Alignment-based and alignment-free methods converge with experimental data on amino acids coded by stop codons at split between nuclear and mitochondrial genetic codes.

PubMed

Seligmann, Hervé

2018-05-01

Genetic codes mainly evolve by reassigning punctuation codons, starts and stops. Previous analyses assuming that undefined amino acids translate stops showed greater divergence between nuclear and mitochondrial genetic codes. Here, three independent methods converge on which amino acids translated stops at split between nuclear and mitochondrial genetic codes: (a) alignment-free genetic code comparisons inserting different amino acids at stops; (b) alignment-based blast analyses of hypothetical peptides translated from non-coding mitochondrial sequences, inserting different amino acids at stops; (c) biases in amino acid insertions at stops in proteomic data. Hence short-term protein evolution models reconstruct long-term genetic code evolution. Mitochondria reassign stops to amino acids otherwise inserted at stops by codon-anticodon mismatches (near-cognate tRNAs). Hence dual function (translation termination and translation by codon-anticodon mismatch) precedes mitochondrial reassignments of stops to amino acids. Stop ambiguity increases coded information, compensates endocellular mitogenome reduction. Mitochondrial codon reassignments might prevent viral infections. Copyright © 2018 Elsevier B.V. All rights reserved.

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream.

PubMed

Xu, Yi; Ju, Ho-Jong; DeBlasio, Stacy; Carino, Elizabeth J; Johnson, Richard; MacCoss, Michael J; Heck, Michelle; Miller, W Allen; Gray, Stewart M

2018-06-01

Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein. IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the Luteoviridae by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed. Copyright © 2018 American Society for Microbiology.

Natural Variant of Collagen-Like Protein A in Serotype M3 Group A Streptococcus Increases Adherence and Decreases Invasive Potential

PubMed Central

Jewell, Brittany E.; Versalovic, Erika M.; Olsen, Randall J.; Bachert, Beth A.; Lukomski, Slawomir; Musser, James M.

2015-01-01

Group A Streptococcus (GAS) predominantly exists as a colonizer of the human oropharynx that occasionally breaches epithelial barriers to cause invasive diseases. Despite the frequency of GAS carriage, few investigations into the contributory molecular mechanisms exist. To this end, we identified a naturally occurring polymorphism in the gene encoding the streptococcal collagen-like protein A (SclA) in GAS carrier strains. All previously sequenced invasive serotype M3 GAS possess a premature stop codon in the sclA gene truncating the protein. The carrier polymorphism is predicted to restore SclA function and was infrequently identified by targeted DNA sequencing in invasive strains of the same serotype. We demonstrate that a strain with the carrier sclA allele expressed a full-length SclA protein, while the strain with the invasive sclA allele expressed a truncated variant. An isoallelic mutant invasive strain with the carrier sclA allele exhibited decreased virulence in a mouse model of invasive disease and decreased multiplication in human blood. Further, the isoallelic invasive strain with the carrier sclA allele persisted in the mouse nasopharynx and had increased adherence to cultured epithelial cells. Repair of the premature stop codon in the invasive sclA allele restored the ability to bind the extracellular matrix proteins laminin and cellular fibronectin. These data demonstrate that a mutation in GAS carrier strains increases adherence and decreases virulence and suggest selection against increased adherence in GAS invasive isolates. PMID:25561712

Stop codon readthrough generates a C-terminally extended variant of the human vitamin D receptor with reduced calcitriol response

PubMed Central

Loughran, Gary; Jungreis, Irwin; Tzani, Ioanna; Power, Michael; Dmitriev, Ruslan I.; Ivanov, Ivaylo P.; Kellis, Manolis; Atkins, John F.

2018-01-01

Although stop codon readthrough is used extensively by viruses to expand their gene expression, verified instances of mammalian readthrough have only recently been uncovered by systems biology and comparative genomics approaches. Previously, our analysis of conserved protein coding signatures that extend beyond annotated stop codons predicted stop codon readthrough of several mammalian genes, all of which have been validated experimentally. Four mRNAs display highly efficient stop codon readthrough, and these mRNAs have a UGA stop codon immediately followed by CUAG (UGA_CUAG) that is conserved throughout vertebrates. Extending on the identification of this readthrough motif, we here investigated stop codon readthrough, using tissue culture reporter assays, for all previously untested human genes containing UGA_CUAG. The readthrough efficiency of the annotated stop codon for the sequence encoding vitamin D receptor (VDR) was 6.7%. It was the highest of those tested but all showed notable levels of readthrough. The VDR is a member of the nuclear receptor superfamily of ligand-inducible transcription factors, and it binds its major ligand, calcitriol, via its C-terminal ligand-binding domain. Readthrough of the annotated VDR mRNA results in a 67 amino acid–long C-terminal extension that generates a VDR proteoform named VDRx. VDRx may form homodimers and heterodimers with VDR but, compared with VDR, VDRx displayed a reduced transcriptional response to calcitriol even in the presence of its partner retinoid X receptor. PMID:29386352

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

PubMed

Rubinstein, M; Mogil, J S; Japón, M; Chan, E C; Allen, R G; Low, M J

1996-04-30

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.

Germline mutations of regulator of telomere elongation helicase 1, RTEL1, in Dyskeratosis congenita.

PubMed

Ballew, Bari J; Yeager, Meredith; Jacobs, Kevin; Giri, Neelam; Boland, Joseph; Burdett, Laurie; Alter, Blanche P; Savage, Sharon A

2013-04-01

Dyskeratosis congenita (DC) is an inherited bone marrow failure and cancer predisposition syndrome caused by aberrant telomere biology. The classic triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia is diagnostic of DC, but substantial clinical heterogeneity exists; the clinically severe variant Hoyeraal Hreidarsson syndrome (HH) also includes cerebellar hypoplasia, severe immunodeficiency, enteropathy, and intrauterine growth retardation. Germline mutations in telomere biology genes account for approximately one-half of known DC families. Using exome sequencing, we identified mutations in RTEL1, a helicase with critical telomeric functions, in two families with HH. In the first family, two siblings with HH and very short telomeres inherited a premature stop codon from their mother who has short telomeres. The proband from the second family has HH and inherited a premature stop codon in RTEL1 from his father and a missense mutation from his mother, who also has short telomeres. In addition, inheritance of only the missense mutation led to very short telomeres in the proband's brother. Targeted sequencing identified a different RTEL1 missense mutation in one additional DC proband who has bone marrow failure and short telomeres. Both missense mutations affect the helicase domain of RTEL1, and three in silico prediction algorithms suggest that they are likely deleterious. The nonsense mutations both cause truncation of the RTEL1 protein, resulting in loss of the PIP box; this may abrogate an important protein-protein interaction. These findings implicate a new telomere biology gene, RTEL1, in the etiology of DC.

Germline Mutations of Regulator of Telomere Elongation Helicase 1, RTEL1, In Dyskeratosis Congenita

PubMed Central

Ballew, Bari J.; Yeager, Meredith; Jacobs, Kevin; Giri, Neelam; Boland, Joseph; Burdett, Laurie; Alter, Blanche P.; Savage, Sharon A.

2013-01-01

Dyskeratosis congenita (DC) is an inherited bone marrow failure and cancer predisposition syndrome caused by aberrant telomere biology. The classic triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia is diagnostic of DC, but substantial clinical heterogeneity exists; the clinically severe variant Hoyeraal Hreidarsson syndrome (HH) also includes cerebellar hypoplasia, severe immunodeficiency, enteropathy, and intrauterine growth retardation. Germline mutations in telomere biology genes account for approximately one-half of known DC families. Using exome sequencing, we identified mutations in RTEL1, a helicase with critical telomeric functions, in two families with HH. In the first family, two siblings with HH and very short telomeres inherited a premature stop codon from their mother who has short telomeres. The proband from the second family has HH and inherited a premature stop codon in RTEL1 from his father and a missense mutation from his mother, who also has short telomeres. Additionally, inheritance of only the missense mutation led to very short telomeres in the proband’s brother. Targeted sequencing identified a different RTEL1 missense mutation in one additional DC proband who has bone marrow failure and short telomeres. Both missense mutations affect the helicase domain of RTEL1, and three in silico prediction algorithms suggest that they are likely deleterious. The nonsense mutations both cause truncation of the RTEL1 protein, resulting in loss of the PIP box; this may abrogate an important protein-protein interaction. These findings implicate a new telomere biology gene, RTEL1, in the etiology of DC. PMID:23329068

Reassigning stop codons via translation termination: How a few eukaryotes broke the dogma.

PubMed

Alkalaeva, Elena; Mikhailova, Tatiana

2017-03-01

The genetic code determines how amino acids are encoded within mRNA. It is universal among the vast majority of organisms, although several exceptions are known. Variant genetic codes are found in ciliates, mitochondria, and numerous other organisms. All revealed genetic codes (standard and variant) have at least one codon encoding a translation stop signal. However, recently two new genetic codes with a reassignment of all three stop codons were revealed in studies examining the protozoa transcriptomes. Here, we discuss this finding and the recent studies of variant genetic codes in eukaryotes. We consider the possible molecular mechanisms allowing the use of certain codons as sense and stop signals simultaneously. The results obtained by studying these amazing organisms represent a new and exciting insight into the mechanism of stop codon decoding in eukaryotes. Also see the video abstract here. © 2017 WILEY Periodicals, Inc.

Rapid Y degeneration and dosage compensation in plant sex chromosomes

PubMed Central

Papadopulos, Alexander S. T.; Chester, Michael; Ridout, Kate; Filatov, Dmitry A.

2015-01-01

The nonrecombining regions of animal Y chromosomes are known to undergo genetic degeneration, but previous work has failed to reveal large-scale gene degeneration on plant Y chromosomes. Here, we uncover rapid and extensive degeneration of Y-linked genes in a plant species, Silene latifolia, that evolved sex chromosomes de novo in the last 10 million years. Previous transcriptome-based studies of this species missed unexpressed, degenerate Y-linked genes. To identify sex-linked genes, regardless of their expression, we sequenced male and female genomes of S. latifolia and integrated the genomic contigs with a high-density genetic map. This revealed that 45% of Y-linked genes are not expressed, and 23% are interrupted by premature stop codons. This contrasts with X-linked genes, in which only 1.3% of genes contained stop codons and 4.3% of genes were not expressed in males. Loss of functional Y-linked genes is partly compensated for by gene-specific up-regulation of X-linked genes. Our results demonstrate that the rate of genetic degeneration of Y-linked genes in S. latifolia is as fast as in animals, and that the evolutionary trajectories of sex chromosomes are similar in the two kingdoms. PMID:26438872

Identification of ALK germline mutation (3605delG) in pediatric anaplastic medulloblastoma.

PubMed

Coco, Simona; De Mariano, Marilena; Valdora, Francesca; Servidei, Tiziana; Ridola, Vita; Andolfo, Immacolata; Oberthuer, André; Tonini, Gian Paolo; Longo, Luca

2012-10-01

The anaplastic lymphoma kinase (ALK) gene has been found either rearranged or mutated in several neoplasms such as anaplastic large-cell lymphoma, non-small-cell lung cancer, neuroblastoma and anaplastic thyroid cancer. Medulloblastoma (MB) is an embryonic pediatric cancer arising from nervous system, a tissue in which ALK is expressed during embryonic development. We performed an ALK mutation screening in 52 MBs and we found a novel heterozygous germline deletion of a single base in exon 23 (3605delG) in a case with marked anaplasia. This G deletion results in a frameshift mutation producing a premature stop codon in exon 25 of ALK tyrosine kinase domain. We also screened three human MB cell lines without finding any mutation of ALK gene. Quantitative expression analysis of 16 out of 52 samples showed overexpression of ALK mRNA in three MBs. In the present study, we report the first mutation of ALK found in MB. Moreover, a deletion of ALK gene producing a stop codon has not been detected in human tumors up to now. Further investigations are now required to elucidate whether the truncated form of ALK may have a role in signal transduction.

Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay

PubMed Central

Ni, Julie Z.; Grate, Leslie; Donohue, John Paul; Preston, Christine; Nobida, Naomi; O’Brien, Georgeann; Shiue, Lily; Clark, Tyson A.; Blume, John E.; Ares, Manuel

2007-01-01

Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified >50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called “ultraconserved” elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression. We suggest that the extreme genomic conservation surrounding these regulatory splicing events within splicing factor genes demonstrates the evolutionary importance of maintaining tightly tuned homeostasis of RNA-binding protein levels in the vertebrate cell. PMID:17369403

CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.

PubMed

Billon, Pierre; Bryant, Eric E; Joseph, Sarah A; Nambiar, Tarun S; Hayward, Samuel B; Rothstein, Rodney; Ciccia, Alberto

2017-09-21

Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP. Copyright © 2017 Elsevier Inc. All rights reserved.

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

PubMed Central

Rubinstein, M; Mogil, J S; Japón, M; Chan, E C; Allen, R G; Low, M J

1996-01-01

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms. Images Fig. 1 Fig. 2 PMID:8633004

Trichomonas vaginalis Metronidazole Resistance Is Associated with Single Nucleotide Polymorphisms in the Nitroreductase Genes ntr4Tv and ntr6Tv

PubMed Central

Paulish-Miller, Teresa E.; Augostini, Peter; Schuyler, Jessica A.; Smith, William L.; Mordechai, Eli; Adelson, Martin E.; Gygax, Scott E.; Secor, William E.

2014-01-01

Metronidazole resistance in the sexually transmitted parasite Trichomonas vaginalis is a problematic public health issue. We have identified single nucleotide polymorphisms (SNPs) in two nitroreductase genes (ntr4Tv and ntr6Tv) associated with resistance. These SNPs were associated with one of two distinct T. vaginalis populations identified by multilocus sequence typing, yet one SNP (ntr6Tv A238T), which results in a premature stop codon, was associated with resistance independent of population structure and may be of diagnostic value. PMID:24550324

Inulin and levan synthesis by probiotic Lactobacillus gasseri strains: characterization of three novel fructansucrase enzymes and their fructan products.

PubMed

Anwar, Munir A; Kralj, Slavko; Piqué, Anna Villar; Leemhuis, Hans; van der Maarel, Marc J E C; Dijkhuizen, Lubbert

2010-04-01

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. Here, we report an evaluation of fructan synthesis in three Lactobacillus gasseri strains, identification of the fructansucrase-encoding genes and characterization of the recombinant proteins and fructan (oligosaccharide) products. High-performance anion-exchange chromatography and nuclear magnetic resonance analysis of the fructo-oligosaccharides (FOS) and polymers produced by the L. gasseri strains and the recombinant enzymes revealed that, in situ, L. gasseri strains DSM 20604 and 20077 synthesize inulin (and oligosaccharides) and levan products, respectively. L. gasseri DSM 20604 is only the second Lactobacillus strain shown to produce inulin polymer and FOS in situ, and is unique in its distribution of FOS synthesized, ranging from DP2 to DP13. The probiotic bacterium L. gasseri DSM 20243 did not produce any fructan, although we identified a fructansucrase-encoding gene in its genome sequence. Further studies showed that this L. gasseri DSM 20243 gene was prematurely terminated by a stop codon. Exchanging the stop codon for a glutamine codon resulted in a recombinant enzyme producing inulin and FOS. The three recombinant fructansucrase enzymes characterized from three different L. gasseri strains have very similar primary protein structures, yet synthesize different fructan products. An interesting feature of the L. gasseri strains is that they were unable to ferment raffinose, whereas their respective recombinant enzymes converted raffinose into fructan and FOS.

Recent evidence for evolution of the genetic code

NASA Technical Reports Server (NTRS)

Osawa, S.; Jukes, T. H.; Watanabe, K.; Muto, A.

1992-01-01

The genetic code, formerly thought to be frozen, is now known to be in a state of evolution. This was first shown in 1979 by Barrell et al. (G. Barrell, A. T. Bankier, and J. Drouin, Nature [London] 282:189-194, 1979), who found that the universal codons AUA (isoleucine) and UGA (stop) coded for methionine and tryptophan, respectively, in human mitochondria. Subsequent studies have shown that UGA codes for tryptophan in Mycoplasma spp. and in all nonplant mitochondria that have been examined. Universal stop codons UAA and UAG code for glutamine in ciliated protozoa (except Euplotes octacarinatus) and in a green alga, Acetabularia. E. octacarinatus uses UAA for stop and UGA for cysteine. Candida species, which are yeasts, use CUG (leucine) for serine. Other departures from the universal code, all in nonplant mitochondria, are CUN (leucine) for threonine (in yeasts), AAA (lysine) for asparagine (in platyhelminths and echinoderms), UAA (stop) for tyrosine (in planaria), and AGR (arginine) for serine (in several animal orders) and for stop (in vertebrates). We propose that the changes are typically preceded by loss of a codon from all coding sequences in an organism or organelle, often as a result of directional mutation pressure, accompanied by loss of the tRNA that translates the codon. The codon reappears later by conversion of another codon and emergence of a tRNA that translates the reappeared codon with a different assignment. Changes in release factors also contribute to these revised assignments. We also discuss the use of UGA (stop) as a selenocysteine codon and the early history of the code.

Genetic, immunological, and clinical features of patients with bacterial and fungal infections due to inherited IL-17RA deficiency

PubMed Central

Lévy, Romain; Okada, Satoshi; Béziat, Vivien; Moriya, Kunihiko; Liu, Caini; Chai, Louis Yi Ann; Migaud, Mélanie; Hauck, Fabian; Al Ali, Amein; Cyrus, Cyril; Vatte, Chittibabu; Patiroglu, Turkan; Unal, Ekrem; Ferneiny, Marie; Hyakuna, Nobuyuki; Nepesov, Serdar; Oleastro, Matias; Ikinciogullari, Aydan; Dogu, Figen; Asano, Takaki; Ohara, Osamu; Yun, Ling; Della Mina, Erika; Bronnimann, Didier; Itan, Yuval; Gothe, Florian; Bustamante, Jacinta; Boisson-Dupuis, Stéphanie; Tahuil, Natalia; Aytekin, Caner; Salhi, Aicha; Al Muhsen, Saleh; Kobayashi, Masao; Toubiana, Julie; Abel, Laurent; Li, Xiaoxia; Camcioglu, Yildiz; Celmeli, Fatih; Klein, Christoph; AlKhater, Suzan A.; Casanova, Jean-Laurent; Puel, Anne

2016-01-01

Chronic mucocutaneous candidiasis (CMC) is defined as recurrent or persistent infection of the skin, nails, and/or mucosae with commensal Candida species. The first genetic etiology of isolated CMC—autosomal recessive (AR) IL-17 receptor A (IL-17RA) deficiency—was reported in 2011, in a single patient. We report here 21 patients with complete AR IL-17RA deficiency, including this first patient. Each patient is homozygous for 1 of 12 different IL-17RA alleles, 8 of which create a premature stop codon upstream from the transmembrane domain and have been predicted and/or shown to prevent expression of the receptor on the surface of circulating leukocytes and dermal fibroblasts. Three other mutant alleles create a premature stop codon downstream from the transmembrane domain, one of which encodes a surface-expressed receptor. Finally, the only known missense allele (p.D387N) also encodes a surface-expressed receptor. All of the alleles tested abolish cellular responses to IL-17A and -17F homodimers and heterodimers in fibroblasts and to IL-17E/IL-25 in leukocytes. The patients are currently aged from 2 to 35 y and originate from 12 unrelated kindreds. All had their first CMC episode by 6 mo of age. Fourteen patients presented various forms of staphylococcal skin disease. Eight were also prone to various bacterial infections of the respiratory tract. Human IL-17RA is, thus, essential for mucocutaneous immunity to Candida and Staphylococcus, but otherwise largely redundant. A diagnosis of AR IL-17RA deficiency should be considered in children or adults with CMC, cutaneous staphylococcal disease, or both, even if IL-17RA is detected on the cell surface. PMID:27930337

Position-dependent termination and widespread obligatory frameshifting in Euplotes translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lobanov, Alexei V.; Heaphy, Stephen M.; Turanov, Anton A.

2016-11-21

The ribosome can change its reading frame during translation in a process known as programmed ribosomal frameshifting. These rare events are supported by complex mRNA signals. However, we found that the ciliates Euplotes crassus and Euplotes focardii exhibit widespread frameshifting at stop codons. 47 different codons preceding stop signals resulted in either +1 or +2 frameshifts, and +1 frameshifting at AAA was the most frequent. The frameshifts showed unusual plasticity and rapid evolution, and had little influence on translation rates. The proximity of a stop codon to the 3' mRNA end, rather than its occurrence or sequence context, appeared tomore » designate termination. Thus, a ‘stop codon’ is not a sufficient signal for translation termination, and the default function of stop codons in Euplotes is frameshifting, whereas termination is specific to certain mRNA positions and probably requires additional factors.« less

RNA editing makes mistakes in plant mitochondria: editing loses sense in transcripts of a rps19 pseudogene and in creating stop codons in coxI and rps3 mRNAs of Oenothera.

PubMed Central

Schuster, W; Brennicke, A

1991-01-01

An intact gene for the ribosomal protein S19 (rps19) is absent from Oenothera mitochondria. The conserved rps19 reading frame found in the mitochondrial genome is interrupted by a termination codon. This rps19 pseudogene is cotranscribed with the downstream rps3 gene and is edited on both sides of the translational stop. Editing, however, changes the amino acid sequence at positions that were well conserved before editing. Other strange editings create translational stops in open reading frames coding for functional proteins. In coxI and rps3 mRNAs CGA codons are edited to UGA stop codons only five and three codons, respectively, downstream to the initiation codon. These aberrant editings in essential open reading frames and in the rps19 pseudogene appear to have been shifted to these positions from other editing sites. These observations suggest a requirement for a continuous evolutionary constraint on the editing specificities in plant mitochondria. Images PMID:1762921

Treacher Collins syndrome with a de Novo 5-bp deletion in the TCOF1 gene.

PubMed

Su, Pen-Hua; Chen, Jia-Yu; Chen, Suh-Jen; Yu, Ju-Shan

2006-06-01

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development with features including malar hypoplasia, micrognathia, microtia, downward slanting palpebral fissures, lower eyelid coloboma, conductive hearing loss, and cleft palate. TCS is caused by mutations in the TCOF1 gene, which encodes the nuclear phosphoprotein treacle. Here, we describe a 1-day-old male infant with classical TCS presentation. A 5-bp deletion in exon 22 of the TCOF1 gene (3469del ACTCT) was found to cause a premature stop codon. This is the first report of TCOF1 gene mutation in the Taiwanese population.

Novel Single-Base Deletional Mutation in Major Intrinsic Protein (MIP) in Autosomal Dominant Cataract

PubMed Central

Geyer, David D.; Spence, M. Anne; Johannes, Meriam; Flodman, Pamela; Clancy, Kevin P.; Berry, Rebecca; Sparkes, Robert S.; Jonsen, Matthew D.; Isenberg, Sherwin J.; Bateman, J. Bronwyn

2006-01-01

PURPOSE To further elucidate the cataract phenotype, and identify the gene and mutation for autosomal dominant cataract (ADC) in an American family of European descent (ADC2) by sequencing the major intrinsic protein gene (MIP), a candidate based on linkage to chromosome 12q13. DESIGN Observational case series and laboratory experimental study. METHODS We examined two at-risk individuals in ADC2. We PCR-amplified and sequenced all four exons and all intron-exon boundaries of the MIP gene from genomic and cloned DNA in affected members to confirm one variant as the putative mutation. RESULTS We found a novel single deletion of nucleotide (nt) 3223 (within codon 235) in exon four, causing a frameshift that alters 41 of 45 subsequent amino acids and creates a premature stop codon. CONCLUSIONS We identified a novel single base pair deletion in the MIP gene and conclude that it is a pathogenic sequence alteration. PMID:16564824

A novel mutation causing complete thyroxine-binding globulin deficiency (TBG-CD-Negev) among the Bedouins in southern Israel.

