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Sample records for dmd gene deletion

  1. Somatic mosaicism for a DMD gene deletion

    SciTech Connect

    Saito, Kayoko; Ikeya, Kiyoko; Kondo, Eri

    1995-03-13

    Mosaicism is a mixed state, with two cell populations of different genetic origins caused by a cell mutation occurring after fertilization. In the present case, DNA analysis of lymphocytes led to a DMD diagnosis before death. Postmortem immunocytochemical and DNA analysis showed somatic mosaicism. At age 18 years, blood lymphocyte DNA analysis showed a DMD gene deletion, upstream from exon 7 to the 5{prime} end containing both muscle and brain promoters. As the patient`s mother and elder sister had no deletions, he was considered to have a new mutation. Immunocytochemical studies of postmortem tissues showed that dystrophin was absent from the tongue, deltoid, intercostal, psoas and rectus femoris muscles, but there was a mix of dystrophin-positive and negative fibers in the rectus abdominis, cardiac, temporalis and sternocleidomastoid muscles. All diaphragm cells were dystrophin positive. Polymerase chain reaction (PCR) amplification from all tissues except the temporalis and sternocleidomastoid muscles, diaphragm and kidney, in which no deletion was found, showed the deletion from at least exon 6 to the 5{prime} end containing both muscle and brain promoters. In this case, a genomic deletion of the DMD gene contributed to the formation of tissues derived from both ectoderm and endoderm, and cells of mesodermal origin showed genotypic and phenotypic heterogeneity. Our results indicate a mutation of the present case may have occurred just before the period of germ layer formation. 34 refs., 7 figs.

  2. Mechanism of Deletion Removing All Dystrophin Exons in a Canine Model for DMD Implicates Concerted Evolution of X Chromosome Pseudogenes.

    PubMed

    VanBelzen, D Jake; Malik, Alock S; Henthorn, Paula S; Kornegay, Joe N; Stedman, Hansell H

    2017-03-17

    Duchenne muscular dystrophy (DMD) is a lethal, X-linked, muscle-wasting disorder caused by mutations in the large, 2.4-Mb dystrophin gene. The majority of DMD-causing mutations are sporadic, multi-exon, frameshifting deletions, with the potential for variable immunological tolerance to the dystrophin protein from patient to patient. While systemic gene therapy holds promise in the treatment of DMD, immune responses to vectors and transgenes must first be rigorously evaluated in informative preclinical models to ensure patient safety. A widely used canine model for DMD, golden retriever muscular dystrophy, expresses detectable amounts of near full-length dystrophin due to alternative splicing around an intronic point mutation, thereby confounding the interpretation of immune responses to dystrophin-derived gene therapies. Here we characterize a naturally occurring deletion in a dystrophin-null canine, the German shorthaired pointer. The deletion spans 5.6 Mb of the X chromosome and encompasses all coding exons of the DMD and TMEM47 genes. The sequences surrounding the deletion breakpoints are virtually identical, suggesting that the deletion occurred through a homologous recombination event. Interestingly, the deletion breakpoints are within loci that are syntenically conserved among mammals, yet the high homology among this subset of ferritin-like loci is unique to the canine genome, suggesting lineage-specific concerted evolution of these atypical sequence elements.

  3. Developmental profile of H19 differentially methylated domain (DMD) deletion alleles reveals multiple roles of the DMD in regulating allelic expression and DNA methylation at the imprinted H19/Igf2 locus.

    PubMed

    Thorvaldsen, Joanne L; Fedoriw, Andrew M; Nguyen, Son; Bartolomei, Marisa S

    2006-02-01

    The differentially methylated domain (DMD) of the mouse H19 gene is a methylation-sensitive insulator that blocks access of the Igf2 gene to shared enhancers on the maternal allele and inactivates H19 expression on the methylated paternal allele. By analyzing H19 DMD deletion alleles H19DeltaDMD and H19Delta3.8kb-5'H19 in pre- and postimplantation embryos, we show that the DMD exhibits positive transcriptional activity and is required for H19 expression in blastocysts and full activation of H19 during subsequent development. We also show that the DMD is required to establish Igf2 imprinting by blocking access to shared enhancers when Igf2 monoallelic expression is initiated in postimplantation embryos and that the single remaining CTCF site of the H19DeltaDMD allele is unable to provide this function. Furthermore, our data demonstrate that sequence outside of the DMD can attract some paternal-allele-specific CpG methylation 5' of H19 in preimplantation embryos, although this methylation is not maintained during postimplantation in the absence of the DMD. Finally, we report a conditional allele floxing the 1.6-kb sequence deleted from the H19DeltaDMD allele and demonstrate that the DMD is required to maintain repression of the maternal Igf2 allele and the full activity of the paternal Igf2 allele in neonatal liver.

  4. Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method

    PubMed Central

    Iyombe-Engembe, Jean-Paul; Ouellet, Dominique L; Barbeau, Xavier; Rousseau, Joël; Chapdelaine, Pierre; Lagüe, Patrick; Tremblay, Jacques P

    2016-01-01

    The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel), a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides. PMID:26812655

  5. Are there ethnic differences in deletions in the dystrophin gene?

    SciTech Connect

    Banerjee, M.; Verma, I.C.

    1997-01-20

    We studied 160 cases of Duchenne muscular dystrophy (DMD) drawn from all parts of India, using multiplex PCR of 27 exons. Of these, 103 (64.4%) showed intragenic deletions. Most (69.7%) of the deletions involved exons 45-51. The phenotype of cases with deletion of single exons did not differ significantly from those with deletion of multiple exons. The distribution of deletions in studies from different countries was variable, but this was accounted for either by the small number of cases studied, or by fewer exons analyzed. It is concluded that there is likely to be no ethnic difference with respect to deletions in the DMD gene. 38 refs., 2 figs., 3 tabs.

  6. Exon deletion patterns of the dystrophin gene in 82 Vietnamese Duchenne/Becker muscular dystrophy patients.

    PubMed

    Tran, Van Khanh; Ta, Van Thanh; Vu, Dung Chi; Nguyen, Suong Thi-Bang; Do, Hai Ngoc; Ta, Minh Hieu; Tran, Thinh Huy; Matsuo, Masafumi

    2013-12-01

    Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common inherited muscle diseases caused by mutations in the dystrophin gene. The reading frame rule explains the genotype-phenotype relationship in DMD/BMD. In Vietnam, extensive mutation analysis has never been conducted in DMD/BMD. Here, 152 Vietnamese muscular dystrophy patients were examined for dystrophin exon deletion by amplifying 19 deletion-prone exons and deletion ends were confirmed by dystrophin cDNA analysis if necessary. The result was that 82 (54%) patients were found to have exon deletions, thus confirming exact deletion ends. A further result was that 37 patterns of deletion were classified. Deletions of exons 45-50 and 49-52 were the most common patterns identified, numbering six cases each (7.3%). The reading frame rule explained the genotype-phenotype relationship, but not five (6.1%) DMD cases. Each of five patients had deletions of exons 11-27 in common. The applicability of the therapy producing semifunctional in frame mRNA in DMD by inducing skipping of a single exon was examined. Induction of exon 51 skipping was ranked at top priority, since 16 (27%) patients were predicted to have semifunctional mRNA skipping. Exons 45 and 53 were the next ranked, with 12 (20%) and 11 (18%) patients, respectively. The largest deletion database of the dystrophin gene, established in Vietnamese DMD/BMD patients, disclosed a strong indication for exon-skipping therapy.

  7. Exon Skipping and Gene Transfer Restore Dystrophin Expression in Human Induced Pluripotent Stem Cells-Cardiomyocytes Harboring DMD Mutations

    PubMed Central

    Dick, Emily; Kalra, Spandan; Anderson, David; George, Vinoj; Ritso, Morten; Laval, Steven H.; Barresi, Rita; Aartsma-Rus, Annemieke; Lochmüller, Hanns

    2013-01-01

    With an incidence of ∼1:3,500 to 5,000 in male children, Duchenne muscular dystrophy (DMD) is an X-linked disorder in which progressive muscle degeneration occurs and affected boys usually die in their twenties or thirties. Cardiac involvement occurs in 90% of patients and heart failure accounts for up to 40% of deaths. To enable new therapeutics such as gene therapy and exon skipping to be tested in human cardiomyocytes, we produced human induced pluripotent stem cells (hiPSC) from seven patients harboring mutations across the DMD gene. Mutations were retained during differentiation and analysis indicated the cardiomyocytes showed a dystrophic gene expression profile. Antisense oligonucleotide-mediated skipping of exon 51 restored dystrophin expression to ∼30% of normal levels in hiPSC-cardiomyocytes carrying exon 47–50 or 48–50 deletions. Alternatively, delivery of a dystrophin minigene to cardiomyocytes with a deletion in exon 35 or a point mutation in exon 70 allowed expression levels similar to those seen in healthy cells. This demonstrates that DMD hiPSC-cardiomyocytes provide a novel tool to evaluate whether new therapeutics can restore dystrophin expression in the heart. PMID:23829870

  8. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations

    PubMed Central

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M.; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A.F.V.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  9. The emerging role of viral vectors as vehicles for DMD gene editing.

    PubMed

    Maggio, Ignazio; Chen, Xiaoyu; Gonçalves, Manuel A F V

    2016-05-23

    Duchenne muscular dystrophy (DMD) is a genetic disorder caused by mutations in the dystrophin-encoding DMD gene. The DMD gene, spanning over 2.4 megabases along the short arm of the X chromosome (Xp21.2), is the largest genetic locus known in the human genome. The size of DMD, combined with the complexity of the DMD phenotype and the extent of the affected tissues, begs for the development of novel, ideally complementary, therapeutic approaches. Genome editing based on the delivery of sequence-specific programmable nucleases into dystrophin-defective cells has recently enriched the portfolio of potential therapies under investigation. Experiments involving different programmable nuclease platforms and target cell types have established that the application of genome-editing principles to the targeted manipulation of defective DMD loci can result in the rescue of dystrophin protein synthesis in gene-edited cells. Looking towards translation into the clinic, these proof-of-principle experiments have been swiftly followed by the conversion of well-established viral vector systems into delivery agents for DMD editing. These gene-editing tools consist of zinc-finger nucleases (ZFNs), engineered homing endoculeases (HEs), transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases (RGNs) based on clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 systems. Here, we succinctly review these fast-paced developments and technologies, highlighting their relative merits and potential bottlenecks, when used as part of in vivo and ex vivo gene-editing strategies.

  10. Relatively low proportion of dystrophin gene deletions in Israeili Duchenne and Becker muscular dystrophy patients

    SciTech Connect

    Shomrat, R.; Gluck, E.; Legum, C.; Shiloh, Y.

    1994-02-15

    Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the X-linked dystrophin gene. The most common mutations in western populations are deletions that are spread non-randomly throughout the gene. Molecular analysis of the dystrophin gene structure by hybridization of the full length cDNA to Southern blots and by PCR in 62 unrelated Israeli male DMD/BMD patients showed deletions in 23 (37%). This proportion is significantly lower than that found in European and North American populations (55-65%). Seventy-eight percent of the deletions were confined to exons 44-52, half of these exons 44-45, and the remaining 22% to exons 1 and 19. There was no correlation between the size of the deletion and the severity of the disease. All the deletions causing frameshift resulted in the DMD phenotypes. 43 refs., 1 fig., 1 tab.

  11. Molecular analysis of the Duchenne muscular dystrophy gene in Spanish individuals: Deletion detection and familial diagnosis

    SciTech Connect

    Patino, A.; Garcia-Delgado, M.; Narbona, J.

    1995-11-06

    Deletion studies were performed in 26 Duchenne muscular dystrophy (DMD) patients through amplification of nine different exons by multiplex polymerase chain reaction (PCR). DNA from paraffin-embedded muscle biopsies was analyzed in 12 of the 26 patients studied. Optimization of this technique is of great utility because it enables analysis of material stored in pathology archives. PCR deletion detection, useful in DMD-affected boys, is problematic in determining the carrier state in female relatives. For this reason, to perform familial linkage diagnosis, we made use of a dinucleotide repeat polymorphism (STRP, or short tandem repeat polymorphism) located in intron 49 of the gene. We designed a new pair of primers that enabled the detection of 22 different alleles in relatives in the 14 DMD families studied. The use of this marker allowed familial diagnosis in 11 of the 14 DMD families and detection of de novo deletions in 3 of the probands. 8 refs., 5 figs., 2 tabs.

  12. Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

    SciTech Connect

    Tennyson, C.N.; Worton, R.G.

    1994-09-01

    The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

  13. Comparative analysis of antisense oligonucleotide sequences targeting exon 53 of the human DMD gene: Implications for future clinical trials.

    PubMed

    Popplewell, Linda J; Adkin, Carl; Arechavala-Gomeza, Virginia; Aartsma-Rus, Annemieke; de Winter, Christa L; Wilton, Steve D; Morgan, Jennifer E; Muntoni, Francesco; Graham, Ian R; Dickson, George

    2010-02-01

    Duchenne muscular dystrophy (DMD) is caused by the lack of functional dystrophin protein, most commonly as a result of a range of out-of-frame mutations in the DMD gene. Modulation of pre-mRNA splicing with antisense oligonucleotides (AOs) to restore the reading frame has been demonstrated in vitro and in vivo, such that truncated but functional dystrophin is expressed. AO-induced skipping of exon 51 of the DMD gene, which could treat 13% of DMD patients, has now progressed to clinical trials. We describe here the methodical, cooperative comparison, in vitro (in DMD cells) and in vivo (in a transgenic mouse expressing human dystrophin), of 24 AOs of the phosphorodiamidate morpholino oligomer (PMO) chemistry designed to target exon 53 of the DMD gene, skipping of which could be potentially applicable to 8% of patients. A number of the PMOs tested should be considered worthy of development for clinical trial.

  14. The AAV-mediated and RNA-guided CRISPR/Cas9 system for gene therapy of DMD and BMD.

    PubMed

    Wang, Jing-Zhang; Wu, Peng; Shi, Zhi-Min; Xu, Yan-Li; Liu, Zhi-Jun

    2017-04-05

    Mutations in the dystrophin gene (Dmd) result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), which afflict many newborn boys. In 2016, Brain and Development published several interesting articles on DMD treatment with antisense oligonucleotide, kinase inhibitor, and prednisolone. Even more strikingly, three articles in the issue 6271 of Science in 2016 provide new insights into gene therapy of DMD and BMD via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). In brief, adeno-associated virus (AAV) vectors transport guided RNAs (gRNAs) and Cas9 into mdx mouse model, gRNAs recognize the mutated Dmd exon 23 (having a stop codon), and Cas9 cut the mutated exon 23 off the Dmd gene. These manipulations restored expression of truncated but partially functional dystrophin, improved skeletal and cardiac muscle function, and increased survival of mdx mice significantly. This review concisely summarized the related advancements and discussed their primary implications in the future gene therapy of DMD, including AAV-vector selection, gRNA designing, Cas9 optimization, dystrophin-restoration efficiency, administration routes, and systemic and long-term therapeutic efficacy. Future orientations, including off-target effects, safety concerns, immune responses, precision medicine, and Dmd-editing in the brain (potentially blocked by the blood-brain barrier) were also elucidated briefly. Collectively, the AAV-mediated and RNA-guided CRISPR/Cas9 system has major superiorities compared with traditional gene therapy, and might contribute to the treatment of DMD and BMD substantially in the near future.

  15. Dissecting the structure and mechanism of a complex duplication-triplication rearrangement in the DMD gene.

    PubMed

    Ishmukhametova, Aliya; Chen, Jian-Min; Bernard, Rafaëlle; de Massy, Bernard; Baudat, Frédéric; Boyer, Amandine; Méchin, Déborah; Thorel, Delphine; Chabrol, Brigitte; Vincent, Marie-Claire; Khau Van Kien, Philippe; Claustres, Mireille; Tuffery-Giraud, Sylvie

    2013-08-01

    Pathogenic complex genomic rearrangements are being increasingly characterized at the nucleotide level, providing unprecedented opportunities to evaluate the complexities of mutational mechanisms. Here, we report the molecular characterization of a complex duplication-triplication rearrangement involving exons 45-60 of the DMD gene. Inverted repeats facilitated this complex rearrangement, which shares common genomic organization with the recently described duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) events; specifically, a 690-kb region comprising DMD exons from 45 to 60 was duplicated in tandem, and another 46-kb segment containing exon 51 was inserted inversely in between them. Taking into consideration (1) the presence of a predicted PRDM9 binding site in the near vicinity of the junction involving two inverted L1 elements and (2) the inherent properties of X-Y chromosome recombination during male meiosis, we proposed an alternative two-step model for the generation of this X-linked DMD DUP-TRP/INV-DUP event.

  16. DMD mutation spectrum analysis in 613 Chinese patients with dystrophinopathy.

    PubMed

    Guo, Ruolan; Zhu, Guosheng; Zhu, Huimin; Ma, Ruiyu; Peng, Ying; Liang, Desheng; Wu, Lingqian

    2015-08-01

    Dystrophinopathy is a group of inherited diseases caused by mutations in the DMD gene. Within the dystrophinopathy spectrum, Duchenne and Becker muscular dystrophies are common X-linked recessive disorders that mainly feature striated muscle necrosis. We combined multiplex ligation-dependent probe amplification with Sanger sequencing to detect large deletions/duplications and point mutations in the DMD gene in 613 Chinese patients. A total of 571 (93.1%) patients were diagnosed, including 428 (69.8%) with large deletions/duplications and 143 (23.3%) with point mutations. Deletion/duplication breakpoints gathered mostly in introns 44-55. Reading frame rules could explain 88.6% of deletion mutations. We identified seventy novel point mutations that had not been previously reported. Spectrum expansion and genotype-phenotype analysis of DMD mutations on such a large sample size in Han Chinese population would provide new insights into the pathogenic mechanism underlying dystrophinopathies.

  17. Xp21 contiguous gene syndromes: Deletion quantitation with bivariate flow karyotyping allows mapping of patient breakpoints

    SciTech Connect

    McCabe, E.R.B.; Towbin, J.A. ); Engh, G. van den; Trask, B.J. )

    1992-12-01

    Bivariate flow karyotyping was used to estimate the deletion sizes for a series of patients with Xp21 contiguous gene syndromes. The deletion estimates were used to develop an approximate scale for the genomic map in Xp21. The bivariate flow karyotype results were compared with clinical and molecular genetic information on the extent of the patients' deletions, and these various types of data were consistent. The resulting map spans >15 Mb, from the telomeric interval between DXS41 (99-6) and DXS68 (1-4) to a position centromeric to the ornithine transcarbamylase locus. The deletion sizing was considered to be accurate to [plus minus]1 Mb. The map provides information on the relative localization of genes and markers within this region. For example, the map suggests that the adrenal hypoplasia congenita and glycerol kinase genes are physically close to each other, are within 1-2 Mb of the telomeric end of the Duchenne muscular dystrophy (DMD) gene, and are nearer to the DMD locus than to the more distal marker DXS28 (C7). Information of this type is useful in developing genomic strategies for positional cloning in Xp21. These investigations demonstrate that the DNA from patients with Xp21 contiguous gene syndromes can be valuable reagents, not only for ordering loci and markers but also for providing an approximate scale to the map of the Xp21 region surrounding DMD. 44 refs., 3 figs.

  18. Direct deletion analysis in two Duchenne muscular dystrophy symptomatic females using polymorphic dinucleotide (CA)n loci within the dystrophin gene.

    PubMed

    Giliberto, Florencia; Ferreiro, Verónica; Dalamón, Viviana; Surace, Ezequiel; Cotignola, Javier; Esperante, Sebastián; Borelina, Daniel; Baranzini, Sergio; Szijan, Irene

    2003-03-31

    Duchenne muscular dystrophy (DMD) is the most common hereditary neuromuscular disease. It is inherited as an X-linked recessive trait in which males show clinical manifestations. In some rare cases, the disease can also be manifested in females. The aim of the present study was to determine the molecular alteration in two cases of nonrelated DMD symptomatic carriers with no previous history of DMD. Multiplex PCR is commonly used to search for deletion in the DMD gene of affected males. This method could not be used in females because the normal X chromosome masks the deletion of the mutated one. Therefore, we used a set of seven highly polymorphic dinucleotide (CA)(n) repeat markers that lie within the human dystrophin gene. The deletions were evidenced by hemizygosity of the loci under study. We localized a deletion in the locus 7A (intron 7) on the maternal X chromosome in one case, and a deletion in the region of introns 49 and 50 on the paternal X chromosome in the other. The use of microsatellite genotyping within the DMD gene enables the detection of the mutant allele in female carriers. It is also a useful method to provide DMD families with more accurate genetic counseling.

  19. Screening of deletions in the dystrophin gene with the cDNA probes Cf23a, Cf56a, and Cf115.

    PubMed Central

    Passos-Bueno, M R; Rapaport, D; Love, D; Flint, T; Bortolini, E R; Zatz, M; Davies, K E

    1990-01-01

    We have analysed 38 DMD patients from 34 families and 30 BMD patients from 12 families using the cDNA probes Cf23a and Cf56a, which map near the centre of the dystrophin gene, and Cf115, which is close to the 3' end of this gene. Together, probes Cf23a and Cf56a detected deletions in 50% of the DMD families and 33% of the BMD families. Probe Cf115 detected a deletion in only one DMD patient, which has not been reported before in severe X linked myopathy. Most of the DMD deletions could be detected with Cf56a while all four BMD deletions were detected with Cf23a. The pattern of deletions could not be used to predict the precise clinical course of the disease and no correlation was found between the severity of the disease and the extent of the gene deletion. A higher frequency of deletions was observed in sporadic (73%) compared with familial DMD (28%) and BMD cases (33%). This result, if confirmed in a larger sample, would have important implications for genetic counselling. Images PMID:2182872

  20. Pattern of deletions of the dystrophin gene in Mexican Duchenne/Becker muscular dystrophy patients: the use of new designed primers for the analysis of the major deletion "hot spot" region.

    PubMed

    Coral-Vazquez, R; Arenas, D; Cisneros, B; Peñaloza, L; Salamanca, F; Kofman, S; Mercado, R; Montañez, C

    1997-06-13

    We have analyzed 59 unrelated Mexican Duchenne/Becker muscular dystrophy patients (DMD/BMD) using PCR analysis of the 2 prone deletion regions in the DMD gene. Thirty one (52%) of the patients had a deletion of one or several of the exons. Most of the alterations (87%) were clustered in exons 44-52, this being the highest percentage reported until now. In order to improve the molecular diagnosis in the Mexican population, we designed a new multiplex assay to PCR amplify exons 44-52. This assay allowed for the identification of a greater number of deletions in this region compared with the 9 and 5-plex assays previously described and to determine most of the deletion end boundaries. This is a reliable alternative for the initial screening of the DMD patients in the Mexican population.

  1. A family with autism and rare copy number variants disrupting the Duchenne/Becker muscular dystrophy gene DMD and TRPM3.

    PubMed

    Pagnamenta, Alistair T; Holt, Richard; Yusuf, Mohammed; Pinto, Dalila; Wing, Kirsty; Betancur, Catalina; Scherer, Stephen W; Volpi, Emanuela V; Monaco, Anthony P

    2011-06-01

    Autism spectrum disorder is a genetically complex and clinically heterogeneous neurodevelopmental disorder. A recent study by the Autism Genome Project (AGP) used 1M single-nucleotide polymorphism arrays to show that rare genic copy number variants (CNVs), possibly acting in tandem, play a significant role in the genetic aetiology of this condition. In this study, we describe the phenotypic and genomic characterisation of a multiplex autism family from the AGP study that was found to harbour a duplication of exons 31-44 of the Duchenne/Becker muscular dystrophy gene DMD and also a rare deletion involving exons 1-9 of TRPM3. Further characterisation of these extremely rare CNVs was carried out using quantitative PCR, fluorescent in situ hybridisation, long-range PCR amplification and sequencing of junction fragments. The maternal chrX:32,097,213-32,321,945 tandem duplication and paternal chr9:72,480,413-73,064,196 deletion (NCBI build 36 coordinates) were transmitted to both affected boys, potentially signifying a multi-hit mechanism. The DMD reading frame rule predicts a Becker phenotype, characterised by later onset and milder symptoms. When last evaluated, neither child had developed signs of muscular dystrophy. These data are consistent with a degree of comorbidity between autism and muscular dystrophy and suggest that genomic background as well as the position of the mutation within the DMD gene may impact on the neurological correlates of Duchenne/Becker muscular dystrophy. Finally, communicating unexpected findings such as these back to families raises a number of ethical questions, which are discussed.

  2. Identification of two point mutations and a one base deletion in exon 19 of the dystrophin gene by heteroduplex formation.

    PubMed

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Sedra, M S; Western, L M; Bartello, C; Mendell, J R

    1993-03-01

    Two thirds of the Duchenne muscular dystrophy population have either gene deletions or duplications. The nondeletion/duplication cases are most likely the result of point mutations or small deletions and duplications that cannot be easily identified by current strategies. The major obstacle in identifying small mutations is due to the large size of the dystrophin gene. We selectively screened 5 DMD exons containing CpG dinucleotides in 110 DMD patients without detectable deletions or duplications. Nonsenses mutations are frequently due to a C- to -T transition within a CG dinucleotide pair. To screen for the nonsense mutations, we used the heteroduplex method. Utilizing this approach, we identified 2 different nonsense mutations and a single base deletion all occurring in exon 19. This is the first report of a clustering of small mutations in the dystrophin gene.

  3. Contrasting evolutionary histories of two introns of the duchenne muscular dystrophy gene, Dmd, in humans.

    PubMed Central

    Nachman, M W; Crowell, S L

    2000-01-01

    The Duchenne muscular dystrophy (Dmd) locus lies in a region of the X chromosome that experiences a high rate of recombination and is thus expected to be relatively unaffected by the effects of selection on nearby genes. To provide a picture of nucleotide variability at a high-recombination locus in humans, we sequenced 5. 4 kb from two introns of Dmd in a worldwide sample of 41 alleles from Africa, Asia, Europe, and the Americas. These same regions were also sequenced in one common chimpanzee and one orangutan. Dramatically different patterns of genetic variation were observed at these two introns, which are separated by >500 kb of DNA. Nucleotide diversity at intron 44 pi = 0.141% was more than four times higher than nucleotide diversity at intron 7 pi = 0.034% despite similar levels of divergence for these two regions. Intron 7 exhibited significant linkage disequilibrium extending over 10 kb and also showed a significant excess of rare polymorphisms. In contrast, intron 44 exhibited little linkage disequilibrium and no skew in the frequency distribution of segregating sites. Intron 7 was much more variable in Africa than in other continents, while intron 44 displayed similar levels of variability in different geographic regions. Comparison of intraspecific polymorphism to interspecific divergence using the HKA test revealed a significant reduction in variability at intron 7 relative to intron 44, and this effect was most pronounced in the non-African samples. These results are best explained by positive directional selection acting at or near intron 7 and demonstrate that even genes in regions of high recombination may be influenced by selection at linked sites. PMID:10924480

  4. Contiguous gene deletion syndrome in a female with ornithine transcarbamylase deficiency.

    PubMed

    Balasubramaniam, S; Rudduck, C; Bennetts, B; Peters, G; Wilcken, B; Ellaway, C

    2010-01-01

    OTC deficiency, a partially dominant X-linked trait, is the most frequent inborn error of the urea cycle. We describe a female patient with a contiguous gene deletion syndrome encompassing the OTC, DMD, RPGR, CYBB and XK genes, amongst others, only manifesting features of OTC deficiency. Molecular characterization was ascertained by MLPA and confirmed by CGH microarray, which revealed an 8.7 Mb deletion of the X-chromosome. Complete de novo deletion of the OTC gene led to a severe clinical phenotype in the proband. The application of high resolution molecular genetic techniques such as MLPA and array CGH, in mutation negative OTC cases allows the identification of chromosomal rearrangements, such as large deletions and provides information for accurate genetic counseling and prenatal diagnosis.

  5. Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells.

    PubMed

    van Deutekom, J C; Bremmer-Bout, M; Janson, A A; Ginjaar, I B; Baas, F; den Dunnen, J T; van Ommen, G J

    2001-07-15

    Due to frame-shifting mutations in the DMD gene that cause dystrophin deficiency, Duchenne muscular dystrophy (DMD) patients suffer from lethal muscle degeneration. In contrast, mutations in the allelic Becker muscular dystrophy (BMD) do not disrupt the translational reading frame, resulting in a less severe phenotype. In this study, we explored a genetic therapy aimed at restoring the reading frame in muscle cells from DMD patients through targeted modulation of dystrophin pre-mRNA splicing. Considering that exon 45 is the single most frequently deleted exon in DMD, whereas exon (45+46) deletions cause only a mild form of BMD, we set up an antisense-based system to induce exon 46 skipping from the transcript in cultured myotubes of both mouse and human origin. In myotube cultures from two unrelated DMD patients carrying an exon 45 deletion, the induced skipping of exon 46 in only approximately 15% of the mRNA led to normal amounts of properly localized dystrophin in at least 75% of myotubes. Our results provide first evidence of highly effective restoration of dystrophin expression from the endogenous gene in DMD patient-derived muscle cells. This strategy may be applicable to not only >65% of DMD mutations, but also many other genetic diseases.

  6. Adeno-Associated Virus (AAV) Mediated Dystrophin Gene Transfer Studies and Exon Skipping Strategies for Duchenne Muscular Dystrophy (DMD).

    PubMed

    Kawecka, Klaudia; Theodoulides, Michael; Hasoglu, Yalin; Jarmin, Susan; Kymalainen, Hanna; Le-Heron, Anita; Popplewell, Linda; Malerba, Alberto; Dickson, George; Athanasopoulos, Takis

    2015-01-01

    Duchenne muscular dystrophy (DMD), an X-linked inherited musclewasting disease primarily affecting young boys with prevalence of between1:3,500- 1:5,000, is a rare genetic disease caused by defects in the gene for dystrophin. Dystrophin protein is critical to the stability of myofibers in skeletal and cardiac muscle. There is currently no cure available to ameliorate DMD and/or its patho-physiology. A number of therapeutic strategies including molecular-based therapeutics that replace or correct the missing or nonfunctional dystrophin protein have been devised to correct the patho-physiological consequences induced by dystrophin absence. We will review the current in vivo experimentation status (including preclinical models and clinical trials) for two of these approaches, namely: 1) Adeno-associated virus (AAV) mediated (micro) dystrophin gene augmentation/ supplementation and 2) Antisense oligonucleotide (AON)-mediated exon skipping strategies.

  7. DMD and IL1RAPL1: two large adjacent genes localized within a common fragile site (FRAXC) have reduced expression in cultured brain tumors.

    PubMed

    McAvoy, S; Ganapathiraju, S; Perez, D S; James, C D; Smith, D I

    2007-01-01

    Common fragile sites (CFSs) are large regions of profound genomic instability found in all individuals. Spanning the center of the two most frequently expressed CFS regions, FRA3B (3p14.3) and FRA16D (16q23.2), are the 1.5 Mb FHIT gene and the 1.0 Mb WWOX gene. These genes are frequently deleted and/or altered in many different cancers. Both FHIT and WWOX have been demonstrated to function as tumor suppressors, both in vitro and in vivo. A number of other large CFS genes have been identified and are also frequently inactivated in multiple cancers. Based on these data, several additional very large genes were tested to determine if they were derived from within CFS regions, but DCC and RAD51L1 were not. However, the 2.0 Mb DMD gene and its immediately distal neighbor, the 1.8 Mb IL1RAPL1 gene are CFS genes contained within the FRAXC CFS region (Xp21.2-->p21.1). They are abundantly expressed in normal brain but were dramatically underexpressed in every brain tumor cell line and xenograft (derived from an intracranial model of glioblastoma multiforme) examined. We studied the expression of eleven other large CFS genes in the same panel of brain tumor cell lines and xenografts and found reduced expression of multiple large CFS genes in these samples. In this report we show that there is selective loss of specific large CFS genes in different cancers that does not appear to be mediated by the relative instability within different CFS regions. Further, the inactivation of multiple large CFS genes in xenografts and brain tumor cell lines may help to explain why this type of cancer is highly aggressive and associated with a poor clinical outcome.

  8. Sequence characterisation of deletion breakpoints in the dystrophin gene by PCR

    SciTech Connect

    Abbs, S.; Sandhu, S.; Bobrow, M.

    1994-09-01

    Partial deletions of the dystrophin gene account for 65% of cases of Duchenne muscular dystrophy. A high proportion of these structural changes are generated by new mutational events, and lie predominantly within two `hotspot` regions, yet the underlying reasons for this are not known. We are characterizing and sequencing the regions surrounding deletion breakpoints in order to: (i) investigate the mechanisms of deletion mutation, and (ii) enable the design of PCR assays to specifically amplify mutant and normal sequences, allowing us to search for the presence of somatic mosaicism in appropriate family members. Using this approach we have been able to demonstrate the presence of somatic mosaicism in a maternal grandfather of a DMD-affected male, deleted for exons 49-50. Three deletions, namely of exons 48-49, 49-50, and 50, have been characterized using a PCR approach that avoids any cloning procedures. Breakpoints were initially localized to within regions of a few kilobases using Southern blot restriction analyses with exon-specific probes and PCR amplification of exonic and intronic loci. Sequencing was performed directly on PCR products: (i) mutant sequences were obtained from long-range or inverse-PCR across the deletion junction fragments, and (ii) normal sequences were obtained from the products of standard PCR, vectorette PCR, or inverse-PCR performed on YACs. Further characterization of intronic sequences will allow us to amplify and sequence across other deletion breakpoints and increase our knowledge of the mechanisms of mutation in the dystophin gene.

  9. Targeted RNA-Seq profiling of splicing pattern in the DMD gene: exons are mostly constitutively spliced in human skeletal muscle

    PubMed Central

    Bougé, Anne-Laure; Murauer, Eva; Beyne, Emmanuelle; Miro, Julie; Varilh, Jessica; Taulan, Magali; Koenig, Michel; Claustres, Mireille; Tuffery-Giraud, Sylvie

    2017-01-01

    We have analysed the splicing pattern of the human Duchenne Muscular Dystrophy (DMD) NB transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3′ splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications. PMID:28045018

  10. Capillary electrophoresis for analysis of deletion and duplication in exon 44-55 of Duchenne muscular dystrophy gene.

    PubMed

    Chen, Chung-An; Chang, Ming-Yuh; Chang, Tung-Ming; Jong, Yuh-Jyh; Wu, Shou-Mei

    2013-09-01

    In this study, a genotyping CGE method was established for analysis of Duchenne muscular dystrophy (DMD) gene deletions and duplications in exon 44-55. A total of 12 DMD exons (exon 44-55) and 2 internal standard gene fragments were simultaneously amplified by using a universal multiplex PCR (UMPCR) and determined by CGE. The conditions of UMPCR and CGE were optimized, including the kinds of polymerase, temperatures in UMPCR, separation matrix, separation temperature, and voltage. Finally, the separation was performed by 1.2% poly(ethylene oxide) in 1× TBE buffer at -6 kV and 25°C. After validation, our results showed the peak patterns for differentiation of genetic deletion or duplication in 27 DMD patients and normal subjects, according to the peak height ratios by comparison of two internal standard peaks. Among the 27 subjects, 23 cases are deletion type and four are duplication type. The data of two patients analyzed by this CGE-PCR method were different from that of multiplex ligation dependent probe amplification method, and the sequencing results demonstrated that our results were correct. This UMPCR-CGE method was considered better than the multiplex ligation dependent probe amplification method. Furthermore, this method can be used for eugenics in clinical applications.

  11. Electroretinographic genotype-phenotype correlations for mouse and man at the dmd/DMD locus

    SciTech Connect

    Millers, D.M.; Weleber, R.G.; Woodward, W.R.

    1994-09-01

    Reduced or absent b-waves in the dark-adapted electroretinogram (ERG) of Duchenne and Becker muscular dystrophy (DMD/BMD) patients led to the identification of dystrophin in human retina and the proposal that it plays a role in retinal electrophysiology. Study of a large group of Duchenne and Becker muscular dystrophy males to determine their ocular characteristics indicated that there were position-specific effects of deletions, with 3{prime} defects associated with severe electroretinographic changes, whereas some 5{prime} patients demonstrated less severe, or even normal, ERGs. We studied the mdx mouse, a model with X-linked muscular dystrophy and defective full-length dystrophin, which failed to show any ERG abnormalities. Given the presence of alternate isoforms of dystrophin in retina, and the 5{prime} deletion DMD/BMD patients with normal ERGs, we studied mouse models with differing dystrophin mutations (mdx{sup Cv3}, mdx{sup Cv5}) to determine the usefulness of alternate strains as models for the visual effects of dystropin. Abnormal ERGs similar to those seen in DMD/BMS patients exist in the mdx{sup Cv3} strain of muscular dystrophy mice. Normal ERGs were found the mdx{sup Cv5} strain. The mutations in the mdx and mdx{sup Cv5} mice have been mapped to the 5{prime} end of the dmd gene, while the mutation in the mdx{sup Cv3} mouse is in the 3{prime} end. Thus, there are position effects of the gene defect on the ERG phenotype that are conserved in the mouse. Such genotype-phenotype correlations may reflect differential expression of shorter isoforms of dystrophin.

  12. Detection of somatic mosaicism in DMD using computer-assisted laser densitometry

    SciTech Connect

    Sutherland, J.E.; Allingham-Hawkins, D.J.; MacKenzie, J.

    1994-09-01

    Approximately two-thirds of Duchenne muscular dystrophy (DMD) patients have a deletion in the dystrophin gene located at Xp21.1. Two PCR-based multiplex systems have been developed which detect 98% of deletions in affected males. Diagnosis of carrier females requires densitometry of PCR products following gel electrophoresis to calculate dosage of specific exons. We have developed a system in which fluorescently labelled PCR products are analysed using a GENESCANNER automated fragment analyser (ABI). Dosage is determined using computer-assisted laser densitometry (CALD). Recently, we diagnosed somatic mosaicism in the mother of an affected boy using this method. PCR analysis showed that the patient had a deletion that included exons 47-51 of his dystrophin gene. CALD analysis on the patient`s 36-year-old mother revealed a 29-34% reduction in the intensity of the bands corresponding to the deleted region of the gene rather than the 50% reduction normally seen in carrier females. A skin biopsy was obtain and monoclonal fibroblast colonies were tested by CALD for the deletion. Four of the twenty colonies screened were found to be deleted while the remaining colonies had two intact copies of the gene. We conclude that this patient is a somatic mosaic for DMD and that the mutation was the result of a post-zygotic event. This is the only case of somatic mosaicism detected among 800 women from 400 DMD families tested using CALD in our laboratory. At least one other case of possible somatic mosaicism has been reported but not confirmed. Germinal mosaicism is thought to occur in approximately 10% of mothers of sporadic DMD patients. Our findings indicate that somatic mosaicism is a much rarer condition among DMD carriers, thus suggesting that mitotic mutations in the dystrophin gene are more likely to occur later in embryogenesis after differentiation of the germline.

  13. Targeted gene deletion in Zygosaccharomyces bailii.

    PubMed

    Mollapour, M; Piper, P

    2001-01-30

    Yeasts of the genus Zygosaccharomyces are notable agents of large-scale food spoilage. Despite the economic importance of these organisms, little is known about the stress adaptations whereby they adapt to many of the more severe conditions of food preservation. In this study it was shown that genes of Z. bailii, a yeast notable for its high resistances to food preservatives and ethanol, can be isolated by complementation of the corresponding mutant strains of Saccharomyces cerevisiae. It was also discovered that the acquisition by S. cerevisiae of a single small Z. bailii gene (ZbYME2) was sufficient for the former yeast to acquire the ability to degrade two major food preservatives, benzoic acid and sorbic acid. Using DNA cassettes containing dominant selectable markers and methods originally developed for performing gene deletions in S. cerevisiae, the two copies of ZbYME2 in the Z. bailii genome were sequentially deleted. The resulting Zbyme2/Zbyme2 homozygous deletant strain had lost any ability to utilize benzoate as sole carbon source and was more sensitive to weak acid preservatives during growth on glucose. Thus, ZbYME2, probably the nuclear gene for a mitochondrial mono-oxygenase function, is essential for Z. bailii to degrade food preservatives. This ability to catabolize weak acid preservatives is a significant factor contributing to the preservative resistance of Z. bailii under aerobic conditions. This study is the first to demonstrate that it is possible to delete in Z. bailii genes that are suspected as being important for growth of this organism in preserved foods and beverages. With the construction of further mutant of Z. bailii strains, a clearer picture should emerge of how this yeast adapts to the conditions of food preservation. This information will, in turn, allow the design of new preservation strategies. GenBank Accession Nos: ZbURA3 (AF279259), ZbTIM9 (AF279260), ZbYME2 (AF279261), ZbTRP1 (AF279262), ZbHHT1(AF296170).

  14. Characterization of five partial deletions of the factor VIII gene

    SciTech Connect

    Youssoufian, H.; Antonarakis, S.E.; Aronis, S.; Tsiftis, G.; Phillips, D.G.; Kazazian, H.H. Jr.

    1987-06-01

    Hemophilia A is an X-linked disorder of coagulation caused by a deficiency of factor VIII. By using cloned DNA probes, the authors have characterized the following five different partial deletions of the factor VIII gene from a panel of 83 patients with hemophilia A: (i) a 7-kilobase (kb) deletion that eliminates exon 6; (ii) a 2.5-kb deletion that eliminates 5' sequences of exon 14; (iii) a deletion of at least 7 kb that eliminates exons 24 and 25; (iv) a deletion of at least 16 kb that eliminates exons 23-25; and (v) a 5.5-kb deletion that eliminates exon 22. The first four deletions are associated with severe hemophilia A. By contrast, the last deletion is associated with moderate disease, possibly because of in-frame splicing from adjacent exons. None of those patients with partial gene deletions had circulating inhibitors to factor VIII. One deletion occurred de novo in a germ cell of the maternal grandmother, while a second deletion occurred in a germ cell of the maternal grandfather. These observations demonstrate that de novo deletions of X-linked genes can occur in either male or female gametes.

  15. Pregnancy after preimplantation diagnosis for a deletion in the dystrophin gene by polymerase chain reaction in embryos obtained after intracytoplasmic sperm injection

    SciTech Connect

    Lissens, W.; Liu, J.; Van Broeckhoven, C.

    1994-09-01

    Duchenne muscular dystrophy (DMD) is one of the most common X-linked recessive diseases. In order to be able to perform a DMD-specific preimplantation diagnosis (PID) in a female carrier of a deletion of exons 3 to 18 in the dystrophin gene, we have developed a PCR assay to detect the deletion based on sequences of exon 17. The efficiency of this PCR was evaluated on 50 single blastomeres from 12 normal control embryos and on 41 blastomeres for 9 male and 3 female embryos from the female DMD carrier, obtained after a first preimplantation diagnosis by sexing. The exon 17 region was amplified with 100% efficiency, except in all 21 blastomeres from 6 male embryos from the carrier where no PCR signals were observed. The negative results in these blastomeres were interpreted as being found only in male embryos carrying the deletion. Intracytoplasmic sperm injection was carried out on the carrier`s metaphase II oocytes retrieved after ovarian stimulation. Embryos were analyzed for the presence of exon 17 and 2 male embryos were found to be deleted, while 4 embryos showed normal amplification signals. Three of the latter embryos were replaced, resulting in a singleton pregnancy. Amniotic cell analysis showed a normal female karyotype and DNA analysis indicated a non-carrier.

  16. Muscular dystrophy in the Japanese Spitz: an inversion disrupts the DMD and RPGR genes.

    PubMed

    Atencia-Fernandez, Sabela; Shiel, Robert E; Mooney, Carmel T; Nolan, Catherine M

    2015-04-01

    An X-linked muscular dystrophy, with deficiency of full-length dystrophin and expression of a low molecular weight dystrophin-related protein, has been described in Japanese Spitz dogs. The aim of this study was to identify the causative mutation and develop a specific test to identify affected cases and carrier animals. Gene expression studies in skeletal muscle of an affected animal indicated aberrant expression of the Duchenne muscular dystrophy (dystrophin) gene and an anomaly in intron 19 of the gene. Genome-walking experiments revealed an inversion that interrupts two genes on the X chromosome, the Duchenne muscular dystrophy gene and the retinitis pigmentosa GTPase regulator gene. All clinically affected dogs and obligate carriers that were tested had the mutant chromosome, and it is concluded that the inversion is the causative mutation for X-linked muscular dystrophy in the Japanese Spitz breed. A PCR assay that amplifies mutant and wild-type alleles was developed and proved capable of identifying affected and carrier individuals. Unexpectedly, a 7-year-old male animal, which had not previously come to clinical attention, was shown to possess the mutant allele and to have a relatively mild form of the disease. This observation indicates phenotypic heterogeneity in Japanese Spitz muscular dystrophy, a feature described previously in humans and Golden Retrievers. With the availability of a simple, fast and accurate test for Japanese Spitz muscular dystrophy, detection of carrier animals and selected breeding should help eliminate the mutation from the breed.

  17. Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics

    PubMed Central

    Calì, Francesco; Ruggeri, Giuseppa; Vinci, Mirella; Meli, Concetta; Carducci, Carla; Leuzzi, Vincenzo; Pozzessere, Simone; Schinocca, Pietro; Ragalmuto, Alda; Chiavetta, Valeria; Miccichè, Salvatore

    2010-01-01

    A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH) alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation- dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8 (systematic name c.353-?_912 + ?del) and exon 6 (systematic name c.510-?_706 + ?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics. PMID:19946181

  18. Comprehensive Analysis of Pathogenic Deletion Variants in Fanconi Anemia Genes

    PubMed Central

    Flynn, Elizabeth K.; Kamat, Aparna; Lach, Francis P.; Donovan, Frank X.; Kimble, Danielle C.; Narisu, Narisu; Sanborn, Erica; Boulad, Farid; Davies, Stella M.; Gillio, Alfred P.; Harris, Richard E.; MacMillan, Margaret L.; Wagner, John E.; Smogorzewska, Agata; Auerbach, Arleen D.; Ostrander, Elaine A.; Chandrasekharappa, Settara C.

    2014-01-01

    Fanconi anemia (FA) is a rare recessive disease resulting from mutations in one of at least 16 different genes. Mutation types and phenotypic manifestations of FA are highly heterogeneous and influence the clinical management of the disease. We analyzed 202 FA families for large deletions, using high-resolution Comparative Genome Hybridization arrays (arrayCGH), Single Nucleotide Polymorphism arrays (SNParrays) and DNA sequencing. We found pathogenic deletions in 88 FANCA, seven FANCC, two FANCD2, and one FANCB families. We find 35% of FA families carry large deletions, accounting for 18% of all FA pathogenic variants. Cloning and sequencing across the deletion breakpoints revealed that 52 FANCA deletion ends, and one FANCC deletion end extended beyond the gene boundaries, potentially affecting neighboring genes with phenotypic consequences. Seventy-five percent of the FANCA deletions are Alu-Alu mediated, predominantly by AluY elements, and appear to be caused by Non-Allelic Homologous Recombination. Individual Alu hotspots were identified. Defining the haplotypes of four FANCA deletions shared by multiple families revealed that three share a common ancestry. Knowing the exact molecular changes that lead to the disease may be critical for a better understanding of the FA phenotype, and to gain insight into the mechanisms driving these pathogenic deletion variants. PMID:25168418

  19. Characterization of a lymphoblastoid line deleted for lambda immunoglobulin genes

    SciTech Connect

    Hough, C.A., White, B.N., Holden, J.A.

    1995-04-01

    While characterizing the cat eye syndrome (CES) supernumerary chromosome for the presence of {lambda} immunoglobulin gene region sequences, a lymphoblastoid cell line from one CES patient was identified in which there was selection of cells deleted from some IGLC and IGLV genes. Two distinct deletions, one on each chromosome 22, were identified, presumably arising from independent somatic recombination events occurring during B-lymphocyte differentiation. The extent of the deleted regions was determined using probes from the various IGLV subgroups and they each covered at least 82 kilobases. The precise definition of the deletions was not possible because of conservation of some restriction sites in the IGLV region. The cell line was used to map putative IGLV genes within the recombinant phage {lambda}V{lambda}135 to the distal part of the IGLV gene region. 35 refs., 4 figs.

  20. Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot

    PubMed Central

    Suzuki, Hitoshi; Aoki, Yoshitsugu; Kameyama, Toshiki; Saito, Takashi; Masuda, Satoru; Tanihata, Jun; Nagata, Tetsuya; Mayeda, Akila; Takeda, Shin’ichi; Tsukahara, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45–55 of the DMD gene, might improve patients’ symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45–55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44–56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5′ splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing. PMID:27754374

  1. A Prospective Study in the Rational Design of Efficient Antisense Oligonucleotides for Exon Skipping in the DMD Gene

    PubMed Central

    Wee, Keng Boon; Wang, Jian Li; Chen, Yi Jun; Xiong, Qian Bin; Lai, Poh San; Yee, Woon Chee

    2012-01-01

    Abstract Antisense oligonucleotide (AON)-mediated exon skipping to restore dystrophin expression in Duchenne muscular dystrophy (DMD) therapy shown promise in a number of human clinical trials. Current AON design methods are semi-empirical, involving either trial-and-error and/or preliminary experimentations. Therefore, a rational approach to design efficient AONs to address the wide spectrum of patients' mutations is desirable. Retrospective studies have extracted many AON design variables, but they were not tested prospectively to design AONs for skipping DMD exons. Not only did the variables differ among the various studies, no numerical cutoff for each variable was inferred, which makes their use in AON design difficult. The challenge is to thus select a minimal set of key independent variables that can consistently design efficient AONs. In this prospective study, a novel set of design variables with respective cutoff values was used to design 23 novel AONs, each to skip one of nine DMD exons. Nineteen AONs were found to be efficacious in inducing specific exon skipping (83% of total), of which 14 were considered efficient (61% of total), i.e., they induced exon skipping in >25% of total transcripts. Notably, the satisfactory success rates were achieved by using only three design variables; namely, co-transcriptional binding accessibility of target site, presence of exonic splicing enhancers, and target length. Retrospective analyses revealed that the most efficient AON in every exon targeted has the lowest average cumulative position (ACP) score. Taking the prospective and retrospective studies together, we propose that design guidelines recommend using the ACP score to select the most efficient AON for each exon. PMID:22486275

  2. Screening Duchenne and Becker muscular dystrophy patients for deletions in 30 exons of the dystrophin gene by three-multiplex PCR

    SciTech Connect

    Risch, N. )

    1992-09-01

    Deletion mutations of the dystrophin gene may cause either the severe Duchenne muscular dystrophy (DMD) or the milder, allelic Becker muscular dystrophy (BMD) and are clustered in two high-frequency-deletion regions (HFDRs) located, respectively, 500 kb and 1,200 kb downstream from the 5[prime] end of the gene. Three PCR reactions described allowed the analysis of a total of 30 exons and led, to the identification of three additional deletions involving the following exons: (a) 42 only, (b) 28-42, and (c) 16 only, none of which were detected with the two original multiplex reactions. Therefore, the three modified multiplexes detected 95 of the 96 deletions identified among the 152 patients studied so far by using Southern analysis and cDNA probes. The only deletion that remained undetected with this system involves exons 22-25 and generates the junction fragment described elsewhere. The percentage of deletion mutations among DMS/BMD patients amounts to 63%, which is in agreement with similar estimates from other laboratories. When field-inversion gel electrophoresis is coupled to Southern analysis, the detection rate of deletion and duplication mutations reaches 65%.

  3. Targeted next-generation sequencing as a comprehensive test for patients with and female carriers of DMD/BMD: a multi-population diagnostic study.

    PubMed

    Wei, Xiaoming; Dai, Yi; Yu, Ping; Qu, Ning; Lan, Zhangzhang; Hong, Xiafei; Sun, Yan; Yang, Guanghui; Xie, Shuqi; Shi, Quan; Zhou, Hanlin; Zhu, Qian; Chu, Yuxing; Yao, Fengxia; Wang, Jinming; He, Jingni; Yang, Yun; Liang, Yu; Yang, Yi; Qi, Ming; Yang, Ling; Wang, Wei; Wu, Haitao; Duan, Jing; Shen, Cheng; Wang, Jun; Cui, Liying; Yi, Xin

    2014-01-01

    Duchenne and Becker muscular dystrophies (DMD/BMD) are the most commonly inherited neuromuscular disease. However, accurate and convenient molecular diagnosis cannot be achieved easily because of the enormous size of the dystrophin gene and complex causative mutation spectrum. Such traditional methods as multiplex ligation-dependent probe amplification plus Sanger sequencing require multiple steps to fulfill the diagnosis of DMD/BMD. Here, we introduce a new single-step method for the genetic analysis of DMD patients and female carriers in real clinical settings and demonstrate the validation of its accuracy. A total of 89 patients, 18 female carriers and 245 non-DMD patients were evaluated using our targeted NGS approaches. Compared with traditional methods, our new method yielded 99.99% specificity and 98.96% sensitivity for copy number variations detection and 100% accuracy for the identification of single-nucleotide variation mutations. Additionally, this method is able to detect partial deletions/duplications, thus offering precise personal DMD gene information for gene therapy. We detected novel partial deletions of exons in nine samples for which the breakpoints were located within exonic regions. The results proved that our new method is suitable for routine clinical practice, with shorter turnaround time, higher accuracy, and better insight into comprehensive genetic information (detailed breakpoints) for ensuing gene therapy.

  4. Gene correction of a duchenne muscular dystrophy mutation by meganuclease-enhanced exon knock-in.

    PubMed

    Popplewell, Linda; Koo, Taeyoung; Leclerc, Xavier; Duclert, Aymeric; Mamchaoui, Kamel; Gouble, Agnés; Mouly, Vincent; Voit, Thomas; Pâques, Frédéric; Cédrone, Frédéric; Isman, Olga; Yáñez-Muñoz, Rafael J; Dickson, George

    2013-07-01

    Duchenne muscular dystrophy (DMD) is a severe inherited, muscle-wasting disorder caused by mutations in the DMD gene. Gene therapy development for DMD has concentrated on vector-based DMD minigene transfer, cell-based gene therapy using genetically modified adult muscle stem cells or healthy wild-type donor cells, and antisense oligonucleotide-induced exon-skipping therapy to restore the reading frame of the mutated DMD gene. This study is an investigation into DMD gene targeting-mediated correction of deletions in human patient myoblasts using a target-specific meganuclease (MN) and a homologous recombination repair matrix. The MN was designed to cleave within DMD intron 44, upstream of a deletion hotspot, and integration-competent lentiviral vectors expressing the nuclease (LVcMN) were generated. MN western blotting and deep gene sequencing for LVcMN-induced non-homologous end-joining InDels (microdeletions or microinsertions) confirmed efficient MN expression and activity in transduced DMD myoblasts. A homologous repair matrix carrying exons 45-52 (RM45-52) was designed and packaged into integration-deficient lentiviral vectors (IDLVs; LVdRM45-52). After cotransduction of DMD myoblasts harboring a deletion of exons 45 to 52 with LVcMN and LVdRM45-52 vectors, targeted knock-in of the RM45-52 region in the correct location in DMD intron 44, and expression of full-length, correctly spliced wild-type dystrophin mRNA containing exons 45-52 were observed. This work demonstrates that genome surgery on human DMD gene mutations can be achieved by MN-induced locus-specific genome cleavage and homologous recombination knock-in of deleted exons. The feasibility of human DMD gene repair in patient myoblasts has exciting therapeutic potential.

  5. Gene deletion in an Italian haemophilia B subject.

    PubMed Central

    Bernardi, F; del Senno, L; Barbieri, R; Buzzoni, D; Gambari, R; Marchetti, G; Conconi, F; Panicucci, F; Positano, M; Pitruzzello, S

    1985-01-01

    DNA from 20 Italian haemophilia B patients was analysed by the Southern blotting technique and hybridisation to a factor IX cDNA probe. A large deletion of factor IX gene was detected in one patient with antibodies to the infused factor; the EcoRI pattern of the other 19 subjects examined was normal. Images PMID:4045960

  6. Efficient sequential repetitive gene deletions in Neurospora crassa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Despite its long-standing history as a model organism, Neurospora crassa has limited tools for repetitive gene deletions utilizing recyclable self-excising marker systems. Here we describe, for the first time, the functionality of a bacterial recombination system employing ß-recombinase acting on si...

  7. A HindIII polymorphism detected by cDMD 4-5a at the DMD locus in a family with Becker muscular dystrophy

    SciTech Connect

    Gibb, M.F.; Greenberg, C.R.; Carson, N.L.

    1994-09-01

    Deletions within the dystrophin gene can be detected by hybridizing a series of cDNA probes to HindIII-digested DNA, with the absence of one or more fragments indicating the presence of a deletion. However, incorrect interpretations can be made if the absence of a fragment is due to a polymorphism rather than a deletion. Otto and Rothbery reported that the 5.2 kb fragment detected by cM 4-5a could be resolved, with extended electrophoresis, into two fragments estimated to be 5.2 and 5.15 kb in size. They concluded that the extra fragment of this doublet appears to be polymorphic, inherited in a Mendelian dominant fashion. The mother, who is an obligate carrier of BMD, does not have the upper fragment as is the case for her normal and affected sons. The father, who clinically has no evidence of neuromuscular disease, does have the upper fragment as do all their daughters. Given a dominant pattern of inheritance, the daughters should be heterozygous. Analysis of one grandson, who was predicted to have inherited the grandpaternal dystrophin gene, showed that he did have the upper fragment, consistent with our conclusions. To date, we have been unable to analyze a grandson that has inherited the grandmaternal allele; however, presuably he would not have the upper fragment of this doublet. We conclude that there likely is a dominant HindIII polymorphism detected with the cDMD 4-5a probe at the DMD locus. Population studies will be required to determine the frequency of this polymorphism; however, it should be noted that absence of the upper fragment of this doublet in a male with BMD/DMD does not necessarily correspond to the presence of a deletion.

  8. IL1RAPL1 is associated with mental retardation in patients with complex glycerol kinase deficiency who have deletions extending telomeric of DAX1.

    PubMed

    Zhang, Yao-Hua; Huang, Bing-Ling; Niakan, Kathy K; McCabe, Linda L; McCabe, Edward R B; Dipple, Katrina M

    2004-09-01

    IL1RAPL1 (interleukin-1 receptor accessory protein-like, gene 1) has recently been shown to be mutated in patients with X-linked mental retardation. Clinical experience has suggested that patients with the contiguous gene syndrome, complex glycerol kinase deficiency (cGKD), will have mental retardation (MR) if they have deletions extending from the GK gene into the DMD gene and/or involving a significant extension telomeric from DAX1. We examined cell lines from patients with cGKD whose clinical features would be informative and would allow us to determine if IL1RAPL1 deletions can help to explain the MR in patients with deletions extending telomeric from DAX1. Our results showed that nearly all patients with deletions involving DAX1, but not DMD, had MR if IL1RAPL1 was deleted. If ILIRAPLI and DMD were intact, the patients with DAX1 deletions only rarely had normal development. Deletions in DNA from patients with cGKD who exhibited MR and had normal IL1RAPL1 all involved the GK and DMD genes. Our data are consistent with the association of IL1RAPL1 gene deletion and MR in the majority of patients with cGKD and deletions extending telomeric from DAX1.

  9. Iron-regulatory proteins DmdR1 and DmdR2 of Streptomyces coelicolor form two different DNA-protein complexes with iron boxes.

    PubMed Central

    Flores, Francisco J; Martín, Juan F

    2004-01-01

    In high G+C Gram-positive bacteria, the control of expression of genes involved in iron metabolism is exerted by a DmdR [divalent (bivalent) metal-dependent regulatory protein] in the presence of Fe2+ or other bivalent ions. The dmdR1 and dmdR2 genes of Streptomyces coelicolor were overexpressed in Escherichia coli and the DmdR1 and DmdR2 proteins were purified to homogeneity. Electrophoretic mobility-shift assays showed that both DmdR1 and DmdR2 bind to the 19-nt tox and desA iron boxes forming two different complexes in each case. Increasing the concentrations of DmdR1 or DmdR2 protein shifted these complexes from their low-molecular-mass form to the high-molecular-mass complexes. Formation of the DNA-protein complexes was prevented by the bivalent metal chelating agent 2,2'-dipyridyl and by antibodies specific against the DmdR proteins. Cross-linking with glutaraldehyde of pure DmdR1 or DmdR2 proteins showed that DmdR1 forms dimers, whereas DmdR2 is capable of forming dimers and probably tetramers. Ten different iron boxes were found in a search for iron boxes in the genome of S. coelicolor. Most of them correspond to putative genes involved in siderophore biosynthesis. Since the nucleotide sequence of these ten boxes is identical (or slightly different) with the synthetic DNA fragment containing the desA box used in the present study, it is proposed that DmdR1 and DmdR2 bind to the iron boxes upstream of at least ten different genes in S. coelicolor. PMID:14960152

  10. Comparison of the phenotypes of patients harboring in-frame deletions starting at exon 45 in the Duchenne muscular dystrophy gene indicates potential for the development of exon skipping therapy.

    PubMed

    Nakamura, Akinori; Shiba, Naoko; Miyazaki, Daigo; Nishizawa, Hitomi; Inaba, Yuji; Fueki, Noboru; Maruyama, Rika; Echigoya, Yusuke; Yokota, Toshifumi

    2017-04-01

    Exon skipping therapy has recently received attention for its ability to convert the phenotype of lethal Duchenne muscular dystrophy (DMD) to a more benign form, Becker muscular dystrophy (BMD), by correcting the open reading frame. This therapy has mainly focused on a hot-spot (exons 45-55) mutation in the DMD gene. Exon skipping of an entire stretch of exons 45-55 is an approach applicable to 46.9% of DMD patients. However, the resulting phenotype is not yet fully understood. Here we examined the clinical profiles of 24 patients with BMD resulting from deletions starting at exon 45. The Δ45-55 group ranged in age from 2 to 87 years; no mortality was observed, and one patient was ambulatory at 79 years of age. The age at which patients became wheelchair-bound in the Δ45-48 group (18-88 years old) was approximately 50 years. Cardiomyopathy was well controlled by pharmaceuticals in both deletion groups. In contrast, the Δ45-47 and Δ45-49 groups exhibited more severe phenotypes than those with other mutations: the age at which patients in the Δ45-49 group became wheelchair-bound was around 30-40 years. Our study shows that clinical severity differs between each hot-spot deletion.

  11. Large deletion in the NF1 gene associated with dysmorphism

    SciTech Connect

    Hughes, H.E.; Maynard, J.; Sourour, E.

    1994-09-01

    Neurofibromatosis type 1 is an autosomal dominant disorder with a prevalence of 1 in 3000. The major clinical features of the disease include cafe-au-lait spots, neurofibromas, Lisch nodules and auxillary freckling. Six sporadic NF1 patients with dysmorphism and intellectual impairment have been described to have a large deletion extending beyond the NF1 gene. We report another spordiac NF1 patient with severe developmental delay, early growth failure and dysmorphism (not Noonan-like) associated with a large deletion involving the NF1 gene. A panel of 12 polymorphic DNA markers within 4 cM of the NF1 gene were used to screen for the NF1 gene rearrangements. With all the polymorphic markers, only a single band was ever observed in this affected individual. However, with DNA probe EW301 which maps to 17p, a biparental inheritance was observed. Analysis with several microsatellite markers indicated that this patient had not inherited an allele from the father. A reduction in the hybridization signal was also observed when DNA from this patient was screened with cDNAs AE25, P5, B3A, and an extragenic marker EW206, clearly indicating hemizygosity at these loci. The combined evidence of dosage reduction and biparental inheritance with DNA marker EW301 indictates that this patient has a deletion of paternal origin rather than uniparental disomy. Pulsed-field gel electrophoresis has not, so far, revealed any evidence of an altered band pattern; however, studies are continuing. FISH analysis is currently in progress using YACs and cosmids to define the extent of this deletion.

  12. Different mosaicism frequencies for proximal and distal Duchenne muscular dystrophy (DMD) mutations indicate difference in etiology and recurrence risk.

    PubMed Central

    Passos-Bueno, M R; Bakker, E; Kneppers, A L; Takata, R I; Rapaport, D; den Dunnen, J T; Zatz, M; van Ommen, G J

    1992-01-01

    In about 65% of the cases of Duchenne muscular dystrophy (DMD) a partial gene deletion or duplication in the dystrophin gene can be detected. These mutations are clustered at two hot spots: 30% at the hot spot in the proximal part of the gene and about 70% at a more distal hot spot. Unexpectedly we observed a higher frequency of proximal gene rearrangements among proved "germ line" mosaic cases. Of the 24 mosaic cases we are aware of, 19 (79%) have a proximal mutation, while only 5 (21%) have a distal mutation. This finding indicates that the mutations at the two hot spots in the dystrophin gene differ in origin. Independent support for the different mosaicism frequency was found by comparing the mutation spectra observed in isolated cases of DMD and familial cases of DMD. In a large two-center study of 473 patients from Brazil and the Netherlands, we detected a significant difference in the deletion distribution of isolated (proximal:distal ratio 1:3) and familial cases (ratio 1:1). We conclude from these data that proximal deletions most likely occur early in embryonic development, causing them to have a higher chance of becoming familial, while distal deletions occur later and have a higher chance of causing only isolated cases. Finally, our findings have important consequences for the calculation of recurrence-risk estimates according to the site of the deletion: a "proximal" new mutant has an increased recurrence risk of approximately 30%, and a "distal" new mutant has a decreased recurrence risk of approximately 4%. PMID:1415256

  13. Duchenne and Becker muscular dystrophy mutations: analysis using 2.6 kb of muscle cDNA from the 5' end of the gene.

    PubMed Central

    Smith, T J; Forrest, S M; Cross, G S; Davies, K E

    1987-01-01

    We have isolated overlapping human fetal muscle cDNAs encompassing 2.6kb which are localised very close to the 5' end of the Duchenne muscular dystrophy (DMD) gene. Using DNA from patients with deletions of previously reported genomic probes, we have mapped the exons across the region. Investigation of deletions in both DMD and Becker muscular dystrophy (BMD) patients shows the deletions to be present in 10% of cases and heterogeneous. Images PMID:3697082

  14. Different mosaicism frequencies for proximal and distal Duchenne muscular dystrophy (DMD) mutations indicate difference in etiology and recurrence risk

    SciTech Connect

    Passos-Bueno, M.R.; Takata, R.I.; Rapaport, D.; Bakker, E.; Kneppers, A.L.J.; Dunnen, J.T. den; Ommen, J.B. van

    1992-11-01

    In about 65% of the cases of Duchenne muscular dystrophy (DMD) a partial gene deletion or duplication in the dystrophin gene can be detected. These mutations are clustered at two hot spots: 30% at the hot spot in the proximal part of the gene and about 70% at a more distal hot spot. Unexpectedly the authors observed a higher frequency of proximal gene rearrangements among proved germ line' mosaic cases. Of the 24 mosaic cases they are aware of, 19 (79%) have a proximal mutation, while only 5 (21%) have a distal mutation. This finding indicates that the mutations at the two hot spots in the dystrophin gene differ in origin. Independent support for the different mosaicism frequency was found by comparing the mutation spectra observed in isolated cases of DMD and familial cases (ratio 1:1). The authors conclude from these data that proximal deletions most likely occur early in embryonic development, causing them to have a higher chance of becoming familial, while distal deletions occur later and have a higher chance of causing only isolated cases. Finally, the findings have important consequences for the calculation of recurrence-risk estimates according to the site of the deletion: a [open quote]proximal[close quote] new mutant has an increased recurrence risk of approximately 30%, and a [open quote]distal[close quote] new mutant has a decreased recurrence risk of approximately 4%. 28 refs., 2 figs., 2 tabs.

  15. Defined single-gene and multi-gene deletion mutant collections in Salmonella enterica sv Typhimurium.

    PubMed

    Porwollik, Steffen; Santiviago, Carlos A; Cheng, Pui; Long, Fred; Desai, Prerak; Fredlund, Jennifer; Srikumar, Shabarinath; Silva, Cecilia A; Chu, Weiping; Chen, Xin; Canals, Rocío; Reynolds, M Megan; Bogomolnaya, Lydia; Shields, Christine; Cui, Ping; Guo, Jinbai; Zheng, Yi; Endicott-Yazdani, Tiana; Yang, Hee-Jeong; Maple, Aimee; Ragoza, Yury; Blondel, Carlos J; Valenzuela, Camila; Andrews-Polymenis, Helene; McClelland, Michael

    2014-01-01

    We constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available.

  16. Gene Deletion in Barley Mediated by LTR-retrotransposon BARE

    PubMed Central

    Shang, Yi; Yang, Fei; Schulman, Alan H.; Zhu, Jinghuan; Jia, Yong; Wang, Junmei; Zhang, Xiao-Qi; Jia, Qiaojun; Hua, Wei; Yang, Jianming; Li, Chengdao

    2017-01-01

    A poly-row branched spike (prbs) barley mutant was obtained from soaking a two-rowed barley inflorescence in a solution of maize genomic DNA. Positional cloning and sequencing demonstrated that the prbs mutant resulted from a 28 kb deletion including the inflorescence architecture gene HvRA2. Sequence annotation revealed that the HvRA2 gene is flanked by two LTR (long terminal repeat) retrotransposons (BARE) sharing 89% sequence identity. A recombination between the integrase (IN) gene regions of the two BARE copies resulted in the formation of an intact BARE and loss of HvRA2. No maize DNA was detected in the recombination region although the flanking sequences of HvRA2 gene showed over 73% of sequence identity with repetitive sequences on 10 maize chromosomes. It is still unknown whether the interaction of retrotransposons between barley and maize has resulted in the recombination observed in the present study. PMID:28252053

  17. Co-occurrence of mutations in both dystrophin- and androgen-receptor genes is a novel cause of female Duchenne muscular dystrophy.

    PubMed

    Katayama, Yoshinori; Tran, Van Khanh; Hoan, Nguyen Thi; Zhang, Zhujun; Goji, Katsumi; Yagi, Mariko; Takeshima, Yasuhiro; Saiki, Kayoko; Nhan, Nguyen Thu; Matsuo, Masafumi

    2006-06-01

    Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder. Here, we report a novel mechanism for the occurrence of DMD in females. In a Vietnamese DMD girl, conventional PCR amplification analysis disclosed a deletion of exons 12-19 of the dystrophin gene on Xp21.2, with a karyotype of 46, XY. Furthermore, a novel mutation in the androgen-receptor gene on Xq11.2-q12 was identified in this girl, which led to male pseudohermaphroditism. Co-occurrence of mutations of these two genes constitutes a novel mechanism underlying female DMD.

  18. DMD and BMD in the same family due to two distinct mutations

    SciTech Connect

    Morandi, L.; Mora, M.; Di Blasi, C.; Brugnoni, R.

    1995-12-04

    We report on a family with a boy affected by Duchenne muscular dystrophy (DMD) and an asymptomatic cousin with a Becker-type dystrophin abnormality, diagnosed by chance. Dystrophin gene analysis showed that these conditions were caused by two distinct deletions with breakpoints in different exons. In Xp21 families, DNA analysis and dystrophin testing of asymptomatic males with high CK plasma levels might detect different dystrophin mutations in separate haplotypes as in our family, although we stress there should be clear clinical or familial indications for such testing. 24 refs., 5 figs.

  19. Effects of G-gene Deletion and Replacement on Rabies Virus Vector Gene Expression

    PubMed Central

    Sato, Sho; Ohara, Shinya; Tsutsui, Ken-Ichiro; Iijima, Toshio

    2015-01-01

    The glycoprotein-gene (G gene) -deleted rabies virus (RV) vector is a powerful tool to examine the function and structure of neural circuits. We previously reported that the deletion of the G gene enhances the transgene expression level of the RV vector. However, the mechanism of this enhancement remains to be clarified. We presume that there are two possible factors for this enhancement. The first factor is the glycoprotein of RV, which shows cytotoxicity; thus, may cause a dysfunction in the translation process of infected cells. The second possible factor is the enhanced expression of the L gene, which encodes viral RNA polymerase. In the RV, it is known that the gene expression level is altered depending on the position of the gene. Since G-gene deletion displaces the L gene in the genome, the expression of the L gene and viral transcription may be enhanced. In this study, we compared the transgene expression level and viral transcription of three recombinant RV vectors. The effect of glycoprotein was examined by comparing the viral gene expression of G-gene-intact RV and G-gene-replaced RV. Despite the fact that the L-gene transcription level of these two RV vectors was similar, the G-gene-replaced RV vector showed higher viral transcription and transgene expression level than the G-gene-intact RV vector. To examine the effect of the position of the L gene, we compared the viral gene expression of the G-gene-deleted RV and G-gene-replaced RV. The G-gene-deleted RV vector showed higher L-gene transcription, viral transcription, and transgene expression level than the G-gene-replaced RV vector. These results indicate that G-gene deletion enhances the transgene expression level through at least two factors, the absence of glycoprotein and enhancement of L-gene expression. These findings enable investigators to design a useful viral vector that shows a controlled desirable transgene expression level in applications. PMID:26023771

  20. VIP Gene Deletion in Mice Causes Cardiomyopathy Associated with Upregulation of Heart Failure Genes

    SciTech Connect

    Szema, Anthony M.; Hamidi, Sayyed A.; Smith, S. David; Benveniste, Helene; Katare, Rajesh Gopalrao

    2013-05-20

    Vasoactive Intestinal Peptide (VIP), a pulmonary vasodilator and inhibitor of vascular smooth muscle proliferation, is absent in pulmonary arteries of patients with idiopathic pulmonary arterial hypertension (PAH). We previously determined that targeted deletion of the VIP gene in mice leads to PAH with pulmonary vascular remodeling and right ventricular (RV) dilatation. Whether the left ventricle is also affected by VIP gene deletion is unknown. In the current study, we examined if VIP knockout mice (VIP-/-) develop both right (RV) and left ventricular (LV) cardiomyopathy, manifested by LV dilatation and systolic dysfunction, as well as overexpression of genes conducive to heart failure.

