regulation cycles in vivo in human xenograft systems; (iii) the level of induction approached three logs using aminoglycoside activators including NB54, a newly synthesized aminoglycoside with significantly reduced toxicity; and (iv) expression levels could be appreciably altered when employing different promoters in a variety of cell types. These results strongly support the contention that this system should have important clinical applications when tight control of gene expression is required.

Stop codon read-through with PTC124 induces palmitoyl-protein thioesterase-1 activity, reduces thioester load and suppresses apoptosis in cultured cells from INCL patients

Infantile neuronal ceroid lipofuscinosis (INCL), a lethal hereditary neurodegenerative lysosomal storage disorder, affects mostly children. It is caused by inactivating mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. Nonsense mutations in a gene generate premature termination codons producing truncated, nonfunctional or deleterious proteins. PPT1 nonsense-mutations account for approximately 31% of INCL patients in the US. Currently, there is no effective treatment for this disease. While aminoglycosides such as gentamycin suppress nonsense mutations, inherent toxicity of aminoglycosides prohibits chronic use in patients. PTC124 is a non-toxic compound that induces ribosomal read-through of premature termination codons. We sought to determine whether PTC124-treatment of cultured cells from INCL patients carrying nonsense mutations in the PPT1 gene would correct PPT1 enzyme-deficiency with beneficial effects. Our results showed that PTC124-treatment of cultured cells from INCL patients carrying PPT1 nonsense-mutations induced PPT1 enzymatic activity in a dose- and time-dependent manner. This low level of PPT1 enzyme activity induced by PTC124 is virtually identical to that induced by gentamycin-treatment. Even though only a modest increase in PPT1 activity was achieved by PTC124-treatment of INCL cells, this treatment reduced the levels of thioester (constituent of ceroid) load. Our results suggest that PTC124-treatment induces PPT1 enzymatic activity in cultured cells from INCL patients carrying PPT1 nonsense-mutations, and this modest enzymatic activity has demonstrable beneficial effects on these cells. The clinical relevance of these effects may be tested in animal models of INCL carrying nonsense mutations in the PPT1 gene.

Treatment of von Willebrand disease with FVIII/VWF concentrates.

Treatment of von Willebrand disease with FVIII/VWF concentrates.