PubMed

Miura, Y; Hershkovitz, E; Inagaki, A; Parvari, R; Oiso, Y; Phillip, M

2000-10-01

T4-binding globulin (TBG) is the major thyroid hormone transport protein in human serum. Inherited TBG abnormalities do not usually alter the metabolic status and are transmitted in X-linked inheritance. A high prevalence of complete TBG deficiency (TBG-CD) has been reported among the Bedouin population in the Negev (southern Israel). In this study we report a novel single mutation causing complete TBG deficiency due to a deletion of the last base of codon 38 (exon 1), which led to a frame shift resulting in a premature stop at codon 51 and a presumed truncated peptide of 50 residues. This new variant of TBG (TBG-CD-Negev) was found among all of the patients studied. We conclude that a single mutation may account for TBG deficiency among the Bedouins in the Negev. This report is the first to describe a mutation in a population with an unusually high prevalence of TBG-CD.

Odilorhabdins, Antibacterial Agents that Cause Miscoding by Binding at a New Ribosomal Site.

PubMed

Pantel, Lucile; Florin, Tanja; Dobosz-Bartoszek, Malgorzata; Racine, Emilie; Sarciaux, Matthieu; Serri, Marine; Houard, Jessica; Campagne, Jean-Marc; de Figueiredo, Renata Marcia; Midrier, Camille; Gaudriault, Sophie; Givaudan, Alain; Lanois, Anne; Forst, Steve; Aumelas, André; Cotteaux-Lautard, Christelle; Bolla, Jean-Michel; Vingsbo Lundberg, Carina; Huseby, Douglas L; Hughes, Diarmaid; Villain-Guillot, Philippe; Mankin, Alexander S; Polikanov, Yury S; Gualtieri, Maxime

2018-04-05

Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome. Copyright © 2018 Elsevier Inc. All rights reserved.

A novel loss-of-function mutation in Npr2 clarifies primary role in female reproduction and reveals a potential therapy for acromesomelic dysplasia, Maroteaux type.

PubMed

Geister, Krista A; Brinkmeier, Michelle L; Hsieh, Minnie; Faust, Susan M; Karolyi, I Jill; Perosky, Joseph E; Kozloff, Kenneth M; Conti, Marco; Camper, Sally A

2013-01-15

We discovered a new spontaneous mutant allele of Npr2 named peewee (pwe) that exhibits severe disproportionate dwarfism and female infertility. The pwe phenotype is caused by a four base-pair deletion in exon 3 that generates a premature stop codon at codon 313 (L313X). The Npr2(pwe/pwe) mouse is a model for the human skeletal dysplasia acromesomelic dysplasia, Maroteaux type (AMDM). We conducted a thorough analysis of the female reproductive tract and report that the primary cause of Npr2(pwe/pwe) female infertility is premature oocyte meiotic resumption, while the pituitary and uterus appear to be normal. Npr2 is expressed in chondrocytes and osteoblasts. We determined that the loss of Npr2 causes a reduction in the hypertrophic and proliferative zones of the growth plate, but mineralization of skeletal elements is normal. Mutant tibiae have increased levels of the activated form of ERK1/2, consistent with the idea that natriuretic peptide receptor type 2 (NPR2) signaling inhibits the activation of the MEK/ERK mitogen activated protein kinase pathway. Treatment of fetal tibiae explants with mitogen activated protein kinase 1 and 2 inhibitors U0126 and PD325901 rescues the Npr2(pwe/pwe) growth defect, providing a promising foundation for skeletal dysplasia therapeutics.

A novel loss-of-function mutation in Npr2 clarifies primary role in female reproduction and reveals a potential therapy for acromesomelic dysplasia, Maroteaux type

PubMed Central

Geister, Krista A.; Brinkmeier, Michelle L.; Hsieh, Minnie; Faust, Susan M.; Karolyi, I. Jill; Perosky, Joseph E.; Kozloff, Kenneth M.; Conti, Marco; Camper, Sally A.

2013-01-01

We discovered a new spontaneous mutant allele of Npr2 named peewee (pwe) that exhibits severe disproportionate dwarfism and female infertility. The pwe phenotype is caused by a four base-pair deletion in exon 3 that generates a premature stop codon at codon 313 (L313X). The Npr2pwe/pwe mouse is a model for the human skeletal dysplasia acromesomelic dysplasia, Maroteaux type (AMDM). We conducted a thorough analysis of the female reproductive tract and report that the primary cause of Npr2pwe/pwe female infertility is premature oocyte meiotic resumption, while the pituitary and uterus appear to be normal. Npr2 is expressed in chondrocytes and osteoblasts. We determined that the loss of Npr2 causes a reduction in the hypertrophic and proliferative zones of the growth plate, but mineralization of skeletal elements is normal. Mutant tibiae have increased levels of the activated form of ERK1/2, consistent with the idea that natriuretic peptide receptor type 2 (NPR2) signaling inhibits the activation of the MEK/ERK mitogen activated protein kinase pathway. Treatment of fetal tibiae explants with mitogen activated protein kinase 1 and 2 inhibitors U0126 and PD325901 rescues the Npr2pwe/pwe growth defect, providing a promising foundation for skeletal dysplasia therapeutics. PMID:23065701

Complete mitochondrial genome of Palawan peacock-pheasant Polyplectron napoleonis (Galliformes, Phasianidae).

PubMed

Quach, Tommy; Brooks, Daniel M; Miranda, Hector C

2016-01-01

The complete mitochondrial genome of the Palawan peacock-pheasant Polyplectron napoleonis is 16,710 bp and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control-region. All protein-coding genes use the standard ATG start codon, except for cox1 which has GTG start codon. Seven out of 13 PCGs have TAA stop codons, two have AGG (cox1 and nd6), and three PCGs (nd2, cox2 and nd4) have incomplete stop codon of just T- - nucleotide.

Mutations in eukaryotic release factors 1 and 3 act as general nonsense suppressors in Drosophila.

PubMed Central

Chao, Anna T; Dierick, Herman A; Addy, Tracie M; Bejsovec, Amy

2003-01-01

In a screen for suppressors of the Drosophila wingless(PE4) nonsense allele, we isolated mutations in the two components that form eukaryotic release factor. eRF1 and eRF3 comprise the translation termination complex that recognizes stop codons and catalyzes the release of nascent polypeptide chains from ribosomes. Mutations disrupting the Drosophila eRF1 and eRF3 show a strong maternal-effect nonsense suppression due to readthrough of stop codons and are zygotically lethal during larval stages. We tested nonsense mutations in wg and in other embryonically acting genes and found that different stop codons can be suppressed but only a subset of nonsense alleles are subject to suppression. We suspect that the context of the stop codon is significant: nonsense alleles sensitive to suppression by eRF1 and eRF3 encode stop codons that are immediately followed by a cytidine. Such suppressible alleles appear to be intrinsically weak, with a low level of readthrough that is enhanced when translation termination is disrupted. Thus the eRF1 and eRF3 mutations provide a tool for identifying nonsense alleles that are leaky. Our findings have important implications for assigning null mutant phenotypes and for selecting appropriate alleles to use in suppressor screens. PMID:14573473

The Role of +4U as an Extended Translation Termination Signal in Bacteria

PubMed Central

Wei, Yulong; Xia, Xuhua

2017-01-01

Termination efficiency of stop codons depends on the first 3′ flanking (+4) base in bacteria and eukaryotes. In both Escherichia coli and Saccharomyces cerevisiae, termination read-through is reduced in the presence of +4U; however, the molecular mechanism underlying +4U function is poorly understood. Here, we perform comparative genomics analysis on 25 bacterial species (covering Actinobacteria, Bacteriodetes, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Proteobacteria, and Spirochaetae) with bioinformatics approaches to examine the influence of +4U in bacterial translation termination by contrasting highly- and lowly-expressed genes (HEGs and LEGs, respectively). We estimated gene expression using the recently formulated Index of Translation Elongation, ITE, and identified stop codon near-cognate transfer RNAs (tRNAs) from well-annotated genomes. We show that +4U was consistently overrepresented in UAA-ending HEGs relative to LEGs. The result is consistent with the interpretation that +4U enhances termination mainly for UAA. Usage of +4U decreases in GC-rich species where most stop codons are UGA and UAG, with few UAA-ending genes, which is expected if UAA usage in HEGs drives up +4U usage. In HEGs, +4U usage increases significantly with abundance of UAA nc_tRNAs (near-cognate tRNAs that decode codons differing from UAA by a single nucleotide), particularly those with a mismatch at the first stop codon site. UAA is always the preferred stop codon in HEGs, and our results suggest that UAAU is the most efficient translation termination signal in bacteria. PMID:27903612

Cryptic splice site in the complementary DNA of glucocerebrosidase causes inefficient expression.

PubMed

Bukovac, Scott W; Bagshaw, Richard D; Rigat, Brigitte A; Callahan, John W; Clarke, Joe T R; Mahuran, Don J

2008-10-15

The low levels of human lysosomal glucocerebrosidase activity expressed in transiently transfected Chinese hamster ovary (CHO) cells were investigated. Reverse transcription PCR (RT-PCR) demonstrated that a significant portion of the transcribed RNA was misspliced owing to the presence of a cryptic splice site in the complementary DNA (cDNA). Missplicing results in the deletion of 179 bp of coding sequence and a premature stop codon. A repaired cDNA was constructed abolishing the splice site without changing the amino acid sequence. The level of glucocerebrosidase expression was increased sixfold. These data demonstrate that for maximum expression of any cDNA construct, the transcription products should be examined.

Peeling skin syndrome: genetic defects in late terminal differentiation of the epidermis.

PubMed

Bowden, Paul E

2011-03-01

In this issue, Israeli and colleagues confirm that homozygous mutations in corneodesmosin (CDSN) cause type B peeling skin syndrome (PSS), an autosomal recessive skin disorder. The deletion mutation described resulted in a frameshift, producing a downstream premature stop codon and early truncation of the protein. The recently described CDSN nonsense mutation in another PSS family also resulted in protein truncation and nonsense-mediated mRNA decay. Type B generalized PSS can now be clearly distinguished from acral PSS, caused by mutations in transglutaminase 5. This directly affects cornified envelope cross-linking rather than corneodesmosome adherence. These observations provide new insight into the molecular defects underlying two closely related forms of PSS.

CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations.

PubMed

Kuscu, Cem; Parlak, Mahmut; Tufan, Turan; Yang, Jiekun; Szlachta, Karol; Wei, Xiaolong; Mammadov, Rashad; Adli, Mazhar

2017-07-01

CRISPR-Cas9-induced DNA damage may have deleterious effects at high-copy-number genomic regions. Here, we use CRISPR base editors to knock out genes by changing single nucleotides to create stop codons. We show that the CRISPR-STOP method is an efficient and less deleterious alternative to wild-type Cas9 for gene-knockout studies. Early stop codons can be introduced in ∼17,000 human genes. CRISPR-STOP-mediated targeted screening demonstrates comparable efficiency to WT Cas9, which indicates the suitability of our approach for genome-wide functional screenings.

L-MPZ, a Novel Isoform of Myelin P0, Is Produced by Stop Codon Readthrough*

PubMed Central

Yamaguchi, Yoshihide; Hayashi, Akiko; Campagnoni, Celia W.; Kimura, Akio; Inuzuka, Takashi; Baba, Hiroko

2012-01-01

Myelin protein zero (P0 or MPZ) is a major myelin protein (∼30 kDa) expressed in the peripheral nervous system (PNS) in terrestrial vertebrates. Several groups have detected a P0-related 36-kDa (or 35-kDa) protein that is expressed in the PNS as an antigen for the serum IgG of patients with neuropathy. The molecular structure and function of this 36-kDa protein are, however, still unknown. We hypothesized that the 36-kDa protein may be derived from P0 mRNA by stop codon readthrough. We found a highly conserved region after the regular stop codon in predicted sequences from the 3′-UTR of P0 in higher animals. MS of the 36-kDa protein revealed that both P0 peptides and peptides deduced from the P0 3′-UTR sequence were found among the tryptic fragments. In transfected cells and in an in vitro transcription/translation system, the 36-kDa molecule was also produced from the identical mRNA that produced P0. We designated this 36-kDa molecule as large myelin protein zero (L-MPZ), a novel isoform of P0 that contains an additional domain at the C terminus. In the PNS, L-MPZ was localized in compact myelin. In transfected cells, just like P0, L-MPZ was localized at cell-cell adhesion sites in the plasma membrane. These results suggest that L-MPZ produced by the stop codon readthrough mechanism is potentially involved in myelination. Since this is the first finding of stop codon readthrough in a common mammalian protein, detailed analysis of L-MPZ expression will help to understand the mechanism of stop codon readthrough in mammals. PMID:22457349

Constructing high complexity synthetic libraries of long ORFs using in vitro selection

NASA Technical Reports Server (NTRS)

Cho, G.; Keefe, A. D.; Liu, R.; Wilson, D. S.; Szostak, J. W.

2000-01-01

We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel. Copyright 2000 Academic Press.

Readthrough of stop codons by use of aminoglycosides in cells from xeroderma pigmentosum group C patients.

PubMed

Kuschal, Christiane; Khan, Sikandar G; Enk, Benedikt; DiGiovanna, John J; Kraemer, Kenneth H

2015-04-01

Readthrough of premature termination (stop) codons (PTC) is a new approach to treatment of genetic diseases. We recently reported that readthrough of PTC in cells from some xeroderma pigmentosum complementation group C (XP-C) patients could be achieved with the aminoglycosides geneticin or gentamicin. We found that the response depended on several factors including the PTC sequence, its location within the gene and the aminoglycoside used. Here, we extended these studies to investigate the effects of other aminoglycosides that are already on the market. We reasoned that topical treatment could deliver much higher concentrations of drug to the skin, the therapeutic target, and thus increase the therapeutic effect while reducing renal or ototoxicity in comparison with systemic treatment. Our prior clinical studies indicated that only a few percent of normal XPC expression was associated with mild clinical disease. We found minimal cell toxicity in the XP-C cells with several aminoglycosides. We found increased XPC mRNA expression in PTC-containing XP-C cells with G418, paromomycin, neomycin and kanamycin and increased XPC protein expression with G418. We conclude that in selected patients with XP, topical PTC therapy can be investigated as a method of personalized medicine to alleviate their cutaneous symptoms. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Using Student Writing and Lexical Analysis to Reveal Student Thinking about the Role of Stop Codons in the Central Dogma

ERIC Educational Resources Information Center

Prevost, Luanna B.; Smith, Michelle K.; Knight, Jennifer K.

2016-01-01

Previous work has shown that students have persistent difficulties in understanding how central dogma processes can be affected by a stop codon mutation. To explore these difficulties, we modified two multiple-choice questions from the Genetics Concept Assessment into three open-ended questions that asked students to write about how a stop codon…

Multiple origins of the phenol reaction negative phenotype in foxtail millet, Setaria italica (L.) P. Beauv., were caused by independent loss-of-function mutations of the polyphenol oxidase (Si7PPO) gene during domestication.

PubMed

Inoue, Takahiko; Yuo, Takahisa; Ohta, Takeshi; Hitomi, Eriko; Ichitani, Katsuyuki; Kawase, Makoto; Taketa, Shin; Fukunaga, Kenji

2015-08-01

Foxtail millet shows variation in positive phenol color reaction (Phr) and negative Phr in grains, but predominant accessions of this crop are negative reaction type, and the molecular genetic basis of the Phr reaction remains unresolved. In this article, we isolated polyphenol oxidase (PPO) gene responsible for Phr using genome sequence information and investigated molecular genetic basis of negative Phr and crop evolution of foxtail millet. First of all, we searched for PPO gene homologs in a foxtail millet genome database using a rice PPO gene as a query and successfully found three copies of the PPO gene. One of the PPO gene homologs on chromosome 7 showed the highest similarity with PPO genes expressed in hulls (grains) of other cereal species including rice, wheat, and barley and was designated as Si7PPO. Phr phenotypes and Si7PPO genotypes completely co-segregated in a segregating population. We also analyzed the genetic variation conferring negative Phr reaction. Of 480 accessions of the landraces investigated, 87 (18.1 %) showed positive Phr and 393 (81.9 %) showed negative Phr. In the 393 Phr negative accessions, three types of loss-of-function Si7PPO gene were predominant and independently found in various locations. One of them has an SNP in exon 1 resulting in a premature stop codon and was designated as stop codon type, another has an insertion of a transposon (Si7PPO-TE1) in intron 2 and was designated as TE1-insertion type, and the other has a 6-bp duplication in exon 3 resulting in the duplication of 2 amino acids and was designated as 6-bp duplication type. As a rare variant of the stop codon type, one accession additionally has an insertion of a transposon, Si7PPO-TE2, in intron 2 and was designated as "stop codon +TE2 insertion type". The geographical distribution of accessions with positive Phr and those with three major types of negative Phr was also investigated. Accessions with positive Phr were found in subtropical and tropical regions at frequencies of ca. 25-67 % and those with negative Phr were broadly found in Europe and Asia. The stop codon type was found in 285 accessions and was broadly distributed in Europe and Asia, whereas the TE-1 insertion type was found in 99 accessions from Europe and Asia but was not found in India. The 6-bp duplication type was found in only 8 accessions from Nansei Islands (Okinawa Prefecture) of Japan. We also analyzed Phr in the wild ancestor and concluded that the negative Phr type was likely to have originated after domestication of foxtail millet. It was also implied that negative Phr of foxtail millet arose by multiple independent loss of function of PPO gene through dispersal because of some advantages under some environmental conditions and human selection as in rice and barley.

Color-deficient cone mosaics associated with Xq28 opsin mutations: A stop codon versus gene deletions

PubMed Central

Wagner-Schuman, Melissa; Neitz, Jay; Rha, Jungtae; Williams, David R.; Neitz, Maureen; Carroll, Joseph

2010-01-01

Our understanding of the etiology of red-green color vision defects is evolving. While missense mutations within the long- (L-) and middle-wavelength sensitive (M-) photopigments and gross rearrangements within the L/M-opsin gene array are commonly associated with red-green defects, recent work using adaptive optics retinal imaging has shown that different genotypes can have distinct consequences for the cone mosaic. Here we examined the cone mosaic in red-green color deficient individuals with multiple X-chromosome opsin genes that encode L opsin, as well as individuals with a single X-chromosome opsin gene that encodes L opsin and a single patient with a novel premature termination codon in his M-opsin gene and a normal L-opsin gene. We observed no difference in cone density between normal trichomats and multiple or single gene dichromats. In addition, we demonstrate different phenotypic effects of a nonsense mutation versus the previously described deleterious polymorphism, (LIAVA), both of which differ from multiple and single gene dichromats. Our results help refine the relationship between opsin genotype and cone photoreceptor mosaic phenotype. PMID:20854834

Novel mutation of OCRL1 in Lowe syndrome.

PubMed

Liu, Ting; Yue, Zhihui; Wang, Haiyan; Tong, Huajuan; Sun, Liangzhong

2015-01-01

Lowe syndrome is a rare, X-linked recessive genetic disease with multi-organ involvement. The pathogenic gene is OCRL1. The authors analyzed the OCRL1 mutation and summarized the clinical features of a Chinese child with Lowe syndrome. The patient is a 3 year 7 mo-old boy. He presented with hypotonia at birth and gradually presented with bilateral congenital cataracts, psychomotor retardation, hypophosphatemic rickets and renal tubular function disorder. Sequence analysis of OCRL1 revealed a novel insertion mutation, c.2367insA (p. Ala813X), in exon 22. This mutation was suspected to cause a premature stop codon of OCRL1 and truncation of the OCRL1 protein. His mother, who carried a heterozygous mutation, had no sign of abnormality.

Compound heterozygous HAX1 mutations in a Swedish patient with severe congenital neutropenia and no neurodevelopmental abnormalities.

PubMed

Carlsson, Göran; Elinder, Göran; Malmgren, Helena; Trebinska, Alicja; Grzybowska, Ewa; Dahl, Niklas; Nordenskjöld, Magnus; Fadeel, Bengt

2009-12-01

Kostmann disease or severe congenital neutropenia (SCN) is an autosomal recessive disorder of neutrophil production. Homozygous HAX1 mutations were recently identified in SCN patients belonging to the original family in northern Sweden described by Kostmann. Moreover, recent studies have suggested an association between neurological dysfunction and HAX1 deficiency. Here we describe a patient with a compound heterozygous HAX1 mutation consisting of a nonsense mutation (c.568C > T, p.Glu190X) and a frame-shift mutation (c.91delG, p.Glu31LysfsX54) resulting in a premature stop codon. The patient has a history of neutropenia and a propensity for infections, but has shown no signs of neurodevelopmental abnormalities.

Does RecA have a role in Borrelia recurrentis?

PubMed

Cutler, S J; Rinky, I J; Bonilla, E M

2011-02-01

Genomic sequencing of two relapsing fever spirochaetes showed truncation of recA in Borrelia recurrentis, but not in Borrelia duttonii. RecA has an important role among bacteria; we investigated whether this characteristic was representative of B. recurrentis, or an artefact following in vitro cultivation. We sequenced recA directly from samples of patient with louse-borne relapsing fever (B. recurrentis) or tick-borne relapsing fever (B. duttonii). We confirmed the premature stop codon in seven louse-borne relapsing fever samples, and its absence from three tick-borne relapsing fever samples. Furthermore, specific signature polymorphisms were found that could differentiate between these highly similar spirochaetes. © 2010 The Authors. Journal Compilation © 2010 European Society of Clinical Microbiology and Infectious Diseases.

A Novel Loss-of-Sclerostin Function Mutation in a First Egyptian Family with Sclerosteosis

PubMed Central

Fayez, Alaaeldin; Aglan, Mona; Esmaiel, Nora; El Zanaty, Taher; Abdel Kader, Mohamed; El Ruby, Mona

2015-01-01

Sclerosteosis is a rare autosomal recessive condition characterized by increased bone density. Mutations in SOST gene coding for sclerostin are linked to sclerosteosis. Two Egyptian brothers with sclerosteosis and their apparently normal consanguineous parents were included in this study. Clinical evaluation and genomic sequencing of the SOST gene were performed followed by in silico analysis of the resulting variation. A novel homozygous frameshift mutation in the SOST gene, characterized as one nucleotide cytosine insertion that led to premature stop codon and loss of functional sclerostin, was identified in the two affected brothers. Their parents were heterozygous for the same mutation. To our knowledge this is the first Egyptian study of sclerosteosis and SOST gene causing mutation. PMID:25984533

Molecular Epidemiology of Colistin-Resistant, Carbapenemase-Producing Klebsiella pneumoniae in Serbia from 2013 to 2016

PubMed Central

Novović, Katarina; Trudić, Anika; Brkić, Snežana; Vasiljević, Zorica; Kojić, Milan; Medić, Deana; Ćirković, Ivana

2017-01-01

ABSTRACT Twenty-seven colistin-resistant, carbapenemase-producing Klebsiella pneumoniae isolates were identified from hospitals in Serbia. All isolates were blaCTX-M-15 positive; ST101, ST888, ST437, ST336, and ST307 were blaOXA-48 positive; and ST340 was blaNDM-1 positive. ST307 had an insertion, and ST336 had a premature stop codon in the mgrB gene. Amino acid substitutions were detected in PmrAB of isolates ST101, ST888, ST336, and ST307. The mcr-1 and mcr-2 were not detected. An increase in phoP, phoQ, and pmrK gene transcription was detected for all sequence types. PMID:28242665

A novel homozygous stop-codon mutation in human HFE responsible for nonsense-mediated mRNA decay.