  1. Deletion and deletion/insertion mutations in the juxtamembrane domain of the FLT3 gene in adult acute myeloid leukemia

    PubMed Central

    Deeb, Kristin K.; Smonskey, Matthew T.; DeFedericis, HanChun; Deeb, George; Sait, Sheila N.J.; Wetzler, Meir; Wang, Eunice S.; Starostik, Petr

    2014-01-01

    In contrast to FLT3 ITD mutations, in-frame deletions in the FLT3 gene have rarely been described in adult acute leukemia. We report two cases of AML with uncommon in-frame mutations in the juxtamembrane domain of the FLT3 gene: a 3-bp (c.1770_1774delCTACGinsGT; p.F590_V592delinsLF) deletion/insertion and a 12-bp (c.1780_1791delTTCAGAGAATAT; p.F594_Y597del) deletion. We verified by sequencing that the reading frame of the FLT3 gene was preserved and by cDNA analysis that the mRNA of the mutant allele was expressed in both cases. Given the recent development of FLT3 inhibitors, our findings may be of therapeutic value for AML patients harboring similar FLT3 mutations. PMID:25379410

  2. Global distribution of the CCR5 gene 32-basepair deletion.

    PubMed

    Martinson, J J; Chapman, N H; Rees, D C; Liu, Y T; Clegg, J B

    1997-05-01

    A mutant allele of the beta-chemokine receptor gene CCR5 bearing a 32-basepair (bp) deletion (denoted delta ccr5) which prevents cell invasion by the primary transmitting strain of HIV-1 has recently been characterized. Homozygotes for the mutation are resistant to infection, even after repeated high-risk exposures, but this resistance appears not to be total, as isolated cases of HIV-positive deletion homozygotes are now emerging. The consequence of the heterozygous state is not clear, but it may delay the progression to AIDS in infected individuals. A gene frequency of approximately 10% was found for delta ccr5 in populations of European descent, but no mutant alleles were reported in indigenous non-European populations. As the total number of non-European samples surveyed was small in comparison with the Europeans the global distribution of this mutation is far from clear. We have devised a rapid PCR assay for delta ccr5 and used it to screen 3,342 individuals from a globally-distributed range of populations. We find that delta ccr5 is not confined to people of European descent but is found at frequencies of 2-5% throughout Europe, the Middle East and the Indian subcontinent (Fig. 1). Isolated occurrences are seen elsewhere throughout the world, but these most likely represent recent European gene flow into the indigenous populations. The inter-population differences in delta ccr5 frequency may influence the pattern of HIV transmission and so will need to be incorporated into future predictions of HIV levels.

  3. More deletions in the 5{prime} region than in the central region of the dystrophin gene were identified among Filipino Duchenne and Becker muscular dystrophy patients

    SciTech Connect

    1995-11-06

    This report describes mutations in the dystrophin gene and the frequency of these mutations in Filipino pedigrees with Duchenne and Becker muscular dystrophy (DMD/BMD). The findings suggest the presence of genetic variability among DMD/BMD patients in different populations. 13 refs., 1 tab.

  4. In vivo gene editing in dystrophic mouse muscle and muscle stem cells.

    PubMed

    Tabebordbar, Mohammadsharif; Zhu, Kexian; Cheng, Jason K W; Chew, Wei Leong; Widrick, Jeffrey J; Yan, Winston X; Maesner, Claire; Wu, Elizabeth Y; Xiao, Ru; Ran, F Ann; Cong, Le; Zhang, Feng; Vandenberghe, Luk H; Church, George M; Wagers, Amy J

    2016-01-22

    Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

  5. Deletion of PLCB1 gene in schizophrenia-affected patients.

    PubMed

    Lo Vasco, Vincenza Rita; Cardinale, Giuseppina; Polonia, Patrizia

    2012-04-01

    A prevalence of 1% in the general population and approximately 50% concordance rate in monozygotic twins was reported for schizophrenia, suggesting that genetic predisposition affecting neurodevelopmental processes might combine with environmental risk factors. A multitude of pathways seems to be involved in the aetiology and/or pathogenesis of schizophrenia, including dopaminergic, serotoninergic, muscarinic and glutamatergic signalling. The phosphoinositide signal transduction system and related phosphoinositide-specific phospholipase C (PI-PLC) enzymes seem to represent a point of convergence in these networking pathways during the development of selected brain regions. The existence of a susceptibility locus on the short arm of chromosome 20 moved us to analyse PLCB1, the gene codifying for PI-PLC β1 enzyme, which maps on 20p12. By using interphase fluorescent in situ hybridization methodology, we found deletions of PLCB1 in orbito-frontal cortex samples of schizophrenia-affected patients.

  6. Deletion of PLCB1 gene in schizophrenia-affected patients

    PubMed Central

    Vasco, Vincenza Rita Lo; Cardinale, Giuseppina; Polonia, Patrizia

    2012-01-01

    Abstract A prevalence of 1% in the general population and approximately 50% concordance rate in monozygotic twins was reported for schizophrenia, suggesting that genetic predisposition affecting neurodevelopmental processes might combine with environmental risk factors. A multitude of pathways seems to be involved in the aetiology and/or pathogenesis of schizophrenia, including dopaminergic, serotoninergic, muscarinic and glutamatergic signalling. The phosphoinositide signal transduction system and related phosphoinositide-specific phospholipase C (PI-PLC) enzymes seem to represent a point of convergence in these networking pathways during the development of selected brain regions. The existence of a susceptibility locus on the short arm of chromosome 20 moved us to analyse PLCB1, the gene codifying for PI-PLC β1 enzyme, which maps on 20p12. By using interphase fluorescent in situ hybridization methodology, we found deletions of PLCB1 in orbito-frontal cortex samples of schizophrenia-affected patients. PMID:22507702

  7. Site-specific deletions involving the tal-1 and sil genes are restricted to cells of the T cell receptor alpha/beta lineage: T cell receptor delta gene deletion mechanism affects multiple genes

    PubMed Central

    1993-01-01

    Site-specific deletions in the tal-1 gene are reported to occur in 12- 26% of T cell acute lymphoblastic leukemias (T-ALL). So far two main types of tal-1 deletions have been described. Upon analysis of 134 T- ALL we have found two new types of tal-1 deletions. These four types of deletions juxtapose the 5' part of the tal-1 gene to the sil gene promoter, thereby deleting all coding sil exons but leaving the coding tal-1 exons undamaged. The recombination signal sequences (RSS) and fusion regions of the tal-1 deletion breakpoints strongly resemble the RSS and junctional regions of immunoglobulin/T cell receptor (TCR) gene rearrangements, which implies that they are probably caused by the same V(D)J recombinase complex. Analysis of the 134 T-ALL suggested that the occurrence of tal-1 deletions is associated with the CD3 phenotype, because no tal-1 deletions were found in 25 TCR-gamma/delta + T-ALL, whereas 8 of the 69 CD3- T-ALL and 11 of the 40 TCR-alpha/beta + T-ALL contained such a deletion. Careful examination of all TCR genes revealed that tal-1 deletions exclusively occurred in CD3- or CD3+ T- ALL of the alpha/beta lineage with a frequency of 18% in T-ALL with one deleted TCR-delta allele, and a frequency of 34% in T-ALL with TCR- delta gene deletions on both alleles. Therefore, we conclude that alpha/beta lineage commitment of the T-ALL and especially the extent of TCR-delta gene deletions determines the chance of a tal-1 deletion. This suggests that tal-1 deletions are mediated via the same deletion mechanism as TCR-delta gene deletions. PMID:8459224

  8. The evolution of an intron: Analysis of a long, deletion-prone intron in the human dystrophin gene

    SciTech Connect

    McNaughton, J.C.; Hughes, G.; Jones, W.A.

    1997-03-01

    The sequence of a 112-kb region of the human dystrophin (DMD/BMD) gene encompassing the deletion prone intron 7 (110 kb) and the much shorter intron 8 (1.1 kb) has been determined. Recognizable insertion sequences account for approximately 40% of intron 7. LINE-1 and THE-1/LTR sequences occur in intron 7 with significantly higher frequency than would be expected statistically while Alu sequences are underrepresented. Intron 7 also contains numerous mammalian-wide interspersed repeats, a diverse range of medium reiteration repeats of unknown origin, and a sequence derived from a mariner transposon. By contrast, the shorter intron 8 contains no detectable insertion sequences. Dating of the L1 and Alu sequences suggests that intron 7 has approximately doubled in size within the past 130 million years, and comparison with the corresponding intron from the pufferfish (Fugu rubripes) suggests that the intron has expanded some 44-fold over a period of 400 million years. The possible contribution of the insertion elements to the instability of intron 7 is discussed. 66 refs., 2 figs., 2 tabs.

  9. Gene deletion in urothelium by specific expression of Cre recombinase.

    PubMed

    Mo, Lan; Cheng, Jin; Lee, Eva Y-H P; Sun, Tung-Tien; Wu, Xue-Ru

    2005-09-01

    Urothelium that lines almost the entire urinary tract acts as a permeability barrier and is involved in the pathogenesis of major urinary diseases, including urothelial carcinoma, urinary tract infection, and interstitial cystitis. However, investigation of urothelial biology and diseases has been hampered by the lack of tissue-specific approaches. To address this deficiency, we sought to develop a urothelium-specific knockout system using the Cre/loxP strategy. Transgenic mouse lines were generated in which a 3.6-kb mouse uroplakin II (UPII) promoter was used to drive the expression of Cre recombinase (Cre). Among the multiple tissues analyzed, Cre was found to be expressed exclusively in the urothelia of the transgenic mice. Crossing a UPII-Cre transgenic line with a ROSA26-LacZ reporter line, in which LacZ expression depends on Cre-mediated deletion of a floxed "stop" sequence, led to LacZ expression only in the urothelium. Gene recombination was also observed when the UPII-Cre line was crossed to an independent line in which a part of the p53 gene was flanked by the loxP sequences (floxed p53). Truncation of the p53 gene and mRNA was observed exclusively in the urothelia of double transgenic mice harboring both the UPII-Cre transgene and the floxed p53 allele. These results demonstrate for the first time the feasibility and potentially wide applicability of the UPII-Cre transgenic mice to inactivate any genes of interest in the urothelium.

  10. The rates and patterns of deletions in the human factor IX gene

    SciTech Connect

    Ketterling, R.P.; Vielhaber, E.L.; Lind, T.J.; Thorland, E.C.; Sommer S.S. )

    1994-02-01

    Deletions are commonly observed in genes with either segments of highly homologous sequences or excessive gene length. However, in the factor IX gene and in most genes, deletions (of [ge]21 bp) are uncommon. The authors have analyzed DNA from 290 families with hemophilia B (203 independent mutations) and have found 12 deletions >20 bp. Eleven of these are >2 kb (range >3-163 kb), and one is 1.1 kb. The junctions of the four deletions that are completely contained within the factor IX gene have been determined. A novel mutation occurred in patient HB128: the data suggest that a 26.8-kb deletion occurred between two segments of alternating purines and pyrimidines and that a 2.3-kb sense strand segment derived from the deleted region was inserted. For a sample of 203 independent mutations, the authors estimate the [open quotes]baseline[close quotes] rates of deletional mutation per base pair per generation as a function of size. The rate for large (>2 kb)I deletions is exceedingly low. For every mutational event in which a given base is at the junction of a large deletion, there are an estimated 58 microdeletions (<20 bp) and 985 single-base substitutions at that base. Analysis of the nine reported deletion junctions in the factor IX gene literature reveals that (i) five are associated with inversion, orphan sequences, or sense strand insertions; (ii) four are simple deletions that display an excess of short direct repeats at their junctions; (iii) there is no dramatic clustering of junctions within the gene; and (iv) with the exception of alternating purines and pyrimidines, deletion junctions are not preferentially associated with repetitive DNA. 58 refs., 5 figs., 5 tabs.

  11. Mucopolysaccharidosis type IVA: Common double deletion in the N-Acetylgalactosamine-6-sulfatase gene (GALNS)

    SciTech Connect

    Hori, Toshinori; Tomatsu, Shunji; Fukuda, Seiji

    1995-04-10

    Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency in N-acetylgalactosamine-6-sulfatase (GALNS). We found two separate deletions of nearly 8.0 and 6.0 kb in the GALNS gene, including some exons. There are Alu repetitive elements near the breakpoints of the 8.0-kb deletion, and this deletion resulted from an Alu-Alu recombination. The other 6.0-kb deletion involved illegitimate recombinational events between incomplete short direct repeats of 8 bp at deletion breakpoints. The same rearrangement has been observed in a heteroallelic state in four unrelated patients. This is the first documentation of a common double deletion a gene that is not a member of a gene cluster. 39 refs., 5 figs.

  12. Mosaic 7q31 deletion involving FOXP2 gene associated with language impairment.

    PubMed

    Palka, Chiara; Alfonsi, Melissa; Mohn, Angelika; Cerbo, Renato; Guanciali Franchi, Paolo; Fantasia, Donatella; Morizio, Elisena; Stuppia, Liborio; Calabrese, Giuseppe; Zori, Roberto; Chiarelli, Francesco; Palka, Giandomenico

    2012-01-01

    We report on a 10-year-old patient with childhood apraxia of speech (CAS) and mild dysmorphic features. Although multiple karyotypes were reported as normal, a bacterial artificial chromosome array comparative genomic hybridization revealed the presence of a de novo 14.8-Mb mosaic deletion of chromosome 7q31. The deleted region involved several genes, including FOXP2, which has been associated with CAS. Interestingly, the deletion reported here was observed in about 50% of cells, which is the first case of mosaicism in a 7q31 deletion. Despite the presence of the deletion in only 50% of cells, the phenotype of the patient was not milder than other published cases. To date, 6 cases with a deletion of 9.1-20 Mb involving the FOXP2 gene have been reported, suggesting a new contiguous gene deletion syndrome characterized mainly by CAS caused by haploinsufficiency of the genes encompassed in the 7q critical region. This report suggests that children found with a deletion involving the FOXP2 region should be evaluated for CAS and that analysis of the FOXP2 gene including array comparative genomic hybridization should be considered in selected patients with CAS. Mosaic deletions in this area may also be considered as causative of CAS.

  13. Deletions of recessive disease genes: CNV contribution to carrier states and disease-causing alleles.

    PubMed

    Boone, Philip M; Campbell, Ian M; Baggett, Brett C; Soens, Zachry T; Rao, Mitchell M; Hixson, Patricia M; Patel, Ankita; Bi, Weimin; Cheung, Sau Wai; Lalani, Seema R; Beaudet, Arthur L; Stankiewicz, Pawel; Shaw, Chad A; Lupski, James R

    2013-09-01

    Over 1200 recessive disease genes have been described in humans. The prevalence, allelic architecture, and per-genome load of pathogenic alleles in these genes remain to be fully elucidated, as does the contribution of DNA copy-number variants (CNVs) to carrier status and recessive disease. We mined CNV data from 21,470 individuals obtained by array-comparative genomic hybridization in a clinical diagnostic setting to identify deletions encompassing or disrupting recessive disease genes. We identified 3212 heterozygous potential carrier deletions affecting 419 unique recessive disease genes. Deletion frequency of these genes ranged from one occurrence to 1.5%. When compared with recessive disease genes never deleted in our cohort, the 419 recessive disease genes affected by at least one carrier deletion were longer and located farther from known dominant disease genes, suggesting that the formation and/or prevalence of carrier CNVs may be affected by both local and adjacent genomic features and by selection. Some subjects had multiple carrier CNVs (307 subjects) and/or carrier deletions encompassing more than one recessive disease gene (206 deletions). Heterozygous deletions spanning multiple recessive disease genes may confer carrier status for multiple single-gene disorders, for complex syndromes resulting from the combination of two or more recessive conditions, or may potentially cause clinical phenotypes due to a multiply heterozygous state. In addition to carrier mutations, we identified homozygous and hemizygous deletions potentially causative for recessive disease. We provide further evidence that CNVs contribute to the allelic architecture of both carrier and recessive disease-causing mutations. Thus, a complete recessive carrier screening method or diagnostic test should detect CNV alleles.

  14. Deletions spanning the neurofibromatosis I gene: Identification and phenotype of five patients

    SciTech Connect

    Kayes, L.M.; Burke, W.; Bennett, R.; Ehrlich, P.; Stephens, K. ); Riccardi, V.M. ); Rubenstein, A. )

    1994-03-01

    Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder characterized by marked variation in clinical severity. To investigate the contribution to variability by genes either contiguous to or contained within the NF1 gene, the authors screened six NF1 patients with mild facial dysmorphology, mental retardation, and/or learning disabilities, for DNA rearrangement of the NF1 region. Five of the six patients had NF1 gene deletions on the basis of quantitative densitometry, locus hemizygosity, and analysis of somatic cell hybrid lines. Analysis of hybrid lines carrying each of the patient's chromosomes 17, with 15 regional DNA markers, demonstrated that each of the five patients carried a deletion >700 kb in size. Minimally, each of the deletions involved the entire 350-kb NF1 gene; the three genes - EVI2A, EVI2B, and OMG-that are contained within an NF1 intron; and considerable flanking DNA. For four of the patients, the deletions mapped to the same interval; the deletion in the fifth patient was larger, extending farther in both directions. The remaining NF1 allele presumably produced functional neurofibromin; no gene rearrangements were detected, and RNA-PCR demonstrated that it was transcribed. These data provide compelling evidence that the NF1 disorder results from haploid insufficiency of neurofibromin. Of the three documented de novo deletion cases, two involved the paternal NF1 allele and one the maternal allele. The parental origin of the single remaining expresses NF1 allele had no dramatic effect on patient phenotype. The deletion patients exhibited a variable number of physical anomalies that were not correlated with the extent of their deletion. All five patients with deletions were remarkable for exhibiting a large number of neurfibromas for their age, suggesting that deletion of an unknown gene in the NF1 region may affect tumor initiation or development. 69 refs., 5 figs., 1 tab.

  15. Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae.

    PubMed

    Park, Yang-Nim; Masison, Daniel; Eisenberg, Evan; Greene, Lois E

    2011-09-01

    The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast.

  16. Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase.

    PubMed Central

    Collis, C M; Hall, R M

    1992-01-01

    Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991) and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed. Images PMID:1311297

  17. Germinal mosaicism for a deletion of the FMR1 gene leading to fragile X syndrome.

    PubMed

    Jiraanont, P; Hagerman, R J; Neri, G; Zollino, M; Murdolo, M; Tassone, F

    2016-09-01

    Aberrant CGG trinucleotide amplification within the FMR1 gene, which spans approximately 38 Kb of genomic DNA is almost always what leads to fragile X syndrome (FXS). However, deletions of part or the entire FMR1 gene can also cause FXS. Both CGG amplification-induced silencing and deletions result in the absence of the FMR1 gene product, FMRP. Here, we report a rare case of germinal mosaicism of a deletion encompassing approximately 300 Kb of DNA, which by removing the entire FMR1 gene led to FXS. The male proband, carrying the deletion, presented in clinic with the typical features of FXS. His mother was analyzed by FISH on metaphase chromosomes with cosmid probe c22.3 spanning the FMR1 locus, and she was found not to carry the deletion on 30 analyzed cells from peripheral blood lymphocytes. Prenatal examination of the mother's third pregnancy showed that the male fetus also had the same deletion as the proband. Following this prenatal diagnosis, FISH analysis in the mother was expanded to 400 metaphases from peripheral lymphocytes, and a heterozygous FMR1 deletion was found in three. Although this result could be considered questionable from a diagnostic point of view, it indicates that the deletion is in the ovary's germinal cells.

  18. Interstitial deletion of 11(p11.2p12): A newly described contiguous gene deletion syndrome involving the gene for hereditary multiple exostoses

    SciTech Connect

    Potocki, L.; Shaffer, L.G.

    1996-03-29

    Individuals with deletions of the proximal portion of the short arm of chromosome 11 share many manifestations including mental retardation, biparietal foramina, minor facial anomalies, and multiple cartilaginous exostoses. The finding of multiple exostoses in these patients is remarkable as the disorder hereditary multiple exostoses, which is inherited in an autosomal dominant manner, has recently been mapped by linkage to three regions, including proximal 11p. We report the clinical and molecular findings in an additional patient with an 11(p11.2p12) deletion. Cytogenetic and molecular analysis demonstrated a de novo, paternally derived deletion for markers which have been shown to be tightly linked to the 11p locus (EXT2). These data support the location of EXT2 within this region and also provide information regarding the ordering of polymorphic markers on 11p. Deletion 11(p11.2p12) is a rare, yet specific, deletion syndrome involving the EXT2 locus, a gene for parietal foramina, and a mental retardation locus, and therefore can be classified as a contiguous gene deletion syndrome. 24 refs., 4 figs., 1 tab.

  19. Discrimination of Deletion and Duplication Subtypes of the Deleted in Azoospermia Gene Family in the Context of Frequent Interloci Gene Conversion

    PubMed Central

    Vaszkó, Tibor; Papp, János; Krausz, Csilla; Casamonti, Elena; Géczi, Lajos; Olah, Edith

    2016-01-01

    Due to its palindromic setup, AZFc (Azoospermia Factor c) region of chromosome Y is one of the most unstable regions of the human genome. It contains eight gene families expressed mainly in the testes. Several types of rearrangement resulting in changes in the cumulative copy number of the gene families were reported to be associated with diseases such as male infertility and testicular germ cell tumors. The best studied AZFc rearrangement is gr/gr deletion. Its carriers show widespread phenotypic variation from azoospermia to normospermia. This phenomenon was initially attributed to different gr/gr subtypes that would eliminate distinct members of the affected gene families. However, studies conducted to confirm this hypothesis have brought controversial results, perhaps, in part, due to the shortcomings of the utilized subtyping methodology. This proof-of-concept paper is meant to introduce here a novel method aimed at subtyping AZFc rearrangements. It is able to differentiate the partial deletion and partial duplication subtypes of the Deleted in Azoospermia (DAZ) gene family. The keystone of the method is the determination of the copy number of the gene family member-specific variant(s) in a series of sequence family variant (SFV) positions. Most importantly, we present a novel approach for the correct interpretation of the variant copy number data to determine the copy number of the individual DAZ family members in the context of frequent interloci gene conversion.Besides DAZ1/DAZ2 and DAZ3/DAZ4 deletions, not yet described rearrangements such as DAZ2/DAZ4 deletion and three duplication subtypes were also found by the utilization of the novel approach. A striking feature is the extremely high concordance among the individual data pointing to a certain type of rearrangement. In addition to being able to identify DAZ deletion subtypes more reliably than the methods used previously, this approach is the first that can discriminate DAZ duplication subtypes as well

  20. Cardiac characterization of 16 patients with large NF1 gene deletions.

    PubMed

    Nguyen, R; Mir, T S; Kluwe, L; Jett, K; Kentsch, M; Mueller, G; Kehrer-Sawatzki, H; Friedman, J M; Mautner, V-F

    2013-10-01

    The aim of this study was to characterize cardiac features of patients with neurofibromatosis 1 (NF1) and large deletions of the NF1 gene region. The study participants were 16 patients with large NF1 deletions and 16 age- and sex-matched NF1 patients without such deletions. All the patients were comprehensively characterized clinically and by echocardiography. Six of 16 NF1 deletion patients but none of 16 non-deletion NF1 patients have major cardiac abnormalities (p = 0.041). Congenital heart defects (CHDs) include mitral insufficiency in two patients and ventricular septal defect, aortic stenosis, and aortic insufficiency in one patient each. Three deletion patients have hypertrophic cardiomyopathy. Two patients have intracardiac tumors. NF1 patients without large deletions have increased left ventricular (LV) diastolic posterior wall thickness (p < 0.001) and increased intraventricular diastolic septal thickness (p = 0.001) compared with a healthy reference population without NF1, suggestive of eccentric LV hypertrophy. CHDs and other cardiovascular anomalies are more frequent among patients with large NF1 deletion and may cause serious clinical complications. Eccentric LV hypertrophy may occur in NF1 patients without whole gene deletions, but the clinical significance of this finding is uncertain. All patients with clinical suspicion for NF1 should be referred to a cardiologist for evaluation and surveillance.

  1. Regulation of DMD pathology by an ankyrin-encoded miRNA

    PubMed Central

    2011-01-01

    Background Duchenne muscular dystrophy (DMD) is an X-linked myopathy resulting from the production of a nonfunctional dystrophin protein. MicroRNA (miRNA) are small 21- to 24-nucleotide RNA that can regulate both individual genes and entire cell signaling pathways. Previously, we identified several mRNA, both muscle-enriched and inflammation-induced, that are dysregulated in the skeletal muscles of DMD patients. One particularly muscle-enriched miRNA, miR-486, is significantly downregulated in dystrophin-deficient mouse and human skeletal muscles. miR-486 is embedded within the ANKYRIN1(ANK1) gene locus, which is transcribed as either a long (erythroid-enriched) or a short (heart muscle- and skeletal muscle-enriched) isoform, depending on the cell and tissue types. Results Inhibition of miR-486 in normal muscle myoblasts results in inhibited migration and failure to repair a wound in primary myoblast cell cultures. Conversely, overexpression of miR-486 in primary myoblast cell cultures results in increased proliferation with no changes in cellular apoptosis. Using bioinformatics and miRNA reporter assays, we have identified platelet-derived growth factor receptor β, along with several other downstream targets of the phosphatase and tensin homolog deleted on chromosome 10/AKT (PTEN/AKT) pathway, as being modulated by miR-486. The generation of muscle-specific transgenic mice that overexpress miR-486 revealed that miR-486 alters the cell cycle kinetics of regenerated myofibers in vivo, as these mice had impaired muscle regeneration. Conclusions These studies demonstrate a link for miR-486 as a regulator of the PTEN/AKT pathway in dystrophin-deficient muscle and an important factor in the regulation of DMD muscle pathology. PMID:21824387

  2. A Low-Order Galerkin Model Based on DMD and Adjoint-DMD modes

    NASA Astrophysics Data System (ADS)

    Zhang, Wei; Wei, Mingjun

    2016-11-01

    Dynamic Mode Decomposition (DMD) has emerged as a new tool for the understanding of flow dynamics associated with frequencies. The DMD modes computed by this process have been considered as an alternative of base functions for model order reduction. However, DMD modes are not orthogonal bases which are usually desired for the simplicity of Galerkin models. Therefore, we used the bi-orthogonal pair of DMD modes and adjoint DMD modes to solve this problem, and introduced an easy approach to derive a simple DMD-Galerkin projection model. The introduction of adjoint DMD modes also provides an easy way to rank DMD modes for order reduction. The approach is applied on a flow-passing-cylinder case in both transition and periodic stages. For the periodic case, DMD-Galerkin model is similar to POD-Galerkin model; and for the transition case, DMD-Galerkin model carries more clear frequency features. Supported by ARL.

  3. DMD transcript imbalance determines dystrophin levels.

    PubMed

    Spitali, Pietro; van den Bergen, Janneke C; Verhaart, Ingrid E C; Wokke, Beatrijs; Janson, Anneke A M; van den Eijnde, Rani; den Dunnen, Johan T; Laros, Jeroen F J; Verschuuren, Jan J G M; 't Hoen, Peter A C; Aartsma-Rus, Annemieke

    2013-12-01

    Duchenne and Becker muscular dystrophies are caused by out-of-frame and in-frame mutations, respectively, in the dystrophin encoding DMD gene. Molecular therapies targeting the precursor-mRNA are in clinical trials and show promising results. These approaches will depend on the stability and expression levels of dystrophin mRNA in skeletal muscles and heart. We report that the DMD gene is more highly expressed in heart than in skeletal muscles, in mice and humans. The transcript mutated in the mdx mouse model shows a 5' to 3' imbalance compared with that of its wild-type counterpart and reading frame restoration via antisense-mediated exon skipping does not correct this event. We also report significant transcript instability in 22 patients with Becker dystrophy, clarifying the fact that transcript imbalance is not caused by premature nonsense mutations. Finally, we demonstrate that transcript stability, rather than transcriptional rate, is an important determinant of dystrophin protein levels in patients with Becker dystrophy. We suggest that the availability of the complete transcript is a key factor to determine protein abundance and thus will influence the outcome of mRNA-targeting therapies.

  4. Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches.

    PubMed

    Lorain, Stéphanie; Peccate, Cécile; Le Hir, Maëva; Griffith, Graziella; Philippi, Susanne; Précigout, Guillaume; Mamchaoui, Kamel; Jollet, Arnaud; Voit, Thomas; Garcia, Luis

    2013-09-01

    RNA-based therapeutic approaches using splice-switching oligonucleotides have been successfully applied to rescue dystrophin in Duchenne muscular dystrophy (DMD) preclinical models and are currently being evaluated in DMD patients. Although the modular structure of dystrophin protein tolerates internal deletions, many mutations that affect nondispensable domains of the protein require further strategies. Among these, trans-splicing technology is particularly attractive, as it allows the replacement of any mutated exon by its normal version as well as introducing missing exons or correcting duplication mutations. We have applied such a strategy in vitro by using cotransfection of pre-trans-splicing molecule (PTM) constructs along with a reporter minigene containing part of the dystrophin gene harboring the stop-codon mutation found in the mdx mouse model of DMD. Optimization of the different functional domains of the PTMs allowed achieving accurate and efficient trans-splicing of up to 30% of the transcript encoded by the cotransfected minigene. Optimized parameters included mRNA stabilization, choice of splice site sequence, inclusion of exon splice enhancers and artificial intronic sequence. Intramuscular delivery of adeno-associated virus vectors expressing PTMs allowed detectable levels of dystrophin in mdx and mdx4Cv, illustrating that a given PTM can be suitable for a variety of mutations.

  5. VIP Gene Deletion in Mice Causes Cardiomyopathy Associated with Upregulation of Heart Failure Genes

    PubMed Central

    Szema, Anthony M.; Hamidi, Sayyed A.; Smith, S. David; Benveniste, Helene

    2013-01-01

    Rationale Vasoactive Intestinal Peptide (VIP), a pulmonary vasodilator and inhibitor of vascular smooth muscle proliferation, is absent in pulmonary arteries of patients with idiopathic pulmonary arterial hypertension (PAH). We previously determined that targeted deletion of the VIP gene in mice leads to PAH with pulmonary vascular remodeling and right ventricular (RV) dilatation. Whether the left ventricle is also affected by VIP gene deletion is unknown. In the current study, we examined if VIP knockout mice (VIP−/−) develop both right (RV) and left ventricular (LV) cardiomyopathy, manifested by LV dilatation and systolic dysfunction, as well as overexpression of genes conducive to heart failure. Methods We examined VIP−/−and wild type (WT) mice using Magnetic Resonance Imaging (MRI) for evidence of cardiomyopathy associated with biventricular dilation and wall thickness changes. Lung tissue from VIP−/− and WT mice was subjected to whole-genome gene microarray analysis. Results Lungs from VIP−/− mice showed overexpression of cardiomyopathy genes: Myh1 was upregulated 224 times over WT, and Mylpf was increased 72 fold. Tnnt3 was increased 105 times and tnnc2 181 fold. Hearts were dilated in VIP−/− mice, with thinning of LV wall and increase in RV and LV chamber size, though RV enlargement varied. Weights of VIP−/− mice were consistently lower. Conclusions Critically-important heart failure-related genes are upregulated in VIP−/− mice associated with the spontaneous cardiomyopathy phenotype, involving both left and right ventricles, suggesting that loss of the VIP gene orchestrates a panoply of pathogenic genes which are detrimental to both left and right cardiac homeostasis. PMID:23700405

  6. Deletion of the "OPHN1" Gene Detected by aCGH

    ERIC Educational Resources Information Center

    Madrigal, I.; Rodriguez-Revenga, L.; Badenas, C.; Sanchez, A.; Mila, M.

    2008-01-01

    Background: The oligophrenin 1 gene ("OPHN1") is an Rho-GTPase-activating protein involved in the regulation of the G-protein cycle required for dendritic spine morphogenesis. Mutations in this gene are implicated in X-linked mental retardation (XLMR). Methods: We report a deletion spanning exons 21 and 22 of the "OPHN1" gene identified by a…

  7. Generation of deletions in pneumococcal mal genes cloned in Bacillus subtilis.

    PubMed Central

    Lopez, P; Espinosa, M; Greenberg, B; Lacks, S A

    1984-01-01

    The pneumococcal recombinant plasmid pLS70, which contains two strong promoters for transcription of the malM and malX genes, is unstable when transferred to Bacillus subtilis, and it gives rise to deleted derivatives. Analysis of proteins produced by the deleted plasmids and restriction mapping of 29 different deletions showed that stabilization in B. subtilis was accompanied by deletions affecting both promoters. Plasmids containing even a single strong promoter were at a selective disadvantage. Nucleotide sequences surrounding the deletions in 10 plasmids were determined. Six different deletions occurred between directly repeated sequences of 3-13 base pairs in length, presumably by a recombination mechanism involving short homologies. Four deletions occurred between sites not contained within repeated sequences. A weak but significant similarity of an 11-base sequence was found surrounding these deletions and the corresponding points of junction in the progenitor plasmids. It is suggested that this sequence may be the recognition site for a topoisomerase-like enzyme that can produce deletions. Images PMID:6089185

  8. Deletions in the CGG repeat region of the FMR1 gene

    SciTech Connect

    Graaff, E. de; Oostra, B.A.; Meijer, H.

    1994-09-01

    The fragile X syndrome is the most frequent cause of inherited mental retardation. A remarkable feature of FMR1, the gene involved in the fragile X syndrome, is the presence of a polymorphic (CGG){sub n} repeat in the first exon of the gene. In patients this repeat is expanded to over 200 repeats. The expansion results in methylation of the CpG island 250 bp upstream of this repeat, leading to the absence of FMR1 mRNA and protein, thus resulting in the fragile X phenotype. We have found that the instability of the repeat is not restricted to the CGG repeat itself but expands to the flanking region as well. Firstly, we have identified a family in which 4 males with the fragile X clinical phenotype had a deletion immediately 5{prime} of the CGG repeat. Sequencing the deletion junction revealed that the AGG triplets that normally intersperse the CGG repeat were lacking. This suggests that prior to the deletion an expansion of the repeat had occured. The male patients with this deletion did not have FMR1 mRNA expression. The deceased grandfather, from whom the deletion originated, was fertile, despite the lack of FMR1 mRNA expression. This indicated that FMR1 expression is not required for spermatogenesis. Other deletions were found in 4 individual patients. These patients were mosaic for both a full mutation and a small deletion in the region surrounding the (CGG){sub n} repeat, present in approximately 5% of their cells. Sequence analysis of the regions surrounding the deletions showed that the (CGG){sub n} repeat was missing in all 4 patients. The 5{prime} endpoints of all deletions were found to be located between 75 to 53 bp proximal to the CGG repeat. This suggests a hot spot region for deletions and emphasizes the instability of this region when the CGG repeat is expanded. Models explaining the occurrence of the deletions will be discussed.

  9. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1994-05-01

    From 1971--1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF{sub 1} mice irradiated with {sup 60}Co {gamma}-rays or JANUS fission-spectrum neutrons. Polymerase chain reaction (PCR) technique was used to detect deletions in the mouse retinoblastoma (mRb) gene. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. Absence of any of these fragments on a Southern blot indicated a deletion of that portion of the mRb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice were analyzed for mRb deletions. In all normal mouse tissues studies all six mRb exon fragments were present on Southern blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, 1 of 6 tumors from {gamma}-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice showed a deletion in one or both mRb alleles. All deletions detected were in the 5{prime} region of the mRb gene.

  10. Identification and molecular characterization of four new large deletions in the beta-globin gene cluster.

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Couprie, Nicole; Francina, Alain

    2009-01-01

    Despite the fact that mutations in the human beta-globin gene cluster are essentially point mutations, a significant number of large deletions have also been described. We present here four new large deletions in the beta-globin gene cluster that have been identified on patients displaying an atypical hemoglobin phenotype (high HbF) at routine analysis. The first deletion, which spreads over 2.0 kb, removes the entire beta-globin gene, including its promoter, and is associated with a typical beta-thal minor phenotype. The three other deletions are larger (19.7 to 23.9 kb) and remove both the delta and beta-globin genes. Phenotypically, they look like an HPFH-deletion as they are associated with normal hematological parameters. The precise localization of their 5' and 3' breakpoints gives new insights about the differences between HPFH and (deltabeta)(0)-thalassemia at the molecular level. The importance of detection of these deletions in prenatal diagnosis and newborn screening of hemoglobinopathies is also discussed.

  11. An atypical case of fragile X syndrome caused by a deletion that includes FMRI gene

    SciTech Connect

    Quan, F.; Zonana, J.; Gunter, K.; Peterson, K.L.; Magenis, R.E., Popovich, B.W.

    1995-05-01

    Fragile X syndrome is the most common form of inherited mental retardation and results from the transcriptional inactivation of the FMR1 gene. In the vast majority of cases, this is caused by the expansion of an unstable CGG repeat in the first exon of the FMR1 gene. We describe here a phenotypically atypical case of fragile X syndrome, caused by a deletion that includes the entire FMR1 gene and {ge}9.0 Mb of flanking DNA. The proband, RK, was a 6-year-old mentally retarded male with obesity and anal atresia. A diagnosis of fragile X syndrome was established by the failure of RK`s DNA to hybridize to a 558-bp PstI-XhoI fragment (pfxa3) specific for the 5{prime}-end of the FMR1 gene. The analysis of flanking markers in the interval from Xq26.3-q28 indicated a deletion extending from between 160-500 kb distal and 9.0 Mb proximal to the FMR1 gene. High-resolution chromosome banding confirmed a deletion with breakpoints in Xq26.3 and Xq27.3. This deletion was maternally transmitted and arose as a new mutation on the grandpaternal X chromosome. The maternal transmission of the deletion was confirmed by FISH using a 34-kb cosmid (c31.4) containing most of the FMR1 gene. These results indicated that RK carried a deletion of the FMR1 region with the most proximal breakpoint described to date. This patient`s unusual clinical presentation may indicate the presence of genes located in the deleted interval proximal to the FMR1 locus that are able to modify the fragile X syndrome phenotype. 36 refs., 7 figs.

  12. Alu-mediated large deletion of the CDSN gene as a cause of peeling skin disease.

    PubMed

    Wada, T; Matsuda, Y; Muraoka, M; Toma, T; Takehara, K; Fujimoto, M; Yachie, A

    2014-10-01

    Peeling skin disease (PSD) is an autosomal recessive skin disorder caused by mutations in CDSN and is characterized by superficial peeling of the upper epidermis. Corneodesmosin (CDSN) is a major component of corneodesmosomes that plays an important role in maintaining epidermis integrity. Herein, we report a patient with PSD caused by a novel homozygous large deletion in the 6p21.3 region encompassing the CDSN gene, which abrogates CDSN expression. Several genes including C6orf15, PSORS1C1, PSORS1C2, CCHCR1, and TCF19 were also deleted, however, the patient showed only clinical features typical of PSD. The deletion size was 59.1 kb. Analysis of the sequence surrounding the breakpoint showed that both telomeric and centromeric breakpoints existed within Alu-S sequences that were oriented in opposite directions. These results suggest an Alu-mediated recombination event as the mechanism underlying the deletion in our patient.

  13. Large-scale Phenotypic Profiling of Gene Deletion Mutants in Candida glabrata

    PubMed Central

    Tscherner, Michael; Kuchler, Karl

    2016-01-01

    Here, we describe a method enabling the phenotypic profiling of genome-scale deletion collections of fungal mutants to detect phenotypes for various stress conditions. These stress conditions include among many others antifungal drug susceptibility, temperature-induced and osmotic as well as heavy metal or oxidative stress. The protocol was extensively used to phenotype a collection of gene deletion mutants in the human fungal pathogen Candida glabrata (C. glabrata) (Schwarzmüller et al., 2014). PMID:27774498

  14. Non-myogenic tumors display altered expression of dystrophin (DMD) and a high frequency of genetic alterations

    PubMed Central

    Luce, Leonela N.; Abbate, Mercedes

    2017-01-01

    DMD gene mutations have been associated with the development of Dystrophinopathies. Interestingly, it has been recently reported that DMD is involved in the development and progression of myogenic tumors, assigning DMD a tumor suppressor activity in these types of cancer. However, there are only few reports that analyze DMD in non-myogenic tumors. Our study was designed to examine DMD expression and genetic alterations in non-myogenic tumors using public repositories. We also evaluated the overall survival of patients with and without DMD mutations. We studied 59 gene expression microarrays (GEO database) and RNAseq (cBioPortal) datasets that included 9817 human samples. We found reduced DMD expression in 15/27 (56%) pairwise comparisons performed (Fold-Change (FC) ≤ 0.70; p-value range = 0.04-1.5×10−20). The analysis of RNAseq studies revealed a median frequency of DMD genetic alterations of 3.4%, higher or similar to other well-known tumor suppressor genes. In addition, we observed significant poorer overall survival for patients with DMD mutations. The analyses of paired tumor/normal tissues showed that the majority of tumor specimens had lower DMD expression compared to their normal adjacent counterpart. Interestingly, statistical significant over-expression of DMD was found in 6/27 studies (FC ≥ 1.4; p-value range = 0.03-3.4×10−15). These results support that DMD expression and genetic alterations are frequent and relevant in non-myogenic tumors. The study and validation of DMD as a new player in tumor development and as a new prognostic factor for tumor progression and survival are warranted. PMID:27391342

  15. Functional profiling in Streptococcus mutans: construction and examination of a genomic collection of gene deletion mutants.

    PubMed

    Quivey, R G; Grayhack, E J; Faustoferri, R C; Hubbard, C J; Baldeck, J D; Wolf, A S; MacGilvray, M E; Rosalen, P L; Scott-Anne, K; Santiago, B; Gopal, S; Payne, J; Marquis, R E

    2015-12-01

    A collection of tagged deletion mutant strains was created in Streptococcus mutans UA159 to facilitate investigation of the aciduric capability of this oral pathogen. Gene-specific barcoded deletions were attempted in 1432 open reading frames (representing 73% of the genome), and resulted in the isolation of 1112 strains (56% coverage) carrying deletions in distinct non-essential genes. As S. mutans virulence is predicated upon the ability of the organism to survive an acidic pH environment, form biofilms on tooth surfaces, and out-compete other oral microflora, we assayed individual mutant strains for the relative fitness of the deletion strain, compared with the parent strain, under acidic and oxidative stress conditions, as well as for their ability to form biofilms in glucose- or sucrose-containing medium. Our studies revealed a total of 51 deletion strains with defects in both aciduricity and biofilm formation. We have also identified 49 strains whose gene deletion confers sensitivity to oxidative damage and deficiencies in biofilm formation. We demonstrate the ability to examine competitive fitness of mutant organisms using the barcode tags incorporated into each deletion strain to examine the representation of a particular strain in a population. Co-cultures of deletion strains were grown either in vitro in a chemostat to steady-state values of pH 7 and pH 5 or in vivo in an animal model for oral infection. Taken together, these data represent a mechanism for assessing the virulence capacity of this pathogenic microorganism and a resource for identifying future targets for drug intervention to promote healthy oral microflora.

  16. A missense mutation in the dystrophin gene in a Duchenne muscular dystrophy patient.

    PubMed

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Bartolo, C; Sedra, M S; Western, L M; Mendell, J R

    1993-08-01

    About two thirds of Duchenne muscular dystrophy (DMD) patients have either gene deletions or duplications. The other DMD cases are most likely the result of point mutations that cannot be easily identified by current strategies. Utilizing a heteroduplex technique and direct sequencing of amplified products, we screened our nondeletion/duplication DMD population for point mutations. We now describe what we believe to be the first dystrophin missense mutation in a DMD patient. The mutation results in the substitution of an evolutionarily conserved leucine to arginine in the actin-binding domain. The patient makes a dystrophin protein which is properly localized and is present at a higher level than is observed in DMD patients. This suggests that an intact actin-binding domain is necessary for protein stability and essential for function.

  17. Is NF-1 gene deletion the molecular mechanism of neurofibromatosis type 1 with destinctive facies?

    SciTech Connect

    Leppig, K.A.; Stephens, K.G.; Viskochill, D.; Kaplan, P.

    1994-09-01

    We have studied a patient with neurofibromatosis type 1 and unusual facial features using fluorescence in situ hybridization (FISH) and found that the patient had a deletion that minimially encompasses exon 2-11 of the NF-1 gene. The patient was one of two individuals initially described by Kaplan and Rosenblatt who suggested that another condition aside from neurofibromatosis type 1 may account for the unusual facial features observed in these patients with neurofibromatosis type 1. FISH studies were performed using a P1 clone probe, P1-9, which contains exons 2-11 of the NF-1 gene on chromosomes prepared from the patients. In all 20 metaphase cells analyzed, one of the chromosome 17 homologues was deleted for the P1-9 probe. Therefore, this patient had neurofibromatosis type 1 and unusual facial features as the result of a deletion which minimally includes exons 2-11 of the NF-1 gene. The extent of the deletion is being mapped by FISH and somatic cell hybrid analysis. The patient studied was a 7-year-old male with mild developmental delays, normal growth parameters, and physical findings consistent with neurofibromatosis type 1, including multiple cafe au lait spots, several curaneous neurofibroma, and speckling of the irises. In addition, his unusual facial features consisted of telecanthus, antimongoloid slant of the palpebral fissures, a broad base of the nose, low set and mildly posteriorly rotated ears, thick helices, high arched palate, short and pointed chin, and low posterior hairline. We propose that deletions of the NF-1 gene and/or contiguous genes are the etiology of neurofibromatosis type 1 and unusual facial features. This particular facial appearance was inherited from the patient`s mother and has been described in other individuals with neurofibromatosis type 1. We are using FISH to rapidly screen patients with this phenotype for large deletions involving the NF-1 gene and flanking DNA sequences.

  18. Distribution of CCR5-delta 32 gene deletion across the Russian part of Eurasia.

    PubMed

    Yudin, N S; Vinogradov, S V; Potapova, T A; Naykova, T M; Sitnikova, V V; Kulikov, I V; Khasnulin, V I; Konchuk, C; Vloschinskii, P E; Ivanov, S V; Kobzev, V F; Romaschenko, A G; Voevoda, M I

    1998-06-01

    32-bp inactivating deletion in the beta-chemokine receptor 5 (CCR5) gene, common in Nothern European populations, is associated with reduced HIV-1 transmission risk and delayed disease progression. We have studied the deletion distribution in many populations in Eurasia by polymerase chain reaction analysis of 531 DNA samples representing West and East Siberian, Central Asian, and Far Eastern parts of Russia. An unusually high frequency (11.1%) of the deleted variant in natives of West Siberia, of Finno-Ugrian descent, was observed. Furthermore, the deletion was infrequent in indigenous populations of Central Asia, East Siberia, the Russian Far East, and Canada. We conclude that the delta(ccr5) distribution is limited primarily to Europeans and related western Siberian Finno-Ugrian populations, with a sharp negative gradient toward the east along the territory of Russian Asia.

  19. Deletion map of the Escherichia coli structural gene for alkaline phosphatase, phoA.

    PubMed Central

    Sarthy, A; Michaelis, S; Beckwith, J

    1981-01-01

    Lambda transducing phages containing portions of the phoA gene have been isolated and used to construct a deletion map of the phoA gene. The isolation of a plaque-forming lambda transducing phage carrying the entire phoA gene is also described. Two new methods for screening or selection of mutants that have altered levels of alkaline phosphatase activity are reported. PMID:6450745

  20. Investigation of TBX1 gene deletion in Iranian children with 22q11.2 deletion syndrome: correlation with conotruncal heart defects

    PubMed Central

    Ganji, Hamid; Salehi, Mansoor; Sedghi, Maryam; Abdali, Hossein; Nouri, Nayereh; Sadri, Leyli; Hosseinzadeh, Majid; Vakili, Bahareh; Lotfi, Mahdi

    2013-01-01

    Background DiGeorge syndrome (DGS) is the result of a microdeletion in chromosome 22q11.2 in over 90% of cases. DGS is the second most frequent syndrome after Down syndrome and has an incidence of 1/4000 births. Unequal crossover between low-copy repeats, on the proximal part of the long arm of chromosome 22, usually results in a 3 Mb deletion in one of the chromosome 22 and a reciprocal and similarly sized duplication on the other one. Several studies have indicated that TBX1 (T-box 1) haploinsufficiency is responsible for many of the phenotypic traits of 22q11.2 deletion syndrome. Conotruncal heart defects (CTDs) are present in 75–85% of patients with 22q11.2 deletion syndrome in Western countries. Methods Among 78 patients fulfilling the criteria for DGS diagnosed by the fluorescence in situ hybridisation test, 24 had 22q11.2 deletion. Screening for TBX1 gene deletion was performed by multiplex ligation-dependent probe amplification (MLPA). Results Our results revealed that of 24 patients with TBX1 gene deletion, 12 had CTDs while 12 did not show any heart defects. Conclusions Our findings indicate that other genes or gene interactions may play a role in penetrance or the severity of heart disease among patients with DGS. PMID:27326128

  1. A catalog of genes homozygously deleted in human lung cancer and the candidacy of PTPRD as a tumor suppressor gene.

    PubMed

    Kohno, Takashi; Otsuka, Ayaka; Girard, Luc; Sato, Masanori; Iwakawa, Reika; Ogiwara, Hideaki; Sanchez-Cespedes, Montse; Minna, John D; Yokota, Jun

    2010-04-01

    A total of 176 genes homozygously deleted in human lung cancer were identified by DNA array-based whole genome scanning of 52 lung cancer cell lines and subsequent genomic PCR in 74 cell lines, including the 52 cell lines scanned. One or more exons of these genes were homozygously deleted in one (1%) to 20 (27%) cell lines. These genes included known tumor suppressor genes, e.g., CDKN2A/p16, RB1, and SMAD4, and candidate tumor suppressor genes whose hemizygous or homozygous deletions were reported in several types of human cancers, such as FHIT, KEAP1, and LRP1B/LRP-DIP. CDKN2A/p16 and p14ARF located in 9p21 were most frequently deleted (20/74, 27%). The PTPRD gene was most frequently deleted (8/74, 11%) among genes mapping to regions other than 9p21. Somatic mutations, including a nonsense mutation, of the PTPRD gene were detected in 8/74 (11%) of cell lines and 4/95 (4%) of surgical specimens of lung cancer. Reduced PTPRD expression was observed in the majority (>80%) of cell lines and surgical specimens of lung cancer. Therefore, PTPRD is a candidate tumor suppressor gene in lung cancer. Microarray-based expression profiling of 19 lung cancer cell lines also indicated that some of the 176 genes, such as KANK and ADAMTS1, are preferentially inactivated by epigenetic alterations. Genetic/epigenetic as well as functional studies of these 176 genes will increase our understanding of molecular mechanisms behind lung carcinogenesis.

  2. A Catalog of Genes Homozygously Deleted in Human Lung Cancer and the Candidacy of PTPRD as a Tumor Suppressor Gene

    PubMed Central

    Kohno, Takashi; Otsuka, Ayaka; Girard, Luc; Sato, Masanori; Iwakawa, Reika; Ogiwara, Hideaki; Sanchez-Cespedes, Montse; Minna, John D.; Yokota, Jun

    2010-01-01

    A total of 176 genes homozygously deleted in human lung cancer were identified by DNA array-based whole genome scanning of 52 lung cancer cell lines and subsequent genomic PCR in 74 cell lines, including the 52 cell lines scanned. One or more exons of these genes were homozygously deleted in one (1%) to 20 (27%) cell lines. These genes included known tumor suppressor genes, e.g., CDKN2A/p16, RB1, and SMAD4, and candidate tumor suppressor genes whose hemizygous or homozygous deletions were reported in several types of human cancers, such as FHIT, KEAP1, and LRP1B/LRP-DIP. CDKN2A/p16 and p14ARF located in 9p21 were most frequently deleted (20/74, 27%). The PTPRD gene was most frequently deleted (8/74, 11%) among genes mapping to regions other than 9p21. Somatic mutations, including a nonsense mutation, of the PTPRD gene were detected in 8/74 (11%) of cell lines and 4/95 (4%) of surgical specimens of lung cancer. Reduced PTPRD expression was observed in the majority (>80%) of cell lines and surgical specimens of lung cancer. Therefore, PTPRD is a candidate tumor suppressor gene in lung cancer. Microarray-based expression profiling of 19 lung cancer cell lines also indicated that some of the 176 genes, such as KANK and ADAMTS1, are preferentially inactivated by epigenetic alterations. Genetic/epigenetic as well as functional studies of these 176 genes will increase our understanding of molecular mechanisms behind lung carcinogenesis. PMID:20073072

  3. Rb and p53 gene deletions in lung adenocarcinomas from irradiated and control mice

    SciTech Connect

    Zhang, Y.; Woloschak, G.E.

    1997-08-01

    This study was conducted on mouse lung adenocarcinoma tissues that were formalin-treated and paraffin-embedded 25 years ago to investigate the large gene deletions of mRb and p53 in B6CF{sub 1} male mice. A total of 80 lung tissue samples from irradiated mice and 40 lung samples from nonirradiated controls were randomly selected and examined in the mRb portion of this study. The results showed a significant (P < 0.05) higher percentage of mRb deletions in lung adenocarcinomas from mice exposed to 60 once-weekly {gamma}-ray doses than those from mice receiving 24 once-weekly {gamma}-ray doses at low doses and low dose rates; however, the percentage was not significantly different (P > 0.05) from that for spontaneous lung adenocarcinomas or lung adenocarcinomas from mice exposed to single-dose {gamma} irradiation at a similar total dose. mRb fragments 3 (71%) and 5 (67%), the parts of the gene that encoded the pocket binding region of Rb protein to adenovirus E1A and SV40 T-antigen, were the most frequently deleted fragments. p53 gene deletion analysis was carried out on normal lungs and lung adenocarcinomas that were initially found to bear mRb deletions. Exons 1,4,5,6, and 9 were chosen to be analyzed.

  4. A 50 kb L1-type deletion mutation of the HEXB gene in Sandhoff disease

    SciTech Connect

    Zhang, Z.X.; Wakamatsu, N.; Akerman, B.R.

    1994-09-01

    Sandhoff disease is an autosomal recessive lysosomal storage disease resulting from mutations of the HEXB gene encoding the {beta}-subunit of {beta}-hexosaminidase A. A 16 kb deletion spanning the promoter region to intron 5 of the HEXB gene, occurring in {approximately}25% of mutant alleles, is the most common mutation known. We have identified a second large deletion in a patient with the severe, infantile form of Sandhoff disease. Single strand conformational polymorphism (SSCP) analysis revealed that the proband, a carrier sister and their mother had one dose of the HEXB gene. This was distinguished through the identification of several polymorphic sites between the promoter and exon 5 (father heterozygous at all sites, others {open_quotes}hemizygous{close_quotes}). Using a combination of pulse field electrophoresis and fine mapping by Southern blot analysis, we found that the deletion begins {approximately}25 kb 5{prime} of the HEXB promoter and ends within a BamHI/MscI fragment in intron 6. Sequence analysis of the region abutting the site of the deletion in intron 6 suggests that the deletion arose from recombination between L1-type sequence repeats. The second mutation, inherited from the father, was found by SSCP analysis and direct sequencing of exon 1 PCR products to be C{sub 185}{yields}T (S62T) and was not present in 60 control chromosomes.

  5. Type I oculocutaneous albinism (OCA1) associated with a large deletion of the tyrosinase (TYR) gene

    SciTech Connect

    Spritz, R.A.; Wick, P.A.; Holmes, S.A.; Schnur, R.E. |

    1994-09-01

    OCA1 is an autosomal recessive disorder in which the biosynthesis of melanin is reduced or absent in skin, hair, and eyes, due to deficient enzymatic activity of tyrosinase. TYR consists of 5 exons spanning over 65 kb at 11q14-q21. Analyses of TYR in >400 unrelated patients with OCA1 have identified more than 50 different point mutations; however, no large deletions have been detected. Here we report a large deletion of TYR in a Caucasian boy with OCA1B. Simultaneous SSCP/heteroduplex screening and DNA sequence analysis indicated that the patient was apparently homozygous for a previously described TYR mutation, adjacent to the 3` splice site of IVS2 (-7, t{r_arrow}a). To distinguish between possible gene deletion vs. maternal uniparental isodisomy, we characterized several chromosome 11 polymorphisms. Maternal uniparental isodisomy was excluded by the patient`s heterozygosity for alleles at D11S35 (11q21-122) and HBG2 (11p15.5). In addition, the patient failed to inherit paternal alleles at an MboI RFLP in exon 1 of TYR and at a TaqI RFLP in the promoter region of the gene. To detect a possible submicroscopic deletion, we performed quantitative Southern blot hybridization using a full length TYR cDNA. Compared with controls, both the patient and his father appeared deleted for two or three TYR-derived PstI fragments; the two TYRL-derived fragments appeared normal. These data indicate that the patient and his father have a partial TYR deletion, including at least exons 1, 2, and IVS2. Based on the organization of the gene, this deletion is at least 50 kb in size. The patient is thus hemizygous for the maternally-inherited mutation in IVS2, accounting for his OCA1B phenotype.

  6. PHLPP1 gene deletion protects the brain from ischemic injury

    PubMed Central

    Chen, Bo; Van Winkle, Jessica A; Lyden, Patrick D; Brown, Joan H; Purcell, Nicole H

    2013-01-01

    A recently discovered protein phosphatase PHLPP (PH domain Leucine-rich repeat Protein Phosphatase) has been shown to dephosphorylate Akt on its hydrophobic motif (Ser473) thereby decreasing Akt kinase activity. We generated PHLPP1 knockout (KO) mice and used them to explore the ability of enhanced in vivo Akt signaling to protect the brain against ischemic insult. Brains from KO mice subjected to middle cerebral artery occlusion (MCAO) for 2 hours showed significantly greater increases in Akt activity and less neurovascular damage after reperfusion than wild-type (WT) mice. Remarkably, infarct volume in the PHLPP1 KO was significantly reduced compared with WT (12.7±2.7% versus 22.9±3.1%) and this was prevented by Akt inhibition. Astrocytes from KO mice and neurons in which PHLPP1 was downregulated showed enhanced Akt activation and diminished cell death in response to oxygen-glucose deprivation. Thus, deletion of PHLPP1 can enhance Akt activation in neurons and astrocytes, and can significantly increase cell survival and diminish infarct size after MCAO. Inhibition of PHLPP could be a therapeutic approach to minimize damage after focal ischemia. PMID:23072745

  7. Insertion/deletion-related polymorphisms in the human T cell receptor beta gene complex

    PubMed Central

    1989-01-01

    Insertion/deletion related polymorphisms (IDRP) involving stretches of 15-30 kb within the human TCR-beta gene complex were revealed by pulse- field gel electrophoresis. Two independent IDRP systems were detected by analysis of Sfi I- and Sal I-digested human DNA samples using probes for TCR C and V region gene segments. The allelic nature of these systems was verified in family studies, and mapping data allowed localization of one area of insertion/deletion among the V gene segments and the other near the C region genes. All but one of 50 individuals tested could be typed for the two allelic systems, and gene frequencies for the two allelic forms were 0.37/0.61 and 0.46/0.54, indicating that these polymorphisms are widespread. PMID:2571667

  8. Immunoglobulin V gene replacement is caused by the intramolecular DNA deletion mechanism.

    PubMed Central

    Usuda, S; Takemori, T; Matsuoka, M; Shirasawa, T; Yoshida, K; Mori, A; Ishizaka, K; Sakano, H

    1992-01-01

    Circular DNA resulting from V gene replacement was studied with an A-MuLV transformed cell line containing ablts. This cell line undergoes V gene replacement at elevated temperatures in the immunoglobulin (Ig) heavy chain (H) gene. Examination of circular DNA revealed that a heptamer-related sequence (TACTGTG) within the coding region of VDJ was joined to the recombination signal sequence (RSS) of a germline VH segment. This provides direct evidence for a intramolecular DNA deletion mechanism for V gene replacement. In the pre-B cell line as well as in in vivo lymphocytes, unusual circular DNAs were found which were structurally similar to the V gene replacement circles. They represented excision products of the deletion type recombination between one complete RSS and a heptamer-like sequence in the Ig H region. PMID:1311252

  9. Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy

    PubMed Central

    Yu, Hong-Hao; Zhao, Heng; Qing, Yu-Bo; Pan, Wei-Rong; Jia, Bao-Yu; Zhao, Hong-Ye; Huang, Xing-Xu; Wei, Hong-Jiang

    2016-01-01

    Dystrophinopathy, including Duchenne muscle dystrophy (DMD) and Becker muscle dystrophy (BMD) is an incurable X-linked hereditary muscle dystrophy caused by a mutation in the DMD gene in coding dystrophin. Advances in further understanding DMD/BMD for therapy are expected. Studies on mdx mice and dogs with muscle dystrophy provide limited insight into DMD disease mechanisms and therapeutic testing because of the different pathological manifestations. Miniature pigs share similar physiology and anatomy with humans and are thus an excellent animal model of human disease. Here, we successfully achieved precise DMD targeting in Chinese Diannan miniature pigs by co-injecting zygotes with Cas9 mRNA and sgRNA targeting DMD. Two piglets were obtained after embryo transfer, one of piglets was identified as DMD-modified individual via traditional cloning, sequencing and T7EN1 cleavage assay. An examination of targeting rates in the DMD-modified piglet revealed that sgRNA:Cas9-mediated on-target mosaic mutations were 70% and 60% of dystrophin alleles in skeletal and smooth muscle, respectively. Meanwhile, no detectable off-target mutations were found, highlighting the high specificity of genetic modification using CRISPR/Cas9. The DMD-modified piglet exhibited degenerative and disordered phenotypes in skeletal and cardiac muscle, and declining thickness of smooth muscle in the stomach and intestine. In conclusion, we successfully generated myopathy animal model by modifying the DMD via CRISPR/Cas9 system in a miniature pig. PMID:27735844

  10. Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy.

    PubMed

    Yu, Hong-Hao; Zhao, Heng; Qing, Yu-Bo; Pan, Wei-Rong; Jia, Bao-Yu; Zhao, Hong-Ye; Huang, Xing-Xu; Wei, Hong-Jiang

    2016-10-09

    Dystrophinopathy, including Duchenne muscle dystrophy (DMD) and Becker muscle dystrophy (BMD) is an incurable X-linked hereditary muscle dystrophy caused by a mutation in the DMD gene in coding dystrophin. Advances in further understanding DMD/BMD for therapy are expected. Studies on mdx mice and dogs with muscle dystrophy provide limited insight into DMD disease mechanisms and therapeutic testing because of the different pathological manifestations. Miniature pigs share similar physiology and anatomy with humans and are thus an excellent animal model of human disease. Here, we successfully achieved precise DMD targeting in Chinese Diannan miniature pigs by co-injecting zygotes with Cas9 mRNA and sgRNA targeting DMD. Two piglets were obtained after embryo transfer, one of piglets was identified as DMD-modified individual via traditional cloning, sequencing and T7EN1 cleavage assay. An examination of targeting rates in the DMD-modified piglet revealed that sgRNA:Cas9-mediated on-target mosaic mutations were 70% and 60% of dystrophin alleles in skeletal and smooth muscle, respectively. Meanwhile, no detectable off-target mutations were found, highlighting the high specificity of genetic modification using CRISPR/Cas9. The DMD-modified piglet exhibited degenerative and disordered phenotypes in skeletal and cardiac muscle, and declining thickness of smooth muscle in the stomach and intestine. In conclusion, we successfully generated myopathy animal model by modifying the DMD via CRISPR/Cas9 system in a miniature pig.

  11. Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

    PubMed Central

    Oh, Man Hwan; Lee, Je Chul; Kim, Jungmin

    2015-01-01

    The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant Acinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of A. baumannii merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in A. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by A. baumannii ATCC 19606 and to ompA gene deletion in other A. baumannii strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant A. baumannii. PMID:25746991

  12. Recurring exon deletions in the haptoglobin (HP) gene associate with lower blood cholesterol levels

    PubMed Central

    Boettger, Linda M.; Salem, Rany M.; Handsaker, Robert E.; Peloso, Gina; Kathiresan, Sekar; Hirschhorn, Joel; McCarroll, Steven A.

    2016-01-01

    Two exons of the human haptoglobin (HP) gene exhibit copy number variation that affects HP multimerization and underlies one of the first protein polymorphisms identified in humans. The evolutionary origins and medical significance of this polymorphism have been uncertain. Here we show that this variation has likely arisen from the recurring reversion of an ancient hominin-specific duplication of these exons. Though this polymorphism has been largely invisible to genome-wide genetic studies to date, we describe a way to analyze it by imputation from SNP haplotypes and find among 22,288 individuals that these HP exonic deletions associate with reduced LDL and total cholesterol levels. We show that these deletions, and a SNP that affects HP expression, are the likely drivers of the strong but complex association of cholesterol levels to SNPs near HP. Recurring exonic deletions in the haptoglobin gene likely enhance human health by lowering cholesterol levels in the blood. PMID:26901066

  13. Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27.

    PubMed

    Wang, Lei; Hoffmann, Jana; Watzlawick, Hildegard; Altenbuchner, Josef

    2015-12-11

    We developed a counterselectable deletion system for Thermus thermophilus HB27 based on cytosine deaminase (encoded by codA) from Thermaerobacter marianensis DSM 12885 and the sensitivity of T. thermophilus HB27 to the antimetabolite 5-fluorocytosine (5-FC). The deletion vector comprises the pUC18 origin of replication, a thermostable kanamycin resistance marker functional in T. thermophilus HB27, and codA under the control of a constitutive putative trehalose promoter from T. thermophilus HB27. The functionality of the system was demonstrated by deletion of the bglT gene, encoding a β-glycosidase, and three carotenoid biosynthesis genes, CYP175A1, crtY, and crtI, from the genome of T. thermophilus HB27.

  14. Deletion of Cmu genes in mouse B lymphocytes upon stimulation with LPS.

    PubMed

    Radbruch, A; Sablitzky, F

    1983-01-01

    Mouse B lymphocytes can be activated polyclonally by bacterial lipopolysaccharide (LPS) to differentiate into plasmablasts. Within several days many cells perform immunoglobulin (Ig) class switching in vitro. We have purified LPS blasts expressing IgM or only IgG3 on the cell surface and analysed the DNA of these cells by Southern hybridisation blotting to detect rearrangement or deletion of CH genes. Quantitative evaluation of the Southern blots suggests that populations of surface IgG3+ (sIgG3+) cells from 6-day and sIgM+ cells from 8-day-old cultures contain only about half as many Cmu genes as spleen cells. Cmu deletion is nearly complete in populations of sIgG3+ cells from 9-day-old cultures. Therefore, upon stimulation with LPS, within a few days Cmu is deleted in most sIgG3+ cells from both chromosomes.

  15. Adenoviral vectors encoding CRISPR/Cas9 multiplexes rescue dystrophin synthesis in unselected populations of DMD muscle cells

    PubMed Central

    Maggio, Ignazio; Liu, Jin; Janssen, Josephine M.; Chen, Xiaoyu; Gonçalves, Manuel A. F. V.

    2016-01-01

    Mutations disrupting the reading frame of the ~2.4 Mb dystrophin-encoding DMD gene cause a fatal X-linked muscle-wasting disorder called Duchenne muscular dystrophy (DMD). Genome editing based on paired RNA-guided nucleases (RGNs) from CRISPR/Cas9 systems has been proposed for permanently repairing faulty DMD loci. However, such multiplexing strategies require the development and testing of delivery systems capable of introducing the various gene editing tools into target cells. Here, we investigated the suitability of adenoviral vectors (AdVs) for multiplexed DMD editing by packaging in single vector particles expression units encoding the Streptococcus pyogenes Cas9 nuclease and sequence-specific gRNA pairs. These RGN components were customized to trigger short- and long-range intragenic DMD excisions encompassing reading frame-disrupting exons in patient-derived muscle progenitor cells. By allowing synchronous and stoichiometric expression of the various RGN components, we demonstrate that dual RGN-encoding AdVs can correct over 10% of target DMD alleles, readily leading to the detection of Becker-like dystrophin proteins in unselected muscle cell populations. Moreover, we report that AdV-based gene editing can be tailored for removing mutations located within the over 500-kb major DMD mutational hotspot. Hence, this single DMD editing strategy can in principle tackle a broad spectrum of mutations present in more than 60% of patients with DMD. PMID:27845387

  16. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1993-04-01

    From 1971 to 1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF{sub 1} mice irradiated with {sup 60}Co {gamma} rays or JANUS fission-spectrum neutrons; normal and tumor tissues from mice in these studies were preserved in paraffin blocks. A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma (mRb) gene in the paraffin-embedded tissues. Microtomed sections were used as the DNA source in PCR reaction mixtures. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments (relative to control PCR products) on a Southern blot indicated a deletion of that portion of the mRb gene. The tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice (569 cGy of {sup 60}Co {gamma} rays or 60 cGy of JANUS neutrons, doses that have been found to have approximately equal biological effectiveness in the BCF, mouse) were analyzed for mRb deletions. In all normal mouse tissues studies, all six mRb exon fragments were present on Southem blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, I of 6 tumors from {gamma}-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice had a deletion in one or both mRb alleles. All deletions detected were in the 5{prime} region of the mRb gene.

  17. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1993-01-01

    From 1971 to 1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF[sub 1] mice irradiated with [sup 60]Co [gamma] rays or JANUS fission-spectrum neutrons; normal and tumor tissues from mice in these studies were preserved in paraffin blocks. A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma (mRb) gene in the paraffin-embedded tissues. Microtomed sections were used as the DNA source in PCR reaction mixtures. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments (relative to control PCR products) on a Southern blot indicated a deletion of that portion of the mRb gene. The tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice (569 cGy of [sup 60]Co [gamma] rays or 60 cGy of JANUS neutrons, doses that have been found to have approximately equal biological effectiveness in the BCF, mouse) were analyzed for mRb deletions. In all normal mouse tissues studies, all six mRb exon fragments were present on Southem blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, I of 6 tumors from [gamma]-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice had a deletion in one or both mRb alleles. All deletions detected were in the 5[prime] region of the mRb gene.

  18. Efficient sequential repetitive gene deletions in Neurospora crassa employing a self-excising β-recombinase/six cassette.

    PubMed

    Szewczyk, Edyta; Kasuga, Takao; Fan, Zhiliang

    2013-03-01

    Despite its long-standing history as a model organism, Neurospora crassa has limited tools for repetitive gene deletions utilizing recyclable self-excising marker systems. Here we describe, for the first time, the functionality of a bacterial recombination system employing β-recombinase acting on six recognition sequences (β-rec/six) in N. crassa, which allowed repetitive site-specific gene deletion and marker recycling. We report generating the mus-51 deletion strain using this system, recycling the marker cassette, and subsequently deleting the global transcriptional regulator gene cre-1.

  19. [Orthopoxvirus genes for kelch-like proteins. III. Construction of mousepox (ectromelia) virus variants with targeted gene deletions].

    PubMed

    Kochneva, G V; Kolosova, I V; Lupan, T A; Sivolobova, G F; Iudin, P V; Grazhdantseva, A A; Riabchikova, E I; Kandrina, N Iu; Shchelkunov, S N

    2009-01-01

    Mousepox (ectromelia) virus genome contains four genes encoding for kelch-like proteins EVM018, EVM027, EVM150 and EVM167. A complete set of insertion plasmids was constructed to allow the production of recombinant ectromelia viruses with targeted deletions of one to four genes of kelch family both individually (single mutants) and in different combinations (double, triple and quadruple mutants). It was shown that deletion of any of the three genes EVMO18, EVM027 or EVM167 resulted in reduction of 50% lethal dose (LD50) by five and more orders in outbred white mice infected intraperitoneally. Deletion of mousepox kelch-gene EVM150 did not influence the virus virulence. Two or more kelch-genes deletion also resulted in high level of attenuation, which could evidently be due to the lack of three genes EVM167, EVM018 and/or EVM027 identified as virulence factors. The local inflammatory process on the model of intradermal injection of mouse ear pinnae (vasodilatation level, hyperemia, cutaneous edema, arterial thrombosis) was significantly more intensive for wild type virus and virulent mutant deltaEVM150 in comparison with avirulent mutant AEVM167.

  20. Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Direct cellobiose production from cellulose by a genetically modified fungus—Neurospora crassa, was explored in this study. A library of N. crassa sextuple beta-glucosidase (bgl) gene deletion strains was constructed. Various concentrations of cellobiose were detected in the culture broth of the N. ...

  1. Soluble epoxide hydrolase: Gene structure, expression and deletion

    PubMed Central

    Harris, Todd R.; Hammock, Bruce D.