PubMed

Padula, Maria Carmela; Martelli, Giuseppe; Larocca, Marilena; Rossano, Rocco; Olivieri, Attilio

2014-09-01

HFE-hemochromatosis (HH) is an autosomal disease characterized by excessive iron absorption. Homozygotes for H63D variant, and still less H63D heterozygotes, generally do not express HH phenotype. The data collected in our previous study in the province of Matera (Basilicata, Italy) underlined that some H63D carriers showed altered iron metabolism, without additional factors. In this study, we selected a cohort of 10/22 H63D carriers with severe biochemical iron overload (BIO). Additional analysis was performed for studying HFE exons, exon-intron boundaries, and untranslated regions (UTRs) by performing DNA extraction, PCR amplification and sequencing. The results showed a novel substitution (NM_000410.3:c.847C>T) in a patient exon 4 (GenBankJQ478433); it introduces a premature stop-codon (PTC). RNA extraction and reverse-transcription were also performed. Quantitative real-time PCR was carried out for verifying if our aberrant mRNA is targeted for nonsense-mediated mRNA decay (NMD); we observed that patient HFE mRNA was expressed much less than calibrator, suggesting that the mutated HFE protein cannot play its role in iron metabolism regulation, resulting in proband BIO. Our finding is the first evidence of a variation responsible for a PTC in iron cycle genes. The genotype-phenotype correlation observed in our cases could be related to the additional mutation. Copyright © 2014 Elsevier Inc. All rights reserved.

A Single Base Pair Mutation Encoding a Premature Stop Codon in the MIS type II receptor is Responsible for Canine Persistent Müllerian Duct Syndrome

PubMed Central

Wu, Xiufeng; Wan, Shengqin; Pujar, Shashikant; Haskins, Mark E.; Schlafer, Donald H.; Lee, Mary M.; Meyers-Wallen, Vicki N.

2008-01-01

Müllerian Inhibiting Substance (MIS), a secreted glycoprotein in the Transforming Growth Factor-beta (TGF-beta) family of growth factors, mediates regression of the Müllerian ducts during embryonic sex differentiation in males. In Persistent Müllerian Duct Syndrome (PMDS), rather than undergoing involution, the Müllerian ducts persist in males, giving rise to the uterus, Fallopian tubes, and upper vagina. Genetic defects in MIS or its receptor (MISRII) have been identified in patients with PMDS. The phenotype in the canine model of PMDS derived from the miniature schnauzer breed is strikingly similar to that of human patients. In this model, PMDS is inherited as a sex-limited autosomal recessive trait. Previous studies indicated that a defect in the MIS receptor or its downstream signaling pathway was likely to be causative of the canine syndrome. In this study the canine PMDS phenotype and clinical sequelae are described in detail. Affected and unaffected members of this pedigree are genotyped, identifying a single base pair substitution in MISRII that introduces a stop codon in exon 3. The homozygous mutation terminates translation at 80 amino acids, eliminating much of the extracellular domain and the entire transmembrane and intracellular signaling domains. Findings in this model may enable insights to be garnered from correlation of detailed clinical descriptions with molecular defects, which are not otherwise possible in the human syndrome. PMID:18723470

WES homozygosity mapping in a recessive form of Charcot-Marie-Tooth neuropathy reveals intronic GDAP1 variant leading to a premature stop codon.

PubMed

Masingue, Marion; Perrot, Jimmy; Carlier, Robert-Yves; Piguet-Lacroix, Guenaelle; Latour, Philippe; Stojkovic, Tanya

2018-05-01

Charcot-Marie-Tooth disease (CMT) refers to a group of clinically and genetically heterogeneous inherited neuropathies. Ganglioside-induced differentiation-associated protein 1 GDAP1-related CMT has been reported in an autosomal dominant or recessive form in patients presenting either axonal or demyelinating neuropathy. We report two Sri Lankan sisters born to consanguineous parents and presenting with a severe axonal sensorimotor neuropathy. The early onset of the disease, the distal and proximal weakness and atrophy leading to major disability, along with areflexia, and, most notably, vocal cord and diaphragm paralysis were highly evocative of a GDAP1-related CMT. However, sequencing of the coding regions of the gene was normal. Whole-exome sequencing (WES) was performed and revealed that the largest region of homozygosity was around GDAP1 with several variants, mostly in non-coding regions. In view of the high clinical suspicion of GDAP1 gene involvement, we examined the variants in this gene and this, along with functional studies, allowed us to identify an alternative splicing site revealing a cryptic in-frame stop codon in intron 4 responsible for a severe loss of wild-type GDAP1. This work is the first to describe a deleterious mutation in GDAP1 gene outside of coding sequences or intronic junctions and emphasizes the importance of interpreting molecular analysis, and in particular WES results, in light of the clinical and electrophysiological phenotype.

New mutations of DAX-1 genes in two Japanese patients with X-linked congenital adrenal hypoplasia and hypogonadotropic hypogonadism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yanase, Toshihiko; Takayanagi, Ryoichi; Oba, Koichi

Congenital adrenal hypoplasia, an X-linked disorder, is characterized by primary adrenal insufficiency and frequent association with hypogonadotropic hypogonadism. The X-chromosome gene DAX-1 has been most recently identified and shown to be responsible for this disorder. We analyzed the DAX-1 genes of two unrelated Japanese patients with congenital adrenal hypoplasia and hypogonadotropic hypogonadism by using PCR amplification of genomic DNA and its complete exonic sequencing. In a family containing several affected individuals, the proband male patient had a stop codon (TGA) in place of tryptophan (TGG) at amino acid position 171. As expected, his mother was a heterozygous carrier for themore » mutation, whereas his father and unaffected brother did not carry this mutation. In another male patient with noncontributory family history, sequencing revealed a 1-bp (T) deletion at amino acid position 280, leading to a frame shift and, subsequently a premature stop codon at amino acid position 371. The presence of this mutation in the patients` genome was further confirmed by digestion of genomic PCR product with MspI created by this mutation. Family studies using MspI digestion of genomic PCR products revealed that neither parent of this individual carried the mutation. These results clearly indicate that congenital adrenal hypoplasia and hypogonadotropic hypogonadism result from not only inherited but also de novo mutation in the DAX-1 gene. 31 refs., 4 figs., 2 tabs.« less

Dural ectasia and FBN1 mutation screening of 40 patients with Marfan syndrome and related disorders: role of dural ectasia for the diagnosis.

PubMed

Attanasio, Monica; Pratelli, Elisa; Porciani, Maria Cristina; Evangelisti, Lucia; Torricelli, Elena; Pellicanò, Giannantonio; Abbate, Rosanna; Gensini, Gian Franco; Pepe, Guglielmina

2013-07-01

Marfan syndrome is an autosomal dominant disorder of connective tissue caused by mutations in the gene encoding fibrillin-1 (FBN1), a matrix component of microfibrils. Dural ectasia, i.e. enlargement of the neural canal mainly located in the lower lumbar and sacral region, frequently occurs in Marfan patients. The aim of our study was to investigate the role of dural ectasia in raising the diagnosis of Marfan syndrome and its association with FBN1 mutations. We studied 40 unrelated patients suspected for MFS, who underwent magnetic resonance imaging searching for dural ectasia. In all of them FBN1 gene analysis was also performed. Thirty-seven patients resulted affected by Marfan syndrome according to the '96 Ghent criteria; in 30 of them the diagnosis was confirmed when revaluated by the recently revised criteria (2010). Thirty-six patients resulted positive for dural ectasia. The degree of dural ectasia was grade 1 in 19 patients, grade 2 in 11 patients, and grade 3 in 6 patients. In 7 (24%) patients, the presence of dural ectasia allowed to reach a positive score for systemic feature criterion. Twenty-four patients carried an FBN1 mutation, that were represented by 13 missense (54%), and 11 (46%) mutations generating a premature termination codon (PTC, frameshifts and stop codons). No mutation was detected in the remaining 16 (6 patients with MFS and 10 with related disorders according to revised Ghent criteria). The prevalence of severe (grade 2 and grade 3) involvement of dura mater was higher in patients harbouring premature termination codon (PTC) mutations than those carrying missense-mutations (8/11 vs 2/13, P = 0.0111). Our data emphasizes the importance of dural ectasia screening to reach the diagnosis of Marfan syndrome especially when it is uncertain and indicates an association between PTC mutations and severe dural ectasia in Marfan patients. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Nonsense mutations in the alcohol dehydrogenase gene of Drosophila melanogaster correlate with an abnormal 3' end processing of the corresponding pre-mRNA.

PubMed Central

Brogna, S

1999-01-01

From bacteria to mammals, mutations that generate premature termination codons have been shown to result in the reduction in the abundance of the corresponding mRNA. In mammalian cells, more often than not, the reduction happens while the RNA is still associated with the nucleus. Here, it is reported that mutations in the alcohol dehydrogenase gene (Adh) of Drosophila melanogaster that generate premature termination codons lead to reduced levels of cytoplasmic and nuclear mRNA. Unexpectedly, it has been found that the poly(A) tails of Adh mRNAs and pre-mRNAs that carry a premature termination codon are longer than in the wild-type transcript. The more 5' terminal the mutation is, the longer is the poly(A) tail of the transcript. These findings suggest that the integrity of the coding region may be required for accurate mRNA 3' end processing. PMID:10199572

GATA3 mutation in a family with hypoparathyroidism, deafness and renal dysplasia syndrome.

PubMed

Zhu, Zi-Yang; Zhou, Qiao-Li; Ni, Shi-Ning; Gu, Wei

2014-08-01

The hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome is an autosomal dominant disorder primarily caused by GATA3 gene mutation. We report here a case that both of a Chinese boy and his father had HDR syndrome which caused by a novel mutation of GATA3. Polymerase chain reaction and DNA sequencing was performed to detect the exons of the GATA3 gene for mutation analysis. Sequence analysis of GATA3 revealed a heterozygous nonsense mutation in this family: a mutation of GATA3 at exon 2 (c.515C >A) that resulted in a premature stop at codon 172 (p.S172X) with a loss of two zinc finger domains. We identified a novel nonsense mutation which will expand the spectrum of HDR-associated GATA3 mutations.

A new nonsense mutation in the NF1 gene with neurofibromatosis-Noonan syndrome phenotype.

PubMed

Yimenicioğlu, Sevgi; Yakut, Ayten; Karaer, Kadri; Zenker, Martin; Ekici, Arzu; Carman, Kürşat Bora

2012-12-01

Neurofibromatosis-Noonan syndrome is a rare autosomal dominant disorder which combines neurofibromatosis type 1 (NF1) features with Noonan syndrome. NF1 gene mutations are reported in the majority of these patients. Sequence analysis of the established genes for Noonan syndrome revealed no mutation; a heterozygous NF1 point mutation c.7549C>T in exon 51, creating a premature stop codon (p.R2517X), had been demonstrated. Neurofibromatosis-Noonan syndrome recently has been considered a subtype of NF1 and caused by different NF1 mutations. We report the case of a 14-year-old boy with neurofibromatosis type 1 with Noonan-like features, who complained of headache with triventricular hydrocephaly and a heterozygous NF1 point mutation c.7549C>T in exon 51.

Emerging genetic therapies to treat Duchenne muscular dystrophy

PubMed Central

Nelson, Stanley F.; Crosbie, Rachelle H.; Miceli, M. Carrie; Spencer, Melissa J.

2010-01-01

Purpose of review Duchenne muscular dystrophy is a progressive muscle degenerative disease caused by dystrophin mutations. The purpose of this review is to highlight two emerging therapies designed to repair the primary genetic defect, called `exon skipping' and `nonsense codon suppression'. Recent findings A drug, PTC124, was identified that suppresses nonsense codon translation termination. PTC124 can lead to restoration of some dystrophin expression in human Duchenne muscular dystrophy muscles with mutations resulting in premature stops. Two drugs developed for exon skipping, PRO051 and AVI-4658, result in the exclusion of exon 51 from mature mRNA. They can restore the translational reading frame to dystrophin transcripts from patients with a particular subset of dystrophin gene deletions and lead to some restoration of dystrophin expression in affected boys' muscle in vivo. Both approaches have concluded phase I trials with no serious adverse events. Summary These novel therapies that act to correct the primary genetic defect of dystrophin deficiency are among the first generation of therapies tailored to correct specific mutations in humans. Thus, they represent paradigm forming approaches to personalized medicine with the potential to lead to life changing treatment for those affected by Duchenne muscular dystrophy. PMID:19745732

Mutation at Tyrosine in AMLRY (GILRY Like) Motif of Yeast eRF1 on Nonsense Codons Suppression and Binding Affinity to eRF3

PubMed Central

Akhmaloka; Susilowati, Prima Endang; Subandi; Madayanti, Fida

2008-01-01

Termination translation in Saccharomyces cerevisiae is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. Two regions in human eRF1, position at 281-305 and position at 411-415, were proposed to be involved on the interaction to eRF3. In this study we have constructed and characterized yeast eRF1 mutant at position 410 (correspond to 415 human eRF1) from tyrosine to serine residue resulting eRF1(Y410S). The mutations did not affect the viability and temperature sensitivity of the cell. The stop codons suppression of the mutant was analyzed in vivo using PGK-stop codon-LACZ gene fusion and showed that the suppression of the mutant was significantly increased in all of codon terminations. The suppression on UAG codon was the highest increased among the stop codons by comparing the suppression of the wild type respectively. In vitro interaction between eRF1 (mutant and wild type) to eRF3 were carried out using eRF1-(His)6 and eRF1(Y410S)-(His)6 expressed in Escherichia coli and indigenous Saccharomyces cerevisiae eRF3. The results showed that the binding affinity of eRF1(Y410S) to eRF3 was decreased up to 20% of the wild type binding affinity. Computer modeling analysis using Swiss-Prot and Amber version 9.0 programs revealed that the overall structure of eRF1(Y410S) has no significant different with the wild type. However, substitution of tyrosine to serine triggered the structural change on the other motif of C-terminal domain of eRF1. The data suggested that increasing stop codon suppression and decreasing of the binding affinity of eRF1(Y410S) were probably due to the slight modification on the structure of the C-terminal domain. PMID:18463713

Termination and read-through proteins encoded by genome segment 9 of Colorado tick fever virus.

PubMed

Mohd Jaafar, Fauziah; Attoui, Houssam; De Micco, Philippe; De Lamballerie, Xavier

2004-08-01

Genome segment 9 (Seg-9) of Colorado tick fever virus (CTFV) is 1884 bp long and contains a large open reading frame (ORF; 1845 nt in length overall), although a single in-frame stop codon (at nt 1052-1054) reduces the ORF coding capacity by approximately 40 %. However, analyses of highly conserved RNA sequences in the vicinity of the stop codon indicate that it belongs to a class of 'leaky terminators'. The third nucleotide positions in codons situated both before and after the stop codon, shows the highest variability, suggesting that both regions are translated during virus replication. This also suggests that the stop signal is functionally leaky, allowing read-through translation to occur. Indeed, both the truncated 'termination' protein and the full-length 'read-through' protein (VP9 and VP9', respectively) were detected in CTFV-infected cells, in cells transfected with a plasmid expressing only Seg-9 protein products, and in the in vitro translation products from undenatured Seg-9 ssRNA. The ratios of full-length and truncated proteins generated suggest that read-through may be down-regulated by other viral proteins. Western blot analysis of infected cells and purified CTFV showed that VP9 is a structural component of the virion, while VP9' is a non-structural protein.

Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in α-spectrin–deficient red cells

PubMed Central

Robledo, Raymond F.; Lambert, Amy J.; Birkenmeier, Connie S.; Cirlan, Marius V.; Cirlan, Andreea Flavia M.; Campagna, Dean R.; Lux, Samuel E.

2010-01-01

Five spontaneous, allelic mutations in the α-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph1J, sph2J, sph2BC, sphDem). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph3J, a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sphIhj, a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent β-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph4J, a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, β-adducin. The severity of anemia in sph4J indicates that the highly conserved cysteine residue at the C-terminus of α-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3. PMID:20056793

Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in alpha-spectrin-deficient red cells.

PubMed

Robledo, Raymond F; Lambert, Amy J; Birkenmeier, Connie S; Cirlan, Marius V; Cirlan, Andreea Flavia M; Campagna, Dean R; Lux, Samuel E; Peters, Luanne L

2010-03-04

Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph(1J), sph(2J), sph(2BC), sph(Dem)). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph(3J), a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent beta-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J), a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.

Using Student Writing and Lexical Analysis to Reveal Student Thinking about the Role of Stop Codons in the Central Dogma

PubMed Central

Prevost, Luanna B.; Smith, Michelle K.; Knight, Jennifer K.

2016-01-01

Previous work has shown that students have persistent difficulties in understanding how central dogma processes can be affected by a stop codon mutation. To explore these difficulties, we modified two multiple-choice questions from the Genetics Concept Assessment into three open-ended questions that asked students to write about how a stop codon mutation potentially impacts replication, transcription, and translation. We then used computer-assisted lexical analysis combined with human scoring to categorize student responses. The lexical analysis models showed high agreement with human scoring, demonstrating that this approach can be successfully used to analyze large numbers of student written responses. The results of this analysis show that students’ ideas about one process in the central dogma can affect their thinking about subsequent and previous processes, leading to mixed models of conceptual understanding. PMID:27909016

Rous Sarcoma Virus RNA Stability Element Inhibits Deadenylation of mRNAs with Long 3′UTRs

PubMed Central

Balagopal, Vidya; Beemon, Karen L.

2017-01-01

All retroviruses use their full-length primary transcript as the major mRNA for Group-specific antigen (Gag) capsid proteins. This results in a long 3′ untranslated region (UTR) downstream of the termination codon. In the case of Rous sarcoma virus (RSV), there is a 7 kb 3′UTR downstream of the gag terminator, containing the pol, env, and src genes. mRNAs containing long 3′UTRs, like those with premature termination codons, are frequently recognized by the cellular nonsense-mediated mRNA decay (NMD) machinery and targeted for degradation. To prevent this, RSV has evolved an RNA stability element (RSE) in the RNA immediately downstream of the gag termination codon. This 400-nt RNA sequence stabilizes premature termination codons (PTCs) in gag. It also stabilizes globin mRNAs with long 3′UTRs, when placed downstream of the termination codon. It is not clear how the RSE stabilizes the mRNA and prevents decay. We show here that the presence of RSE inhibits deadenylation severely. In addition, the RSE also impairs decapping (DCP2) and 5′-3′ exonucleolytic (XRN1) function in knockdown experiments in human cells. PMID:28763028

Complete mitochondrial genome of the Yellownose skate: Zearaja chilensis (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Lee, Youn-Ho

2016-01-01

The complete sequence of mitochondrial DNA of a Yellownose skate, Zearaja chilensis was determined for the first time. It is 16,909 bp in length covering 2 rRNA, 22 tRNA and 13 protein coding genes with the identical gene order and structure as those of other Rajidae species. The nucleotide of L-strand is composed of low G (14.3%), and slightly high A + T (58.9%) nucleotides. The strong codon usage bias against the use of G (6.0%) is found at the third codon positions. Twelve of the 13 protein coding genes use ATG as the start codon while COX1 starts with GTG. As for the stop codon, only ND4 shows an incomplete stop codon TA. This is the first report of the mitogenome for a species in the genus Zearaja, providing a valuable source of genetic information on the evolution of the family Rajidae and the genus Zearaja as well as for establishment of a sustainble fishery management plan of the species.

Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

PubMed

Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

2016-02-19

Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.

PubMed

Gan, Qinglei; Fan, Chenguang

2017-11-01

Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNA Lys , tRNA Tyr , and tRNA Gln (CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2016 Elsevier B.V. All rights reserved.

Origin of noncoding DNA sequences: molecular fossils of genome evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Naora, H.; Miyahara, K.; Curnow, R.N.

The total amount of noncoding sequences on chromosomes of contemporary organisms varies significantly from species to species. The authors propose a hypothesis for the origin of these noncoding sequences that assumes that (i) an approx. 0.55-kilobase (kb)-long reading frame composed the primordial gene and (ii) a 20-kb-long single-stranded polynucleotide is the longest molecule (as a genome) that was polymerized at random and without a specific template in the primordial soup/cell. The statistical distribution of stop codons allows examination of the probability of generating reading frames of approx. 0.55 kb in this primordial polynucleotide. This analysis reveals that with three stopmore » codons, a run of at least 0.55-kb equivalent length of nonstop codons would occur in 4.6% of 20-kb-long polynucleotide molecules. They attempt to estimate the total amount of noncoding sequences that would be present on the chromosomes of contemporary species assuming that present-day chromosomes retain the prototype primordial genome structure. Theoretical estimates thus obtained for most eukaryotes do not differ significantly from those reported for these specific organisms, with only a few exceptions. Furthermore, analysis of possible stop-codon distributions suggests that life on earth would not exist, at least in its present form, had two or four stop codons been selected early in evolution.« less

Rules of UGA-N decoding by near-cognate tRNAs and analysis of readthrough on short uORFs in yeast.

PubMed

Beznosková, Petra; Gunišová, Stanislava; Valášek, Leoš Shivaya

2016-03-01

The molecular mechanism of stop codon recognition by the release factor eRF1 in complex with eRF3 has been described in great detail; however, our understanding of what determines the difference in termination efficiencies among various stop codon tetranucleotides and how near-cognate (nc) tRNAs recode stop codons during programmed readthrough in Saccharomyces cerevisiae is still poor. Here, we show that UGA-C as the only tetranucleotide of all four possible combinations dramatically exacerbated the readthrough phenotype of the stop codon recognition-deficient mutants in eRF1. Since the same is true also for UAA-C and UAG-C, we propose that the exceptionally high readthrough levels that all three stop codons display when followed by cytosine are partially caused by the compromised sampling ability of eRF1, which specifically senses cytosine at the +4 position. The difference in termination efficiencies among the remaining three UGA-N tetranucleotides is then given by their varying preferences for nc-tRNAs. In particular, UGA-A allows increased incorporation of Trp-tRNA whereas UGA-G and UGA-C favor Cys-tRNA. Our findings thus expand the repertoire of general decoding rules by showing that the +4 base determines the preferred selection of nc-tRNAs and, in the case of cytosine, it also genetically interacts with eRF1. Finally, using an example of the GCN4 translational control governed by four short uORFs, we also show how the evolution of this mechanism dealt with undesirable readthrough on those uORFs that serve as the key translation reinitiation promoting features of the GCN4 regulation, as both of these otherwise counteracting activities, readthrough versus reinitiation, are mediated by eIF3. © 2016 Beznosková et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology.

PubMed

Jones, Jennifer E; Long, Kristin M; Whitmore, Alan C; Sanders, Wes; Thurlow, Lance R; Brown, Julia A; Morrison, Clayton R; Vincent, Heather; Peck, Kayla M; Browning, Christian; Moorman, Nathaniel; Lim, Jean K; Heise, Mark T

2017-11-14

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4 + T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes significant outbreaks of viral arthralgia. Studies with CHIKV and other alphaviruses demonstrated that the opal termination codon within nsP3 is highly conserved. However, some strains of CHIKV and other alphaviruses contain mutations in the opal termination codon. These mutations alter the virulence of related alphaviruses in mammalian and mosquito hosts. Here, we report that a clinical isolate of a CHIKV strain from the recent outbreak in the Caribbean islands contains a mixture of viruses encoding either the opal termination codon or an arginine mutation. Mutating the opal stop codon to an arginine residue attenuates CHIKV-induced disease in a mouse model. Compared to infection with the opal-containing parental virus, infection with the arginine mutant causes limited swelling and inflammation, as well as dampened recruitment of immune mediators of pathology, including CD4 + T cells and NK cells. We propose that the opal termination codon plays an essential role in the induction of severe CHIKV disease. Copyright © 2017 Jones et al.

Thought stopping and supportive therapy can reduce postpartum blues and anxiety parents of premature babies.

PubMed

Laela, Sri; Anna Keliat, Budi; Mustikasari

2018-02-01

The parents of premature baby tend to be at risk undergoing postpartum blues and anxiety. It is due to many problems faced by postpartum mother of premature baby. This research is aim to identifying influence of thought stopping and supportive therapy of postpartum blues and anxiety parents of premature babies. This is quantitative with quasi-experiment with control group pretest-posttest design and consecutive sampling method. Sample in this research are 62 postpartum mothers of premature babies in perinatal NICU (neonatal intensive care unit). The results show that there is significant decrease of postpartum blues and anxiety (p value = 0.000) in the group that was treated by using nursing intervention, thought stopping and supportive therapy and greater significant decrease than the group that was only treated by nursing intervention. Thought stopping and supportive therapy are able to decrease postpartum blues and anxiety parents of premature babies. Copyright © 2018 Elsevier España, S.L.U. All rights reserved.

Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology

PubMed Central

Jones, Jennifer E.; Long, Kristin M.; Whitmore, Alan C.; Sanders, Wes; Thurlow, Lance R.; Brown, Julia A.; Morrison, Clayton R.; Vincent, Heather; Browning, Christian; Moorman, Nathaniel; Lim, Jean K.

2017-01-01

ABSTRACT Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo. Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4+ T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication. PMID:29138302

Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster

PubMed Central

Dunn, Joshua G; Foo, Catherine K; Belletier, Nicolette G; Gavis, Elizabeth R; Weissman, Jonathan S

2013-01-01

Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution. DOI: http://dx.doi.org/10.7554/eLife.01179.001 PMID:24302569

Mutation in the 5' alternatively spliced region of the XNP/ATR-X gene causes Chudley-Lowry syndrome.

PubMed

Abidi, Fatima E; Cardoso, Carlos; Lossi, Anne-Marie; Lowry, Robert Brian; Depetris, Danielle; Mattéi, Marie-Geneviève; Lubs, Herbert A; Stevenson, Roger E; Fontes, Michel; Chudley, Albert E; Schwartz, Charles E

2005-02-01

The Chudley-Lowry syndrome (ChLS, MIM 309490) is an X-linked recessive condition characterized by moderate to severe mental retardation, short stature, mild obesity, hypogonadism, and distinctive facial features characterized by depressed nasal bridge, anteverted nares, inverted-V-shaped upper lip, and macrostomia. The original Chudley-Lowry family consists of three affected males in two generations. Linkage analysis had localized the gene to a large interval, Xp21-Xq26 and an obligate carrier was demonstrated to have highly skewed X inactivation. The combination of the clinical phenotype, consistent with that of the patients with ATR-X syndrome, the skewed X-inactivation pattern in a carrier female, as well as the mapping interval including band Xq13.3, prompted us to consider the XNP/ATR-X gene being involved in this syndrome. Using RT-PCR analysis, we screened the entire XNP/ATR-X gene and found a mutation in exon 2 (c.109C > T) giving rise to a stop codon at position 37 (p.R37X). Western blot and immunocytochemical analyses using a specific monoclonal antibody directed against XNP/ATR-X showed the protein to be present in lymphoblastoid cells from one affected male, despite the premature stop codon. To explain these discordant results, we further analyzed the 5' region of the XNP/ATR-X gene and found three alternative transcripts, which differ in the presence or absence of exon 2, and the length of exon 1. Our data suggest that ChLS is allelic to the ATR-X syndrome with its less severe phenotype being due to the presence of some XNP/ATR-X protein.

Tissue- and case-specific retention of intron 40 in mature dystrophin mRNA.

PubMed

Nishida, Atsushi; Minegishi, Maki; Takeuchi, Atsuko; Niba, Emma Tabe Eko; Awano, Hiroyuki; Lee, Tomoko; Iijima, Kazumoto; Takeshima, Yasuhiro; Matsuo, Masafumi

2015-06-01

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), comprises 79 exons that show multiple alternative splicing events. Intron retention, a type of alternative splicing, may control gene expression. We examined intron retention in dystrophin introns by reverse-transcription PCR from skeletal muscle, focusing on the nine shortest (all <1000 bp), because these are more likely to be retained. Only one, intron 40, was retained in mRNA; sequencing revealed insertion of a complete intron 40 (851 nt) between exons 40 and 41. The intron 40 retention product accounted for 1.2% of the total product but had a premature stop codon at the fifth intronic codon. Intron 40 retention was most strongly observed in the kidney (36.6%) and was not obtained from the fetal liver, lung, spleen or placenta. This indicated that intron retention is a tissue-specific event whose level varies among tissues. In two DMD patients, intron 40 retention was observed in one patient but not in the other. Examination of splicing regulatory factors revealed that intron 40 had the highest guanine-cytosine content of all examined introns in a 30-nt segment at its 3' end. Further studies are needed to clarify the biological role of intron 40-retained dystrophin mRNA.

Whole Genome Sequencing Reveals a De Novo SHANK3 Mutation in Familial Autism Spectrum Disorder

PubMed Central

Nemirovsky, Sergio I.; Córdoba, Marta; Zaiat, Jonathan J.; Completa, Sabrina P.; Vega, Patricia A.; González-Morón, Dolores; Medina, Nancy M.; Fabbro, Mónica; Romero, Soledad; Brun, Bianca; Revale, Santiago; Ogara, María Florencia; Pecci, Adali; Marti, Marcelo; Vazquez, Martin; Turjanski, Adrián; Kauffman, Marcelo A.

2015-01-01

Introduction Clinical genomics promise to be especially suitable for the study of etiologically heterogeneous conditions such as Autism Spectrum Disorder (ASD). Here we present three siblings with ASD where we evaluated the usefulness of Whole Genome Sequencing (WGS) for the diagnostic approach to ASD. Methods We identified a family segregating ASD in three siblings with an unidentified cause. We performed WGS in the three probands and used a state-of-the-art comprehensive bioinformatic analysis pipeline and prioritized the identified variants located in genes likely to be related to ASD. We validated the finding by Sanger sequencing in the probands and their parents. Results Three male siblings presented a syndrome characterized by severe intellectual disability, absence of language, autism spectrum symptoms and epilepsy with negative family history for mental retardation, language disorders, ASD or other psychiatric disorders. We found germline mosaicism for a heterozygous deletion of a cytosine in the exon 21 of the SHANK3 gene, resulting in a missense sequence of 5 codons followed by a premature stop codon (NM_033517:c.3259_3259delC, p.Ser1088Profs*6). Conclusions We reported an infrequent form of familial ASD where WGS proved useful in the clinic. We identified a mutation in SHANK3 that underscores its relevance in Autism Spectrum Disorder. PMID:25646853

Whole genome sequencing reveals a de novo SHANK3 mutation in familial autism spectrum disorder.

PubMed

Nemirovsky, Sergio I; Córdoba, Marta; Zaiat, Jonathan J; Completa, Sabrina P; Vega, Patricia A; González-Morón, Dolores; Medina, Nancy M; Fabbro, Mónica; Romero, Soledad; Brun, Bianca; Revale, Santiago; Ogara, María Florencia; Pecci, Adali; Marti, Marcelo; Vazquez, Martin; Turjanski, Adrián; Kauffman, Marcelo A

2015-01-01

Clinical genomics promise to be especially suitable for the study of etiologically heterogeneous conditions such as Autism Spectrum Disorder (ASD). Here we present three siblings with ASD where we evaluated the usefulness of Whole Genome Sequencing (WGS) for the diagnostic approach to ASD. We identified a family segregating ASD in three siblings with an unidentified cause. We performed WGS in the three probands and used a state-of-the-art comprehensive bioinformatic analysis pipeline and prioritized the identified variants located in genes likely to be related to ASD. We validated the finding by Sanger sequencing in the probands and their parents. Three male siblings presented a syndrome characterized by severe intellectual disability, absence of language, autism spectrum symptoms and epilepsy with negative family history for mental retardation, language disorders, ASD or other psychiatric disorders. We found germline mosaicism for a heterozygous deletion of a cytosine in the exon 21 of the SHANK3 gene, resulting in a missense sequence of 5 codons followed by a premature stop codon (NM_033517:c.3259_3259delC, p.Ser1088Profs*6). We reported an infrequent form of familial ASD where WGS proved useful in the clinic. We identified a mutation in SHANK3 that underscores its relevance in Autism Spectrum Disorder.

Introduction of translation stop codons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity

USGS Publications Warehouse

Garver, K.A.; Conway, C.M.; Kurath, G.

2006-01-01

A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine. ?? Springer Science+Business Media, Inc. 2006.

Confirmation of RAX gene involvement in human anophthalmia

PubMed Central

Lequeux, L.; Rio, Marlène; Vigouroux, Armelle; Titeux, Matthias; Etchevers, Heather; Malecaze, François; Chassaing, Nicolas; Calvas, Patrick

2008-01-01

Microphthalmia and anophthalmia are at the severe end of the spectrum of abnormalities in ocular development. Mutations in several genes have been involved in syndromic and non-syndromic anophthalmia. Previously, RAX recessive mutations were implicated in a single patient with right anophthalmia and left microphthalmia and sclerocornea. Here, we report the findings of novel compound heterozygous RAX mutations in a child with bilateral anophthalmia. Both mutations are located in exon 3. c.664delT is a frameshifting deletion predicted to introduce a premature stop codon (p.Ser222ArgfsX62), and c.909 C>G is a nonsense mutation with similar consequences (p.Tyr303X). This is the second report of a patient with anophthalmia caused by RAX mutations. These findings confirm that RAX plays a major role in the early stages of eye development and is involved in human anophthalmia. PMID:18783408

Confirmation of RAX gene involvement in human anophthalmia.

PubMed

Lequeux, L; Rio, M; Vigouroux, A; Titeux, M; Etchevers, H; Malecaze, F; Chassaing, N; Calvas, P

2008-10-01

Microphthalmia and anophthalmia are at the severe end of the spectrum of abnormalities in ocular development. Mutations in several genes have been involved in syndromic and non-syndromic anophthalmia. Previously, RAX recessive mutations were implicated in a single patient with right anophthalmia, left microphthalmia and sclerocornea. In this study, we report the findings of novel compound heterozygous RAX mutations in a child with bilateral anophthalmia. Both mutations are located in exon 3. c.664delT is a frameshifting deletion predicted to introduce a premature stop codon (p.Ser222ArgfsX62), and c.909C>G is a nonsense mutation with similar consequences (p.Tyr303X). This is the second report of a patient with anophthalmia caused by RAX mutations. These findings confirm that RAX plays a major role in the early stages of eye development and is involved in human anophthalmia.

Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja

PubMed Central

Lozano, Roberto; Ponce, Olga; Ramirez, Manuel; Mostajo, Nelly; Orjeda, Gisella

2012-01-01

The majority of disease resistance (R) genes identified to date in plants encode a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain containing protein. Additional domains such as coiled-coil (CC) and TOLL/interleukin-1 receptor (TIR) domains can also be present. In the recently sequenced Solanum tuberosum group phureja genome we used HMM models and manual curation to annotate 435 NBS-encoding R gene homologs and 142 NBS-derived genes that lack the NBS domain. Highly similar homologs for most previously documented Solanaceae R genes were identified. A surprising ∼41% (179) of the 435 NBS-encoding genes are pseudogenes primarily caused by premature stop codons or frameshift mutations. Alignment of 81.80% of the 577 homologs to S. tuberosum group phureja pseudomolecules revealed non-random distribution of the R-genes; 362 of 470 genes were found in high density clusters on 11 chromosomes. PMID:22493716

Hearing loss in a patient with the myopathic form of mitochondrial DNA depletion syndrome and a novel mutation in the TK2 gene.

PubMed

Martí, Ramon; Nascimento, Andrés; Colomer, Jaume; Lara, Mari C; López-Gallardo, Ester; Ruiz-Pesini, Eduardo; Montoya, Julio; Andreu, Antoni L; Briones, Paz; Pineda, Mercè

2010-08-01

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a devastating disorder of infancy caused by a significant reduction of the number of copies of mitochondrial DNA in one or more tissues. We report a Spanish patient with the myopathic form of MDS, harboring two mutations in the thymidine kinase 2 gene (TK2): a previously reported deletion (p.K244del) and a novel nucleotide duplication in the exon 2, generating a frameshift and premature stop codon. Sensorineural hearing loss was a predominant symptom in the patient and a novel feature of MDS due to TK2 mutations. The patient survived up to the age of 8.5 y, which confirms that survival above the age of 5 y is not infrequent in patients with MDS due to TK2 deficiency.

The SNP g.1311T>C associated with the absence of β-casein in goat milk influences CSN2 promoter activity.

PubMed

Cosenza, G; Iannaccone, M; Pico, B A; Ramunno, L; Capparelli, R

2016-10-01

Quantitative individual differences in the amount of β-casein in goat milk are determined by at least nine alleles. In particular, two alleles (CSN2(0) and CSN2(01) ) are associated with an undetectable amount of this protein in milk. The CSN2(01) allele is characterized by a single nucleotide substitution at position 373 of the seventh exon (AJ011018:g.8915C>T), responsible for the formation of a premature stop codon at the 182 position. Herein, we report the contribution of the SNP g.1311T>C, which demonstrates a linkage with the SNP AJ011018:g.8915C>T, to the promoter transcriptional activity. Particularly, we indicate that the nucleotide C at position 1311 negatively affects the promoter activity of the CSN2 gene. © 2016 Stichting International Foundation for Animal Genetics.

A novel Werner Syndrome mutation: pharmacological treatment by read-through of nonsense mutations and epigenetic therapies

PubMed Central

Agrelo, Ruben; Sutz, Miguel Arocena; Setien, Fernando; Aldunate, Fabian; Esteller, Manel; Da Costa, Valeria; Achenbach, Ricardo

2015-01-01

Werner Syndrome (WS) is a rare inherited disease characterized by premature aging and increased propensity for cancer. Mutations in the WRN gene can be of several types, including nonsense mutations, leading to a truncated protein form. WRN is a RecQ family member with both helicase and exonuclease activities, and it participates in several cell metabolic pathways, including DNA replication, DNA repair, and telomere maintenance. Here, we reported a novel homozygous WS mutation (c.3767 C > G) in 2 Argentinian brothers, which resulted in a stop codon and a truncated protein (p.S1256X). We also observed increased WRN promoter methylation in the cells of patients and decreased messenger WRN RNA (WRN mRNA) expression. Finally, we showed that the read-through of nonsense mutation pharmacologic treatment with both aminoglycosides (AGs) and ataluren (PTC-124) in these cells restores full-length protein expression and WRN functionality. PMID:25830902

Balancing Selection of a Frame-Shift Mutation in the MRC2 Gene Accounts for the Outbreak of the Crooked Tail Syndrome in Belgian Blue Cattle

PubMed Central

Li, Wanbo; Dive, Marc; Tamma, Nico; Michaux, Charles; Druet, Tom; Huijbers, Ivo J.; Isacke, Clare M.; Coppieters, Wouter; Georges, Michel; Charlier, Carole

2009-01-01

We herein describe the positional identification of a 2-bp deletion in the open reading frame of the MRC2 receptor causing the recessive Crooked Tail Syndrome in cattle. The resulting frame-shift reveals a premature stop codon that causes nonsense-mediated decay of the mutant messenger RNA, and the virtual absence of functional Endo180 protein in affected animals. Cases exhibit skeletal anomalies thought to result from impaired extracellular matrix remodeling during ossification, and as of yet unexplained muscular symptoms. We demonstrate that carrier status is very significantly associated with desired characteristics in the general population, including enhanced muscular development, and that the resulting heterozygote advantage caused a selective sweep which explains the unexpectedly high frequency (25%) of carriers in the Belgian Blue Cattle Breed. PMID:19779552

Ribosome rearrangements at the onset of translational bypassing

PubMed Central

Agirrezabala, Xabier; Samatova, Ekaterina; Klimova, Mariia; Zamora, Miguel; Gil-Carton, David; Rodnina, Marina V.; Valle, Mikel

2017-01-01

Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNAGly to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap. PMID:28630923

Methylation of class I translation termination factors: structural and functional aspects.

PubMed

Graille, Marc; Figaro, Sabine; Kervestin, Stéphanie; Buckingham, Richard H; Liger, Dominique; Heurgué-Hamard, Valérie

2012-07-01

During protein synthesis, release of polypeptide from the ribosome occurs when an in frame termination codon is encountered. Contrary to sense codons, which are decoded by tRNAs, stop codons present in the A-site are recognized by proteins named class I release factors, leading to the release of newly synthesized proteins. Structures of these factors bound to termination ribosomal complexes have recently been obtained, and lead to a better understanding of stop codon recognition and its coordination with peptidyl-tRNA hydrolysis in bacteria. Release factors contain a universally conserved GGQ motif which interacts with the peptidyl-transferase centre to allow peptide release. The Gln side chain from this motif is methylated, a feature conserved from bacteria to man, suggesting an important biological role. However, methylation is catalysed by completely unrelated enzymes. The function of this motif and its post-translational modification will be discussed in the context of recent structural and functional studies. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

The complete mitochondrial genome of the Longnose skate: Raja rhina (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Lee, Youn-Ho

2015-02-01

The complete sequence of mitochondrial DNA of a longnose skate, Raja rhina was determined for the first time. It is 16,910 bp in length containing 2 rRNA, 22 tRNA and 13 protein coding genes with the same gene order and structure as those of other Rajidae species. The nucleotide of L-strand is composed of 30.1% A, 27.2% C, 28.5% T and 14.2% G, showing a slight A + T bias. The G is the least used base and markedly lower at the third codon position (5.4%). Twelve of the 13 protein coding genes use ATG as their start codon while the COX1 starts with GTG. As for stop codon, only ND4 shows incomplete stop codon TA. This mitogenome is the first report for a species of the genus Raja, and providing a valuable resource of genetic information for understanding the phylogenetic relationship and the evolution of the genus Raja as well as the family, Rajidae.

A second mutation in the type II procollagen gene (COL2AI) causing stickler syndrome (arthro-ophthalmopathy) is also a premature termination codon.

PubMed Central

Ahmad, N N; McDonald-McGinn, D M; Zackai, E H; Knowlton, R G; LaRossa, D; DiMascio, J; Prockop, D J

1993-01-01

Genetic linkage analyses suggest that mutations in type II collagen may be responsible for Stickler syndrome, or arthro-ophthalmopathy (AO), in many families. In the present study oligonucleotide primers were developed to amplify and directly sequence eight of the first nine exons of the gene for type II procollagen (COL2A1). Analysis of the eight exons in 10 unrelated probands with AO revealed that one had a single-base mutation in one allele that changed the codon of -CGA- for arginine at amino acid position alpha 1-9 in exon 7 to a premature termination signal for translation. The second mutation found to cause AO was, therefore, similar to the first in that both created premature termination signals in the COL2A1 gene. Since mutations producing premature termination signals have not previously been detected in genes for fibrillar collagens, the results raise the possibility that such mutations in the COL2A1 gene are a common cause of AO. Images Figure 2 Figure 3 PMID:8434604

Evolution of CCL11: genetic characterization in lagomorphs and evidence of positive and purifying selection in mammals.

PubMed

Neves, Fabiana; Abrantes, Joana; Esteves, Pedro J

2016-07-01

The interactions between chemokines and their receptors are crucial for differentiation and activation of inflammatory cells. CC chemokine ligand 11 (CCL11) binds to CCR3 and to CCR5 that in leporids underwent gene conversion with CCR2. Here, we genetically characterized CCL11 in lagomorphs (leporids and pikas). All lagomorphs have a potentially functional CCL11, and the Pygmy rabbit has a mutation in the stop codon that leads to a longer protein. Other mammals also have mutations at the stop codon that result in proteins with different lengths. By employing maximum likelihood methods, we observed that, in mammals, CCL11 exhibits both signatures of purifying and positive selection. Signatures of purifying selection were detected in sites important for receptor binding and activation. Of the three sites detected as under positive selection, two were located close to the stop codon. Our results suggest that CCL11 is functional in all lagomorphs, and that the signatures of purifying and positive selection in mammalian CCL11 probably reflect the protein's biological roles. © The Author(s) 2016.

Immune-escape mutations and stop-codons in HBsAg develop in a large proportion of patients with chronic HBV infection exposed to anti-HBV drugs in Europe.

PubMed

Colagrossi, Luna; Hermans, Lucas E; Salpini, Romina; Di Carlo, Domenico; Pas, Suzan D; Alvarez, Marta; Ben-Ari, Ziv; Boland, Greet; Bruzzone, Bianca; Coppola, Nicola; Seguin-Devaux, Carole; Dyda, Tomasz; Garcia, Federico; Kaiser, Rolf; Köse, Sukran; Krarup, Henrik; Lazarevic, Ivana; Lunar, Maja M; Maylin, Sarah; Micheli, Valeria; Mor, Orna; Paraschiv, Simona; Paraskevis, Dimitros; Poljak, Mario; Puchhammer-Stöckl, Elisabeth; Simon, François; Stanojevic, Maja; Stene-Johansen, Kathrine; Tihic, Nijaz; Trimoulet, Pascale; Verheyen, Jens; Vince, Adriana; Lepej, Snjezana Zidovec; Weis, Nina; Yalcinkaya, Tülay; Boucher, Charles A B; Wensing, Annemarie M J; Perno, Carlo F; Svicher, Valentina

2018-06-01

HBsAg immune-escape mutations can favor HBV-transmission also in vaccinated individuals, promote immunosuppression-driven HBV-reactivation, and increase fitness of drug-resistant strains. Stop-codons can enhance HBV oncogenic-properties. Furthermore, as a consequence of the overlapping structure of HBV genome, some immune-escape mutations or stop-codons in HBsAg can derive from drug-resistance mutations in RT. This study is aimed at gaining insight in prevalence and characteristics of immune-associated escape mutations, and stop-codons in HBsAg in chronically HBV-infected patients experiencing nucleos(t)ide analogues (NA) in Europe. This study analyzed 828 chronically HBV-infected European patients exposed to ≥ 1 NA, with detectable HBV-DNA and with an available HBsAg-sequence. The immune-associated escape mutations and the NA-induced immune-escape mutations sI195M, sI196S, and sE164D (resulting from drug-resistance mutation rtM204 V, rtM204I, and rtV173L) were retrieved from literature and examined. Mutations were defined as an aminoacid substitution with respect to a genotype A or D reference sequence. At least one immune-associated escape mutation was detected in 22.1% of patients with rising temporal-trend. By multivariable-analysis, genotype-D correlated with higher selection of ≥ 1 immune-associated escape mutation (OR[95%CI]:2.20[1.32-3.67], P = 0.002). In genotype-D, the presence of ≥ 1 immune-associated escape mutations was significantly higher in drug-exposed patients with drug-resistant strains than with wild-type virus (29.5% vs 20.3% P = 0.012). Result confirmed by analysing drug-naïve patients (29.5% vs 21.2%, P = 0.032). Strong correlation was observed between sP120T and rtM204I/V (P < 0.001), and their co-presence determined an increased HBV-DNA. At least one NA-induced immune-escape mutation occurred in 28.6% of patients, and their selection correlated with genotype-A (OR[95%CI]:2.03[1.32-3.10],P = 0.001). Finally, stop-codons are present in 8.4% of patients also at HBsAg-positions 172 and 182, described to enhance viral oncogenic-properties. Immune-escape mutations and stop-codons develop in a large fraction of NA-exposed patients from Europe. This may represent a potential threat for horizontal and vertical HBV transmission also to vaccinated persons, and fuel drug-resistance emergence.