    2013-01-01

    Mammalian soluble epoxide hydrolase (sEH) converts epoxides to their corresponding diols through the addition of a water molecule. sEH readily hydrolyzes lipid signaling molecules, including the epoxyeicosatrienoic acids (EETs), epoxidized lipids produced from arachidonic acid by the action of cytochrome p450s. Through its metabolism of the EETs and other lipid mediators, sEH contributes to the regulation of vascular tone, nociception, angiogenesis and the inflammatory response. Because of its central physiological role in disease states such as cardiac hypertrophy, diabetes, hypertension, and pain sEH is being investigated as a therapeutic target. This review begins with a brief introduction to sEH protein structure and function. sEH evolution and gene structure are then discussed before human small nucleotide polymorphisms and mammalian gene expression are described in the context of several disease models. The review ends with an overview of studies that have employed the sEH knockout mouse model. PMID:23701967

  2. Translocation (X;6) in a female with Duchenne muscular dystrophy: implications for the localisation of the DMD locus.

    PubMed Central

    Zatz, M; Vianna-Morgante, A M; Campos, P; Diament, A J

    1981-01-01

    A female with Duchenne muscular dystrophy who was a carrier of a balanced translocation t(X;6)(p21;q21) is reported. Four other previously described (X;A) translocations associated with DMD share with the present case a breakpoint at Xp21. The extremely low probability of five independent (X;A) translocations having a breakpoint at Xp21 points to a non-rand association of this site with the DMD phenotype. A DMD locus at Xp21 could be damaged by the translocation, giving rise to Duchenne muscular dystrophy. Alternatively, a pre-existing DMD gene could weaken the chromosome, favouring breaks at Xp21. Images PMID:7334502

  3. Interstitial deletion of 11q-implicating the KIRREL3 gene in the neurocognitive delay associated with Jacobsen syndrome.

    PubMed

    Guerin, Andrea; Stavropoulos, Dimitri J; Diab, Yaser; Chénier, Sébastien; Christensen, Hilary; Kahr, Walter H A; Babul-Hirji, Riyana; Chitayat, David

    2012-10-01

    Jacobsen syndrome (JS) is a rare contiguous gene disorder characterized by a deletion within the distal part of the long arm of chromosome 11 ranging in size from 7 to 20 Mb. The clinical findings include characteristic dysmorphic features, growth and psychomotor delays and developmental anomalies involving the brain, eyes, heart, kidneys, immune, hematologic, endocrine, and gastrointestinal systems. The majority of cases are due to a terminal deletion of 11q; however interstitial deletions have also been reported. We report on a child with clinical manifestations consistent with JS who had a 2.899 Mb interstitial deletion at 11q24.2-q24.3 which is the smallest interstitial deletion reported so far to our knowledge. This deletion includes the KIRREL3 gene, and given our patient's history of neurocognitive delay and autism spectrum disorder, it raises the possibility that this gene is a candidate for the social and expressive language delay observed in our patient.

  4. Deletion of Interstitial Genes between TMPRSS2 and ERG Promotes Prostate Cancer Progression.

    PubMed

    Linn, Douglas E; Penney, Kathryn L; Bronson, Roderick T; Mucci, Lorelei A; Li, Zhe

    2016-04-01

    TMPRSS2-ERG gene fusions that occur frequently in human prostate cancers can be generated either through insertional chromosomal rearrangement or by intrachromosomal deletion. Genetically, a key difference between these two mechanisms is that the latter results in deletion of a ∼3-Mb interstitial region containing genes with unexplored roles in prostate cancer. In this study, we characterized two mouse models recapitulating TMPRSS2-ERG insertion or deletion events in the background of prostate-specific PTEN deficiency. We found that only the mice that lacked the interstitial region developed prostate adenocarcinomas marked by poor differentiation and epithelial-to-mesenchymal transition. Mechanistic investigations identified several interstitial genes, including Ets2 and Bace2, whose reduced expression correlated in the gene homologs in human prostate cancer with biochemical relapse and lethal disease. Accordingly, PTEN-deficient mice with prostate-specific knockout of Ets2 exhibited marked progression of prostate adenocarcinomas that was partly attributed to activation of MAPK signaling. Collectively, our findings established that Ets2 is a tumor suppressor gene in prostate cancer, and its loss along with other genes within the TMPRSS2-ERG interstitial region contributes to disease progression. Cancer Res; 76(7); 1869-81. ©2016 AACR.

  5. Human cone visual pigment deletions spare sufficient photoreceptors to warrant gene therapy.

    PubMed

    Cideciyan, Artur V; Hufnagel, Robert B; Carroll, Joseph; Sumaroka, Alexander; Luo, Xunda; Schwartz, Sharon B; Dubra, Alfredo; Land, Megan; Michaelides, Michel; Gardner, Jessica C; Hardcastle, Alison J; Moore, Anthony T; Sisk, Robert A; Ahmed, Zubair M; Kohl, Susanne; Wissinger, Bernd; Jacobson, Samuel G

    2013-12-01

    Human X-linked blue-cone monochromacy (BCM), a disabling congenital visual disorder of cone photoreceptors, is a candidate disease for gene augmentation therapy. BCM is caused by either mutations in the red (OPN1LW) and green (OPN1MW) cone photoreceptor opsin gene array or large deletions encompassing portions of the gene array and upstream regulatory sequences that would predict a lack of red or green opsin expression. The fate of opsin-deficient cone cells is unknown. We know that rod opsin null mutant mice show rapid postnatal death of rod photoreceptors. Using in vivo histology with high-resolution retinal imaging, we studied a cohort of 20 BCM patients (age range 5-58) with large deletions in the red/green opsin gene array. Already in the first years of life, retinal structure was not normal: there was partial loss of photoreceptors across the central retina. Remaining cone cells had detectable outer segments that were abnormally shortened. Adaptive optics imaging confirmed the existence of inner segments at a spatial density greater than that expected for the residual blue cones. The evidence indicates that human cones in patients with deletions in the red/green opsin gene array can survive in reduced numbers with limited outer segment material, suggesting potential value of gene therapy for BCM.

  6. FOXP2 gene deletion and infant feeding difficulties: a case report

    PubMed Central

    Zimmerman, Emily; Maron, Jill L.

    2016-01-01

    Forkhead box protein P2 (FOXP2) is a well-studied gene known to play an essential role in normal speech development. Deletions in the gene have been shown to result in developmental speech disorders and regulatory disruption of downstream gene targets associated with common forms of language impairments. Despite similarities in motor planning and execution between speech development and oral feeding competence, there have been no reports to date linking deletions within the FOXP2 gene to oral feeding impairments in the newborn. The patient was a nondysmorphic, appropriately and symmetrically grown male infant born at 35-wk gestational age. He had a prolonged neonatal intensive care unit stay because of persistent oral feeding incoordination requiring gastrostomy tube placement. Cardiac and neurological imagings were within normal limits. A microarray analysis found an ∼9-kb loss within chromosome band 7q3.1 that contains exon 2 of FOXP2, demonstrating a single copy of this region instead of the normal two copies per diploid gene. This case study expands our current understanding of the role FOXP2 exerts on motor planning and coordination necessary for both oral feeding success and speech–language development. This case report has important consequences for future diagnosis and treatment for infants with FOXP2 deletions, mutations, and varying levels of gene expression. PMID:27148578

  7. Tackling the issue of environmental survival of live Salmonella Typhimurium vaccines: deletion of the lon gene.

    PubMed

    Leyman, Bregje; Boyen, Filip; Van Parys, Alexander; Verbrugghe, Elin; Haesebrouck, Freddy; Pasmans, Frank

    2012-12-01

    Vaccination is an important measure to control Salmonella contamination in the meat production chain. A previous study showed that both the ΔrfaJ and ΔrfaL strains are suitable markers and allow serological differentiation of infected and vaccinated animals. The aim of this study was to verify whether deletion of the lon gene in a Salmonella Typhimurium ΔrfaJ marker strain resulted in decreased environmental survival. Our results indicate that deletion of the lon gene in the ΔrfaJ strain did not affect invasiveness in IPEC-J2 cells and resulted in an increased susceptibility to UV, disinfectants (such as hydrogen peroxide and tosylchloramide sodium) and citric acid. Immunization of pigs with inactivated ΔrfaJ or ΔlonΔrfaJ vaccines allowed differentiation of infected and vaccinated pigs. Furthermore, deletion of the lon gene did not reduce the protection conferred by live wild type or ΔrfaJ vaccines against subsequent challenge with a virulent Salmonella Typhimurium strain in BALB/c mice. Based on our results in mice, we conclude that deletion of lon in ΔrfaJ contributes to environmental safety of the ΔrfaJ DIVA strain.

  8. Large exonic deletions in POLR3B gene cause POLR3-related leukodystrophy.

    PubMed

    Gutierrez, Mariana; Thiffault, Isabelle; Guerrero, Kether; Martos-Moreno, Gabriel Á; Tran, Luan T; Benko, William; van der Knaap, Marjo S; van Spaendonk, Rosalina M L; Wolf, Nicole I; Bernard, Geneviève

    2015-06-05

    POLR3-related (or 4H) leukodystrophy is an autosomal recessive disorder caused by mutations in POLR3A or POLR3B and is characterized by neurological and non-neurological features. In a small proportion of patients, no mutation in either gene or only one mutation is found. Analysis of the POLR3B cDNA revealed a large deletion of exons 21-22 in one case and of exons 26-27 in another case. These are the first reports of long deletions causing POLR3-related leukodystrophy, suggesting that deletions and duplications in POLR3A or POLR3B should be investigated in patients with a compatible phenotype, especially if one pathogenic variant has been identified.

  9. Phosphoinositide-specific Phospholipase C β1 gene deletion in bipolar disorder affected patient.

    PubMed

    Lo Vasco, Vincenza Rita; Longo, Lucia; Polonia, Patrizia

    2013-03-01

    The involvement of phosphoinositides (PI) signal transduction pathway and related molecules, such as the Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes, in the pathophysiology of mood disorders is corroborated by a number of recent evidences. Our previous works identified the deletion of PLCB1 gene, which codifies for the PI-PLC β1 enzyme, in 4 out 15 patients affected with schizophrenia, and no deletion both in major depression affected patients and in normal controls. By using interphase fluorescent in situ hybridization methodology, we analyzed PLCB1 in paraffin embedded samples of orbito-frontal cortex of 15 patients affected with bipolar disorder. Deletion of PLCB1 was identified in one female patient.

  10. Phenotype-Genotype Discrepancy Due to a 5.5-kb Deletion in the GALT Gene.

    PubMed

    González-del Angel, Ariadna; Velázquez-Aragón, José; Alcántara-Ortigoza, Miguel A; Vela-Amieva, Marcela; Hernández-Martínez, Nancy

    2012-01-01

    Classical galactosemia is an autosomal recessive inborn error of metabolism caused by a deficiency of the galactose-1-phosphate uridyltransferase (GALT). More than 200 mutations have been described in the GALT gene. A 5.5-kb GALT deletion, first described in patients of Ashkenazi Jewish ancestry, may lead either to an erroneous genotype assignment of classical galactosemia or to discrepancies with parental genotypes and the expected biochemical phenotype. The presence of the 5.5-kb deletion was examined in 27 Mexican nonrelated families with at least one child with reduced GALT activity in erythrocytes and it was detected in the 5.5% (n=3) of the 54 alleles tested. The first molecular studies in three of our families showed that the genotypes of the parents were inconsistent with those of their children, which were considered initially as homozygous p.N314D-Duarte 2, but after analyzing for the presence of the 5.5-kb deletion, were reassigned as compound heterozygotes [5.5-kb deletion]+[p.N314D-Duarte 2]. Identification of the 5.5-kb deletion in Mexican patients suggests that this mutation might not be exclusive to a given ethnic group and should be tested in other populations, especially when there is a discrepancy between the genotypes of patients and parents or by incongruence between biochemical phenotype and GALT genotype. Establishing a genotype-phenotype correlation for the 5.5-kb GALT deletion and determining the appropriate management will require additional studies in patients with a G/G genotype bearing the 5.5-kb GALT deletion.

  11. Total alpha-globin gene cluster deletion has high frequency in Filipinos

    SciTech Connect

    Hunt, J.A.; Haruyama, A.Z.; Chu, B.M.

    1994-09-01

    Most {alpha}-thalassemias [Thal] are due to large deletions. In Southeast Asians, the (--{sup SEA}) double {alpha}-globin gene deletion is common, 3 (--{sup Tot}) total {alpha}-globin cluster deletions are known: Filipino (--{sup Fil}), Thai (--{sup Thai}), and Chinese (--{sup Chin}). In a Hawaii Thal project, provisional diagnosis of {alpha}-Thal-1 heterozygotes was based on microcytosis, normal isoelectric focusing, and no iron deficiency. One in 10 unselected Filipinos was an {alpha}-Thal-1 heterozygote, 2/3 of these had a (--{sup Tot}) deletion: a {var_sigma}-cDNA probe consistently showed fainter intensity of the constant 5.5 kb {var_sigma}{sub 2} BamHI band, with no heterzygosity for {var_sigma}-globin region polymorphisms; {alpha}-cDNA or {var_sigma}-cDNA probes showed no BamHI or BglII bands diagnostic of the (--{sup SEA}) deletion; bands for the (-{alpha}) {alpha}-Thal-2 single {alpha}-globin deletions were only seen in Hb H cases. A reliable monoclonal anti-{var_sigma}-peptide antibody test for the (--{sup SEA}) deletion was always negative in (--{sup Tot}) samples. Southern digests with the Lo probe, a gift from D. Higgs of Oxford Univ., confirmed that 49 of 50 (--{sup Tot}) chromosomes in Filipinos were (--{sup Fil}). Of 20 {alpha}-Thal-1 hydrops born to Filipinos, 11 were (--{sup Fil}/--{sup SEA}) compound heterozygotes; 9 were (--{sup SEA}/--{sup SEA}) homozygotes, but none was a (--{sup Fil}/--{sup Fil}).

  12. Pex gene deletions in Gy and Hyp mice provide mouse models for X-linked hypophosphatemia.

    PubMed

    Strom, T M; Francis, F; Lorenz, B; Böddrich, A; Econs, M J; Lehrach, H; Meitinger, T

    1997-02-01

    X-linked hypophosphatemic rickets in humans is caused by mutations in the PEX gene which codes for a protein homologous to neutral endopeptidases. Hyp and Gy mice both have X-linked hypophosphatemic rickets, although genetic data and the different phenotypic spectra observed have previously suggested that two different genes are mutated. In addition to the metabolic disorder observed in Hyp mice, male Gy mice are sterile and show circling behavior and reduced viability. We now report the cloning of the mouse homolog of PEX which is highly conserved between man and mouse. The 3' end of this gene is deleted in Hyp mice. In Gy mice, the first three exons and the promotor region are deleted. Thus, Hyp and Gy are allelic mutations and both provide mouse models for X-linked hypophosphatemia.

  13. Deletion in the FMR1 gene in a fragile-X male

    SciTech Connect

    Mannermaa, A.; Pulkkinen, L.; Kajanoja, E.

    1996-08-09

    The pathogenesis of fragile-X syndrome is a consequence of absence of the FMR1 gene product associated with expansion of the CGG repeat and abnormal methylation of this and a CpG island 250 hp proximal to the CGG repeat located at exon 1 in the FMR1 gene. While this is usually the case, some suspected fragile-X syndrome patients have been described with a mutation other than CGG expansion. We describe here an affected fragile-X male, who was found to be mosaic of a full mutation of the CGG expansion and a deletion in the FMR1 gene. The patient`s phenotype is probably mainly due to the effect of the full mutation of the repeat sequence. Thus, the influence of the deletion is difficult to evaluate. 20 refs., 2 figs.

  14. Molecular basis and consequences of a deletion in the amelogenin gene, analyzed by capture PCR

    SciTech Connect

    Lagerstroem-Fermer, M.; Pettersson, U.; Landegren, U. )

    1993-07-01

    A mutation that disrupts the gene for one of the major proteins in tooth enamel has been investigated. The mutation is located in the amelogenin gene and causes X-linked amelogenesis imperfecta, characterized by defective mineralization of tooth enamel. The authors have isolated the breakpoints of a 5-kb deletion in the amelogenin gene on the basis of nucleotide sequence information located upstream of the lesion, using a technique termed capture PCR. The deletion removes five of the seven exons, spanning from the second intron to the last exon. Only the first two codons for the mature protein remain, consistent with the relatively severe phenotype of affected individuals in the present family. The mutation appears to have arisen as an illegitimate recombination event since of 11 nucleotide positions immediately surrounding the two breakpoints, 9 are identical. 17 refs., 3 figs., 1 tab.

  15. A common deletion at D6S265 in the hemochromatosis gene region

    SciTech Connect

    Pyper, W.R.; Burt, M.J.; Powell, L.W.

    1994-09-01

    Positional cloning of the hemochromatosis (HC) gene on chromosome 6p has utilized a number of highly polymorphic microsatellite markers. While the putative HC gene has been localized within 1 cM of HLA-A, definition of the genetic limits of the HC locus has been controversial. Isolation and characterization of additional markers within this region will enable construction of a physical map upon which the HC gene can located. D6S265 is one such microsatellite, physically mapped within 120 kb centromeric of HLA-A. Recombinant and linkage analysis of this dinucleotide repeat in 24 Australian families segregating for HC positioned D6S265 within 1 cM of the HC gene, while allele association analysis showed allele 1 to be significantly increased in HC patients ({chi}{sup 2}=41.4, p<0.001, RR=5.75). In 6 of the 24 HC families, a D6265 locus deletion was found to segregate with HLA-A25 and HLA-A26 alleles. The D6S265 locus deletion was not associated with expression of HC. This study enables us to exclude candidate HC genes from the deleted region involving D6S265, and gives further support for an area of instability in the HLA class I region.

  16. Engineering validamycin production by tandem deletion of γ-butyrolactone receptor genes in Streptomyces hygroscopicus 5008.

    PubMed

    Tan, Gao-Yi; Peng, Yao; Lu, Chenyang; Bai, Linquan; Zhong, Jian-Jiang

    2015-03-01

    Paired homologs of γ-butyrolactone (GBL) biosynthesis gene afsA and GBL receptor gene arpA are located at different positions in genome of Streptomyces hygroscopicus 5008. Inactivation of afsA homologs dramatically decreased biosynthesis of validamycin, an important anti-fungal antibiotic and a critical substrate for antidiabetic drug synthesis, and the deletion of arpA homologs increased validamycin production by 26% (ΔshbR1) and 20% (ΔshbR3). By double deletion, the ΔshbR1/R3 mutant showed higher transcriptional levels of adpA-H (the S. hygroscopicus ortholog of the global regulatory gene adpA) and validamycin biosynthetic genes, and validamycin production increased by 55%. Furthermore, by engineering a high-producing industrial strain via tandem deletion of GBL receptor genes, validamycin production and productivity were enhanced from 19 to 24 g/L (by 26%) and from 6.7 to 9.7 g/L(-1) d(-1) (by 45%), respectively, which was the highest ever reported. The strategy demonstrated here may be useful to engineering other Streptomyces spp. with multiple pairs of afsA-arpA homologs.

  17. Efficient BLG-Cre mediated gene deletion in the mammary gland.

    PubMed

    Selbert, S; Bentley, D J; Melton, D W; Rannie, D; Lourenço, P; Watson, C J; Clarke, A R

    1998-09-01

    Using the phage P1-derived Cre/loxP recombination system, we have developed a strategy for efficient mammary tissue specific inactivation of floxed genes. Transgenic mice were generated which express Cre DNA-recombinase under the control of the mammary gland specific promoter of the ovine beta-lactoglobulin (BLG) gene. To test the specificity of Cre mediated recombination, we crossed these mice to animals harbouring a floxed DNA ligase I allele. We show that the BLG-Cre construct specifies mammary specific gene deletion, and furthermore that it is temporally regulated, predominantly occurring during lactation. We fully characterised the extent of gene deletion in one line (line 74). In this strain the virgin gland is characterised by low levels (7%) of Cre mediated deletion, whereas 70-80% of cells within the lactating mammary gland have undergone recombination. Immunohistochemistry and indirect in situ PCR were used respectively to demonstrate that both Cre protein and Cre activity were evenly distributed throughout the population of secretory epithelial cells. The level of background recombination in non-mammary tissues was found to be < or = 1.1%, irrespective of mammary gland developmental status. Crossing the transgenic BLG-Cre strain described here to mice harbouring other floxed alleles will facilitate the functional analysis of those genes during differentiation and development of the mammary gland.

  18. A Human Minor Histocompatibility Antigen Resulting from Differential Expression due to a Gene Deletion

    PubMed Central

    Murata, Makoto; Warren, Edus H.; Riddell, Stanley R.

    2003-01-01

    Minor histocompatibility antigens (minor H antigens) are targets of graft-versus-host disease and graft-versus-leukemia responses after allogeneic human leukocyte antigen identical hematopoietic stem cell transplantation. Only a few human minor H antigens have been molecularly characterized and in all cases, amino acid differences between homologous donor and recipient proteins due to nucleotide polymorphisms in the respective genes were responsible for immunogenicity. Here, we have used cDNA expression cloning to identify a novel human minor H antigen encoded by UGT2B17, an autosomal gene in the multigene UDP-glycosyltransferase 2 family that is selectively expressed in liver, intestine, and antigen-presenting cells. In contrast to previously defined human minor H antigens, UGT2B17 is immunogenic because of differential expression of the protein in donor and recipient cells as a consequence of a homozygous gene deletion in the donor. Deletion of individual members of large gene families is a common form of genetic variation in the population and our results provide the first evidence that differential protein expression as a consequence of gene deletion is a mechanism for generating minor H antigens in humans. PMID:12743171

  19. Comparative transcriptome analysis of muscular dystrophy models Large(myd), Dmd(mdx)/Large(myd) and Dmd(mdx): what makes them different?

    PubMed

    Almeida, Camila F; Martins, Poliana Cm; Vainzof, Mariz

    2016-08-01

    Muscular dystrophies (MD) are a clinically and genetically heterogeneous group of Mendelian diseases. The underlying pathophysiology and phenotypic variability in each form are much more complex, suggesting the involvement of many other genes. Thus, here we studied the whole genome expression profile in muscles from three mice models for MD, at different time points: Dmd(mdx) (mutation in dystrophin gene), Large(myd-/-) (mutation in Large) and Dmd(mdx)/Large(myd-/-) (both mutations). The identification of altered biological functions can contribute to understand diseases and to find prognostic biomarkers and points for therapeutic intervention. We identified a substantial number of differentially expressed genes (DEGs) in each model, reflecting diseases' complexity. The main biological process affected in the three strains was immune system, accounting for the majority of enriched functional categories, followed by degeneration/regeneration and extracellular matrix remodeling processes. The most notable differences were in 21-day-old Dmd(mdx), with a high proportion of DEGs related to its regenerative capacity. A higher number of positive embryonic myosin heavy chain (eMyHC) fibers confirmed this. The new Dmd(mdx)/Large(myd-/-) model did not show a highly different transcriptome from the parental lineages, with a profile closer to Large(myd-/-), but not bearing the same regenerative potential as Dmd(mdx). This is the first report about transcriptome profile of a mouse model for congenital MD and Dmd(mdx)/Large(myd). By comparing the studied profiles, we conclude that alterations in biological functions due to the dystrophic process are very similar, and that the intense regeneration in Dmd(mdx) involves a large number of activated genes, not differentially expressed in the other two strains.

  20. [Treatment progress of Duchenne Muscular Dystrophy (DMD)].

    PubMed

    Smogorzewska, Elzbieta Monika; Weinberg, Kenneth I

    2004-01-01

    Duchenne muscular dystrophy (DMD) is a common lethal disease for which no effective treatment is currently available. There exists a mouse model of the disease in which the usefulness of gene therapy was established. However, no progress towards human application was made due to the lack of a proper method for gene delivery. During the past several years, researchers acquired data which led them to believe that bone marrow stem cells are capable of generating not only blood cells, but also liver, heart, skin, muscle, and other tissue. Although the term "stem cell plasticity" became very popular, other studies have suggested that bone marrow might contain different types of stem cells that can produce non-hematopoietic cells. For example, mesenchymal stem cell (MSC) in bone marrow give rise to osteocytes, chondrocytes, adipocytes, and skeletal muscle. Recently, researchers have been able to show that transplanted bone marrow cells can contribute to muscle cells in a human patient who was diagnosed with two genetic diseases: severe combined immunodeficiency (SCID) and Duchenne muscular dystrophy. The odds of this happening is estimated at one in seven million. The results of studying this patient's medical history were reported by collaborating researchers at Children's Hospital, Los Angeles and Children's Hospital, Boston in an article titled "Long-term persistence of donor nuclei in a Duchenne muscular dystrophy (DMD) patient receiving bone marrow transplantation" published in the September 2002 issue of the Journal of Clinical Investigation. This patient was transplanted 15 years ago at Children's Hospital Los Angeles with paternal HLA-haploidentical T cell-depleted bone marrow. He engrafted and became a hematopoietic chimera having T and NK lymphocytes of donor origin. Studies performed on the muscle biopsy from the patient 13 years after transplantation demonstrated that the muscle showed evidence of donor derived nuclei. In addition, analysis of his bone marrow

  1. A new deletion in autosomal dominant guanosine triphosphate cyclohydrolase I deficiency gene--Segawa disease.

    PubMed

    Bianca, S; Bianca, M

    2006-02-01

    Hereditary Progressive Dystonia with marked diurnal fluctuation (HPD) is an autosomally dominantly inherited dystonia which is characterized by marked diurnal fluctuation of symptoms and by marked and sustained response to levodopa associated with mutations in guanosine triphosphate cyclohydrolase (GCH-1) deficiency gene. We report an italian patient with a new 18 bp deletion at 267 in exon 1 in the GCH-1 gene. The peculiarity of our patient is the new mutations never reported and mnemonic disturbances that are also not reported in the classical HPD.A genotype-phenotype relationship may be suggested between different gene mutations and non classical clinical manifestations.

  2. Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis

    PubMed Central

    Mueller, Konrad E.; Wolf, Katerina

    2016-01-01

    ABSTRACT Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation. PMID:26787828

  3. SHOX gene and conserved noncoding element deletions/duplications in Colombian patients with idiopathic short stature

    PubMed Central

    Sandoval, Gloria Tatiana Vinasco; Jaimes, Giovanna Carola; Barrios, Mauricio Coll; Cespedes, Camila; Velasco, Harvy Mauricio

    2014-01-01

    SHOX gene mutations or haploinsufficiency cause a wide range of phenotypes such as Leri Weill dyschondrosteosis (LWD), Turner syndrome, and disproportionate short stature (DSS). However, this gene has also been found to be mutated in cases of idiopathic short stature (ISS) with a 3–15% frequency. In this study, the multiplex ligation-dependent probe amplification (MLPA) technique was employed to determine the frequency of SHOX gene mutations and their conserved noncoding elements (CNE) in Colombian patients with ISS. Patients were referred from different centers around the county. From a sample of 62 patients, 8.1% deletions and insertions in the intragenic regions and in the CNE were found. This result is similar to others published in other countries. Moreover, an isolated case of CNE 9 duplication and a new intron 6b deletion in another patient, associated with ISS, are described. This is one of the first studies of a Latin American population in which deletions/duplications of the SHOX gene and its CNE are examined in patients with ISS. PMID:24689071

  4. Metabolic network analysis revealed distinct routes of deletion effects between essential and non-essential genes.

    PubMed

    Ma, Jing; Zhang, Xun; Ung, Choong Yong; Chen, Yu Zong; Li, Baowen

    2012-04-01

    Interest in essential genes has arisen recently given their importance in antimicrobial drug development. Although knockouts of essential genes are commonly known to cause lethal phenotypes, there is insufficient understanding on the intermediate changes followed by genetic perturbation and to what extent essential genes correlate to other genes. Here, we characterized the gene knockout effects by using a list of affected genes, termed as 'damage lists'. These damage lists were identified through a refined cascading failure approach that was based on a previous topological flux balance analysis. Using an Escherichia coli metabolic network, we incorporated essentiality information into damage lists and revealed that the knockout of an essential gene mainly affects a large range of other essential genes whereas knockout of a non-essential gene only interrupts other non-essential genes. Also, genes sharing common damage lists tend to have the same essentiality. We extracted 72 core functional modules from the common damage lists of essential genes and demonstrated their ability to halt essential metabolites production. Overall, our network analysis revealed that essential and non-essential genes propagated their deletion effects via distinct routes, conferring mechanistic explanation to the observed lethality phenotypes of essential genes.

  5. Correlations between long inverted repeat (LIR) features, deletion size and distance from breakpoint in human gross gene deletions

    PubMed Central

    Aygun, Nevim

    2015-01-01

    Long inverted repeats (LIRs) have been shown to induce genomic deletions in yeast. In this study, LIRs were investigated within ±10 kb spanning each breakpoint from 109 human gross deletions, using Inverted Repeat Finder (IRF) software. LIR number was significantly higher at the breakpoint regions, than in control segments (P < 0.001). In addition, it was found that strong correlation between 5′ and 3′ LIR numbers, suggesting contribution to DNA sequence evolution (r = 0.85, P < 0.001). 138 LIR features at ±3 kb breakpoints in 89 (81%) of 109 gross deletions were evaluated. Significant correlations were found between distance from breakpoint and loop length (r = −0.18, P < 0.05) and stem length (r = −0.18, P < 0.05), suggesting DNA strands are potentially broken in locations closer to bigger LIRs. In addition, bigger loops cause larger deletions (r = 0.19, P < 0.05). Moreover, loop length (r = 0.29, P < 0.02) and identity between stem copies (r = 0.30, P < 0.05) of 3′ LIRs were more important in larger deletions. Consequently, DNA breaks may form via LIR-induced cruciform structure during replication. DNA ends may be later repaired by non-homologous end-joining (NHEJ), with following deletion. PMID:25657065

  6. EPHA7, a new target gene for 6q deletion in T-cell lymphoblastic lymphomas.

    PubMed

    López-Nieva, Pilar; Vaquero, Concepción; Fernández-Navarro, Pablo; González-Sánchez, Laura; Villa-Morales, María; Santos, Javier; Esteller, Manel; Fernández-Piqueras, José

    2012-02-01

    Cryptic deletions at chromosome 6q are common cytogenetic abnormalities in T-cell lymphoblastic leukemia/lymphoma (T-LBL), but the target genes have not been formally identified. Our results build on detection of specific chromosomal losses in a mouse model of γ-radiation-induced T-LBLs and provide interesting clues for new putative susceptibility genes in a region orthologous to human 6q15-6q16.3. Among these, Epha7 emerges as a bona fide candidate tumor suppressor gene because it is inactivated in practically all the T-LBLs analyzed (100% in mouse and 95.23% in human). We provide evidence showing that Epha7 downregulation may occur, at least in part, by loss of heterozygosity (19.35% in mouse and 12.5% in human) or promoter hypermethylation (51.61% in mouse and 43.75% in human) or a combination of both mechanisms (12.90% in mouse and 6.25% in human). These results indicate that EPHA7 might be considered a new tumor suppressor gene for 6q deletions in T-LBLs. Notably, this gene is located in 6q16.1 proximal to GRIK2 and CASP8AP2, other candidate genes identified in this region. Thus, del6q seems to be a complex region where inactivation of multiple genes may cooperatively contribute to the onset of T-cell lymphomas.

  7. Isolation of a zinc finger gene consistently deleted in DiGeorge syndrome.

    PubMed

    Aubry, M; Demczuk, S; Desmaze, C; Aikem, M; Aurias, A; Julien, J P; Rouleau, G A

    1993-10-01

    DiGeorge syndrome is a human developmental disorder resulting in hypoplasia of the thymus and parathyroids, and conotruncal heart defects. We recently isolated four genes with zinc finger DNA binding motifs mapping to chromosome 22q11.2 DiGeorge critical region. We now report that one of them, ZNF74 gene, is hemizygously deleted in 23 out of 24 DiGeorge syndrome patients tested. ZNF74 mRNA transcripts are detected in human and mouse embryos but not in adult tissues. Sequence analysis of a corresponding cDNA reveals an an open reading frame encoding 12 zinc finger motifs of the Kruppel/TFIIIA type as well as N-terminal and C-terminal non-zinc finger domains. These results suggest that changes in the dosage of a putative transcription factor through ZNF74 hemizygous deletion may be critical for DiGeorge developmental anomalies.

  8. A 65 bp deletion in band 3 gene of beta-thalassemia patients in Indonesia.

    PubMed

    Dewajanthi, Anna Maria; Lubis, Vita K; Wanandi, Septelia Inawati; Gatot, Djajadiman; Soegianto, Rondang R; Freisleben, Seruni K U; Wahidiyat, Iskandar; Freisleben, Hans-Joachim

    2014-01-01

    We investigated whether in addition to the well known genetic alteration in red blood cell membrane band 3 protein, a deletion of 9 amino acids leading to ovalocytosis, other mutations to band 3 also exist. In 12% of our thalassemia major patients investigated, we found two bands in the agarose gel-electrophoresis of PCR products from band 3 gene with a difference of 65 +/- 10 bp, equivalent to a deletion of 20 to 25 amino acids in band 3 protein. Thus, a co-existing band 3-mutant allele in addition to the thalassemic globin gene defects, could also contribute to erythrocyte membrane defects and to the spectrum of clinical symptoms of these thalassemia major patients.

  9. Absence of human chorionic somatomammotropin during pregnancy associated with two types of gene deletion.

    PubMed

    Simon, P; Decoster, C; Brocas, H; Schwers, J; Vassart, G

    1986-11-01

    Complete absence of human somatomammotropin (hCS) was demonstrated in two patients experiencing an otherwise uneventful pregnancy. After delivery, DNA was prepared from the neonate blood or from the placenta and the integrity of the hCS-hGH gene cluster was investigated by Southern blotting and hybridization with an hCS cDNA probe. Patient 1 was found to be homozygous for a deletion involving hCS-A, hGH-V, and hCS-B. Patient 2 was a double heterozygote, with one chromosome bearing the same deletion as that of patient 1, while in the other, only the hCS-A gene was missing. Considerations relative to the frequency of the defect are derived from the present results.

  10. Deletion of the meq gene significantly decreases immunosuppression in chickens caused by pathogenic marek's disease virus

    PubMed Central

    2011-01-01

    Background Marek's disease virus (MDV) causes an acute lymphoproliferative disease in chickens, resulting in immunosuppression, which is considered to be an integral aspect of the pathogenesis of Marek's disease (MD). A recent study showed that deletion of the Meq gene resulted in loss of transformation of T-cells in chickens and a Meq-null virus, rMd5ΔMeq, could provide protection superior to CVI988/Rispens. Results In the present study, to investigate whether the Meq-null virus could be a safe vaccine candidate, we constructed a Meq deletion strain, GX0101ΔMeq, by deleting both copies of the Meq gene from a pathogenic MDV, GX0101 strain, which was isolated in China. Pathogenesis experiments showed that the GX0101ΔMeq virus was fully attenuated in specific pathogen-free chickens because none of the infected chickens developed Marek's disease-associated lymphomas. The study also evaluated the effects of GX0101ΔMeq on the immune system in chickens after infection with GX0101ΔMeq virus. Immune system variables, including relative lymphoid organ weight, blood lymphocytes and antibody production following vaccination against AIV and NDV were used to assess the immune status of chickens. Experimental infection with GX0101ΔMeq showed that deletion of the Meq gene significantly decreased immunosuppression in chickens caused by pathogenic MDV. Conclusion These findings suggested that the Meq gene played an important role not only in tumor formation but also in inducing immunosuppressive effects in MDV-infected chickens. PMID:21205328

  11. Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

    PubMed Central

    Li, Nan; Qin, Ting; Zhang, Xiao Lin; Huang, Bei; Liu, Zhi Xin; Xie, Hai Xia; Zhang, Jin; McBride, Mark J.

    2015-01-01

    Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ΔcslA ΔcslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes. PMID:26253667

  12. Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence.

    PubMed

    Li, Nan; Qin, Ting; Zhang, Xiao Lin; Huang, Bei; Liu, Zhi Xin; Xie, Hai Xia; Zhang, Jin; McBride, Mark J; Nie, Pin

    2015-11-01

    Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ΔcslA ΔcslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes.

  13. Deletion of the APOBEC3B gene strongly impacts susceptibility to falciparum malaria.

    PubMed

    Jha, Pankaj; Sinha, Swapnil; Kanchan, Kanika; Qidwai, Tabish; Narang, Ankita; Singh, Prashant Kumar; Pati, Sudhanshu S; Mohanty, Sanjib; Mishra, Saroj K; Sharma, Surya K; Awasthi, Shally; Venkatesh, Vimala; Jain, Sanjeev; Basu, Analabha; Xu, Shuhua; Mukerji, Mitali; Habib, Saman

    2012-01-01

    APOBEC3B, a gene involved in innate response, exhibits insertion-deletion polymorphism across world populations. We observed the insertion allele to be nearly fixed in malaria endemic regions of sub-Saharan Africa as well as populations with high malaria incidence in the past. This prompted us to investigate the possible association of the polymorphism with falciparum malaria. We studied the distribution of APOBEC3B, in 25 diverse Indian populations comprising of 500 samples and 176 severe or non-severe Plasmodium falciparum patients and 174 ethnically-matched uninfected individuals from a P. falciparum endemic and a non-endemic region of India. The deletion frequencies ranged from 0% to 43% in the Indian populations. The frequency of the insertion allele strikingly correlated with the endemicity map of P. falciparum malaria in India. A strong association of the deletion allele with susceptibility to falciparum malaria in the endemic region (non-severe vs. control, Odds ratio=4.96, P value=9.5E(-06); severe vs. control, OR=4.36, P value=5.76E(-05)) was observed. Although the frequency of deletion allele was higher in the non-endemic region, there was a significant association of the homozygous deletion genotype with malaria (OR=3.17, 95% CI=1.10-10.32, P value=0.0177). Our study also presents a case for malaria as a positive selection force for the APOBEC3B insertion and suggests a major role for this gene in innate immunity against malaria.

  14. Attenuation of classical swine fever virus by deletion of the viral N(pro) gene.

    PubMed

    Mayer, Daniel; Hofmann, Martin A; Tratschin, Jon Duri

    2004-01-02

    We have reported earlier that replacement of the N(pro) gene of classical swine fever virus (CSFV) by the murine ubiquitin gene only slightly affects the characteristics of virus replication in the porcine kidney cell line SK-6 [J. Virol. 72 (1998) 7681]. Here, for the moderately virulent CSFV strain Alfort/187 as well as for the highly virulent strain Eystrup we show that the respective N(pro)-deleted viruses are attenuated. Vaccination of pigs with either of the two deletion mutants resulted in the induction of a strong antibody response. Animals were protected against challenge with a lethal dose of highly virulent CSFV indicating that N(pro) deletion mutants are excellent candidates for a modified live virus vaccine. A chimeric virus obtained by replacement of the N(pro) gene in the Eystrup virus by the corresponding sequence of the avirulent CSFV vaccine strain Riems resulted in a virus that was highly virulent. This indicates that the virulence of CSFV correlates with the presence of N(pro) and also suggests that N(pro) is not responsible for the varying virulence observed between individual strains of CSFV.

  15. Germline deletions in the tumour suppressor gene FOCAD are associated with polyposis and colorectal cancer development.

    PubMed

    Weren, Robbert D A; Venkatachalam, Ramprasath; Cazier, Jean-Baptiste; Farin, Henner F; Kets, C Marleen; de Voer, Richarda M; Vreede, Lilian; Verwiel, Eugène T P; van Asseldonk, Monique; Kamping, Eveline J; Kiemeney, Lambertus A; Neveling, Kornelia; Aben, Katja K H; Carvajal-Carmona, Luis; Nagtegaal, Iris D; Schackert, Hans K; Clevers, Hans; van de Wetering, Marc; Tomlinson, Ian P; Ligtenberg, Marjolijn J L; Hoogerbrugge, Nicoline; Geurts van Kessel, Ad; Kuiper, Roland P

    2015-06-01

    Heritable genetic variants can significantly affect the lifetime risk of developing cancer, including polyposis and colorectal cancer (CRC). Variants in genes currently known to be associated with a high risk for polyposis or CRC, however, explain only a limited number of hereditary cases. The identification of additional genetic causes is, therefore, crucial to improve CRC prevention, detection and treatment. We have performed genome-wide and targeted DNA copy number profiling and resequencing in early-onset and familial polyposis/CRC patients, and show that deletions affecting the open reading frame of the tumour suppressor gene FOCAD are recurrent and significantly enriched in CRC patients compared with unaffected controls. All patients carrying FOCAD deletions exhibited a personal or family history of polyposis. RNA in situ hybridization revealed FOCAD expression in epithelial cells in the colonic crypt, the site of tumour initiation, as well as in colonic tumours and organoids. Our data suggest that monoallelic germline deletions in the tumour suppressor gene FOCAD underlie moderate genetic predisposition to the development of polyposis and CRC.

  16. Deletion of Indian hedgehog gene causes dominant semi-lethal Creeper trait in chicken

    PubMed Central

    Jin, Sihua; Zhu, Feng; Wang, Yanyun; Yi, Guoqiang; Li, Junying; Lian, Ling; Zheng, Jiangxia; Xu, Guiyun; Jiao, Rengang; Gong, Yu; Hou, Zhuocheng; Yang, Ning

    2016-01-01

    The Creeper trait, a classical monogenic phenotype of chicken, is controlled by a dominant semi-lethal gene. This trait has been widely cited in the genetics and molecular biology textbooks for illustrating autosomal dominant semi-lethal inheritance over decades. However, the genetic basis of the Creeper trait remains unknown. Here we have utilized ultra-deep sequencing and extensive analysis for targeting causative mutation controlling the Creeper trait. Our results indicated that the deletion of Indian hedgehog (IHH) gene was only found in the whole-genome sequencing data of lethal embryos and Creeper chickens. Large scale segregation analysis demonstrated that the deletion of IHH was fully linked with early embryonic death and the Creeper trait. Expression analysis showed a much lower expression of IHH in Creeper than wild-type chickens. We therefore suggest the deletion of IHH to be the causative mutation for the Creeper trait in chicken. Our findings unravel the genetic basis of the longstanding Creeper phenotype mystery in chicken as the same gene also underlies bone dysplasia in human and mouse, and thus highlight the significance of IHH in animal development and human haploinsufficiency disorders. PMID:27439785

  17. Exon skipping and translation in patients with frameshift deletions in the dystrophin gene

    SciTech Connect

    Sherratt, T.G.; Dubowitz, V.; Sewry, C.A.; Strong, P.N. ); Vulliamy, T. )

    1993-11-01

    Although many Duchenne muscular dystrophy patients have a deletion in the dystrophin gene which disrupts the translational reading frame, they express dystrophin in a small proportion of skeletal muscle fibers ([open quotes]revertant fibers[close quotes]). Antibody studies have shown, indirectly, that dystrophin synthesis in revertant fibers is facilitated by a frame-restoring mechanism; in the present study, the feasibility of mRNA splicing was investigated. Dystrophin transcripts were analyzed in skeletal muscle from individuals possessing revertant fibers and a frameshift deletion in the dystrophin gene. In each case a minor in-frame transcript was detected, in which exons adjacent to those deleted from the genome had been skipped. There appeared to be some correlation between the levels of in-frame transcripts and the predicted translation products. Low levels of alternatively spliced transcripts were also present in normal muscle. The results provide further evidence of exon skipping in the dystrophin gene and indicate that this may be involved in the synthesis of dystrophin by revertant fibers. 44 refs., 12 figs.

  18. Recurrent deletions of puroindoline genes at the grain hardness locus in four independent lineages of polyploid wheat.

    PubMed

    Li, Wanlong; Huang, Li; Gill, Bikram S

    2008-01-01

    Polyploidy is known to induce numerous genetic and epigenetic changes but little is known about their physiological bases. In wheat, grain texture is mainly determined by the Hardness (Ha) locus consisting of genes Puroindoline a (Pina) and b (Pinb). These genes are conserved in diploid progenitors but were deleted from the A and B genomes of tetraploid Triticum turgidum (AB). We now report the recurrent deletions of Pina-Pinb in other lineages of polyploid wheat. We analyzed the Ha haplotype structure in 90 diploid and 300 polyploid accessions of Triticum and Aegilops spp. Pin genes were conserved in all diploid species and deletion haplotypes were detected in all polyploid Triticum and most of the polyploid Aegilops spp. Two Pina-Pinb deletion haplotypes were found in hexaploid wheat (Triticum aestivum; ABD). Pina and Pinb were eliminated from the G genome, but maintained in the A genome of tetraploid Triticum timopheevii (AG). Subsequently, Pina and Pinb were deleted from the A genome but retained in the A(m) genome of hexaploid Triticum zhukovskyi (A(m)AG). Comparison of deletion breakpoints demonstrated that the Pina-Pinb deletion occurred independently and recurrently in the four polyploid wheat species. The implications of Pina-Pinb deletions for polyploid-driven evolution of gene and genome and its possible physiological significance are discussed.

  19. Possible deletion of a developmentally regulated heavy-chain variable region gene in autoimmune diseases

    SciTech Connect

    Yang, Pei-Ming; Olee, Tsaiwei; Kozin, F.; Carson, D.A.; Chen, P.P. ); Olsen, N.J. ); Siminovitch, K.A. )

    1990-10-01

    Several autoantibody-associated variable region (V) genes are preferentially expressed during early ontogenic development, suggesting strongly that they are of developmental and physiological importance. As such, it is possible that polymorphisms in one or more of these genes may alter susceptibility to autoimmune disease. The authors have searched extensively for a probe related to a developmentally regulated V gene that has the power to differentiate among highly homologous V genes in human populations. Using such a probe (i.e., Humhv3005/P1) related to both anti-DNA and anti-IgG autoantibodies, they studied restriction fragment length polymorphisms in patients with rheumatoid arthritis and systemic lupus erythematosus and found an apparent heavy-chain V (V{sub H}) gene deletion that was nearly restricted to the autoimmune patients. These data suggest that deletions of physiologically important V{sub H} genes may increase the risk of autoimmunity through indirect effects on the development and homeostasis of the B-cell repertoire.

  20. Genetic characterization in symptomatic female DMD carriers: lack of relationship between X-inactivation, transcriptional DMD allele balancing and phenotype

    PubMed Central

    2012-01-01

    Background Although Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly affect males, a clinically significant proportion of females manifesting symptoms have also been reported. They represent an heterogeneous group characterized by variable degrees of muscle weakness and/or cardiac involvement. Though preferential inactivation of the normal X chromosome has long been considered the principal mechanism behind disease manifestation in these females, supporting evidence is controversial. Methods Eighteen females showing a mosaic pattern of dystrophin expression on muscle biopsy were recruited and classified as symptomatic (7) or asymptomatic (11), based on the presence or absence of muscle weakness. The causative DMD gene mutations were identified in all cases, and the X-inactivation pattern was assessed in muscle DNA. Transcriptional analysis in muscles was performed in all females, and relative quantification of wild-type and mutated transcripts was also performed in 9 carriers. Dystrophin protein was quantified by immunoblotting in 2 females. Results The study highlighted a lack of relationship between dystrophic phenotype and X-inactivation pattern in females; skewed X-inactivation was found in 2 out of 6 symptomatic carriers and in 5 out of 11 asymptomatic carriers. All females were characterized by biallelic transcription, but no association was found between X-inactivation pattern and allele transcriptional balancing. Either a prevalence of wild-type transcript or equal proportions of wild-type and mutated RNAs was observed in both symptomatic and asymptomatic females. Moreover, very similar levels of total and wild-type transcripts were identified in the two groups of carriers. Conclusions This is the first study deeply exploring the DMD transcriptional behaviour in a cohort of female carriers. Notably, no relationship between X-inactivation pattern and transcriptional behaviour of DMD gene was observed, suggesting that the

  1. Spontaneous deletion in the FMR-1 gene in a patient with fragile X syndrome and cherubism

    SciTech Connect

    Popovich, B.W.; Anoe, K.S.; Johnson, D.B.

    1994-09-01

    Fragile X mental retardation results from the transcriptional inactivation of the FMR-1 gene and is commonly caused by the expansion of an unstable CGG trinucleotide repeat located in the first exon of the FMR-1 gene. We describe here a two generation fragile X family in which expansion of the CGG repeat may have resulted in a deletion of a least portion of the FMR-1 gene. One member of this family, AB, carries an apparent deletion of the FMR-1 gene and presents with mental retardation and also cherubism, a feature not usually associated with fragile X syndrome. Cherubism is a condition characterized by a swelling of the lower face and is caused by giant cell lesions of the mandible and maxilla, and often the anterior ends of the ribs. The size of the CGG repeat region in this family was determined by Southern analysis of BglII, EcoRI, and PstI digested genomic DNA, isolated from peripheral blood lymphocytes, using a 558 bp PstI-Xhol fragment specific for the 5{prime}-end of the FMR-1 gene. SB and TB, the mother and maternal half-brother of AB, respectively, were both found to carry an expanded FMR-1 allele with greater than 200 CGG repeats. Negligible hybridization was observed in the DNA of AB. In addition, no amplification was observed when the polymerase chain reaction (PCR) was performed using primers flanking the CGG repeat region. These results are consistent with a deletion of at least the 5{prime} portion of the FMR-1 gene in the majority of peripheral blood lymphocytes. Further work is underway using FMR-1 cDNA probes and additional PCR primers to determine the nature of the molecular lesion in AB`s DNA and determine the relationship of this lesion to his cherubism.

  2. Refinement of causative genes in monosomy 1p36 through clinical and molecular cytogenetic characterization of small interstitial deletions.

    PubMed

    Rosenfeld, Jill A; Crolla, John A; Tomkins, Susan; Bader, Patricia; Morrow, Bernice; Gorski, Jerome; Troxell, Robin; Forster-Gibson, Cynthia; Cilliers, Deirdre; Hislop, R Gordon; Lamb, Allen; Torchia, Beth; Ballif, Blake C; Shaffer, Lisa G

    2010-08-01

    Monosomy 1p36 is the most common terminal deletion syndrome seen in humans, occurring in approximately 1 in 5,000 live births. Common features include mental retardation, characteristic dysmorphic features, hypotonia, seizures, hearing loss, heart defects, cardiomyopathy, and behavior abnormalities. Similar phenotypes are seen among patients with a variety of deletion sizes, including terminal and interstitial deletions, complex rearrangements, and unbalanced translocations. Consequently, critical regions harboring causative genes for each of these features have been difficult to identify. Here we report on five individuals with 200-823 kb overlapping deletions of proximal 1p36.33, four of which are apparently de novo. They present with features of monosomy 1p36, including developmental delay and mental retardation, dysmorphic features, hypotonia, behavioral abnormalities including hyperphagia, and seizures. The smallest region of deletion overlap is 174 kb and contains five genes; these genes are likely candidates for some of the phenotypic features in monosomy 1p36. Other genes deleted in a subset of the patients likely play a contributory role in the phenotypes, including GABRD and seizures, PRKCZ and neurologic features, and SKI and dysmorphic and neurologic features. Characterization of small deletions is important for narrowing critical intervals and for the identification of causative or candidate genes for features of monosomy 1p36 syndrome.

  3. Partial gene deletion in LEC rat: An animal model for Wilson disease

    SciTech Connect

    Wu, J.; Forbes, J.R.; Cox, D.W.

    1994-09-01

    Wilson disease is an inherited disorder of copper transport in which incorporation of copper into ceruloplasmin and excretion of copper into bile are greatly reduced. Copper accumulates to a toxic level in the liver and also in the brain and kidney, causing a spectrum of hepatic and neurological abnormalities. We have recently cloned the gene for Wilson disease (designated ATP7B), which encodes a putative copper-transporting P-type ATPase. The inbred mutant Long-Evans Cinnamon (LEC) rat strain shows similarity to Wilson disease in many clinical and biochemical features. We have cloned cDNAs for the rat homologue (Atp7b) of the human Wilson disease gene (ATP7B) and have shown that the two genes have {approximately}82% identity at the amino acid sequence level. Rat cDNA sequences were used to identify a partial deletion in the Atp7b gene in the LEC rat. The deletion removes at least 750 bp of the coding region at the 3{prime} end, which includes the crucial ATP binding domain and extends downstream of the gene. The proximal breakpoint has been precisely localized at the cDNA level. Our results provide convincing evidence that the LEC rat is an animal model for Wilson disease. This model will be important for studying liver pathophysiology, for developing therapy for Wilson disease, and for studying the pathway of copper transport and its possible interaction with other heavy metals.

  4. Systematic Phenotyping of a Large-Scale Candida glabrata Deletion Collection Reveals Novel Antifungal Tolerance Genes

    PubMed Central

    Hiller, Ekkehard; Istel, Fabian; Tscherner, Michael; Brunke, Sascha; Ames, Lauren; Firon, Arnaud; Green, Brian; Cabral, Vitor; Marcet-Houben, Marina; Jacobsen, Ilse D.; Quintin, Jessica; Seider, Katja; Frohner, Ingrid; Glaser, Walter; Jungwirth, Helmut; Bachellier-Bassi, Sophie; Chauvel, Murielle; Zeidler, Ute; Ferrandon, Dominique; Gabaldón, Toni; Hube, Bernhard; d'Enfert, Christophe; Rupp, Steffen; Cormack, Brendan; Haynes, Ken; Kuchler, Karl

    2014-01-01

    The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes. PMID:24945925

  5. UFD1L, a developmentally expressed ubiquitination gene, is deleted in CATCH 22 syndrome.

    PubMed

    Pizzuti, A; Novelli, G; Ratti, A; Amati, F; Mari, A; Calabrese, G; Nicolis, S; Silani, V; Marino, B; Scarlato, G; Ottolenghi, S; Dallapiccola, B

    1997-02-01

    The CATCH 22 acronym outlines the main clinical features of 22q11.2 deletions (cardiac defects, abnormal facies, thymic hypoplasia, cleft palate and hypocalcemia), usually found in DiGeorge (DGS) and velo-cardio-facial (VCFS) syndromes. Hemizygosity of this region may also be the cause of over 100 different clinical signs. The CATCH 22 locus maps within a 1.5 Mb region, which encompasses several genes. However, no single defect in 22q11.2 hemizygous patients can be ascribed to any gene so far isolated from the critical region of deletion. We have identified a gene in the CATCH 22 critical region, whose functional features and tissue-specific expression suggest a distinct role in embryogenesis. This gene, UFD1L, encodes the human homolog of the yeast ubiquitin fusion degradation 1 protein (UFD1p), involved in the degradation of ubiquitin fusion proteins. Cloning and characterization of the murine homolog (Ufd1l) showed it to be expressed during embryogenesis in the eyes and in the linear ear primordia. These data suggest that the proteolytic pathway that recognizes ubiquitin fusion proteins for degradation is conserved in vertebrates and that the UFD1L gene hemizygosity is the cause of some of the CATCH 22-associated developmental defects.

  6. Histone Modifier Genes Alter Conotruncal Heart Phenotypes in 22q11.2 Deletion Syndrome.

    PubMed

    Guo, Tingwei; Chung, Jonathan H; Wang, Tao; McDonald-McGinn, Donna M; Kates, Wendy R; Hawuła, Wanda; Coleman, Karlene; Zackai, Elaine; Emanuel, Beverly S; Morrow, Bernice E

    2015-12-03

    We performed whole exome sequence (WES) to identify genetic modifiers on 184 individuals with 22q11.2 deletion syndrome (22q11DS), of whom 89 case subjects had severe congenital heart disease (CHD) and 95 control subjects had normal hearts. Three genes including JMJD1C (jumonji domain containing 1C), RREB1 (Ras responsive element binding protein 1), and SEC24C (SEC24 family member C) had rare (MAF < 0.001) predicted deleterious single-nucleotide variations (rdSNVs) in seven case subjects and no control subjects (p = 0.005; Fisher exact and permutation tests). Because JMJD1C and RREB1 are involved in chromatin modification, we investigated other histone modification genes. Eighteen case subjects (20%) had rdSNVs in four genes (JMJD1C, RREB1, MINA, KDM7A) all involved in demethylation of histones (H3K9, H3K27). Overall, rdSNVs were enriched in histone modifier genes that activate transcription (Fisher exact p = 0.0004, permutations, p = 0.0003, OR = 5.16); however, rdSNVs in control subjects were not enriched. This implicates histone modification genes as influencing risk for CHD in presence of the deletion.

  7. Novel deletions involving the USH2A gene in patients with Usher syndrome and retinitis pigmentosa

    PubMed Central

    García-García, Gema; Jaijo, Teresa; Aparisi, Maria J.; Larrieu, Lise; Faugère, Valérie; Blanco-Kelly, Fiona; Ayuso, Carmen; Roux, Anne-Francoise; Millán, José M.

    2014-01-01

    Purpose The aim of the present work was to identify and characterize large rearrangements involving the USH2A gene in patients with Usher syndrome and nonsyndromic retinitis pigmentosa. Methods The multiplex ligation-dependent probe amplification (MLPA) technique combined with a customized array-based comparative genomic hybridization (aCGH) analysis was applied to 40 unrelated patients previously screened for point mutations in the USH2A gene in which none or only one pathologic mutation was identified. Results We detected six large deletions involving USH2A in six out of the 40 cases studied. Three of the patients were homozygous for the deletion, and the remaining three were compound heterozygous with a previously identified USH2A point mutation. In five of these cases, the patients displayed Usher type 2, and the remaining case displayed nonsyndromic retinitis pigmentosa. The exact breakpoint junctions of the deletions found in USH2A in four of these cases were characterized. Conclusions Our study highlights the need to develop improved efficient strategies of mutation screening based upon next generation sequencing (NGS) that reduce cost, time, and complexity and allow simultaneous identification of all types of disease-causing mutations in diagnostic procedures. PMID:25352746

  8. The TREAT-NMD DMD Global Database: Analysis of More than 7,000 Duchenne Muscular Dystrophy Mutations

    PubMed Central

    Bladen, Catherine L; Salgado, David; Monges, Soledad; Foncuberta, Maria E; Kekou, Kyriaki; Kosma, Konstantina; Dawkins, Hugh; Lamont, Leanne; Roy, Anna J; Chamova, Teodora; Guergueltcheva, Velina; Chan, Sophelia; Korngut, Lawrence; Campbell, Craig; Dai, Yi; Wang, Jen; Barišić, Nina; Brabec, Petr; Lahdetie, Jaana; Walter, Maggie C; Schreiber-Katz, Olivia; Karcagi, Veronika; Garami, Marta; Viswanathan, Venkatarman; Bayat, Farhad; Buccella, Filippo; Kimura, En; Koeks, Zaïda; van den Bergen, Janneke C; Rodrigues, Miriam; Roxburgh, Richard; Lusakowska, Anna; Kostera-Pruszczyk, Anna; Zimowski, Janusz; Santos, Rosário; Neagu, Elena; Artemieva, Svetlana; Rasic, Vedrana Milic; Vojinovic, Dina; Posada, Manuel; Bloetzer, Clemens; Jeannet, Pierre-Yves; Joncourt, Franziska; Díaz-Manera, Jordi; Gallardo, Eduard; Karaduman, A Ayşe; Topaloğlu, Haluk; El Sherif, Rasha; Stringer, Angela; Shatillo, Andriy V; Martin, Ann S; Peay, Holly L; Bellgard, Matthew I; Kirschner, Jan; Flanigan, Kevin M; Straub, Volker; Bushby, Kate; Verschuuren, Jan; Aartsma-Rus, Annemieke; Béroud, Christophe; Lochmüller, Hanns

    2015-01-01

    Analyzing the type and frequency of patient-specific mutations that give rise to Duchenne muscular dystrophy (DMD) is an invaluable tool for diagnostics, basic scientific research, trial planning, and improved clinical care. Locus-specific databases allow for the collection, organization, storage, and analysis of genetic variants of disease. Here, we describe the development and analysis of the TREAT-NMD DMD Global database (http://umd.be/TREAT_DMD/). We analyzed genetic data for 7,149 DMD mutations held within the database. A total of 5,682 large mutations were observed (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions and 199 (14%) affected the splice sites. Point mutations totalled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations). PMID:25604253

  9. Interleukin 3 gene is located on human chromosome 5 and is deleted in myeloid leukemias with a deletion of 5q

    SciTech Connect

    Le Beau, M.M.; Epstein, N.D.; O'Brien, S.J.; Nienhuis, A.W.; Yang, Y.C.; Clark, S.C.; Rowley, J.D.

    1987-08-01

    The gene IL-3 encodes interleukin 3, a hematopoietic colony-stimulating factor (CSF) that is capable of supporting the proliferation of a broad range of hematopoietic cell types. By using somatic cell hybrids and in situ chromosomal hybridization, the authors localized this gene to human chromosome 5 at bands q23-31, a chromosomal region that is frequently deleted (del(5q)) in patients with myeloid disorders. By in situ hybridization, IL-3 was found to be deleted in the 5q-chromosome of one patient with refractory anemia who had a del(5)(q15q33.3), of three patients with refractory anemia (two patients) or acute nonlymphocytic leukemia (ANLL) de novo who had a similar distal breakpoint (del(5)(q13q33.3)), and of a fifth patient, with therapy-related ANLL, who had a similar distal breakpoint in band q33(del(5)(q14q33.3)). Southern blot analysis of somatic cell hybrids retaining the normal or the deleted chromosome 5 from two patients with the refractory anemia 5q- syndrome indicated that IL-3 sequences were absent from the hybrids retaining the deleted chromosome 5 but not from hybrids that had a cytologically normal chromosome 5. Thus, a small segment of chromosome 5 contains IL-3, GM-CSF, CSF-1, and FMS. The findings and earlier results indicating that GM-CSF, CSF-1, and FMS were deleted in the 5q- chromosome, suggest that loss of IL-3 or of other CSF genes may play an important role in the pathogenesis of hematologic disorders associated with a del(5q).

  10. Deletion and aberrant CpG island methylation of Caspase 8 gene in medulloblastoma.

    PubMed

    Gonzalez-Gomez, Pilar; Bello, M Josefa; Inda, M Mar; Alonso, M Eva; Arjona, Dolores; Amiñoso, Cinthia; Lopez-Marin, Isabel; de Campos, Jose M; Sarasa, Jose L; Castresana, Javier S; Rey, Juan A

    2004-09-01

    Aberrant methylation of promoter CpG islands in human genes is an alternative genetic inactivation mechanism that contributes to the development of human tumors. Nevertheless, few studies have analyzed methylation in medulloblastomas. We determined the frequency of aberrant CpG island methylation for Caspase 8 (CASP8) in a group of 24 medulloblastomas arising in 8 adult and 16 pediatric patients. Complete methylation of CASP8 was found in 15 tumors (62%) and one case displayed hemimethylation. Three samples amplified neither of the two primer sets for methylated or unmethylated alleles, suggesting that genomic deletion occurred in the 5' flanking region of CASP8. Our findings suggest that methylation commonly contributes to CASP8 silencing in medulloblastomas and that homozygous deletion or severe sequence changes involving the promoter region may be another mechanism leading to CASP8 inactivation in this neoplasm.

  11. Immunogenic response induced by wzm and wzt gene deletion mutants from Brucella abortus S19.

    PubMed

    Wang, Xiu-Ran; Yan, Guang-Mou; Zhang, Rui; Lang, Xu-Long; Yang, Yan-Ling; Li, Xiao-Yan; Chen, Si; Qian, Jing; Wang, Xing-Long

    2014-02-01

    Brucellosis is an infectious disease affecting humans and animals worldwide. Effective methods of control include inducing immunity in animals by vaccination and elimination. Brucella abortus S19 is one of the popular vaccines for control of cattle brucellosis, as it has low virulence. In this paper, allelic exchange plasmids of wzm and wzt genes were constructed and partially knocked out to evaluate the effects on the induction of immunity to Brucella abortus S19 mutants. Cytokine secretion in vitro, INF-γ induction in vivo and antibody dynamics were evaluated. These data suggested that the immunity-eliciting ability of the wzm and wzt gene deletion mutants was similar, although reduced compared with the S19 strain. The results demonstrated that the wzt gene may be more important in the regulation of the induction of immunity than the wzm gene.

  12. The evolution of small insertions and deletions in the coding genes of Drosophila melanogaster.

    PubMed

    Chong, Zechen; Zhai, Weiwei; Li, Chunyan; Gao, Min; Gong, Qiang; Ruan, Jue; Li, Juan; Jiang, Lan; Lv, Xuemei; Hungate, Eric; Wu, Chung-I

    2013-12-01

    Studies of protein evolution have focused on amino acid substitutions with much less systematic analysis on insertion and deletions (indels) in protein coding genes. We hence surveyed 7,500 genes between Drosophila melanogaster and D. simulans, using D. yakuba as an outgroup for this purpose. The evolutionary rate of coding indels is indeed low, at only 3% of that of nonsynonymous substitutions. As coding indels follow a geometric distribution in size and tend to fall in low-complexity regions of proteins, it is unclear whether selection or mutation underlies this low rate. To resolve the issue, we collected genomic sequences from an isogenic African line of D. melanogaster (ZS30) at a high coverage of 70× and analyzed indel polymorphism between ZS30 and the reference genome. In comparing polymorphism and divergence, we found that the divergence to polymorphism ratio (i.e., fixation index) for smaller indels (size ≤ 10 bp) is very similar to that for synonymous changes, suggesting that most of the within-species polymorphism and between-species divergence for indels are selectively neutral. Interestingly, deletions of larger sizes (size ≥ 11 bp and ≤ 30 bp) have a much higher fixation index than synonymous mutations and 44.4% of fixed middle-sized deletions are estimated to be adaptive. To our surprise, this pattern is not found for insertions. Protein indel evolution appear to be in a dynamic flux of neutrally driven expansion (insertions) together with adaptive-driven contraction (deletions), and these observations provide important insights for understanding the fitness of new mutations as well as the evolutionary driving forces for genomic evolution in Drosophila species.

  13. Isolation and characterization of a novel gene deleted in DiGeorge syndrome.

    PubMed

    Kurahashi, H; Akagi, K; Inazawa, J; Ohta, T; Niikawa, N; Kayatani, F; Sano, T; Okada, S; Nishisho, I

    1995-04-01

    The region commonly deleted in DiGeorge syndrome (DGS) has been localized at 22q11.1-q11.2 with the aid of a high resolution banding technique. A 22q11 specific plasmid library was constructed with a microdissection and microcloning method. Dosage analysis proved three of 144 randomly selected microclones to detect hemizygosity in two patients with DGS. Two of the clones were found to contain independent low-copy-number repetitive sequences, all of which were included in the region deleted in the DGS patients. Screening of the cosmid library and subsequent cosmid walking allowed us to obtain two cosmid contigs corresponding to the microclones within the deletion (contig 1 and contig 2), whose order fluorescence in situ hybridization identified as centromere-contig 1-contig 2-telomere on 22q. By direct selection strategy using one of the cosmids of contig 1, a 4.3 kb cDNA was obtained from fetal brain cDNA library. Sequence analysis of the cDNA revealed an open reading frame encoding 552 amino acids which had several characteristics of DNA-binding proteins. The gene, designated LZTR-1, which was transcribed in several essential fetal organs, proved to be hemizygously deleted in seven of eight DGS patients or its variants, but not in one DGS patient and GM00980. Although LZTR-1 does not locate in the shortest region of overlap, several of its structural characteristics identifying it as transcriptional regulator suggest that it plays a crucial role in embryogenesis and that haploinsufficiency of this gene may be partly related to the development of DGS.

  14. Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies

    PubMed Central

    Toh, Zhi Yon Charles; Thandar Aung-Htut, May; Pinniger, Gavin; Adams, Abbie M.; Krishnaswarmy, Sudarsan; Wong, Brenda L.; Fletcher, Sue; Wilton, Steve D.

    2016-01-01

    Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes. PMID:26745801

  15. Identification of a PKP2 gene deletion in a family with arrhythmogenic right ventricular cardiomyopathy

    PubMed Central

    Li Mura, Ilena Egle Astrid; Bauce, Barbara; Nava, Andrea; Fanciulli, Manuela; Vazza, Giovanni; Mazzotti, Elisa; Rigato, Ilaria; De Bortoli, Marzia; Beffagna, Giorgia; Lorenzon, Alessandra; Calore, Martina; Dazzo, Emanuela; Nobile, Carlo; Luisa Mostacciuolo, Maria; Corrado, Domenico; Basso, Cristina; Daliento, Luciano; Thiene, Gaetano; Rampazzo, Alessandra

    2013-01-01

    Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disease characterized by progressive myocardial loss, with fibro-fatty replacement, and high frequency of ventricular arrhythmias that can lead to sudden cardiac death. ARVC is a genetically determined disorder, usually caused by point mutations in components of the cardiac desmosome. Conventional mutation screening of ARVC genes fails to detect causative mutations in about 50% of index cases, suggesting a further genetic heterogeneity. We performed a genome-wide linkage study and a copy number variations (CNVs) analysis, using high−density SNP arrays, in an ARVC family showing no mutations in any of the desmosomal genes. The CNVs analysis identified a heterozygous deletion of about 122 kb on chromosome 12p11.21, including the entire plakophilin-2 gene and shared by all affected family members. It was not listed on any of available public CNVs databases and was confirmed by quantitative real-time PCR. This is the first SNP array-based genome-wide study leading to the identification of a CNV segregating with the disease phenotype in an ARVC family. This result underscores the importance of performing additional analysis for possible genomic deletions/duplications in ARVC patients without point mutations in known disease genes. PMID:23486541

  16. Mouse model of inducible nephrogenic diabetes insipidus produced by floxed aquaporin-2 gene deletion.

    PubMed

    Yang, Baoxue; Zhao, Dan; Qian, Liman; Verkman, A S

    2006-08-01

    Transgenic mouse models of defective urinary concentrating ability produced by deletion of various membrane transport or receptor proteins, including aquaporin-2 (AQP2), are associated with neonatal mortality from polyuria. Here, we report an inducible mouse model of AQP2 gene deletion with severe polyuria in adult mice. LoxP sequences were inserted into introns 1 and 2 in the mouse AQP2 gene by homologous recombination in embryonic stem cells. Mating of germ-line AQP2-loxP mice with tamoxifen-inducible Cre-expressing mice produced offspring with inducible homozygous Cre-AQP2-loxP, which had a normal phenotype. Tamoxifen injections over 10 days resulted in AQP2 gene excision, with undetectable full-length AQP2 transcript in kidney and a >95% reduction in immunoreactive AQP2 protein. Urine osmolality decreased from approximately 2,000 to <500 mosmol/kgH(2)O after 4-5 days, with urine output increasing from 2 to 25 ml/day. Urine osmolality did not increase after water deprivation. Interestingly, AQP3 protein expression in the collecting duct was increased by about fivefold after AQP2 gene excision. Mild renal damage was seen after 6 wk of polyuria, with collecting duct dilatation, yet normal creatinine clearance and serum chemistries. These results establish the first adult model of nephrogenic diabetes insipidus (NDI) caused by AQP2 deficiency, with daily urine output comparable to body weight, although remarkable preservation of renal function compared with non-inducible NDI models.

  17. Deletion of cdvB paralogous genes of Sulfolobus acidocaldarius impairs cell division.

    PubMed

    Yang, Nuan; Driessen, Arnold J M

    2014-03-01

    The majority of Crenarchaeota utilize the cell division system (Cdv) to divide. This system consists of three highly conserved genes, cdvA, cdvB and cdvC that are organized in an operon. CdvC is homologous to the AAA-type ATPase Vps4, involved in multivesicular body biogenesis in eukaryotes. CdvA is a unique archaeal protein that interacts with the membrane, while CdvB is homologous to the eukaryal Vps24 and forms helical filaments. Most Crenarcheota contain additional CdvB paralogs. In Sulfolobus acidocaldarius these are termed CdvB1-3. We have used a gene inactivation approach to determine the impact of these additional cdvB genes on cell division. Independent deletion mutants of these genes were analyzed for growth and protein localization. One of the deletion strains (ΔcdvB3) showed a severe growth defect on plates and delayed growth on liquid medium. It showed the formation of enlarged cells and a defect in DNA segregation. Since these defects are accompanied with an aberrant localization of CdvA and CdvB, we conclude that CdvB3 fulfills an important accessory role in cell division.

  18. Infectious bronchitis viruses with naturally occurring genomic rearrangement and gene deletion.

    PubMed

    Hewson, Kylie A; Ignjatovic, Jagoda; Browning, Glenn F; Devlin, Joanne M; Noormohammadi, Amir H

    2011-02-01

    Infectious bronchitis viruses (IBVs) are group III coronaviruses that infect poultry worldwide. Genetic variations, including whole-gene deletions, are key to IBV evolution. Australian subgroup 2 IBVs contain sequence insertions and multiple gene deletions that have resulted in a substantial genomic divergence from international IBVs. The genomic variations present in Australian IBVs were investigated and compared to those of another group III coronavirus, turkey coronavirus (TCoV). Open reading frames (ORFs) found throughout the genome of Australian IBVs were analogous in sequence and position to TCoV ORFs, except for ORF 4b, which appeared to be translocated to a different position in the subgroup 2 strains. Subgroup 2 strains were previously reported to lack genes 3a, 3b and 5a, with some also lacking 5b. Of these, however, genes 3b and 5b were found to be present but contained various mutations that may affect transcription. In this study, it was found that subgroup 2 IBVs have undergone a more substantial genomic rearrangements than previously thought.