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs

PubMed Central

Gubbens, Jacob; Kim, Soo Jung; Yang, Zhongying; Johnson, Arthur E.; Skach, William R.

2010-01-01

Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNACys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies. PMID:20581130

Identification of a de novo mutation of SOX10 in a Chinese patient with Waardenburg syndrome type IV.

PubMed

Liang, Fenghe; Zhao, Min; Fan, Lynn; Zhang, Hongyan; Shi, Yang; Han, Rui; Qu, Chunyan

2016-12-01

Waardenburg syndrome is a rare genetic disorder, characterized by the association of sensorineural hearing loss and pigmentation abnormalities. Four subtypes have been classified. The present study aimed to analyze the clinical feature and investigate the genetic cause for a Chinese case of Waardenburg type IV (WS4). The patient and his family members were subjected to mutation detection in the candidate gene SOX10 by Sanger sequencing. The patient has the clinical features of WS4, including sensorineural hearing loss, bright blue irides, premature graying of the hair and Hirschsprung disease. A novel heterozygous frameshift mutation, c.752_753ins7 (p.Gly252Alafs*31) in the exon 5 of SOX10 was detected in the patient, but not found in the unaffected family members and 100 normal controls. This mutation results in a premature stop codon 31 amino acid downstream. The novel mutation c.752_753ins7 (p.Gly252Alafs*31) arose de novo and was considered as the cause of WS4 in the proband. This study further characterized the molecular complexity of WS4 and provided a clinical case for genotype-phenotype correlation studies of different phenotypes caused by SOX10 mutations. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Molecular diagnosis of analbuminemia: a new case caused by a nonsense mutation in the albumin gene.

PubMed

Dagnino, Monica; Caridi, Gianluca; Haenni, Ueli; Duss, Adrian; Aregger, Fabienne; Campagnoli, Monica; Galliano, Monica; Minchiotti, Lorenzo

2011-01-01

Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin (ALB). We report here a new case diagnosed in a 45 years old man of Southwestern Asian origin, living in Switzerland, on the basis of his low ALB concentration (0.9 g/L) in the absence of renal or gastrointestinal protein loss, or liver dysfunction. The clinical diagnosis was confirmed by a mutational analysis of the albumin (ALB) gene, carried out by single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), and DNA sequencing. This screening of the ALB gene revealed that the proband is homozygous for two mutations: the insertion of a T in a stretch of eight Ts spanning positions c.1289 + 23-c.1289 + 30 of intron 10 and a c.802 G > T transversion in exon 7. Whereas the presence of an additional T in the poly-T tract has no direct deleterious effect, the latter nonsense mutation changes the codon GAA for Glu244 to the stop codon TAA, resulting in a premature termination of the polypeptide chain. The putative protein product would have a length of only 243 amino acid residues instead of the normal 585 found in the mature serum albumin, but no evidence for the presence in serum of such a truncated polypeptide chain could be obtained by two dimensional electrophoresis and western blotting analysis.

Pyviko: an automated Python tool to design gene knockouts in complex viruses with overlapping genes.

PubMed

Taylor, Louis J; Strebel, Klaus

2017-01-07

Gene knockouts are a common tool used to study gene function in various organisms. However, designing gene knockouts is complicated in viruses, which frequently contain sequences that code for multiple overlapping genes. Designing mutants that can be traced by the creation of new or elimination of existing restriction sites further compounds the difficulty in experimental design of knockouts of overlapping genes. While software is available to rapidly identify restriction sites in a given nucleotide sequence, no existing software addresses experimental design of mutations involving multiple overlapping amino acid sequences in generating gene knockouts. Pyviko performed well on a test set of over 240,000 gene pairs collected from viral genomes deposited in the National Center for Biotechnology Information Nucleotide database, identifying a point mutation which added a premature stop codon within the first 20 codons of the target gene in 93.2% of all tested gene-overprinted gene pairs. This shows that Pyviko can be used successfully in a wide variety of contexts to facilitate the molecular cloning and study of viral overprinted genes. Pyviko is an extensible and intuitive Python tool for designing knockouts of overlapping genes. Freely available as both a Python package and a web-based interface ( http://louiejtaylor.github.io/pyViKO/ ), Pyviko simplifies the experimental design of gene knockouts in complex viruses with overlapping genes.

A novel germline inactivating mutation in the CASR gene in an Italian kindred affected by familial hypocalciuric hypercalcemia.

PubMed

Falchetti, Alberto; Gozzini, Alessia; Terranegra, Annalisa; Soldati, Laura; Vezzoli, Giuseppe; Leoncini, Gigliola; Giusti, Francesca; Franceschelli, Francesco; Masi, Laura; Tanini, Annalisa; Cavalli, Loredana; Brandi, Maria Luisa

2012-05-01

Familial hypocalciuric hypercalcemia (FHH) syndrome is a rare benign condition, inherited as an autosomal dominant trait, in which inactivating mutations of the calcium-sensing receptor (CASR) gene affects the body's ability to regulate calcium homeostasis. Its outcome is featured by increased levels of serum calcium, moderate hypophosphatemia, and inadequately normal or elevated circulating parathyroid hormone levels. Affected patients are mostly asymptomatic and do not benefit from surgical resection of their mildly enlarged parathyroids. We evaluated for hypercalcemia an Italian family that was identified via a young adult male proband referred to our center for parathyroidectomy. The patients and the family members were evaluated both biochemically and genetically as suspected FHH subjects. An in vitro functional study was performed by site-directed mutagenesis, and CASR activity was monitored by measuring intracellular calcium ([Ca(2)(+)](i)). The patient had a novel germline heterozygous CASR mutation (c.361_364GATT; p.D121del/fsX122). The mutation caused a premature stop codon at codon 122, exiting a truncated protein. The biochemical phenotype of all family members carrying the heterozygous deletion was concordant with classic FHH syndrome. Our findings confirm the role of CASR gene mutational analysis to offer a valuable addition for the recognition of FHH in hypercalcemic patients not yet characterized for a positive familial history of hypercalcemia, the only condition that identifies CASR gene mutations in hypercalcemia.

Functional Analyses of c.2268dup in Thyroid Peroxidase Gene Associated with Goitrous Congenital Hypothyroidism

PubMed Central

Harun, Fatimah; Jalaludin, Muhammad Yazid; Lim, Chor Yin; Ng, Khoon Leong

2014-01-01

The c.2268dup mutation in thyroid peroxidase (TPO) gene was reported to be a founder mutation in Taiwanese patients with dyshormonogenetic congenital hypothyroidism (CH). The functional impact of the mutation is not well documented. In this study, homozygous c.2268dup mutation was detected in two Malaysian-Chinese sisters with goitrous CH. Normal and alternatively spliced TPO mRNA transcripts were present in thyroid tissues of the two sisters. The abnormal transcript contained 34 nucleotides originating from intron 12. The c.2268dup is predicted to generate a premature termination codon (PTC) at position 757 (p.Glu757X). Instead of restoring the normal reading frame, the alternatively spliced transcript has led to another stop codon at position 740 (p.Asp739ValfsX740). The two PTCs are located at 116 and 201 nucleotides upstream of the exons 13/14 junction fulfilling the requirement for a nonsense-mediated mRNA decay (NMD). Quantitative RT-PCR revealed an abundance of unidentified transcripts believed to be associated with the NMD. TPO enzyme activity was not detected in both patients, even though a faint TPO band of about 80 kD was present. In conclusion, the c.2268dup mutation leads to the formation of normal and alternatively spliced TPO mRNA transcripts with a consequential loss of TPO enzymatic activity in Malaysian-Chinese patients with goitrous CH. PMID:24745015

The complete mitochondrial genome of the Korean skate: Hongeo koreana (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Kim, Sung; Kim, Choong-Gon; Lee, Youn-Ho

2014-12-01

The complete mitochondrial genome of the Korean skate, Hongeo koreana, the sole member of its genus, is investigated for the first time. The genome consists of 16,906 bp in length including 2 rRNA, 22 tRNA and 13 protein coding genes with the same gene order and structure of the genome as those of other Rajidae species. The overall nucleotide composition of the L-strand is A = 29.8%, C = 27.9%, T = 27.9% and G = 14.3%, showing a high A + T bias. The anti-G bias (6.0%) is more significant in the third codon position. Twelve of the 13 protein-coding genes use ATG as their start codon while the COX1 gene starts with GTG. For stop codon, ND3 and ND4 genes show incomplete stop codon T. The mitogenome sequence of H. koreana will provide important information on the evolution and the phylogenetic relation of the genus Hongeo in relation to the other genera of the family Rajidae.

Identification of a novel exonic mutation at -13 from 5' splice site causing exon skipping in a girl with mitochondrial acetoacetyl-coenzyme A thiolase deficiency.

PubMed Central

Fukao, T; Yamaguchi, S; Wakazono, A; Orii, T; Hoganson, G; Hashimoto, T

1994-01-01

We identified a novel exonic mutation which causes exon skipping in the mitochondrial acetoacetyl-CoA thiolase (T2) gene from a girl with T2 deficiency (GK07). GK07 is a compound heterozygote; the maternal allele has a novel G to T transversion at position 1136 causing Gly379 to Val substitution (G379V) of the T2 precursor. In case of in vivo expression analysis, cells transfected with this mutant cDNA showed no evidence of restored T2 activity. The paternal allele was associated with exon 8 skipping at the cDNA level. At the gene level, a C to T transition causing Gln272 to termination codon (Q272STOP) was identified within exon 8, 13 bp from the 5' splice site of intron 8 in the paternal allele. The mRNA with Q272STOP could not be detected in GK07 fibroblasts, presumably because pre-mRNA with Q272STOP was unstable because of the premature termination. In vivo splicing experiments revealed that the exonic mutation caused partial skipping of exon 8. This substitution was thought to alter the secondary structure of T2 pre-mRNA around exon 8 and thus impede normal splicing. The role of exon sequences in the splicing mechanism is indicated by the exon skipping which occurred with an exonic mutation. Images PMID:7907600

Somatic mutations in cancer: Stochastic versus predictable.

PubMed

Gold, Barry

2017-02-01

The origins of human cancers remain unclear except for a limited number of potent environmental mutagens, such as tobacco and UV light, and in rare cases, familial germ line mutations that affect tumor suppressor genes or oncogenes. A significant component of cancer etiology has been deemed stochastic and correlated with the number of stem cells in a tissue, the number of times the stem cells divide and a low incidence of random DNA polymerase errors that occur during each cell division. While somatic mutations occur during each round of DNA replication, mutations in cancer driver genes are not stochastic. Out of a total of 2843 codons, 1031 can be changed to stop codons by a single base substitution in the tumor suppressor APC gene, which is mutated in 76% of colorectal cancers (CRC). However, the nonsense mutations, which comprise 65% of all the APC driver mutations in CRC, are not random: 43% occur at Arg CGA codons, although they represent <3% of the codons. In TP53, CGA codons comprise <3% of the total 393 codons but they account for 72% and 39% of the mutations in CRC and ovarian cancer OVC, respectively. This mutation pattern is consistent with the kinetically slow, but not stochastic, hydrolytic deamination of 5-methylcytosine residues at specific methylated CpG sites to afford T·G mismatches that lead to C→T transitions and stop codons at CGA. Analysis of nonsense mutations in CRC, OVC and a number of other cancers indicates the need to expand the predictable risk factors for cancer to include, in addition to random polymerase errors, the methylation status of gene body CGA codons in tumor suppressor genes. Copyright © 2017. Published by Elsevier B.V.

A common periodic table of codons and amino acids.

PubMed

Biro, J C; Benyó, B; Sansom, C; Szlávecz, A; Fördös, G; Micsik, T; Benyó, Z

2003-06-27

A periodic table of codons has been designed where the codons are in regular locations. The table has four fields (16 places in each) one with each of the four nucleotides (A, U, G, C) in the central codon position. Thus, AAA (lysine), UUU (phenylalanine), GGG (glycine), and CCC (proline) were placed into the corners of the fields as the main codons (and amino acids) of the fields. They were connected to each other by six axes. The resulting nucleic acid periodic table showed perfect axial symmetry for codons. The corresponding amino acid table also displaced periodicity regarding the biochemical properties (charge and hydropathy) of the 20 amino acids and the position of the stop signals. The table emphasizes the importance of the central nucleotide in the codons and predicts that purines control the charge while pyrimidines determine the polarity of the amino acids. This prediction was experimentally tested.

Stop Codon Reassignment in the Wild

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ivanova, Natalia; Schwientek, Patrick; Tripp, H. James

Since the discovery of the genetic code and protein translation mechanisms (1), a limited number of variations of the standard assignment between unique base triplets (codons) and their encoded amino acids and translational stop signals have been found in bacteria and phages (2-3). Given the apparent ubiquity of the canonical genetic code, the design of genomically recoded organisms with non-canonical codes has been suggested as a means to prevent horizontal gene transfer between laboratory and environmental organisms (4). It is also predicted that genomically recoded organisms are immune to infection by viruses, under the assumption that phages and their hostsmore » must share a common genetic code (5). This paradigm is supported by the observation of increased resistance of genomically recoded bacteria to phages with a canonical code (4). Despite these assumptions and accompanying lines of evidence, it remains unclear whether differential and non-canonical codon usage represents an absolute barrier to phage infection and genetic exchange between organisms. Our knowledge of the diversity of genetic codes and their use by viruses and their hosts is primarily derived from the analysis of cultivated organisms. Advances in single-cell sequencing and metagenome assembly technologies have enabled the reconstruction of genomes of uncultivated bacterial and archaeal lineages (6). These initial findings suggest that large scale systematic studies of uncultivated microorganisms and viruses may reveal the extent and modes of divergence from the canonical genetic code operating in nature. To explore alternative genetic codes, we carried out a systematic analysis of stop codon reassignments from the canonical TAG amber, TGA opal, and TAA ochre codons in assembled metagenomes from environmental and host-associated samples, single-cell genomes of uncultivated bacteria and archaea, and a collection of phage sequences« less

Novel nonsense mutation of the endothelin-B receptor gene in a family with Waardenburg-Hirschsprung disease.

PubMed

Syrris, P; Carter, N D; Patton, M A

1999-11-05

Waardenburg syndrome (WS) comprises sensorineural hearing loss, hypopigmentation of skin and hair, and pigmentary disturbances of the irides. Four types of WS have been classified to date; in WS type IV (WS4), patients additionally have colonic aganglionosis (Hirschsprung disease, HSCR). Mutations in the endothelin-3 (EDN3), endothelin-B receptor (EDNRB), and Sox10 genes have been identified as causative for WS type IV. We screened a family with a combined WS-HSCR phenotype for mutations in the EDNRB locus using standard DNA mutation analysis and sequencing techniques. We have identified a novel nonsense mutation at codon 253 (CGA-->TGA, Arg-->STOP). This mutation leads to a premature end of the translation of EDNRB at exon 3, and it is predicted to produce a truncated and nonfunctional endothelin-B receptor. All affected relatives were heterozygous for the Arg(253)-->STOP mutation, whereas it was not observed in over 50 unrelated individuals used as controls. These data confirm the role of EDNRB in the cause of the Waardenburg-Hirschsprung syndrome and demonstrate that in WS-HSCR there is a lack of correlation between phenotype and genotype and a variable expression of disease even within the same family. Copyright 1999 Wiley-Liss, Inc.

Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion

PubMed Central

O’Donoghue, Patrick; Prat, Laure; Heinemann, Ilka U.; Ling, Jiqiang; Odoi, Keturah; Liu, Wenshe R.; Söll, Dieter

2012-01-01

Over 300 amino acids are found in proteins in nature, yet typically only 20 are genetically encoded. Reassigning stop codons and use of quadruplet codons emerged as the main avenues for genetically encoding non-canonical amino acids (NCAAs). Canonical aminoacyl-tRNAs with near-cognate anticodons also read these codons to some extent. This background suppression leads to ‘statistical protein’ that contains some natural amino acid(s) at a site intended for NCAA. We characterize near-cognate suppression of amber, opal and a quadruplet codon in common Escherichia coli laboratory strains and find that the PylRS/tRNAPyl orthogonal pair cannot completely outcompete contamination by natural amino acids. PMID:23036644

Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway

PubMed Central

Ge, Zhiyun; Quek, Bao Lin; Beemon, Karen L; Hogg, J Robert

2016-01-01

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD. DOI: http://dx.doi.org/10.7554/eLife.11155.001 PMID:26744779

Structural insights into eRF3 and stop codon recognition by eRF1

PubMed Central

Cheng, Zhihong; Saito, Kazuki; Pisarev, Andrey V.; Wada, Miki; Pisareva, Vera P.; Pestova, Tatyana V.; Gajda, Michal; Round, Adam; Kong, Chunguang; Lim, Mengkiat; Nakamura, Yoshikazu; Svergun, Dmitri I.; Ito, Koichi; Song, Haiwei

2009-01-01

Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1's M domain contacts eRF3's GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1's stimulatory effect on eRF3's GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1's putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1. PMID:19417105

The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code

PubMed Central

Hofhuis, Julia; Schueren, Fabian; Nötzel, Christopher; Lingner, Thomas; Gärtner, Jutta; Jahn, Olaf

2016-01-01

Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (extended protein) to ‘normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases. PMID:27881739

Neutral lipid-storage disease with myopathy and extended phenotype with novel PNPLA2 mutation.

PubMed

Massa, Roberto; Pozzessere, Simone; Rastelli, Emanuele; Serra, Laura; Terracciano, Chiara; Gibellini, Manuela; Bozzali, Marco; Arca, Marcello

2016-04-01

Neutral lipid-storage disease with myopathy is caused by mutations in PNPLA2, which produce skeletal and cardiac myopathy. We report a man with multiorgan neutral lipid storage and unusual multisystem clinical involvement, including cognitive impairment. Quantitative brain MRI with voxel-based morphometry and extended neuropsychological assessment were performed. In parallel, the coding sequences and intron/exon boundaries of the PNPLA2 gene were screened by direct sequencing. Neuropsychological assessment revealed global cognitive impairment, and brain MRI showed reduced gray matter volume in the temporal lobes. Molecular characterization revealed a novel homozygous mutation in exon 5 of PNPLA2 (c.714C>A), resulting in a premature stop codon (p.Cys238*). Some PNPLA2 mutations, such as the one described here, may present with an extended phenotype, including brain involvement. In these cases, complete neuropsychological testing, combined with quantitative brain MRI, may help to characterize and quantify cognitive impairment. © 2016 Wiley Periodicals, Inc.

Identification of a novel NHS mutation in a Chinese family with Nance-Horan syndrome.

PubMed

Li, Aijun; Li, Bingzhen; Wu, Lemeng; Yang, Liping; Chen, Ningning; Ma, Zhizhong

2015-04-01

To identiy the disease causing mutation in a Chinese family presenting with early-onset cataract and dental anomalies. A specific Hereditary Eye Disease Enrichment Panel (HEDEP) (personalized customization by MyGenostics, Baltimore, MD) based on targeted exome capture technology was used to collect the protein coding regions of 30 early-onset cataract associated genes, and high throughput sequencing was done with Illumina HiSeq 2000 platform. The identified variant was confirmed with Sanger sequencing. A novel deletion in exon 4 (c.852delG) of NHS gene was identified; the identified 1 bp deletion altered the reading frame and was predicted to result in a premature stop codon after the addition of twelve novel amino acid (p.S285PfsX13). This mutation co-segregated in affected males and obligate female carriers, but was absent in 100 matched controls. Our findings broaden the spectrum of NHS mutations causing Nance-Horan syndrome and phenotypic spectrum of the disease in Chinese patients.

Absence of the HLA-G*0113N allele in Amerindian populations from the Brazilian Amazon region.

PubMed

Mendes-Junior, Celso T; Castelli, Erick C; Moreau, Philippe; Simões, Aguinaldo L; Donadi, Eduardo A

2010-04-01

The HLA-G gene is predominantly expressed at the maternal-fetal interface and has been associated with maternal-fetal tolerance. The HLA-G*0113N is a null allele defined by the insertion of a premature stop codon at exon 2, observed in a single Ghanaian individual. Likewise the G*0105N allele, the occurrence of the HLA-G*0113N in a population from an area with high pathogen load suggests that the reduced HLA-G expression in G*0113N heterozygous placentas could improve the intrauterine defense against infections. The presence of the G*0113N allele here was investigated in 150 Amerindians from five isolated tribes that inhabit the Central Amazon and in 295 admixed individuals from the State of São Paulo, Southeastern Brazil, previously genotyped for HLA-G. No copy of the G*0113N null allele was found in both population samples by exon 2 sequence-based analysis, reinforcing its restricted occurrence in Africa.

Deep intronic GPR143 mutation in a Japanese family with ocular albinism

PubMed Central

Naruto, Takuya; Okamoto, Nobuhiko; Masuda, Kiyoshi; Endo, Takao; Hatsukawa, Yoshikazu; Kohmoto, Tomohiro; Imoto, Issei

2015-01-01

Deep intronic mutations are often ignored as possible causes of human disease. Using whole-exome sequencing, we analysed genomic DNAs of a Japanese family with two male siblings affected by ocular albinism and congenital nystagmus. Although mutations or copy number alterations of coding regions were not identified in candidate genes, the novel intronic mutation c.659-131 T > G within GPR143 intron 5 was identified as hemizygous in affected siblings and as heterozygous in the unaffected mother. This mutation was predicted to create a cryptic splice donor site within intron 5 and activate a cryptic acceptor site at 41nt upstream, causing the insertion into the coding sequence of an out-of-frame 41-bp pseudoexon with a premature stop codon in the aberrant transcript, which was confirmed by minigene experiments. This result expands the mutational spectrum of GPR143 and suggests the utility of next-generation sequencing integrated with in silico and experimental analyses for improving the molecular diagnosis of this disease. PMID:26061757

Deep intronic GPR143 mutation in a Japanese family with ocular albinism.

PubMed

Naruto, Takuya; Okamoto, Nobuhiko; Masuda, Kiyoshi; Endo, Takao; Hatsukawa, Yoshikazu; Kohmoto, Tomohiro; Imoto, Issei

2015-06-10

Deep intronic mutations are often ignored as possible causes of human disease. Using whole-exome sequencing, we analysed genomic DNAs of a Japanese family with two male siblings affected by ocular albinism and congenital nystagmus. Although mutations or copy number alterations of coding regions were not identified in candidate genes, the novel intronic mutation c.659-131 T > G within GPR143 intron 5 was identified as hemizygous in affected siblings and as heterozygous in the unaffected mother. This mutation was predicted to create a cryptic splice donor site within intron 5 and activate a cryptic acceptor site at 41nt upstream, causing the insertion into the coding sequence of an out-of-frame 41-bp pseudoexon with a premature stop codon in the aberrant transcript, which was confirmed by minigene experiments. This result expands the mutational spectrum of GPR143 and suggests the utility of next-generation sequencing integrated with in silico and experimental analyses for improving the molecular diagnosis of this disease.

The first de novo mutation of the connexin 32 gene associated with X linked Charcot-Marie-Tooth disease.