  19. BMP antagonists enhance myogenic differentiation and ameliorate the dystrophic phenotype in a DMD mouse model.

    PubMed

    Shi, SongTing; Hoogaars, Willem M H; de Gorter, David J J; van Heiningen, Sandra H; Lin, Herbert Y; Hong, Charles C; Kemaladewi, Dwi U; Aartsma-Rus, Annemieke; ten Dijke, Peter; 't Hoen, Peter A C

    2011-02-01

    Duchenne Muscular Dystrophy (DMD) is an X-linked lethal muscle wasting disease characterized by muscle fiber degeneration and necrosis. The progressive pathology of DMD can be explained by an insufficient regenerative response resulting in fibrosis and adipose tissue formation. BMPs are known to inhibit myogenic differentiation and in a previous study we found an increased expression of a BMP family member BMP4 in DMD myoblasts. The aim of the current study was therefore to investigate whether inhibition of BMP signaling could be beneficial for myoblast differentiation and muscle regeneration processes in a DMD context. All tested BMP inhibitors, Noggin, dorsomorphin and LDN-193189, were able to accelerate and enhance myogenic differentiation. However, dorsomorphin repressed both BMP and TGFβ signaling and was found to be toxic to primary myoblast cell cultures. In contrast, Noggin was found to be a potent and selective BMP inhibitor and was therefore tested in vivo in a DMD mouse model. Local adenoviral-mediated overexpression of Noggin in muscle resulted in an increased expression of the myogenic regulatory genes Myog and Myod1 and improved muscle histology. In conclusion, our results suggest that repression of BMP signaling may constitute an attractive adjunctive therapy for DMD patients.

  20. The chromosomal arrangement of human alpha-like globin genes: sequence homology and alpha-globin gene deletions.

    PubMed

    Lauer, J; Shen, C K; Maniatis, T

    1980-05-01

    We report the isolation of a cluster of four alpha-like globin genes from a bacteriophage lambda library of human DNA (Lawn et al., 1978). Analysis of the cloned DNA confirms the linkage arrangement of the two adult alpha-globin genes (alpha 1 and alpha 2) previously derived from genomic blotting experiments (Orkin, 1978) and identifies two additional closely linked alpha-like genes. The nucleotide sequence of a portion of each of these alpha-like genes was determined. One of these sequences is tentatively identified as an embryonic zeta-globin gene (zeta 1) by comparison with structural data derived from purified zeta-globin protein (J. Clegg, personal communication), while the other sequence cannot be matched with any known alpha-like polypeptide sequence (we designate this sequence phi alpha 1). Localization of the four alpha-like sequences on a restriction map of the gene cluster indicates that the genes have the same transcriptional orientation and are arranged in the order 5'-zeta 1-phi alpha 1-alpha 2-alpha 1-3'. Genomic blotting experiments identified a second, nonallelic zeta-like globin gene (phi 2) located 10-12 kb 5' to the cloned zeta-globin gene. Comparison of the locations of restriction sites within alpha 1 and alpha 2 and heteroduplex studies reveal extensive sequence homology within and flanking the two genes. The homologous sequences, which are interrupted by two blocks of nonhomology, span a region of approximately 4 kb. This extensive sequence homology between two genes which are thought to be the products of an ancient duplication event suggests the existence of a mechanism for sequence matching during evolution. One consequence of this arrangement of homologous sequences is the occurrence of two types of deletions in recombinant phage DNA during propagation in E. coli. The locations and sizes of the two types of deletions are indistinguishable from those of the two types of deletions associated with alpha-thalassemia 2 (Embury et al., 1979

  1. An atypical case of fragile X syndrome caused by a deletion that includes the FMR-1 gene

    SciTech Connect

    Quan, F.; Johnson, D.B.; Anoe, K.S.

    1994-09-01

    Fragile X syndrome results from the transcriptional inactivation of the FMR-1 gene. This is commonly caused by the expansion of an unstable CGG trinucleotide repeat in the first exon of the FMR-1 gene. We describe here an atypical case of fragile X syndrome caused by a deletion that includes the FMR-1 gene. RK is a 6-year-old hyperactive, mentally retarded male. Southern analysis of PstI digested genomic DNA was performed using a 558 bp XhoI-PstI fragment specific for the 5`-end of the FMR-1 gene. This analysis revealed the absence of the normal 1.0 kb PstI fragment, indicating the deletion of at least a portion of the FMR-1 gene. PCR analysis using Xq27.3 microsatellite and STS markers confirmed the presence of a deletion of at least 600 kb encompassing the FMR-1 gene. Southern blot and PCR analysis demonstrated that this deletion was maternally transmitted and arose as a new mutation on the grandpaternal X-chromosome. High resolution chromosome banding revealed an extremely small deletion of a portion of band Xq27 which was confirmed by fluorescent in situ hybridrization (FISH) analysis using a 34 kb cosmid containing the FMR-1 gene. As expected, RK manifests physical features typical of fragile X syndrome, including a high arched palate, prognathism, and large ears. Interestingly, RK also presents with anal atresia, obesity and short stature, features not part of fragile X syndrome. In addition, RK has normal sized testicles and does not exhibit the characteristic gaze avoidance, hand-flapping, and crowd anxiety behaviors. These atypical features may result from the deletion of additional genes in the vicinity of the FMR-1 gene. Further work is underway to determine more precisely the extent of the deletion in RK`s DNA.

  2. Inflammatory peeling skin syndrome caused by homozygous genomic deletion in the PSORS1 region encompassing the CDSN gene.

    PubMed

    Ishida-Yamamoto, Akemi; Furio, Laetitia; Igawa, Satomi; Honma, Masaru; Tron, Elodie; Malan, Valerie; Murakami, Masamoto; Hovnanian, Alain

    2014-01-01

    Peeling skin syndrome (PSS) type B is a rare recessive genodermatosis characterized by lifelong widespread, reddish peeling of the skin with pruritus. The disease is caused by small-scale mutations in the Corneodesmosin gene (CDSN) leading to premature termination codons. We report for the first time a Japanese case resulting from complete deletion of CDSN. Corneodesmosin was undetectable in the epidermis, and CDSN was unamplifiable by PCR. QMPSF analysis demonstrated deletion of CDSN exons inherited from each parent. Deletion mapping using microsatellite haplotyping, CGH array and PCR analysis established that the genomic deletion spanned 49-72 kb between HCG22 and TCF19, removing CDSN as well as five other genes within the psoriasis susceptibility region 1 (PSORS1) on 6p21.33. This observation widens the spectrum of molecular defects underlying PSS type B and shows that loss of these five genes from the PSORS1 region does not result in an additional cutaneous phenotype.

  3. Independent nonframeshift deletions in the MC1R gene are not associated with melanistic coat coloration in three mustelid lineages.

    PubMed

    Hosoda, T; Sato, J J; Shimada, T; Campbell, K L; Suzuki, H

    2005-01-01

    Sequence variation within the 5' flanking (about 240 bp) and exon regions (426 bp) of the melanocortin-1 receptor (MC1R) gene was examined to determine the potential role of this protein in the melanistic coat coloration of 17 mustelid species in four genera: Gulo (wolverines), Martes (martens), Mustela (weasels), and Meles (badgers). Members of the genera Mustela and Meles, together with Martes flavigula and Martes pennanti, were shown to have intact gene sequences. However, several "in frame" deletions of the MC1R gene region implicated in melanism of other species were detected within members of the genera Martes and Gulo. For instance, Gulo gulo possessed a 15 bp deletion in the second transmembrane domain coding region, while Martes americana, Martes melampus, Martes zibellina, and Martes martes shared a 45 bp deletion overlapping this area. In addition, Martes foina was found to possess a 10 bp insertion followed closely by a 28 bp deletion immediately downstream of the deletion found in other martens. Notably, none of these indels was associated with a melanistic phenotype. Phylogenetic analysis revealed that each of these nonrandomly distributed deletions arose independently during the evolution of this family. Specific indel-neighboring motifs appear to largely account for the biased and repeated occurrence of deletion events in the Martes/Gulo clade.

  4. R3-R4 deletion in the PRNP gene is associated with Creutzfeldt-Jakob disease (CJD)

    SciTech Connect

    Cervenakova, L.; Brown, P.; Nagle, J.

    1994-09-01

    There are conflicting reports on the association of deletions in the PRNP gene on chromosome 20 with CJD, a rapidly progressive fatal spongiform encephalopathy. We accumulated data suggesting that a deletion of R3-R4 type (parts of the third and fourth repeats are deleted from the area of four repeating 24 bp sequences in the 5{prime} region of the gene) is causing CJD. Screening of 129 unaffected control individuals demonstrated presence of a deletion of R2 type in four (1.55% of the studied chromosomes), but none of them had the R3-R4 type. Of 181 screened patients with spongiform encephalopathies, two had a deletion of R3-R4 type with no other mutations in the coding sequence. Both patients had a classical rapidly progressive dementing disease and diffuse spongiform degeneration, and both cases were apparently sporadic. The same R3-R4 type of deletion was detected in three additional neuropathologically confirmed spongiform encephalopathy patients, of which two had other known pathogenic mutations in the PRNP gene: at codon 178 on the methionine allele exhibiting the phenotype of fatal familial insomnia, and codon 200 causing CJD with severe dementia; the third was a patient with iatrogenic CJD who developed the disease after treatment with growth hormone extracted from cadaveric human pituitary glands. In all cases the deletion coincided with a variant sequence at position 129 coding for methionine.

  5. Distribution of the CCR5 gene 32-basepair deletion in 11 Chinese populations.

    PubMed

    Zhang, Chunyu; Fu, Songbin; Xue, Yali; Wang, Qi; Huang, Xiaoyi; Wang, Baiqiu; Liu, An; Ma, Linlin; Yu, Yang; Shi, Rongqian; Lv, Fuqu; Shi, Zhongcheng; Zhang, Yu; Cheng, Wenhong; Ai, Qionghua; Xu, Fang; Huang, Chengbin; Chen, Baibin; Kang, Xianghua; Sun, Yanyang; Zhang, Guiyin; Li, Pu

    2002-09-01

    A mutant allele of the chemokine receptor gene CCR5 bearing a 32-basepair deletion (delta 32CCR5) could increase the resistance to HIV-1 infection or delayed progression to AIDS. The frequency of this mutation is higher in Europeans than in Asians. To investigate the distribution of this polymorphism in China, 715 individuals from 11 Chinese populations were screened by PCR, including the Han and 10 other ethnic groups. The delta 32CCR5 gene was found in 16 individuals from 5 ethnic groups. All of them were heterozygous. The frequency of the mutant alleles of delta 32CCR5 is low in China and reflects (or might reflect) ancestral gene flow from Europe to Chinese ethnic groups and recent intermarriage within the ethnic groups.

  6. Exploration of geosmin synthase from Streptomyces peucetius ATCC 27952 by deletion of doxorubicin biosynthetic gene cluster.

    PubMed

    Singh, Bijay; Oh, Tae-Jin; Sohng, Jae Kyung

    2009-10-01

    Thorough investigation of Streptomyces peucetius ATCC 27952 genome revealed a sesquiterpene synthase, named spterp13, which encodes a putative protein of 732 amino acids with significant similarity to S. avermitilis MA-4680 (SAV2163, GeoA) and S. coelicolor A3(2) (SCO6073). The proteins encoded by SAV2163 and SCO6073 produce geosmin in the respective strains. However, the spterp13 gene seemed to be silent in S. peucetius. Deletion of the doxorubicin gene cluster from S. peucetius resulted in increased cell growth rate along with detectable production of geosmin. When we over expressed the spterp13 gene in S. peucetius DM07 under the control of an ermE* promoter, 2.4 +/- 0.4-fold enhanced production of geosmin was observed.

  7. A 57-bp deletion in the ovine KAP6-1 gene affects wool fibre diameter.

    PubMed

    Zhou, H; Gong, H; Li, S; Luo, Y; Hickford, J G H

    2015-08-01

    High glycine-tyrosine keratin-associated proteins (HGT-KAPs) are predominantly present in the orthocortex of wool fibres. They vary in abundance in different wools and have been implicated in regulating wool fibre properties, but little is known about the functional roles of these proteins in the fibre matrix. In this study, we used polymerase chain reaction--single-strand conformational polymorphism (PCR-SSCP) analysis to screen for variation in a gene encoding the ovine HGT-KAP6-1 protein. We identified three gene variants (A, B and C). Variants A and B were similar to each other, with only three nucleotide differences occurring downstream of the coding sequence. However, variant C had a 57-bp deletion that would notionally result in a loss of 19 amino acids in the protein. The presence of C was found to be associated with an increase in mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), coefficient of variation of fibre diameter (CVFD) and prickle factor (percentage of fibres over 30 microns; PF). Sheep of genotype BC produced wool of greater MFD, FDSD and PF than sheep of genotypes AA, AB and BB. The CVFD was greater in the BC sheep than the AB sheep. The results suggest that variation in ovine KRTAP6-1 affects wool fibre diameter-associated traits and that the 57-bp deletion in this gene would lead to coarser wool with greater FDSD, CVFD and PF.

  8. An S receptor kinase gene in self-compatible Brassica napus has a 1-bp deletion.

    PubMed Central

    Goring, D R; Glavin, T L; Schafer, U; Rothstein, S J

    1993-01-01

    S locus glycoprotein (SLG) and S locus receptor kinase (SRK) cDNAs were isolated from an S allele present in a number of self-compatible Brassica napus lines. This A10 allele did not segregate with self-incompatibility in crosses involving other self-incompatible B. napus lines. The SLG-A10 cDNA was found to contain an intact open reading frame and was predicted to encode an SLG protein with sequence similarities to those previously associated with phenotypically strong self-incompatibility reactions. SLG-A10 transcripts were detected in the developing stigma at steady state levels even higher than those detected for SLG alleles linked with self-incompatibility. Analysis of the corresponding SRK-A10 cDNA showed that it was very similar to other S locus receptor kinase genes and was expressed predominantly in the stigma. However, a 1-bp deletion was detected in the SRK gene toward the 3' end of the SLG homology domain. This deletion would lead to premature termination of translation and the production of a truncated SRK protein. The A10 allele was determined to represent a B. oleracea S allele based on its segregation pattern with the B. oleracea S24 allele when both these alleles were present in the same B. napus background. These results suggest that a functional SRK gene is required for Brassica self-incompatibility. PMID:8518554

  9. A next-generation genetically attenuated Plasmodium falciparum parasite created by triple gene deletion.

    PubMed

    Mikolajczak, Sebastian A; Lakshmanan, Viswanathan; Fishbaugher, Matthew; Camargo, Nelly; Harupa, Anke; Kaushansky, Alexis; Douglass, Alyse N; Baldwin, Michael; Healer, Julie; O'Neill, Matthew; Phuong, Thuan; Cowman, Alan; Kappe, Stefan H I

    2014-09-01

    Immunization with live-attenuated Plasmodium sporozoites completely protects against malaria infection. Genetic engineering offers a versatile platform to create live-attenuated sporozoite vaccine candidates. We previously generated a genetically attenuated parasite (GAP) by deleting the P52 and P36 genes in the NF54 wild-type (WT) strain of Plasmodium falciparum (Pf p52(-)/p36(-) GAP). Preclinical assessment of p52(-)/p36(-) GAP in a humanized mouse model indicated an early and severe liver stage growth defect. However, human exposure to >200 Pf p52(-)/p36(-) GAP-infected mosquito bites in a safety trial resulted in peripheral parasitemia in one of six volunteers, revealing that this GAP was incompletely attenuated. We have now created a triple gene deleted GAP by additionally removing the SAP1 gene (Pf p52(-)/p36(-)/sap1(-) GAP) and employed flippase (FLP)/flippase recognition target (FRT) recombination for drug selectable marker cassette removal. This next-generation GAP was indistinguishable from WT parasites in blood stage and mosquito stage development. Using an improved humanized mouse model transplanted with human hepatocytes and human red blood cells, we show that despite a high-dose sporozoite challenge, Pf p52(-)/p36(-)/sap1(-) GAP did not transition to blood stage infection and appeared to be completely attenuated. Thus, clinical testing of Pf p52(-)/p36(-)/sap1(-) GAP assessing safety, immunogenicity, and efficacy against sporozoite challenge is warranted.

  10. Microevolution of Duplications and Deletions and Their Impact on Gene Expression in the Nematode Pristionchus pacificus

    PubMed Central

    2015-01-01

    The evolution of diversity across the animal kingdom has been accompanied by tremendous gene loss and gain. While comparative genomics has been fruitful to characterize differences in gene content across highly diverged species, little is known about the microevolution of structural variations that cause these differences in the first place. In order to investigate the genomic impact of structural variations, we made use of genomic and transcriptomic data from the nematode Pristionchus pacificus, which has been established as a satellite model to Caenorhabditis elegans for comparative biology. We exploit the fact that P. pacificus is a highly diverse species for which various genomic data including the draft genome of a sister species P. exspectatus is available. Based on resequencing coverage data for two natural isolates we identified large (> 2kb) deletions and duplications relative to the reference strain. By restriction to completely syntenic regions between P. pacificus and P. exspectatus, we were able to polarize the comparison and to assess the impact of structural variations on expression levels. We found that while loss of genes correlates with lack of expression, duplication of genes has virtually no effect on gene expression. Further investigating expression of individual copies at sites that segregate between the duplicates, we found in the majority of cases only one of the copies to be expressed. Nevertheless, we still find that certain gene classes are strongly depleted in deletions as well as duplications, suggesting evolutionary constraint acting on synteny. In summary, our results are consistent with a model, where most structural variations are either deleterious or neutral and provide first insights into the microevolution of structural variations in the P. pacificus genome. PMID:26125626

  11. A Genetic Screen for Fission Yeast Gene Deletion Mutants Exhibiting Hypersensitivity to Latrunculin A

    PubMed Central

    Asadi, Farzad; Michalski, Dorothy; Karagiannis, Jim

    2016-01-01

    Fission yeast cells treated with low doses of the actin depolymerizing drug, latrunculin A (LatA), delay entry into mitosis via a mechanism that is dependent on both the Clp1p and Rad24p proteins. During this delay, cells remain in a cytokinesis-competent state that is characterized by continuous repair and/or reestablishment of the actomyosin ring. In this manner, cells ensure the faithful completion of the preceding cytokinesis in response to perturbation of the cell division machinery. To uncover other genes with a role in this response, or simply genes with roles in adapting to LatA-induced stress, we carried out a genome-wide screen and identified a group of 38 gene deletion mutants that are hyper-sensitive to the drug. As expected, we found genes affecting cytokinesis and/or the actin cytoskeleton within this set (ain1, acp2, imp2). We also identified genes with roles in histone modification (tra1, ngg1), intracellular transport (apl5, aps3), and glucose-mediated signaling (git3, git5, git11, pka1, cgs2). Importantly, while the identified gene deletion mutants are prone to cytokinesis failure in the presence of LatA, they are nevertheless fully capable of cell division in the absence of the drug. These results indicate that fission yeast cells make use of a diverse set of regulatory modules to counter abnormal cytoskeletal perturbations, and furthermore, that these modules act redundantly to ensure cell survival and proliferation. PMID:27466272

  12. Total beta-globin gene deletion has high frequency in Filipinos

    SciTech Connect

    Patrick, N.; Miyakawa, F.; Hunt, J.A.

    1994-09-01

    The distribution of {beta}-thalassemia [{beta}{sup Th}] mutations is unique to each ethnic group. Most mutations affect one or a few bases; large deletions have been rare. Among families screened in Hawaii, [{beta}{sup Th}] heterozygotes were diagnosed by microcytosis, absence of abnormal hemoglobins on isoelectric focusing, and raised Hb A{sub 2} by chromatography. Gene frequency for {beta}{sup Th} was 0.02 in Filipinos. In Filipinos, polymerase chain reaction [PCR] with denaturing gradient gel electrophoresis for {beta}{sup Th} mutations detected a mutation in only 6 of 42 {beta}{sup Th} heterozygotes; an IVS2-666 C/T polymorphism showed non-heterozygosity in 37 and heterozygosity in only 5 of these {beta}{sup Th} heterozygotes. One {beta}{sup Th}/{beta}{sup Th} major patient and his mother had no mutation detected by allele-specific oligomer hybridization; PCR failed to amplify any DNA from his {beta}-globin gene. After a total {beta}-globin gene deletion [{beta}{sup Del}] was found in a Filipino family in Ontario, specific PCR amplification for {beta}{sup Del} detected this in 43 of 53 {beta}{sup Th} Filipino samples tested; the above {beta}{sup Th}/{beta}{sup Th} patient was a ({beta}{sup Del}/{beta}{sup Del}) homozygote. The {beta}{sup Del} may account for over 60% of all {beta}{sup Th} alleles in Filipinos; this is the highest proportion of a deletion {beta}{sup Th} mutation reported from any population. Most but not all {beta}{sup Del} heterozygotes had high Hb F [5.13 {plus_minus} 3.94 mean {plus_minus} 1 s.d.] compared to the codon 41/42 four base deletion common in Chinese [2.30 {plus_minus} 0.86], or to {beta}{sup Th} heterozygotes with normal {alpha}-globin genes [2.23 {plus_minus} 0.80].

  13. Familial spinal neurofibromatosis due to a multiexonic NF1 gene deletion.

    PubMed

    Pizzuti, Antonio; Bottillo, Irene; Inzana, Francesca; Lanari, Valentina; Buttarelli, Francesca; Torrente, Isabella; Giallonardo, Anna Teresa; De Luca, Alessandro; Dallapiccola, Bruno

    2011-08-01

    We report the detailed clinical presentation and molecular features of a spinal neurofibromatosis familial case where a 40-year-old woman, presenting with multiple bilateral spinal neurofibromas and no other clinical feature of neurofibromatosis type 1 (NF1), inherited a paternal large multiexonic deletion (c.5944-?_7126+?del) which resulted in NF1 gene haploinsufficiency at the RNA level. In the clinically unaffected 73-year-old father, spinal cord MRI disclosed bilateral and symmetrical hypertrophy of spinal lumbosacral roots. Our study widens the phenotypic and mutational spectrum of NF1 and illustrates the difficulties of counseling patients with border-line or atypical presentation of this disorder.

  14. Homozygous deletion of the. alpha. - and. beta. sub 1 -interferon genes in human leukemia and derived cell lines

    SciTech Connect

    Diaz, M.O.; Ziemin, S.; Le Beau, M.M.; Pitha, P.; Smith, S.D.; Chilcote, R.R.; Rowley, J.D. )

    1988-07-01

    The loss of bands p21-22 from one chromosome 9 homologue as a consequence of a deletion of the short arm (del(9p)), unbalanced translocation, or monosomy 9 is frequently observed in the malignant cells of patients with lymphoid neoplasias, including acute lymphoblastic leukemia and non-Hodgkin lymphoma. The {alpha}- and {beta}{sub 1}-interferon genes have been assigned to this chromosome region (9p21-22). The authors now present evidence of the homozygous deletion of the interferon genes in neoplastic hematopoietic cell lines and primary leukemia cells in the presence or absence of chromosomal deletions that are detectable at the level of the light microscope. In these cell lines, the deletion of the interferon genes is accompanied by a deficiency of 5{prime}-methylthioadenosine phosphorylase, an enzyme of purine metabolism. These homozygous deletions may be associated with the loss of a tumor-suppressor gene that is involved in the development of these neoplasias. The relevant genes may be either the interferon genes themselves or a gene that has a tumor-suppressor function and is closely linked to them.

  15. Large deletion of the GJB6 gene in deaf patients heterozygous for the GJB2 gene mutation: genotypic and phenotypic analysis.

    PubMed

    Feldmann, Delphine; Denoyelle, Françoise; Chauvin, Pierre; Garabédian, Eréa-Noël; Couderc, Rémy; Odent, Sylvie; Joannard, Alain; Schmerber, Sébastien; Delobel, Bruno; Leman, Jacques; Journel, Hubert; Catros, Hélène; Le Maréchal, Cédric; Dollfus, Hélène; Eliot, Marie-Madeleine; Delaunoy, Jean-Pierre; David, Albert; Calais, Catherine; Drouin-Garraud, Valérie; Obstoy, Marie-Françoise; Bouccara, Didier; Sterkers, Olivier; Huy, Patrice Tran Ba; Goizet, Cyril; Duriez, Françoise; Fellmann, Florence; Hélias, Jocelyne; Vigneron, Jacqueline; Montaut, Bétina; Lewin, Patricia; Petit, Christine; Marlin, Sandrine

    2004-06-15

    Recent investigations identified a large deletion of the GJB6 gene in trans to a mutation of GJB2 in deaf patients. We looked for GJB2 mutations and GJB6 deletions in 255 French patients presenting with a phenotype compatible with DFNB1. 32% of the patients had biallelic GJB2 mutations and 6% were a heterozygous for a GJB2 mutation and a GJB6 deletion. Biallelic GJB2 mutations and combined GJB2/GJB6 anomalies were more frequent in profoundly deaf children. Based on these results, we are now assessing GJB6 deletion status in cases of prelingual hearing loss.

  16. Pathogenicity and immunogenicity of recombinant Tiantan Vaccinia Virus with deleted C12L and A53R genes.

    PubMed

    Dai, Kaifan; Liu, Ying; Liu, Mingjie; Xu, Jianqing; Huang, Wei; Huang, Xianggang; Liu, Lianxing; Wan, Yanmin; Hao, Yanling; Shao, Yiming

    2008-09-15

    Interest is increasing regarding replicating poxvirus as HIV vaccine vector. In China, the Tiantan Vaccinia Virus (TV) has been used most extensively in the battle of eradicating smallpox. Recently, TV was developing as vaccine vector to fight against infectious diseases such as human immunodeficiency virus (HIV). However, replicating vaccinia virus sometimes may pose serious post-vaccination complications, especially in immunosuppressed individuals. To develop a safer and more effective TV-based vector, we constructed C12L (vIL-18 binding protein) and A53R (vTNF receptor homolog) gene-deleted mutants which are based on parental TV and VTKgpe (TV expressing HIV gagpol and env gene), respectively. The pathogenicity and immunogenicity were also evaluated. Deleting these two immunomodulatory genes lessened the virulence of the parental virus in both mice and rabbit models. Notably, C12L deletion mutant attenuated the skin virulence of parental virus by as high as approximate 2 logs. Furthermore, VTKgpe with A53R and C12L gene deletion retains the high immunogenicity of the parental virus to elicit strong humoral and cellular responses to the HIV target genes despite the remarkable attenuation. These data suggest that deletion of the cytokine viroceptor gene is feasible to obtain a safer and replication-competent TV vector for vaccination and immunotherapy.

  17. [Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System].

    PubMed

    Li, Lili; Wang, Zhan; Zhou, Yubai; Zhang, Fang; Shen, Sisi; Li, Zelin; Zeng, Yi

    2015-09-01

    For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.

  18. An atypical deletion of the Williams–Beuren syndrome interval implicates genes associated with defective visuospatial processing and autism

    PubMed Central

    Edelmann, Lisa; Prosnitz, Aaron; Pardo, Sherly; Bhatt, Jahnavi; Cohen, Ninette; Lauriat, Tara; Ouchanov, Leonid; González, Patricia J; Manghi, Elina R; Bondy, Pamela; Esquivel, Marcela; Monge, Silvia; Delgado, Marietha F; Splendore, Alessandra; Francke, Uta; Burton, Barbara K; McInnes, L Alison

    2007-01-01

    Background During a genetic study of autism, a female child who met diagnostic criteria for autism spectrum disorder, but also exhibited the cognitive–behavioural profile (CBP) associated with Williams–Beuren syndrome (WBS) was examined. The WBS CBP includes impaired visuospatial ability, an overly friendly personality, excessive non‐social anxiety and language delay. Methods Using array‐based comparative genomic hybridisation (aCGH), a deletion corresponding to BAC RP11‐89A20 in the distal end of the WBS deletion interval was detected. Hemizygosity was confirmed using fluorescence in situ hybridisation and fine mapping was performed by measuring the copy number of genomic DNA using quantitative polymerase chain reaction. Results The proximal breakpoint was mapped to intron 1 of GTF2IRD1 and the distal breakpoint lies 2.4–3.1 Mb towards the telomere. The subject was completely hemizygous for GTF2I, commonly deleted in carriers of the classic ∼1.5 Mb WBS deletion, and GTF2IRD2, deleted in carriers of the rare ∼1.84 Mb WBS deletion. Conclusion Hemizygosity of the GTF2 family of transcription factors is sufficient to produce many aspects of the WBS CBP, and particularly implicate the GTF2 transcription factors in the visuospatial construction deficit. Symptoms of autism in this case may be due to deletion of additional genes outside the typical WBS interval or remote effects on gene expression at other loci. PMID:16971481

  19. Carotenoid-based phenotypic screen of the yeast deletion collection reveals new genes with roles in isoprenoid production.

    PubMed

    Özaydın, Bilge; Burd, Helcio; Lee, Taek Soon; Keasling, Jay D

    2013-01-01

    Beside their essential cellular functions, isoprenoids have value as pharmaceuticals, nutriceuticals, pesticides, and fuel alternatives. Engineering microorganisms for production of isoprenoids is relatively easy, sustainable, and cost effective in comparison to chemical synthesis or extraction from natural producers. We introduced genes encoding carotenoid biosynthetic enzymes into the haploid yeast deletion collection to identify gene deletions that improved isoprenoid production. Deletions that showed significant improvement in carotenoid production were further screened for production of bisabolene, an isoprenoid alternative to petroleum-derived diesel. Combining those deletions with other mevalonate pathway modifications increased production of bisabolene from 40mg/L to 800mg/L in shake-flask cultures. In a fermentation process, this engineered strain produced 5.2g/L of bisabolene.

  20. [Repression of the enzyme inducible syntheses in Escherichia coli K12 mutant with a deleted ptsH gene].

    PubMed

    Gershanovich, V N; Il'ina, T S; Rusina, O Iu; Iurovitskaia, N V; Bol'shakova, T N

    1977-01-01

    The genome of lambda phage with thermosensitive repressor was integrated into the pts region of the E. coli chromosome. Such a lysogenic culture behaves as a pts mutant at 30 degrees. Heating of cells of this strain leads to the induction of lambda prophage and formation of deletions in the pts region. A mutant with a deletion covering ptsH gene was isolated after prophage induction. The deletion nature of pts mutation was confirmed in genetic and biochemical experiments. It was shown that the deletion is small and does not involve ptsI and lig genes. The isolated deltaptsH mutant possesses all characteristics of pts mutants: pleiotropic impairment of transport and utilization of a number of carbohydrates, repression of the enzyme inducible synthesis and resistance to catabolite repression with glucose. These data (together with earlier ones) allow us to conclude that the phosphorylated form of HPr is involved (in direct of indirect manner/ in activation of DNA transcription.

  1. Correction of a deletion mutant by gene targeting with an adenovirus vector.

    PubMed Central

    Wang, Q; Taylor, M W

    1993-01-01

    The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter. Images PMID:8423811

  2. Improved Techniques for Endogenous Epitope Tagging and Gene Deletion in Toxoplasma gondii

    PubMed Central

    Upadhya, Rajendra; Kim, Kami; Hogue-Angeletti, Ruth; Weiss, Louis M

    2011-01-01

    Toxoplasma gondii is an excellent model organism for studies on the biology of the Apicomplexa due to its ease of in vitro cultivation and genetic manipulation. Large-scale reverse genetic studies in T. gondii have, however, been difficult due to the low frequency of homologous recombination. Efforts to ensure homologous recombination have necessitated engineering long flanking regions in the targeting construct. This requirement makes it difficult to engineer chromosomally targeted epitope tags or gene knock out constructs only by restriction enzyme mediated cloning steps. To address this issue we employed multisite Gateway® recombination techniques to generate chromosomal gene manipulation targeting constructs. Incorporation of 1.5 to 2.0 kb flanking homologous sequences in PCR generated targeting constructs resulted in 90% homologous recombination events in wild type T. gondii (RH strain) as determined by epitope tagging and target gene deletion experiments. Furthermore, we report that split marker constructs were equally efficient for targeted gene disruptions using the T. gondii UPRT gene locus as a test case. The methods described in this paper represent an improved strategy for efficient epitope tagging and gene disruptions in T. gondii. PMID:21352857

  3. Targeted gene deletion of miRNAs in mice by TALEN system.

    PubMed

    Takada, Shuji; Sato, Tempei; Ito, Yoshiaki; Yamashita, Satoshi; Kato, Tomoko; Kawasumi, Miyuri; Kanai-Azuma, Masami; Igarashi, Arisa; Kato, Tomomi; Tamano, Moe; Asahara, Hiroshi

    2013-01-01

    Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.

  4. Deletion of AS87_03730 gene changed the bacterial virulence and gene expression of Riemerella anatipestifer

    PubMed Central

    Wang, Xiaolan; Yue, Jiaping; Ding, Chan; Wang, Shaohui; Liu, Beibei; Tian, Mingxing; Yu, Shengqing

    2016-01-01

    Riemerella anatipestifer is an important pathogen of waterfowl, which causes septicemia anserum exsudativa in ducks. In this study, an AS87_03730 gene deletion R. anatipestifer mutant Yb2ΔAS87_03730 was constructed to investigate the role of AS87_03730 on R. anatipestifer virulence and gene regulation. By deleting a 708-bp fragment from AS87_03730, the mutant Yb2ΔAS87_03730 showed a significant decreased growth rate in TSB and invasion capacity in Vero cells, compared to wild-type strain Yb2. Moreover, the median lethal dose (LD50) of Yb2ΔAS87_03730 was 1.24 × 107 colony forming units (CFU), which is about 80-fold attenuated than that of Yb2 (LD50 = 1.53 × 105 CFU). Furthermore, RNA-Seq analysis and Real-time PCR indicated 19 up-regulated and two down-regulated genes in Yb2ΔAS87_03730. Functional analysis revealed that 12 up-regulated genes were related to “Translation, ribosomal structure and biogenesis”, two were classified into “Cell envelope biogenesis, outer membrane”, one was involved in “Amino acid transport and metabolism”, and the other four had unknown functions. Polymerase chain reaction and sequence analysis indicated that the AS87_03730 gene is highly conserved among R. anatipestifer strains, as the percent sequence identity was over 93.5%. This study presents evidence that AS87_03730 gene is involved in bacterial virulence and gene regulation of R. anatipestifer. PMID:26928424

  5. Spermine deficiency in Gy mice caused by deletion of the spermine synthase gene.

    PubMed

    Lorenz, B; Francis, F; Gempel, K; Böddrich, A; Josten, M; Schmahl, W; Schmidt, J; Lehrach, H; Meitinger, T; Strom, T M

    1998-03-01

    Two mouse mutations gyro (Gy) and hypophosphatemia (Hyp) are mouse models for X-linked hypophosphatemic rickets and have been shown to be deleted for the 5' and 3' end of the mouse homolog of PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome; formerly called PEX), respectively. In addition to the metabolic disorder observed in Hyp mice, male Gy mice are sterile and show circling behavior and reduced viability. The human SMS (spermine synthase) gene maps approximately 39 kb upstream of PHEX and is transcribed in the same direction. To elucidate the complex phenotype of Gy mice, we characterized the genomic region upstream of Phex. By establishing the genomic structure of mouse Sms, a 160-190 kb deletion was shown in Gy mice, which includes both Phex and Sms. There are several pseudogenes of SMS / Sms in man and mouse. Northern analysis revealed three different Sms transcripts which are absent in Gy mice. Measurement of polyamine levels revealed a marked decrease in spermine in liver and pancreas of affected male Gy mice. Analysis of brain tissue revealed no gross or histological abnormalities. Gy provides a mouse model for a defect in the polyamine pathway, which is known to play a key role in cell proliferation.

  6. Detection of APC gene deletions in colorectal malignancies using quantitative PCR in a Chinese population.

    PubMed

    Fang, Zhengyu; Xiong, Yi; Li, Jiana; Liu, Li; Li, Manhui; Zhang, Wei; Shi, Lei; Wan, Jun

    2011-09-01

    The adenomatous polyposis coli (APC) gene has been shown to be involved in genetic instability and to be downregluated in several human carcinomas. The chromosome locus of APC, 5q21-22, is frequently deleted in colorectal cancers (CRCs). The functional impact of such regions needs to be extensively investigated in large amount of clinical samples. Case-matched tissues of CRC and adjacent normal epithelium (n = 134) were included in this study. Quantitative PCR was carried out to examine the copy number as well as mRNA expression of APC gene in colorectal malignancies. Our results showed that copy number deletions of APC were present in a relatively high percentage of colorectal cancer samples (26.1%, 35 out of 134). There was a positive correlation between copy number decrease of APC and tumor progression in CRCs. Furthermore, copy number loss of APC was correlated with decreased mRNA expression. However, mRNA levels of APC were also impaired in CRC samples with unaltered copy numbers, indicating that sporadic CRCs exhibit different mechanisms of APC regulation.

  7. The sebaceous nevus: a nevus with deletions of the PTCH gene.

    PubMed

    Xin, H; Matt, D; Qin, J Z; Burg, G; Böni, R

    1999-04-15

    Sebaceous nevi (SN) are congenital malformations of the skin with the potential to develop into basal cell carcinoma (BCC). To date, the molecular basis for their carcinogenic potential remains unknown. The genetic defect in BCC is known and involves the human homologue of Drosophila patched (PTCH) on chromosome 9q22.3. The objective of this study was to test whether allelic deletion of the PTCH gene could already be detected in SN. Twenty-one paraffin-embedded SN were investigated in this study. Basaloid cells in conjunction with mature sebaceous glands as well as epidermal layer apart from SN were microdissected and subjected to single-step DNA extraction. We performed the analysis with polymorphic markers at 9q22.3 (D9S15, D9S252, D9S287, and D9S303). Of the 20 informative SN, 8 (40%) exhibited loss of heterozygosity at least at one locus. Here, we provide the first evidence of the involvement of the tumor suppressor gene PTCH in SN. Whether PTCH deletion in SN is associated with progression to BCC and/or other appendageal tumors should be addressed in future studies.

  8. Ring chromosome 20 syndrome without deletions of the subtelomeric and CHRNA4--KCNQ2 genes loci.

    PubMed

    Elghezal, Hatem; Hannachi, Hanene; Mougou, Soumaya; Kammoun, Hassene; Triki, Chahnez; Saad, Ali

    2007-01-01

    Ring chromosome 20 (r(20)) syndrome is a rare disease characterized by refractory epilepsy, moderate mental retardation and particular electroencephalographic disorder with non-convulsive status epilepticus. Here, we report a new case of r(20) syndrome in a 12 year old female who presented minimal dysmorphism, generalised tonic-clonic and absence seizures refractory to medical therapy and behavioural troubles. Among 20 cytogenetically analysed cells, 14 (70%) exhibited a 46,XX,r(20)(p13q13.3) karyotype and 6 (30%) showed a normal 46,XX caryotype. Interphasic FISH using centromeric probe of chromosome 20 detects the presence of a chromosome 20 monosomy in 7% and a duplicated ring chromosome 20 in 8% of studied cells. Metaphase FISH using chromosome 20 telomeric probes and specific probes of CHRNA4 and KCNQ2 genes detects the absence of any deletion in the ring chromosome 20. Clinical symptoms of r(20) syndrome are attributed to telomeric partial monosomy generated by ring chromosome and causing an haploinsufficiency of two epilepsy genes CHRNA4 and KCNQ2. However, our patient presents the typical epilepsy disorder but no detectable deletion in the ring chromosome 20. We speculate that clinical features of ring chromosome 20 syndrome are caused by low mosaicism of chromosome 20 monosomy caused by the loss of the ring chromosome 20.

  9. Neurogenin 3-directed cre deletion of Tsc1 gene causes pancreatic acinar carcinoma.

    PubMed

    Ding, Li; Han, Lingling; Li, Yin; Zhao, Jing; He, Ping; Zhang, Weizhen

    2014-11-01

    The role of tuberous sclerosis complex (TSC) in the pathogenesis of pancreatic cancers remains largely unknown. The present study shows that neurogenin 3 directed Cre deletion of Tsc1 gene induces the development of pancreatic acinar carcinoma. By cross-breeding the Neurog3-cre mice with Tsc1 (loxp/loxp) mice, we generated the Neurog3-Tsc1-/- transgenic mice in which Tsc1 gene is deleted and mTOR signaling activated in the pancreatic progenitor cells. All Neurog3-Tsc1-/- mice developed notable adenocarcinoma-like lesions in pancreas starting from the age of 100 days old. The tumor lesions are composed of cells with morphological and molecular resemblance to acinar cells. Metastasis of neoplasm to liver and lung was detected in 5% of animals. Inhibition of mTOR signaling by rapamycin significantly attenuated the growth of the neoplasm. Relapse of the neoplasm occurred within 14 days upon cessation of rapamycin treatment. Our studies indicate that activation of mTOR signaling in the pancreatic progenitor cells may trigger the development of acinar carcinoma. Thus, mTOR may serve as a potential target for treatment of pancreatic acinar carcinoma.

  10. Deletion of pigR gene in Monascus ruber leads to loss of pigment production.

    PubMed

    Xie, Nana; Liu, Qingpei; Chen, Fusheng

    2013-09-01

    Pigments produced by Monascus are traditional food colorants and are widely used as dietary supplements. Since genes involving in pigment biosynthesis have not been reported, we describe the identification of a putative pigment-regulatory gene (pigR) obtained by molecular analysis of an albino strain of Monascus ruber M7. In the pigR-deleted strain (ΔpigR), neither the pigments nor pigR expression were detected by HPLC or reverse-transcription PCR, respectively, whereas the introduction of the pigR, together with a constitutive trpC promoter into ΔpigR, caused it to produce 5.4 U of red pigments/g dry mycelia, about 12-fold higher than Monascus ruber M7 (0.46 U/g dry mycelia). Thus pigR up-regulates pigment production in Monascus ruber M7.

  11. Pentoxifylline as a rescue treatment for DMD

    PubMed Central

    Zimmerman, A.; Bertorini, T.; Clemens, P.R.; Connolly, A.M.; Mesa, L.; Gorni, K.; Kornberg, A.; Kolski, H.; Kuntz, N.; Nevo, Y.; Tesi-Rocha, C.; Nagaraju, K.; Rayavarapu, S.; Hache, L.P.; Mayhew, J.E.; Florence, J.; Hu, F.; Arrieta, A.; Henricson, E.; Leshner, R.T.; Mah, J.K.; Igarashi, Masanori; Abdel-Hamid, Hoda; Pestronk, Alan; Dubroski, Alberto; Ryan, Monique; Kaminski, Sarah; Bartczak, Marisa; Parker, Katherine; Duong, Tina; Thannhauser, Jennifer; Goia, Edit; Chiu, Angela; Caton, Megan; Rashed, Hani; Feliciano, Casandra; Clifft, Judy; Coleman, Ann; Bise, Christopher; Paulukonis, Kara; Wulf, Charlie; Renna, Renee; Malkus, Betsy; Siener, Catherine; Corderi, Jose; Capone, Luca; Ferretti, Marco; Villano, Dani; Carroll, Kate; Kennedy, Rachel; Kennedy, Cam; Chen, Lucia; Peterson, Wendy Korn; Coleman-Wood, Krista; Kotakarvi, Brian; Yaffe, Debbie; Weisband, Elana

    2012-01-01

    Objective: To determine whether pentoxifylline (PTX) slows the decline of muscle strength and function in ambulatory boys with Duchenne muscular dystrophy (DMD). Methods: This was a multicenter, randomized, double-blinded, controlled trial comparing 12 months of daily treatment with PTX or placebo in corticosteroid-treated boys with DMD using a slow-release PTX formulation (∼20 mg/kg/day). The primary outcome was the change in mean total quantitative muscle testing (QMT) score. Secondary outcomes included changes in QMT subscales, manual muscle strength, pulmonary function, and timed function tests. Outcomes were compared using Student t tests and a linear mixed-effects model. Adverse events (AEs) were compared using the Fisher exact test. Results: A total of 64 boys with DMD with a mean age of 9.9 ± 2.9 years were randomly assigned to PTX or placebo in 11 participating Cooperative International Neuromuscular Research Group centers. There was no significant difference between PTX and the placebo group in total QMT scores (p = 0.14) or in most of the secondary outcomes after a 12-month treatment. The use of PTX was associated with mild to moderate gastrointestinal or hematologic AEs. Conclusion: The addition of PTX to corticosteroid-treated boys with DMD at a moderate to late ambulatory stage of disease did not improve or halt the deterioration of muscle strength and function over a 12-month study period. Classification of evidence: This study provides Class I evidence that treatment with PTX does not prevent deterioration in muscle function or strength in corticosteroid-treated boys with DMD. PMID:22402864

  12. Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition.

    PubMed Central

    Tuddenham, E G; Schwaab, R; Seehafer, J; Millar, D S; Gitschier, J; Higuchi, M; Bidichandani, S; Connor, J M; Hoyer, L W; Yoshioka, A

    1994-01-01

    A large number of different mutations in the factor VIII (F8) gene have been identified as a cause of haemophilia A. This compilation lists known single base-pair substitutions, deletions and insertions in the F8 gene and reviews the status of the inversional events which account for a substantial proportion of mutations causing severe haemophilia A. PMID:7984443

  13. Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition.

    PubMed Central

    Tuddenham, E G; Schwaab, R; Seehafer, J; Millar, D S; Gitschier, J; Higuchi, M; Bidichandani, S; Connor, J M; Hoyer, L W; Yoshioka, A

    1994-01-01

    A large number of different mutations in the factor VIII (F8) gene have been identified as a cause of haemophilia A. This compilation lists known single base-pair substitutions, deletions and insertions in the F8 gene and reviews the status of the inversional events which account for a substantial proportion of mutations causing severe haemophilia A. PMID:7937051

  14. Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Δ48-50 DMD cells

    PubMed Central

    De Angelis, Fernanda Gabriella; Sthandier, Olga; Berarducci, Barbara; Toso, Silvia; Galluzzi, Giuliana; Ricci, Enzo; Cossu, Giulio; Bozzoni, Irene

    2002-01-01

    Deletions and point mutations in the dystrophin gene cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy, depending on whether the translational reading frame is lost or maintained. Because internal in-frame deletions in the protein produce only mild myopathic symptoms, it should be possible, by preventing the inclusion of specific mutated exon(s) in the mature dystrophin mRNA, to restore a partially corrected phenotype. Such control has been previously accomplished by the use of synthetic oligonucleotides; nevertheless, a significant drawback to this approach is caused by the fact that oligonucleotides would require periodic administrations. To circumvent this problem, we have produced several constructs able to express in vivo, in a stable fashion, large amounts of chimeric RNAs containing antisense sequences. In this paper we show that antisense molecules against exon 51 splice junctions are able to direct skipping of this exon in the human DMD deletion 48–50 and to rescue dystrophin synthesis. We also show that the highest skipping activity was found when antisense constructs against the 5′ and 3′ splice sites are coexpressed in the same cell. PMID:12077324

  15. A Population of Deletion Mutants and an Integrated Mapping and Exome-seq Pipeline for Gene Discovery in Maize

    PubMed Central

    Jia, Shangang; Li, Aixia; Morton, Kyla; Avoles-Kianian, Penny; Kianian, Shahryar F.; Zhang, Chi; Holding, David

    2016-01-01

    To better understand maize endosperm filling and maturation, we used γ-irradiation of the B73 maize reference line to generate mutants with opaque endosperm and reduced kernel fill phenotypes, and created a population of 1788 lines including 39 Mo17 × F2s showing stable, segregating, and viable kernel phenotypes. For molecular characterization of the mutants, we developed a novel functional genomics platform that combined bulked segregant RNA and exome sequencing (BSREx-seq) to map causative mutations and identify candidate genes within mapping intervals. To exemplify the utility of the mutants and provide proof-of-concept for the bioinformatics platform, we present detailed characterization of line 937, an opaque mutant harboring a 6203 bp in-frame deletion covering six exons within the Opaque-1 gene. In addition, we describe mutant line 146 which contains a 4.8 kb intragene deletion within the Sugary-1 gene and line 916 in which an 8.6 kb deletion knocks out a Cyclin A2 gene. The publically available algorithm developed in this work improves the identification of causative deletions and its corresponding gaps within mapping peaks. This study demonstrates the utility of γ-irradiation for forward genetics in large nondense genomes such as maize since deletions often affect single genes. Furthermore, we show how this classical mutagenesis method becomes applicable for functional genomics when combined with state-of-the-art genomics tools. PMID:27261000

  16. Dopamine D2 receptor gene -141C Insertion/Deletion polymorphism in Turkish schizophrenic patients.

    PubMed

    Kurt, Hulyam; Dikmen, Miris; Basaran, Ayşe; Yenilmez, Cinar; Ozdemir, Figen; Degirmenci, Irfan; Gunes, Hasan Veysi; Kucuk, Meral Urhan; Mutlu, Fezan

    2011-02-01

    Schizophrenia is a chronic and neuropsychiatric disease that affects about 0.5-1% of the world's population. An increase in dopamine and dopamine D2 receptor (DRD2) gene products has been well described in schizophrenic patients. Several groups have studied the relationship between dopaminergic hyperactivity and cellular communications have obtained discordant results. Studies searching for the relationship between the schizophrenia and DRD2 gene have gained more interest. Our objective was to determine the relationships among schizophrenic symptoms in schizophrenia subtypes and severity of symptoms in terms of DRD2 gene -141C Insertion/Deletion [Ins/Del; I/D] polymorphism by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) assay method. Genomic DNA was prepared from peripheral blood by using salt extraction method. After amplification of genomic DNA, PCR products were digested with BstNI restriction enzyme for the detection of DRD2 gene -141C Ins/Del polymorphism in 73 schizophrenic patients and 60 healthy control subjects. The allelic frequencies of the DRD2 gene -141C Ins/Del polymorphism in case and control groups were 79.5 and 77.5% for I allele; 20.5 and 22.5% for D allele respectively. There was no significant difference in frequencies of genotypes and alleles between the two groups. In schizophrenic and control subjects, there were no significant relationship in severity of the disease and schizophrenia types among the -141C Ins/Del genotypes and alleles.

  17. Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene

    SciTech Connect

    Karl, M.; Lamberts, S.W.J.; Detera-Wadleigh, S.D.; Encio, I.J.; Stratakis, C.A.; Hurley, D.M.; Accili, D.; Chrousos, G.P. Erasmus Univ. of Rotterdam )

    1993-03-01

    The clinical syndrome of generalized, compensated glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hyper- or hypocortisolism, and manifestations of androgen and/or mineralocorticoid excess. This condition results from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. The authors studied the molecular mechanisms of this syndrome in a Dutch kindred, whose affected members had hypercortisolism and approximately half of normal GRs, and whose proband was a young woman with manifestations of hyperandrogenism. Using the polymerase chain reaction to amplify and sequence each of the nine exons of the GR gene [alpha], along with their 5[prime]- and 3[prime]-flanking regions, the authors identified a 4-base deletion at the 3[prime]-boundary of exon 6 in one GR allele ([Delta][sub 4]), which removed a donor splice site in all three affected members studied. In contrast, the sequence of exon 6 in the two unaffected siblings was normal. A single nucleotide substitution causing an amino acid substitution in the amino terminal domain of the GR (asparagine to serine, codon 363) was also discovered in exon 2 of the other allele (G[sub 1220]) in the proband, in one of her affected brothers and in her unaffected sister. This deletion in the glucocorticoid receptor gene was associated with the expression of only one allele and a decrease of GR protein by 50% in affected members of this glucocorticoid resistant family. The mutation identified in exon 2 did not segregate with the disease and appears to be of no functional significance. The presence of the null allele was apparently compensated for by increased cortisol production at the expense of concurrent hyperandrogenism. 40 refs., 3 figs.

  18. A Next-generation Genetically Attenuated Plasmodium falciparum Parasite Created by Triple Gene Deletion

    PubMed Central

    Mikolajczak, Sebastian A; Lakshmanan, Viswanathan; Fishbaugher, Matthew; Camargo, Nelly; Harupa, Anke; Kaushansky, Alexis; Douglass, Alyse N; Baldwin, Michael; Healer, Julie; O'Neill, Matthew; Phuong, Thuan; Cowman, Alan; Kappe, Stefan H I

    2014-01-01

    Immunization with live-attenuated Plasmodium sporozoites completely protects against malaria infection. Genetic engineering offers a versatile platform to create live-attenuated sporozoite vaccine candidates. We previously generated a genetically attenuated parasite (GAP) by deleting the P52 and P36 genes in the NF54 wild-type (WT) strain of Plasmodium falciparum (Pf p52−/p36− GAP). Preclinical assessment of p52−/p36− GAP in a humanized mouse model indicated an early and severe liver stage growth defect. However, human exposure to >200 Pf p52−/p36− GAP-infected mosquito bites in a safety trial resulted in peripheral parasitemia in one of six volunteers, revealing that this GAP was incompletely attenuated. We have now created a triple gene deleted GAP by additionally removing the SAP1 gene (Pf p52−/p36−/sap1− GAP) and employed flippase (FLP)/flippase recognition target (FRT) recombination for drug selectable marker cassette removal. This next-generation GAP was indistinguishable from WT parasites in blood stage and mosquito stage development. Using an improved humanized mouse model transplanted with human hepatocytes and human red blood cells, we show that despite a high-dose sporozoite challenge, Pf p52−/p36−/sap1− GAP did not transition to blood stage infection and appeared to be completely attenuated. Thus, clinical testing of Pf p52−/p36−/sap1− GAP assessing safety, immunogenicity, and efficacy against sporozoite challenge is warranted. PMID:24827907

  19. Deletion of psychiatric risk gene Cacna1c impairs hippocampal neurogenesis in cell-autonomous fashion.

    PubMed

    Völkening, Bianca; Schönig, Kai; Kronenberg, Golo; Bartsch, Dusan; Weber, Tillmann

    2017-05-01

    Ca(2+) is a universal signal transducer which fulfills essential functions in cell development and differentiation. CACNA1C, the gene encoding the alpha-1C subunit (i.e., Cav 1.2) of the voltage-dependent l-type calcium channel (LTCC), has been implicated as a risk gene in a variety of neuropsychiatric disorders. To parse the role of Cav 1.2 channels located on astrocyte-like stem cells and their descendants in the development of new granule neurons, we created Tg(GLAST-CreERT2) /Cacna1c(fl/fl) /RCE:loxP mice, a transgenic tool that allows cell-type-specific inducible deletion of Cacna1c. The EGFP reporter was used to trace the progeny of recombined type-1 cells. FACS-sorted Cacna1c-deficient neural precursor cells from the dentate gyrus showed reduced proliferative activity in neurosphere cultures. Moreover, under differentiation conditions, Cacna1c-deficient NPCs gave rise to fewer neurons and more astroglia. Similarly, under basal conditions in vivo, Cacna1c gene deletion in type-1 cells decreased type-1 cell proliferation and reduced the neuronal fate-choice decision of newly born cells, resulting in reduced net hippocampal neurogenesis. Unexpectedly, electroconvulsive seizures completely compensated for the proliferation deficit of Cacna1c deficient type-1 cells, indicating that there must be Cav 1.2-independent mechanisms of controlling proliferation related to excitation. In the aggregate, this is the first report demonstrating the presence of functional L-type 1.2 channels on type-1 cells. Cav 1.2 channels promote type-1 cell proliferation and push the glia-to-neuron ratio in the direction of a neuronal fate choice and subsequent neuronal differentiation. Cav 1.2 channels expressed on NPCs and their progeny possess the ability to shape neurogenesis in a cell-autonomous fashion.

  20. Study of six patients with complete F9 deletion characterized by cytogenetic microarray: role of the SOX3 gene in intellectual disability.

    PubMed

    Jourdy, Y; Chatron, N; Carage, M-L; Fretigny, M; Meunier, S; Zawadzki, C; Gay, V; Negrier, C; Sanlaville, D; Vinciguerra, C

    2016-10-01

    Essentials Some hemophilia B (HB) patients with complete F9 deletion present with intellectual disability (ID). We delineate six F9 complete deletions and investigate genotype/phenotype correlation. We identify SOX3 as a candidate gene for ID, acting through haploinsufficiency, in HB patients. All complete F9 deletions in ID patients should be explored with cytogenetic microarrays.

  1. Deletion of the BCSP31 gene of Brucella abortus by replacement.

    PubMed Central

    Halling, S M; Detilleux, P G; Tatum, F M; Judge, B A; Mayfield, J E

    1991-01-01

    The 31-kDa salt-extractable immunogenic protein, BCSP31, was deleted from several Brucella abortus strains by replacement with a marker gene encoding resistance to the antibiotics kanamycin and neomycin. The BCSP31 gene replacement plasmids, constructed with ColE1-derived vectors, were introduced by electroporation into B. abortus strain 19 (S19), into a rough variant of B. abortus S19, and into B. abortus S2308, and antibiotic-resistant transformants were isolated. B. abortus S19 is an attenuated strain used as a vaccine for prevention of bovine brucellosis in the United States, and B. abortus S2308 is a commonly used challenge strain. The antibiotic-resistant isolates were all obtained by recombination; none were spontaneous mutants. Loss of the gene encoding BCSP31 and presence of the marker gene were confirmed by Southern analysis. Vector sequences were either absent or linked to the genome, indicating that ColE1-derived plasmids are not maintained in B. abortus. Survival of B. abortus mutant strains in the macrophagelike cell line J774 and in HeLa cells was examined and shown to be indistinguishable from that of the parental strain. Images PMID:1937745

  2. Deletion of Rictor in brain and fat alters peripheral clock gene expression and increases blood pressure.

    PubMed

    Drägert, Katja; Bhattacharya, Indranil; Pellegrini, Giovanni; Seebeck, Petra; Azzi, Abdelhalim; Brown, Steven A; Georgiopoulou, Stavroula; Held, Ulrike; Blyszczuk, Przemyslaw; Arras, Margarete; Humar, Rok; Hall, Michael N; Battegay, Edouard; Haas, Elvira

    2015-08-01

    The mammalian target of rapamycin complex 2 (mTORC2) contains the essential protein RICTOR and is activated by growth factors. mTORC2 in adipose tissue contributes to the regulation of glucose and lipid metabolism. In the perivascular adipose tissue, mTORC2 ensures normal vascular reactivity by controlling expression of inflammatory molecules. To assess whether RICTOR/mTORC2 contributes to blood pressure regulation, we applied a radiotelemetry approach in control and Rictor knockout (Rictor(aP2KO)) mice generated using adipocyte protein-2 gene promoter-driven CRE recombinase expression to delete Rictor. The 24-hour mean arterial pressure was increased in Rictor(aP2KO) mice, and the physiological decline in mean arterial pressure during the dark period was impaired. In parallel, heart rate and locomotor activity were elevated during the dark period with a pattern similar to blood pressure changes. This phenotype was associated with mild cardiomyocyte hypertrophy, decreased cardiac natriuretic peptides, and their receptor expression in adipocytes. Moreover, clock gene expression was reduced or phase-shifted in perivascular adipose tissue. No differences in clock gene expression were observed in the master clock suprachiasmatic nucleus, although Rictor gene expression was also lower in brain of Rictor(aP2KO) mice. Thus, this study highlights the importance of RICTOR/mTORC2 for interactions between vasculature, adipocytes, and brain to tune physiological outcomes, such as blood pressure and locomotor activity.

  3. Sensitivity of hematopoietic stem cells to mitochondrial dysfunction by SdhD gene deletion

    PubMed Central

    Bejarano-García, José Antonio; Millán-Uclés, África; Rosado, Iván V; Sánchez-Abarca, Luís Ignacio; Caballero-Velázquez, Teresa; Durán-Galván, María José; Pérez-Simón, José Antonio; Piruat, José I

    2016-01-01

    It is established that hematopoietic stem cells (HSC) in the hypoxic bone marrow have adapted their metabolism to oxygen-limiting conditions. This adaptation includes suppression of mitochondrial activity, induction of anerobic glycolysis, and activation of hypoxia-inducible transcription factor 1α (Hif1α)-dependent gene expression. During progression of hematopoiesis, a metabolic switch towards mitochondrial oxidative phosphorylation is observed, making this organelle essential for determining cell fate choice in bone marrow. However, given that HSC metabolism is essentially oxygen-independent, it is still unclear whether functional mitochondria are absolutely required for their survival. To assess the actual dependency of these undifferentiated cells on mitochondrial function, we have performed an analysis of the hematopoiesis in a mouse mutant, named SDHD-ESR, with inducible deletion of the mitochondrial protein-encoding SdhD gene. This gene encodes one of the subunits of the mitochondrial complex II (MCII). In this study, we demonstrate that, in contrast to what has been previously established, survival of HSC, and also myeloid and B-lymphoid progenitors, depends on proper mitochondrial activity. In addition, gene expression analysis of these hematopoietic lineages in SDHD-ESR mutants calls into question the proposed activation of Hif1α in response to MCII dysfunction. PMID:27929539

  4. Overlapping submicroscopic deletions in Xq28 in two unrelated boys with developmental disorders: Identification of a gene near FRAXE

    SciTech Connect

    Gedeon, A.K.; Sutherland, G.R. |; Ades, L.C.; Gecz, J.; Baker, E.; Mulley, J.C.; Keinaenen, M.; Kaeaeriaeinen, H.

    1995-04-01

    Two unrelated boys are described with delay in development and submicroscopic deletions in Xq28, near FRAXE. Molecular diagnosis to exclude the fragile X (FRAXA) syndrome used the direct probe pfxa3, together with a control probe pS8 (DXS296), against PstI restriction digests of DNA. Deletions were detected initially by the control probe pS8, which is an anonymous fragment subcloned from YAC 539, within 1 Mb distal to FRAXA. Further molecular analyses determined that the maximum size of the deletion is <100 kb in one boy (MK) and is wholly overlapped by the deletion of up to {approximately}200 kb in the other (CB). These deletions lie between the sequences detected by the probe VK21C (DXS296) and a dinucleotide repeat VK18AC (DXS295). The patient MK had only speech delay with otherwise normal development, while patient CB had global developmental delay that included speech delay. Detection of overlapping deletions in these two cases led to speculation that coding sequences of a gene(s) important in language development may be affected. Hybridization of the pS8 and VK21A probes to zooblots revealed cross-species homology. This conservation during evolution suggested that this region contains sequences with functional significance in normal development. The VK21A probe detected a 9.5-kb transcript in placenta and brain and a smaller, 2.5-kb, transcript in other tissues analyzed. 26 refs., 6 figs.

  5. Deletion and methylation of the tumour suppressor gene p16/CDKN2 in primary head and neck squamous cell carcinoma.

    PubMed Central

    González, M V; Pello, M F; López-Larrea, C; Suárez, C; Menéndez, M J; Coto, E

    1997-01-01

    AIMS: To study the homozygous deletion and methylation status of the 5' CpG island of the p16 and p15 genes (9p21) in a set of primary advanced head and neck squamous cell carcinomas (SCC) and to test whether inactivation of these genes by these mechanisms contributes to head and neck SCC development. METHODS: DNA was extracted from fresh tumours. Homozygous deletion was determined by the polymerase chain reaction (PCR) followed by hybridisation with the corresponding probe, radioactively labelled by the random priming method. Methylation status of the CpG island of the 5' region of these genes was assessed by digestion with the appropriate restriction enzymes followed by PCR and subsequent hybridisation with the corresponding probe. The presence of point mutations was determined by PCR-SSCP (single strand conformation polymorphism). RESULTS: The p16 and p15 genes were homozygously deleted in 20% and 10% of the tumours, respectively. No point mutations were found at p16 and p15. The 5' CpG island at the p16 gene was methylated in 20% of the cases. CONCLUSIONS: The tumour suppressor gene p16 is inactivated through homozygous deletion or methylation in a significant proportion of cases of head and neck SCC. Images PMID:9378820

  6. Color hologram reconstruction based on single DMD

    NASA Astrophysics Data System (ADS)

    Xing, Jiang; Zhou, Hao; Wu, Dan; Hou, Jun-jian; Gu, Ji-hua

    2016-09-01

    Because of the magnification chromatic aberration and the transverse chromatic aberration caused from different wavelengths of color lasers in the process of color holographic optoelectronic reconstruction based on DMD, the reconstructed holograms of three color components can not coincide. Firstly, on the reference of blue color component, the magnification chromatic aberration of the original image is eliminated. Secondly, according to the analysis of the incident angles of three lasers, the transverse chromatic aberration is eliminated by adjusting the incident angles. At last, the synthesized color hologram is obtained by means of the experiments based on DMD. The method proposed in this paper does not use any lens, so there is no axial chromatic aberration.

  7. A deletion in the bovine FANCI gene compromises fertility by causing fetal death and brachyspina.

    PubMed

    Charlier, Carole; Agerholm, Jorgen Steen; Coppieters, Wouter; Karlskov-Mortensen, Peter; Li, Wanbo; de Jong, Gerben; Fasquelle, Corinne; Karim, Latifa; Cirera, Susanna; Cambisano, Nadine; Ahariz, Naima; Mullaart, Erik; Georges, Michel; Fredholm, Merete

    2012-01-01

    Fertility is one of the most important traits in dairy cattle, and has been steadily declining over the last decades. We herein use state-of-the-art genomic tools, including high-throughput SNP genotyping and next-generation sequencing, to identify a 3.3 Kb deletion in the FANCI gene causing the brachyspina syndrome (BS), a rare recessive genetic defect in Holstein dairy cattle. We determine that despite the very low incidence of BS (<1/100,000), carrier frequency is as high as 7.4% in the Holstein breed. We demonstrate that this apparent discrepancy is likely due to the fact that a large proportion of homozygous mutant calves die during pregnancy. We postulate that several other embryonic lethals may segregate in livestock and significantly compromise fertility, and propose a genotype-driven screening strategy to detect the corresponding deleterious mutations.

  8. Delta 32 deletion of CCR5 gene and association with asthma or atopy.

    PubMed

    Mitchell, T J; Walley, A J; Pease, J E; Venables, P J; Wiltshire, S; Williams, T J; Cookson, W O

    2000-10-28

    The CCR5-delta32 deletion polymorphism (CCR5-delta32) was investigated for linkage and association to asthma and atopy using two panels of nuclear families containing 1284 individuals. No statistically significant linkage to asthma/wheeze or atopy was observed in either of the two panels of families. Multiallelic transmission disequilibrium tests (TDT) of the combined data found no significant association for atopy (52 independent alleles transmitted, 51 non-transmitted) or asthma/wheeze (39 transmitted, 44 non-transmitted). Although functional evidence might suggest that CCR5 is a good candidate gene for atopic asthma, this study provides no genetic evidence from CCR5-delta32 polymorphism to support this hypothesis.

  9. Sensitive detection of deletions of one or more exons in the neurofibromatosis type 2 (NF2) gene by multiplexed gene dosage polymerase chain reaction.

    PubMed

    Diebold, Ruth; Bartelt-Kirbach, Britta; Evans, D Gareth; Kaufmann, Dieter; Hanemann, C Oliver

    2005-02-01

    Mutation detection in the neurofibromatosis type 2 (NF2) gene is challenging because when combining mutation detection methods such as single-strand conformational polymorphism and heteroduplex analysis, denaturing gradient gel electrophoresis, and direct sequencing of aberrant polymerase chain reaction (PCR) fragments only 30 to 60% of the constitutional mutations are detected. Because large deletions and complete chromosome rearrangements are also described methods such as microarray-comparative genomic hybridization and fluorescence in situ hybridization are also used. The one type of mutation often missed corresponds to deletions encompassing one or few exons. To detect this type we have developed a swift and reliable method. We perform a gene dosage analysis with two fluorescent multiplex PCR assays that amplify 15 of the 17 NF2 exons. The labeled PCR products are quantified and gene dose is calculated with respect to controls. We tested the reliability of this method with DNA from eight NF2 patients with known heterozygous NF2 deletions, eight controls and four unknown NF2 patients. In all of the patients with known heterozygous deletions we found in several exons a reduction of gene dosage to 50 to 69%. In one NF2 patient with previously unknown mutation and a severe phenotype we found the gene dosage of two exons reduced by 50% indicating a deletion of these two exons on one allele. This finding was validated by reverse transcriptase-PCR on fibroblast and schwannoma cell cultures of this patient and cDNA sequencing. Our gene dosage assay will detect deletions of one or more exons as well as gross deletions of the whole coding region of the gene. It can complement the existing screening methods because it is faster and easier.

  10. Following Tetraploidy in Maize, a Short Deletion Mechanism Removed Genes Preferentially from One of the Two Homeologs

    PubMed Central

    Pedersen, Brent S.; Lyons, Eric; Lisch, Damon; Subramaniam, Shabarinath; Freeling, Michael

    2010-01-01

    Previous work in Arabidopsis showed that after an ancient tetraploidy event, genes were preferentially removed from one of the two homeologs, a process known as fractionation. The mechanism of fractionation is unknown. We sought to determine whether such preferential, or biased, fractionation exists in maize and, if so, whether a specific mechanism could be implicated in this process. We studied the process of fractionation using two recently sequenced grass species: sorghum and maize. The maize lineage has experienced a tetraploidy since its divergence from sorghum approximately 12 million years ago, and fragments of many knocked-out genes retain enough sequence similarity to be easily identifiable. Using sorghum exons as the query sequence, we studied the fate of both orthologous genes in maize following the maize tetraploidy. We show that genes are predominantly lost, not relocated, and that single-gene loss by deletion is the rule. Based on comparisons with orthologous sorghum and rice genes, we also infer that the sequences present before the deletion events were flanked by short direct repeats, a signature of intra-chromosomal recombination. Evidence of this deletion mechanism is found 2.3 times more frequently on one of the maize homeologs, consistent with earlier observations of biased fractionation. The over-fractionated homeolog is also a greater than 3-fold better target for transposon removal, but does not have an observably higher synonymous base substitution rate, nor could we find differentially placed methylation domains. We conclude that fractionation is indeed biased in maize and that intra-chromosomal or possibly a similar illegitimate recombination is the primary mechanism by which fractionation occurs. The mechanism of intra-chromosomal recombination explains the observed bias in both gene and transposon loss in the maize lineage. The existence of fractionation bias demonstrates that the frequency of deletion is modulated. Among the

  11. Deletions of multidrug resistance gene loci in breast cancer leads to the down-regulation of its expression and predict tumor response to neoadjuvant chemotherapy

    PubMed Central

    Litviakov, Nikolai V.; Cherdyntseva, Nadezhda V.; Tsyganov, Matvey M.; Slonimskaya, Elena M.; Ibragimova, Marina K.; Kazantseva, Polina V.; Kzhyshkowska, Julia; Choinzonov, Eugeniy L.

    2016-01-01

    Neoadjuvant chemotherapy (NAC) is intensively used for the treatment of primary breast cancer. In our previous studies, we reported that clinical tumor response to NAC is associated with the change of multidrug resistance (MDR) gene expression in tumors after chemotherapy. In this study we performed a combined analysis of MDR gene locus deletions in tumor DNA, MDR gene expression and clinical response to NAC in 73 BC patients. Copy number variations (CNVs) in biopsy specimens were tested using high-density microarray platform CytoScanTM HD Array (Affymetrix, USA). 75%–100% persons having deletions of MDR gene loci demonstrated the down-regulation of MDR gene expression. Expression of MDR genes was 2–8 times lower in patients with deletion than in patients having no deletion only in post-NAC tumors samples but not in tumor tissue before chemotherapy. All patients with deletions of ABCB1 ABCB 3 ABCC5 gene loci – 7q21.1, 6p21.32, 3q27 correspondingly, and most patients having deletions in ABCC1 (16p13.1), ABCC2 (10q24), ABCG1 (21q22.3), ABCG2 (4q22.1), responded favorably to NAC. The analysis of all CNVs, including both amplification and deletion showed that the frequency of 13q14.2 deletion was 85% among patients bearing tumor with the deletion at least in one MDR gene locus versus 9% in patients with no deletions. Differences in the frequency of 13q14.2 deletions between the two groups were statistically significant (p = 2.03 ×10−11, Fisher test, Bonferroni-adjusted p = 1.73 × 10−8). In conclusion, our study for the first time demonstrates that deletion MDR gene loci can be used as predictive marker for tumor response to NAC. PMID:26799285

  12. Deletion of exon 3 of the insulin receptor gene in a kindred with a familial form of insulin resistance

    SciTech Connect

    Wertheimer, E.; Barbetti, F.; Accili, D.; Taylor, S.I.; Litvin, Y.; Ebstein, R.P.; Bennet, E.R.

    1994-05-01

    Molecular scanning techniques, such as denaturing gradient gel electrophoresis (DGGE), greatly facilitate screening candidate genes for mutations. The authors have used DGGE to screen for mutations in the insulin receptor gene in a family in which four of five daughters were affected by type A insulin resistance in association with acanthosis nigricans and hyperandrogenism. DGGE did not detect mutations in any of the 22 exons of the insulin receptor gene. Nevertheless, Southern blot analysis suggested that there was a deletion of exon 3 in the other paternal allele of the insulin receptor gene. Analysis of the father`s cDNA confirmed that exon 3 was deleted from mRNA molecules derived from one of his two alleles of the insulin receptor gene. Furthermore, the father was found to be hemizygous for a polymorphic sequence (GAC{sup Asp} at codon 234) in exon 3 that was not inherited by any of the five daughters. Instead, all five daughters inherited the paternal allele with the deletion mutation. They did not detect mutations in the mother`s insulin receptor gene. Furthermore, the clinical syndrome did not segregate with either of the mother`s two alleles of the insulin receptor gene. Although the youngest daughter inherited the mutant allele from her father, she was not clinically affected. The explanation for the incomplete penetrance is not known. These results emphasize the importance of specifically searching for deletion mutations when screening candidate genes for mutations. Furthermore, the existence of apparently asymptomatic carriers of mutations in the insulin receptor gene, such as the father in the present study, suggests that the prevalence of mutations in the insulin receptor gene may be higher than would be predicted on the basis of the observed prevalence of patients with extreme insulin resistance. 34 refs., 6 figs., 1 tab.

  13. Analysis of crossover breakpoints yields new insights into the nature of the gene conversion events associated with large NF1 deletions mediated by nonallelic homologous recombination.

    PubMed

    Bengesser, Kathrin; Vogt, Julia; Mussotter, Tanja; Mautner, Victor-Felix; Messiaen, Ludwine; Cooper, David N; Kehrer-Sawatzki, Hildegard

    2014-02-01

    Large NF1 deletions are mediated by nonallelic homologous recombination (NAHR). An in-depth analysis of gene conversion operating in the breakpoint-flanking regions of large NF1 deletions was performed to investigate whether the rate of discontinuous gene conversion during NAHR with crossover is increased, as has been previously noted in NAHR-mediated rearrangements. All 20 germline type-1 NF1 deletions analyzed were mediated by NAHR associated with continuous gene conversion within the breakpoint-flanking regions. Continuous gene conversion was also observed in 31/32 type-2 NF1 deletions investigated. In contrast to the meiotic type-1 NF1 deletions, type-2 NF1 deletions are predominantly of post-zygotic origin. Our findings therefore imply that the mitotic as well as the meiotic NAHR intermediates of large NF1 deletions are processed by long-patch mismatch repair (MMR), thereby ensuring gene conversion tract continuity instead of the discontinuous gene conversion that is characteristic of short-patch repair. However, the single type-2 NF1 deletion not exhibiting continuous gene conversion was processed without MMR, yielding two different deletion-bearing chromosomes, which were distinguishable in terms of their breakpoint positions. Our findings indicate that MMR failure during NAHR, followed by post-meiotic/mitotic segregation, has the potential to give rise to somatic mosaicism in human genomic rearrangements by generating breakpoint heterogeneity.

  14. A large AZFc deletion removes DAZ3/DAZ4 and nearby genes from men in Y haplogroup N.

    PubMed

    Fernandes, S; Paracchini, S; Meyer, L H; Floridia, G; Tyler-Smith, C; Vogt, P H

    2004-01-01

    Deletion of the entire AZFc locus on the human Y chromosome leads to male infertility. The functional roles of the individual gene families mapped to AZFc are, however, still poorly understood, since the analysis of the region is complicated by its repeated structure. We have therefore used single-nucleotide variants (SNVs) across approximately 3 Mb of the AZFc sequence to identify 17 AZFc haplotypes and have examined them for deletion of individual AZFc gene copies. We found five individuals who lacked SNVs from a large segment of DNA containing the DAZ3/DAZ4 and BPY2.2/BPY2.3 gene doublets in distal AZFc. Southern blot analyses showed that the lack of these SNVs was due to deletion of the underlying DNA segment. Typing 118 binary Y markers showed that all five individuals belonged to Y haplogroup N, and 15 of 15 independently ascertained men in haplogroup N carried a similar deletion. Haplogroup N is known to be common and widespread in Europe and Asia, and there is no indication of reduced fertility in men with this Y chromosome. We therefore conclude that a common variant of the human Y chromosome lacks the DAZ3/DAZ4 and BPY2.2/BPY2.3 doublets in distal AZFc and thus that these genes cannot be required for male fertility; the gene content of the AZFc locus is likely to be genetically redundant. Furthermore, the observed deletions cannot be derived from the GenBank reference sequence by a single recombination event; an origin by homologous recombination from such a sequence organization must be preceded by an inversion event. These data confirm the expectation that the human Y chromosome sequence and gene complement may differ substantially between individuals and more variations are to be expected in different Y chromosomal haplogroups.

  15. A self-excising beta-recombinase/six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a previous study we developed a cassette employing a bacterial beta-recombinase acting on six recognition sequences (beta-rec/six), which allowed repetitive site-specific gene deletion and marker recycling in Neurospora crassa. However, only one positive selection marker was used in the cassette...

  16. Robust Parameter Identification to Perform the Modeling of pta and poxB Genes Deletion Effect on Escherichia Coli.

    PubMed

    Guerrero-Torres, V; Rios-Lozano, M; Badillo-Corona, J A; Chairez, I; Garibay-Orijel, C

    2016-08-01

    The aim of this study was to design a robust parameter identification algorithm to characterize the effect of gene deletion on Escherichia coli (E. coli) MG1655. Two genes (pta and poxB) in the competitive pathways were deleted from this microorganism to inhibit pyruvate consumption. This condition deviated the E. coli metabolism toward the Krebs cycle. As a consequence, the biomass, substrate (glucose), lactic, and acetate acids as well as ethanol concentrations were modified. A hybrid model was proposed to consider the effect of gene deletion on the metabolism of E. coli. The model parameters were estimated by the application of a least mean square method based on the instrument variable technique. To evaluate the parametric identifier method, a set of robust exact differentiators, based on the super-twisting algorithm, was implemented. The hybrid model was successfully characterized by the parameters obtained from experimental information of E. coli MG1655. The significant difference between parameters obtained with wild-type strain and the modified (with deleted genes) justifies the application of the parametric identification algorithm. This characterization can be used to optimize the production of different byproducts of commercial interest.

  17. DDX3Y gene rescue of a Y chromosome AZFa deletion restores germ cell formation and transcriptional programs

    PubMed Central

    Ramathal, Cyril; Angulo, Benjamin; Sukhwani, Meena; Cui, Jun; Durruthy-Durruthy, Jens; Fang, Fang; Schanes, Paula; Turek, Paul J.; Orwig, Kyle E.; Reijo Pera, Renee

    2015-01-01

    Deletions of the AZFa region (AZoospermia Factor-a) region of the human Y chromosome cause irreversible spermatogenic failure that presents clinically in men as Sertoli-cell only (SCO) pathology of the testis. Deletions of the AZFa region typically encompass two genes: DDX3Y and USP9Y. However, human genetic evidence indicates that SCO is most tightly linked to deletion of DDX3Y and that deletions/mutations of USP9Y can be transmitted from one generation to the next. Here, we generated stable iPSC lines with AZFa deletions, tested complementation via introduction of DDX3Y, and assessed ability to form germ cells in vivo in a xenotransplantation model. We observed a quantifiable improvement in formation of germ cell like cells (GCLCs) from complemented donor iPSCs. Moreover, expression of UTF1, a prospermatogonial protein, was restored in cells complemented by introduction of DDX3Y on the AZFa background. Whole-genome RNA sequencing of purified GCLCs revealed an enrichment of genes involved in translational suppression and transcriptional control in DDX3Y-rescued GCLCs over mutant GCLCs, which maintained a molecular phenotype more similar to undifferentiated iPSCs. This study demonstrates the ability to probe fundamental genetics of human germ cell formation by complementation and indicates that DDX3Y functions in the earliest stages of human germ cell development. PMID:26456624

  18. Identification of a Novel Deletion in AVP-NPII Gene in a Patient with Central Diabetes Insipidus.

    PubMed

    Deniz, Ferhat; Acar, Ceren; Saglar, Emel; Erdem, Beril; Karaduman, Tugce; Yonem, Arif; Cagiltay, Eylem; Ay, Seyit Ahmet; Mergen, Hatice

    2015-01-01

    Central Diabetes Insipidus (CDI) is caused by a deficiency of antidiuretic hormone and characterized by polyuria, polydipsia and inability to concentrate urine. Our objective was to present the results of the molecular analyses of AVP-neurophysin II (AVP-NPII) gene in a large familial neurohypophyseal (central) DI pedigree. A male patient and his family members were analyzed and the prospective clinical data were collected. The proband applied to hospital for eligibility to be a recruit in Armed Forces. The patient had severe polyuria (20 L/day), polydipsia (20.5 L/day), fatique, and deep thirstiness. CDI was confirmed with the water deprivation-desmopressin test according to an increase in urine osmolality from 162 mOsm/kg to 432 mOsm/kg after desmopressin acetate injection. To evaluate the coding regions of AVP-NPII gene, polymerase chain reactions were performed and amplified regions were submitted to direct sequence analysis. We detected a heterozygous three base pair deletion at codon 69-70 (207_209delGGC) in exon 2, which lead to a deletion of the amino acid alanine. A three-dimensional protein structure prediction was shown for the deleted AVP-NPII and compared with the wild type. The three base pair deletion may yield an abnormal AVP precursor in neurophysin moiety, but further functional analyses are needed to understand the function of the deleted protein.

  19. Increased frequency of DNA deletions in pink-eyed unstable mice carrying a mutation in the Werner syndrome gene homologue.

    PubMed

    Lebel, Michel

    2002-01-01

    Werner syndrome (WS) is a rare autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases, including cancers. Accumulating evidence indicates that the WS gene product is involved in resolving aberrant DNA structures that may arise during the process of DNA replication and/or transcription. To estimate the frequency of DNA deletions directly in the skin of mouse embryos, mice with a deletion of part of the murine WRN helicase domain were created. These mutant mice were then crossed to the pink-eyed unstable animals, which have a 70 kb internal duplication at the pink-eyed dilution (p) gene. This report indicates that the frequency of deletion of the duplicated sequence at the p locus is elevated in mice with a mutation in the WRN allele when compared with wild-type mice. In addition, the inhibitor of topoisomerase I camptothecin also increases the frequency of deletion at the p locus. This frequency is even more elevated in WRN mutant mice treated with camptothecin. In contrast, while the inhibition of poly(ADP-ribose) polymerase (PARP) activity by 3-aminobenzamide increases the frequency of DNA deletion, mutant WRN mice are not significantly more sensitive to the inhibition of PARP activity than wild-type animals.

  20. Deletion of a region that is a candidate for the difference between the deletion forms of hereditary persistence of fetal hemoglobin and deltabeta-thalassemia affects beta- but not gamma-globin gene expression.

    PubMed Central

    Calzolari, R; McMorrow, T; Yannoutsos, N; Langeveld, A; Grosveld, F

    1999-01-01

    The analysis of a number of cases of beta-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the beta-globin locus has led to the identification of several DNA elements that have been implicated in the switch from human fetal gamma- to adult beta-globin gene expression. We have tested this hypothesis for an element that covers the minimal distance between the thalassemia and HPFH deletions and is thought to be responsible for the difference between a deletion HPFH and deltabeta-thalassemia, located 5' of the delta-globin gene. This element has been deleted from a yeast artificial chromosome (YAC) containing the complete human beta-globin locus. Analysis of this modified YAC in transgenic mice shows that early embryonic expression is unaffected, but in the fetal liver it is subject to position effects. In addition, the efficiency of transcription of the beta-globin gene is decreased, but the developmental silencing of the gamma-globin genes is unaffected by the deletion. These results show that the deleted element is involved in the activation of the beta-globin gene perhaps through the loss of a structural function required for gene activation by long-range interactions. PMID:10022837

  1. Large genomic deletions inactivate the BRCA2 gene in breast cancer families

    PubMed Central

    Agata, S; Dalla, P; Callegaro, M; Scaini, M; Menin, C; Ghiotto, C; Nicoletto, O; Zavagno, G; Chieco-Bianchi, L; D'Andrea, E; Montagna, M

    2005-01-01

    Background: BRCA1 and BRCA2 are the two major genes responsible for the breast and ovarian cancers that cluster in families with a genetically determined predisposition. However, regardless of the mutation detection method employed, the percentage of families without identifiable alterations of these genes exceeds 50%, even when applying stringent criteria for family selection. A small but significant increase in mutation detection rate has resulted from the discovery of large genomic alterations in BRCA1. A few studies have addressed the question of whether BRCA2 might be inactivated by the same kinds of alteration, but most were either done on a relatively small number of samples or employed cumbersome mutation detection methods of variable sensitivity. Objective: To analyse 121 highly selected families using the recently available BRCA2 multiplex ligation dependent probe amplification (MLPA) technique. Results: Three different large genomic deletions were identified and confirmed by analysis of the mutant transcript and genomic characterisation of the breakpoints. Conclusions: Contrary to initial suggestions, the presence of BRCA2 genomic rearrangements is worth investigating in high risk breast or ovarian cancer families. PMID:16199546

  2. Deletion in the EVC2 gene causes chondrodysplastic dwarfism in Tyrolean Grey cattle.

    PubMed

    Murgiano, Leonardo; Jagannathan, Vidhya; Benazzi, Cinzia; Bolcato, Marilena; Brunetti, Barbara; Muscatello, Luisa Vera; Dittmer, Keren; Piffer, Christian; Gentile, Arcangelo; Drögemüller, Cord

    2014-01-01

    During the summer of 2013 seven Italian Tyrolean Grey calves were born with abnormally short limbs. Detailed clinical and pathological examination revealed similarities to chondrodysplastic dwarfism. Pedigree analysis showed a common founder, assuming autosomal monogenic recessive transmission of the defective allele. A positional cloning approach combining genome wide association and homozygosity mapping identified a single 1.6 Mb genomic region on BTA 6 that was associated with the disease. Whole genome re-sequencing of an affected calf revealed a single candidate causal mutation in the Ellis van Creveld syndrome 2 (EVC2) gene. This gene is known to be associated with chondrodysplastic dwarfism in Japanese Brown cattle, and dwarfism, abnormal nails and teeth, and dysostosis in humans with Ellis-van Creveld syndrome. Sanger sequencing confirmed the presence of a 2 bp deletion in exon 19 (c.2993_2994ACdel) that led to a premature stop codon in the coding sequence of bovine EVC2, and was concordant with the recessive pattern of inheritance in affected and carrier animals. This loss of function mutation confirms the important role of EVC2 in bone development. Genetic testing can now be used to eliminate this form of chondrodysplastic dwarfism from Tyrolean Grey cattle.

  3. Autosomal recessive hypercholesterolemia in Spanish kindred due to a large deletion in the ARH gene.

    PubMed

    Quagliarini, Fabiana; Vallvé, Joan-Carles; Campagna, Filomena; Alvaro, Adriana; Fuentes-Jimenez, Francisco José; Sirinian, Maria Isabella; Meloni, Francesca; Masana, Luis; Arca, Marcello

    2007-11-01

    Autosomal recessive hypercholesterolemia (ARH) is a rare genetic defect that causes marked elevation of plasma low-density lipoprotein cholesterol (LDL-C) and premature atherosclerosis. It is due to mutations in the ARH gene that plays a critical role in the internalization of LDL receptor (LDLR) in liver cells. We describe a Spanish family where a 24-year-old proband and his 13-year-old sister showed the typical characteristics of ARH. The proband's LDLR activity in peripheral lymphocytes was 14% of normal and his in vivo LDL catabolism was reduced by 64% compared to normal. Notably, the sister showed normal lipid levels when her umbilical cord blood was tested. In this family, ARH was due to homozygosity for a large approximately 1.6kb deletion that eliminates exon 4 of ARH gene. Analysis of ARH mRNA demonstrated that the fusion of exon 3 to exon 5 during the splicing of the primary transcript changes the reading frame leading to stop codon 7 amino acids downstream in exon 5. No protein product was detected in affected individuals by immunoblot analysis. This novel mutation adds new support to the molecular heterogeneity of ARH in the Mediterranean basin.

  4. A novel heterozygous deletion in the EVC2 gene causes Weyers acrofacial dysostosis.

    PubMed

    Ye, Xiaoqian; Song, Guangtai; Fan, Mingwen; Shi, Lisong; Jabs, Ethylin Wang; Huang, Shangzhi; Guo, Ruiqiang; Bian, Zhuan

    2006-03-01

    Weyers acrofacial dysostosis (MIM 193530) is an autosomal dominant disorder clinically characterized by mild short stature, postaxial polydactyly, nail dystrophy and dysplastic teeth. Ellis-van Creveld syndrome (EvC, MIM 225500) is an autosomal recessive disorder with a similar, but more severe phenotype. Mutations in the EVC have been identified in both syndromes. However, the EVC mutations only occur in a small proportion of EvC patients. Recently, mutations in a new gene, EVC2, were found to be associated with other EvC cases. The EVC and EVC2 are located close to each other in a head-to-head configuration and may be functionally related. In this study, we report identification of a novel heterozygous deletion in the EVC2 that is responsible for autosomal dominant Weyers acrofacial dysostosis in a large Chinese family. This constitutes the first report of Weyers acrofacial dysostosis caused by this gene. Hence, the spectrum of malformation syndromes due to EVC2 mutations is further extended. Our data provides conclusive evidence that Weyers acrofacial dysostosis and EvC syndrome are allelic and genetically heterogeneous conditions.

  5. Global Deletion of TSPO Does Not Affect the Viability and Gene Expression Profile

    PubMed Central

    Wang, Huaishan; Yang, Jia; Yang, Qi; Fu, Yi; Hu, Yu; Liu, Fang; Wang, Weiqing; Cui, Lianxian; Chen, Hui; Zhang, Jianmin; He, Wei

    2016-01-01

    Translocator Protein (18kDa, TSPO) is a mitochondrial outer membrane transmembrane protein. Its expression is elevated during inflammation and injury. However, the function of TSPO in vivo is still controversial. Here, we constructed a TSPO global knockout (KO) mouse with a Cre-LoxP system that abolished TSPO protein expression in all tissues and showed normal phenotypes in the physiological condition. The birth rates of TSPO heterozygote (Het) x Het or KO x KO breeding were consistent with Mendel’s Law, suggesting a normal viability of TSPO KO mice at birth. RNA-seq analysis showed no significant difference in the gene expression profile of lung tissues from TSPO KO mice compared with wild type mice, including the genes associated with bronchial alveoli immune homeostasis. The alveolar macrophage population was not affected by TSPO deletion in the physiological condition. Our findings contradict the results of Papadopoulos, but confirmed Selvaraj’s findings. This study confirms TSPO deficiency does not affect viability and bronchial alveolar immune homeostasis. PMID:27907096

  6. A heterozygous deletion in the glutamate decarboxylase 67 gene enhances maternal and fetal stress vulnerability.

    PubMed

    Uchida, Taku; Oki, Yutaka; Yanagawa, Yuchio; Fukuda, Atsuo

    2011-04-01

    Both down-regulation of glutamate decarboxylase 67 (GAD67) and maternal exposure to severe stress during pregnancy can increase the risk of schizophrenia and related psychotic disorders in the offspring. To investigate a gene-environment interaction, we performed the restraint-and-light stress to pregnant GAD67-GFP knock-in (GAD67(+/GFP)) and wild-type (GAD67(+/+)) mice three times a day for 45 min per session during gestational day (G) 15.0-17.5. The stress hormone (corticosterone) level of pregnant GAD67(+/GFP) mice (the overall GABA content is reduced because of the destruction of one allele of the endogenous GAD67 gene) was higher than that of GAD67(+/+), even without stress. The fetal body weights (GAD67(+/+)) in the GAD67(+/GFP) mothers were lower than those in the GAD67(+/+) mothers. GAD67(+/GFP) fetuses exhibited higher corticosterone (CORT) levels than GAD67(+/+) fetuses, even in non-stressed GAD67(+/+) mothers. Fetal body weight-decreases and CORT-increases by maternal stress (GAD67(+/+) mother) were significantly more in the GAD67(+/GFP) fetuses than the GAD67(+/+) fetuses. These results indicate that a GAD67 heterozygous deletion itself enhances vulnerability by many aspects, e.g., maternal stress, maternity, and being in utero. Thus, an abnormality in GAD67 could interact with environmental risk factors of psychiatric disorders, including schizophrenia.

  7. Entire CAPN3 gene deletion in a patient with limb-girdle muscular dystrophy type 2A.

    PubMed

    Jaka, Oihane; Azpitarte, Margarita; Paisán-Ruiz, Coro; Zulaika, Miren; Casas-Fraile, Leire; Sanz, Raúl; Trevisiol, Nathalie; Levy, Nicolas; Bartoli, Marc; Krahn, Martin; López de Munain, Adolfo; Sáenz, Amets

    2014-09-01

    Limb-girdle muscular dystrophy type 2A (LGMD2A) due to mutations in the CAPN3 gene is one of the most common of autosomal recessive limb-girdle muscular dystrophies. We describe a patient who had a typical LGMD2A phenotype and posterior compartment involvement on MRI. Different genetic analyses were performed, including microarray analysis. There was an apparently homozygous mutation in exon 24, c.2465G>T, p.(*822Leuext62*), and a lack of correlation in the disease segregation analyses. This suggested the presence of a genomic rearrangement. In fact, a heterozygous deletion of the entire CAPN3 gene was found. This novel deletion comprised the terminal region of the GANC gene and the entire CAPN3 gene. This finding points out the need to reconsider and adapt our current strategy of molecular diagnosis in order to detect these types of genomic rearrangements that escape standard mutation screening procedures.

  8. A deletion in the N-myc downstream regulated gene 1 (NDRG1) gene in Greyhounds with polyneuropathy.

    PubMed

    Drögemüller, Cord; Becker, Doreen; Kessler, Barbara; Kemter, Elisabeth; Tetens, Jens; Jurina, Konrad; Jäderlund, Karin Hultin; Flagstad, Annette; Perloski, Michele; Lindblad-Toh, Kerstin; Matiasek, Kaspar

    2010-06-22

    The polyneuropathy of juvenile Greyhound show dogs shows clinical similarities to the genetically heterogeneous Charcot-Marie-Tooth (CMT) disease in humans. The pedigrees containing affected dogs suggest monogenic autosomal recessive inheritance and all affected dogs trace back to a single male. Here, we studied the neuropathology of this disease and identified a candidate causative mutation. Peripheral nerve biopsies from affected dogs were examined using semi-thin histology, nerve fibre teasing and electron microscopy. A severe chronic progressive mixed polyneuropathy was observed. Seven affected and 17 related control dogs were genotyped on the 50k canine SNP chip. This allowed us to localize the causative mutation to a 19.5 Mb interval on chromosome 13 by homozygosity mapping. The NDRG1 gene is located within this interval and NDRG1 mutations have been shown to cause hereditary motor and sensory neuropathy-Lom in humans (CMT4D). Therefore, we considered NDRG1 a positional and functional candidate gene and performed mutation analysis in affected and control Greyhounds. A 10 bp deletion in canine NDRG1 exon 15 (c.1080_1089delTCGCCTGGAC) was perfectly associated with the polyneuropathy phenotype of Greyhound show dogs. The deletion causes a frame shift (p.Arg361SerfsX60) which alters several amino acids before a stop codon is encountered. A reduced level of NDRG1 transcript could be detected by RT-PCR. Western blot analysis demonstrated an absence of NDRG1 protein in peripheral nerve biopsy of an affected Greyhound. We thus have identified a candidate causative mutation for polyneuropathy in Greyhounds and identified the first genetically characterized canine CMT model which offers an opportunity to gain further insights into the pathobiology and therapy of human NDRG1 associated CMT disease. Selection against this mutation can now be used to eliminate polyneuropathy from Greyhound show dogs.

  9. Mapping of the spontaneous deletion in the Ap3d1 gene of mocha mice: fast and reliable genotyping

    PubMed Central

    Drasbek, Kim Ryun; Holm, Mai Marie; Delenclos, Marion; Jensen, Kimmo

    2008-01-01

    Background The mocha mouse carries a spontaneous deletion in the Ap3d1 gene, encoding the delta 1 subunit of the adaptor related protein complex 3, (Ap3d1), and subsequently lack the expression of functional AP-3. This leads to a deficiency in vesicle transport and storage, which affects neurotransmitter vesicle turnover and release in the central nervous system. Since the genomic sequence of the Ap3d1 gene of mocha mouse is not known, precise mapping of the deletion as well as reliable genotyping protocols are lacking. Findings We sequenced the Ap3d1 gene (HGNC GeneID: 8943) around the deletion site in the mocha mouse and revealed a 10639 bp deletion covering exon 2 to 6. Subsequently, new PCR primers were designed yielding a reliable genotyping protocol of both newborn and adult tissue. To examine the genotypes further, hippocampal neurons were cultured from mocha and control mice. Patch-clamp recordings showed that mocha neurons had a higher input resistance, and that autaptic EPSC in mocha cultures depressed faster and stronger as compared with control cultures. Conclusion Our study reports the sequence of the deleted part of the Ap3d1 gene in mocha mice, as well as a reliable PCR-based genotyping protocol. We cultured hippocampal neurons from control and mocha mice, and found a difference in input resistance of the neurons, and in the synaptic short-term plasticity of glutamatergic autapses showing a larger synaptic depression than controls. The described procedures may be useful for the future utilization of the mocha mouse as a model of defective vesicle biogenesis. Importantly, as genotyping by eye color is complicated in newborn mice, the designed protocol is so fast and reliable that newborn mice could rapidly be genotyped and hippocampal neurons dissociated and cultured, which is normally best done at P0-P2. PMID:19032734

  10. A Self-deleting Cre-lox-ermAM Cassette, CHESHIRE, for marker-less gene deletion in Streptococcus pneumoniae

    PubMed Central

    Weng, Liming; Biswas, Indranil; Morrison, Donald A.

    2009-01-01

    Although targeted mutagenesis of Streptococcus pneumoniae is readily accomplished with the aid of natural genetic transformation and chimeric donor DNA constructs assembled in vitro, the drug resistance markers often employed for selection of recombinant products can themselves be undesirable by-products of the genetic manipulation. A new cassette carrying the erythromycin-resistance marker ermAM is described that can be used as a temporary marker for selection of desired recombinants. The cassette may subsequently be removed at will by virtue of an embedded fucose-regulated Cre recombinase gene and terminal lox66 and lox71 Cre recognition sites, with retention of 34 bp from the cassette as an inert residual double-mutant lox72 site. PMID:19850089

  11. Molecular demonstration of SLC4A1 gene deletion in two Mexican patients with Southeast Asian ovalocytosis.

    PubMed

    Ramos-Kuri, Manuel; Carrillo Farga, Joaquín; Zúñiga, Joaquín; Amador Guerrero, María Teresa; Granados, Julio; Estrada, Francisco J

    2005-06-01

    We describe the finding of two Mexican patients with a specific 27-bp deletion in the solute carrier family 4 gene (SLC4A1delta27) (also known as the band 3 gene found on chromosome 17q21-q22), characteristic of Southeast Asian ovalocytosis (SAO). The patients were asymptomatic, and the initial diagnosis was made by microscopic observation of the presence of typical stomatocytic ovalocytes. The gene deletion was confirmed by PCR and DNA sequencing. Both patients were heterozygous for the deletion. One patient is from Tabasco state, in southeastern Mexico, a malaria-endemic zone. The other patient is from Mexico City, which is not a malaria-endemic area. Their families have no non-Mexican ancestors and their previous generations were born in Mexico. Both patients carry the HLA-B*3501 subtype, characteristic of Amerindians and Asian populations. Familial and HLA data led us to conclude that these two patients are the first report of SLC4A1delta27 in Amerindians. The nucleotide analysis showing a perfect match sequence between Southeast Asian and Mexican patients suggests, but does not prove, that the Mexican gene is not a de novo mutation. Instead, this gene might be the result of migration of individuals with Asian ancestry into the Mexican gene pool. We are looking for other families with the mutation to detect, by HLA analysis, the ancient ethnic origin of these patients.

  12. Common fragile site profiling in epithelial and erythroid cells reveals that most recurrent cancer deletions lie in fragile sites hosting large genes.

    PubMed

    Le Tallec, Benoît; Millot, Gaël Armel; Blin, Marion Esther; Brison, Olivier; Dutrillaux, Bernard; Debatisse, Michelle

    2013-08-15

    Cancer genomes exhibit numerous deletions, some of which inactivate tumor suppressor genes and/or correspond to unstable genomic regions, notably common fragile sites (CFSs). However, 70%-80% of recurrent deletions cataloged in tumors remain unexplained. Recent findings that CFS setting is cell-type dependent prompted us to reevaluate the contribution of CFS to cancer deletions. By combining extensive CFS molecular mapping and a comprehensive analysis of CFS features, we show that the pool of CFSs for all human cell types consists of chromosome regions with genes over 300 kb long, and different subsets of these loci are committed to fragility in different cell types. Interestingly, we find that transcription of large genes does not dictate CFS fragility. We further demonstrate that, like CFSs, cancer deletions are significantly enriched in genes over 300 kb long. We now provide evidence that over 50% of recurrent cancer deletions originate from CFSs associated with large genes.

  13. A partial gene deletion of SLC45A2 causes oculocutaneous albinism in Doberman pinscher dogs.

    PubMed

    Winkler, Paige A; Gornik, Kara R; Ramsey, David T; Dubielzig, Richard R; Venta, Patrick J; Petersen-Jones, Simon M; Bartoe, Joshua T

    2014-01-01

    The first white Doberman pinscher (WDP) dog was registered by the American Kennel Club in 1976. The novelty of the white coat color resulted in extensive line breeding of this dog and her offspring. The WDP phenotype closely resembles human oculocutaneous albinism (OCA) and clinicians noticed a seemingly high prevalence of pigmented masses on these dogs. This study had three specific aims: (1) produce a detailed description of the ocular phenotype of WDPs, (2) objectively determine if an increased prevalence of ocular and cutaneous melanocytic tumors was present in WDPs, and (3) determine if a genetic mutation in any of the genes known to cause human OCA is causal for the WDP phenotype. WDPs have a consistent ocular phenotype of photophobia, hypopigmented adnexal structures, blue irides with a tan periphery and hypopigmented retinal pigment epithelium and choroid. WDPs have a higher prevalence of cutaneous melanocytic neoplasms compared with control standard color Doberman pinschers (SDPs); cutaneous tumors were noted in 12/20 WDP (<5 years of age: 4/12; >5 years of age: 8/8) and 1/20 SDPs (p<0.00001). Using exclusion analysis, four OCA causative genes were investigated for their association with WDP phenotype; TYR, OCA2, TYRP1 and SLC45A2. SLC45A2 was found to be linked to the phenotype and gene sequencing revealed a 4,081 base pair deletion resulting in loss of the terminus of exon seven of SLC45A2 (chr4∶77,062,968-77,067,051). This mutation is highly likely to be the cause of the WDP phenotype and is supported by a lack of detectable SLC45A2 transcript levels by reverse transcriptase PCR. The WDP provides a valuable model for studying OCA4 visual disturbances and melanocytic neoplasms in a large animal model.

  14. A Partial Gene Deletion of SLC45A2 Causes Oculocutaneous Albinism in Doberman Pinscher Dogs

    PubMed Central

    Winkler, Paige A.; Gornik, Kara R.; Ramsey, David T.; Dubielzig, Richard R.; Venta, Patrick J.; Petersen-Jones, Simon M.; Bartoe, Joshua T.

    2014-01-01

    The first white Doberman pinscher (WDP) dog was registered by the American Kennel Club in 1976. The novelty of the white coat color resulted in extensive line breeding of this dog and her offspring. The WDP phenotype closely resembles human oculocutaneous albinism (OCA) and clinicians noticed a seemingly high prevalence of pigmented masses on these dogs. This study had three specific aims: (1) produce a detailed description of the ocular phenotype of WDPs, (2) objectively determine if an increased prevalence of ocular and cutaneous melanocytic tumors was present in WDPs, and (3) determine if a genetic mutation in any of the genes known to cause human OCA is causal for the WDP phenotype. WDPs have a consistent ocular phenotype of photophobia, hypopigmented adnexal structures, blue irides with a tan periphery and hypopigmented retinal pigment epithelium and choroid. WDPs have a higher prevalence of cutaneous melanocytic neoplasms compared with control standard color Doberman pinschers (SDPs); cutaneous tumors were noted in 12/20 WDP (<5 years of age: 4/12; >5 years of age: 8/8) and 1/20 SDPs (p<0.00001). Using exclusion analysis, four OCA causative genes were investigated for their association with WDP phenotype; TYR, OCA2, TYRP1 and SLC45A2. SLC45A2 was found to be linked to the phenotype and gene sequencing revealed a 4,081 base pair deletion resulting in loss of the terminus of exon seven of SLC45A2 (chr4∶77,062,968–77,067,051). This mutation is highly likely to be the cause of the WDP phenotype and is supported by a lack of detectable SLC45A2 transcript levels by reverse transcriptase PCR. The WDP provides a valuable model for studying OCA4 visual disturbances and melanocytic neoplasms in a large animal model. PMID:24647637

  15. Programmable CGH on photochromic material using DMD

    NASA Astrophysics Data System (ADS)

    Alata, Romain; Pariani, Giorgio; Zamkotsian, Frederic; Lanzoni, Patrick; Bianco, Andrea; Bertarelli, Chiara

    2016-07-01

    Computer Generated Holograms (CGHs) are useful for wavefront shaping and complex optics testing, including aspherical and free-form optics. Today, CGHs are recorded directly with a laser or intermediates masks but allows only recording binary CGHs; binary CGHs are efficient but can reconstruct only pixilated images. We propose to use a Digital Micro-mirror Device (DMD) for writing binary CGHs as well as grayscale CGHs, able to reconstruct fulfilled images. DMD is actually studied at LAM, for generating programmable slit masks in multi-object spectrographs. It is composed of 2048x1080 individually controllable micro-mirrors, with a pitch of 13.68 μm. This is a real-time reconfigurable mask, perfect for recording CGHs. A first setup has been developed for hologram recording, where the DMD is enlightened with a collimated beam and illuminates a photosensible plate through an Offner relay, with a magnification of 1:1. Our set up resolution is 2-3 μm, leading to a CGH resolution equal to the DMD micro mirror size. In order to write and erase CGHs during test procedure or on request, we use a photochromic plate called PUR-GD71-50-ST developed at Politecnico di Milano. It is opaque at rest, and becomes transparent when it is illuminated with visible light, between 500 and 700 nm; then it can be erased by a UV flash. We choose to code the CGHs in equally spaced levels, so called stepped CGH. We recorded up to 1000x1000 pixels CGHs with a contrast greater than 50, knowing that the material is able to reach an ultimate contrast of 1000. A second bench has also been developed, dedicated to the reconstruction of the recorded images with a 632.8nm He-Ne laser beam. Very faithful reconstructions have been obtained. Thanks to our recording and reconstruction set-ups, we have been able to successfully record binary and stepped CGHs, and reconstruct them with a high fidelity, revealing the potential of this method for generating programmable/rewritable stepped CGHs on

  16. Gap-PCR Screening for Common Large Deletional Mutations of β-Globin Gene Cluster Revealed a Higher Prevalence of the Turkish Inversion/Deletion (δβ)0 Mutation in Antalya

    PubMed Central

    Bilgen, Türker; Altıok Clark, Özden; Öztürk, Zeynep; Yeşilipek, M. Akif; Keser, İbrahim

    2016-01-01

    Objective: Although the calculated carrier frequency for point mutations of the β-globin gene is around 10% for Antalya Province, nothing is known about the profile of large deletional mutations involving the β-globin gene. In this study, we aimed to screen common deletional mutations in the β-globin gene cluster in patients for whom direct DNA sequencing was not able to demonstrate the mutation(s) responsible for the disease phenotype. Materials and Methods: Thirty-one index cases selected with a series of selection events among 60 cases without detected β-globin gene mutation from 580 thalassemia-related cases tested by direct sequencing over the last 4 years in our diagnostic center were screened for the most common 8 different large deletional mutations of the β-globin gene cluster by gap-PCR. Results: We detected 1 homozygous and 9 heterozygous novel unrelated cases for the Turkish inversion/deletion (δβ)0 mutation in our series of 31 cases. Our study showed that the Turkish inversion/deletion (δβ)0 mutation per se accounts for 16.6% of the unidentified causative alleles and also accounts for 1.5% of all detected mutations over the last 4 years in our laboratory. Conclusion: Since molecular diagnosis of deletional mutations in the β-globin gene cluster warrants different approaches, it deserves special attention in order to provide prenatal diagnosis and prevention opportunities to the families involved. We conclude that the Turkish inversion/deletion (δβ)0, as the most prevalent deletional mutation detected so far, has to be routinely tested for in Antalya, and the gap-PCR approach has valuable diagnostic potential in the patients at risk. PMID:26377447

  17. A large germline deletion in the Chek2 kinase gene is associated with an increased risk of prostate cancer

    PubMed Central

    Cybulski, C; Wokołorczyk, D; Huzarski, T; Byrski, T; Gronwald, J; Górski, B; Dębniak, T; Masojć, B; Jakubowska, A; Gliniewicz, B; Sikorski, A; Stawicka, M; Godlewski, D; Kwias, Z; Antczak, A; Krajka, K; Lauer, W; Sosnowski, M; Sikorska‐Radek, P; Bar, K; Klijer, R; Zdrojowy, R; Małkiewicz, B; Borkowski, A; Borkowski, T; Szwiec, M; Narod, S A; Lubiński, J

    2006-01-01

    Background Germline mutations in the Chek2 kinase gene (CHEK2) have been associated with a range of cancer types. Recently, a large deletion of exons 9 and 10 of CHEK2 was identified in several unrelated patients with breast cancer of Czech or Slovak origin. The geographical and ethnic extent of this founder allele has not yet been determined. Participants and methods We assayed for the presence of this deletion, and of three other CHEK2 founder mutations, in 1864 patients with prostate cancer and 5496 controls from Poland. Results The deletion was detected in 24 of 5496 (0.4%) controls from the general population, and is the most common CHEK2 truncating founder allele in Polish patients. The deletion was identified in 15 of 1864 (0.8%) men with unselected prostate cancer (OR 1.9; 95% CI 0.97 to 3.5; p = 0.09) and in 4 of 249 men with familial prostate cancer (OR 3.7; 95% CI 1.3 to 10.8; p = 0.03). These ORs were similar to those associated with the other truncating mutations (IVS2+1G→A, 1100delC). Conclusion A large deletion of exons 9 and 10 of CHEK2 confers an increased risk of prostate cancer in Polish men. The del5395 founder deletion might be present in other Slavic populations, including Ukraine, Belarus, Russia, Baltic and Balkan countries. It will be of interest to see to what extent this deletion is responsible for the burden of prostate cancer in other populations. PMID:17085682

  18. Familiar Hypopigmentation Syndrome in Sheep Associated with Homozygous Deletion of the Entire Endothelin Type-B Receptor Gene

    PubMed Central

    Lühken, Gesine; Fleck, Katharina; Pauciullo, Alfredo; Huisinga, Maike; Erhardt, Georg

    2012-01-01

    In humans, rodents and horses, pigmentary anomalies in combination with other disorders, notably intestinal aganglionosis, are associated with variants of the endothelin type-B receptor gene (EDNRB). In an inbred Cameroon sheep flock, five white lambs with light blue eyes were sired from the same ram and died within a few hours up to a few days after birth, some of them with signs of intestinal obstruction. The aim of this study was to investigate if the observed hypopigmentation and a possible lethal condition were associated with a molecular change at the ovine EDNRB locus, and to check if such a genetic alteration also occurs in other Cameroon sheep flocks. Sequence analysis revealed a deletion of about 110 kb on sheep chromosome 10, comprising the entire EDNRB gene, on both chromosomes in the two available hypopigmented lambs and on a single chromosome in the two dams and three other unaffected relatives. This micro-chromosomal deletion was also confirmed by quantitative real-time PCR and by fluorescence in situ hybridization. Genotyping of a total of 127 Cameroon sheep in 7 other flocks by duplex PCR did not identify additional carriers of the deletion. Although both hypopigmented lambs available for post-mortem examination had a considerably dilated cecum and remaining meconium, histopathological examination of intestinal samples showed morphologically normal ganglion cells in appropriate number and distribution. This is to our knowledge the first description of an ENDRB gene deletion and associated clinical signs in a mammalian species different from humans and rodents. In humans and rats it is postulated that the variable presence and severity of intestinal aganglionosis and other features in individuals with EDNRB deletion is due to a variable genetic background and multiple gene interactions. Therefore the here analyzed sheep are a valuable animal model to test these hypotheses in another species. PMID:23300849

  19. Evolutionary pathway of the Beijing lineage of Mycobacterium tuberculosis based on genomic deletions and mutT genes polymorphisms.

    PubMed

    Rindi, Laura; Lari, Nicoletta; Cuccu, Barbara; Garzelli, Carlo

    2009-01-01

    Among the genotypes that prevail in the modern spectrum of Mycobacterium tuberculosis strains, the Beijing genotype is the one that causes major concern, as it is geographically widespread and it is considered hypervirulent. Comparative genomic studies have shown that Beijing strains have principally evolved through mechanisms of deletion of chromosomal regions, designated regions of difference (RD), and mutations. In this paper, we aimed to determine the evolutionary history of Beijing strains through the analysis of polymorphisms generated by deletions of large specific sequences, i.e., RD105, RD181, RD150, and RD142, and by single nucleotide substitutions in genes mutT4 and mutT2, coding for DNA repair enzymes. Based on the molecular characteristics of a collection of Beijing strains recently isolated in Tuscany, Italy, we propose a phylogenetic reconstruction of the Beijing family. According to our model, the Beijing family evolved from a M. tuberculosis progenitor following deletion of the RD207 region, an event responsible for the loss of spacers 1-34 in the direct repeat (DR) locus. The major lineages of the Beijing family then evolved via subsequent deletions of regions RD105, RD181 and RD150. In the most ancient evolutionary lineages genes mutT4 and mutT2 were in wild type configuration; the mutT4 mutation was acquired subsequent to the RD181 deletion in a progenitor strain that, in turn, gave rise to a sublineage bearing the mutT2 mutation. Within the major branches of the Beijing family, deletion of additional spacers in the DR locus led to evolution of sublineages characterized by different spoligotypes. Our evolutionary model of the Beijing family provides a deeper framework than previously proposed for epidemiologic and phylogenetic studies of circulating M. tuberculosis Beijing strains, thus allowing a more systematic and comprehensive evaluation of the relevance of Beijing strain variability.

  20. In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology.

    PubMed

    Fazli, Mustafa; Harrison, Joe J; Gambino, Michela; Givskov, Michael; Tolker-Nielsen, Tim

    2015-06-01

    Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generation of deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counterselectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counterselectable pheS gene and the I-SceI homing endonuclease. This system provides efficiency in cloning homology regions of target genes and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a cyclic di-GMP (c-di-GMP)-responsive regulator protein important for biofilm formation.

  1. In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology

    PubMed Central

    Fazli, Mustafa; Harrison, Joe J.; Gambino, Michela; Givskov, Michael

    2015-01-01

    Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generation of deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counterselectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counterselectable pheS gene and the I-SceI homing endonuclease. This system provides efficiency in cloning homology regions of target genes and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a cyclic di-GMP (c-di-GMP)-responsive regulator protein important for biofilm formation. PMID:25795676

  2. Greig cephalopolysyndactyly syndrome: Altered phenotype of a contiguous gene syndrome by the presence of a chromosomal deletion

    SciTech Connect

    Hersh, J.H.; Williams, P.G.; Yen, F.F.

    1994-09-01

    Greig cephalopolysyndactyly syndrome (GCPS) is characterized by craniofacial anomalies, broad thumbs and halluces, polydactyly of the hands and feet, and variable syndactyly. Intellectual abilities are usually normal. Inheritance is in an autosomal dominant fashion. The disorder has been mapped to chromosome 7p13, suggesting that the condition represents a contiguous gene syndrome (CGS). A male infant presented with multiple congenital anomalies, including omphalocele, dysgenesis of the corpus callosum, hydrocephalus, esotropia, broad thumbs and halluces, syndactyly, polydactyly of one foot, hypotonia and developmental delay. A de novo interstitial deletion of chromosome 7p was detected, 46,XY,del(7)(p13p15). Although clinical findings in this case were reminiscent of GCPS, and the chromosomal abnormality included the region assigned to the candidate gene for this syndrome, additional physical abnormalities were present, as well as cognitive deficits. Some of these features have been previously described in patients with chromosomal deletions of 7p. The chromosomal abnormality in our case provides supportive evidence of the gene locus in GCPS, and that GCPS represents a new CGS. However, a larger deletion, extending beyond the limits of the gene, significantly altered the phenotype. Isolation of the gene responsible for GCPS, and identification of additional patients with chromosomal abnormalities in this region of chromosome 7, should help to provide more accurate genotype-phenotype correlations.

  3. Hereditary fructose intolerance: functional study of two novel ALDOB natural variants and characterization of a partial gene deletion.

    PubMed

    Esposito, Gabriella; Imperato, Maria Rosaria; Ieno, Luigi; Sorvillo, Rosa; Benigno, Vincenzo; Parenti, Giancarlo; Parini, Rossella; Vitagliano, Luigi; Zagari, Adriana; Salvatore, Francesco

    2010-12-01

    Hereditary fructose intolerance (HFI) is an autosomal recessive metabolic disease caused by impaired functioning of human liver aldolase (ALDOB). At least 54 subtle/point mutations and only two large intragenic deletions have been found in the ALDOB gene. Here we report two novel ALDOB variants (p.R46W and p.Y343H) and an intragenic deletion that we found in patients with suspected HFI. The residual catalytic activity of the recombinant p.R46W and p.Y343H variants toward F1P was particularly altered. We also characterized a large intragenic deletion that we found in six unrelated patients. This is the first report of six unrelated patients sharing the same ALDOB deletion, thus indicating a founder effect for this allele in our geographic area. Because this deletion involves ALDOB exon 5, it can mimic worldwide common pathogenic genotypes, that is, homozygous p.A150P and p.A175D. Finally, the identification of only one ALDOB mutation in symptomatic patients suggests that HFI symptoms can, albeit rarely, appear also in heterozygotes. Therefore, an excessive and continuous fructose dietary intake may have deleterious effects even in apparently asymptomatic HFI carriers.

  4. A novel deletion/insertion caused by a replication error in the β-globin gene locus control region.

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Meley, Roland; Pondarré, Corinne; Francina, Alain

    2011-01-01

    Deletions in the β-globin locus control region (β-LCR) lead to (εγδβ)(0)-thalassemia [(εγδβ)(0)-thal]. In patients suffering from these rare deletions, a normal hemoglobin (Hb), phenotype is found, contrasting with a hematological thalassemic phenotype. Multiplex-ligation probe amplification (MLPA) is an efficient tool to detect β-LCR deletions combined with long-range polymerase chain reaction (PCR) and DNA sequencing to pinpoint deletion breakpoints. We present here a novel 11,155 bp β-LCR deletion found in a French Caucasian patient which removes DNase I hypersensitive site 2 (HS2) to HS4 of the β-LCR. Interestingly, a 197 bp insertion of two inverted sequences issued from the HS2-HS3 inter-region is present and suggests a complex rearrangement during replication. Carriers of this type of thalassemia can be misdiagnosed as an α-thal trait. Consequently, a complete α- and β-globin gene cluster analysis is required to prevent a potentially damaging misdiagnosis in genetic counselling.

  5. Homozygous deletion of TRMT10A as part of a contiguous gene deletion in a syndrome of failure to thrive, delayed puberty, intellectual disability and diabetes mellitus.

    PubMed

    Zung, Amnon; Kori, Michal; Burundukov, Ella; Ben-Yosef, Tamar; Tatoor, Yasmin; Granot, Esther

    2015-12-01

    Two recent reports describe a new syndrome of intellectual disability, short stature, microcephaly, and young onset diabetes or disturbed glucose metabolism in association with inactivating mutations in the TRMT10A gene. We investigated the clinical spectrum presented by a 17-year-old female with a homozygous contiguous gene deletion involving the TRMT10A gene. From infancy, she presented with failure to thrive and microcephaly. Puberty was characterized by a slow and an inconsistent course of progression. Concomitantly, gonadotropin levels fluctuated between low and high levels which were compatible with gonadal failure. Unlike the previous reports, the patient had ketoacidosis at onset of diabetes and islet cell autoantibodies. Nevertheless, glycemic control was excellent (HbA1C 5.0%-6.2%). RT-PCR and Western blot analysis demonstrated a complete abolishment of TRMT10A mRNA and its translated protein. In order to elucidate the nature of diabetes in this patient, endogenous insulin secretion and glycemic control were evaluated by a glucagon stimulation test and continuous glucose monitoring both during insulin treatment and off therapy. Endogenous insulin secretion still persisted 22 months after onset of diabetes and relatively normal glucose levels were kept over 3 days without insulin treatment. The fluctuating course of puberty and diabetes may reflect intermittent apoptotic damages due to sensitization of the relevant cells to various stress agents in the absence of functional TRMT10A.

  6. Detection of a functional insertion sequence responsible for deletion of the thermostable direct hemolysin gene (tdh) in Vibrio parahaemolyticus.

    PubMed

    Kamruzzaman, Muhammad; Bhoopong, Phuangthip; Vuddhakul, Varaporn; Nishibuchi, Mitsuaki

    2008-09-15

    The thermostable direct hemolysin coded by the tdh gene is a marker of virulent strains of Vibrio parahaemolyticus. The tdh genes are flanked by insertion sequences collectively named as ISVs or their remnants; but the ISVs so far examined have accumulated mutations in the transposase genes and underwent structural arrangements and their transposition activity could not be expected; the tdh gene was thus considered to have been acquired by V. parahaemolyticus through horizontal transfer in the past during evolution. We recently isolated from the same patient tdh+ strains and a tdh(-) strain (PCR examination) that were otherwise indistinguishable. The purpose of this study was to examine the hypothesis that the tdh(-) strain was derived from the tdh+ strain by a deletion of the tdh gene mediated by a functional ISV. Southern blot hybridization showed tdh+ sequences in the tdh(-) strain (PSU-1466). Nucleotide sequence analysis of the tdh and its flanking sequences revealed the tdh gene was split into two parts and they were located 3182-bp apart in PSU-1466. The two tdh sequences were flanked by one of the ISVs, named as ISVpa3, in PSU-1466. This genetic structure could be explained by an ISVpa3-mediated partial tdh deletion from a tdh+ strain followed by transposition of the duplicated ISVpa3 and the deleted tdh sequence into a neighboring location. The ISVpa3 of PSU-1466 coded for a full-length transposase and a DDE motif. We were able to demonstrate transposition activity of the ISVpa3 cloned from PSU-1466 using the replicon fusion assay with the conjugal transfer of a cointegrate from Escherichia coli to V. parahaemolyticus. Our data support ISVpa3-mediated partial tdh deletion resulted in the emergence of the tdh(-) strain.

  7. Virulence characteristics of extraintestinal pathogenic Escherichia coli deletion of gene encoding the outer membrane protein X

    PubMed Central

    MENG, Xianrong; LIU, Xueling; ZHANG, Liyuan; HOU, Bo; LI, Binyou; TAN, Chen; LI, Zili; ZHOU, Rui; LI, Shaowen

    2016-01-01

    Outer membrane protein X (OmpX) and its homologues have been proposed to contribute to the virulence in various bacterial species. But, their role in virulence of extraintestinal pathogenic Escherichia coli (ExPEC) is yet to be determined. This study evaluates the role of OmpX in ExPEC virulence in vitro and in vivo using a clinical strain PPECC42 of porcine origin. The ompX deletion mutant exhibited increased swimming motility and decreased adhesion to, and invasion of pulmonary epithelial A549 cell, compared to the wild-type strain. A mild increase in LD50 and distinct decrease in bacterial load in such organs as heart, liver, spleen, lung and kidney were observed in mice infected with the ompX mutant. Complementation of the complete ompX gene in trans restored the virulence of mutant strain to the level of wild-type strain. Our results reveal that OmpX contributes to ExPEC virulence, but may be not an indispensable virulence determinant. PMID:27149893

  8. Goldenhar and cri-du-chat syndromes: a contiguous gene deletion syndrome?

    PubMed

    Choong, Yee Fong; Watts, Patrick; Little, Elizabeth; Beck, Lyn

    2003-06-01

    We report a full-term male infant born to nonconsanguinous parents who had clinical features of Goldenhar syndrome and cri du chat syndrome. At birth, the infant was noted to have dysmorphic features with bilateral preauricular tags, rotated ears, bilateral epicanthic folds, a left epibulbar lipodermoid, and an accessory left nipple. After he was assessed for feeding difficulty and tachypnea, he was found to have esophageal atresia with tracheoesophageal fistula. In addition, he had a high-pitched, cat-like cry, characteristic of cri-du-chat syndrome. He also failed a hearing test. Chromosomal analysis and fluorescence in situ hybridisation studies showed an unbalanced karyotype with a terminal deletion of the segment p14 on the short arm of chromosome 5, which is consistent with the cri-du-chat locus. The association of Goldenhar syndrome and cri-du-chat syndrome in this patient suggests that the chromosome 5p14 locus may harbor a gene implicated with Goldenhar syndrome.

  9. Immunoglobulin gene insertions and deletions in the affinity maturation of HIV-1 broadly reactive neutralizing antibodies.

    PubMed

    Kepler, Thomas B; Liao, Hua-Xin; Alam, S Munir; Bhaskarabhatla, Rekha; Zhang, Ruijun; Yandava, Chandri; Stewart, Shelley; Anasti, Kara; Kelsoe, Garnett; Parks, Robert; Lloyd, Krissey E; Stolarchuk, Christina; Pritchett, Jamie; Solomon, Erika; Friberg, Emma; Morris, Lynn; Karim, Salim S Abdool; Cohen, Myron S; Walter, Emmanuel; Moody, M Anthony; Wu, Xueling; Altae-Tran, Han R; Georgiev, Ivelin S; Kwong, Peter D; Boyd, Scott D; Fire, Andrew Z; Mascola, John R; Haynes, Barton F

    2014-09-10

    Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events.

  10. Deletions in the COL4A5 collagen gene in X-linked Alport syndrome. Characterization of the pathological transcripts in nonrenal cells and correlation with disease expression.

    PubMed Central

    Antignac, C; Knebelmann, B; Drouot, L; Gros, F; Deschênes, G; Hors-Cayla, M C; Zhou, J; Tryggvason, K; Grünfeld, J P; Broyer, M

    1994-01-01

    The type IV collagen alpha 5 chain (COL4A5) gene of 88 unrelated male patients with X-linked Alport syndrome was tested for major gene rearrangements by Southern blot analysis, using COL4A5 cDNA probes. 14 different deletions were detected, providing a 16% deletion rate in the COL4A5 gene in the patient population. The deletions are dispersed all over the gene with different sizes, ranging from 1 kb to the complete absence of the gene (> 250 kb) in one patient. In four patients with intragenic deletions, absence of the alpha 3 (IV) chain in the glomerular basement membrane was demonstrated by immunohistochemical studies. This finding supports the hypothesis that abnormalities in the alpha 5 (IV) chain may prevent normal incorporation of the alpha 3 (IV) chain into the glomerular basement membrane. Direct sequencing of cDNA amplified from lymphoblast mRNA of four patients with internal gene deletions, using appropriate combinations of primers amplifying across the predicted boundaries of the deletions, allowed us to determine the effect of the genomic rearrangements on the transcripts and, by inference, on the alpha 5 (IV) chain. Regardless of the extent of deletion and of the putative protein product, the 14 deletions occur in patients with juvenile-type Alport syndrome. Images PMID:8132760

  11. Detection of large gene rearrangements in X-linked genes by dosage analysis: identification of novel α-galactosidase A (GLA) deletions causing Fabry disease.

    PubMed

    Dobrovolny, Robert; Nazarenko, Irina; Kim, Jungmin; Doheny, Dana; Desnick, Robert J

    2011-06-01

    For most Mendelian disorders, targeted genome sequencing is an effective method to detect causative mutations. However, sequencing PCR-amplified exonic regions and their intronic boundaries can miss large deletions or duplications and mutations that lead to PCR failures in autosomal dominant disorders and in heterozygote detection for X-linked diseases. Here, a method is described for detecting large (>50 bp) deletions/duplications in the X-linked α-galactosidase A (GLA) gene, which cause Fabry disease. Briefly, multiplex PCR mixtures were designed to amplify each GLA exon and an unrelated internal control exon to normalize GLA exonic amplicon peak heights. For each normalized GLA amplicon, the normal control female to male peak-height ratios were 1.8 to 2.2 (expected 2.0), whereas the expected ratios for deletions or duplications would be ∼1.0 or 3.0, respectively. Using this method, three novel deletions, c.369+3_547+954del4096insT, c.194+2049_369+773del2619insCG, and c.207_369+651del814ins231, were detected in unrelated women with signs and/or symptoms suggestive of Fabry disease, but no "sequencing-detectable" mutations. The deletions were confirmed by sequencing their respective GLA RT-PCR products. This method can identify gene rearrangements that may be cryptic to genomic DNA sequencing and can be readily adapted to other X-linked or autosomal dominant genes.

  12. Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

    PubMed Central

    Eberhart, Lauren J.; Ohlert, Janet M.; Knutson, Carolann M.; Plunkett, Mary H.

    2015-01-01

    Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes. PMID:25888177

  13. Characterization of a de novo 43-bp deletion of the Gs[alpha] gene (GNAS1) in Albright hereditary osteodystrophy

    SciTech Connect

    Luttikhuis, M.E.M.O.; Trembath, R.C. ); Wilson, L.C. Institute of Child Health, London ); Leonard, J.V. )

    1994-05-15

    Albright hereditary osteodystrophy (AHO) is an autosomal dominant disorder characterized by short stature, obesity, mental retardation, subcutaneous calcification, and brachy-metaphalangia. Two distinct forms of AHO exist; pseudohypoparathyroidism type I (PHPI) and pseudopseudohypoparathyrodism (PPHP). The classification is dependent upon the presence or absence, respectively, of resistance to parathyroid and other hormones that bind to Gs-protein-coupled membrane receptors stimulating adenylyl cyclase. Gs is a heterotrimeric protein comprising [alpha], [beta], and [gamma]-subunits encoded by separate genes. Genomic DNA was isolated from peripheral leukocytes from 13 unrelated AHO patients. Exon 4 and flanking intronic sequence of GNAS1 were PCR amplified. A single PCR product corresponding to the expected 159-bp fragment was identified in 12 affected individuals with either PHPIa or PPHP. In patient 10285 an additional smaller fragment was detected but was not present in either of the unaffected parents. These two fragments were isolated from a 2% agarose gel. Direct sequencing of the smaller fragment revealed a 43-bp deletion comprising at least 35 hp of the 3[prime] end of exon 4 and the donor splice site of intron 4 and extending into the following intro. The 43-bp deletion would lead to a premature stop codon, 62 codons downstream of the deletion. The de novo mutation reported here is the largest deletion in the Gs[alpha] gene described so far for AHO patients.

  14. Functional analysis of the PsbX protein by deletion of the corresponding gene in Synechocystis sp. PCC 6803.

    PubMed

    Funk, C

    2000-12-01

    The psbX gene (sml0002) coding for a 4.1 kDa protein in Photosystem II of plants and cyanobacteria was deleted in both wild type and in a Photosystem I-less mutant of the cyanobacterium Synechocystis sp. PCC 6803. Polymerase chain reaction and sequencing analysis showed that the mutants had completely segregated. Deletion of the PsbX protein does not seem to influence growth rate, electron transport or water oxidation ability. Whereas a high light induction of the psbX mRNA could be observed in wild type, deletion of the gene did not lead to high light sensibility. Light saturation measurements and 77K fluorescence measurements indicated a minor disconnection of the antenna in the deletion mutant. Furthermore, fluorescence induction measurements as well as immuno-staining of the D1 protein showed that the amount of Photosystem II complexes in the mutants was reduced by 30%. Therefore, PsbX does not seem to be necessary for the Photosystem II electron transport, but directly or indirectly involved in the regulation of the amount of functionally active Photosystem II centres in Synechocystis sp. PCC 6803.

  15. Delineation of candidate genes responsible for structural brain abnormalities in patients with terminal deletions of chromosome 6q27

    PubMed Central

    Peddibhotla, Sirisha; Nagamani, Sandesh CS; Erez, Ayelet; Hunter, Jill V; Holder Jr, J Lloyd; Carlin, Mary E; Bader, Patricia I; Perras, Helene MF; Allanson, Judith E; Newman, Leslie; Simpson, Gayle; Immken, LaDonna; Powell, Erin; Mohanty, Aaron; Kang, Sung-Hae L; Stankiewicz, Pawel; Bacino, Carlos A; Bi, Weimin; Patel, Ankita; Cheung, Sau W

    2015-01-01

    Patients with terminal deletions of chromosome 6q present with structural brain abnormalities including agenesis of corpus callosum, hydrocephalus, periventricular nodular heterotopia, and cerebellar malformations. The 6q27 region harbors genes that are important for the normal development of brain and delineation of a critical deletion region for structural brain abnormalities may lead to a better genotype–phenotype correlation. We conducted a detailed clinical and molecular characterization of seven unrelated patients with deletions involving chromosome 6q27. All patients had structural brain abnormalities. Using array comparative genomic hybridization, we mapped the size, extent, and genomic content of these deletions. The smallest region of overlap spans 1.7 Mb and contains DLL1, THBS2, PHF10, and C6orf70 (ERMARD) that are plausible candidates for the causation of structural brain abnormalities. Our study reiterates the importance of 6q27 region in normal development of brain and helps identify putative genes in causation of structural brain anomalies. PMID:24736736

  16. Role of ISKpn7 and deletions in blaKPC gene expression.

    PubMed

    Naas, Thierry; Cuzon, Gaelle; Truong, Ha-Vy; Nordmann, Patrice

    2012-09-01

    The carbapenemase-encoding bla(KPC) gene, which is rapidly spreading in Gram-negative rods, is located on a Tn3-based transposon, Tn4401, which carries a polymorphic region giving rise to five isoforms (a, b, c, d, and e) that is located immediately upstream of the bla(KPC) gene and thus likely involved in its expression. Using 5' rapid amplification of cDNA ends (5'RACE), we identified three potential promoter sequences (P1, P2, and P3) upstream of the bla(KPC) gene, of which only P1 (absent from isoforms c and d) and P2 (present in all isoforms, with a -35 box located inside the right inverted repeat of ISKpn7) were shown to be true promoters involved in expression. One representative of each different promoter combination of Tn4401, i.e., P2 alone (isoform c), P1-P2 (isoform a), and P1-P2-P3 (isoform b), was cloned into an Escherichia coli plasmid vector. Using reverse transcription-PCR (RT-PCR), the highest level of expression was obtained with isoform a (P1 and P2), which is also the most commonly encountered form in enterobacterial clinical isolates, followed by isoforms b (P1, P2, and P3) and c (P2 only). These differences in expression led to slight differences in MIC values of carbapenems. In silico analysis of the DNA sequence of isoform b revealed a stem-loop structure that is likely responsible for strong stops observed in 5'RACE experiments and for decreased expression compared to that with isoform a (P1 and P2). In addition, such structures could also be at the origin for the deletions observed in isoforms a and c. Taken together, these results indicate that the P1 and P2 promoters both contribute to the expression of the bla(KPC) gene and that the construct with the highest level of expression is that possessing isoform a, which is also the most commonly encountered form in clinical isolates.

  17. Screening the dystrophin gene suggests a high rate of polymorphism in general but no exonic deletions in schizophrenics

    SciTech Connect

    Lindor, N.M.; Sobell, J.L.; Thibodeau, S.N.

    1994-03-15

    The dystrophin gene, located at chromosome Xp21, was evaluated as a candidate gene in chronic schizophrenia in response to the report of a large family in which schizophrenia cosegregated with Becker muscular dystrophy. Genomic DNA from 94 men with chronic schizophrenia was evaluated by Southern blot analysis using cDNA probes that span exons 1-59. No exonic deletions were identified. An unexpectedly high rate of polymorphism was calculated in this study and two novel polymorphisms were found, demonstrating the usefulness of the candidate gene approach even when results of the original study are negative. 41 refs., 1 fig., 4 tabs.

  18. Human homologue sequences to the Drosophila dishevelled segment-polarity gene are deleted in the DiGeorge syndrome

    SciTech Connect

    Pizzuti, A.; Ratti, A.; Penso, D.; Silani, V.; Scarlato, G.

    1996-04-01

    DiGeorge syndrome (DGS) is a developmental defect of some of the neural crest derivatives. Most DGS patients show haploinsufficiency due to interstitial deletions of the proximal long arm of chromosome 22. Deletions of 22q11 have also been reported in patients with the velo-cardio-facial syndrome and familial conotruncal heart defects. It has been suggested that the wide phenotype spectrum associated with 22q11 monosomy is a consequence of contiguous-gene deletions. We report the isolation of human cDNAs homologous to the Drosophila dishevelled (dsh) segment-polarity gene. Sequences homologous to the 3{prime} UTR of these transcripts (DVL-22) were positioned within the DGS critical region and were found to be deleted in DGS patients. Human DVL mRNAs are expressed in several fetal and adult tissues, including the thymus and, at high levels, the heart. Two transcripts, 3.2 and 5 kb, were detected, in Northern blot analysis, with different expression patterns in the surveyed tissues when different cDNAs were used. The isolated cDNAs exhibit high amino acid homology with the mouse and Xenopus Dvl-1 gene, the only other vertebrate dsh homologues so far isolated. The pivotal role of dsh in fly development suggests an analogous key function in vertebrate embryogenesis of its homologue genes. Since DGS may be due to perturbation of differentiation mechanisms at decisive embryological stages, a Dsh-like gene in the small-region overlap (SRO) might be a candidate for the pathogenesis of this disorder. 52 refs., 3 figs.

  19. Human homologue sequences to the Drosophila dishevelled segment-polarity gene are deleted in the DiGeorge syndrome.

    PubMed

    Pizzuti, A; Novelli, G; Mari, A; Ratti, A; Colosimo, A; Amati, F; Penso, D; Sangiuolo, F; Calabrese, G; Palka, G; Silani, V; Gennarelli, M; Mingarelli, R; Scarlato, G; Scambler, P; Dallapiccola, B

    1996-04-01

    DiGeorge syndrome (DGS) is a developmental defect of some of the neural crest derivatives. Most DGS patients show haploinsufficiency due to interstitial deletions of the proximal long arm of chromosome 22. Deletions of 22q11 have also been reported with patients with the velocardio-facial syndrome and familial conotruncal heart defects. It has been suggested that the wide phenotype spectrum associated with 22q11 monosomy is a consequence of contiguous-gene deletions. We report the isolation of human cDNAs homologous to the Drosophila dishevelled (dsh) segment-polarity gene. Sequences homologous to the 3' UTR of these transcripts (DVL-22) were positioned within the DGS critical region and were found to be deleted in DGS patients. Human DVL mRNAs are expressed in several fetal and adult tissues, including the thymus and, at high levels, the heart. Two transcripts, 3.2 and 5kb, were detected, in northern blot analysis, with different expression patterns in the surveyed tissues when different cDNAs were used. The isolated cDNAs exhibit high amino acid homology with the mouse and Xenopus Dvl-1 gene, the only other vertebrate dsh homologues so far isolated. The pivotal role of dsh in fly development suggests an analogous key function in vertebrate embryogenesis of its homologue genes. Since DGS may be due to perturbation of differentiation mechanisms at decisive embryological stages, a Dsh-like gene in the small-region overlap (SRO) might be a candidate for the pathogenesis of this disorder.

  20. Human homologue sequences to the Drosophila dishevelled segment-polarity gene are deleted in the DiGeorge syndrome.

    PubMed Central

    Pizzuti, A.; Novelli, G.; Mari, A.; Ratti, A.; Colosimo, A.; Amati, F.; Penso, D.; Sangiuolo, F.; Calabrese, G.; Palka, G.; Silani, V.; Gennarelli, M.; Mingarelli, R.; Scarlato, G.; Scambler, P.; Dallapiccola, B.

    1996-01-01

    DiGeorge syndrome (DGS) is a developmental defect of some of the neural crest derivatives. Most DGS patients show haploinsufficiency due to interstitial deletions of the proximal long arm of chromosome 22. Deletions of 22q11 have also been reported with patients with the velocardio-facial syndrome and familial conotruncal heart defects. It has been suggested that the wide phenotype spectrum associated with 22q11 monosomy is a consequence of contiguous-gene deletions. We report the isolation of human cDNAs homologous to the Drosophila dishevelled (dsh) segment-polarity gene. Sequences homologous to the 3' UTR of these transcripts (DVL-22) were positioned within the DGS critical region and were found to be deleted in DGS patients. Human DVL mRNAs are expressed in several fetal and adult tissues, including the thymus and, at high levels, the heart. Two transcripts, 3.2 and 5kb, were detected, in northern blot analysis, with different expression patterns in the surveyed tissues when different cDNAs were used. The isolated cDNAs exhibit high amino acid homology with the mouse and Xenopus Dvl-1 gene, the only other vertebrate dsh homologues so far isolated. The pivotal role of dsh in fly development suggests an analogous key function in vertebrate embryogenesis of its homologue genes. Since DGS may be due to perturbation of differentiation mechanisms at decisive embryological stages, a Dsh-like gene in the small-region overlap (SRO) might be a candidate for the pathogenesis of this disorder. Images Figure 1 Figure 2 Figure 3 PMID:8644734

  1. IL1RAPL1 gene deletion as a cause of X-linked intellectual disability and dysmorphic features.

    PubMed

    Youngs, Erin L; Henkhaus, Rebecca; Hellings, Jessica A; Butler, Merlin G

    2012-01-01

    Intellectual disability affects approximately 2% of the population with males outnumbering females due to involvement of over 300 genes on the X chromosome. The most common form of X-linked intellectual disability (XLID) is fragile X syndrome. We report a family with an apparent XLID pattern with the proband, his mother and maternal half brother having an Xp21.3 deletion detected with chromosomal microarray analysis involving the interleukin 1 receptor accessory protein-like 1 (IL1RAPL1) gene. IL1RAPL1 is highly expressed in the postnatal brain, specifically hippocampus suggesting a specialized role in memory and learning abilities. The proband presented with intellectual disability, a broad face, prominent and wide nasal root, ptosis, a wide philtrum and a small mouth. XLID due to involvement of the IL1RAPL1 gene has been reported to cause nonsyndromic XLID. We report a new family with XLID due to partial deletion of IL1RAPL1, summarize reported literature and describe similar phenotypic similarities among the affected individuals in this family and those reported in the literature proposing that deletion of IL1RAPL1 may cause syndromic XLID. Additional reports are needed to further characterize whether syndromic features are related to disturbances of this gene.

  2. Identification of a De Novo 3bp Deletion in CRYBA1/A3 Gene in Autosomal Dominant Congenital Cataract.

    PubMed

    Mohebi, Masoumeh; Akbari, Abolfazl; Babaei, Nahid; Sadeghi, Abdolrahim; Heidari, Mansour

    2016-12-01

    Autosomal dominant congenital cataract (ADCC) is the most common form of inherited cataracts and accounts for one-third of congenital cataracts. Heterozygous null mutations in the crystallin genes are the major cause of the ADCC. This study aims to detect the mutational spectrum of four crystallin genes, CRYBA1/A3, CRYBB1, CRYBB2 and CRYGD in an Iranian family. Genomic DNA was isolated from whole blood cells from theproband and other family members. The coding regions and flanking intronicsequences of crystalline genes were analyzed by Sanger sequencing in aproband with ADCC. The identified mutation was further evaluated in available family members. To predict the potential protein partners of CRYBA1/A3, we also used an in-silico analysis. A de novo heterozygous deletion (c.272-274delGAG, p.G91del) in exon 4 of CRYBA1/A3 gene, leading to a deletion of Glycine at codon 91 was found. This genetic variation did not change the reading frame of CRYBA1 protein. In conclusion, we identified a de novo in-frame 3-bp deletion in the proband with an autosomal dominant congenital cataract, but not in her parents, in an Iranian family. This mutation has occurred de novo on a paternal gamete during spermatogenesis. The in-silico results predicted the interaction of CRYBA1 protein with the other CRY as well as proteins responsible for eye cell signaling.

  3. Association of deletion allele of insertion/deletion polymorphism in α2B adrenoceptor gene and hypertension with or without type 2 diabetes mellitus

    PubMed Central

    Tayel, Safaa I; Khader, Heba F; El-Helbawy, Nesreen G; Ibrahim, Waleed A

    2012-01-01

    Background Vascular α2B-adrenoreceptors have the potential to increase blood pressure by mediating vasoconstriction. A nine-nucleotide deletion in the receptor enhances vasoconstriction and exacerbates hypertension. The aim of this study was to determine the association between insertion/deletion (I/D) polymorphism of the α2B-adrenoceptor and hypertension with and without diabetes. Methods The study was carried out in 35 hypertensive patients with diabetes, 35 hypertensive patients without diabetes, and 30 healthy controls. Clinical data, blood lipid profiles, and I/D polymorphism were assessed. Results Hypertensive patients were significantly older, with significantly higher systolic/diastolic blood pressures and worse plasma lipid profiles than controls. The frequency of the DD genotype was significantly higher in both hypertensive patients with (77.14%, P < 0.01) and without (71.43%, P < 0.05) diabetes versus controls (40%). Also, the D allele was significantly more common in both hypertensive patients with (84.29%, P < 0.01) and without (80%, P < 0.05) diabetes versus controls (58.33%). Hypertensive patients were more likely to have the D allele with (3.83-fold) and without (2.85-fold) diabetes. The frequencies of the DD genotype and the D allele were not significantly (P > 0.05) different between the patient groups. The DD genotype was associated with significantly lower high-density lipoprotein (P = 0.001) and significantly higher low-density lipoprotein (P = 0.017) levels versus the II and ID genotypes in the hypertensive group without diabetes. Conclusion A marked and statistically significant association between DD genotype and D allele of I/D polymorphism in the α2B-adrenoceptor gene may be a risk factor for hypertension ± diabetes. The association between the DD genotype and dyslipidemia may partially explain its role in precipitating hypertension. PMID:23776387

  4. Ventilatory Chemosensory Drive Is Blunted in the mdx Mouse Model of Duchenne Muscular Dystrophy (DMD)

    PubMed Central

    Mosqueira, Matias; Baby, Santhosh M.; Khurana, Tejvir S.