PubMed Central

Meggouh, F; Benomar, A; Rouger, H; Tardieu, S; Birouk, N; Tassin, J; Barhoumi, C; Yahyaoui, M; Chkili, T; Brice, A; LeGuern, E

1998-01-01

X linked Charcot-Marie-Tooth disease (CMTX) is a hereditary motor and sensory neuropathy caused by mutations in the connexin 32 gene (Cx32). Using the SSCP technique and direct sequencing of PCR amplified genomic DNA fragments of the Cx32 gene from a Moroccan patient and her relatives, we identified the first de novo mutation of the Cx32 gene, consisting of a deletion of a G residue at position 499 in the Cx32 open reading frame. This previously unreported mutation produces a frameshift at position 147 in the protein and introduces a premature stop codon (TAG) at nucleotide 643, which results in the production of a truncated Cx32 molecule. This mutation illustrates the risk of an erroneous diagnosis of autosomal recessive CMT, especially in populations where consanguineous unions are frequent, and its consequences for genetic counselling, which can be avoided by molecular analysis. Images PMID:9541114

Establishment of an Immortalized Skin Keratinocyte Cell Line Derived from the Animal Model Mastomys coucha.

PubMed

Hasche, Daniel; Stephan, Sonja; Savelyeva, Larissa; Westermann, Frank; Rösl, Frank; Vinzón, Sabrina E

2016-01-01

In the present report we describe the establishment of a spontaneous immortalized skin keratinocyte cell line derived from the skin of the multimammate rodent Mastomys coucha. These animals are used in preclinical studies for a variety of human diseases such as infections with nematodes, bacteria and papillomaviruses, especially regarding cutaneous manifestations such as non-melanoma skin cancer. Here we characterize the cells in terms of their origin and cytogenetic features. Searching for genomic signatures, a spontaneous mutation in the splicing donor sequence of Trp53 (G to A transition at the first position of intron 7) could be detected. This point mutation leads to alternative splicing and to a premature stop codon, resulting in a truncated and, in turn, undetectable form of p53, probably contributing to the process of immortalization. Mastomys coucha-derived skin keratinocytes can be used as an in vitro system to investigate molecular and immunological aspects of infectious agent interactions with their host cells.

Identification of the Bovine Arachnomelia Mutation by Massively Parallel Sequencing Implicates Sulfite Oxidase (SUOX) in Bone Development

PubMed Central

Drögemüller, Cord; Tetens, Jens; Sigurdsson, Snaevar; Gentile, Arcangelo; Testoni, Stefania; Lindblad-Toh, Kerstin; Leeb, Tosso

2010-01-01

Arachnomelia is a monogenic recessive defect of skeletal development in cattle. The causative mutation was previously mapped to a ∼7 Mb interval on chromosome 5. Here we show that array-based sequence capture and massively parallel sequencing technology, combined with the typical family structure in livestock populations, facilitates the identification of the causative mutation. We re-sequenced the entire critical interval in a healthy partially inbred cow carrying one copy of the critical chromosome segment in its ancestral state and one copy of the same segment with the arachnomelia mutation, and we detected a single heterozygous position. The genetic makeup of several partially inbred cattle provides extremely strong support for the causality of this mutation. The mutation represents a single base insertion leading to a premature stop codon in the coding sequence of the SUOX gene and is perfectly associated with the arachnomelia phenotype. Our findings suggest an important role for sulfite oxidase in bone development. PMID:20865119

A de novo mutation in KIT causes white spotting in a subpopulation of German Shepherd dogs.

PubMed

Wong, A K; Ruhe, A L; Robertson, K R; Loew, E R; Williams, D C; Neff, M W

2013-06-01

Although variation in the KIT gene is a common cause of white spotting among domesticated animals, KIT has not been implicated in the diverse white spotting observed in the dog. Here, we show that a loss-of-function mutation in KIT recapitulates the coat color phenotypes observed in other species. A spontaneous white spotting observed in a pedigree of German Shepherd dogs was mapped by linkage analysis to a single locus on CFA13 containing KIT (pairwise LOD = 15). DNA sequence analysis identified a novel 1-bp insertion in the second exon that co-segregated with the phenotype. The expected frameshift and resulting premature stop codons predicted a severely truncated c-Kit receptor with presumably abolished activity. No dogs homozygous for the mutation were recovered from multiple intercrosses (P = 0.01), suggesting the mutation is recessively embryonic lethal. These observations are consistent with the effects of null alleles of KIT in other species. © 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.

Titin truncating variants affect heart function in disease cohorts and the general population

PubMed Central

Schafer, Sebastian; de Marvao, Antonio; Adami, Eleonora; Fiedler, Lorna R; Ng, Benjamin; Khin, Ester; Rackham, Owen J L; van Heesch, Sebastiaan; Pua, Chee J; Kui, Miao; Walsh, Roddy; Tayal, Upasana; Prasad, Sanjay K; Dawes, Timothy J W; Ko, Nicole S J; Sim, David; Chan, Laura L; Chin, Calvin W L; Mazzarotto, Francesco; Barton, Paul J; Kreuchwig, Franziska; de Kleijn, Dominique P V; Totman, Teresa; Biffi, Carlo; Tee, Nicole; Rueckert, Daniel; Schneider, Valentin; Faber, Allison; Regitz-Zagrosek, Vera; Seidman, Jonathan G; Seidman, Christine E; Linke, Wolfgang A; Kovalik, Jean-Paul; O’Regan, Declan Patrick; Ware, James S; Hubner, Norbert; Cook, Stuart A

2016-01-01

Titin truncating variants (TTNtv) commonly cause dilated cardiomyopathy (DCM). TTNtv are also encountered in ~1% of the general population where they may be silent, perhaps reflecting allelic factors. To better understand TTNtv we integrated TTN allelic series, cardiac imaging and genomic data in humans and studied rat models with disparate TTNtv. In patients with DCM, TTNtv throughout TTN were significantly associated with DCM. Ribosomal profiling in rat revealed the translational footprint of premature stop codons in Ttn, TTNtv position-independent nonsense-mediated degradation of the mutant allele and a signature of perturbed cardiac metabolism. Heart physiology in rats with TTNtv was unremarkable at baseline but became impaired during cardiac stress. In healthy humans, machine-based analysis of high-resolution cardiac scans showed TTNtv to be associated with eccentric cardiac remodelling. These data show that TTNtv have molecular and physiological effects on the heart across species, with a continuum of expressivity in health and disease. PMID:27869827

Ataluren treatment of patients with nonsense mutation dystrophinopathy.

PubMed

Bushby, Katharine; Finkel, Richard; Wong, Brenda; Barohn, Richard; Campbell, Craig; Comi, Giacomo P; Connolly, Anne M; Day, John W; Flanigan, Kevin M; Goemans, Nathalie; Jones, Kristi J; Mercuri, Eugenio; Quinlivan, Ros; Renfroe, James B; Russman, Barry; Ryan, Monique M; Tulinius, Mar; Voit, Thomas; Moore, Steven A; Lee Sweeney, H; Abresch, Richard T; Coleman, Kim L; Eagle, Michelle; Florence, Julaine; Gappmaier, Eduard; Glanzman, Allan M; Henricson, Erik; Barth, Jay; Elfring, Gary L; Reha, Allen; Spiegel, Robert J; O'donnell, Michael W; Peltz, Stuart W; Mcdonald, Craig M

2014-10-01

Dystrophinopathy is a rare, severe muscle disorder, and nonsense mutations are found in 13% of cases. Ataluren was developed to enable ribosomal readthrough of premature stop codons in nonsense mutation (nm) genetic disorders. Randomized, double-blind, placebo-controlled study; males ≥ 5 years with nm-dystrophinopathy received study drug orally 3 times daily, ataluren 10, 10, 20 mg/kg (N=57); ataluren 20, 20, 40 mg/kg (N=60); or placebo (N=57) for 48 weeks. The primary endpoint was change in 6-Minute Walk Distance (6MWD) at Week 48. Ataluren was generally well tolerated. The primary endpoint favored ataluren 10, 10, 20 mg/kg versus placebo; the week 48 6MWD Δ=31.3 meters, post hoc P=0.056. Secondary endpoints (timed function tests) showed meaningful differences between ataluren 10, 10, 20 mg/kg, and placebo. As the first investigational new drug targeting the underlying cause of nm-dystrophinopathy, ataluren offers promise as a treatment for this orphan genetic disorder with high unmet medical need. Copyright © 2014 Wiley Periodicals, Inc.

Mutations of the RTEL1 Helicase in a Hoyeraal-Hreidarsson Syndrome Patient Highlight the Importance of the ARCH Domain.

PubMed

Jullien, Laurent; Kannengiesser, Caroline; Kermasson, Laetitia; Cormier-Daire, Valérie; Leblanc, Thierry; Soulier, Jean; Londono-Vallejo, Arturo; de Villartay, Jean-Pierre; Callebaut, Isabelle; Revy, Patrick

2016-05-01

The DNA helicase RTEL1 participates in telomere maintenance and genome stability. Biallelic mutations in the RTEL1 gene account for the severe telomere biology disorder characteristic of the Hoyeraal-Hreidarsson syndrome (HH). Here, we report a HH patient (P4) carrying two novel compound heterozygous mutations in RTEL1: a premature stop codon (c.949A>T, p.Lys317*) and an intronic deletion leading to an exon skipping and an in-frame deletion of 25 amino-acids (p.Ile398_Lys422). P4's cells exhibit short and dysfunctional telomeres similarly to other RTEL1-deficient patients. 3D structure predictions indicated that the p.Ile398_Lys422 deletion affects a part of the helicase ARCH domain, which lines the pore formed with the core HD and the iron-sulfur cluster domains and is highly specific of sequences from the eukaryotic XPD family members. © 2016 WILEY PERIODICALS, INC.

Hyperphenylalaninemia due to defects in tetrahydrobiopterin metabolism: Molecular characterization of mutations in 6-pyruvoyl-tetrahydropterin synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thoeny, B.; Leimbacher, W.; Blau, N.

1994-05-01

A variant type of hyperphenylalaninemia is caused by a deficiency of tetrahydrobiopterin (BH[sub 4]), the obligatory cofactor for phenylalanine hydroxylase. The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity, the second enzyme in the biosynthetic pathway for BH[sub 4]. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was found to be active when expressed in Escherichia coli. The authors now have investigated two patients for their molecular nature of this autosomal recessive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one withmore » the peripheral form, on the basis of an elevated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular analysis was performed on the patients' cultured primary skin fibroblasts. PTPS activities were found in vitro to be reduced to background activity. Direct cDNA sequence analysis using reverse transcriptase-PCR technology showed for the patient with the central form a homozygous G-to-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E.coli revealed 14% activity when compared with the wild-type enzyme. The patient with the peripheral form exhibited compound heteroxygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion ([Delta]14bp), leading to a frameshift at lysine 120 and a premature stop codon (K120[yields]Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzyme activity, whereas expression of the deletion allele [Delta]14bp exhibited no detectable activity. All three mutations result in reduced enzymatic activity when reconstituted in E. coli.« less

Use of ade1 and ade2 mutations for development of a versatile red/white colour assay of amyloid-induced oxidative stress in saccharomyces cerevisiae.

PubMed

Bharathi, Vidhya; Girdhar, Amandeep; Prasad, Archana; Verma, Meenkshi; Taneja, Vibha; Patel, Basant K

2016-12-01

Mutations in adenine biosynthesis pathway genes ADE1 and ADE2 have been conventionally used to score for prion [PSI + ] in yeast. If ade1-14 mutant allele is present, which contains a premature stop codon, [psi - ] yeast appear red on YPD medium owing to accumulation of a red intermediate compound in vacuoles. In [PSI + ] yeast, partial inactivation of the translation termination factor, Sup35 protein, owing to its amyloid aggregation allows for read-through of the ade1-14 stop codon and the yeast appears white as the red intermediate pigment is not accumulated. The red colour development in ade1 and ade2 mutant yeast requires reduced-glutathione, which helps in transport of the intermediate metabolite P-ribosylaminoimidazole carboxylate into vacuoles, which develops the red colour. Here, we hypothesize that amyloid-induced oxidative stress would deplete reduced-glutathione levels and thus thwart the development of red colour in ade1 or ade2 yeast. Indeed, when we overexpressed amyloid-forming human proteins TDP-43, Aβ-42 and Poly-Gln-103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast yielded some white colonies. Further, the white colour eventually reverted back to red upon turning off the amyloid protein's expression. Also, the aggregate-bearing yeast have increased oxidative stress and white phenotype yeast revert to red when grown on media with reducing agent. Furthermore, the red/white assay could also be emulated in ade2-1, ade2Δ, and ade1Δ mutant yeast and also in an ade1-14 mutant with erg6 gene deletion that increases cell-wall permeability. This model would be useful tool for drug-screening against general amyloid-induced oxidative stress and toxicity. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

A decamer duplication in the 3′ region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred

PubMed Central

Vidal, Ruben; Révész, Tamas; Rostagno, Agueda; Kim, Eugene; Holton, Janice L.; Bek, Toke; Bojsen-Møller, Marie; Braendgaard, Hans; Plant, Gordon; Ghiso, Jorge; Frangione, Blas

2000-01-01

Familial Danish dementia (FDD), also known as heredopathia ophthalmo-oto-encephalica, is an autosomal dominant disorder characterized by cataracts, deafness, progressive ataxia, and dementia. Neuropathological findings include severe widespread cerebral amyloid angiopathy, hippocampal plaques, and neurofibrillary tangles, similar to Alzheimer's disease. N-terminal sequence analysis of isolated leptomeningeal amyloid fibrils revealed homology to ABri, the peptide originated by a point mutation at the stop codon of gene BRI in familial British dementia. Molecular genetic analysis of the BRI gene in the Danish kindred showed a different defect, namely the presence of a 10-nt duplication (795–796insTTTAATTTGT) between codons 265 and 266, one codon before the normal stop codon 267. The decamer duplication mutation produces a frame-shift in the BRI sequence generating a larger-than-normal precursor protein, of which the amyloid subunit (designated ADan) comprises the last 34 C-terminal amino acids. This de novo-created amyloidogenic peptide, associated with a genetic defect in the Danish kindred, stresses the importance of amyloid formation as a causative factor in neurodegeneration and dementia. PMID:10781099

Characterization of the complete mitochondrial genome of the Grey-backed Shrike, Lanius tephronotus (Aves: Passeriformes): the first representative of the family Laniidae with a novel CAA stop codon at the end of cox2 gene.

PubMed

Qian, Chaoju; Yan, Xia; Guo, Zhichun; Wang, Yuanxiu; Li, Xixi; Yang, Jianke; Kan, Xianzhao

2013-08-01

The complete Grey-backed Shrike mitochondrial genome has been sequenced to be 16,820 bp in length, consisting of 37 encode genes: 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. In addition, a single control region was also observed. Compared with other reported Passeriformes mtgenome sequences, three bases CAA were detected at the end of Lanius tephronotus cox2 gene with the downstream adjacent base T. The first base of CAA probably occurred C to U transcript editing event resulting in a normal stop codon UAA.

Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation.

PubMed

Banik, Mitali; Duguid, Scott; Cloutier, Sylvie

2011-06-01

Three genes encoding fatty acid desaturase 3 (fad3a, fad3b, and a novel fad3c) were cloned from four flax genotypes varying in linolenic acid content. Real-time PCR was used to quantify expression levels of the three fad3 genes during seed development. High amounts of both fad3a and fad3b transcripts were observed and reached their peak levels at 20 days after anthesis, except for fad3a from SP2047 where only low level expression was observed throughout seed development. Transcript accumulation of the novel fad3c gene was at similar background levels. The fatty acid composition was analysed for all genotypes and stages of development and compared with the fad3 gene expression patterns. α-Linolenic acid gradually accumulated during seed development, while linoleic acid was transient and decreased in M5791, UGG5-5, and AC McDuff. In contrast, the linolenic acid present in the early stages of development nearly completely disappeared in SP2047, while linoleic acid steadily accumulated. fad3a of the low linolenic acid line SP2047 encoded a truncated protein caused by a premature stop codon resulting from a single point mutation, and the low level of transcript accumulation in this genotype is likely due to nonsense-mediated mRNA decay caused by the premature termination of translation as a result of this early stop codon. Although substantial amounts of transcript accumulation occurred with fad3b of SP2047 genotype, cloning of the gene revealed a mutation in the first histidine box causing an amino acid change. Heterologous expression in yeast of the SP2047 and UGG5-5 fad3b genes showed that the mutation in the histidine box in SP2047 caused the enzyme inactivity. Taken together, these results showed that fad3a and fad3b are responsible for linolenic acid accumulation in flax seeds but did not support a major role for the novel fad3c. These observations were further supported by phenotypic and genotypic assessment of a doubled haploid population. Expression patterns of fad3a and fad3b were highly correlated with linolenic acid accumulation during seed development, with the exception of fad3b in SP2047 whose lack of activity was caused by the histidine box mutation despite its transcript accumulation being similar to that of the fad3b of the other genotypes.

Sequence Analysis of Mitochondrial Genome of Toxascaris leonina from a South China Tiger.

PubMed

Li, Kangxin; Yang, Fang; Abdullahi, A Y; Song, Meiran; Shi, Xianli; Wang, Minwei; Fu, Yeqi; Pan, Weida; Shan, Fang; Chen, Wu; Li, Guoqing

2016-12-01

Toxascaris leonina is a common parasitic nematode of wild mammals and has significant impacts on the protection of rare wild animals. To analyze population genetic characteristics of T. leonina from South China tiger, its mitochondrial (mt) genome was sequenced. Its complete circular mt genome was 14,277 bp in length, including 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. The nucleotide composition was biased toward A and T. The most common start codon and stop codon were TTG and TAG, and 4 genes ended with an incomplete stop codon. There were 13 intergenic regions ranging 1 to 10 bp in size. Phylogenetically, T. leonina from a South China tiger was close to canine T. leonina . This study reports for the first time a complete mt genome sequence of T. leonina from the South China tiger, and provides a scientific basis for studying the genetic diversity of nematodes between different hosts.

Using Student Writing and Lexical Analysis to Reveal Student Thinking about the Role of Stop Codons in the Central Dogma.

PubMed

Prevost, Luanna B; Smith, Michelle K; Knight, Jennifer K

2016-01-01

Previous work has shown that students have persistent difficulties in understanding how central dogma processes can be affected by a stop codon mutation. To explore these difficulties, we modified two multiple-choice questions from the Genetics Concept Assessment into three open-ended questions that asked students to write about how a stop codon mutation potentially impacts replication, transcription, and translation. We then used computer-assisted lexical analysis combined with human scoring to categorize student responses. The lexical analysis models showed high agreement with human scoring, demonstrating that this approach can be successfully used to analyze large numbers of student written responses. The results of this analysis show that students' ideas about one process in the central dogma can affect their thinking about subsequent and previous processes, leading to mixed models of conceptual understanding. © 2016 L. B. Prevost et al. CBE—Life Sciences Education © 2016 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Discovery of Clinically Approved Agents That Promote Suppression of Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations.

PubMed

Mutyam, Venkateshwar; Du, Ming; Xue, Xiaojiao; Keeling, Kim M; White, E Lucile; Bostwick, J Robert; Rasmussen, Lynn; Liu, Bo; Mazur, Marina; Hong, Jeong S; Falk Libby, Emily; Liang, Feng; Shang, Haibo; Mense, Martin; Suto, Mark J; Bedwell, David M; Rowe, Steven M

2016-11-01

Premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Several agents are known to suppress PTCs but are poorly efficacious or toxic. To determine whether there are clinically available agents that elicit translational readthrough and improve CFTR function sufficient to confer therapeutic benefit to patients with CF with PTCs. Two independent screens, firefly luciferase and CFTR-mediated transepithelial chloride conductance assay, were performed on a library of 1,600 clinically approved compounds using fisher rat thyroid cells stably transfected with stop codons. Select agents were further evaluated using secondary screening assays including short circuit current analysis on primary cells from patients with CF. In addition, the effect of CFTR modulators (ivacaftor) was tested in combination with the most efficacious agents. From the primary screen, 48 agents were selected as potentially active. Following confirmatory tests in the transepithelial chloride conductance assay and prioritizing agents based on favorable pharmacologic properties, eight agents were advanced for secondary screening. Ivacaftor significantly increased short circuit current following forskolin stimulation in cells treated with pyranoradine tetraphosphate, potassium p-aminobenzoate, and escin as compared with vehicle control. Escin, an herbal agent, consistently induced readthrough activity as demonstrated by enhanced CFTR expression and function in vitro. Clinically approved drugs identified as potential readthrough agents, in combination with ivacaftor, may induce nonsense suppression to restore therapeutic levels of CFTR function. One or more agents may be suitable to advance to human testing.

Molecular Diagnosis of Analbuminemia: A New Case Caused by a Nonsense Mutation in the Albumin Gene

PubMed Central

Dagnino, Monica; Caridi, Gianluca; Haenni, Ueli; Duss, Adrian; Aregger, Fabienne; Campagnoli, Monica; Galliano, Monica; Minchiotti, Lorenzo

2011-01-01

Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin (ALB). We report here a new case diagnosed in a 45 years old man of Southwestern Asian origin, living in Switzerland, on the basis of his low ALB concentration (0.9 g/L) in the absence of renal or gastrointestinal protein loss, or liver dysfunction. The clinical diagnosis was confirmed by a mutational analysis of the albumin (ALB) gene, carried out by single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), and DNA sequencing. This screening of the ALB gene revealed that the proband is homozygous for two mutations: the insertion of a T in a stretch of eight Ts spanning positions c.1289 + 23–c.1289 + 30 of intron 10 and a c.802 G > T transversion in exon 7. Whereas the presence of an additional T in the poly-T tract has no direct deleterious effect, the latter nonsense mutation changes the codon GAA for Glu244 to the stop codon TAA, resulting in a premature termination of the polypeptide chain. The putative protein product would have a length of only 243 amino acid residues instead of the normal 585 found in the mature serum albumin, but no evidence for the presence in serum of such a truncated polypeptide chain could be obtained by two dimensional electrophoresis and western blotting analysis. PMID:22174600

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

PubMed Central

Jacinto-Loeza, Eva; Vivanco-Domínguez, Serafín; Guarneros, Gabriel; Hernández-Sánchez, Javier

2008-01-01

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3–8 codon minigenes containing AGAAGA codons before the stop codon. β-Galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, β-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 μg/μl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of β-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNAArg4. These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA. PMID:18583364

The Mitochondrial Genome and Transcriptome of the Basal Dinoflagellate Hematodinium sp.: Character Evolution within the Highly Derived Mitochondrial Genomes of Dinoflagellates

PubMed Central

Gornik, S. G.; Waller, R. F.

2012-01-01

The sister phyla dinoflagellates and apicomplexans inherited a drastically reduced mitochondrial genome (mitochondrial DNA, mtDNA) containing only three protein-coding (cob, cox1, and cox3) genes and two ribosomal RNA (rRNA) genes. In apicomplexans, single copies of these genes are encoded on the smallest known mtDNA chromosome (6 kb). In dinoflagellates, however, the genome has undergone further substantial modifications, including massive genome amplification and recombination resulting in multiple copies of each gene and gene fragments linked in numerous combinations. Furthermore, protein-encoding genes have lost standard stop codons, trans-splicing of messenger RNAs (mRNAs) is required to generate complete cox3 transcripts, and extensive RNA editing recodes most genes. From taxa investigated to date, it is unclear when many of these unusual dinoflagellate mtDNA characters evolved. To address this question, we investigated the mitochondrial genome and transcriptome character states of the deep branching dinoflagellate Hematodinium sp. Genomic data show that like later-branching dinoflagellates Hematodinium sp. also contains an inflated, heavily recombined genome of multicopy genes and gene fragments. Although stop codons are also lacking for cox1 and cob, cox3 still encodes a conventional stop codon. Extensive editing of mRNAs also occurs in Hematodinium sp. The mtDNA of basal dinoflagellate Hematodinium sp. indicates that much of the mtDNA modification in dinoflagellates occurred early in this lineage, including genome amplification and recombination, and decreased use of standard stop codons. Trans-splicing, on the other hand, occurred after Hematodinium sp. diverged. Only RNA editing presents a nonlinear pattern of evolution in dinoflagellates as this process occurs in Hematodinium sp. but is absent in some later-branching taxa indicating that this process was either lost in some lineages or developed more than once during the evolution of the highly unusual dinoflagellate mtDNA. PMID:22113794

The mitochondrial genome and transcriptome of the basal dinoflagellate Hematodinium sp.: character evolution within the highly derived mitochondrial genomes of dinoflagellates.