    2013-01-01

    Duchenne Muscular Dystrophy (DMD) is caused by mutations in the DMD gene resulting in an absence of dystrophin in neurons and muscle. Respiratory failure is the most common cause of mortality and previous studies have largely concentrated on diaphragmatic muscle necrosis and respiratory failure component. Here, we investigated the integrity of respiratory control mechanisms in the mdx mouse model of DMD. Whole body plethysmograph in parallel with phrenic nerve activity recordings revealed a lower respiratory rate and minute ventilation during normoxia and a blunting of the hypoxic ventilatory reflex in response to mild levels of hypoxia together with a poor performance on a hypoxic stress test in mdx mice. Arterial blood gas analysis revealed low PaO2 and pH and high PaCO2 in mdx mice. To investigate chemosensory respiratory drive, we analyzed the carotid body by molecular and functional means. Dystrophin mRNA and protein was expressed in normal mice carotid bodies however, they are absent in mdx mice. Functional analysis revealed abnormalities in Dejours test and the early component of the hypercapnic ventilatory reflex in mdx mice. Together, these results demonstrate a malfunction in the peripheral chemosensory drive that would be predicted to contribute to the respiratory failure in mdx mice. These data suggest that investigating and monitoring peripheral chemosensory drive function may be useful for improving the management of DMD patients with respiratory failure. PMID:23922741

  5. Mutational Spectrum of DMD Mutations in Dystrophinopathy Patients: Application of Modern Diagnostic Techniques to a Large Cohort

    PubMed Central

    Flanigan, Kevin M.; Dunn, Diane; von Niederhausern, Andrew; Soltanzadeh, Payam; Gappmaier, Eduard; Howard, Michael T.; Sampson, Jacinda; Mendell, Jerry; Wall, Cheryl; King, Wendy; Pestronk, Alan; Florence, Julaine; Connolly, Anne; Mathews, Katherine D.; Stephan, Carrie; Laubenthal, Karla; Wong, Brenda; Morehart, Paula; Meyer, Amy; Finkel, Richard; Bonnemann, Carsten G.; Medne, Livija; Day, John W.; Dalton, Joline C.; Margolis, Marcia; Hinton, Veronica; Weiss, Robert B.

    2010-01-01

    Mutations in the DMD gene, encoding the dystrophin protein, are responsible for the dystrophinopathies Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), and X-linked Dilated Cardiomyopathy (XLDC). Mutation analysis has traditionally been challenging, due to the large gene size (79 exons over 2.2 Mb of genomic DNA). We report a very large aggregate data set comprised of DMD mutations detected in samples from patients enrolled in the United Dystrophinopathy Project, a multicenter research consortium, and in referral samples submitted for mutation analysis with a diagnosis of dystrophinopathy. We report 1111 mutations in the DMD gene, including 891 mutations with associated phenotypes. These results encompass 506 point mutations (including 294 nonsense mutations) and significantly expand the number of mutations associated with the dystrophinopathies, highlighting the utility of modern diagnostic techniques. Our data supports the uniform hypermutability of CGA>TGA mutations, establishes the frequency of polymorphic muscle (Dp427m) protein isoforms and reveals unique genomic haplotypes associated with `private' mutations. We note that 60% of these patients would be predicted to benefit from skipping of a single DMD exon using antisense oligonucleotide therapy, and 62% would be predicted to benefit from an inclusive multi-exon skipping approach directed toward exons 45 through 55. PMID:19937601

  6. Deletion of the RING-finger peroxin 2 gene in Aspergillus nidulans does not affect meiotic development.

    PubMed

    Hynes, Michael J; Murray, Sandra L; Kahn, Freya K

    2010-05-01

    Peroxins are required for protein import into peroxisomes as well as for peroxisome biogenesis and proliferation. Loss-of-function mutations in genes for the RING-finger peroxins Pex2, Pex10 and Pex12 lead to a specific block in meiosis in the ascomycete Podospora anserina. However, loss of protein import into peroxisomes does not result in this meiotic defect. Therefore, it has been suggested that these peroxins have a specific function required for meiosis. To determine whether this role is conserved in other filamentous fungi, we have deleted the gene encoding Pex2 in Aspergillus nidulans. The phenotypes resulting from this deletion are no different from those of previously isolated pex mutants affected in peroxisomal protein import, and viable ascospores are produced in selfed crosses. Therefore, the role of the RING-finger peroxins in meiosis is not conserved in filamentous ascomycetes.

  7. Acetylcholinesterase (AChE) gene modification in transgenic animals: functional consequences of selected exon and regulatory region deletion.

    PubMed

    Camp, Shelley; Zhang, Limin; Marquez, Michael; de la Torre, Brian; Long, Jeffery M; Bucht, Goran; Taylor, Palmer

    2005-12-15

    AChE is an alternatively spliced gene. Exons 2, 3 and 4 are invariantly spliced, and this sequence is responsible for catalytic function. The 3' alternatively spliced exons, 5 and 6, are responsible for AChE disposition in tissue [J. Massoulie, The origin of the molecular diversity and functional anchoring of cholinesterases. Neurosignals 11 (3) (2002) 130-143; Y. Li, S. Camp, P. Taylor, Tissue-specific expression and alternative mRNA processing of the mammalian acetylcholinesterase gene. J. Biol. Chem. 268 (8) (1993) 5790-5797]. The splice to exon 5 produces the GPI anchored form of AChE found in the hematopoietic system, whereas the splice to exon 6 produces a sequence that binds to the structural subunits PRiMA and ColQ, producing AChE expression in brain and muscle. A third alternative RNA species is present that is not spliced at the 3' end; the intron 3' of exon 4 is used as coding sequence and produces the read-through, unanchored form of AChE. In order to further understand the role of alternative splicing in the expression of the AChE gene, we have used homologous recombination in stem cells to produce gene specific deletions in mice. Alternatively and together exon 5 and exon 6 were deleted. A cassette containing the neomycin gene flanked by loxP sites was used to replace the exon(s) of interest. Tissue analysis of mice with exon 5 deleted and the neomycin cassette retained showed very low levels of AChE expression, far less than would have been anticipated. Only the read-through species of the enzyme was produced; clearly the inclusion of the selection cassette disrupted splicing of exon 4 to exon 6. The selection cassette was then deleted in exon 5, exon 6 and exons 5 + 6 deleted mice by breeding to Ella-cre transgenic mice. AChE expression in serum, brain and muscle has been analyzed. Another AChE gene targeted mouse strain involving a region in the first intron, found to be critical for AChE expression in muscle cells [S. Camp, L. Zhang, M. Marquez, B

  8. A novel AAT-deletion mutation in the coding sequence of the BCO2 gene in yellow-fat rabbits.

    PubMed

    Strychalski, Janusz; Brym, Paweł; Czarnik, Urszula; Gugołek, Andrzej

    2015-11-01

    The carcasses of yellow-fat rabbits may be attractive to modern consumers, because they have a relatively high content of biologically active compounds. One of the main candidate genes associated with the yellow-fat trait is β-carotene 9',10'-oxygenase (BCO2). This study is the first report of the novel AAT-deletion mutation at codon 248 of the BCO2 gene, which has been found in homozygous yellow-fat rabbits. The deletion mutation, located at the beginning of exon 6, results in the absence of asparagine in protein. We also developed a PCR-RFLP test that supports intravital genotyping of indel polymorphism based on genomic DNA.

  9. Scattered light in a DMD based multi-object spectrometer

    NASA Astrophysics Data System (ADS)

    Fourspring, Kenneth D.; Ninkov, Zoran; Kerekes, John P.

    2010-07-01

    The DMD (Digital Micromirror Device) has an important future in both ground and space based multi-object spectrometers. A series of laboratory measurements have been performed to determine the scattered light properties of a DMD. The DMD under test had a 17 μm pitch and 1 μm gap between adjacent mirrors. Prior characterization of this device has focused on its use in DLP (TI Digital Light Processing) projector applications in which a whole pixel is illuminated by a uniform collimated source. The purpose of performing these measurements is to determine the limiting signal to noise ratio when utilizing the DMD as a slit mask in a spectrometer. The DMD pixel was determined to scatter more around the pixel edge and central via, indicating the importance of matching the telescope point spread function to the DMD. Also, the generation of DMD tested here was determined to have a significant mirror curvature. A maximum contrast ratio was determined at several wavelengths. Further measurements are underway on a newer generation DMD device, which has a smaller mirror pitch and likely different scatter characteristics. A previously constructed instrument, RITMOS (RIT Multi-Object Spectrometer) will be used to validate these scatter models and signal to noise ratio predications through imaging a star field.

  10. A large deletion of the AVPR2 gene causing severe nephrogenic diabetes insipidus in a Turkish family.

    PubMed

    Saglar, Emel; Deniz, Ferhat; Erdem, Beril; Karaduman, Tugce; Yönem, Arif; Cagiltay, Eylem; Mergen, Hatice

    2014-05-01

    X-linked nephrogenic diabetes insipidus (NDI) is a rare hereditary disease caused by mutations in arginine vasopressin type 2 receptor (AVPR2) and characterized by the production of large amounts of urine and an inability to concentrate urine in response to the antidiuretic hormone vasopressin. We have identified a novel 388 bp deletion starting in intron 1 and ending in exon 2 in the AVPR2 gene in a patient with NDI and in his family. We have revealed that this mutation is a de novo mutation for the mother of the proband patient. Prospective clinical data were collected for all family members. The water deprivation test confirmed the diagnosis of diabetes insipidus. The patient has severe symptoms like deep polyuria nocturia, polydipsia, and fatigue. He was given arginine vasopressin treatment while he was a child. However, he could not get well due to his nephrogenic type of illness. Both of his nephews have the same complains in addition to failure to grow. We have sequenced all exons and intron-exon boundaries of the AVPR2 gene of all family members. The analyses of bioinformatics and comparative genomics of the deletion were done via considering the DNA level damage. AVPR2 gene mutation results in the absence of the three transmembrane domains, two extracellular domains, and one cytoplasmic domain. Three-dimensional protein structure prediction was shown. We concluded that X-linked NDI and severity of illness in this family is caused by a novel 388 bp deletion in the AVPR2 gene that is predicted to truncate the receptor protein, and also this deletion may lead to dysfunctioning in protein activity and inefficient or inadequate binding abilities.

  11. Rapid Diagnostic Tests for Malaria Diagnosis in the Peruvian Amazon: Impact of pfhrp2 Gene Deletions and Cross-Reactions

    PubMed Central

    Maltha, Jessica; Gamboa, Dionicia; Bendezu, Jorge; Sanchez, Luis; Cnops, Lieselotte; Gillet, Philippe; Jacobs, Jan

    2012-01-01

    Background In the Peruvian Amazon, Plasmodium falciparum and Plasmodium vivax malaria are endemic in rural areas, where microscopy is not available. Malaria rapid diagnostic tests (RDTs) provide quick and accurate diagnosis. However, pfhrp2 gene deletions may limit the use of histidine-rich protein-2 (PfHRP2) detecting RDTs. Further, cross-reactions of P. falciparum with P. vivax-specific test lines and vice versa may impair diagnostic specificity. Methods Thirteen RDT products were evaluated on 179 prospectively collected malaria positive samples. Species diagnosis was performed by microscopy and confirmed by PCR. Pfhrp2 gene deletions were assessed by PCR. Results Sensitivity for P. falciparum diagnosis was lower for PfHRP2 compared to P. falciparum-specific Plasmodium lactate dehydrogenase (Pf-pLDH)- detecting RDTs (71.6% vs. 98.7%, p<0.001). Most (19/21) false negative PfHRP2 results were associated with pfhrp2 gene deletions (25.7% of 74 P. falciparum samples). Diagnostic sensitivity for P. vivax (101 samples) was excellent, except for two products. In 10/12 P. vivax-detecting RDT products, cross-reactions with the PfHRP2 or Pf-pLDH line occurred at a median frequency of 2.5% (range 0%–10.9%) of P. vivax samples assessed. In two RDT products, two and one P. falciparum samples respectively cross-reacted with the Pv-pLDH line. Two Pf-pLDH/pan-pLDH-detecting RDTs showed excellent sensitivity with few (1.0%) cross-reactions but showed faint Pf-pLDH lines in 24.7% and 38.9% of P. falciparum samples. Conclusion PfHRP2-detecting RDTs are not suitable in the Peruvian Amazon due to pfhrp2 gene deletions. Two Pf-pLDH-detecting RDTs performed excellently and are promising RDTs for this region although faint test lines are of concern. PMID:22952633

  12. Candidate Genes and the Behavioral Phenotype in 22q11.2 Deletion Syndrome

    ERIC Educational Resources Information Center

    Prasad, Sarah E.; Howley, Sarah; Murphy, Kieran C.

    2008-01-01

    There is an overwhelming evidence that children and adults with 22q11.2 deletion syndrome (22q11.2DS) have a characteristic behavioral phenotype. In particular, there is a growing body of evidence that indicates an unequivocal association between 22q11.2DS and schizophrenia, especially in adulthood. Deletion of 22q11.2 is the third highest risk…

  13. Analysis of sporadic tuberous sclerosis patients with the TSC2 cDNA reveals several gene rearrangements and deletions

    SciTech Connect

    Wilson, P.J.; Short, M.P.; Bove, C.

    1994-09-01

    Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by hamartomas and hamartias in many organs including brain, skin, heart and kidneys. Two TSC genes have been localized through linkage analysis, TSC1 to 9q34 and TSC2 to 16p13.3. TSC2 was recently cloned. The distribution of sporadic TSC patients between TSC1 and TSC2 is at present unknown, but tests of genetic heterogeneity in families suggest that each is equally represented. Genetic heterogeneity may account for some of the variation in clinical expression; however, there is no evidence at present to support differences in clinical phenotypes between the 2 genetic loci. With the isolation of the TSC2 gene we have commenced mutation studies of our familial and sporadic TSC patients. Thus far six chromosome 16-linked families have been screened with the TSC2 cDNA and no detectable changes were observed using Southern analysis. In addition, 85 sporadic TSC patients were analyzed by Southern analysis. Using multiple restriction digests, nine patients revealed altered patterns, including three patients that appeared to have complete deletions. RT-PCR was performed on these patients confirming that the TSC2 gene was deleted. However, the remaining patients showed normal patterns, indicating that they either have TSC1 mutations or they possess more subtle small deletions or point mutations. At present we are designing an SSCP-based approach to determine the nature of the mutations in our 16 linked TSC families.

  14. Identification of a new human catenin gene family member (ARVCF) from the region deleted in velo-cardio-facial syndrome.

    PubMed

    Sirotkin, H; O'Donnell, H; DasGupta, R; Halford, S; St Jore, B; Puech, A; Parimoo, S; Morrow, B; Skoultchi, A; Weissman, S M; Scambler, P; Kucherlapati, R

    1997-04-01

    Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are characterized by a wide spectrum of phenotypes, including conotruncal heart defects, cleft palate, and facial dysmorphology. Hemizygosity for a portion of chromosome 22q11 has been detected in 80-85% of VCFS/DGS patients. Both syndromes are thought to be the result of a developmental field defect. Using two independent gene-isolation procedures, we isolated a new catenin family member termed ARVCF (armadillo repeat gene deleted in VCFS) from the interval deleted in VCFS. ARVCF encodes a protein of 962 amino acids that contains a coiled coil domain and 10 tandem armadillo repeats. The primary structure of the protein is most closely related to the murine catenin p120CAS, which suggests a role for ARVCF in protein-protein interactions at adherens junctions. ARVCF is expressed ubiquitously in all fetal and adult tissues examined. This gene is hemizygous in all VCFS patients with interstitial deletions. Based on the physical location and potential functions of ARVCF, we suggest that hemizygosity at this locus may play a role in the etiology of some of the phenotypes associated with VCFS.

  15. Recurrent Deletions of Puroindoline Genes at the Grain Hardness Locus in Four Independent Lineages of Polyploid Wheat1[W][OA

    PubMed Central

    Li, Wanlong; Huang, Li; Gill, Bikram S.

    2008-01-01

    Polyploidy is known to induce numerous genetic and epigenetic changes but little is known about their physiological bases. In wheat, grain texture is mainly determined by the Hardness (Ha) locus consisting of genes Puroindoline a (Pina) and b (Pinb). These genes are conserved in diploid progenitors but were deleted from the A and B genomes of tetraploid Triticum turgidum (AB). We now report the recurrent deletions of Pina-Pinb in other lineages of polyploid wheat. We analyzed the Ha haplotype structure in 90 diploid and 300 polyploid accessions of Triticum and Aegilops spp. Pin genes were conserved in all diploid species and deletion haplotypes were detected in all polyploid Triticum and most of the polyploid Aegilops spp. Two Pina-Pinb deletion haplotypes were found in hexaploid wheat (Triticum aestivum; ABD). Pina and Pinb were eliminated from the G genome, but maintained in the A genome of tetraploid Triticum timopheevii (AG). Subsequently, Pina and Pinb were deleted from the A genome but retained in the Am genome of hexaploid Triticum zhukovskyi (AmAG). Comparison of deletion breakpoints demonstrated that the Pina-Pinb deletion occurred independently and recurrently in the four polyploid wheat species. The implications of Pina-Pinb deletions for polyploid-driven evolution of gene and genome and its possible physiological significance are discussed. PMID:18024553

  16. Encephalopathy and bilateral cataract in a boy with an interstitial deletion of Xp22 comprising the CDKL5 and NHS genes.

    PubMed

    Van Esch, Hilde; Jansen, Anna; Bauters, Marijke; Froyen, Guy; Fryns, Jean-Pierre

    2007-02-15

    We describe a male patient with a deletion at Xp22, detected by high resolution X-array CGH. The clinical phenotype present in this infant boy, consists of severe encephalopathy, congenital cataracts and tetralogy of Fallot and can be attributed to the deletion of the genes within the interval. Among these deleted genes are the gene for Nance-Horan syndrome and the cyclin-dependent kinase-like 5 gene (CDKL5), responsible for the early seizure variant of Rett syndrome. This is the first description of a male patient with a deletion of these genes, showing the involvement of CDKL5 in severe epileptic encephalopathy in males. Moreover it illustrates the added value of high resolution array-CGH in molecular diagnosis of mental retardation-multiple congenital anomaly cases.

  17. Deletion analysis of Streptococcus pneumoniae late competence genes distinguishes virulence determinants that are dependent or independent of competence induction

    PubMed Central

    Zhu, Luchang; Lin, Jingjun; Kuang, Zhizhou; Vidal, Jorge E.; Lau, Gee W.

    2015-01-01

    Summary The competence regulon of Streptococcus pneumoniae (pneumococcus) is crucial for genetic transformation. During competence development, the alternative sigma factor ComX is activated, which in turn, initiates transcription of 80 “late” competence genes. Interestingly, only 16 late genes are essential for genetic transformation. We hypothesized that these late genes that are dispensable for competence are beneficial to pneumococcal fitness during infection. These late genes were systematically deleted, and the resulting mutants were examined for their fitness during mouse models of bacteremia and acute pneumonia. Among these, 14 late genes were important for fitness in mice. Significantly, deletion of some late genes attenuated pneumococcal fitness to the same level in both wild-type and ComX-null genetic backgrounds, suggesting that the constitutive baseline expression of these genes was important for bacterial fitness. In contrast, some mutants were attenuated only in the wild-type genetic background but not in the ComX-null background, suggesting that specific expression of these genes during competence state contributed to pneumococcal fitness. Increased virulence during competence state was partially caused by the induction of allolytic enzymes that enhanced pneumolysin release. These results distinguish the role of basal expression versus competence induction in virulence functions encoded by ComX-regulated late competence genes. Graphical abstract During genetic transformation of pneumococcus, the alternative sigma factor ComX regulates expression of 14 late competence genes important for virulence. The constitutive baseline expression of some of these genes is important for bacteremia and acute pneumonia infections. In contrast, elevated expression of DprA, CbpD, CibAB, and Cinbox are dependent on competence development, enhancing the release of pneumolysin. These results distinguish the role of basal expression versus competence induction in

  18. Identification of two rare and novel large deletions in ITGB4 gene causing epidermolysis bullosa with pyloric atresia.

    PubMed

    Mencía, Ángeles; García, Marta; García, Eva; Llames, Sara; Charlesworth, Alexandra; de Lucas, Raúl; Vicente, Asunción; Trujillo-Tiebas, María José; Coto, Pablo; Costa, Marta; Vera, Ángel; López-Pestaña, Arantxa; Murillas, Rodolfo; Meneguzzi, Guerrino; Jorcano, José Luis; Conti, Claudio J; Escámez Toledano, María José; del Río Nechaevsky, Marcela

    2016-04-01

    Epidermolysis bullosa with pyloric atresia (EB-PA) is a rare autosomal recessive hereditary disease with a variable prognosis from lethal to very mild. EB-PA is classified into Simplex form (EBS-PA: OMIM #612138) and Junctional form (JEB-PA: OMIM #226730), and it is caused by mutations in ITGA6, ITGB4 and PLEC genes. We report the analysis of six patients with EB-PA, including two dizygotic twins. Skin immunofluorescence epitope mapping was performed followed by PCR and direct sequencing of the ITGB4 gene. Two of the patients presented with non-lethal EB-PA associated with missense ITGB4 gene mutations. For the other four, early postnatal demise was associated with complete lack of β4 integrin due to a variety of ITGB4 novel mutations (2 large deletions, 1 splice-site mutation and 3 missense mutations). One of the deletions spanned 278 bp, being one of the largest reported to date for this gene. Remarkably, we also found for the first time a founder effect for one novel mutation in the ITGB4 gene. We have identified 6 novel mutations in the ITGB4 gene to be added to the mutation database. Our results reveal genotype-phenotype correlations that contribute to the molecular understanding of this heterogeneous disease, a pivotal issue for prognosis and for the development of novel evidence-based therapeutic options for EB management.

  19. Contribution of Scaffoldins to Biomass Degradation by Clostridium Thermocellum: The Effect of Scaffoldin-Deletions on Expression of Other Genes

    SciTech Connect

    Xu, Qi; Podkaminer, Kara; Resch, Michael G.; Donohoe, Bryon; Olson, Daniel G.; Baker, John O.; Klingeman, Dawn M.; Syed, Mustafa; Wilson, Charlotte M.; Brown, Steven D.; Yang, Shihui; Magnusson, Lauren; Maness, Pin-Ching; Decker, Steve R.; Lynd, Lee R.; Bomble, Yannick J.; Himmel, Michael E.

    2014-04-28

    The cellulosome system contributes greatly to the extreme efficiency of C. thermocellum cellulose degradation. In order to further understand the cellulosome working mechanism, we have knocked out C. thermocellum scaffoldin genes to generate a variety of deletion mutants. The knockout most detrimental to enzymatic hydrolysis by the secretome is that of the primary scaffoldin CipA. Deletion of multiple secondary scaffoldins results in secretome activities intermediate between those of the parent strain and the CipA-knockout mutants. The order of relative secretome activities is the same, whether the cellulosic substrate is microcrystalline cellulose (Avicel) or deacetylated acid-pretreated corn stover (DACS), but the relative magnitudes of the deletion effects are strongly substrate-dependent. Similar trends are observed in fermentation studies of the abilities of the parent and knockout strains themselves to utilize Avicel and DACS. Data from transcriptomic and proteomic studies of these strains when grown on both substrates are used to relate the activity and growth effects of the deletions to their effects on the overall expression of lignocellulose-degrading enzymes by C. thermocellum.

  20. Differential Gene Expression Reveals Mitochondrial Dysfunction in an Imprinting Center Deletion Mouse Model of Prader-Willi Syndrome

    PubMed Central

    Fan, Weiwei; Coskun, Pinar E.; Nalbandian, Angèle; Knoblach, Susan; Resnick, James L.; Hoffman, Eric; Wallace, Douglas C.; Kimonis, Virginia E.

    2013-01-01

    Prader-Willi syndrome (PWS) is a genetic disorder caused by deficiency of imprinted gene expression from the paternal chromosome 15q11-15q13 and clinically characterized by neonatal hypotonia, short stature, cognitive impairment, hypogonadism, hyperphagia, morbid obesity and diabetes. Previous clinical studies suggest that a defect in energy metabolism may be involved in the pathogenesis of PWS. We focused our attention on the genes associated with energy metabolism and found that there were 95 and 66 mitochondrial genes differentially expressed in PWS muscle and brain, respectively. Assessment of enzyme activities of mitochondrial oxidative phosphorylation (OXPHOS) complexes in the brain, heart, liver and muscle were assessed. We found the enzyme activities of the cardiac mitochondrial complexes II+III were upregulated in the imprinting center deletion (PWS-IC) mice compared to the wild type littermates. These studies suggest that differential gene expression, especially of the mitochondrial genes may contribute to the pathophysiology of PWS. PMID:24127921

  1. The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase, is caused by a deletion in the VRN1 gene.

    PubMed

    Shitsukawa, Naoki; Ikari, Chihiro; Shimada, Sanae; Kitagawa, Satoshi; Sakamoto, Koichi; Saito, Hiroyuki; Ryuto, Hiromichi; Fukunishi, Nobuhisa; Abe, Tomoko; Takumi, Shigeo; Nasuda, Shuhei; Murai, Koji

    2007-04-01

    The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase (mvp), was induced by nitrogen ion-beam treatment and was identified by its inability to transit from the vegetative to reproductive phase. In our previous study, we showed that WAP1 (wheat APETALA1) is a key gene in the regulatory pathway that controls phase transition from vegetative to reproductive growth in common wheat. WAP1 is an ortholog of the VRN1 gene that is responsible for vernalization insensitivity in einkorn wheat. The mvp mutation resulted from deletion of the VRN1 coding and promoter regions, demonstrating that WAP1/VRN1 is an indispensable gene for phase transition in wheat. Expression analysis of flowering-related genes in mvp plants indicated that wheat GIGANTIA (GI), CONSTANS (CO) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) genes either act upstream of or in a different pathway to WAP1/VRN1.

  2. Central precocious puberty in a patient with X-linked adrenal hypoplasia congenita and Xp21 contiguous gene deletion syndrome.

    PubMed

    Koh, Ji Won; Kang, So Young; Kim, Gu Hwan; Yoo, Han Wook; Yu, Jeesuk

    2013-06-01

    X-linked adrenal hypoplasia congenita is caused by the mutation of DAX-1 gene (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1), and can occur as part of a contiguous gene deletion syndrome in association with glycerol kinase (GK) deficiency, Duchenne muscular dystrophy and X-linked interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) gene deficiency. It is usually associated with hypogonadotropic hypogonadism, although in rare cases, it has been reported to occur in normal puberty or even central precocious puberty. This study addresses a case in which central precocious puberty developed in a boy with X-linked adrenal hypoplasia congenita who had complete deletion of the genes DAX-1, GK and IL1RAPL1 (Xp21 contiguous gene deletion syndrome). Initially he was admitted for the management of adrenal crisis at the age of 2 months, and managed with hydrocortisone and florinef. At 45 months of age, his each testicular volumes of 4 mL and a penile length of 5 cm were noted, with pubic hair of Tanner stage 2. His bone age was advanced and a gonadotropin-releasing hormone (GnRH) stimulation test showed a luteinizing hormone peak of 8.26 IU/L, confirming central precocious puberty. He was then treated with a GnRH agonist, as well as steroid replacement therapy. In Korea, this is the first case of central precocious puberty developed in a male patient with X-linked adrenal hypoplasia congenita.

  3. Clinically relevant single gene or intragenic deletions encompassing critical neurodevelopmental genes in patients with developmental delay, mental retardation, and/or autism spectrum disorders.

    PubMed

    Mikhail, Fady M; Lose, Edward J; Robin, Nathaniel H; Descartes, Maria D; Rutledge, Katherine D; Rutledge, S Lane; Korf, Bruce R; Carroll, Andrew J

    2011-10-01

    Recent studies suggest that copy number variations (CNVs) encompassing several genes involved in neurodevelopmental pathways are associated with a variety of neuropsychiatric phenotypes, including developmental delay (DD), mental retardation (MR), and autism spectrum disorders (ASDs). Here we present eight patients in a cohort of approximately 1,200 patients referred for clinical array CGH testing for various neurodevelopmental phenotypes,whowere identified to carry small (<1.0Mb with the majority <500 kb) either total gene or intragenic deletions encompassing critical synaptic and other neurodevelopmental genes. The presentations of these patients included variable degrees of DD, speech problems, learning disabilities, MR, autistic-like features, and mild non-specific dysmorphic features. These genes belong to four functional categories, including neuronal transcription factor genes (NFIA at 1p31.3, MEF2C at 5q14.3, andCAMAT1at 1p36.23p36.31), neuron-specific splicing factor genes (RBFOX1 at 16p13.2p13.3), genes involved in synapse formation and maintenance (CNTNAP2 at 7q35 and LRFN5 at 14q21.2), and genes involved in neurotransmission (CHRNA7 at 15q13.3 and IL1RAPL1 at Xp21.2p21.3). Our report expands the list of neurodevelopmental genes deleted in various neurobehavioral phenotypes, expands the phenotypes caused by haploinsufficiency of previously reported critical neurodevelopmental genes, and elucidates the clinical relevance and need for careful clinical interpretation of some small CNVs<500 kb. This report also suggests that small clinically relevant deletions encompassing critical synaptic and other neurodevelopmental genes can present clinically with various neurobehavioral phenotypes, which implies the existence of overlapping neuronal pathways in the pathogenesis of these phenotypes.

  4. Unusual presentation of pelizaeus-merzbacher disease: female patient with deletion of the proteolipid protein 1 gene.

    PubMed

    Brender, Teva; Wallerstein, Donna; Sum, John; Wallerstein, Robert

    2015-01-01

    Pelizaeus-Merzbacher disease (PMD) is neurodegenerative leukodystrophy caused by dysfunction of the proteolipid protein 1 (PLP1) gene on Xq22, which codes for an essential myelin protein. As an X-linked condition, PMD primarily affects males; however there have been a small number of affected females reported in the medical literature with a variety of different mutations in this gene. No affected females to date have a deletion like our patient. In addition to this, our patient has skewed X chromosome inactivation which adds to her presentation as her unaffected mother also carries the mutation.

  5. Unusual Presentation of Pelizaeus-Merzbacher Disease: Female Patient with Deletion of the Proteolipid Protein 1 Gene

    PubMed Central

    Brender, Teva; Wallerstein, Donna; Sum, John; Wallerstein, Robert

    2015-01-01

    Pelizaeus-Merzbacher disease (PMD) is neurodegenerative leukodystrophy caused by dysfunction of the proteolipid protein 1 (PLP1) gene on Xq22, which codes for an essential myelin protein. As an X-linked condition, PMD primarily affects males; however there have been a small number of affected females reported in the medical literature with a variety of different mutations in this gene. No affected females to date have a deletion like our patient. In addition to this, our patient has skewed X chromosome inactivation which adds to her presentation as her unaffected mother also carries the mutation. PMID:25789183

  6. Transposition of a duplicate antibiotic resistance gene and generation of deletions in plasmid R6K.

    PubMed Central

    Holmans, P L; Clowes, R C

    1979-01-01

    Transformation experiments showed that spontaneous deletions which result in loss of streptomycin resistance and an increase in conjugal transfer efficiency are present at a frequency of about 10(-4) in plasmid molecules of R6K. Similar deletions were thus readily selected by conjugal transfer of R6K, and their appearance was dependent upon recA+ activity in either donor or recipient host. The deoxyribonucleic acid segment deleted in four mutants examined was concluded to extend from the same terminus of the transposon, TnA, in the same direction, but to different extents, and to retain the TnA region intact. Insertions of a duplicate TnA element were found in R6K plasmids isolated from strains selected for increased ampicillin resistance, which were unstable in recA+ strains. In four plasmids examined after transfer to a recA host, an inverted repeat of the preexisting TnA element was shown to have been inserted at a similar location and was in two instances associated with deletions which extended from the same direction as those described above. The deletions are ascribed to the result of recA+-dependent recombination between direct repeats of TnA. Images PMID:370107

  7. 1p13.2 deletion displays clinical features overlapping Noonan syndrome, likely related to NRAS gene haploinsufficiency

    PubMed Central

    Linhares, Natália Duarte; Freire, Maíra Cristina Menezes; Cardenas, Raony Guimarães Corrêa do Carmo Lisboa; Pena, Heloisa Barbosa; Lachlan, Katherine; Dallapiccola, Bruno; Bacino, Carlos; Delobel, Bruno; James, Paul; Thuresson, Ann-Charlotte; Annerén, Göran; Pena, Sérgio D. J.

    2016-01-01

    Abstract Deletion-induced hemizygosity may unmask deleterious autosomal recessive variants and be a cause of the phenotypic variability observed in microdeletion syndromes. We performed complete exome sequencing (WES) analysis to examine this possibility in a patient with 1p13.2 microdeletion. Since the patient displayed clinical features suggestive of Noonan Syndrome (NS), we also used WES to rule out the presence of pathogenic variants in any of the genes associated with the different types of NS. We concluded that the clinical findings could be attributed solely to the 1p13.2 haploinsufficiency. Retrospective analysis of other nine reported patients with 1p13.2 microdeletions showed that six of them also presented some characteristics of NS. In all these cases, the deleted segment included the NRAS gene. Gain-of-function mutations of NRAS gene are causally related to NS type 6. Thus, it is conceivable that NRAS haploinsufficiency and gain-of-function mutations may have similar clinical consequences. The same phenomenon has been described for two other genes belonging to the Ras/MAPK pathway: MAP2K2 and SHOC2. In conclusion, we here report genotype-phenotype correlations in patients with chromosome 1p13.2 microdeletions and we propose that NRAS may be a critical gene for the NS characteristics in the patients. PMID:27561113

  8. AVAQ 594-597 deletion of the TfR2 gene in a Japanese family with hemochromatosis.

    PubMed

    Hattori, Ai; Wakusawa, Shinnya; Hayashi, Hisao; Harashima, Ai; Sanae, Fujiko; Kawanaka, Miwa; Yamada, Gohtaro; Yano, Motoyashi; Yoshioka, Kenntaro

    2003-06-01

    The majority of Caucasian patients with hemochromatosis are homozygous for C282Y mutation of the HFE gene. In contrast to its high prevalence in Caucasians, hemochromatosis is a rare disorder in Japan. This may be due to the low prevalence of the C282Y mutation of the HFE gene in Japanese. Recent reports suggest that the mutations of transferrin receptor 2 (TfR2) gene may be involved in non-HFE hemochromatosis. Therefore, we investigated the TfR2 gene of 6 sporadic and 5 familiar cases of Japanese hemochromatosis. Three siblings in one family were found to be homozygous for an AVAQ 594-597 deletion. All three had severe iron deposits in the hepatocytes and bile ducts, but none was affected by diabetes mellitus. This mutation was not detected in 100 control individuals. Further study was undertaken to investigate whether the large deletion of the TfR2 gene is the mutation responsible for some of the Japanese hemochromatosis cases.

  9. Targeted deletion of Hand2 in cardiac neural crest-derived cells influences cardiac gene expression and outflow tract development

    PubMed Central

    Holler, Kristen L.; Hendershot, Tyler J.; Troy, Sophia E.; Vincentz, Joshua W.; Firulli, Anthony B.; Howard, Marthe J.

    2010-01-01

    The basic helix-loop-helix DNA binding protein Hand2 has critical functions in cardiac development both in neural crest-derived and mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest has allowed us to genetically dissect Hand2-dependent defects specifically in outflow tract and cardiac cushion independent of Hand2 functions in mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest results in misalignment of the aortic arch arteries and outflow tract, contributing to development of double outlet right ventricle (DORV) and ventricular septal defects (VSD). These neural crest-derived developmental anomalies are associated with altered expression of Hand2-target genes we have identified by gene profiling. A number of Hand2 direct target genes have been identified using ChIP and ChIP-on-chip analyses. We have identified and validated a number of genes related to cell migration, proliferation/cell cycle and intracellular signaling whose expression is affected by Hand2 deletion in the neural crest and which are associated with development of VSD and DORV. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting expression of target genes associated with a number of functional interactions in neural crest-derived cells required for proper patterning of the outflow tract, generation of the appropriate number of neural crest-derived cells for elongation of the conotruncus and cardiac cushion organization. Our genetic model has made it possible to investigate the molecular genetics of neural crest contributions to outflow tract morphogenesis and cell differentiation. PMID:20144608

  10. Improved α-amylase production by Aspergillus oryzae after a double deletion of genes involved in carbon catabolite repression.

    PubMed

    Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

    2014-01-01

    In filamentous fungi, the expression of secretory glycoside hydrolase encoding genes, such as those for amylases, cellulases, and xylanases, is generally repressed in the presence of glucose. CreA and CreB have been observed to be regulating factors for carbon catabolite repression. In this study, we generated single and double deletion creA and/or creB mutants in Aspergillus oryzae. The α-amylase activities of each strain were compared under various culture conditions. For the wild-type strain, mRNA levels of α-amylase were markedly decreased in the later stage of submerged culture under inducing conditions, whereas this reduced expression was not observed for single creA and double creA/creB deletion mutants. In addition, α-amylase activity of the wild-type strain was reduced in submerged culture containing high concentrations of inducing sugars, whereas all constructed mutants showed higher α-amylase activities. In particular, the α-amylase activity of the double deletion mutant in a medium containing 5% starch was >10-fold higher than that of the wild-type strain under the same culture conditions. In solid-state cultures using wheat bran as a substrate, the α-amylase activities of single creA and double deletion mutants were >2-fold higher than that of the wild-type strain. These results suggested that deleting both creA and creB resulted in dramatic improvements in the production of secretory glycoside hydrolases in filamentous fungi.

  11. A Case of 9.7 Mb Terminal Xp Deletion Including OA1 Locus Associated with Contiguous Gene Syndrome

    PubMed Central

    Cho, Eun-Hae; Kim, Sook-Young

    2012-01-01

    Terminal or interstitial deletions of Xp (Xp22.2→Xpter) in males have been recognized as a cause of contiguous gene syndromes showing variable association of apparently unrelated clinical manifestations such as Leri-Weill dyschondrosteosis (SHOX), chondrodysplasia punctata (CDPX1), mental retardation (NLGN4), ichthyosis (STS), Kallmann syndrome (KAL1), and ocular albinism (GPR143). Here we present a case of a 13.5 yr old boy and sister with a same terminal deletion of Xp22.2 resulting in the absence of genes from the telomere of Xp to GPR143 of Xp22. The boy manifested the findings of all of the disorders mentioned above. We began a testosterone enanthate monthly replacement therapy. His sister, 11 yr old, manifested only Leri-Weill dyschondrosteosis, and had engaged in growth hormone therapy for 3 yr. To the best of our knowledge, this is the first report of a male with a 9.7 Mb terminal Xp deletion including the OA1 locus in Korea. PMID:23091330

  12. Effect of deletion and overexpression of tryptophan metabolism genes on growth and fermentation capacity at low temperature in wine yeast.

    PubMed

    López-Malo, María; García-Rios, Estefani; Chiva, Rosana; Guillamon, José Manuel; Martí-Raga, María

    2014-01-01

    Low-temperature fermentations produce wines with greater aromatic complexity, but the success of these fermentations greatly depends on the adaptation of yeast cells to cold. Tryptophan has been previously reported to be a limiting amino acid during Saccharomyces cerevisiae growth at low temperature. The objective of this study was to determine the influence of the tryptophan metabolism on growth and fermentation performance during low-temperature wine fermentation. To this end, we constructed the deletion mutants of the TRP1 and TAT2 genes in a derivative haploid of a commercial wine strain, and the TAT2 gene was overexpressed in the prototroph and auxotroph (Δtrp1) backgrounds. Then we characterized growth and fermentation activity during wine fermentation at low and optimum temperatures. Our results partially support the role of this amino acid in cold yeast growth. Although deletion of TRP1 impaired amino acid uptake and the growth rate at low temperature in synthetic must, this growth impairment did not affect the fermentation rate. Deletion of TAT2 endorsed this strain with the highest nitrogen consumption capacity and the greatest fermentation activity at low temperature. Our results also evidenced reduced ammonium consumption in all the strains at low temperature.

  13. Central precocious puberty in a boy with 22q13 deletion syndrome and NOTCH-1 gene duplication.

    PubMed

    Giannakopoulos, Aris; Fryssira, Helen; Tzetis, Maria; Xaidara, Athina; Kanaka-Gantenbein, Christina

    2016-11-01

    The 22q13 deletion syndrome or Phelan-McDermid syndrome is a neurodevelopmental disorder associated with developmental delay, hypotonia, delayed or absent speech, autistic-like behavior, normal to accelerated growth and dysmorphic faces. We report the occurrence of central precocious puberty in a boy diagnosed with Phelan-McDermid syndrome. At the age of 1 year, our patient presented with increased testicular volume for his age, bone age advancement and growth acceleration. Stimulated gonadotropin levels demonstrated a premature activation of the hypothalamic-pituitary-gonadal (HPG) axis. Central precocious puberty was treated with gonadotropin-releasing hormone (GnRH) analog. Molecular diagnosis with array-comparative genomic hybridization (CGH) revealed a major deletion of 5.8 Mb at the 22q13 chromosomal region and a 25 kb duplication at the 9q34.3 region that included the NOTCH-1 gene. On the background of 22q13 deletion syndrome and data from animals on the effect of abnormal NOTCH-1 gene expression on kisspeptin neuron formation, we discuss the probable role of Notch signaling in the premature activation of the HPG axis.

  14. Unexpected effects of gene deletion on mercury interactions with the methylation-deficient mutant hgcAB

    SciTech Connect

    Lin, Hui; Hurt, Jr., Richard Ashley; Johs, Alexander; Parks, Jerry M; Morrell-Falvey, Jennifer L; Liang, Liyuan; Elias, Dwayne A; Gu, Baohua

    2014-01-01

    The hgcA and hgcB gene pair is essential for mercury (Hg) methylation by certain anaerobic bacteria,1 but little is known about how deletion of hgcAB affects cell surface interactions and intracellular uptake of Hg. Here, we compare hgcAB mutants with the wild-type (WT) strains of both Geobacter sulfurreducens PCA and Desulfovibrio desulfuricans ND132 and observe differences in Hg redox transformations, adsorption, and uptake in laboratory incubation studies. In both strains, deletion of hgcAB increased the reduction of Hg(II) but decreased the oxidation of Hg(0) under anaerobic conditions. The measured cellular thiol content in hgcAB mutants was lower than the WT, accounting for decreased adsorption and uptake of Hg. Despite the lack of methylation activity, Hg uptake by the hgcAB continued, albeit at a slower rate than the WT. These findings demonstrate that deletion of the hgcAB gene not only eliminates Hg methylation but also alters cell physiology, resulting in changes to Hg redox reactions, sorption, and uptake by cells.

  15. EZH2 expression in gliomas: Correlation with CDKN2A gene deletion/ p16 loss and MIB-1 proliferation index.

    PubMed

    Purkait, Suvendu; Sharma, Vikas; Jha, Prerana; Sharma, Mehar Chand; Suri, Vaishali; Suri, Ashish; Sharma, B S; Sarkar, Chitra

    2015-10-01

    Enhancer of zeste homolog 2 (EZH2) mediated down-regulation of CDKN2A/p16 has been observed in cell lines as well as in a few carcinomas. However, there is no study correlating EZH2 expression with CDKN2A/p16 status in gliomas. Hence, the present study was conducted to evaluate EZH2 expression in astrocytic and oligodendroglial tumors and correlate with CDKN2A/p16 status as well as MIB-1 labeling index (LI). Gliomas of all grades (n = 118) were studied using immunohistochemistry to assess EZH2, p16 and MIB-1 LI and fluorescence in situ hybrization to evaluate CDKN2A gene status. EZH2 expression and CDKN2A homozygous deletion (HD) were both significantly more frequent in high-grade gliomas (HGG). Further, strong EZH2 expression (LI ≥ 25%) was significantly more common in HGGs without CDKN2A HD (48.7%; 19/39) as compared to cases with deletion (15.8%; 3/19). Loss of p16 expression was noted in 100% and 51.3% of CDKN2A deleted and non-deleted tumors, respectively. Notably, 80% (16/20) of the CDKN2A non-deleted HGGs with p16 loss had strong EZH2 expression, in contrast to only 15.8% (3/19) in the deleted group. Loss of p16 expression significantly correlated with MIB-1 LI, irrespective of EZH2 status. Thus, this study shows that EZH2 expression correlates with tumor grade in both astrocytic and oligodendroglial tumors and hence can be used as a diagnostic marker to differentiate between low and HGGs. Further, this is the first report demonstrating an inverse correlation of strong EZH2 expression with CDKN2A HD in HGGs. Loss of p16 protein expression is mostly attributable to CDKN2A HD and correlates significantly with MIB-1 LI. Notably, our study for the first time suggests a possible epigenetic mechanism of p16 loss in CDKN2A non-deleted HGGs mediated by strong EZH2 expression. A hypothetical model for control of proliferative activity in low versus HGGs is therefore proposed.

  16. Understanding the Role of Tbx1 as a Candidate Gene for 22q11.2 Deletion Syndrome

    PubMed Central

    Gao, Shan; Li, Xiao; Amendt, Brad A.

    2013-01-01

    22q11.2 deletion syndrome (22q11.2DS) is caused by a commonly occurring microdeletion on chromosome 22. Clinical findings include cardiac malformations, thymic and parathyroid hypoplasia, craniofacial dysmorphisms, and dental defects. These phenotypes are due mainly to abnormal development of the pharyngeal apparatus. Targeted deletion studies in mice and analysis of naturally occurring mutations in humans have implicated Tbx1 as a candidate gene for 22q11.2DS. Tbx1 belongs to an evolutionarily conserved T-box family of transcription factors, whose expression is precisely regulated during embryogenesis, and it appears to regulate the proliferation and differentiation of various progenitor cells during organogenesis. In this review, we discuss the mechanisms of Tbx1 during development of the heart, thymus and parathyroid glands, as well as during formation of the palate, teeth, and other craniofacial features. PMID:23996541

  17. Deletion/duplication mutation screening of TP53 gene in patients with transitional cell carcinoma of urinary bladder using multiplex ligation-dependent probe amplification.

    PubMed

    Bazrafshani, Mohammad Reza R; Nowshadi, Pouriaali A; Shirian, Sadegh; Daneshbod, Yahya; Nabipour, Fatemeh; Mokhtari, Maral; Hosseini, Fatemehsadat; Dehghan, Somayeh; Saeedzadeh, Abolfazl; Mosayebi, Ziba

    2016-02-01

    Bladder cancer is a molecular disease driven by the accumulation of genetic, epigenetic, and environmental factors. The aim of this study was to detect the deletions/duplication mutations in TP53 gene exons using multiplex ligation-dependent probe amplification (MLPA) method in the patients with transitional cell carcinoma (TCC). The achieved formalin-fixed paraffin-embedded tissues from 60 patients with TCC of bladder were screened for exonal deletions or duplications of every 12 TP53 gene exons using MLPA. The pathological sections were examined by three pathologists and categorized according to the WHO scoring guideline as 18 (30%) grade I, 22 (37%) grade II, 13 (22%) grade III, and 7 (11%) grade IV cases of TCC. None mutation changes of TP53 gene were detected in 24 (40%) of the patients. Furthermore, mutation changes including, 15 (25%) deletion, 17 (28%) duplication, and 4 (7%) both deletion and duplication cases were observed among 60 samples. From 12 exons of TP53 gene, exon 1 was more subjected to exonal deletion. Deletion of exon 1 of TP53 gene has occurred in 11 (35.4%) patients with TCC. In general, most mutations of TP53, either deletion or duplication, were found in exon 1, which was statistically significant. In addition, no relation between the TCC tumor grade and any type of mutation were observed in this research. MLPA is a simple and efficient method to analyze genomic deletions and duplications of all 12 exons of TP53 gene. The finding of this report that most of the mutations of TP53 occur in exon 1 is in contrast to that of the other reports suggesting that exons 5-8 are the most (frequently) mutated exons of TP53 gene. The mutations of exon 1 of TP53 gene may play an important role in the tumorogenesis of TCC.

  18. The major and minor chicken vitellogenin genes are each adjacent to partially deleted pseudogene copies of the other.

    PubMed Central

    Silva, R; Fischer, A H; Burch, J B

    1989-01-01

    The major chicken vitellogenin gene (VTGII) has previously been cloned and sequenced. We now report the isolation of genomic clones that encompass a minor chicken vitellogenin gene (VTGIII) which is also expressed in the liver in response to estradiol. Our analysis reveals that a pseudogene for VTGII (psi VTGII) lies 1,426 base pairs upstream of this VTGIII gene. A reevaluation of published sequence data reveals that the converse is also true, namely, that a pseudogene for VTGIII (psi VTGIII) lies 1,345 base pairs downstream of the VTGII gene. Our results show that a 335-base-pair deletion has removed the psi VTGIII promoter and cap site but left residual estrogen response element in a region where nuclease-hypersensitive sites have been reported to be induced in response to estradiol. Images PMID:2796998

  19. Targeted viral delivery of Cre recombinase induces conditional gene deletion in cardiovascular circuits of the mouse brain.

    PubMed

    Sinnayah, Puspha; Lindley, Timothy E; Staber, Patrick D; Davidson, Beverly L; Cassell, Martin D; Davisson, Robin L

    2004-06-17

    The Cre/loxP system has shown promise for investigating genes involved in nervous system function and pathology, although its application for studying central neural regulation of cardiovascular function and disease has not been explored. Here, we report for the first time that recombination of loxP-flanked genes can be achieved in discrete cardiovascular regulatory nuclei of adult mouse brain using targeted delivery of adenovirus (Ad) or feline immunodeficiency virus (FIV) bearing Cre recombinase (Ad-Cre, FIV-Cre). Single stereotaxic microinjections of Ad-Cre or FIV-Cre into specific nuclei along the subfornical organ-hypothalamic-hypophysial and brain stem-parabrachial axes resulted in robust and highly localized gene deletion as early as 7 days and for as long as 3 wk in a reporter mouse model in which Cre recombinase activates beta-galactosidase expression. An even greater selectivity in Cre-mediated gene deletion could be achieved in unique subpopulations of cells, such as vasopressin-synthesizing magnocellular neurons, by delivering Ad-Cre via retrograde transport. Moreover, Ad-Cre and FIV-Cre induced gene recombination in differential cell populations within these cardiovascular nuclei. FIV-Cre infection resulted in LacZ activation selectively in neurons, whereas both neuronal and glial cell types underwent gene recombination upon infection with Ad-Cre. These results establish the feasibility of using a combination of viral and Cre/loxP technologies to target specific cardiovascular nuclei in the brain for conditional gene modification and suggest the potential of this approach for determining the functional role of genes within these sites.

  20. The First Report of a 290-bp Deletion in β-Globin Gene in the South of Iran

    PubMed Central

    Hamid, Mohammad; Nejad, Ladan Dawoody; Shariati, Gholamreza; Galehdari, Hamid; Saberi, Alihossein; Mohammadi-Anaei, Marziye

    2017-01-01

    Background: β-thalassemia is one of the most widespread diseases in the world, including Iran. In this study, we reported, for the first time, a 290-bp β-globin gene deletion in the south of Iran. Methods: Four individuals from three unrelated families with Arabic ethnic background were studied in Khuzestan Province. Red blood cell indices and hemoglobin analysis were carried out according to the standard methods. Genomic DNA was obtained from peripheral blood cells by salting out procedures. β-globin gene amplification, multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing were performed. Results: The PCR followed by sequencing and MLPA test of the β-globin gene confirmed the presence of a 290-bp deletion in the heterozygous form, along with -88C>A mutation. All the individuals had elevated hemoglobin A2 and normal fetal hemoglobin levels. Conclusions: This mutation causes β0-thalassemia and can be highly useful for prenatal diagnosis in compound heterozygous condition with different β-globin gene mutations. PMID:26948378

  1. Microarray-based mutation detection in the dystrophin gene

    PubMed Central

    Hegde, Madhuri R.; Chin, Ephrem L.H.; Mulle, Jennifer G.; Okou, David T.; Warren, Stephen T.; Zwick, Michael E.

    2008-01-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked recessive neuromuscular disorders caused by mutations in the dystrophin gene affecting approximately 1 in 3,500 males. The human dystrophin gene spans > 2,200 kb, or roughly 0.1% of the genome, and is composed of 79 exons. The mutational spectrum of disease-causing alleles, including exonic copy number variations (CNVs), is complex. Deletions account for approximately 65% of DMD mutations and 85% of BMD mutations. Duplications occur in approximately 6–10% of males with either DMD or BMD. The remaining 30–35% of mutations consist of small deletions, insertions, point mutations, or splicing mutations, most of which introduce a premature stop codon. Laboratory analysis of dystrophin can be used to confirm a clinical diagnosis of DMD, characterize the type of dystrophin mutation, and perform prenatal testing and carrier testing for females. Current dystrophin diagnostic assays involve a variety of methodologies, including multiplex PCR, Southern blot analysis, MLPA, DOVAM-S, and SCAIP; however, these methods are time-consuming, laborious, and do not accurately detect duplication mutations in the dystrophin gene. Furthermore, carrier testing in females is often difficult when a related affected male is unavailable. Here we describe the development, design, validation, and implementation of a high-resolution CGH microarray-based approach capable of accurately detecting both deletions and duplications in the dystrophin gene. This assay can be readily adopted by clinical molecular testing laboratories and represents a rapid, cost-effective approach for screening a large gene, such as dystrophin. PMID:18663755

  2. Microarray-based mutation detection in the dystrophin gene.

    PubMed

    Hegde, Madhuri R; Chin, Ephrem L H; Mulle, Jennifer G; Okou, David T; Warren, Stephen T; Zwick, Michael E

    2008-09-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked recessive neuromuscular disorders caused by mutations in the dystrophin gene affecting approximately 1 in 3,500 males. The human dystrophin gene spans>2,200 kb, or roughly 0.1% of the genome, and is composed of 79 exons. The mutational spectrum of disease-causing alleles, including exonic copy number variations (CNVs), is complex. Deletions account for approximately 65% of DMD mutations and 85% of BMD mutations. Duplications occur in approximately 6 to 10% of males with either DMD or BMD. The remaining 30 to 35% of mutations consist of small deletions, insertions, point mutations, or splicing mutations, most of which introduce a premature stop codon. Laboratory analysis of dystrophin can be used to confirm a clinical diagnosis of DMD, characterize the type of dystrophin mutation, and perform prenatal testing and carrier testing for females. Current dystrophin diagnostic assays involve a variety of methodologies, including multiplex PCR, Southern blot analysis, multiplex ligation-dependent probe amplification (MLPA), detection of virtually all mutations-SSCP (DOVAM-S), and single condition amplification/internal primer sequencing (SCAIP); however, these methods are time-consuming, laborious, and do not accurately detect duplication mutations in the dystrophin gene. Furthermore, carrier testing in females is often difficult when a related affected male is unavailable. Here we describe the development, design, validation, and implementation of a high-resolution comparative genomic hybridization (CGH) microarray-based approach capable of accurately detecting both deletions and duplications in the dystrophin gene. This assay can be readily adopted by clinical molecular testing laboratories and represents a rapid, cost-effective approach for screening a large gene, such as dystrophin.

  3. Angiotensin-converting enzyme (ACE) gene insertion/deletion polymorphism is not a risk factor for hypertension in SLE nephritis.

    PubMed

    Negi, Vir S; Devaraju, Panneer; Gulati, Reena

    2015-09-01

    SLE is a systemic autoimmune disease with high prevalence of hypertension. Around 40-75 % of SLE patients develop nephritis, a major cause of hypertension and mortality. Angiotensin-converting enzyme (ACE) maintains the blood pressure and blood volume homeostasis. An insertion/deletion (I/D) polymorphism in intron 16 of ACE gene was reported to influence the development of hypertension, nephritis, and cardiovascular diseases in different ethnic populations. Despite compelling evidence for the high prevalence of hypertension in individuals with SLE, underlying factors for its development are not well studied. With this background, we analyzed the influence of ACE insertion/deletion polymorphism on susceptibility to SLE, development of nephritis and hypertension, other clinical features and autoantibody phenotype in South Indian SLE patients. Three hundred patients with SLE and 460 age and sex similar ethnicity matched individuals were included as patients and healthy controls, respectively. The ACE gene insertion/deletion polymorphism was analyzed by PCR. Insertion (I) and deletion (D) alleles were observed to be equally distributed among patients (57 and 43 %) and controls (59 and 41 %), respectively. The mutant (D) allele did not confer significant risk for SLE (II vs. ID: p = 0.4, OR 1.15, 95 % CI 0.8-1.6; II vs. DD: p = 0.34, OR 1.22, 95 % CI 0.8-1.85). There was no association of the ACE genotype or the allele with development of lupus nephritis (II vs. ID: p = 0.19, OR 1.41, 95 % CI 0.84-2.36; II vs. DD: p = 0.41, OR 0.74, 95 % CI 0.38-1.41) or hypertension (II vs. ID: p = 0.85, OR 0.9, 95 % CI 0.43-1.8; II vs. DD: p = 0.66, OR 1.217, 95 % CI 0.5-2.8). The presence of mutant allele (D) was not found to influence any clinical features or autoantibody phenotype. The insertion/deletion polymorphism of the ACE gene is not a genetic risk factor for SLE and does not influence development of hypertension or lupus nephritis in South Indian

  4. Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site.

    PubMed Central

    Solera, J; Magallón, M; Martin-Villar, J; Coloma, A

    1992-01-01

    DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment. Images Figure 1 PMID:1346483

  5. Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site.

    PubMed

    Solera, J; Magallón, M; Martin-Villar, J; Coloma, A

    1992-02-01

    DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.

  6. Identification of candidate tumour suppressor gene loci for Hodgkin and Reed-Sternberg cells by characterisation of homozygous deletions in classical Hodgkin lymphoma cell lines.

    PubMed

    Giefing, Maciej; Arnemann, Joachim; Martin-Subero, Jose Ignacio; Nieländer, Inga; Bug, Stefanie; Hartmann, Sylvia; Arnold, Norbert; Tiacci, Enrico; Frank, Matthias; Hansmann, Martin-Leo; Küppers, Ralf; Siebert, Reiner

    2008-09-01

    Several tumour suppressor genes (TSG) have been identified as a result of mapping homozygous deletions in cancer cells. To identify putative TSG involved in the pathogenesis of classical Hodgkin lymphoma (cHL), we investigated four cHL cell lines (L428, HDLM2, KMH2, L1236) using four different array-Comparative Genomic Hybridisation (array-CGH) platforms and focused on high resolution identification of homozygous deletions. Out of 79 candidate regions of bi-allelic loss identified by array-CGH, besides previously described regions, 28 novel regions of homozygous deletions could be verified by polymerase chain reaction. These regions ranged from 13 kb to 619 kb in size. Eleven of the 28 novel bi-allelic losses were putative copy number polymorphisms. This left 17 regions that might harbour novel tumour suppressors involved in Hodgkin lymphoma. Expression profiling with two different platforms confirmed lack of expression of the majority of the genes located in the homozygous deletions. Furthermore, analysis of ontology annotations of genes located in the homozygously deleted regions indicated an enrichment of genes involved in apoptosis and cell death. In summary, through the mapping of homozygous deletions in cell lines this study identified a series of genes, such as SEPT9, GNG7 and CYBB, which might encode candidate tumour suppressors involved in the pathogenesis of cHL.

  7. Novel deletion of the E3A ubiquitin protein ligase gene detected by multiplex ligation-dependent probe amplification in a patient with Angelman syndrome

    PubMed Central

    Calì, Francesco; Ragalmuto, Alda; Chiavetta, Valeria; Calabrese, Giuseppe; Fichera, Marco; Vinci, Mirella; Ruggeri, Giuseppa; Schinocca, Pietro; Sturnio, Maurizio; Romano, Salvatore; Elia, Maurizio

    2010-01-01

    Angelman syndrome (AS) is a severe neurobehavioural disorder caused by failure of expression of the maternal copy of the imprinted domain located on 15q11-q13. There are different mechanisms leading to AS: maternal microdeletion, uniparental disomy, defects in a putative imprinting centre, mutations of the E3 ubiquitin protein ligase (UBE3A) gene. However, some of suspected cases of AS are still scored negative to all the latter mutations. Recently, it has been shown that a proportion of negative cases bear large deletions overlapping one or more exons of the UBE3A gene. These deletions are difficult to detect by conventional gene-scanning methods due to the masking effect by the non-deleted allele. In this study, we have used for the first time multiplex ligation-dependent probe amplification (MLPA) and comparative multiplex dosage analysis (CMDA) to search for large deletions affecting the UBE3A gene. Using this approach, we identified a novel causative deletion involving exon 8 in an affected sibling. Based on our results, we propose the use of MLPA as a fast, accurate and inexpensive test to detect large deletions in the UBE3A gene in a small but significant percentage of AS patients. PMID:21072004

  8. Radiation-induced total-deletion mutations in the human hprt gene: a biophysical model based on random walk interphase chromatin geometry

    NASA Technical Reports Server (NTRS)

    Wu, H.; Sachs, R. K.; Yang, T. C.

    1998-01-01

    PURPOSE: To develop a biophysical model that explains the sizes of radiation-induced hprt deletions. METHODS: Key assumptions: (1) Deletions are produced by two DSB that are closer than an interaction distance at the time of DSB induction; (2) Interphase chromatin is modelled by a biphasic random walk distribution; and (3) Misrejoining of DSB from two separate tracks dominates at low-LET and misrejoining of DSB from a single track dominates at high-LET. RESULTS: The size spectra for radiation-induced total deletions of the hprt gene are calculated. Comparing with the results of Yamada and coworkers for gamma-irradiated human fibroblasts the study finds that an interaction distance of 0.75 microm will fit both the absolute frequency and the size spectrum of the total deletions. It is also shown that high-LET radiations produce, relatively, more total deletions of sizes below 0.5 Mb. The model predicts an essential gene to be located between 2 and 3 Mb from the hprt locus towards the centromere. Using the same assumptions and parameters as for evaluating mutation frequencies, a frequency of intra-arm chromosome deletions is calculated that is in agreement with experimental data. CONCLUSIONS: Radiation-induced total-deletion mutations of the human hprt gene and intrachange chromosome aberrations share a common mechanism for their induction.

  9. A 66-bp deletion in growth hormone releasing hormone gene 5'-flanking region with largemouth bass recessive embryonic lethal.

    PubMed

    Ma, D M; Han, L Q; Bai, J J; Li, S J; Fan, J J; Yu, L Y; Quan, Y C

    2014-06-01

    Growth hormone releasing hormone (GHRH) regulates the secretion of growth hormone (GH) in the pituitary gland. A 66-bp deletion (c.-923_-858del) was detected in the 5'-flanking sequence of the largemouth bass (Micropterus salmoides) GHRH gene. In two cultured random populations of adult individuals (A: n = 170 and B: n = 150), the genotype ratios of +/+:+/- were 2.5:1 and 2.8:1 respectively. Only one -/- fish was detected. A Largemouth bass family was constructed with two heterozygous individuals (+/-) as parents. The genotype ratio of +/+:+/-:-/- in the filial generation embryos was 1:1.6:0.1 at the neurula and 1:2:0 at hatched larvae stages. This indicated that the 66-bp deletion was a recessive lethal site and that homozygous individuals (-/-) died off in embryonic development. The growth traits (body weight, body length and body depth) were measured, and the GHRH mRNA expression levels in brain tissue were detected using real-time PCR. The effects of genotype (+/-) on growth traits and GHRH mRNA expression were not significant. Although the cause of death was not clear, the results hint that the 66-bp deletion site in GHRH 5'-flanking sequence significantly affects the livability in largemouth bass embryonic development.

  10. A 4-bp Deletion in the Birt-Hogg-Dubé Gene (FLCN) Causes Dominantly Inherited Spontaneous Pneumothorax

    PubMed Central

    Painter, Jodie N.; Tapanainen, Hanna; Somer, Mirja; Tukiainen, Pentti; Aittomäki, Kristiina

    2005-01-01

    Primary spontaneous pneumothorax (PSP), a condition in which air enters the pleural space and causes secondary lung collapse, is mostly sporadic but also occurs in families. The precise etiology of PSP remains unknown, although it is associated with emphysemalike changes (bullae) in the lungs of almost all patients. We describe the results of a genetic study of a large Finnish family with a dominantly inherited tendency to PSP. A genomewide scan suggested linkage to chromosome 17p11. Screening of the best candidate gene, FLCN, revealed a 4-bp deletion in the first coding exon, which causes a frameshift that predicts a protein truncation 50 missense amino acids downstream. All carriers of the deletion had bullous lung lesions. Mutations in FLCN are also responsible for Birt-Hogg-Dubé (BHD) syndrome (a dominantly inherited disease characterized by benign skin tumors, PSP, and diverse types of renal cancer) and, rarely, are detected in sporadic renal and colorectal tumors. Unlike other FLCN mutations, the exon 4 deletion seems to be associated with bullous lung changes only with 100% penetrance. These results suggest that changes in FLCN may have an important role in the development of PSP and, more importantly, of emphysema, a chronic pulmonary disease that often leads to formation of bullous lesions and lowered pulmonary function. Additionally, given the strong association of PSP and BHD, the connection between these conditions needs to be investigated further, particularly in patients with familial PSP, who may be at a greater risk of developing renal cancer. PMID:15657874

  11. Deletion of the Saccharomyces cerevisiae ARO8 gene, encoding an aromatic amino acid transaminase, enhances phenylethanol production from glucose.

    PubMed

    Romagnoli, Gabriele; Knijnenburg, Theo A; Liti, Gianni; Louis, Edward J; Pronk, Jack T; Daran, Jean-Marc

    2015-01-01

    Phenylethanol has a characteristic rose-like aroma that makes it a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbour an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source, such as glucose, provides an economically attractive alternative for phenylalanine bioconversion. In this study, synthetic genetic array (SGA) screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to underlie the transcriptional upregulation of ARO10 during growth, with ammonium sulphate as the sole nitrogen source. Physiological characterization revealed that the aro8Δ mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8Δ mutation also stimulated phenylethanol production when combined with other, previously documented, mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced >3 mm phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products.

  12. Deletion of 5'-coding sequences of the cellular p53 gene in mouse erythroleukemia: a novel mechanism of oncogene regulation.

    PubMed Central

    Rovinski, B; Munroe, D; Peacock, J; Mowat, M; Bernstein, A; Benchimol, S

    1987-01-01

    The p53 gene is rearranged in an erythroleukemic cell line (DP15-2) transformed by Friend retrovirus. Here, we characterize the mutation and identify a deletion of approximately equal to 3.0 kilobases that removes exon 2 coding sequences. The gene is expressed in DP15-2 cells and results in synthesis of a 44,000-dalton protein that is missing the N-terminal amino acid residues of p53. The truncated protein is unusually stable and accumulates to high levels intracellularly. Moreover, it appears to have undergone a change in conformation as revealed by epitope mapping studies. This study represents the first description of an altered p53 gene product arising by mutation during neoplastic progression and identifies a region in the p53 protein molecule that plays a role in determining p53 stability in vivo. Images PMID:3547084

  13. A 45 X male patient with 7q distal deletion and rearrangement with SRY gene translocation: a case report.

    PubMed

    Bilen, S; Okten, A; Karaguzel, G; Ikbal, M; Aslan, Y

    2013-01-01

    Here we present a male newborn with multiple congenital anomalies who also has an extremely rare form of testicular disorder of sex development (DSD). His karyotype was 45X, without any mosaicism. SRY gene was positive by polymerase chain reaction (PCR), and rearranged on distal part of the 7th chromosome by fluorescence in situ hybridization (FISH) analysis. SRY, normally located on the Y chromosome, is the most important gene that plays a role in the development of male sex. SRY gen may be translocated onto another chromosome, mostly X chromosome in the XX testicular DSD. On the other hand very few cases of 45 X testicular DSD were published to date. Other clinical manifestations of our patient were compatible with distal 7 q deletion syndrome. To the best of our knowledge this is the first case of 45 X testicular DSD with SRY gene rearranged on the 7th autosomal chromosome.

  14. Recombination of the bph (Biphenyl) Catabolic Genes from Plasmid pWW100 and Their Deletion during Growth on Benzoate

    PubMed Central

    Lloyd-Jones, Gareth; de Jong, Caroline; Ogden, Richard C.; Duetz, Wouter A.; Williams, Peter A.

    1994-01-01

    Pseudomonas sp. strain CB406 was isolated from polychlorinated biphenyl-contaminated soil and harbors a nontransmissible plasmid, pWW100, of approximately 200 kb which carries the genes required for biphenyl and 4-chlorobiphenyl catabolism. The catabolic phenotype was mobilized following the construction in vivo of a cointegrate plasmid containing functional upper and lower biphenyl operons inserted into the broad-host-range R plasmid RP4. The Bph+ phenotype carried by pWW100 was stable in nonselective media but was unstable during growth on benzoate, where the sequential selection of two species of bph deletion derivatives occurs at high frequency. This mirrors observations made with TOL plasmids (encoding toluene and xylene catabolism) grown under similar conditions. Subcloning of dioxygenase genes involved in biphenyl catabolism confirmed the localization of the bph genes on the wild-type plasmid and the RP4 cointegrate plasmid. Images PMID:16349195

  15. Tph2 gene deletion enhances amphetamine-induced hypermotility: effect of 5-HT restoration and role of striatal noradrenaline release.

    PubMed

    Carli, Mirjana; Kostoula, Chrysaugi; Sacchetti, Giuseppina; Mainolfi, Pierangela; Anastasia, Alessia; Villani, Claudia; Invernizzi, Roberto William

    2015-11-01

    Variants of tryptophan hydroxylase-2 (Tph2), the gene encoding enzyme responsible for the synthesis of brain serotonin (5-HT), have been associated with neuropsychiatric disorders, substance abuse and addiction. This study assessed the effect of Tph2 gene deletion on motor behavior and found that motor activity induced by 2.5 and 5 mg/kg amphetamine was enhanced in Tph2(-/-) mice. Using the in vivo microdialysis technique we found that the ability of amphetamine to stimulate noradrenaline (NA) release in the striatum was reduced by about 50% in Tph2(-/-) mice while the release of dopamine (DA) was not affected. Tph2 deletion did not affect the release of NA and DA in the prefrontal cortex. The role of endogenous 5-HT in enhancing the effect of amphetamine was confirmed showing that treatment with the 5-HT precursor 5-hydroxytryptophan (10 mg/kg) restored tissue and extracellular levels of brain 5-HT and the effects of amphetamine on striatal NA release and motor activity in Tph2(-/-) mice. Treatment with the NA precursor dihydroxyphenylserine (400 mg/kg) was sufficient to restore the effect of amphetamine on striatal NA release and motor activity in Tph2(-/-) mice. These findings indicate that amphetamine-induced hyperactivity is attenuated by endogenous 5-HT through the inhibition of striatal NA release. Tph2(-/-) mice may be a useful preclinical model to assess the role of 5-HT-dependent mechanisms in the action of psychostimulants. Acute sensitivity to the motor effects of amphetamine has been associated to increased risk of psychostimulant abuse. Here, we show that deletion of Tph2, the gene responsible for brain 5-HT synthesis, enhances the motor effect of amphetamine in mice through the inhibition of striatal NA release. This suggests that Tph2(-/-) mice is a useful preclinical model to assess the role of 5-HT-dependent mechanisms in psychostimulants action. Tph2, tryptophan hydroxylase-2.

  16. Metabolic and developmental effects resulting from deletion of the citA gene encoding citrate synthase in Aspergillus nidulans.

    PubMed

    Murray, Sandra L; Hynes, Michael J

    2010-04-01

    Citrate synthase is a central activity in carbon metabolism. It is required for the tricarboxylic acid (TCA) cycle, respiration, and the glyoxylate cycle. In Saccharomyces cerevisiae and Arabidopsis thaliana, there are mitochondrial and peroxisomal isoforms encoded by separate genes, while in Aspergillus nidulans, a single gene, citA, encodes a protein with predicted mitochondrial and peroxisomal targeting sequences (PTS). Deletion of citA results in poor growth on glucose but not on derepressing carbon sources, including those requiring the glyoxylate cycle. Growth on glucose is restored by a mutation in the creA carbon catabolite repressor gene. Methylcitrate synthase, required for propionyl-coenzyme A (CoA) metabolism, has previously been shown to have citrate synthase activity. We have been unable to construct the mcsADelta citADelta double mutant, and the expression of mcsA is subject to CreA-mediated carbon repression. Therefore, McsA can substitute for the loss of CitA activity. Deletion of citA does not affect conidiation or sexual development but results in delayed conidial germination as well as a complete loss of ascospores in fruiting bodies, which can be attributed to loss of meiosis. These defects are suppressed by the creA204 mutation, indicating that McsA activity can substitute for the loss of CitA. A mutation of the putative PTS1-encoding sequence in citA had no effect on carbon source utilization or development but did result in slower colony extension arising from single conidia or ascospores. CitA-green fluorescent protein (GFP) studies showed mitochondrial localization in conidia, ascospores, and hyphae. Peroxisomal localization was not detected. However, a very low and variable detection of punctate GFP fluorescence was sometimes observed in conidia germinated for 5 h when the mitochondrial targeting sequence was deleted.