PubMed

Jackson, C J; Gornik, S G; Waller, R F

2012-01-01

The sister phyla dinoflagellates and apicomplexans inherited a drastically reduced mitochondrial genome (mitochondrial DNA, mtDNA) containing only three protein-coding (cob, cox1, and cox3) genes and two ribosomal RNA (rRNA) genes. In apicomplexans, single copies of these genes are encoded on the smallest known mtDNA chromosome (6 kb). In dinoflagellates, however, the genome has undergone further substantial modifications, including massive genome amplification and recombination resulting in multiple copies of each gene and gene fragments linked in numerous combinations. Furthermore, protein-encoding genes have lost standard stop codons, trans-splicing of messenger RNAs (mRNAs) is required to generate complete cox3 transcripts, and extensive RNA editing recodes most genes. From taxa investigated to date, it is unclear when many of these unusual dinoflagellate mtDNA characters evolved. To address this question, we investigated the mitochondrial genome and transcriptome character states of the deep branching dinoflagellate Hematodinium sp. Genomic data show that like later-branching dinoflagellates Hematodinium sp. also contains an inflated, heavily recombined genome of multicopy genes and gene fragments. Although stop codons are also lacking for cox1 and cob, cox3 still encodes a conventional stop codon. Extensive editing of mRNAs also occurs in Hematodinium sp. The mtDNA of basal dinoflagellate Hematodinium sp. indicates that much of the mtDNA modification in dinoflagellates occurred early in this lineage, including genome amplification and recombination, and decreased use of standard stop codons. Trans-splicing, on the other hand, occurred after Hematodinium sp. diverged. Only RNA editing presents a nonlinear pattern of evolution in dinoflagellates as this process occurs in Hematodinium sp. but is absent in some later-branching taxa indicating that this process was either lost in some lineages or developed more than once during the evolution of the highly unusual dinoflagellate mtDNA.

Metabolic characterization of a mouse deficient in all known leptin receptor isoforms.

PubMed

Osborn, Olivia; Sanchez-Alavez, Manuel; Brownell, Sara E; Ross, Brendon; Klaus, Joe; Dubins, Jeffrey; Beutler, Bruce; Conti, Bruno; Bartfai, Tamas

2010-01-01

We have characterized a newly generated mouse model of obesity, a mouse strain deficient in all five previously described leptin receptor isoforms. These transgenic mice, named the db (333)/db (333) mice, were identified from an ENU mutagenesis screen and carry a point mutation in the seventh exon of the db gene encoding the leptin receptor, resulting in a premature stop codon (Y(333)Stop) and gene product that lacks STAT signaling domains. db (333)/db (333) mice have a morbidly obese phenotype, with body weights diverging from wild type as early as 4 weeks of age (P < 0.05). To determine the contribution of the short isoforms of the leptin receptor in this metabolic phenotype, we performed an extensive metabolic characterization of the db (333)/db (333) mouse in relation to the well-characterized db/db mouse lacking only the long form of the leptin receptor. db (333)/db (333) mice have similar endocrine and metabolic parameters as previously described in other leptin receptor transgenic mice including db/db mice that lack only the long isoform of the leptin receptor. However, db (333)/db (333) mice show a subtle trend toward higher body weight and insulin levels, lower oxygen, carbon dioxide production, respiratory exchange ratio (RER), and temperature than db/db mice suggesting the short isoforms may play an additional role in energy homeostasis.

Co-expression of the Thermotoga neapolitana aglB gene with an upstream 3'-coding fragment of the malG gene improves enzymatic characteristics of recombinant AglB cyclomaltodextrinase.

PubMed

Lunina, Natalia A; Agafonova, Elena V; Chekanovskaya, Lyudmila A; Dvortsov, Igor A; Berezina, Oksana V; Shedova, Ekaterina N; Kostrov, Sergey V; Velikodvorskaya, Galina A

2007-07-01

A cluster of Thermotoga neapolitana genes participating in starch degradation includes the malG gene of sugar transport protein and the aglB gene of cyclomaltodextrinase. The start and stop codons of these genes share a common overlapping sequence, aTGAtg. Here, we compared properties of expression products of three different constructs with aglB from T. neapolitana. The first expression vector contained the aglB gene linked to an upstream 90-bp 3'-terminal region of the malG gene with the stop codon overlapping with the start codon of aglB. The second construct included the isolated coding sequence of aglB with two tandem potential start codons. The expression product of this construct in Escherichia coli had two tandem Met residues at its N terminus and was characterized by low thermostability and high tendency to aggregate. In contrast, co-expression of aglB and the 3'-terminal region of malG (the first construct) resulted in AglB with only one N-terminal Met residue and a much higher specific activity of cyclomaltodextrinase. Moreover, the enzyme expressed by such a construct was more thermostable and less prone to aggregation. The third construct was the same as the second one except that it contained only one ATG start codon. The product of its expression had kinetic and other properties similar to those of the enzyme with only one N-terminal Met residue.

Examination of food chain-derived Listeria monocytogenes strains of different serotypes reveals considerable diversity in inlA genotypes, mutability, and adaptation to cold temperatures.

PubMed

Kovacevic, Jovana; Arguedas-Villa, Carolina; Wozniak, Anna; Tasara, Taurai; Allen, Kevin J

2013-03-01

Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37°C to 4°C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.

Complete mitochondrial genome of Chocolate Pansy, Junonia iphita (Lepidoptera: Nymphalidae: Nymphalinae).

PubMed

Vanlalruati, Catherine; Mandal, Surajit De; Gurusubramanian, Guruswami; Senthil Kumar, Nachimuthu

2016-07-01

The complete mitochondrial genome of Junonia iphita was determined to be 15,433 bp in length, including 37 typical mitochondrial genes and an AT-rich region. All the protein coding genes (PCGs) are initiated by typical ATN codons, except cox1 gene that is by CGA codon. Eight genes use complete termination codon (TAA), whereas the cox1, cox2 and nad5 genes end with single T; nad4 and nad1 ends with stop codon TA. All the tRNA show secondary cloverleaf structures except trnS1 (AGN). The A + T rich region is 546 bp in length containing ATAGA motif followed by a 18 bp poly-T stretch, two microsatellite-like (TA)9 elements and 8 bp poly-A stretch immediately upstream of trnM gene.

Codes in the codons: construction of a codon/amino acid periodic table and a study of the nature of specific nucleic acid-protein interactions.

PubMed

Benyo, B; Biro, J C; Benyo, Z

2004-01-01

The theory of "codon-amino acid coevolution" was first proposed by Woese in 1967. It suggests that there is a stereochemical matching - that is, affinity - between amino acids and certain of the base triplet sequences that code for those amino acids. We have constructed a common periodic table of codons and amino acids, where the nucleic acid table showed perfect axial symmetry for codons and the corresponding amino acid table also displayed periodicity regarding the biochemical properties (charge and hydrophobicity) of the 20 amino acids and the position of the stop signals. The table indicates that the middle (2/sup nd/) amino acid in the codon has a prominent role in determining some of the structural features of the amino acids. The possibility that physical contact between codons and amino acids might exist was tested on restriction enzymes. Many recognition site-like sequences were found in the coding sequences of these enzymes and as many as 73 examples of codon-amino acid co-location were observed in the 7 known 3D structures (December 2003) of endonuclease-nucleic acid complexes. These results indicate that the smallest possible units of specific nucleic acid-protein interaction are indeed the stereochemically compatible codons and amino acids.

Complete mitochondrial genome of Germain's Peacock-Pheasant Polyplectron germaini (Aves, Galliformes, Phasianidae).

PubMed

Omeire, Destiny; Abdin, Shaunte; Brooks, Daniel M; Miranda, Hector C

2015-04-01

The Germain's Peacock-Pheasant Polyplectron germaini (Aves, Galliformes, Phasianidae) is classified as Near Threatened on the IUCN Red List. The complete mitochondrial genome of P. germaini is 16,699 bp, consisting of 13 protein-coding genes, 2 rRNA, 22 tRNA genes and 1 control region. All of the 13 protein-coding genes have ATG as start codon. Eight of the 13 protein-coding genes have TAA as stop codon.

Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

NASA Technical Reports Server (NTRS)

Kim, Chun-Hyung; Zabetian, Cyrus P.; Cubells, Joseph F.; Cho, Sonhae; Biaggioni, Italo; Cohen, Bruce M.; Robertson, David; Kim, Kwang-Soo

2002-01-01

Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

A novel non-sense mutation in the SLC2A10 gene of an arterial tortuosity syndrome patient of Kurdish origin.

PubMed

Zaidi, Syed H E; Meyer, Sascha; Peltekova, Vanya D; Lindinger, Angelika; Teebi, Ahmad S; Faiyaz-Ul-Haque, Muhammad

2009-07-01

Arterial tortuosity syndrome (ATS) is a rare autosomal recessive disorder in which patients display tortuosity of arteries in addition to hyperextensible skin, joint laxity, and other connective tissue features. This syndrome is caused by mutations in the SLC2A10 gene. In this article we describe an ATS girl of Kurdish origin who, in addition to arterial tortuosity and connective tissue features, displays stomach displacement within the thorax and bilateral hip dislocation. Clinical details of this patient have been reported previously. Sequencing of the SLC2A10 gene identified a novel homozygous non-sense c.756C>A mutation in this patient's DNA. This mutation in the SLC2A10 gene replaces a cysteine encoding codon with a stop signal. This is believed to cause a premature truncation of GLUT10 protein in this patient. We conclude that patients of Kurdish origin who display arterial tortuosity associated with skin hyperextensibility, joint hypermobility, and characteristic facial features may carry mutations in the SLC2A10 gene.

Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms

PubMed Central

Oh, Stephen T.; Simonds, Erin F.; Jones, Carol; Hale, Matthew B.; Goltsev, Yury; Gibbs, Kenneth D.; Merker, Jason D.; Zehnder, James L.; Nolan, Garry P.

2010-01-01

Dysregulated Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F–negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis. PMID:20404132

Cloning and Characterization of a Critical Regulator for Preharvest Sprouting in Wheat

PubMed Central

Liu, Shubing; Sehgal, Sunish K.; Li, Jiarui; Lin, Meng; Trick, Harold N.; Yu, Jianming; Gill, Bikram S.; Bai, Guihua

2013-01-01

Sprouting of grains in mature spikes before harvest is a major problem in wheat (Triticum aestivum) production worldwide. We cloned and characterized a gene underlying a wheat quantitative trait locus (QTL) on the short arm of chromosome 3A for preharvest sprouting (PHS) resistance in white wheat using comparative mapping and map-based cloning. This gene, designated TaPHS1, is a wheat homolog of a MOTHER OF FLOWERING TIME (TaMFT)-like gene. RNA interference-mediated knockdown of the gene confirmed that TaPHS1 positively regulates PHS resistance. We discovered two causal mutations in TaPHS1 that jointly altered PHS resistance in wheat. One GT-to-AT mutation generates a mis-splicing site, and the other A-to-T mutation creates a premature stop codon that results in a truncated nonfunctional transcript. Association analysis of a set of wheat cultivars validated the role of the two mutations on PHS resistance. The molecular characterization of TaPHS1 is significant for expediting breeding for PHS resistance to protect grain yield and quality in wheat production. PMID:23821595

Identification of Mutations Underlying 20 Inborn Errors of Metabolism in the United Arab Emirates Population

PubMed Central

Ben-Rebeh, Imen; Hertecant, Jozef L.; Al-Jasmi, Fatma A.; Aburawi, Hanan E.; Al-Yahyaee, Said A.; Al-Gazali, Lihadh

2012-01-01

Inborn errors of metabolism (IEM) are frequently encountered by physicians in the United Arab Emirates (UAE). However, the mutations underlying a large number of these disorders have not yet been determined. Therefore, the objective of this study was to identify the mutations underlying a number of IEM disorders among UAE residents from both national and expatriate families. A case series of patients from 34 families attending the metabolic clinic at Tawam Hospital were clinically evaluated, and molecular testing was carried out to determine their causative mutations. The mutation analysis was carried out at molecular genetics diagnostic laboratories. Thirty-eight mutations have been identified as responsible for twenty IEM disorders, including in the metabolism of amino acids, lipids, steroids, metal transport and mitochondrial energy metabolism, and lysosomal storage disorders. Nine of the identified mutations are novel, including two missense mutations, three premature stop codons and four splice site mutations. Mutation analysis of IEM disorders in the UAE population has an important impact on molecular diagnosis and genetic counseling for families affected by these disorders. PMID:22106832

Autosomal Recessive Oculodentodigital Dysplasia: A Case Report and Review of the Literature.

PubMed

Taşdelen, Elifcan; Durmaz, Ceren D; Karabulut, Halil G

2018-06-15

Oculodentodigital dysplasia (ODDD) is a rare condition characterized by a typical facial appearance and variable findings of the eyes, teeth, and fingers. ODDD is caused by mutations in the GJA1 gene in chromosome 6q22 and inherited in an autosomal dominant manner in the majority of the patients. However, in recent clinical reports, autosomal recessive ODDD cases due to by GJA1 mutations were also described. Here, we report on a 14-year-old boy with microphthalmia, microcornea, narrow nasal bridge, hypoplastic alae nasi, prominent columnella, hypodontia, dental caries, and partial syndactyly of the 2nd and 3rd toes. These clinical findings were concordant with the diagnosis of ODDD, and a novel homozygous mutation (c.442C>T, p.Arg148Ter) was determined in the GJA1 gene leading to a premature stop codon. His phenotypically normal parents were found to be carriers of the same mutation. This is the third family in the literature in which ODDD segregates in an autosomal recessive manner. © 2018 S. Karger AG, Basel.

Hd6, a rice quantitative trait locus involved in photoperiod sensitivity, encodes the α subunit of protein kinase CK2

PubMed Central

Takahashi, Yuji; Shomura, Ayahiko; Sasaki, Takuji; Yano, Masahiro

2001-01-01

Hd6 is a quantitative trait locus involved in rice photoperiod sensitivity. It was detected in backcross progeny derived from a cross between the japonica variety Nipponbare and the indica variety Kasalath. To isolate a gene at Hd6, we used a large segregating population for the high-resolution and fine-scale mapping of Hd6 and constructed genomic clone contigs around the Hd6 region. Linkage analysis with P1-derived artificial chromosome clone-derived DNA markers delimited Hd6 to a 26.4-kb genomic region. We identified a gene encoding the α subunit of protein kinase CK2 (CK2α) in this region. The Nipponbare allele of CK2α contains a premature stop codon, and the resulting truncated product is undoubtedly nonfunctional. Genetic complementation analysis revealed that the Kasalath allele of CK2α increases days-to-heading. Map-based cloning with advanced backcross progeny enabled us to identify a gene underlying a quantitative trait locus even though it exhibited a relatively small effect on the phenotype. PMID:11416158

A recognizable type of syndromic short stature with arthrogryposis caused by bi-allelic SEMA3A loss-of-function variants.

PubMed

Baumann, M; Steichen-Gersdorf, E; Krabichler, B; Müller, T; Janecke, A R

2017-07-01

The semaphorins constitute a large family of secreted and membrane-associated proteins that regulate many developmental processes, including neural circuit assembly, bone formation and angiogenesis. Recently, bi-allelic loss-of-function variants in SEMA3A (semaphorin 3A) were identified in a single patient with a particular pattern of multiple congenital anomalies (MCA). Using homozygosity mapping combined with exome sequencing, we identified a homozygous SEMA3A variant causing a premature stop codon in an 8 year old boy with the same pattern of MCA. The phenotype of these patients is characterized by postnatal short stature, skeletal anomalies of the thorax, a minor congenital heart or vascular defect, camptodactyly, micropenis, and variable additional anomalies. Motor development is delayed in both patients, and intellectual development is delayed in one patient. Our observation of a second case supports the notion that bi-allelic mutations in SEMA3A cause an autosomal recessive type of syndromic short stature. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mechanisms responsible for imipenem resistance among Pseudomonas aeruginosa clinical isolates exposed to imipenem concentrations within the mutant selection window.

PubMed

Vassilara, Foula; Galani, Irene; Souli, Maria; Papanikolaou, Konstantinos; Giamarellou, Helen; Papadopoulos, Antonios

2017-07-01

The aim of this study was to determine the propensities of imipenem to select for resistant Pseudomonas aeruginosa mutants by determining the mutant prevention concentrations (MPCs) for 9 unrelated clinical isolates and the accession of any relationship with mechanisms of resistance development. The MPC/MIC ratios ranged from 4 to 16. Detection of resistance mechanisms in the mutant derivatives of the nine isolates mainly revealed inactivating mutations in the gene coding for outer membrane protein OprD. Point mutations leading to premature stop codons or amino acid substitution S278P, ≥1bp deletion leading to frameshift mutations and interruption of the oprD by an insertion sequence, were observed. MPC and mutant selection window (MSW) are unique parameters that may guide the implementation of antimicrobial treatment, providing useful information about the necessary imipenem concentration needed in the infection area, in order to avoid the emergence of resistance, especially in clinical situations with high bacterial load. Copyright © 2017 Elsevier Inc. All rights reserved.

Correlations of HBV Genotypes, Mutations Affecting HBeAg Expression and HBeAg/ anti-HBe Status in HBV Carriers

PubMed Central

Lim, Chee Kent; Tan, Joanne Tsui Ming; Khoo, Jason Boo Siang; Ravichandran, Aarthi; Low, Hsin Mei; Chan, Yin Chyi; Ton, So Har

2006-01-01

This study was carried out to determine the effects of hepatitis B virus genotypes, core promoter mutations (A1762G1764→T1762A1764) as well as precore stop codon mutations (TGG→TAG) on HBeAg expression and HBeAg/ anti-HBe status. Study was also performed on the effects of codon 15 variants (C1858/ T1858) on the predisposition of precore stop codon mutations (TGG→TAG). A total of 77 sera samples were analyzed. Fifty one samples were successfully genotyped of which the predominant genotype was genotype B (29/ 51, 56.9 %), followed by genotype C (16/ 51, 31.4 %). Co-infections by genotypes B and C were observed in four samples (7.8 %). To a lesser degree, genotypes D and E (2.0 % each) were also observed. For core promoter mutations, the prevalence was 68.8 % (53/ 77) for A1762G1764 wild-type and 14.3 % (11/ 77) for T1762A1764 mutant while 9.1 % (7/ 77) was co-infected by both strains. The prevalence of codon 15 variants was found to be 42.9 % (33/ 77) for T1858 variant and 16.9 % (13/ 77) for C1858 variant. No TAG mutation was found. In our study, no associations were found between genotypes (B and C) and core promoter mutations as well as codon 15 variants. Also no correlation was observed between HBeAg/ anti-HBe status with genotypes (B and C) and core promoter mutations. PMID:16421626

Control of total GFP expression by alterations to the 3′ region nucleotide sequence

PubMed Central

2013-01-01

Background Previously, we distinguished the Escherichia coli type II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for unfolded and folded soluble target proteins. The translocation of folded protein to the periplasm for soluble expression via the Tat pathway was controlled by an N-terminal hydrophilic leader sequence. In this study, we investigated the effect of the hydrophilic C-terminal end and its nucleotide sequence on total and soluble protein expression. Results The native hydrophilic C-terminal end of GFP was obtained by deleting the C-terminal peptide LeuGlu-6×His, derived from pET22b(+). The corresponding clones induced total and soluble GFP expression that was either slightly increased or dramatically reduced, apparently through reconstruction of the nucleotide sequence around the stop codon in the 3′ region. In the expression-induced clones, the hydrophilic C-terminus showed increased Tat pathway specificity for soluble expression. However, in the expression-reduced clone, after analyzing the role of the 5′ poly(A) coding sequence with a substituted synonymous codon, we proved that the longer 5′ poly(A) coding sequence interacted with the reconstructed 3′ region nucleotide sequence to create a new mRNA tertiary structure between the 5′ and 3′ regions, which resulted in reduced total GFP expression. Further, to recover the reduced expression by changing the 3′ nucleotide sequence, after replacing selected C-terminal 5′ codons and the stop codon in the ORF with synonymous codons, total GFP expression in most of the clones was recovered to the undeleted control level. The insertion of trinucleotides after the stop codon in the 3′-UTR recovered or reduced total GFP expression. RT-PCR revealed that the level of total protein expression was controlled by changes in translational or transcriptional regulation, which were induced or reduced by the substitution or insertion of 3′ region nucleotides. Conclusions We found that the hydrophilic C-terminal end of GFP increased Tat pathway specificity and that the 3′ nucleotide sequence played an important role in total protein expression through translational and transcriptional regulation. These findings may be useful for efficiently producing recombinant proteins as well as for potentially controlling the expression level of specific genes in the body for therapeutic purposes. PMID:23834827

Mutational spectrum of Xeroderma pigmentosum group A in Egyptian patients.

PubMed

Amr, Khalda; Messaoud, Olfa; El Darouti, Mohamad; Abdelhak, Sonia; El-Kamah, Ghada

2014-01-01

Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disease characterized by hyperphotosensitivity, DNA repair defects and a predisposition to skin cancers. The most frequently occurring type worldwide is the XP group A (XPA). There is a close relationship between the clinical features that ranged from severe to mild form and the mutational site in XPA gene. The aim of this study is to carry out the mutational analysis in Egyptian patients with XP-A. This study was carried out on four unrelated Egyptian XP-A families. Clinical features were examined and direct sequencing of the coding region of XPA gene was performed in patients and their parents. Direct sequencing of the whole coding region of the XPA gene revealed the identification of two homozygous nonsense mutations: (c.553C >T; p.(Gln185)) and (c.331G>T; p.(Glu111)), which create premature, stop codon and a homodeletion (c.374delC: p.Thr125Ilefs 15) that leads to frameshift and premature translation termination. We report the identification of one novel XPA gene mutation and two known mutations in four unrelated Egyptian families with Xermoderma pigmentosum. All explored patients presented severe neurological abnormalities and have mutations located in the DNA binding domain. This report gives insight on the mutation spectrum of XP-A in Egypt. This would provide a valuable tool for early diagnosis of this severe disease. © 2013 Elsevier B.V. All rights reserved.

Comparative Analysis of Syntenic Genes in Grass Genomes Reveals Accelerated Rates of Gene Structure and Coding Sequence Evolution in Polyploid Wheat1[W][OA

PubMed Central

Akhunov, Eduard D.; Sehgal, Sunish; Liang, Hanquan; Wang, Shichen; Akhunova, Alina R.; Kaur, Gaganpreet; Li, Wanlong; Forrest, Kerrie L.; See, Deven; Šimková, Hana; Ma, Yaqin; Hayden, Matthew J.; Luo, Mingcheng; Faris, Justin D.; Doležel, Jaroslav; Gill, Bikram S.