  17. Pleiotropy in microdeletion syndromes: neurologic and spermatogenic abnormalities in mice homozygous for the p6H deletion are likely due to dysfunction of a single gene.

    PubMed

    Rinchik, E M; Carpenter, D A; Handel, M A

    1995-07-03

    Variability and complexity of phenotypes observed in microdeletion syndromes can be due to deletion of a single gene whose product participates in several aspects of development or can be due to the deletion of a number of tightly linked genes, each adding its own effect to the syndrome. The p6H deletion in mouse chromosome 7 presents a good model with which to address this question of multigene vs. single-gene pleiotropy. Mice homozygous for the p6H deletion are diluted in pigmentation, are smaller than their littermates, and manifest a nervous jerky-gait phenotype. Male homozygotes are sterile and exhibit profound abnormalities in spermiogenesis. By using N-ethyl-N-nitrosourea (EtNU) mutagenesis and a breeding protocol designed to recover recessive mutations expressed hemizygously opposite a large p-locus deletion, we have generated three noncomplementing mutations that map to the p6H deletion. Each of these EtNU-induced mutations has adverse effects on the size, nervous behavior, and progression of spermiogenesis that characterize p6H deletion homozygotes. Because EtNU is thought to induce primarily intragenic (point) mutations in mouse stem-cell spermatogonia, we propose that the trio of phenotypes (runtiness, nervous jerky gait, and male sterility) expressed in p6H deletion homozygotes is the result of deletion of a single highly pleiotropic gene. We also predict that a homologous single locus, quite possibly tightly linked and distal to the D15S12 (P) locus in human chromosome 15q11-q13, may be associated with similar developmental abnormalities in humans.

  18. Genes associated with the genesis of leiomyoma of the uterus in a commonly deleted chromosomal region at 7q22.

    PubMed

    Saito, Emi; Okamoto, Aikou; Saito, Misato; Shinozaki, Hideo; Takakura, Satoshi; Yanaihara, Nozomu; Ochiai, Kazunori; Tanaka, Tadao

    2005-03-01

    Uterine leiomyoma occurs in about 20-30% of women over the age of 30, and is the most frequent benign tumor in gynecology. Despite its benign status, leiomyoma of the uterus has been reported to involve chromosomal abnormalities on chromosome 7. To search for genes associated with the genesis and development of this disease, we examined microsatellite alterations on chromosome 7 in 41 uterine leiomyomas, and identified a commonly-deleted region. Allelic imbalance on chromosome 7 was detected with an incidence of 7% (3/41), with the D7S501 locus being the most frequently affected (13%). The commonly deleted region was between D7S2545 and D7S2420. We examined alterations in the expression of genes located within this region by RT-PCR. Only the LAMB1 (Laminin beta1) gene showed a variable expression. Of the 21 cases, 12 showed an increase, and 5 (24%) a decrease in the expression of LAMB1 in the leiomyomatous region. These results suggested that alteration of LAMB1 expression is associated with the genesis and development of uterine leiomyoma.

  19. Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo

    PubMed Central

    Németh, Tamás; Futosi, Krisztina; Sitaru, Cassian; Ruland, Jürgen; Mócsai, Attila

    2016-01-01

    Neutrophils are terminally differentiated cells with limited transcriptional activity. The biological function of their gene expression changes is poorly understood. CARD9 regulates transcription during antifungal immunity but its role in sterile inflammation is unclear. Here we show that neutrophil CARD9 mediates pro-inflammatory chemokine/cytokine but not lipid mediator release during non-infectious inflammation. Genetic deficiency of CARD9 suppresses autoantibody-induced arthritis and dermatitis in mice. Neutrophil-specific deletion of CARD9 is sufficient to induce that phenotype. Card9−/− neutrophils show defective immune complex-induced gene expression changes and pro-inflammatory chemokine/cytokine release but normal LTB4 production and other short-term responses. In vivo deletion of CARD9 reduces tissue levels of pro-inflammatory chemokines and cytokines but not LTB4. The CARD9-mediated signalling pathway involves Src-family kinases, Syk, PLCγ2, Bcl10/Malt1 and NFκB. Collectively, CARD9-mediated gene expression changes within neutrophils play important roles during non-infectious inflammation in vivo and CARD9 acts as a divergence point between chemokine/cytokine and lipid mediator release. PMID:27032818

  20. Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus.

    PubMed

    Sanford, B; Holinka, L G; O'Donnell, V; Krug, P W; Carlson, J; Alfano, M; Carrillo, C; Wu, Ping; Lowe, Andre; Risatti, G R; Gladue, D P; Borca, M V

    2016-02-02

    African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically deleting virus genes involved in virulence, including the thymidine kinase (TK) gene. TK has been shown to be involved in the virulence of several viruses, including ASFV. Here we report the construction of a recombinant virus (ASFV-G/V-ΔTK) obtained by deleting the TK gene in a virulent strain of ASFV Georgia adapted to replicate in Vero cells (ASFV-G/VP30). ASFV-G/P-ΔTK demonstrated decreased replication both in primary swine macrophage cell cultures and in Vero cells compared with ASFV-G/VP30. In vivo, intramuscular administration of up to 10(6) TCID50 of ASFV-G/V-ΔTK does not result in ASF disease. However, these animals are not protected when challenged with the virulent parental Georgia strain.

  1. Autosomal Dominant Retinal Degeneration and Bone Loss in Patients with a 12-bp Deletion in the CRX Gene

    PubMed Central

    Tzekov, Radouil T.; Liu, Yuhui; Sohocki, Melanie M.; Zack, Donald J.; Daiger, Stephen P.; Heckenlively, John R.; Birch, David G.

    2008-01-01

    Purpose To define the phenotypic expression of a deletion in the gene encoding the transcription factor CRX in a large, seven-generation, white family. Methods Fourteen affected individuals, all heterozygous for the Leu146del12 mutation in the cone-rod homeobox gene (CRX), and four nonaffected relatives from the same family were examined with visual function tests, and 10 underwent bone mineral density (BMD) measurement. Results The ability of the mutated CRX protein to transactivate rhodopsin promoter was decreased by approximately 25%, and its ability to react synergistically with neural retinal leucine zipper (NRL) was reduced by more than 30%. The affected members of the family had an autosomal dominant ocular condition most closely resembling Leber congenital amaurosis (LCA) with severe visual impairment at an early age. Depending on age, affected members showed varying degrees of significant visual acuity loss, elevated dark-adaptation thresholds, significantly reduced cone and rod electroretinogram (ERG) amplitudes, and progressive constriction of the visual fields, in most cases leading to complete blindness. Six affected members had reduced levels of BMD in the spine and the hip (osteopenia). Four affected female members who were receiving long-term hormonal replacement therapy (HRT) demonstrated normal values of BMD. Conclusions This large deletion of the CRX gene is associated with a severe form of autosomal dominant retinal degeneration. Affected members not receiving HRT showed reduced BMD (osteopenia). This phenotype may reflect the abnormal influence of mutant CRX on both retinal and pineal development. PMID:11328746

  2. New variants, challenges and pitfalls in DMD genotyping: implications in diagnosis, prognosis and therapy.

    PubMed

    Santos, Rosário; Gonçalves, Ana; Oliveira, Jorge; Vieira, Emília; Vieira, José Pedro; Evangelista, Teresinha; Moreno, Teresa; Santos, Manuela; Fineza, Isabel; Bronze-da-Rocha, Elsa

    2014-08-01

    Molecular characterization of patients with Duchenne or Becker muscular dystrophies is essential for establishing a differential diagnosis, allowing appropriate clinical follow-up, patient management and genetic counseling. In light of the recent mutation-based therapeutic approaches, DMD gene analysis has gained further relevance. Owing to the size and complexity of the DMD gene and the diversity of mutation types, molecular analysis is not always a straightforward task requiring the combination of several methodologies. Our national genetic diagnostic service genetically characterized 308 dystrophinopathy patients (284 unrelated families), leading to the identification of 175 distinct mutations, including 39 unpublished variants. These studies revealed several potential diagnostic pitfalls (because of technical limitations or related with DMD's genetic heterogeneity) that may be overlooked even considering the international disease-specific diagnostic guidelines. Comprehensive analysis involved expression studies at the mRNA level, the identification of splicing changes and ultimately providing evidence for apparent exceptions to the reading-frame rule. Besides increasing the mutation detection rate, this detailed molecular characterization is indispensable for the identification of suitable candidates for the new mutation-centered therapies. As patient registries are internationally recognized as essential for clinical trial recruitment, this led us to develop the Portuguese Duchenne and Becker Muscular Dystrophy registry in collaboration with the Translational Research in Europe-Assessment and Treatment of Neuromuscular Diseases network.

  3. Isolation of precise plastid deletion mutants by homology-based excision: a resource for site-directed mutagenesis, multi-gene changes and high-throughput plastid transformation.

    PubMed

    Kode, Vasumathi; Mudd, Elisabeth A; Iamtham, Siriluck; Day, Anil

    2006-06-01

    We describe a simple and efficient homology-based excision method to delete plastid genes. The procedure allows one or more adjacent plastid genes to be deleted without the retention of a marker gene. We used aadA-based transformation to duplicate a 649 bp region of plastid DNA corresponding to the atpB promoter region. Efficient recombination between atpB repeats deletes the intervening foreign genes and 1,984 bp of plastid DNA (co-ordinates 57,424-59,317) containing the rbcL gene. Only five foreign bases are present in DeltarbcL plants illustrating the precision of homology-based excision. Sequence analysis of non-functional rbcL-related sequences in DeltarbcL plants indicated an extra-plastidic origin. Mutant DeltarbcL plants were heterotrophic, pale-green and contained round plastids with reduced amounts of thylakoids. Restoration of autotrophy and leaf pigmentation following aadA-based transformation with the wild-type rbcL gene ruled out mutations in other genes. Excision and re-use of aadA shows that, despite the multiplicity of plastid genomes, homology-based excision ensures complete removal of functional aadA genes. Rescue of the DeltarbcL mutation and autotrophic growth stabilizes transgenic plastids in heteroplasmic transformants following antibiotic withdrawal, enhancing the overall efficiency of plastid transformation. Unlike the available set of homoplasmic knockout mutants in 25 plastid genes, the rbcL deletion mutant isolated here is readily transformed with the efficient aadA marker gene. This improvement in deletion design facilitates advanced studies that require the isolation of double mutants in distant plastid genes and the replacement of the deleted locus with site-directed mutant alleles and is not easily achieved using other methods.

  4. The sub-optimal phenotypes of double-knockout mutants of Escherichia coli depend on the order of gene deletions.†

    PubMed Central

    Gawand, Pratish; Abukar, Fatumina Said; Venayak, Naveen; Partow, Siavash; Motter, Adilson E.; Mahadevan, Radhakrishnan

    2016-01-01

    Metabolic networks are characterized by multiple redundant reactions that do not have a clear biological function. The redundancies in the metabolic networks are implicated in adaptation to random mutations and survival under different environmental conditions. Reactions that are not active under wild-type growth conditions, but get transiently activated after a mutation event such as gene deletion are known as latent reactions. Characterization of multiple-gene knockout mutants can identify the physiological roles of latent reactions. In this study, we characterized double-gene deletion mutants of E. coli with an aim to investigate the sub-optimal physiology of the mutants and the plausible roles of latent reactions. Specifically, we investigated the effects of deletion of the glyoxylate-shunt gene aceA (encoding a latent reaction enzyme, isocitrate lyase) on the growth characteristics of the mutant E. coli Δpgi. The deletion of aceA reduced the growth rate of E. coli Δpgi, indicating that the activation of the glyoxylate shunt plays an important role in adaptation of the mutant E. coli Δpgi. We also investigated the effect of the order of the gene deletions on the growth rates and substrate uptake rates of the double-gene deletion mutants. The results indicate that the order in which genes are deleted determines the phenotype of the mutants during the sub-optimal growth phase. To elucidate the mechanism behind the difference between the observed phenotypes, we carried out transcriptomic analysis and constraint-based modeling of the mutants. Transcriptomic analysis showed differential expression of the gene aceK (encoding the protein isocitrate dehydrogenase kinase) involved in controlling the isocitrate flux through the TCA cycle and the glyoxylate shunt. Higher acetate production in the E. coli ΔaceA1 Δpgi2 mutant was consistent with the increased aceK expression, which limits the TCA cycle flux and causes acetate production via overflow metabolism. PMID

  5. Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector

    SciTech Connect

    Watanabe, Daisuke; Brockman, Mark A.; Ndung'u, Thumbi; Mathews, Lydia; Lucas, William T.; Murphy, Cynthia G.; Felber, Barbara K.; Pavlakis, George N.; Deluca, Neal A.; Knipe, David M. . E-mail: david_knipe@hms.harvard.edu

    2007-01-20

    Herpes simplex virus (HSV) recombinants induce durable immune responses in rhesus macaques and mice and have induced partial protection in rhesus macaques against mucosal challenge with virulent simian immunodeficiency virus (SIV). In this study, we evaluated the properties of a new generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE) gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27, ICP22, and ICP47 genes. Because several of the HSV IE genes have been implicated in immune evasion, inactivation of the genes encoding these proteins was expected to result in enhanced immunogenicity. The d106 virus expresses few HSV gene products and shows minimal cytopathic effect in cultured cells. When d106 was inoculated into mice, viral DNA accumulated at high levels in draining lymph nodes, consistent with an ability to transduce dendritic cells and activate their maturation and movement to lymph nodes. A d106 recombinant expressing Escherichia coli {beta}-galactosidase induced durable {beta}-gal-specific IgG and CD8{sup +} T cell responses in naive and HSV-immune mice. Finally, d106-based recombinants have been constructed that express simian immunodeficiency virus (SIV) gag, env, or a rev-tat-nef fusion protein for several days in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine vector: limited expression of HSV gene products and cytopathogenicity, high level expression of transgenes, ability to induce durable immune responses, and an ability to transduce dendritic cells and induce their maturation and migration to lymph nodes.

  6. The synergy of tobacco and alcohol and glutathione S-transferase θ 1 gene deletion and oral squamous cell carcinoma

    PubMed Central

    D’ Mello, Sarah; Bavle, Radhika Manoj; Paremala, K; Makarla, Soumya; Sudhakara, M; Bhatt, Madhura

    2016-01-01

    Background: Oral squamous cell carcinoma (OSCC) is the leading cancer among males in India. It is related to tobacco habits and alcohol consumption as well as the individual susceptibility for xenobiotic metabolizing enzyme polymorphisms. Glutathione S-transferase θ 1 (GSTT1) is a Phase II metabolic enzyme which is directly involved in catalyzing chemicals to mutagenic intermediates. This gene is characterized by genetic polymorphism resulting in complete gene deletion and subsequent absence of the enzyme, which ultimately dictates the risk of cancer development. Scraping buccal mucosa to obtain DNA from the cells is a simple, readily acceptable and rapid method to detect and assess the gene. Aim: To assess GSTT1 gene deletion in individuals giving a history of tobacco smoking and/or chewing and alcohol consumption and absence of clinically detectable lesions; and in OSCC cases to gauge if GSTT1 gene deletion confers protection to an individual and whether it can be used as a “single” marker to arrive at this conclusion. To validate the use of buccal scrape for determining the genotype of an individual by assessing the polymorphism at GSTT1 gene locus (22q11.2). Materials and Methods: Fifty-two cases were evaluated using buccal mucosal scrapes of tobacco habituates for 8 or more years, without clinically evident lesion (Group I) and from mucosa of tobacco habituates with clinically evident and histopathologically confirmed OSCC (Group II). DNA extraction and genotype at GSTT1 gene locus was determined by polymerase chain reaction assay. Statistical Analysis: The results were statistically analyzed using Chi-square test. Results: 90.66% of subjects had GSTT1 null genotype in Group I subjects. In Group II, subjects with both clinically and histopathologically diagnosed oral cancer, about 76.96% had GSTT1 null genotype. Conclusion: GSTT1 null genotype confers protection to individuals with tobacco habits and alcohol consumption, predominantly to those who used

  7. Molecular cloning and chromosomal localization of the human cyclin C (CCNC) and cyclin E (CCNE) genes: Deletion of the CCNC gene in human tumors

    SciTech Connect

    Li, Haimin; Lahti, J.M.; Kidd, V.J.

    1996-03-01

    The human G1-phase cyclins are important regulators of cell cycle progression that interact with various cyclin-dependent kinases and facilitate entry into S-phase. We have confirmed the localization of the human cyclin C (CCNC) gene to chromosome 6q21 and of human cyclin E (CCNE to 19q12). The CCNC gene structure was also determined, and we have shown that it is deleted in a subset of acute lymphoblastic leukemias, including a patient sample containing a t(2;6)(p21;q15), with no apparent cytogenetic deletion. Single-strand conformational polymorphism analysis of the remaining CCNC allele from patients with a deletion of one allele established that there were no further mutations within the exons or the flanking intronic sequences. These results suggest either that haploinsufficiency of the cyclin C protein is sufficient to promote tumorigenesis or that the important tumor suppressor gene is linked to the CCNC locus. 48 refs., 4 figs., 1 tab.

  8. CNS-restricted Transduction and CRISPR/Cas9-mediated Gene Deletion with an Engineered AAV Vector

    PubMed Central

    Murlidharan, Giridhar; Sakamoto, Kensuke; Rao, Lavanya; Corriher, Travis; Wang, Dan; Gao, Guangping; Sullivan, Patrick; Asokan, Aravind

    2016-01-01

    Gene therapy using recombinant adeno-associated viral (AAV) vectors is emerging as a promising approach to treat central nervous system disorders such as Spinal muscular atrophy, Batten, Parkinson and Alzheimer disease amongst others. A critical remaining challenge for central nervous system-targeted gene therapy, silencing or gene editing is to limit potential vector dose-related toxicity in off-target cells and organs. Here, we characterize a lab-derived AAV chimeric (AAV2g9), which displays favorable central nervous system attributes derived from both parental counterparts, AAV2 and AAV9. This synthetic AAV strain displays preferential, robust, and widespread neuronal transduction within the brain and decreased glial tropism. Importantly, we observed minimal systemic leakage, decreased sequestration and gene transfer in off-target organs with AAV2g9, when administered into the cerebrospinal fluid. A single intracranial injection of AAV2g9 vectors encoding guide RNAs targeting the schizophrenia risk gene MIR137 (encoding MIR137) in CRISPR/Cas9 knockin mice resulted in brain-specific gene deletion with no detectable events in the liver. This engineered AAV vector is a promising platform for treating neurological disorders through gene therapy, silencing or editing modalities. PMID:27434683

  9. Impact of deletion of envelope-related genes of recombinant Sendai viruses on immune responses following pulmonary gene transfer of neonatal mice.

    PubMed

    Tanaka, S; Yonemitsu, Y; Yoshida, K; Okano, S; Kondo, H; Inoue, M; Hasegawa, M; Masumoto, K; Suita, S; Taguchi, T; Sueishi, K

    2007-07-01

    We demonstrated previously that the additive-type recombinant Sendai virus (rSeV) is highly efficient for use in pulmonary gene transfer; however, rSeV exhibits inflammatory responses. To overcome this problem, we tested newly developed non-transmissible constructs, namely, temperature-sensitive F-deleted vector, rSeV/dF (ts-rSeV/dF) and a rSeV with all the envelope-related genes deleted (rSeV/dFdMdHN), for pulmonary gene transfer in neonatal mice, by assessing their toxicity and immune responses. The gene expression in the lungs of neonatal ICR mice peaked on day 2, then gradually decreased until almost disappearing at 14 days after infection in all constructs. Loss of body weight and mortality rate, however, were dramatically improved in mice treated with SeV/dFdMdHN (mortality=0%, n=41) and ts-rSeV/dF (24.2%, n=33) compared with additive rSeV (70.7%, n=58). Although the deletion of envelope-related genes of SeV had a small impact on the production of antibody and cytotoxic T-lymphocyte activity in both adults and neonates, a dramatic reduction was found in the events related to innate responses, including the production of proinflammatory cytokines, particularly in the case of neonates. These results indicate that pulmonary gene transfer using SeV/dFdMdHN warrants further investigation for its possible use in developing safer therapeutics for neonatal lung diseases, including cystic fibrosis.

  10. Clinical and genetic characterization of chanarin-dorfman syndrome patients: first report of large deletions in the ABHD5 gene

    PubMed Central

    2010-01-01

    Background Chanarin-Dorfman syndrome (CDS) is a rare autosomal recessive disorder characterized by nonbullous congenital ichthyosiform erythroderma (NCIE) and an intracellular accumulation of triacylglycerol (TG) droplets in most tissues. The clinical phenotype involves multiple organs and systems, including liver, eyes, ears, skeletal muscle and central nervous system (CNS). Mutations in ABHD5/CGI58 gene are associated with CDS. Methods Eight CDS patients belonging to six different families from Mediterranean countries were enrolled for genetic study. Molecular analysis of the ABHD5 gene included the sequencing of the 7 coding exons and of the putative 5' regulatory regions, as well as reverse transcript-polymerase chain reaction analysis and sequencing of normal and aberrant ABHD5 cDNAs. Results Five different mutations were identified, four of which were novel, including two splice-site mutations (c.47+1G>A and c.960+5G>A) and two large deletions (c.898_*320del and c.662-1330_773+46del). All the reported mutations are predicted to be pathogenic because they lead to an early stop codon or a frameshift producing a premature termination of translation. While nonsense, missense, frameshift and splice-site mutations have been identified in CDS patients, large genomic deletions have not previously been described. Conclusions These results emphasize the need for an efficient approach for genomic deletion screening to ensure an accurate molecular diagnosis of CDS. Moreover, in spite of intensive molecular screening, no mutations were identified in one patient with a confirmed clinical diagnosis of CDS, appointing to genetic heterogeneity of the syndrome. PMID:21122093

  11. Mesomelia-Synostoses Syndrome Results from Deletion of SULF1 and SLCO5A1 Genes at 8q13

    PubMed Central

    Isidor, Bertrand; Pichon, Olivier; Redon, Richard; Day-Salvatore, Debra; Hamel, Antoine; Siwicka, Karolina A.; Bitner-Glindzicz, Maria; Heymann, Dominique; Kjellén, Lena; Kraus, Cornelia; Leroy, Jules G.; Mortier, Geert R.; Rauch, Anita; Verloes, Alain; David, Albert; Le Caignec, Cédric

    2010-01-01

    Mesomelia-synostoses syndrome (MSS) or mesomelic dysplasia with acral synostoses Verloes-David-Pfeiffer type is a rare autosomal-dominant disorder characterized by mesomelic limb shortening, acral synostoses, and multiple congenital malformations. So far, five patients in four unrelated families have been reported worldwide with MMS. By using whole-genome oligonucleotide array CGH, we have identified an interstitial deletion at 8q13 in all patients. The deletions vary from 582 Kb to 738 Kb in size, but invariably encompass only two genes: SULF1, encoding the heparan sulfate 6-O-endosulfatase 1, and SLCO5A1, encoding the solute carrier organic anion transporter family member 5A1. SULF1 acts as a regulator of numerous growth factors in skeletal embryonic development whereas the function of SLCO5A1 is yet unknown. Breakpoint sequence analyses performed in two families showed nonrecurrent deletions. Real-time quantitative RT-PCR analysis showed the highest levels of SULF1 transcripts in human osteoblasts and cartilage whereas SLCO5A1 was highly expressed in human fetal and adult brain and heart. Our results strongly suggest that haploinsufficiency of SULF1 contributes to this mesomelic chondrodysplasia, highlighting the critical role of endosulfatase in human skeletal development. Codeletion of SULF1 and SLCO5A1—which does not result from a low-copy repeats (LCRs)-mediated recombination event in at least two families—was found in all patients, so we suggest that haploinsufficiency of SULF1 combined with haploinsufficiency of SLCO5A1 (or the altered expression of a neighboring gene through position effect) could be necessary in the pathogenesis of MSS. PMID:20602915

  12. Homozygous deletion in the SMN1 gene in asymptomatic individual - genetic counselling issues in SMA-risk families.

    PubMed

    Jędrzejowska, Maria; Szczałuba, Krzysztof; Sielska, Danuta

    2011-01-01

    Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The mode of inheritance of SMA is what determines the relatively low risk to off spring of affected persons, sibs of carriers of the pathogenic mutation or their more distant relatives. Nonetheless, the risk in question is increased beyond that in the population in general, thereby indicating the need for preventative measures to be taken in respect of SMA families. We present clinical characteristics of such a SMA-risk family. An apparently unaffected brother (patient) of the proband together with his pregnant wife sought genetic counselling about the SMA risk in their off spring. The estimated a priori risk was of about 0.5% (1 in 200). Molecular diagnostic tests performed in the patient indicated the presence of a homozygous deletion of the SMN1 gene identical to the one detected in the affected proband. The patient's wife was identified as a carrier of the deletion. The conditional risk for off spring of the couple was thus recalculated as 50% (1 in 2). Homozygous deletion of the SMN1 gene in unaffected individuals is a relatively rare event, yet one that nevertheless has significant impact on genetic counselling in SMA families. In these circumstances, molecular confirmation of SMA in such families allows for the provision of competent and reliable genetic advice, as well as for the introduction of secondary preventative measures. It also meets inclusion criteria as regards optional invasive prenatal diagnostic testing in families with a high (.25%) risk of the condition being present in the off spring.

  13. 3p14 deletion is a rare contiguous gene syndrome: report of 2 new patients and an overview of 14 patients.

    PubMed

    Dimitrov, B I; Ogilvie, C; Wieczorek, D; Wakeling, E; Sikkema-Raddatz, B; van Ravenswaaij-Arts, C M A; Josifova, D

    2015-06-01

    Interstitial deletions of chromosome 3p14p12 are a rare chromosome rearrangement. Twenty-six patients have been reported in the literature to date, however, a specific clinical phenotype has not yet been delineated. We describe three patients (two new) with overlapping chromosome 3p14p12 deletions and review the clinical and molecular data of 11 well-characterized, published cases. These patients had a number of features in common, such as short stature, failure to thrive, facial dysmorphism, congenital heart defects, urogenital abnormalities, neurological problems, hearing loss, and global developmental delay, suggesting that the interstitial chromosome 3p14p12 deletion gives rise to a multiple congenital anomaly syndrome. Some of the patients show clinical overlap with other complex syndromes such as CHARGE syndrome. Genotype-phenotype analysis revealed candidate genes for parts of the clinical features suggesting that the 3p14 deletion is a contiguous gene syndrome.

  14. Identification and characterization of a novel homozygous deletion in the alpha-N-acetylglucosaminidase gene in a patient with Sanfilippo type B syndrome (mucopolysaccharidosis IIIB).

    PubMed

    Champion, Kristen J; Basehore, Monica J; Wood, Tim; Destrée, Anne; Vannuffel, Pascal; Maystadt, Isabelle

    2010-05-01

    Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disease that is caused by a deficiency of the lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). Over 100 different mutations in the NAGLU gene have been identified in Sanfilippo syndrome type B patients; however, no large deletions have been reported. Here we present the first case of a large homozygous intragenic NAGLU gene deletion identified in an affected child of consanguineous parents. Long range and multiplex PCR methods were used to characterize this deletion which encompasses exons 3 and 4 and is 1146 base pairs long. We propose that Alu element-mediated unequal homologous recombination between an Alu-Y in intron 2 and an Alu-Sx in intron 4 is the likely mechanism for this deletion, thereby contributing further insight into the molecular etiology of this disorder and providing additional evidence of its allelic heterogeneity.

  15. Exclusion of APC and VHL gene deletions by array-based comparative hybridization in two patients with microscopically visible chromosomal aberrations.

    PubMed

    Wallerstein, Robert J; Brooks, Susan Sklower; Streck, Deanna L; Kurvathi, Rohini; Toruner, Gokce A

    2007-10-15

    Karyotyping is a major component of the genetic work-up of patients with dysmorphism. Cytogenetic aberrations close to a known tumor suppressor gene raise important clinical issues because deletion of that tumor suppressor gene can cause genetic predisposition to cancer. We present two cancer-free dysmorphic patients with karyotypes of 46,XX,del(5)(q15q22.3) and 46,XX,del(3)(p25.2~pter). These deletions are close to the APC and VHL genes that confer susceptibility to familial Adenomatous polyposis (OMIM #17510) and von-Hippel-Lindau syndrome (OMIM #193300), respectively. The array-based comparative genomic hybridization (array-CGH) analysis using a custom Agilent 44K oligonucleotide array demonstrated an interstitial 20.7-megabase (Mb) deletion on 5q (chr5: 89,725,638-110,491,345) and a terminal 9.45-Mb deletion on 3p (chr3:pter-9,450,984). According to the March 2006 human reference sequence, the APC gene is located at chr5: 112,101,483-112,209,835 and the VHL gene is located at chr3: 10,158,319-10,168,746. These results indicate that the APC gene is 2,300 kilobases (kb) and the VHL gene is 700 kb away from deleted regions. Southern blot analysis for APC and VHL genes were negative, consistent with array-CGH findings. These results demonstrate the power of array-CCH to assess potential tumor suppressor gene involvement and cancer risk in patients with microscopically visible deletions in areas near tumor suppressors.

  16. Enhanced freeze tolerance of baker's yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough.

    PubMed

    Tan, Haigang; Dong, Jian; Wang, Guanglu; Xu, Haiyan; Zhang, Cuiying; Xiao, Dongguang

    2014-08-01

    Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker's yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301(TPS1) overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301(TPS1) were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301(TPS1) was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker's yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker's yeast.

  17. Homozygous Deletion of the Very Low Density Lipoprotein Receptor Gene Causes Autosomal Recessive Cerebellar Hypoplasia with Cerebral Gyral Simplification

    PubMed Central

    Boycott, Kym M.; Flavelle, Shauna; Bureau, Alexandre; Glass, Hannah C.; Fujiwara, T. Mary; Wirrell, Elaine; Davey, Krista; Chudley, Albert E.; Scott, James N.; McLeod, D. Ross; Parboosingh, Jillian S.

    2005-01-01

    An autosomal recessive syndrome of nonprogressive cerebellar ataxia and mental retardation is associated with inferior cerebellar hypoplasia and mild cerebral gyral simplification in the Hutterite population. An identity-by-descent mapping approach using eight patients from three interrelated Hutterite families localized the gene for this syndrome to chromosome region 9p24. Haplotype analysis identified familial and ancestral recombination events and refined the minimal region to a 2-Mb interval between markers D9S129 and D9S1871. A 199-kb homozygous deletion encompassing the entire very low density lipoprotein receptor (VLDLR) gene was present in all affected individuals. VLDLR is part of the reelin signaling pathway, which guides neuroblast migration in the cerebral cortex and cerebellum. To our knowledge, this syndrome represents the first human lipoprotein receptor malformation syndrome and the second human disease associated with a reelin pathway defect. PMID:16080122

  18. Frequent intragenic deletion of the P gene in Tanzanian patients with Type II oculocutaneous albinism (OCA2)

    SciTech Connect

    Spritz, R.; Fukai, K.; Holmes, S.A.

    1995-06-01

    Type II oculocutaneous albinism (OCA2) is an autosomal recessive disorder in which the biosynthesis of melanin pigment is reduced in the skin, hair, and eyes. OCA2, which results from mutations of the P gene, is the most frequent type of albinism in African and African-American patients. OCA2 is especially frequent in Tanzania, where it occurs with an incidence of {approximately}1/1,400. We have identified abnormalities of the P gene in each of 13 unrelated patients with OCA2 from Tanzania. One of these, a deletion of exon 7, is strongly predominant, accounting for {approximately}77% of mutant alleles in this group of patients. 20 refs., 2 figs.

  19. A novel and de novo deletion in the OCRL1 gene associated with a severe form of Lowe syndrome.

    PubMed

    Peces, Ramón; Peces, Carlos; de Sousa, Erika; Vega, Cristina; Selgas, Rafael; Nevado, Julián

    2013-12-01

    The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked disorder. The mutation of the gene OCRL1 localized at Xq26.1, coding for the enzyme phosphatidylinositol (4,5) bisphosphate (PIP2P) 5-phosphatase, is responsible for the phenotypic characteristics of the disease. We report a 22-year-old male with a severe form of OCRL syndrome, diagnosed on the basis of congenital cataracts, severe psychomotor and cognitive deficits, and renal tubular dysfunction without Fanconi syndrome. The patient presented low molecular weight proteinuria, nephrocalcinosis, nephrolithiasis, rickets, and growth retardation and developed progressive renal failure. Genetic analysis showed a novel and de novo deletion of exons 10-13 in the OCRL1 gene.

  20. Symptomatic female carriers of Duchenne muscular dystrophy (DMD): genetic and clinical characterization.

    PubMed

    Giliberto, Florencia; Radic, Claudia Pamela; Luce, Leonela; Ferreiro, Verónica; de Brasi, Carlos; Szijan, Irene

    2014-01-15

    Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the dystrophin gene and is characterized by muscle degeneration and death. DMD affects males; females being asymptomatic carriers of mutations. However, some of them manifest symptoms due to a translocation between X chromosome and an autosome or to a heterozygous mutation leading to inactivation of most of their normal X chromosome. Six symptomatic female carriers and two asymptomatic were analyzed by: I) Segregation of STRs-(CA)n and MLPA assays to detect a hemizygous alteration, and II) X chromosome inactivation pattern to uncover the reason for symptoms in these females. The symptomatic females shared mild but progressive muscular weakness and increased serum creatin kinase (CK) levels. Levels of dystrophin protein were below normal or absent in many fibers. Segregation of STRs-(CA)n revealed hemizygous patterns in three patients, which were confirmed by MLPA. In addition, this analysis showed a duplication in another patient. X chromosome inactivation assay revealed a skewed X inactivation pattern in the symptomatic females and a random inactivation pattern in the asymptomatic ones. Our results support the hypothesis that the DMD phenotype in female carriers of a dystrophin mutation has a direct correlation with a skewed X-chromosome inactivation pattern.

  1. Contiguous Xp11.4 Gene Deletion Leading to Ornithine Transcarbamylase Deficiency Detected by High-density Single-nucleotide Array

    PubMed Central

    Ono, Mizuho; Tsuda, Junnosuke; Mouri, Yoko; Arai, Junichi; Arinami, Tadao; Noguchi, Emiko

    2010-01-01

    Ornithine transcarbamylase (OTC) is one of the enzymes involved in the urea cycle. OTC deficiency, which is caused by impaired synthesis of OTC in the liver, is the most common inherited disease of urea cycle disorders. In this paper, we describe the case of an OTC-deficient Japanese boy wherein an analysis based on high-density single-nucleotide polymorphisms (SNPs) revealed the absence of the entire OTC locus and nearby genes. We identified a deletion on Xp11.4; the size of the deletion fragment was approximately 1 Mb. The deleted region included genes encoding transmembrane 4 superfamily member 2 (TSPAN7), MID1 interacting protein 1 (MID1IP1) and part of the retinitis pigmentosa GTPase regulator (RPGR) in addition to OTC. The results of a high-density SNP assay and PCR confirmed that the mother of the patient was a carrier of the mutation. Previously, determination of breakpoints for large unknown deletions was timeconsuming and laborintensive. However, the use of the widely available DNA chip technology allows for rapid determination of deletion breakpoints; therefore, it will become a standard technique in study of patients with a large genomic deletion of contiguous genes for provision of comprehensive genetic counseling and initiation of clinical management. PMID:23926375

  2. Isolation of a gene expressed during early embryogenesis from the region of 22q11 commonly deleted in DiGeorge syndrome.

    PubMed

    Halford, S; Wilson, D I; Daw, S C; Roberts, C; Wadey, R; Kamath, S; Wickremasinghe, A; Burn, J; Goodship, J; Mattei, M G

    1993-10-01

    DiGeorge syndrome (DGS) is one of several syndromes associated with deletions within the proximal long-arm of chromosome 22. The region of chromosome 22q11 responsible for the haploinsufficiency syndromes (the DiGeorge Critical Region or DGCR) has been mapped using RFLPs, quantitative Southern blotting and FISH. Similar deletions are seen in the velo-cardio-facial syndrome (VCFS) and familial congenital heart defects. It is not known whether the phenotypic spectrum is the result of the hemizygosity of one gene or whether it is a consequence of contiguous genes being deleted. However, the majority of patients have a large (> = 2Mb deletion). In this paper we report the isolation of a gene, lab name T10, encoding a serine/threonine rich protein of unknown function which maps to the commonly deleted region of chromosome 22q11. Studies in the mouse indicate that it maps to MMU16 and is expressed during early embryogenesis. Although not mapping within the shortest region of overlap for DGS/VCFS, and therefore not the major gene involved in DGS, the expression pattern suggests that this gene may be involved in modifying the haploinsufficient phenotype of hemizygous patients.

  3. Intragenic deletions of IL1RAPL1: Report of two cases and review of the literature.

    PubMed

    Behnecke, Anne; Hinderhofer, Katrin; Bartsch, Oliver; Nümann, Astrid; Ipach, Marie-Luise; Damatova, Natalja; Haaf, Thomas; Dufke, Andreas; Riess, Olaf; Moog, Ute

    2011-02-01

    IL1RAPL1 (interleukin-1 receptor accessory protein-like 1) located at Xp21.3-22.1 has repeatedly been shown to be deleted in patients with a contiguous gene syndrome also affecting neighboring genes, in particular DMD (dystrophin), DAX-1 (NR0B1, nuclear receptor subfamily 0, group B, member 1), and GK (glycerol kinase). In contrast, intragenic deletions of IL1RAPL1 or other mutations or cytogenetic aberrations affecting IL1RAPL1 have only rarely been identified. Up to date, they have mostly been associated with nonspecific mental retardation (MRX). We report on two nonrelated patients with MR and additional dysmorphic features who both show intragenic deletions of IL1RAPL1, one of them being de novo (exon 2) and the other one being inherited from his mother (exons 3-5). Deletions were identified by microarray-based chromosome analysis and confirmed by multiplex PCR and FISH, respectively. These data, along with recent functional studies indicating its role in neuronal development, provide further evidence for the relevance of IL1RAPL1 in the pathogenesis of X-linked MR and add knowledge to the phenotypic spectrum of IL1RAPL1 mutations.

  4. Deletion of mouse FXR gene disturbs multiple neurotransmitter systems and alters neurobehavior

    PubMed Central

    Huang, Fei; Wang, Tingting; Lan, Yunyi; Yang, Li; Pan, Weihong; Zhu, Yonghui; Lv, Boyang; Wei, Yuting; Shi, Hailian; Wu, Hui; Zhang, Beibei; Wang, Jie; Duan, Xiaofeng; Hu, Zhibi; Wu, Xiaojun

    2015-01-01

    Farnesoid X receptor (FXR) is a nuclear hormone receptor involved in bile acid synthesis and homeostasis. Dysfunction of FXR is involved in cholestasis and atherosclerosis. FXR is prevalent in liver, gallbladder, and intestine, but it is not yet clear whether it modulates neurobehavior. In the current study, we tested the hypothesis that mouse FXR deficiency affects a specific subset of neurotransmitters and results in an unique behavioral phenotype. The FXR knockout mice showed less depressive-like and anxiety-related behavior, but increased motor activity. They had impaired memory and reduced motor coordination. There were changes of glutamatergic, GABAergic, serotoninergic, and norepinephrinergic neurotransmission in either hippocampus or cerebellum. FXR deletion decreased the amount of the GABA synthesis enzyme GAD65 in hippocampus but increased GABA transporter GAT1 in cerebral cortex. FXR deletion increased serum concentrations of many bile acids, including taurodehydrocholic acid, taurocholic acid, deoxycholic acid (DCA), glycocholic acid (GCA), tauro-α-muricholic acid, tauro-ω-muricholic acid, and hyodeoxycholic acid (HDCA). There were also changes in brain concentrations of taurocholic acid, taurodehydrocholic acid, tauro-ω-muricholic acid, tauro-β-muricholic acid, deoxycholic acid, and lithocholic acid (LCA). Taken together, the results from studies with FXR knockout mice suggest that FXR contributes to the homeostasis of multiple neurotransmitter systems in different brain regions and modulates neurobehavior. The effect appears to be at least partially mediated by bile acids that are known to cross the blood-brain barrier (BBB) inducing potential neurotoxicity. PMID:25870546

  5. A 76-bp deletion in the Mip gene causes autosomal dominant cataract in Hfi mice.

    PubMed

    Sidjanin, D J; Parker-Wilson, D M; Neuhäuser-Klaus, A; Pretsch, W; Favor, J; Deen, P M; Ohtaka-Maruyama, C; Lu, Y; Bragin, A; Skach, W R; Chepelinsky, A B; Grimes, P A; Stambolian, D E

    2001-06-15

    Hfi is a dominant cataract mutation where heterozygotes show hydropic lens fibers and homozygotes show total lens opacity. The Hfi locus was mapped to the distal part of mouse chromosome 10 close to the major intrinsic protein (Mip), which is expressed only in cell membranes of lens fibers. Molecular analysis of Mip revealed a 76-bp deletion that resulted in exon 2 skipping in Mip mRNA. In Hfi/Hfi this deletion resulted in a complete absence of the wildtype Mip. In contrast, Hfi/+ animals had the same amount of wildtype Mip as +/+. Results from pulse-chase expression studies excluded hetero-oligomerization of wildtype and mutant Mip as a possible mechanism for cataract formation in the Hfi/+. We propose that the cataract phenotype in the Hfi heterozygote mutant is due to a detrimental gain of function by the mutant Mip resulting in either cytotoxicity or disruption in processing of other proteins important for the lens. Cataract formation in the Hfi/Hfi mouse is probably a combined result of both the complete loss of wildtype Mip and a gain of function of the mutant Mip.

  6. Lentiviral vectors can be used for full-length dystrophin gene therapy

    PubMed Central

    Counsell, John R.; Asgarian, Zeinab; Meng, Jinhong; Ferrer, Veronica; Vink, Conrad A.; Howe, Steven J.; Waddington, Simon N.; Thrasher, Adrian J.; Muntoni, Francesco; Morgan, Jennifer E.; Danos, Olivier

    2017-01-01

    Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a ‘template-switching’ lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD. PMID:28303972

  7. Lentiviral vectors can be used for full-length dystrophin gene therapy.

    PubMed

    Counsell, John R; Asgarian, Zeinab; Meng, Jinhong; Ferrer, Veronica; Vink, Conrad A; Howe, Steven J; Waddington, Simon N; Thrasher, Adrian J; Muntoni, Francesco; Morgan, Jennifer E; Danos, Olivier

    2017-12-01

    Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

  8. Homozygous deletion but not mutation of exons 5 and 8 of the fragile histidine triad (FHIT) gene is associated with features of differentiated thyroid carcinoma.

    PubMed

    Yin, De-Tao; Wang, Lin; Sun, Jianrei; Yin, Fengyan; Yan, Qingtao; Shen, Ru-Long; Gao, Jian-Xin; He, Gang

    2010-01-01

    The fragile histidine triad (FHIT) gene encompasses the most common human fragile site, FRA3B at 3p14.2, a region that is involved in homozygous deletions in a variety of human tumors. FHIT is considered to be a tumor suppressor gene that is frequently inactivated in various types of cancer. To study the role of the FHIT gene in thyroid tumorigenesis, we looked for homozygous deletions or mutations of exons 5 and 8 of the FHIT gene in 65 cases of differentiated thyroid carcinoma (DTC) and their matched non-cancerous epithelium (NCE), using exon-specific PCR amplification and PCR single strand conformation polymorphism (PCR-SSCP) techniques. In DTC, the incidence of homozygous deletion of exon 5 was 30.8% (20/65), and it was associated with tumor metastasis to lymph nodes (p <0.05). The incidence of homozygous deletion of exon 8 was 29.2% (19/65), and it was associated with the tumor pathological grade, TNM stage, and lymph node metastasis (p <0.05). There was strong correlation between homozygous deletions of exon 5 and exon 8 (p <0.01). No point mutations were observed in either exon 5 or exon 8. These findings suggest that: (a) exons 5 and 8 of FHIT are key target regions of deletion, (b) homozygous deletions of exon 5 and exon 8 may be good biomarkers for the biological behavior of DTC, and (c) point mutation of these exons may not be involved in the inactivation of the FHIT gene in DTC.

  9. Combined deletion of DAZ2 and DAZ4 copies of Y chromosome DAZ gene is associated with male infertility in Tunisian men.

    PubMed

    Ghorbel, Myriam; Baklouti-Gargouri, Siwar; Keskes, Rim; Chakroun, Nozha; Sellami, Afifa; Fakhfakh, Faiza; Ammar-Keskes, Leila

    2014-09-01

    The relationship between male infertility and AZFc micro-deletions that remove multiple genes of the Y chromosome varies among countries and populations. The purpose of this study was to analyze the prevalence and the characteristics of different Deleted in azoospermia (DAZ) gene copy deletions and their association with spermatogenic failure and male infertility in Tunisian men. 241 infertile men (30.7% azoospermic (n=74), 31.5% oligozoospermic (n=76) and 37.7% normozoospermic (n=91)) and 115 fertile healthy males who fathered at least one child were included in the study. Three DAZ-specific single nucleotide variant loci and six bi-allelic DAZ-SNVs (I-VI) were analyzed using polymerase chain reaction (PCR)-restriction fragment length polymorphism and PCR. Our findings showed high frequencies of infertile men (73.85%) and controls (78.26%) having only three DAZ gene copies (DAZ1/DAZ2/DAZ3 or DAZ1/DAZ3/DAZ4 variants); so deletion of DAZ2 or DAZ4 were frequent both in infertile (36.5% and 37.3%, respectively) and fertile groups (33.9% and 44.3%, respectively) and removing DAZ4 copy was significantly more frequent in oligospermic than in normospermic men (p=0.04) in infertile group. We also report for the first time that simultaneous deletion of both DAZ2 and DAZ4 copies was significantly more common in infertile men (12.4%) than in fertile men (4.3%) (p=0.01). However, deletions of DAZ1/DAZ2 and DAZ3/DAZ4 clusters were very rare. Analysis of DAZ gene copies in Tunisian population, suggested that the simultaneous deletion of DAZ2 and DAZ4 gene copies is associated with male infertility, and that oligospermia seems to be promoted by removing DAZ4 copy.

  10. Schizophrenia and chromosomal deletions

    SciTech Connect

    Lindsay, E.A.; Baldini, A.; Morris, M. A.

    1995-06-01

    Recent genetic linkage analysis studies have suggested the presence of a schizophrenia locus on the chromosomal region 22q11-q13. Schizophrenia has also been frequently observed in patients affected with velo-cardio-facial syndrome (VCFS), a disorder frequently associated with deletions within 22q11.1. It has been hypothesized that psychosis in VCFS may be due to deletion of the catechol-o-methyl transferase gene. Prompted by these observations, we screened for 22q11 deletions in a population of 100 schizophrenics selected from the Maryland Epidemiological Sample. Our results show that there are schizophrenic patients carrying a deletion of 22q11.1 and a mild VCFS phenotype that might remain unrecognized. These findings should encourage a search for a schizophrenia-susceptibility gene within the deleted region and alert those in clinical practice to the possible presence of a mild VCFS phenotype associated with schizophrenia. 9 refs.

  11. Dietary MicroRNA Database (DMD): An Archive Database and Analytic Tool for Food-Borne microRNAs.

    PubMed

    Chiang, Kevin; Shu, Jiang; Zempleni, Janos; Cui, Juan

    2015-01-01

    With the advent of high throughput technology, a huge amount of microRNA information has been added to the growing body of knowledge for non-coding RNAs. Here we present the Dietary MicroRNA Databases (DMD), the first repository for archiving and analyzing the published and novel microRNAs discovered in dietary resources. Currently there are fifteen types of dietary species, such as apple, grape, cow milk, and cow fat, included in the database originating from 9 plant and 5 animal species. Annotation for each entry, a mature microRNA indexed as DM0000*, covers information of the mature sequences, genome locations, hairpin structures of parental pre-microRNAs, cross-species sequence comparison, disease relevance, and the experimentally validated gene targets. Furthermore, a few functional analyses including target prediction, pathway enrichment and gene network construction have been integrated into the system, which enable users to generate functional insights through viewing the functional pathways and building protein-protein interaction networks associated with each microRNA. Another unique feature of DMD is that it provides a feature generator where a total of 411 descriptive attributes can be calculated for any given microRNAs based on their sequences and structures. DMD would be particularly useful for research groups studying microRNA regulation from a nutrition point of view. The database can be accessed at http://sbbi.unl.edu/dmd/.

  12. Dietary MicroRNA Database (DMD): An Archive Database and Analytic Tool for Food-Borne microRNAs

    PubMed Central

    Chiang, Kevin; Shu, Jiang; Zempleni, Janos; Cui, Juan

    2015-01-01

    With the advent of high throughput technology, a huge amount of microRNA information has been added to the growing body of knowledge for non-coding RNAs. Here we present the Dietary MicroRNA Databases (DMD), the first repository for archiving and analyzing the published and novel microRNAs discovered in dietary resources. Currently there are fifteen types of dietary species, such as apple, grape, cow milk, and cow fat, included in the database originating from 9 plant and 5 animal species. Annotation for each entry, a mature microRNA indexed as DM0000*, covers information of the mature sequences, genome locations, hairpin structures of parental pre-microRNAs, cross-species sequence comparison, disease relevance, and the experimentally validated gene targets. Furthermore, a few functional analyses including target prediction, pathway enrichment and gene network construction have been integrated into the system, which enable users to generate functional insights through viewing the functional pathways and building protein-protein interaction networks associated with each microRNA. Another unique feature of DMD is that it provides a feature generator where a total of 411 descriptive attributes can be calculated for any given microRNAs based on their sequences and structures. DMD would be particularly useful for research groups studying microRNA regulation from a nutrition point of view. The database can be accessed at http://sbbi.unl.edu/dmd/. PMID:26030752

  13. Identification of two novel deletion mutations within the Gs alpha gene (GNAS1) in Albright hereditary osteodystrophy.

    PubMed

    Yu, D; Yu, S; Schuster, V; Kruse, K; Clericuzio, C L; Weinstein, L S

    1999-09-01

    Albright hereditary osteodystrophy (AHO) is a genetic disorder characterized by short stature, skeletal defects, and obesity. Within AHO kindreds, some affected family members have only the somatic features of AHO [pseudopseudohypoparathyroidism (PPHP)], whereas others have these features in association with resistance to multiple hormones that stimulate adenylyl cyclase within their target tissues [pseudohypoparathyroidism type Ia (PHP Ia)]. Affected members of most AHO kindreds (both those with PPHP and those with PHP Ia) have a partial deficiency of Gs alpha, the alpha-subunit of the G protein that couples receptors to adenylyl cyclase stimulation, and in a number of cases heterozygous loss of function mutations within the Gs alpha gene (GNAS1) have been identified. Using PCR with the attachment of a high melting domain (GC-clamp) and temperature gradient gel electrophoresis, two novel heterozygous frameshift mutations within GNAS1 were found in two AHO kindreds. In one kindred all affected members (both PHP Ia and PPHP) had a heterozygous 2-bp deletion in exon 8, whereas in the second kindred a heterozygous 2-bp deletion in exon 4 was identified in all affected members examined. In both cases the frameshift encoded a premature termination codon several codons downstream of the deletion. In the latter kindred affected members were previously shown to have decreased levels of GNAS1 messenger ribonucleic acid expression. These results further underscore the genetic heterogeneity of AHO and provides further evidence that PHP Ia and PPHP are two clinical presentations of a common genetic defect. Serial measurements of thyroid function in members of kindred 1 indicate that TSH resistance progresses with age and becomes more evident after the first year of life.

  14. Adipose-specific deletion of autophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis.

    PubMed

    Zhang, Yong; Goldman, Scott; Baerga, Rebecca; Zhao, Yun; Komatsu, Masaaki; Jin, Shengkan

    2009-11-24

    White adipocytes have a unique structure in which nearly the entire cell volume is occupied by one large lipid droplet. However, the molecular and cellular processes involved in the cytoplasmic remodeling necessary to create this structure are poorly defined. Autophagy is a membrane trafficking process leading to lysosomal degradation. Here, we investigated the effect of the deletion of an essential autophagy gene, autophagy-related gene 7 (atg7), on adipogenesis. A mouse model with a targeted deletion of atg7 in adipose tissue was generated. The mutant mice were slim and contained only 20% of the mass of white adipose tissue (WAT) found in wild-type mice. Interestingly, approximately 50% of the mutant white adipocytes were multilocular. The mutant white adipocytes were smaller with a larger volume of cytosol and contained more mitochondria. These cells exhibited altered fatty acid metabolism with increased rates of beta-oxidation and reduced rates of hormone-induced lipolysis. Consistently, the mutant mice had lower fed plasma concentrations of fatty acids and the levels decreased at faster rates upon insulin stimuli. These mutant mice exhibited increased insulin sensitivity. The mutant mice also exhibited markedly decreased plasma concentrations of leptin but not adiponectin, lower plasma concentrations of triglyceride and cholesterol, and they had higher levels of basal physical activity. Strikingly, these mutant mice were resistant to high-fat-diet-induced obesity. Taken together, our results indicate that atg7, and by inference autophagy, plays an important role in normal adipogenesis and that inhibition of autophagy by disrupting the atg7 gene has a unique anti-obesity and insulin sensitization effect.

  15. Diminished dosage of 22q11 genes disrupts neurogenesis and cortical development in a mouse model of 22q11 deletion/DiGeorge syndrome.

    PubMed

    Meechan, Daniel W; Tucker, Eric S; Maynard, Thomas M; LaMantia, Anthony-Samuel

    2009-09-22

    The 22q11 deletion (or DiGeorge) syndrome (22q11DS), the result of a 1.5- to 3-megabase hemizygous deletion on human chromosome 22, results in dramatically increased susceptibility for "diseases of cortical connectivity" thought to arise during development, including schizophrenia and autism. We show that diminished dosage of the genes deleted in the 1.5-megabase 22q11 minimal critical deleted region in a mouse model of 22q11DS specifically compromises neurogenesis and subsequent differentiation in the cerebral cortex. Proliferation of basal, but not apical, progenitors is disrupted, and subsequently, the frequency of layer 2/3, but not layer 5/6, projection neurons is altered. This change is paralleled by aberrant distribution of parvalbumin-labeled interneurons in upper and lower cortical layers. Deletion of Tbx1 or Prodh (22q11 genes independently associated with 22q11DS phenotypes) does not similarly disrupt basal progenitors. However, expression analysis implicates additional 22q11 genes that are selectively expressed in cortical precursors. Thus, diminished 22q11 gene dosage disrupts cortical neurogenesis and interneuron migration. Such developmental disruption may alter cortical circuitry and establish vulnerability for developmental disorders, including schizophrenia and autism.

  16. Evidence of gene deletion of p21 (WAF1/CIP1), a cyclin-dependent protein kinase inhibitor, in thyroid carcinomas.

    PubMed Central

    Shi, Y.; Zou, M.; Farid, N. R.; al-Sedairy, S. T.

    1996-01-01

    Eukaryotic cell cycle progression is controlled by a host of cyclin/cyclin-dependent kinases (Cdks), that are themselves regulated by multiple factors, including a group of small cyclin-Cdk inhibitor proteins (p15, p16, p21 and p27). The involvement of Cdk inhibitors in carcinogenesis has been demonstrated by the studies of p16. p53 is frequently mutated in thyroid carcinomas and p21/Waf1 is a downstream effector of p53. It is conceivable that genetic defects of genes downstream in the p53 pathway could also be oncogenic. We, therefore, examined a series of 57 thyroid tumour specimens (eight follicular adenomas and 49 carcinomas) for deletion and point mutation of the p21/Waf1 gene. Three different kinds of deletions ranging from 349 to 450 bp were detected in five papillary carcinoma specimens by reverse transcription-polymerase chain reaction (RT-PCR). All the deletions were involved in the second exon of the p21/Waf1 gene. RT-PCR single strand conformational polymorphism (SSCP) analysis of remaining samples failed to reveal any point mutations in the coding region of the gene, except for a polymorphism at codon 31 (Ser to Arg). Genomic Southern blot analysis did not demonstrate any gene deletion or rearrangement in these samples, indicating abnormal RNA splicing may be involved. Analysis of intron-exon boundary and the coding region of the second exon did not reveal any mutation except for a point mutation (C to G) located 16 bp downstream from the splice donor site of the second intron in three out of five samples with p21/Waf1 deletions. Whether the mutation plays any role in aberrant RNA splicing remains to be determined. Among the five samples with p21/Waf1 gene deletions, none of them simultaneously carried a p53 or retinoblastoma (Rb) gene mutation. No p21/Waf1 abnormality was found in the benign adenomas. Thus, 12.5% (5/40) of thyroid papillary carcinoma specimens harboured p21/Waf1 gene deletions. Our data suggest that p21/Waf1 gene deletion is involved

  17. Deletion of the telomerase reverse transcriptase gene and haploinsufficiency of telomere maintenance in Cri du chat syndrome.

    PubMed

    Zhang, Anju; Zheng, Chengyun; Hou, Mi; Lindvall, Charlotta; Li, Ke-Jun; Erlandsson, Fredrik; Björkholm, Magnus; Gruber, Astrid; Blennow, Elisabeth; Xu, Dawei

    2003-04-01

    Cri du chat syndrome (CdCS) results from loss of the distal portion of chromosome 5p, where the telomerase reverse transcriptase (hTERT) gene is localized (5p15.33). hTERT is the rate-limiting component for telomerase activity that is essential for telomere-length maintenance and sustained cell proliferation. Here, we show that a concomitant deletion of the hTERT allele occurs in all 10 patients with CdCS whom we examined. Induction of hTERT mRNA in proliferating lymphocytes derived from five of seven patients was lower than that in unaffected control individuals (P<.05). The patient lymphocytes exhibited shorter telomeres than age-matched unaffected individuals (P<.0001). A reduction in replicative life span and a high rate of chromosome fusions were observed in cultured patient fibroblasts. Reconstitution of telomerase activity by ectopic expression of hTERT extended the telomere length, increased the population doublings, and prevented the end-to-end fusion of chromosomes. We conclude that hTERT is limiting and haploinsufficient for telomere maintenance in humans in vivo. Accordingly, the hTERT deletion may be one genetic element contributing to the phenotypic changes in CdCS.

  18. Trisomy of the short stature homeobox-containing gene (SHOX), resulting from a duplication-deletion of the X chromosome.

    PubMed

    Adamson, K A; Cross, I; Batch, J A; Rappold, G A; Glass, I A; Ball, S G

    2002-05-01

    The Turner syndrome (TS) is a complex disorder associated with almost invariant short stature and gonadal dysgenesis, as well as a variety of other major organ malformations. Recently, a homeobox-containing gene entitled short-stature homeobox-containing gene (SHOX), was isolated from a minimal short stature gene interval from the pseudoautosomal region of Xp (and Yp). Together with the demonstrable escape of SHOX from X-inactivation, this suggested SHOX to be a strong candidate gene for the short stature component of TS, and as SHOX haploinsufficiency appears to be the molecular basis of a mesomelic short statured skeletal dysplasia (Leri-Weill syndrome), this suggested that SHOX protein expression levels may confer a dosage effect on human stature. However, in this communication we report a normal statured female with gonadal dysgenesis, due to the inheritance of a recombinant duplication-deletion X-chromosome. The karyotype of the proband was 46,X,rec(X)dup(Xp)inv(X)(p11.22q21.2)mat and fluorescent in situ hybridization of her metaphases with a SHOX cosmid confirmed the proband to be trisomic for SHOX. This communication suggests the relationship between levels of SHOX expression and human stature to be more complex than envisaged previously. The presence of normal stature in our patient rather than tall stature is likely to represent the natural variation seen in patients with transcription factor disorders.

  19. Gene expression of key regulators of mitochondrial biogenesis is sex dependent in mice with growth hormone receptor deletion in liver

    PubMed Central

    Zawada, Ilona; Masternak, Michal M.; List, Edward O.; Stout, Michael B.; Berryman, Darlene E.; Lewinski, Andrzej; Kopchick, John J.; Bartke, Andrzej; Karbownik-Lewinska, Malgorzata; Gesing, Adam

    2015-01-01

    Mitochondrial biogenesis is an essential process for cell viability. Mice with disruption of the growth hormone receptor (GHR) gene (Ghr gene) in the liver (LiGHRKO), in contrast to long-lived mice with global deletion of the Ghr gene (GHRKO), are characterized by lack of improved insulin sensitivity and severe hepatic steatosis. Tissue-specific disruption of the GHR in liver results in a mouse model with dramatically altered GH/IGF1 axis. We have previously shown increased levels of key regulators of mitochondrial biogenesis in insulin-sensitive GHRKO mice. The aim of the present study is to assess, using real-time PCR, the gene expression of key regulators of mitochondrial biogenesis (Pgc1α, Ampk, Sirt1, Nrf2 and Mfn2) and a marker of mitochondrial activity (CoxIV) in brains, kidneys and livers of male and female LiGHRKO and wild-type (WT) mice. There were significant differences between males and females. In the brain, expression of Pgc1α, Ampk, Sirt1, Nrf2 and Mfn2 was lower in pooled females compared to pooled males. In the kidneys, expression of Ampk and Sirt1 was also lower in female mice. In the liver, no differences between males and females were observed. Sexual dimorphism may play an important role in regulating the biogenesis of mitochondria. PMID:25855408

  20. Gene expression of key regulators of mitochondrial biogenesis is sex dependent in mice with growth hormone receptor deletion in liver.

    PubMed

    Zawada, Ilona; Masternak, Michal M; List, Edward O; Stout, Michael B; Berryman, Darlene E; Lewinski, Andrzej; Kopchick, John J; Bartke, Andrzej; Karbownik-Lewinska, Malgorzata; Gesing, Adam

    2015-03-01

    Mitochondrial biogenesis is an essential process for cell viability. Mice with disruption of the growth hormone receptor (GHR) gene (Ghr gene) in the liver (LiGHRKO), in contrast to long-lived mice with global deletion of the Ghr gene (GHRKO), are characterized by lack of improved insulin sensitivity and severe hepatic steatosis. Tissue-specific disruption of the GHR in liver results in a mouse model with dramatically altered GH/IGF1 axis. We have previously shown increased levels of key regulators of mitochondrial biogenesis in insulin-sensitive GHRKO mice. The aim of the present study is to assess, using real-time PCR, the gene expression of key regulators of mitochondrial biogenesis (Pgc1α, Ampk, Sirt1, Nrf2 and Mfn2) and a marker of mitochondrial activity (CoxIV) in brains, kidneys and livers of male and female LiGHRKO and wild-type (WT) mice. There were significant differences between males and females. In the brain, expression of Pgc1α, Ampk, Sirt1, Nrf2 and Mfn2 was lower in pooled females compared to pooled males. In the kidneys, expression of Ampk and Sirt1 was also lower in female mice. In the liver, no differences between males and females were observed. Sexual dimorphism may play an important role in regulating the biogenesis of mitochondria.

  1. A novel deletion and two recurrent substitutions on type VII collagen gene in seven Iranian patients with epidermolysis bullosa

    PubMed Central

    Hamidi, Armita Kakavand; Moghaddam, Mohammad; Hatamnejadian, Nasim; Ebrahimi, Ahmad

    2016-01-01

    Objective(s): Epidermolysis bullosa is one of the most important series of mechano-bullous heritable skin disorders which is categorized into four major types according to the layer that bullae forms within basement membrane zone. In dystrophic form of the disease, blisters are made in the sublamina densa zone, at the level of type VII collagen protein which produce anchoring fibrils. Type VII collagen gene is the only responsible gene for this form. The aim of this study was to survey causative mutations of type VII collagen gene among Iranian patients with epidermolysis bullosa. Materials and Methods: For this purpose, exons 73-75 were investigated by polymerase chain reaction followed by direct sequencing. Results: In current study, we found three different point mutations in type VII collagen alleles in 7 out of 50 patients. Four patients were homozygous for a new deletion which resulted in frame shift (p.Pro2089fs). Two patients were homozygous for a recurrent glycine substitution (p.G2031S) and one patient was detected with an allele carrying a substitution (p.R2069C). Conclusion: The results emphasized heterogeneity in the type VII collagen gene and will provide a sign for early diagnosis and future study of the disease pathogenesis. PMID:27746867

  2. High association of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism with recurrent aphthous stomatitis.

    PubMed

    Karakus, Nevin; Yigit, Serbulent; Kalkan, Goknur; Sezer, Saime

    2013-08-01

    Recurrent aphthous stomatitis (RAS) is a common ulcerative disease of the oral mucosa. Oral ulcers are also the most common feature of Behçet's disease (BD). Association of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism with BD has been reported in Turkish population. The aim of the present study was to investigate the possible association between ACE gene I/D polymorphism and RAS, and evaluate if there was an association with clinical features in a relatively large cohort of Turkish patients. The study included 198 patients affected by RAS and 214 healthy controls. ACE gene I/D polymorphism genotypes were determined using polymerase chain reaction with I and D allele-specific primers. The genotype and allele frequencies of I/D polymorphism showed statistically significant differences between RAS patients and controls (p < 0.0001 and p < 0.0001, respectively). After stratifying RAS patients according to clinical and demographical characteristics, no significant association was observed. In conclusion, the results of this study suggest that I/D polymorphism of the ACE gene was positively associated with predisposition to develop RAS in Turkish population. Further studies with larger populations are recommended.

  3. Deletion of the mouse homolog of KCNAB2, a gene linked to monosomy 1p36, results in associative memory impairments and amygdala hyperexcitability.

    PubMed

    Perkowski, John J; Murphy, Geoffrey G

    2011-01-05

    Ablation of the distal end of the short arm of chromosome 1 [1p36 deletion syndrome (1p36DS)] is one of the most commonly occurring terminal deletion syndromes in humans, occurring in ∼1 in 5000 newborns. Subjects with 1p36DS manifest a wide range of clinical features including growth delay, congenital heart defects, and craniofacial dysmorphism. In addition, individuals with 1p36DS often exhibit some form of neurological abnormality and are typically cognitively impaired. Although there is significant variability with regard to the extent of the deletion, several genes have been mapped to region 1p36 that are known to regulate neuronal function. One such gene--KCNAB2--encodes the potassium channel auxiliary subunit Kvβ2, which has been previously shown to modulate voltage-gated potassium currents in heterologous expression systems. Here, we present experiments characterizing mice in which the ortholog of KCNAB2 was deleted. We find that deletion of Kcnab2 in mice leads to deficits in associative learning and memory. In addition, using whole-cell current-clamp, we find that deletion of Kcnab2 leads to a reduction in the slow afterhyperpolarization following a burst of action potentials and a concomitant increase in neuronal excitability in projection neurons in the lateral nucleus of the amygdala. Our results suggest that loss of Kvβ2 likely contributes to the cognitive and neurological impairments observed in 1p36DS patients.

  4. Projection collimator optics for DMD-based infrared scene simulator

    NASA Astrophysics Data System (ADS)

    Zheng, Yawei; Hu, Yu; Li, Junnan; Huang, Meili; Gao, Jiaobo; Wang, Jun; Sun, Kefeng; Li, Jianjun; Zhang, Fang

    2016-10-01

    The design of the collimator for dynamic infrared (IR) scene simulation based on the digital micro-mirror devices (DMD) is present in this paper. The collimator adopts a reimaging configuration to limit in physical size availability and cost. The aspheric lens is used in the relay optics to improve the image quality and simplify the optics configuration. The total internal reflection (TIR) prisms is located between the last surface of the optics and the DMD to fold the raypaths of the IR light source. The optics collimates the output from 1024×768 element DMD in the 8 10.3μm waveband and enables an imaging system to be tested out of 8° Field Of View (FOV). The long pupil distance of 800mm ensures the remote location seekers under the test.

  5. The effects of upaB deletion and the double/triple deletion of upaB, aatA, and aatB genes on pathogenicity of avian pathogenic Escherichia coli.

    PubMed

    Zhu-Ge, Xiang-Kai; Pan, Zi-Hao; Tang, Fang; Mao, Xiang; Hu, Lin; Wang, Shao-Hui; Xu, Bin; Lu, Cheng-Ping; Fan, Hong-Jie; Dai, Jian-Jun

    2015-12-01

    Autotransporters (ATs) are associated with pathogenesis of Avian Pathogenic Escherichia coli (APEC). The molecular characterization of APEC ATs can provide insights about their relevance to APEC pathogenesis. Here, we characterized a conventional autotransporter UpaB in APEC DE205B genome. The upaB existed in 41.9 % of 236 APEC isolates and was predominantly associated with ECOR B2 and D. Our studies showed that UpaB mediates the DE205B adhesion in DF-1 cells, and enhances autoaggregation and biofilm formation of fimbria-negative E. coli AAEC189 (MG1655Δfim) in vitro. Deletion of upaB of DE205B attenuates the virulence in duck model and early colonization in the duck lungs during APEC systemic infection. Furthermore, double and triple deletion of upaB, aatA, and aatB genes cumulatively attenuated DE205B adhesion in DF-1 cells, accompanying with decreased 50 % lethal dose (LD50) in duck model and the early colonization in the duck lungs. However, DE205BΔupaB/ΔaatA/ΔaatB might "compensate" the influence of gene deletion by upregulating the expression of fimbrial adhesin genes yqiL, yadN, and vacuolating autotransporter vat during early colonization of APEC. Finally, we demonstrated that vaccination with recombinant UpaB, AatA, and AatB proteins conferred protection against colisepticemia caused by DE205B infection in duck model.

  6. X-linked late-onset sensorineural deafness caused by a deletion involving OA1 and a novel gene containing WD-40 repeats.

    PubMed Central

    Bassi, M T; Ramesar, R S; Caciotti, B; Winship, I M; De Grandi, A; Riboni, M; Townes, P L; Beighton, P; Ballabio, A; Borsani, G

    1999-01-01

    We have identified a novel gene, transducin (beta)-like 1 (TBL1), in the Xp22.3 genomic region, that shows high homology with members of the WD-40-repeat protein family. The gene contains 18 exons spanning approximately 150 kb of the genomic region adjacent to the ocular albinism gene (OA1) on the telomeric side. However, unlike OA1, TBL1 is transcribed from telomere to centromere. Northern analysis indicates that TBL1 is ubiquitously expressed, with two transcripts of approximately 2.1 kb and 6.0 kb. The open reading frame encodes a 526-amino acid protein, which shows the presence of six beta-transducin repeats (WD-40 motif) in the C-terminal domain. The homology with known beta-subunits of G proteins and other WD-40-repeat containing proteins is restricted to the WD-40 motif. Genomic analysis revealed that the gene is either partly or entirely deleted in patients carrying Xp22.3 terminal deletions. The complexity of the contiguous gene-syndrome phenotype shared by these patients depends on the number of known disease genes involved in the deletions. Interestingly, one patient carrying a microinterstitial deletion involving the 3' portion of both TBL1 and OA1 shows the OA1 phenotype associated with X-linked late-onset sensorineural deafness. We postulate an involvement of TBL1 in the pathogenesis of the ocular albinism with late-onset sensorineural deafness phenotype. PMID:10330347

  7. Male with typical fragile X phenotype is deleted for part of the FMR1 gene and for about 100 kb of upstream region

    SciTech Connect

    Trottier, Y.; Imbert, G.; Mandel, J.L.; Fryns, J.P.; Poustka, A.

    1994-07-15

    We report on a patient with moderate mental retardation and a typical fragile X phenotype, with no family history and no fragile X site on cytogenetic analysis. The patient was found to have a deletion encompassing part of the FMR1 gene and a 70-100 kb region upstream of the FMR1 promotor region. This deletion is smaller than those previously reported and confirms that FMR1 is the major and probably the only gene implicated in the phenotype of the fragile X syndrome. 16 refs., 3 figs.

  8. Genome-Wide Deletion Screening with the Array CGH Method in Mouse Offspring Derived from Irradiated Spermatogonia Indicates that Mutagenic Responses are Highly Variable among Genes.

    PubMed

    Asakawa, Jun-Ichi; Kodaira, Mieko; Miura, Akiko; Tsuji, Takahiro; Nakamoto, Yoshiko; Imanaka, Masaaki; Kitamura, Jun; Cullings, Harry; Nishimura, Mayumi; Shimada, Yoshiya; Nakamura, Nori

    2016-12-01

    Until the end of the 20th century, mouse germ cell data on induced mutation rates, which were collected using classical genetic methods at preselected specific loci, provided the principal basis for estimates of genetic risks from radiation in humans. The work reported on here is an extension of earlier efforts in this area using molecular methods. It focuses on validating the use of array comparative genomic hybridization (array CGH) methods for identifying radiation-induced copy number variants (CNVs) and specifically for DNA deletions. The emphasis on deletions stems from the view that it constitutes the predominant type of radiation-induced genetic damage, which is relevant for estimating genetic risks in humans. In the current study, deletion mutations were screened in the genomes of F1 mice born to unirradiated or 4 Gy irradiated sires at the spermatogonia stage (100 offspring each). The array CGH analysis was performed using a "2M array" with over 2 million probes with a mean interprobe distance of approximately 1 kb. The results provide evidence of five molecularly-confirmed paternally-derived deletions in the irradiated group (5/100) and one in the controls (1/100). These data support a calculation, which estimates that the mutation rate is 1 × 10(-2)/Gy per genome for induced deletions; this is much lower than would be expected if one assumes that the specific locus rate of 1 × 10(-5)/locus per Gy (at 34 loci) is applicable to other genes in the genome. The low observed rate of induced deletions suggests that the effective number of genes/genomic regions at which recoverable deletions could be induced would be only approximately 1,000. This estimate is far lower than expected from the size of the mouse genome (>20,000 genes). Such a discrepancy between observation and expectation can occur if the genome contains numerous genes that are far less sensitive to radiation-induced deletions, if many deletion-bearing offspring are not viable or if the current

  9. A recombination outside the BB deletion refines the location of the X-linked retinitis pigmentosa locus RP3

    SciTech Connect

    Fujita, R.; Bingham, E.; Forsythe, P.; McHenry, C.

    1996-07-01

    Genetic loci for X-linked retinitis pigmentosa (XLRP) have been mapped between Xp11.22 and Xp22.13 (RP2, RP3, RP6, and RP15). The RP3 gene, which is responsible for the predominant form of XLRP in most Caucasian populations, has been localized to Xp21.1 by linkage analysis and the map positions of chromosomal deletions associated with the disease. Previous linkage studies have suggested that RP3 is flanked by the markers DXS1110 (distal) and OTC (proximal). Patient BB was though to have RP because of a lesion at the RP3 locus, in addition to chronic granulomatous disease, Duchenne muscular dystrophy (DMD), mild mental retardation, and the McLeod phenotype. This patient carried a deletion extending {approximately}3 Mb from DMD in Xp21.3 to Xp21.1, with the proximal breakpoint located {approximately}40 kb centromeric to DXS1110. The RP3 gene, therefore, is believed to reside between DXS1110 and the proximal breakpoint of the BB deletion. In order to refine the location of RP3 and to ascertain patients with RP3, we have been analyzing several XLRP families for linkage to Xp markers. Linkage analysis in an American family of 27 individuals demonstrates segregation of XLRP with markers in Xp21.1, consistent with the RP3 subtype. One affected male shows a recombination event proximal to DXS1110. Additional markers within the DXS1110-OTC interval show that the crossover is between two novel polymorphic markers, DXS8349 and M6, both of which are present in BB DNA and lie centromeric to the proximal breakpoint. This recombination places the XLRP mutation in this family outside the BB deletion and redefines the location of RP3. 22 refs., 3 figs., 2 tabs.

  10. Deletion of the X-linked Opsin Gene Array Locus Control Region (LCR) Results in Disruption of the Cone Mosaic

    PubMed Central

    Carroll, Joseph; Rossi, Ethan A.; Porter, Jason; Neitz, Jay; Roorda, Austin; Williams, David; Neitz, Maureen

    2010-01-01

    Blue-cone monochromacy (BCM) is an X-linked condition in which long- (L−) and middle- (M−) wavelength-sensitive cone function is absent. Due to the X-linked nature of the condition, female carriers are spared from a full manifestation of the associated defects but can show visual symptoms, including abnormal cone electroretinograms. Here we imaged the cone mosaic in four females carrying an L/M array with deletion of the locus control region, resulting in an absence of L/M opsin gene expression (effectively acting as a cone opsin knockout). On average, they had cone mosaics with reduced density and disrupted organization compared to normal trichromats. This suggests that the absence of opsin in a subset of cones results in their early degeneration, with X-inactivation the likely mechanism underlying phenotypic variability in BCM carriers. PMID:20638402

  11. The Fundamentals of Using the Digital Micromirror Device (DMD(TM)) for Projection Display

    NASA Technical Reports Server (NTRS)

    Yoder, Lars A.

    1995-01-01

    Developed by Texas Instruments (TI) the digital micromirror device (DMD(tm)) is a quickly emerging and highly useful micro-electro-mechanical structures (MEMS) device. Using standard semiconductor fabrication technology, the DMD's simplicity in concept and design will provide advantageous solutions for many different applications. At the rudimentary level, the DMD is a precision, semiconductor light switch. In the initial commercial development of DMD technology, TI has concentrated on projection display and hardcopy. This paper will focus on how the DMD is used for projection display. Other application areas are being explored and evaluated to find appropriate and beneficial uses for the DMD.

  12. Transcriptome analysis of acetic-acid-treated yeast cells identifies a large set of genes whose overexpression or deletion enhances acetic acid tolerance.

    PubMed

    Lee, Yeji; Nasution, Olviyani; Choi, Eunyong; Choi, In-Geol; Kim, Wankee; Choi, Wonja

    2015-08-01

    Acetic acid inhibits the metabolic activities of Saccharomyces cerevisiae. Therefore, a better understanding of how S. cerevisiae cells acquire the tolerance to acetic acid is of importance to develop robust yeast strains to be used in industry. To do this, we examined the transcriptional changes that occur at 12 h post-exposure to acetic acid, revealing that 56 and 58 genes were upregulated and downregulated, respectively. Functional categorization of them revealed that 22 protein synthesis genes and 14 stress response genes constituted the largest portion of the upregulated and downregulated genes, respectively. To evaluate the association of the regulated genes with acetic acid tolerance, 3 upregulated genes (DBP2, ASC1, and GND1) were selected among 34 non-protein synthesis genes, and 54 viable mutants individually deleted for the downregulated genes were retrieved from the non-essential haploid deletion library. Strains overexpressing ASC1 and GND1 displayed enhanced tolerance to acetic acid, whereas a strain overexpressing DBP2 was sensitive. Fifty of 54 deletion mutants displayed enhanced acetic acid tolerance. Three chosen deletion mutants (hsps82Δ, ato2Δ, and ssa3Δ) were also tolerant to benzoic acid but not propionic and sorbic acids. Moreover, all those five (two overexpressing and three deleted) strains were more efficient in proton efflux and lower in membrane permeability and internal hydrogen peroxide content than controls. Individually or in combination, those physiological changes are likely to contribute at least in part to enhanced acetic acid tolerance. Overall, information of our transcriptional profile was very useful to identify molecular factors associated with acetic acid tolerance.

  13. Upon Accounting for the Impact of Isoenzyme Loss, Gene Deletion Costs Anticorrelate with Their Evolutionary Rates.

    PubMed

    Jacobs, Christopher; Lambourne, Luke; Xia, Yu; Segrè, Daniel

    2017-01-01

    System-level metabolic network models enable the computation of growth and metabolic phenotypes from an organism's genome. In particular, flux balance approaches have been used to estimate the contribution of individual metabolic genes to organismal fitness, offering the opportunity to test whether such contributions carry information about the evolutionary pressure on the corresponding genes. Previous failure to identify the expected negative correlation between such computed gene-loss cost and sequence-derived evolutionary rates in Saccharomyces cerevisiae has been ascribed to a real biological gap between a gene's fitness contribution to an organism "here and now" and the same gene's historical importance as evidenced by its accumulated mutations over millions of years of evolution. Here we show that this negative correlation does exist, and can be exposed by revisiting a broadly employed assumption of flux balance models. In particular, we introduce a new metric that we call "function-loss cost", which estimates the cost of a gene loss event as the total potential functional impairment caused by that loss. This new metric displays significant negative correlation with evolutionary rate, across several thousand minimal environments. We demonstrate that the improvement gained using function-loss cost over gene-loss cost is explained by replacing the base assumption that isoenzymes provide unlimited capacity for backup with the assumption that isoenzymes are completely non-redundant. We further show that this change of the assumption regarding isoenzymes increases the recall of epistatic interactions predicted by the flux balance model at the cost of a reduction in the precision of the predictions. In addition to suggesting that the gene-to-reaction mapping in genome-scale flux balance models should be used with caution, our analysis provides new evidence that evolutionary gene importance captures much more than strict essentiality.

  14. Virtual reality 3D headset based on DMD light modulators

    SciTech Connect

    Bernacki, Bruce E.; Evans, Allan; Tang, Edward

    2014-06-13

    We present the design of an immersion-type 3D headset suitable for virtual reality applications based upon digital micro-mirror devices (DMD). Our approach leverages silicon micro mirrors offering 720p resolution displays in a small form-factor. Supporting chip sets allow rapid integration of these devices into wearable displays with high resolution and low power consumption. Applications include night driving, piloting of UAVs, fusion of multiple sensors for pilots, training, vision diagnostics and consumer gaming. Our design is described in which light from the DMD is imaged to infinity and the user’s own eye lens forms a real image on the user’s retina.

  15. Screening of point mutations by multiple SSCP analysis in the dystrophin gene

    SciTech Connect

    Lasa, A.; Baiget, M.; Gallano, P.

    1994-09-01

    Duchenne muscular dystrophy (DMD) is a lethal, X-linked neuromuscular disorder. The population frequency of DMD is one in approximately 3500 boys, of which one third is thought to be a new mutant. The DMD gene is the largest known to date, spanning over 2,3 Mb in band Xp21.2; 79 exons are transcribed into a 14 Kb mRNA coding for a protein of 427 kD which has been named dystrophin. It has been shown that about 65% of affected boys have a gene deletion with a wide variation in localization and size. The remaining affected individuals who have no detectable deletions or duplications would probably carry more subtle mutations that are difficult to detect. These mutations occur in several different exons and seem to be unique to single patients. Their identification represents a formidable goal because of the large size and complexity of the dystrophin gene. SSCP is a very efficient method for the detection of point mutations if the parameters that affect the separation of the strands are optimized for a particular DNA fragment. The multiple SSCP allows the simultaneous study of several exons, and implies the use of different conditions because no single set of conditions will be optimal for all fragments. Seventy-eight DMD patients with no deletion or duplication in the dystrophin gene were selected for the multiple SSCP analysis. Genomic DNA from these patients was amplified using the primers described for the diagnosis procedure (muscle promoter and exons 3, 8, 12, 16, 17, 19, 32, 45, 48 and 51). We have observed different mobility shifts in bands corresponding to exons 8, 12, 43 and 51. In exons 17 and 45, altered electrophoretic patterns were found in different samples identifying polymorphisms already described.

  16. Deletion of the Mouse Slc30a8 Gene Encoding Zinc Transporter-8 Results in Impaired Insulin Secretion

    PubMed Central

    Pound, Lynley D.; Sarkar, Suparna; Benninger, Richard K. P.; Wang, Yingda; Suwanichkul, Adisak; Shadoan, Melanie K.; Printz, Richard L.; Oeser, James K.; Lee, Catherine E.; Piston, David W.; McGuinness, Owen P.; Hutton, John C.; Powell, David R.; O’Brien, Richard M.

    2010-01-01

    Synopsis The Slc30a8 gene encodes the islet-specific zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Polymorphic variants in amino acid 325 of human ZnT-8 are associated with altered susceptibility to type 2 diabetes and ZnT-8 autoantibody epitope specificity changes in type 1 diabetes. To assess the physiological importance of ZnT-8, mice carrying a Slc30a8 exon 3 deletion were analyzed histologically and phenotyped for energy metabolism and pancreatic hormone secretion. No gross anatomical or behavioral changes or differences in body weight were observed between wild type and ZnT-8 −/− mice and ZnT-8 −/− mouse islets were indistinguishable from wild type in terms of their numbers, size and cellular composition. However, total zinc content was markedly reduced in ZnT-8 −/− mouse islets, as evaluated both by Timm’s histochemical staining of pancreatic sections and direct measurements in isolated islets. Blood glucose levels were unchanged in 16 week old, 6 hr fasted animals of either gender, however, plasma insulin concentrations were reduced in both female (~31%) and male (~47%) ZnT-8 −/− mice. Intraperitoneal glucose tolerance tests demonstrated no impairment in glucose clearance in male ZnT-8 −/− mice but glucose-stimulated insulin secretion from isolated islets was reduced ~33% relative to wild type littermates. In summary, Slc30a8 gene deletion is accompanied by a modest impairment in insulin secretion without major alterations in glucose metabolism. PMID:19450229

  17. Na+ dependent acid-base transporters in the choroid plexus; insights from slc4 and slc9 gene deletion studies

    PubMed Central

    Christensen, Henriette L.; Nguyen, An T.; Pedersen, Fredrik D.; Damkier, Helle H.

    2013-01-01

    The choroid plexus epithelium (CPE) is located in the ventricular system of the brain, where it secretes the majority of the cerebrospinal fluid (CSF) that fills the ventricular system and surrounds the central nervous system. The CPE is a highly vascularized single layer of cuboidal cells with an unsurpassed transepithelial water and solute transport rate. Several members of the slc4a family of bicarbonate transporters are expressed in the CPE. In the basolateral membrane the electroneutral Na+ dependent Cl−/HCO3− exchanger, NCBE (slc4a10) is expressed. In the luminal membrane, the electrogenic Na+:HCO3− cotransporter, NBCe2 (slc4a5) is expressed. The electroneutral Na+:HCO3− cotransporter, NBCn1 (slc4a7), has been located in both membranes. In addition to the bicarbonate transporters, the Na+/H+ exchanger, NHE1 (slc9a1), is located in the luminal membrane of the CPE. Genetically modified mice targeting slc4a2, slc4a5, slc4a7, slc4a10, and slc9a1 have been generated. Deletion of slc4a5, 7 or 10, or slc9a1 has numerous impacts on CP function and structure in these mice. Removal of the transporters affects brain ventricle size (slc4a5 and slc4a10) and intracellular pH regulation (slc4a7 and slc4a10). In some instances, removal of the proteins from the CPE (slc4a5, 7, and 10) causes changes in abundance and localization of non-target transporters known to be involved in pH regulation and CSF secretion. The focus of this review is to combine the insights gathered from these knockout mice to highlight the impact of slc4 gene deletion on the CSF production and intracellular pH regulation resulting from the deletion of slc4a5, 7 and 10, and slc9a1. Furthermore, the review contains a comparison of the described human mutations of these genes to the findings in the knockout studies. Finally, the future perspective of utilizing these proteins as potential targets for the treatment of CSF disorders will be discussed. PMID:24155723

  18. Deletion of virulence associated genes from attenuated African swine fever virus isolate OUR T88/3 decreases its ability to protect against challenge with virulent virus.

    PubMed

    Abrams, Charles C; Goatley, Lynnette; Fishbourne, Emma; Chapman, David; Cooke, Lyndsay; Oura, Christopher A; Netherton, Christopher L; Takamatsu, Haru-Hisa; Dixon, Linda K

    2013-08-15

    African swine fever virus (ASFV) causes an acute haemorrhagic disease of domestic pigs against which there is no effective vaccine. The attenuated ASFV strain OUR T88/3 has been shown previously to protect vaccinated pigs against challenge with some virulent strains including OUR T88/1. Two genes, DP71L and DP96R were deleted from the OUR T88/3 genome to create recombinant virus OUR T88/3ΔDP2. Deletion of these genes from virulent viruses has previously been shown to reduce ASFV virulence in domestic pigs. Groups of 6 pigs were immunised with deletion virus OUR T88/3ΔDP2 or parental virus OUR T88/3 and challenged with virulent OUR T88/1 virus. Four pigs (66%) were protected by inoculation with the deletion virus OUR T88/3ΔDP2 compared to 100% protection with the parental virus OUR T88/3. Thus the deletion of the two genes DP71L and DP96R from OUR T88/3 strain reduced its ability to protect pigs against challenge with virulent virus.

  19. Heterozygous Hfe gene deletion leads to impaired glucose homeostasis, but not liver injury in mice fed a high-calorie diet.

    PubMed

    Britton, Laurence; Jaskowski, Lesley; Bridle, Kim; Santrampurwala, Nishreen; Reiling, Janske; Musgrave, Nick; Subramaniam, V Nathan; Crawford, Darrell

    2016-06-01

    Heterozygous mutations of the Hfe gene have been proposed as cofactors in the development and progression of nonalcoholic fatty liver disease (NAFLD). Homozygous Hfe deletion previously has been shown to lead to dysregulated hepatic lipid metabolism and accentuated liver injury in a dietary mouse model of NAFLD We sought to establish whether heterozygous deletion of Hfe is sufficient to promote liver injury when mice are exposed to a high-calorie diet (HCD). Eight-week-old wild-type and Hfe(+/-) mice received 8 weeks of a control diet or HCD Liver histology and pathways of lipid and iron metabolism were analyzed. Liver histology demonstrated that mice fed a HCD had increased NAFLD activity score (NAS), steatosis, and hepatocyte ballooning. However, liver injury was unaffected by Hfe genotype. Hepatic iron concentration (HIC) was increased in Hfe(+/-) mice of both dietary groups. HCD resulted in a hepcidin-independent reduction in HIC Hfe(+/-) mice demonstrated raised fasting serum glucose concentrations and HOMA-IR score, despite unaltered serum adiponectin concentrations. Downstream regulators of hepatic de novo lipogenesis (pAKT, SREBP-1, Fas, Scd1) and fatty acid oxidation (AdipoR2, Pparα, Cpt1) were largely unaffected by genotype. In summary, heterozygous Hfe gene deletion is associated with impaired iron and glucose metabolism. However, unlike homozygous Hfe deletion, heterozygous gene deletion did not affect lipid metabolism pathways or liver injury in this model.

  20. Prevalence of pfhrp2 and/or pfhrp3 Gene Deletion in Plasmodium falciparum Population in Eight Highly Endemic States in India

    PubMed Central

    Bharti, Praveen Kumar; Chandel, Himanshu Singh; Ahmad, Amreen; Krishna, Sri; Udhayakumar, Venkatachalam; Singh, Neeru

    2016-01-01

    Background Plasmodium falciparum encoded histidine rich protein (HRP2) based malaria rapid diagnostic tests (RDTs) are used in India. Deletion of pfhrp2 and pfhrp3 genes contributes to false negative test results, and large numbers of such deletions have been reported from South America, highlighting the importance of surveillance to detect such deletions. Methods This is the first prospective field study carried out at 16 sites located in eight endemic states of India to assess the performance of PfHRP2 based RDT kits used in the national malaria control programme. In this study, microscopically confirmed P. falciparum but RDT negative samples were assessed for presence of pfhrp2, pfhrp3, and their flanking genes using PCR. Results Among 1521 microscopically positive P. falciparum samples screened, 50 were negative by HRP2 based RDT test. Molecular testing was carried out using these 50 RDT negative samples by assuming that 1471 RDT positive samples carried pfhrp2 gene. It was found that 2.4% (36/1521) and 1.8% (27/1521) of samples were negative for pfhrp2 and pfhrp3 genes, respectively. However, the frequency of pfhrp2 deletions varied between the sites ranging from 0–25% (2.4, 95% CI; 1.6–3.3). The frequency of both pfhrp2 and pfhrp3 gene deletion varied from 0–8% (1.6, 95% CI; 1.0–2.4). Conclusion This study provides evidence for low level presence of pfhrp2 and pfhrp3 deleted P. falciparum parasites in different endemic regions of India, and periodic surveillance is warranted for reliable use of PfHRP2 based RDTs. PMID:27518538

  1. Two novel members of the interleukin-1 receptor gene family, one deleted in Xp22.1-Xp21.3 mental retardation.

    PubMed

    Jin, H; Gardner, R J; Viswesvaraiah, R; Muntoni, F; Roberts, R G

    2000-02-01

    X-linked mental retardation is estimated to affect approximately 1 in 600 males. Although numerous genes responsible for syndromic mental retardation have been identified, the study of non-syndromic mental retardation suffers from intrinsic issues of genetic heterogeneity. During the investigation of three brothers with a contiguous gene deletion syndrome of Becker muscular dystrophy, glycerol kinase deficiency, congenital adrenal hypoplasia, and mental retardation, we found their dystrophin gene to be fused tail-to-tail with a gene encoding a novel member of the interleukin-1 receptor family, IL1RAPL1. This gene has a close relative in Xq22, which we call IL1RAPL2. Both IL1RAPL1 and IL1RAPL2 have novel C-terminal sequences not present in other related proteins, and are encoded by very large genes. The 1.8-megabase deletion in these patients removes not only the last exon of the dystrophin gene, the entire glycerol kinase and DAX-1 genes, and the MAGE-B gene cluster, but also three exons encoding the intracellular signalling domain of IL1RAPL1. The literature contains multiple reports of patients with non-syndromic mental retardation in association with an Xp22.1-Xp21.3 microdeletion of a marker which lies within the IL1RAPL1 gene. The gene is also wholly or partially deleted in patients with mental retardation as part of a contiguous deletion syndrome. We suggest that IL1RAPL1, and perhaps IL1RAPL2, are strong candidates for X-linked non-syndromic mental retardation loci, and that molecules resembling IL-1 and IL-18 play a role in the development or function of the central nervous system.

  2. T lymphocytes and dendritic cells are activated by the deletion of peroxiredoxin II (Prx II) gene.

    PubMed

    Moon, Eun-Yi; Noh, Young-Wook; Han, Ying-Hao; Kim, Sun-Uk; Kim, Jin-Man; Yu, Dae-Yeul; Lim, Jong-Seok

    2006-02-15

    Peroxiredoxin II (Prx II) is a member of antioxidant enzyme family and it plays a protective role against oxidative damage. Constitutive production of endogenous reactive oxygen species was detected in spleen and bone marrow cells lacking Prx II. Here, we investigated the role of Prx II in immune responses. The total number of splenocytes (especially, the population of S-phase cells and CD3(+) T cells) was significantly higher in Prx II(-/-) mice than in wild type. Number of peripheral blood mononuclear cells (PBMCs) in Prx II(-/-) mice was also higher than wild type. Differentiation of Prx II(-/-) mouse bone marrow cells into CD11c-positive dendritic cells was greater than that of wild type. Transplantation of Prx II(-/-) bone marrow cells into wild type mice increased PBMCs in blood and bone marrow-derived dendritic cells. Prx II deletion enhances concanavalin A (ConA)-induced splenocyte proliferation and mixed lymphocyte reaction (MLR) activity of bone marrow-derived CD11c-positive dendritic cells to stimulate recipient splenocytes. Collectively, these data suggest that Prx II inhibits the immune cell responsiveness, which may be regulated by scavenging the low amount of reactive oxygen species (ROS).

  3. The BCG Moreau RD16 deletion inactivates a repressor reshaping transcription of an adjacent gene.

    PubMed

    Galvão, Teca Calcagno; Lima, Cristiane Rodrigues; Gomes, Leonardo Henrique Ferreira; Pagani, Talita Duarte; Ferreira, Marcelo Alves; Gonçalves, Antonio S; Correa, Paloma Rezende; Degrave, Wim Maurits; Mendonça-Lima, Leila

    2014-01-01

    The Brazilian anti-tuberculosis vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG) BCG Moreau is unique in having a deletion of 7608 bp (RD16) that results in the truncation of a putative TetR transcriptional regulator, the ortholog of Mycobacterium tuberculosis rv3405c, BCG_M3439c. We investigated the effect of this truncation on the expression of the rv3406 ortholog (BCG_M3440), lying 81 bp downstream in the opposite orientation. RT-PCR and western blot experiments show that rv3406 mRNA and Rv3406 accumulate in BCG Moreau but not in BCG Pasteur (strain that bears an intact rv3405c), suggesting this to be a result of rv3405c truncation. Recombinant Rv3405c forms a complex with the rv3405c-rv3406 intergenic region, which contains a characteristic transcription factor binding site, showing it to have DNA binding activity. Complementation of M. bovis BCG Moreau with an intact copy of rv3405c abolishes Rv3406 accumulation. These results show that Rv3405c is a DNA binding protein that acts as a transcriptional repressor of rv3406.

  4. A candidate gene approach to identify modifiers of the palatal phenotype in 22q11.2 deletion syndrome patients

    PubMed Central

    Widdershoven, Josine C.C.; Bowser, Mark; Sheridan, Molly B.; McDonald-McGinn, Donna M.; Zackai, Elaine H.; Solot, Cynthia B.; Kirschner, Richard E.; Beemer, Frits A.; Morrow, Bernice E.; Devoto, Marcella; Emanuel, Beverly S.

    2014-01-01

    Objective Palatal anomalies are one of the identifying features of 22q11.2 deletion syndrome (22q11.2DS) affecting about one third of patients. To identify genetic variants that increase the risk of cleft or palatal anomalies in 22q11.2DS patients, we performed a candidate gene association study in 101 patients with 22q11.2DS genotyped with the Affymetrix genome-wide human SNP array 6.0. Methods Patients from Children's Hospital of Philadelphia, USA and Wilhelmina Children's Hospital Utrecht, The Netherlands were stratified based on palatal phenotype (overt cleft, submucosal cleft, bifid uvula). SNPs in 21 candidate genes for cleft palate were analyzed for genotype-phenotype association. In addition, TBX1 sequencing was carried out. Quality control and association analyses were conducted using the software package PLINK. Results Genotype and phenotype data of 101 unrelated patients (63 non-cleft subjects (62.4%), 38 cleft subjects (37.6%)) were analyzed. A Total of 39 SNPs on 10 genes demonstrated a p-value ≤0.05 prior to correction. The most significant SNPs were found on FGF10. However none of the SNPs remained significant after correcting for multiple testing. Conclusions Although these results are promising, analysis of additional samples will be required to confirm that variants in these regions influence risk for cleft palate or palatal anomalies in 22q11.2DS patients. PMID:23121717

  5. Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3.

    PubMed

    Zhang, Wei; He, Yulian; Gao, Weixia; Feng, Jun; Cao, Mingfeng; Yang, Chao; Song, Cunjiang; Wang, Shufang

    2015-02-01

    Here, we attempted to elevate poly-gamma-glutamic acid (γ-PGA) production by modifying genes involved in glutamate metabolism in Bacillus amyloliquefaciens LL3. Products of rocR, rocG and gudB facilitate the conversion from glutamate to 2-oxoglutarate in Bacillus subtillis. The gene odhA is responsible for the synthesis of a component of the 2-oxoglutarate dehydrogenase complex that catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl coenzyme A. In-frame deletions of these four genes were performed. In shake flask experiments the gudB/rocG double mutant presented enhanced production of γ-PGA, a 38 % increase compared with wild type. When fermented in a 5-L fermenter with pH control, the γ-PGA yield of the rocR mutant was increased to 5.83 g/L from 4.55 g/L for shake flask experiments. The gudB/rocG double mutant produced 5.68 g/L γ-PGA compared with that of 4.03 g/L for the wild type, a 40 % increase. Those results indicated the possibility of improving γ-PGA production by modifying glutamate metabolism, and identified potential genetic targets to improve γ-PGA production.

  6. Association of angiotensin converting enzyme gene insertion/deletion polymorphism and familial hypercholesterolemia in the Saudi population

    PubMed Central

    2013-01-01

    Background The study of the association between genotype and phenotype is of great importance for the prediction of multiple diseases and pathophysiological conditions. The relationship between angiotensin converting enzyme (ACE) Insertion/Deletion (I/D) polymorphism and Familial Hypercholesterolemia (FH) has been not fully investigated in all the ethnicities. In this study we sought to determine the frequency of I/D polymorphism genotypes of ACE gene in Saudi patients with FH. Results This is a case–control study carried out purely in Saudi population. Genomic DNA was isolated from 128 subjects who have participated in this study. ACE gene I/D polymorphism was analyzed by polymerase chain reaction in 64 FH cases and 64 healthy controls. There was no statistically significant difference between the groups with respect to genotype distribution. Furthermore, we did not find any significant difference in the frequency of ACE I/D polymorphism in FH subjects when stratified by gender (p = 0.43). Conclusion Our data suggest that ACE gene I/D polymorphism examined in this study has no role in predicting the occurrence and diagnosis of FH. PMID:24289455

  7. An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

    PubMed Central

    Netzker, Tina; Schroeckh, Volker; Gregory, Matthew A.; Flak, Michal; Krespach, Mario K. C.; Leadlay, Peter F.

    2016-01-01

    ABSTRACT Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces rapamycinicus. Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca2+ ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate. IMPORTANCE The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species. PMID:27037115

  8. Isolation and characterization of a novel transcript embedded within HIRA, a gene deleted in DiGeorge syndrome.

    PubMed

    Pizzuti, A; Novelli, G; Ratti, A; Amati, F; Bordoni, R; Mandich, P; Bellone, E; Conti, E; Bengala, M; Mari, A; Silani, V; Dallapiccola, B

    1999-07-01

    We have isolated a few cDNAs from different human tissues, transcribed from the first intron of HIRA, a gene deleted in the DiGeorge syndrome. These cDNAs are produced by an intronic gene (22k48) which is transcribed by the HIRA opposite strand and is itself arranged in exons and subjected to alternative splicing. The longest continuum cDNA sequence we obtained is 3.6 kb long and contains 3 different exons and 2 introns. 22k48 cDNA is composed of several tandemly arranged repeated elements (Alu, LINEs, CAn) surrounding a unique sequence. In situ hybridization showed the presence of 22k48 RNA in the cytoplasm of CNS and PNS neurons. 22k48 RNA is able to bind cytoplasmic proteins in the range of 45 to 60 kDa. 22k48 is a new member of the small group of genes that are transcribed but not translated, and its haploinsufficiency could contribute to the pathogenesis of the DiGeorge syndrome.

  9. Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice.

    PubMed

    Wein, Nicolas; Vulin, Adeline; Falzarano, Maria S; Szigyarto, Christina Al-Khalili; Maiti, Baijayanta; Findlay, Andrew; Heller, Kristin N; Uhlén, Mathias; Bakthavachalu, Baskar; Messina, Sonia; Vita, Giuseppe; Passarelli, Chiara; Brioschi, Simona; Bovolenta, Matteo; Neri, Marcella; Gualandi, Francesca; Wilton, Steve D; Rodino-Klapac, Louise R; Yang, Lin; Dunn, Diane M; Schoenberg, Daniel R; Weiss, Robert B; Howard, Michael T; Ferlini, Alessandra; Flanigan, Kevin M

    2014-09-01

    Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject-derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5' exons of DMD.

  10. An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology

    SciTech Connect

    Geller, A.I.; Keyomarsi, K.; Bryan, J.; Pardee, A.B. )

    1990-11-01

    The authors have previously described a defective herpes simplex virus (HSV-1) vector system that permits that introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli {beta}-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; ts mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. They now report an efficient packaging system for HSV-1 VECTORS USING A DELETION MUTANT, d30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses {beta}-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.

  11. A mouse gene (Dgcr6) related to the Drosophila gonadal gene is expressed in early embryogenesis and is the homolog of a human gene deleted in DiGeorge syndrome.

    PubMed

    Lindsay, E A; Baldini, A

    1997-01-01

    We report the identification of a mouse gene, Dgcr6, which shows high sequence similarity to gonadal (gdl), a Drosophila gene of unknown function. Dgcr6 is the mouse homolog of human DGCR6, previously shown to be deleted in DiGeorge syndrome, a developmental field defect affecting the derivatives of the pharyngeal arches which is associated with 22q11.2 deletions. The Dgcr6 transcript has a 594 nucleotide open reading frame (ORF) encoding 198 amino acids. We previously mapped Dgcr6 to mouse chromosome 16B1-B3, a region known to contain other mouse homologs of genes deleted in DiGeorge syndrome. Expression studies were performed by Northern blotting analysis on mouse embryo and adult tissues and by RNA in situ hybridization on mouse embryo sections. Results show that Dgcr6 transcripts are abundant during mouse embryogenesis, from at least 7 days post coitum. In particular, high expression was detected in the brain, spinal cord and pharyngeal arches. On adult tissues high expression was detected in testis. The function of Dgcr6 is to be determined, but its developmental expression suggests that this gene may play a role in the developmental defects associated with 22q11.2 deletions.

  12. Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia.

    PubMed

    Rachid Viana, Giselle Maria; Akinyi Okoth, Sheila; Silva-Flannery, Luciana; Lima Barbosa, Danielle Regina; Macedo de Oliveira, Alexandre; Goldman, Ira F; Morton, Lindsay C; Huber, Curtis; Anez, Arletta; Dantas Machado, Ricardo Luiz; Aranha Camargo, Luís Marcelo; Costa Negreiros do Valle, Suiane; Marins Póvoa, Marinete; Udhayakumar, Venkatachalam; Barnwell, John W

    2017-01-01

    More than 80% of available malaria rapid diagnostic tests (RDTs) are based on the detection of histidine-rich protein-2 (PfHRP2) for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3), are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para) and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2%) of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients). In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples) to 50.9% (30 out of 59 samples). In Bolivia, only one of 25 samples (4%) tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples) were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region.

  13. Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia

    PubMed Central

    Rachid Viana, Giselle Maria; Akinyi Okoth, Sheila; Silva-Flannery, Luciana; Lima Barbosa, Danielle Regina; Macedo de Oliveira, Alexandre; Goldman, Ira F.; Morton, Lindsay C.; Huber, Curtis; Anez, Arletta; Dantas Machado, Ricardo Luiz; Aranha Camargo, Luís Marcelo; Costa Negreiros do Valle, Suiane; Marins Póvoa, Marinete; Barnwell, John W.

    2017-01-01

    More than 80% of available malaria rapid diagnostic tests (RDTs) are based on the detection of histidine-rich protein-2 (PfHRP2) for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3), are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para) and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2%) of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients). In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples) to 50.9% (30 out of 59 samples). In Bolivia, only one of 25 samples (4%) tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples) were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region. PMID:28301474

  14. Delimitation of the Earliness per se D1 (Eps-D1) flowering gene to a subtelomeric chromosomal deletion in bread wheat (Triticum aestivum).

    PubMed

    Zikhali, Meluleki; Wingen, Luzie U; Griffiths, Simon

    2016-01-01

    Earliness per se (Eps) genes account for the variation in flowering time when vernalization and photoperiod requirements are satisfied. Genomics and bioinformatics approaches were used to describe allelic variation for 40 Triticum aestivum genes predicted, by synteny with Brachypodium distachyon, to be in the 1DL Eps region. Re-sequencing 1DL genes revealed that varieties carrying early heading alleles at this locus, Spark and Cadenza, carry a subtelomeric deletion including several genes. The equivalent region in Rialto and Avalon is intact. A bimodal distribution in the segregating Spark X Rialto single seed descent (SSD) populations enabled the 1DL QTL to be defined as a discrete Mendelian factor, which we named Eps-D1. Near isogenic lines (NILs) and NIL derived key recombinants between markers flanking Eps-D1 suggest that the 1DL deletion contains the gene(s) underlying Eps-D1. The deletion spans the equivalent of the Triticum monoccocum Eps-A (m) 1 locus, and hence includes MODIFIER OF TRANSCRIPTION 1 (MOT1) and FTSH PROTEASE 4 (FTSH4), the candidates for Eps-A (m) 1. The deletion also contains T. aestivum EARLY FLOWERING 3-D1 (TaELF3-D1) a homologue of the Arabidopsis thaliana circadian clock gene EARLY FLOWERING 3. Eps-D1 is possibly a homologue of Eps-B1 on chromosome 1BL. NILs carrying the Eps-D1 deletion have significantly reduced total TaELF3 expression and altered TaGIGANTEA (TaGI) expression compared with wild type. Altered TaGI expression is consistent with an ELF3 mutant, hence we propose TaELF3-D1 as the more likely candidate for Eps-D1. This is the first direct fine mapping of Eps effect in bread wheat.

  15. Delimitation of the Earliness per se D1 (Eps-D1) flowering gene to a subtelomeric chromosomal deletion in bread wheat (Triticum aestivum)

    PubMed Central

    Zikhali, Meluleki; Wingen, Luzie U.; Griffiths, Simon

    2016-01-01

    Earliness per se (Eps) genes account for the variation in flowering time when vernalization and photoperiod requirements are satisfied. Genomics and bioinformatics approaches were used to describe allelic variation for 40 Triticum aestivum genes predicted, by synteny with Brachypodium distachyon, to be in the 1DL Eps region. Re-sequencing 1DL genes revealed that varieties carrying early heading alleles at this locus, Spark and Cadenza, carry a subtelomeric deletion including several genes. The equivalent region in Rialto and Avalon is intact. A bimodal distribution in the segregating Spark X Rialto single seed descent (SSD) populations enabled the 1DL QTL to be defined as a discrete Mendelian factor, which we named Eps-D1. Near isogenic lines (NILs) and NIL derived key recombinants between markers flanking Eps-D1 suggest that the 1DL deletion contains the gene(s) underlying Eps-D1. The deletion spans the equivalent of the Triticum monoccocum Eps-A m 1 locus, and hence includes MODIFIER OF TRANSCRIPTION 1 (MOT1) and FTSH PROTEASE 4 (FTSH4), the candidates for Eps-A m 1. The deletion also contains T. aestivum EARLY FLOWERING 3-D1 (TaELF3-D1) a homologue of the Arabidopsis thaliana circadian clock gene EARLY FLOWERING 3. Eps-D1 is possibly a homologue of Eps-B1 on chromosome 1BL. NILs carrying the Eps-D1 deletion have significantly reduced total TaELF3 expression and altered TaGIGANTEA (TaGI) expression compared with wild type. Altered TaGI expression is consistent with an ELF3 mutant, hence we propose TaELF3-D1 as the more likely candidate for Eps-D1. This is the first direct fine mapping of Eps effect in bread wheat. PMID:26476691

  16. Upon Accounting for the Impact of Isoenzyme Loss, Gene Deletion Costs Anticorrelate with Their Evolutionary Rates

    PubMed Central

    Xia, Yu; Segrè, Daniel

    2017-01-01

    System-level metabolic network models enable the computation of growth and metabolic phenotypes from an organism’s genome. In particular, flux balance approaches have been used to estimate the contribution of individual metabolic genes to organismal fitness, offering the opportunity to test whether such contributions carry information about the evolutionary pressure on the corresponding genes. Previous failure to identify the expected negative correlation between such computed gene-loss cost and sequence-derived evolutionary rates in Saccharomyces cerevisiae has been ascribed to a real biological gap between a gene’s fitness contribution to an organism “here and now” and the same gene’s historical importance as evidenced by its accumulated mutations over millions of years of evolution. Here we show that this negative correlation does exist, and can be exposed by revisiting a broadly employed assumption of flux balance models. In particular, we introduce a new metric that we call “function-loss cost”, which estimates the cost of a gene loss event as the total potential functional impairment caused by that loss. This new metric displays significant negative correlation with evolutionary rate, across several thousand minimal environments. We demonstrate that the improvement gained using function-loss cost over gene-loss cost is explained by replacing the base assumption that isoenzymes provide unlimited capacity for backup with the assumption that isoenzymes are completely non-redundant. We further show that this change of the assumption regarding isoenzymes increases the recall of epistatic interactions predicted by the flux balance model at the cost of a reduction in the precision of the predictions. In addition to suggesting that the gene-to-reaction mapping in genome-scale flux balance models should be used with caution, our analysis provides new evidence that evolutionary gene importance captures much more than strict essentiality. PMID:28107392

  17. A new case of interstitial 6q16.2 deletion in a patient with Prader-Willi-like phenotype and investigation of SIM1 gene deletion in 87 patients with syndromic obesity.

    PubMed

    Varela, Monica C; Simões-Sato, Alex Y; Kim, Chong A; Bertola, Débora R; De Castro, Claudia I E; Koiffmann, Celia P

    2006-01-01

    The association of obesity, phenotypic abnormalities and mental retardation characterizes syndromic obesity. Its most common form is the Prader-Willi syndrome (PWS-- neonatal hypotonia, poor sucking, delayed psychomotor development, hyperphagia, severe obesity, short stature, small hands and feet, hypogonadism, mild to moderate mental retardation and behavioral disorders). A PWS-like phenotype has been described in patients with chromosome abnormalities involving the chromosome region 6q16.2 that includes the SIM1 gene. Herein we report cytogenetic and gene studies including a screening for the SIM1 gene deletion, performed on 87 patients with PWS-like phenotype, and describe the fifth case of syndromic obesity with an interstitial deletion of the chromosome segment 6q16-q21 and suggest that mutational analysis and further studies of the parental origin of chromosome alterations of 6q16.2 in patients with and without PWS-like phenotype are needed to evaluate possible imprinting effects of SIM1 gene and establish the contribution that alterations in this gene makes to the etiology of syndromic and non-syndromic obesity.

  18. Cloning and comparative mapping of a gene from the commonly deleted region of DiGeorge and Velocardiofacial syndromes conserved in C. elegans.

    PubMed

    Rizzu, P; Lindsay, E A; Taylor, C; O'Donnell, H; Levy, A; Scambler, P; Baldini, A

    1996-09-01

    We have identified and cloned a gene, ES2, encoding a putative 476 amino acid protein with a predicted Mr of 52,568. The gene is localized within the DiGeorge/Velocardiofacial syndrome locus on 22q11.2 and is deleted in all the patients in which a deletion within 22q11 could be demonstrated, with the exception of one patient. ES2 is expressed in all the tissues studied. Sequence comparison showed identity with five ESTs and at the amino acid level the sequence was highly similar to, and collinear with, a hypothetical C. elegans protein of unknown function. Mutation analysis was performed in 16 patients without deletion, but no mutation has been found. The cDNA sequence is conserved in mouse and is localized on MMU16B1-B3, known to contain a syntenic group in common with HSA 22q11.2.

  19. Detection of large scale 3' deletions in the PMS2 gene amongst Colon-CFR participants: have we been missing anything?

    PubMed

    Clendenning, Mark; Walsh, Michael D; Gelpi, Judith Balmana; Thibodeau, Stephen N; Lindor, Noralane; Potter, John D; Newcomb, Polly; LeMarchand, Loic; Haile, Robert; Gallinger, Steve; Hopper, John L; Jenkins, Mark A; Rosty, Christophe; Young, Joanne P; Buchanan, Daniel D

    2013-09-01

    Current screening practices have been able to identify PMS2 mutations in 78 % of cases of colorectal cancer from the Colorectal Cancer Family Registry (Colon CFR) which showed solitary loss of the PMS2 protein. However the detection of large-scale deletions in the 3' end of the PMS2 gene has not been possible due to technical difficulties associated with pseudogene sequences. Here, we utilised a recently described MLPA/long-range PCR-based approach to screen the remaining 22 % (n = 16) of CRC-affected probands for mutations in the 3' end of the PMS2 gene. No deletions encompassing any or all of exons 12 through 15 were identified; therefore, our results suggest that 3' deletions in PMS2 are not a frequent occurrence in such families.

  20. Prolidase Deficiency in a Mexican-American Patient Identified by Array CGH Reveals a Novel and the Largest PEPD Gene Deletion

    PubMed Central

    Hintze, Jonathan P.; Kirby, Amelia; Torti, Erin; Batanian, Jacqueline R.

    2016-01-01

    Prolidase deficiency (PD) is a rare genetic disorder caused by mutations in the peptidase D (PEPD) gene, affecting collagen degradation. Features include lower extremity ulcers, facial dysmorphism, frequent respiratory infections, and intellectual disability, though there is significant intra- and interfamilial variability. Twenty-eight mutations have been previously reported, all either small deletions/duplications or point mutations discovered by enzyme or DNA assays. PD has been reported in patients of various ethnic backgrounds, but never in the Mexican-American population. We describe the first Mexican-American patient with PD, who presented with typical facial features, developmental delay, microcephaly, and xerosis. Chromosome microarray analysis (CMA) revealed a homozygous deletion in the region of 19q13.11, estimated to be between 124.79 and 195.72 kb in size, representing the largest PEPD gene deletion reported to date and the first discovered by CMA. PMID:27385964

  1. Let-7b regulates the expression of the growth hormone receptor gene in deletion-type dwarf chickens

    PubMed Central

    2012-01-01

    Background A deletion mutation in the growth hormone receptor (GHR) gene results in the inhibition of skeletal muscle growth and fat deposition in dwarf chickens. We used microarray techniques to determine microRNA (miRNA) and mRNA expression profiles of GHR in the skeletal muscles of 14-day-old embryos as well as 7-week-old deletion-type dwarf and normal-type chickens. Our aim was to elucidate the miRNA regulation of GHR expression with respect to growth inhibition and fat deposition. Results At the same developmental stages, different expression profiles in skeletal muscles of dwarf and normal chickens occurred for four miRNAs (miR-1623, miR-181b, let-7b, and miR-128). At different developmental stages, there was a significant difference in the expression profiles of a greater number of miRNAs. Eleven miRNAs were up-regulated and 18 down-regulated in the 7-week-old dwarf chickens when compared with profiles in 14-day-old embryos. In 7-week-old normal chickens, seven miRNAs were up-regulated and nine down-regulated compared with those in 14-day-old embryos. In skeletal muscles, 22 genes were up-regulated and 33 down-regulated in 14-day-old embryos compared with 7-week-old dwarf chickens. Sixty-five mRNAs were up-regulated and 108 down-regulated in 14-day-old embryos as compared with 7-week-old normal chickens. Thirty-four differentially expressed miRNAs were grouped into 18 categories based on overlapping seed and target sequences. Only let-7b was found to be complementary to its target in the 3′ untranslated region of GHR, and was able to inhibit its expression. Kyoto Encyclopedia of Genes and Genomes pathway analysis and quantitative polymerase chain reactions indicated there were three main signaling pathways regulating skeletal muscle growth and fat deposition of chickens. These were influenced by let-7b-regulated GHR. Suppression of the cytokine signaling 3 (SOCS3) gene was found to be involved in the signaling pathway of adipocytokines. Conclusions There

  2. Proteomics and bioinformatics analysis of mouse hypothalamic neurogenesis with or without EPHX2 gene deletion

    PubMed Central

    Zhong, Lijun; Zhou, Juntuo; Wang, Dawei; Zou, Xiajuan; Lou, Yaxin; Liu, Dan; Yang, Bin; Zhu, Yi; Li, Xiaoxia

    2015-01-01

    The aim of this study was to identify differently expressed proteins in the presence and absence of EPHX2 gene in mouse hypothalamus using proteomics profiling and bioinformatics analysis. This study was performed on 3 wild type (WT) and 3 EPHX2 gene global knockout (KO) mice (EPHX2 -/-). Using the nano- electrospray ionization (ESI)-LC-MS/MS detector, we identified 31 over-expressed proteins in WT mouse hypothalamus compared to the KO counterparts. Gene Ontology (GO) annotation in terms of the protein-protein interaction network indicated that cellular metabolic process, protein metabolic process, signaling transduction and protein post-translation biological processes involved in EPHX2 -/- regulatory network. In addition, signaling pathway enrichment analysis also highlighted chronic neurodegenerative diseases and some other signaling pathways, such as TGF-beta signaling pathway, T cell receptor signaling pathway, ErbB signaling pathway, Neurotrophin signaling pathway and MAPK signaling pathway, were strongly coupled with EPHX2 gene knockout. Further studies into the molecular functions of EPHX2 gene in hypothalamus will help to provide new perspective in neurogenesis. PMID:26722453

  3. The effect of CSE gene deletion in caerulein-induced acute pancreatitis in the mouse.

    PubMed

    Ang, Abel D; Rivers-Auty, Jack; Hegde, Akhil; Ishii, Isao; Bhatia, Madhav

    2013-11-15

    Hydrogen sulfide (H2S) has been reported to be involved in the signaling of the inflammatory response; however, there are differing views as to whether it is pro- or anti-inflammatory. In this study, we sought to determine whether endogenously synthesized H2S via cystathionine-γ-lyase (CSE) plays a pro- or anti-inflammatory role in caerulein-induced pancreatitis. To investigate this, we used mice genetically deficient in CSE to elucidate the function of CSE in caerulein-induced acute pancreatitis. We compared the inflammatory response and tissue damage of wild-type (WT) and CSE knockout (KO) mice following 10 hourly administrations of 50 μg/kg caerulein or saline control. From this, we found that the CSE KO mice showed significantly less local pancreatic damage as well as acute pancreatitis-associated lung injury compared with the WT mice. There were also lower levels of pancreatic eicosanoid and cytokines, as well as reduced acinar cell NF-κB activation in the CSE KO mice compared with WT mice. Additionally, in WT mice, there was a greater level of pancreatic CSE expression and sulfide-synthesizing activity in caerulein-induced pancreatitis compared with the saline control. When comparing the two saline-treated control groups, we noted that the CSE KO mice showed significantly less pancreatic H2S-synthesizing activity relative to the WT mice. These results indicate that endogenous H2S generated by CSE plays a key proinflammatory role via NF-κB activation in caerulein-induced pancreatitis, and its genetic deletion affords significant protection against acute pancreatitis and associated lung injury.

  4. Large Deletions in the pAtC58 Megaplasmid of Agrobacterium tumefaciens Can Confer Reduced Carriage Cost and Increased Expression of Virulence Genes

    PubMed Central

    Morton, Elise R.; Merritt, Peter M.; Bever, James D.; Fuqua, Clay

    2013-01-01

    The accessory plasmid pAtC58 of the common laboratory strain of Agrobacterium tumefaciens confers numerous catabolic functions and has been proposed to play a role in virulence. Genomic sequencing of evolved laboratory strains of A. tumefaciens revealed the presence of multiple deletion events in the At plasmid, with reductions in plasmid size ranging from 25% to 30% (115–194 kb). Flanking both ends of the sites of these deletions is a short-nucleotide repeat sequence that is in a single copy in the deleted plasmids, characteristic of a phage- or transposon-mediated deletion event. This repeat sequence is widespread throughout the C58 genome, but concentrated on the At plasmid, suggesting its frequency to be nonrandom. In this study, we assess the prevalence of the larger of these deletions in multiple C58 derivatives and characterize its functional significance. We find that in addition to elevating virulence gene expression, this deletion is associated with a significantly reduced carriage cost to the cell. These observations are a clear demonstration of the dynamic nature of the bacterial genome and suggest a mechanism for genetic plasticity of these costly but otherwise stable plasmids. Additionally, this phenomenon could be the basis for some of the dramatic recombination events so ubiquitous within and among megaplasmids. PMID:23783172

  5. Murine fumarylacetoacetate hydrolase (Fah) gene is disrupted by a neonatally lethal albino deletion that defines the hepatocyte-specific developmental regulation 1 (hsdr-1) locus

    SciTech Connect

    Klebig, M.L. Oak Ridge National Lab., TN ); Russell, L.B.; Rinchik, E.M. )

    1992-02-15

    Homozygous deletion of the hepatocyte-specific developmental regulation 1 (hsdr-1) locus in mouse chromosome 7 results in perinatal death and a pleiotropic syndrome characterized by ultrastructural abnormalities of the liver and kidney, failure of induction of a number of specific transcription units in the liver and kidney during late gestation, and marked overexpression of an enzyme that defends against oxidative stress. Previously, the breakpoints of two albino (c) deletions (c{sup 14CoS} and c{sup IFAFyh}) that genetically define hsdr-1 were localized, on a long-range map, in the vicinity of the distal breakpoint of a viable albino deletion (c{sup 24R75M}) that breaks proximally within the c locus. Here the authors report the use of a probe derived from a deletion breakpoint fusion fragment cloned from c{sup 24R75M}/c{sup 24R75M} DNA to clone a breakpoint fusion fragment caused by the c{sup 14CoS} deletion. The proximal breakpoint of the c{sup 14CoS} deletion was discovered to disrupt a gene (Fah) encoding fumarylacetoacetate hydrolase, the last enzyme in the tyrosine degradation pathway. These mouse mutants may also provide models for the human genetic disorder hereditary tyrosinemia, which is associated with fumarylacetoacetate hydrolase deficiency and liver and kidney dysfunction.

  6. Structure and methylation-associated silencing of a gene within a homozygously deleted region of human chromosome band 8p22

    SciTech Connect

    MacGrogan, D.; Levy, A.; Bookstein, R.

    1996-07-01

    The structure and expression pattern of a human gene located within a homozygously deleted region of a metastatic prostate cancer have been characterized. Multiple cDNA fragments of this gene were isolated by hybrid capture with yeast artificial chromosome hybrid capture with yeast artificial chromosome clones covering the deletion region. Eleven coding exons spanned 205-220 kb of the 730- to 970-kb deletion. The predicted amino acid sequence was 43% identical to that of an accessory or regulatory subunit of the oligosaccharyltransferase enzyme complex in Saccharomyces cerevisiae. Hydrophobicity profiles of all three gene products were similar and showed four putative membrane-spanning domains in the molecules` C-terminal halves, suggesting a general conservation of function. The gene was expressed as an {approximately}1.5-kb mRNA in most nonlymphoid human cells/tissues including prostate, lung, liver, and colon. Expression was detected in many epithelial tumor cell lines, but was undetectable by Northern blot or RT-PCR in 14 of 15 colorectal, 1 of 8 lung, and 1 of 4 liver cancer cell lines. Lack of expression in tumor cell lines was highly correlated with hypermethylation of a CpG island located at the gene`s 5{prime} end. These findings form a basis for further work on this candidate tumor suppressor gene. 36 refs., 9 figs., 2 tabs.

  7. Three phases of DiGeorge/22q11 deletion syndrome pathogenesis during brain development: patterning, proliferation, and mitochondrial functions of 22q11 genes.

    PubMed

    Meechan, D W; Maynard, T M; Tucker, E S; LaMantia, A-S

    2011-05-01

    DiGeorge, or 22q11 deletion syndrome (22q11DS), the most common survivable human genetic deletion disorder, is caused by deletion of a minimum of 32 contiguous genes on human chromosome 22, and presumably results from diminished dosage of one, some, or all of these genes--particularly during development. Nevertheless, the normal functions of 22q11 genes in the embryo or neonate, and their contribution to developmental pathogenesis that must underlie 22q11DS are not well understood. Our data suggests that a substantial number of 22q11 genes act specifically and in concert to mediate early morphogenetic interactions and subsequent cellular differentiation at phenotypically compromised sites--the limbs, heart, face and forebrain. When dosage of a broad set of these genes is diminished, early morphogenesis is altered, and initial 22q11DS phenotypes are established. Thereafter, functionally similar subsets of 22q11 genes--especially those that influence the cell cycle or mitochondrial function--remain expressed, particularly in the developing cerebral cortex, to regulate neurogenesis and synaptic development. When dosage of these genes is diminished, numbers, placement and connectivity of neurons and circuits essential for normal behavior may be disrupted. Such disruptions likely contribute to vulnerability for schizophrenia, autism, or attention deficit/hyperactivity disorder seen in most 22q11DS patients.

  8. Virulence Associated Genes-Deleted Salmonella Montevideo Is Attenuated, Highly Immunogenic and Confers Protection against Virulent Challenge in Chickens

    PubMed Central

    Lalsiamthara, Jonathan; Lee, John H.

    2016-01-01

    To construct a novel live vaccine against Salmonella enterica serovar Montevideo (SM) infection in chickens, two important bacterial regulatory genes, lon and cpxR, which are associated with invasion and virulence, were deleted from the wild type SM genome. Attenuated strains, JOL1625 (Δlon), JOL1597 (ΔcpxR), and JOL1599 (ΔlonΔcpxR) were thereby generated. Observations with scanning electron microscopy suggested that JOL1625 and JOL1599 cells showed increased ruffled surface which may be related to abundant extracellular polysaccharide (EPS) production. JOL1597 depicted milder ruffled surface but showed increased surface corrugation. ConA affinity-based fluorometric quantification and fluorescence microscopy revealed significant increases in EPS production in JOL1625 and JOL1599. Four weeks old chickens were used for safety and immunological studies. The mutants were not observed in feces beyond day 3 nor in spleen and cecum beyond day 7, whereas wild type SM was detected for at least 2 weeks in spleen and cecum. JOL1599 was further evaluated as a vaccine candidate. Chickens immunized with JOL1599 showed strong humoral responses, as indicated by systemic IgG and secretory IgA levels, as well as strong cell-mediated immune response, as indicated by increased lymphocyte proliferation. JOL1599-immunized groups also showed significant degree of protection against wild type challenge. Our results indicate that Δlon- and/or ΔcpxR-deleted SM exhibited EPS-enhanced immunogenicity and attenuation via reduced bacterial cell intracellular replication, conferred increased protection, and possess safety qualities favorable for effective vaccine development against virulent SM infections. PMID:27785128

  9. Improving the MVA Vaccine Potential by Deleting the Viral Gene Coding for the IL-18 Binding Protein

    PubMed Central

    Pascutti, María Fernanda; Rodríguez, Ana María; Maeto, Cynthia; Perdiguero, Beatriz; Gómez, Carmen E.; Esteban, Mariano; Calamante, Gabriela; Gherardi, María Magdalena

    2012-01-01

    Background Modified Vaccinia Ankara (MVA) is an attenuated strain of Vaccinia virus (VACV) currently employed in many clinical trials against HIV/AIDS and other diseases. MVA still retains genes involved in host immune response evasion, enabling its optimization by removing some of them. The aim of this study was to evaluate cellular immune responses (CIR) induced by an IL-18 binding protein gene (C12L) deleted vector (MVAΔC12L). Methodology/Principal Findings BALB/c and C57BL/6 mice were immunized with different doses of MVAΔC12L or MVA wild type (MVAwt), then CIR to VACV epitopes in immunogenic proteins were evaluated in spleen and draining lymph nodes at acute and memory phases (7 and 40 days post-immunization respectively). Compared with parental MVAwt, MVAΔC12L immunization induced a significant increase of two to three-fold in CD8+ and CD4+ T-cell responses to different VACV epitopes, with increased percentage of anti-VACV cytotoxic CD8+ T-cells (CD107a/b+) during the acute phase of the response. Importantly, the immunogenicity enhancement was also observed after MVAΔC12L inoculation with different viral doses and by distinct routes (systemic and mucosal). Potentiation of MVA's CIR was also observed during the memory phase, in correlation with a higher protection against an intranasal challenge with VACV WR. Of note, we could also show a significant increase in the CIR against HIV antigens such as Env, Gag, Pol and Nef from different subtypes expressed from two recombinants of MVAΔC12L during heterologous DNA prime/MVA boost vaccination regimens. Conclusions/Significance This study demonstrates the relevance of IL-18 bp contribution in the immune response evasion during MVA infection. Our findings clearly show that the deletion of the viral IL-18 bp gene is an effective approach to increase MVA vaccine efficacy, as immunogenicity improvements were observed against vector antigens and more importantly to HIV antigens. PMID:22384183

  10. Deletion of the Chemokine Binding Protein Gene from the Parapoxvirus Orf Virus Reduces Virulence and Pathogenesis in Sheep

    PubMed Central

    Fleming, Stephen B.; McCaughan, Catherine; Lateef, Zabeen; Dunn, Amy; Wise, Lyn M.; Real, Nicola C.; Mercer, Andrew A.

    2017-01-01

    Orf virus (ORFV) is the type species of the Parapoxvirus genus of the family Poxviridae and infects sheep and goats, often around the mouth, resulting in acute pustular skin lesions. ORFV encodes several secreted immunomodulators including a broad-spectrum chemokine binding protein (CBP). Chemokines are a large family of secreted chemotactic proteins that activate and regulate inflammation induced leukocyte recruitment to sites of infection. In this study we investigated the role of CBP in vivo in the context of ORFV infection of sheep. The CBP gene was deleted from ORFV strain NZ7 and infections of sheep used to investigate the effect of CBP on pathogenesis. Animals were either infected with the wild type (wt) virus, CBP-knockout virus or revertant strains. Sheep were infected by scarification on the wool-less area of the hind legs at various doses of virus. The deletion of the CBP gene severely attenuated the virus, as only few papules formed when animals were infected with the CBP-knock-out virus at the highest dose (107 p.f.u). In contrast, large pustular lesions formed on almost all animals infected with the wt and revertant strains at 107 p.f.u. The lesions for the CBP-knock-out virus resolved approximately 5–6 days p.i, at a dose of 107 pfu whereas in animals infected with the wt and revertants at this dose, lesions began to resolve at approximately 10 days p.i. Few pustules developed at the lowest dose of 103 p.f.u. for all viruses. Immunohistochemistry of biopsy skin-tissue from pustules showed that the CBP-knockout virus replicated in all animals at the highest dose and was localized to the skin epithelium while haematoxylin and eosin staining showed histological features of the CBP-knockout virus typical of the parent virus with acanthosis, elongated rete ridges and orthokeratotic hyperkeratosis. MHC-II immunohistochemistry analysis for monocytes and dendritic cells showed greater staining within the papillary dermis of the CBP-knock-out virus compared

  11. Effects of deletion of different PP2C protein phosphatase genes on stress responses in Saccharomyces cerevisiae.

    PubMed

    Sharmin, Dilruba; Sasano, Yu; Sugiyama, Minetaka; Harashima, Satoshi

    2014-10-01

    A key mechanism of signal transduction in eukaryotes is reversible protein phosphorylation, mediated through protein kinases and protein phosphatases (PPases). Modulation of signal transduction by this means regulates many biological processes. Saccharomyces cerevisiae has 40 PPases, including seven protein phosphatase 2C (PP2C PPase) genes (PTC1-PTC7). However, their precise functions remain poorly understood. To elucidate their cellular functions and to identify those that are redundant, we constructed 127 strains with deletions of all possible combinations of the seven PP2C PPase genes. All 127 disruptants were viable under nutrient-rich conditions, demonstrating that none of the combinations induced synthetic lethality under these conditions. However, several combinations exhibited novel phenotypes, e.g. the Δptc5Δptc7 double disruptant and the Δptc2Δptc3Δptc5Δptc7 quadruple disruptant exhibited low (13°C) and high (37°C) temperature-sensitive growth, respectively. Interestingly, the septuple disruptant Δptc1Δptc2Δptc3Δptc4Δptc5Δptc6Δptc7 showed an essentially normal growth phenotype at 37°C. The Δptc2Δptc3Δptc5Δptc7 quadruple disruptant was sensitive to LiCl (0.4 m). Two double disruptants, Δptc1Δptc2 and Δptc1Δptc4, displayed slow growth and Δptc1Δptc2Δptc4 could not grow on medium containing 1.5 m NaCl. The Δptc1Δptc6 double disruptant showed increased sensitivity to caffeine, congo red and calcofluor white compared to each single deletion. Our observations indicate that S. cerevisiae PP2C PPases have a shared and important role in responses to environmental stresses. These disruptants also provide a means for exploring the molecular mechanisms of redundant PTC gene functions under defined conditions.

  12. Micro-dystrophin and follistatin co-delivery restores muscle function in aged DMD model.

    PubMed

    Rodino-Klapac, Louise R; Janssen, Paul M L; Shontz, Kimberly M; Canan, Benjamin; Montgomery, Chrystal L; Griffin, Danielle; Heller, Kristin; Schmelzer, Leah; Handy, Chalonda; Clark, K Reed; Sahenk, Zarife; Mendell, Jerry R; Kaspar, Brian K

    2013-12-15

    Pharmacologic strategies have provided modest improvement in the devastating muscle-wasting disease, Duchenne muscular dystrophy (DMD). Pre-clinical gene therapy studies have shown promise in the mdx mouse model; however, studies conducted after disease onset fall short of fully correcting muscle strength or protecting against contraction-induced injury. Here we examine the treatment effect on muscle physiology in aged dystrophic mice with significant disease pathology by combining two promising therapies: micro-dystrophin gene replacement and muscle enhancement with follistatin, a potent myostatin inhibitor. Individual treatments with micro-dystrophin and follistatin demonstrated marked improvement in mdx mice but were insufficient to fully restore muscle strength and response to injury to wild-type levels. Strikingly, when combined, micro-dystrophin/follistatin treatment restored force generation and conferred resistance to contraction-induced injury in aged mdx mice. Pre-clinical studies with miniature dystrophins have failed to demonstrate full correction of the physiological defects seen in mdx mice. Importantly, the addition of a muscle enhancement strategy with delivery of follistatin in combination with micro-dystrophin gene therapy completely restored resistance to eccentric contraction-induced injury and improved force. Eccentric contraction-induced injury is a pre-clinical parameter relevant to the exercise induced injury that occurs in DMD patients, and herein, we demonstrate compelling evidence for the therapeutic potential of micro-dystrophin/follistatin combinatorial therapy.

  13. Conditional Deletion of the Phd2 Gene in Articular Chondrocytes Accelerates Differentiation and Reduces Articular Cartilage Thickness

    PubMed Central

    Cheng, Shaohong; Pourteymoor, Sheila; Alarcon, Catrina; Mohan, Subburaman

    2017-01-01

    Based on our findings that PHD2 is a negative regulator of chondrocyte differentiation and that hypoxia signaling is implicated in the pathogenesis of osteoarthritis, we investigated the consequence of disruption of the Phd2 gene in chondrocytes on the articular cartilage phenotype in mice. Immunohistochemistry detected high expression of PHD2 in the superficial zone (SZ), while PHD3 and HIF-1α (target of PHD2) are mainly expressed in the middle-deep zone (MDZ). Conditional deletion of the Phd2 gene (cKO) in chondrocytes accelerated the transition of progenitors to hypertrophic (differentiating) chondrocytes as revealed by reduced SZ thickness, and increased MDZ thickness, as well as increased chondrocyte hypertrophy. Immunohistochemistry further revealed decreased levels of progenitor markers but increased levels of hypertrophy markers in the articular cartilage of the cKO mice. Treatment of primary articular chondrocytes, in vitro, with IOX2, a specific inhibitor of PHD2, promoted articular chondrocyte differentiation. Knockdown of Hif-1α expression in primary articular chondrocytes using lentiviral vectors containing Hif-1α shRNA resulted in reduced expression levels of Vegf, Glut1, Pgk1, and Col10 compared to control shRNA. We conclude that Phd2 is a key regulator of articular cartilage development that acts by inhibiting the differentiation of articular cartilage progenitors via modulating HIF-1α signaling. PMID:28349987

  14. pNEB193-derived suicide plasmids for gene deletion and protein expression in the methane-producing archaeon, Methanosarcina acetivorans

    PubMed Central

    Shea, Mitchell T.; Walter, Mary E.; Duszenko, Nikolas; Ducluzeau, Anne-Lise; Aldridge, Jared; King, Shannon K.; Buan, Nicole R.

    2016-01-01

    Gene deletion and protein expression are cornerstone procedures for studying metabolism in any organism, including methane-producing archaea (methanogens). Methanogens produce coenzymes and cofactors not found in most bacteria, therefore it is sometimes necessary to express and purify methanogen proteins from the natural host. Protein expression in the native organism is also useful when studying post-translational modifications and their effect on gene expression or enzyme activity. We have created several new suicide plasmids to complement existing genetic tools for use in the methanogen, Methanosarcina acetivorans. The new plasmids are derived from the commercially available E. coli plasmid, pNEB193, and cannot replicate autonomously in methanogens. The designed plasmids facilitate markerless gene deletion, gene transcription, protein expression, and purification of proteins with cleavable affinity tags from the methanogen, Methanosarcina acetivorans. PMID:26876941

  15. EUCLID: design of the prism DMD NIR spectrograph

    NASA Astrophysics Data System (ADS)

    Content, Robert; Sharples, Ray M.; Blake, Simon; Talbot, R. Gordon

    2010-07-01

    EUCLID, the ESA Dark Energy Mission, contains a NIR and a visible imagers (NIP & VIS), and an NIR spectrograph (NIS). Different designs of the NIS have been studied especially a slitless design, a Digital Micromirror Device (DMD) design using grisms and another using prisms, and more recently a combination of the NIP and NIS into one instrument. We present the design of the prism DMD NIS. This design has the advantage over the slitless design of having a DMD mask which reduces the background by a factor of more than 100 and all the advantages over the grism DMD NIS that a prism gives over a grism as a higher and more uniform transmission, the absence of parasite orders, and a choice of the slope of the spectral resolution with wavelength. The field per spectrograph was made sufficiently large to reduce the number of spectrographs to two. The design was made so that the mapping of the sky of the NIS is easily compatible with the mapping strategy of the NIP and VIS. Two designs were made. In one, the field is larger but the surface shapes of the optics are complex which makes manufacturing more challenging. In the other, the design was made to be fully compatible with the manufacturing criteria of SESO after extensive discussions to carefully understand the manufacturing limitations especially the formula for highly aspheric surface shapes as biconics. This was done by directly integrating the criteria into the optimization process of ZEMAX. A calibration system that uses the DMD with the micromirrors in their OFF positions was also developed.

  16. Heterozygous deletion of the LRFN2 gene is associated with working memory deficits.

    PubMed

    Thevenon, Julien; Souchay, Céline; Seabold, Gail K; Dygai-Cochet, Inna; Callier, Patrick; Gay, Sébastien; Corbin, Lucie; Duplomb, Laurence; Thauvin-Robinet, Christel; Masurel-Paulet, Alice; El Chehadeh, Salima; Avila, Magali; Minot, Delphine; Guedj, Eric; Chancenotte, Sophie; Bonnet, Marlène; Lehalle, Daphne; Wang, Ya-Xian; Kuentz, Paul; Huet, Frédéric; Mosca-Boidron, Anne-Laure; Marle, Nathalie; Petralia, Ronald S; Faivre, Laurence

    2016-06-01

    Learning disabilities (LDs) are a clinically and genetically heterogeneous group of diseases. Array-CGH and high-throughput sequencing have dramatically expanded the number of genes implicated in isolated intellectual disabilities and LDs, highlighting the implication of neuron-specific post-mitotic transcription factors and synaptic proteins as candidate genes. We report a unique family diagnosed with autosomal dominant learning disability and a 6p21 microdeletion segregating in three patients. The 870 kb microdeletion encompassed the brain-expressed gene LRFN2, which encodes for a synaptic cell adhesion molecule. Neuropsychological assessment identified selective working memory deficits, with borderline intellectual functioning. Further investigations identified a defect in executive function, and auditory-verbal processes. These data were consistent with brain MRI and FDG-PET functional brain imaging, which, when compared with controls, revealed abnormal brain volume and hypometabolism of gray matter structures implicated in working memory. We performed electron microscopy immunogold labeling demonstrating the localization of LRFN2 at synapses of cerebellar and hippocampal rat neurons, often associated with the NR1 subunit of N-methyl-D-aspartate receptors (NMDARs). Altogether, the combined approaches imply a role for LRFN2 in LD, specifically for working memory processes and executive function. In conclusion, the identification of familial cases of clinically homogeneous endophenotypes of LD might help in both the management of patients and genetic counseling for families.

  17. Molecular characterization of the breakpoints of a 12-kb deletion in the NF1 gene in a family showing germ-line mosaicism

    SciTech Connect

    Lazaro, C.; Gaona, A.; Lynch, M.

    1995-11-01

    Neurofibromatosis type 1 (NF1) is caused by deletions, insertions, translocations, and point mutations in the NF1 gene, which spans 350 kb on the long arm of human chromosome 17. Although several point mutations have been described, large molecular abnormalities have rarely been characterized in detail. We describe here the molecular breakpoints of a 12-kb deletion of the NF1 gene, which is responsible for the NF1 phenotype in a kindred with two children affected because of germ-line mosaicism in the unaffected father, who has the mutation in 10% of his spermatozoa. The mutation spans introns 31-39, removing 12,021 nt and inserting 30 bp, of which 19 bp are a direct repetition of a sequence located in intron 31, just 4 bp before the 5{prime} breakpoint. The 5{prime} and 3{prime} breakpoints contain the sequence TATTTTA, which could be involved in the generation of the deletion. The most plausible explanation for the mechanism involved in the generation of this 12-kb deletion is homologous/nonhomologous recombination. Since sperm of the father does not contain the corresponding insertion of the 12-kb deleted sequence, this deletion could have occurred within the NF1 chromosome through loop formation. RNA from lymphocytes of one of the NF1 patients showed similar levels of the mutated and normal transcripts, suggesting that the NF1-mRNA from mutations causing frame shifts of the reading frame or stop codons in this gene is not degraded during its processing. The mutation was not detected in fresh lymphocytes from the unaffected father by PCR analysis, supporting the case for true germ-line mosaicism. 30 refs., 3 figs.

  18. Polymorphism of the human complement C4 and steroid 21-hydroxylase genes. Restriction fragment length polymorphisms revealing structural deletions, homoduplications, and size variants.

    PubMed Central

    Schneider, P M; Carroll, M C; Alper, C A; Rittner, C; Whitehead, A S; Yunis, E J; Colten, H R

    1986-01-01

    Several autoimmune disorders as well as congenital adrenal hyperplasia (CAH) are either associated or closely linked with genetic variants of the fourth component of complement (C4A and C4B) and the enzyme steroid 21-hydroxylase (21-OH). These proteins are encoded by genes that are located downstream from the genes for complement proteins, C2 and factor B (BF) between HLA-B and -DR in the major histocompatibility complex (MHC). Previous studies of variants and null alleles were based on electrophoretic mobility of C4 protein and linkage with disease phenotypes. These data did not permit analysis of the basis for the observed null alleles and duplicated variants. We studied this region of the MHC in 126 haplotypes for a structural analysis of the four adjacent loci, C4A, 21-OHA, C4B, and 21-OHB. About half of the C4 genes typed as C4 null are deleted and several unrecognized homoduplicated C4 alleles were detected. Hence the frequencies of different C4 structural variants must be recalculated based on a direct analysis of the genes. Analysis of the C4/21-OH genes of patients with the classical (salt-wasting) form of CAH showed that some involve a deletion of the C4B and 21-OHB genes; whereas for two only the 21-OHB gene is deleted, i.e., the C4B gene is present. Together, these data provide a better understanding of the mechanisms generating and importance of deleted C4 and 21-OH null alleles in human disease. Images PMID:3018042

  19. FBXW12, a novel F box p