2013-01-01

Cycles of whole-genome duplication (WGD) and diploidization are hallmarks of eukaryotic genome evolution and speciation. Polyploid wheat (Triticum aestivum) has had a massive increase in genome size largely due to recent WGDs. How these processes may impact the dynamics of gene evolution was studied by comparing the patterns of gene structure changes, alternative splicing (AS), and codon substitution rates among wheat and model grass genomes. In orthologous gene sets, significantly more acquired and lost exonic sequences were detected in wheat than in model grasses. In wheat, 35% of these gene structure rearrangements resulted in frame-shift mutations and premature termination codons. An increased codon mutation rate in the wheat lineage compared with Brachypodium distachyon was found for 17% of orthologs. The discovery of premature termination codons in 38% of expressed genes was consistent with ongoing pseudogenization of the wheat genome. The rates of AS within the individual wheat subgenomes (21%–25%) were similar to diploid plants. However, we uncovered a high level of AS pattern divergence between the duplicated homeologous copies of genes. Our results are consistent with the accelerated accumulation of AS isoforms, nonsynonymous mutations, and gene structure rearrangements in the wheat lineage, likely due to genetic redundancy created by WGDs. Whereas these processes mostly contribute to the degeneration of a duplicated genome and its diploidization, they have the potential to facilitate the origin of new functional variations, which, upon selection in the evolutionary lineage, may play an important role in the origin of novel traits. PMID:23124323

A Frameshift Mutation in the Cubilin Gene (CUBN) in Border Collies with Imerslund-Gräsbeck Syndrome (Selective Cobalamin Malabsorption)

PubMed Central

Owczarek-Lipska, Marta; Jagannathan, Vidhya; Drögemüller, Cord; Lutz, Sabina; Glanemann, Barbara

2013-01-01

Imerslund-Gräsbeck syndrome (IGS) or selective cobalamin malabsorption has been described in humans and dogs. IGS occurs in Border Collies and is inherited as a monogenic autosomal recessive trait in this breed. Using 7 IGS cases and 7 non-affected controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 3.53 Mb interval on chromosome 2. We re-sequenced the genome of one affected dog at ∼10× coverage and detected 17 non-synonymous variants in the critical interval. Two of these non-synonymous variants were in the cubilin gene (CUBN), which is known to play an essential role in cobalamin uptake from the ileum. We tested these two CUBN variants for association with IGS in larger cohorts of dogs and found that only one of them was perfectly associated with the phenotype. This variant, a single base pair deletion (c.8392delC), is predicted to cause a frameshift and premature stop codon in the CUBN gene. The resulting mutant open reading frame is 821 codons shorter than the wildtype open reading frame (p.Q2798Rfs*3). Interestingly, we observed an additional nonsense mutation in the MRC1 gene encoding the mannose receptor, C type 1, which was in perfect linkage disequilibrium with the CUBN frameshift mutation. Based on our genetic data and the known role of CUBN for cobalamin uptake we conclude that the identified CUBN frameshift mutation is most likely causative for IGS in Border Collies. PMID:23613799

Novel BRCA1 splice-site mutation in ovarian cancer patients of Slavic origin.

PubMed

Krivokuca, Ana; Dragos, Vita Setrajcic; Stamatovic, Ljiljana; Blatnik, Ana; Boljevic, Ivana; Stegel, Vida; Rakobradovic, Jelena; Skerl, Petra; Jovandic, Stevo; Krajc, Mateja; Magic, Mirjana Brankovic; Novakovic, Srdjan

2018-04-01

Mutations in breast cancer susceptibility gene 1 (BRCA1) lead to defects in a number of cellular pathways including DNA damage repair and transcriptional regulation, resulting in the elevated genome instability and predisposing to breast and ovarian cancers. We report a novel mutation LRG_292t1:c.4356delA,p.(Ala1453Glnfs*3) in the 12th exon of BRCA1, in the splice site region near the donor site of intron 12. It is a frameshift mutation with the termination codon generated on the third amino acid position from the site of deletion. Human Splice Finder 3.0 and MutationTaster have assessed this variation as disease causing, based on the alteration of splicing, creation of premature stop codon and other potential alterations initiated by nucleotide deletion. Among the most important alterations are frameshift and splice site changes (score of the newly created donor splice site: 0.82). c.4356delA was associated with two ovarian cancer cases in two families of Slavic origin. It was detected by next generation sequencing, and confirmed with Sanger sequencing in both cases. Because of the fact that it changes the reading frame of the protein, novel mutation c.4356delA p.(Ala1453Glnfs*3) in BRCA1 gene might be of clinical significance for hereditary ovarian cancer. Further functional as well as segregation analyses within the families are necessary for appropriate clinical classification of this variant. Since it has been detected in two ovarian cancer patients of Slavic origin, it is worth investigating founder effect of this mutation in Slavic populations.

The Complete Mitochondrial Genome of the Rice Moth, Corcyra cephalonica

PubMed Central

Wu, Yu-Peng; Li, Jie; Zhao, Jin-Liang; Su, Tian-Juan; Luo, A-Rong; Fan, Ren-Jun; Chen, Ming-Chang; Wu, Chun-Sheng; Zhu, Chao-Dong

2012-01-01

The complete mitochondrial genome (mitogenome) of the rice moth, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) was determined as a circular molecular of 15,273 bp in size. The mitogenome composition (37 genes) and gene order are the same as the other lepidopterans. Nucleotide composition of the C. cephalonica mitogenome is highly A+T biased (80.43%) like other insects. Twelve protein-coding genes start with a typical ATN codon, with the exception of coxl gene, which uses CGA as the initial codon. Nine protein-coding genes have the common stop codon TAA, and the nad2, cox1, cox2, and nad4 have single T as the incomplete stop codon. 22 tRNA genes demonstrated cloverleaf secondary structure. The mitogenome has several large intergenic spacer regions, the spacer1 between trnQ gene and nad2 gene, which is common in Lepidoptera. The spacer 3 between trnE and trnF includes microsatellite-like repeat regions (AT)18 and (TTAT)3. The spacer 4 (16 bp) between trnS2 gene and nad1 gene has a motif ATACTAT; another species, Sesamia inferens encodes ATCATAT at the same position, while other lepidopteran insects encode a similar ATACTAA motif. The spacer 6 is A+T rich region, include motif ATAGA and a 20-bp poly(T) stretch and two microsatellite (AT)9, (AT)8 elements. PMID:23413968

The complete mitochondrial genome of the rice moth, Corcyra cephalonica.

PubMed

Wu, Yu-Peng; Li, Jie; Zhao, Jin-Liang; Su, Tian-Juan; Luo, A-Rong; Fan, Ren-Jun; Chen, Ming-Chang; Wu, Chun-Sheng; Zhu, Chao-Dong

2012-01-01

The complete mitochondrial genome (mitogenome) of the rice moth, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) was determined as a circular molecular of 15,273 bp in size. The mitogenome composition (37 genes) and gene order are the same as the other lepidopterans. Nucleotide composition of the C. cephalonica mitogenome is highly A+T biased (80.43%) like other insects. Twelve protein-coding genes start with a typical ATN codon, with the exception of coxl gene, which uses CGA as the initial codon. Nine protein-coding genes have the common stop codon TAA, and the nad2, cox1, cox2, and nad4 have single T as the incomplete stop codon. 22 tRNA genes demonstrated cloverleaf secondary structure. The mitogenome has several large intergenic spacer regions, the spacer1 between trnQ gene and nad2 gene, which is common in Lepidoptera. The spacer 3 between trnE and trnF includes microsatellite-like repeat regions (AT)18 and (TTAT)(3). The spacer 4 (16 bp) between trnS2 gene and nad1 gene has a motif ATACTAT; another species, Sesamia inferens encodes ATCATAT at the same position, while other lepidopteran insects encode a similar ATACTAA motif. The spacer 6 is A+T rich region, include motif ATAGA and a 20-bp poly(T) stretch and two microsatellite (AT)(9), (AT)(8) elements.

Different inactivating mutations of the mineralocorticoid receptor in fourteen families affected by type I pseudohypoaldosteronism.

PubMed

Sartorato, Paola; Lapeyraque, Anne-Laure; Armanini, Decio; Kuhnle, Ursula; Khaldi, Yasmina; Salomon, Rémi; Abadie, Véronique; Di Battista, Eliana; Naselli, Arturo; Racine, Alain; Bosio, Maurizio; Caprio, Massimiliano; Poulet-Young, Véronique; Chabrolle, Jean-Pierre; Niaudet, Patrick; De Gennes, Christiane; Lecornec, Marie-Hélène; Poisson, Elodie; Fusco, Anna Maria; Loli, Paola; Lombès, Marc; Zennaro, Maria-Christina

2003-06-01

We have analyzed the human mineralocorticoid receptor (hMR) gene in 14 families with autosomal dominant or sporadic pseudohypoaldosteronism (PHA1), a rare form of mineralocorticoid resistance characterized by neonatal renal salt wasting and failure to thrive. Six heterozygous mutations were detected. Two frameshift mutations in exon 2 (insT1354, del8bp537) and one nonsense mutation in exon 4 (C2157A, Cys645stop) generate truncated proteins due to premature stop codons. Three missense mutations (G633R, Q776R, L979P) differently affect hMR function. The DNA binding domain mutant R633 exhibits reduced maximal transactivation, although its binding characteristics and ED(50) of transactivation are comparable with wild-type hMR. Ligand binding domain mutants R776 and P979 present reduced or absent aldosterone binding, respectively, which is associated with reduced or absent ligand-dependent transactivation capacity. Finally, P979 possesses a transdominant negative effect on wild-type hMR activity, whereas mutations G633R and Q776R probably result in haploinsufficiency in PHA1 patients. We conclude that hMR mutations are a common feature of autosomal dominant PHA1, being found in 70% of our familial cases. Their absence in some families underscores the importance of an extensive investigation of the hMR gene and the role of precise diagnostic procedures to allow for identification of other genes potentially involved in the disease.

Male gender identity in complete androgen insensitivity syndrome.

PubMed

T'Sjoen, Guy; De Cuypere, Griet; Monstrey, Stan; Hoebeke, Piet; Freedman, F Kenneth; Appari, Mahesh; Holterhus, Paul-Martin; Van Borsel, John; Cools, Martine

2011-06-01

Women and girls with complete androgen insensitivity syndrome (CAIS) invariably have a female typical core gender identity. In this case report, we describe the first case of male gender identity in a CAIS individual raised female leading to complete sex reassignment involving both androgen treatment and phalloplasty. CAIS was diagnosed at age 17, based on an unambiguously female phenotype, a 46,XY karyotype, and a 2660delT androgen receptor (AR) gene mutation, leading to a premature stop in codon 807. Bilateral gonadectomy was performed but a short period of estrogen treatment induced a negative emotional reaction and treatment was stopped. Since the age of 3, childhood-onset cross gender behavior had been noticed. After a period of psychotherapy, persisting male gender identity was confirmed. There was no psychiatric co-morbidity and there was an excellent real life experience. Testosterone substitution was started, however without inducing any of the desired secondary male characteristics. A subcutaneous mastectomy was performed and the patient received phalloplasty by left forearm free flap and scrotoplasty. Testosterone treatment was continued, without inducing virilization, and bone density remained normal. The patient qualifies as female-to-male transsexual and was treated according to the Standards of Care by the World Professional Association for Transgender Health with good outcome. However, we do not believe that female sex of rearing as a standard procedure should be questioned in CAIS. Our case challenges the role of a functional AR pathway in the development of male gender identity.

Apnea of Prematurity (For Parents)

MedlinePlus

... large bursts of breath followed by periods of shallow breathing or stopped breathing. Apnea of prematurity usually ... seconds and is followed by several fast and shallow breaths. Periodic breathing doesn't cause a change ...

A novel mutation in the FGB: c.1105C>T turns the codon for amino acid Bβ Q339 into a stop codon causing hypofibrinogenemia.

PubMed

Marchi, Rita; Brennan, Stephen; Meyer, Michael; Rojas, Héctor; Kanzler, Daniela; De Agrela, Marisela; Ruiz-Saez, Arlette

2013-03-01

Routine coagulation tests on a 14year-old male with frequent epistaxis showed a prolonged thrombin time together with diminished functional (162mg/dl) and gravimetric (122mg/dl) fibrinogen concentrations. His father showed similar aberrant results and sequencing of the three fibrinogen genes revealed a novel heterozygous nonsense mutation in the FGB gene c.1105C>T, which converts the codon for residue Bβ 339Q to stop, causing deletion of Bβ chain residues 339-461. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and RP-HPLC (reverse-phase high-pressure liquid chromatography) of purified fibrinogen showed only normal Aα, Bβ, and γ chains, indicating that molecules with the truncated 37,990Da β chain were not secreted into plasma. Functional analysis showed impaired fibrin polymerization, fibrin porosity, and elasticity compared to controls. By laser scanning confocal microscopy the patient's fibers were slightly thinner than normal. Electrospray ionization mass spectrometry (ESI MS) presented normal sialylation of the oligosaccharide chains, and liver function tests showed no evidence of liver dysfunction that might explain the functional abnormalities. Copyright © 2012 Elsevier Inc. All rights reserved.

Representation mutations from standard genetic codes

NASA Astrophysics Data System (ADS)

Aisah, I.; Suyudi, M.; Carnia, E.; Suhendi; Supriatna, A. K.

2018-03-01

Graph is widely used in everyday life especially to describe model problem and describe it concretely and clearly. In addition graph is also used to facilitate solve various kinds of problems that are difficult to be solved by calculation. In Biology, graph can be used to describe the process of protein synthesis in DNA. Protein has an important role for DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). Proteins are composed of amino acids. In this study, amino acids are related to genetics, especially the genetic code. The genetic code is also known as the triplet or codon code which is a three-letter arrangement of DNA nitrogen base. The bases are adenine (A), thymine (T), guanine (G) and cytosine (C). While on RNA thymine (T) is replaced with Urasil (U). The set of all Nitrogen bases in RNA is denoted by N = {C U, A, G}. This codon works at the time of protein synthesis inside the cell. This codon also encodes the stop signal as a sign of the stop of protein synthesis process. This paper will examine the process of protein synthesis through mathematical studies and present it in three-dimensional space or graph. The study begins by analysing the set of all codons denoted by NNN such that to obtain geometric representations. At this stage there is a matching between the sets of all nitrogen bases N with Z 2 × Z 2; C=(\\overline{0},\\overline{0}),{{U}}=(\\overline{0},\\overline{1}),{{A}}=(\\overline{1},\\overline{0}),{{G}}=(\\overline{1},\\overline{1}). By matching the algebraic structure will be obtained such as group, group Klein-4,Quotien group etc. With the help of Geogebra software, the set of all codons denoted by NNN can be presented in a three-dimensional space as a multicube NNN and also can be represented as a graph, so that can easily see relationship between the codon.

Conserved nonsense-prone CpG sites in apoptosis-regulatory genes: conditional stop signs on the road to cell death.

PubMed

Zhao, Yongzhong; Epstein, Richard J

2013-01-01

Methylation-prone CpG dinucleotides are strongly conserved in the germline, yet are also predisposed to somatic mutation. Here we quantify the relationship between germline codon mutability and somatic carcinogenesis by comparing usage of the nonsense-prone CGA (→TGA) codons in gene groups that differ in apoptotic function; to this end, suppressor genes were subclassified as either apoptotic (gatekeepers) or repair (caretakers). Mutations affecting CGA codons in sporadic tumors proved to be highly asymmetric. Moreover, nonsense mutations were 3-fold more likely to affect gatekeepers than caretakers. In addition, intragenic CGA clustering nonrandomly affected functionally critical regions of gatekeepers. We conclude that human gatekeeper suppressor genes are enriched for nonsense-prone codons, and submit that this germline vulnerability to tumors could reflect in utero selection for a methylation-dependent capability to short-circuit environmental insults that otherwise trigger apoptosis and fetal loss.

Codon Optimization of the Human Papillomavirus E7 Oncogene Induces a CD8+ T Cell Response to a Cryptic Epitope Not Harbored by Wild-Type E7

PubMed Central

Lorenz, Felix K. M.; Wilde, Susanne; Voigt, Katrin; Kieback, Elisa; Mosetter, Barbara; Schendel, Dolores J.; Uckert, Wolfgang

2015-01-01

Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine. PMID:25799237

A protocol for isolating insect mitochondrial genomes: a case study of NUMT in Melipona flavolineata (Hymenoptera: Apidae).

PubMed

Françoso, Elaine; Gomes, Fernando; Arias, Maria Cristina

2016-07-01

Nuclear mitochondrial DNA insertions (NUMTs) are mitochondrial DNA sequences that have been transferred into the nucleus and are recognized by the presence of indels and stop codons. Although NUMTs have been identified in a diverse range of species, their discovery was frequently accidental. Here, our initial goal was to develop and standardize a simple method for isolating NUMTs from the nuclear genome of a single bee. Subsequently, we tested our new protocol by determining whether the indels and stop codons of the cytochrome c oxidase subunit I (COI) sequence of Melipona flavolineata are of nuclear origin. The new protocol successfully demonstrated the presence of a COI NUMT. In addition to NUMT investigations, the protocol described here will also be very useful for studying mitochondrial mutations related to diseases and for sequencing complete mitochondrial genomes with high read coverage by Next-Generation technology.

Analysis of the DNA sequence of a 15,500 bp fragment near the left telomere of chromosome XV from Saccharomyces cerevisiae reveals a putative sugar transporter, a carboxypeptidase homologue and two new open reading frames.

PubMed

Gamo, F J; Lafuente, M J; Casamayor, A; Ariño, J; Aldea, M; Casas, C; Herrero, E; Gancedo, C

1996-06-15

We report the sequence of a 15.5 kb DNA segment located near the left telomere of chromosome XV of Saccharomyces cerevisiae. The sequence contains nine open reading frames (ORFs) longer than 300 bp. Three of them are internal to other ones. One corresponds to the gene LGT3 that encodes a putative sugar transporter. Three adjacent ORFs were separated by two stop codons in frame. These ORFs presented homology with the gene CPS1 that encodes carboxypeptidase S. The stop codons were not found in the same sequence derived from another yeast strain. Two other ORFs without significant homology in databases were also found. One of them, O0420, is very rich in serine and threonine and presents a series of repeated or similar amino acid stretches along the sequence.

Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals

PubMed Central

Schueren, Fabian; Lingner, Thomas; George, Rosemol; Hofhuis, Julia; Dickel, Corinna; Gärtner, Jutta; Thoms, Sven

2014-01-01

Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes. DOI: http://dx.doi.org/10.7554/eLife.03640.001 PMID:25247702

Evaluating Sense Codon Reassignment with a Simple Fluorescence Screen.

PubMed

Biddle, Wil; Schmitt, Margaret A; Fisk, John D

2015-12-22

Understanding the interactions that drive the fidelity of the genetic code and the limits to which modifications can be made without breaking the translational system has practical implications for understanding the molecular mechanisms of evolution as well as expanding the set of encodable amino acids, particularly those with chemistries not provided by Nature. Because 61 sense codons encode 20 amino acids, reassigning the meaning of sense codons provides an avenue for biosynthetic modification of proteins, furthering both fundamental and applied biochemical research. We developed a simple screen that exploits the absolute requirement for fluorescence of an active site tyrosine in green fluorescent protein (GFP) to probe the pliability of the degeneracy of the genetic code. Our screen monitors the restoration of the fluorophore of GFP by incorporation of a tyrosine in response to a sense codon typically assigned another meaning in the genetic code. We evaluated sense codon reassignment at four of the 21 sense codons read through wobble interactions in Escherichia coli using the Methanocaldococcus jannaschii orthogonal tRNA/aminoacyl tRNA synthetase pair originally developed and commonly used for amber stop codon suppression. By changing only the anticodon of the orthogonal tRNA, we achieved sense codon reassignment efficiencies between 1% (Phe UUU) and 6% (Lys AAG). Each of the orthogonal tRNAs preferentially decoded the codon traditionally read via a wobble interaction in E. coli with the exception of the orthogonal tRNA with an AUG anticodon, which incorporated tyrosine in response to both the His CAU and His CAC codons with approximately equal frequencies. We applied our screen in a high-throughput manner to evaluate a 10(9)-member combined tRNA/aminoacyl tRNA synthetase library to identify improved sense codon reassigning variants for the Lys AAG codon. A single rapid screen with the ability to broadly evaluate reassignable codons will facilitate identification and improvement of the combinations of sense codons and orthogonal pairs that display efficient reassignment.

Generation of Esr1-Knockout Rats Using Zinc Finger Nuclease-Mediated Genome Editing

PubMed Central

Dhakal, Pramod; Kubota, Kaiyu; Chakraborty, Damayanti; Lei, Tianhua; Larson, Melissa A.; Wolfe, Michael W.; Roby, Katherine F.; Vivian, Jay L.

2014-01-01

Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action. PMID:24506075

Generation of Esr1-knockout rats using zinc finger nuclease-mediated genome editing.

PubMed

Rumi, M A Karim; Dhakal, Pramod; Kubota, Kaiyu; Chakraborty, Damayanti; Lei, Tianhua; Larson, Melissa A; Wolfe, Michael W; Roby, Katherine F; Vivian, Jay L; Soares, Michael J

2014-05-01

Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action.

Mutation in and lack of expression of tyrosinase-related protein-1 (TRP-1) in melanocytes from an individual with brown oculocutaneous albinism: a new subtype of albinism classified as "OCA3".

PubMed Central

Boissy, R. E.; Zhao, H.; Oetting, W. S.; Austin, L. M.; Wildenberg, S. C.; Boissy, Y. L.; Zhao, Y.; Sturm, R. A.; Hearing, V. J.; King, R. A.; Nordlund, J. J.

1996-01-01

Most types of human oculocutaneous albinism (OCA) result from mutations in the gene for tyrosinase (OCA1) or the P protein (OCA2), although other types of OCA have been described but have not been mapped to specific loci. Melanocytes were cultured from an African-American with OCA, who exhibited the phenotype of Brown OCA, and his normal fraternal twin. Melanocytes cultured from the patient with OCA and the normal twin appeared brown versus black, respectively. Melanocytes from both the patient with OCA and the normal twin demonstrated equal amounts of NP-40-soluble melanin; however, melanocytes from the patient with OCA contained only 7% of the amount of insoluble melanin found from the normal twin. Tyrosinase- related protein-1 (TRP-1) was not detected in the OCA melanocytes by use of various anti-TRP-1 probes. Furthermore, transcripts for TRP-1 were absent in cultured OCA melanocytes. The affected twin was homozygous for a single-bp deletion in exon 6, removing an A in codon 368 and leading to a premature stop at codon 384. Tyrosine hydroxylase activity of the OCA melanocytes was comparable to controls when assayed in cell lysates but was only 30% of controls when assayed in intact cells. We conclude that this mutation of the human TRP-1 gene affects its interaction with tyrosinase, resulting in dysregulation of tyrosinase activity, promotes the synthesis of brown versus black melanin, and is responsible for a third genetic type of OCA in humans, which we classify as "OCA3." Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8651291

Can We Stop the Current Epidemic of Blindness From Retinopathy of Prematurity?

PubMed

Dutta, Sourabh; Raghuveer, Talkad; Vinekar, Anand; Dogra, Mangat Ram

2016-11-07

Retinopathy of Prematurity (ROP) is a retinal vascular disease of the premature infant. There have been significant advances in the understanding of the pathogenesis, prevention, detection and treatment of severe ROP. In developed countries, all premature infants at risk for ROP are screened routinely based on national guidelines and treatment is prompt, resulting in good visual outcomes. Blindness from ROP is a rare event in these countries. However, there is an ongoing epidemic of ROP- induced blindness in India. The reasons for this epidemic of ROP blindness are many and include increasing survival of premature infants, lack of equipment such as oxygen-blenders and pulse oximeters that are necessary for safe use of supplemental oxygen in premature infants, lack of routine screening for ROP, lack of trained personnel for ROP screening, and lack of Ophthalmologists to promptly initiate treatment when infants develop severe ROP. All the identified causes can be resolved with a concerted national co-operative effort of Pediatricians, Neonatologists, Ophthalmologists, Indian Academy of Pediatrics, National Neonatology Forum, Indian Academy of Ophthalmologists, Government of India, and parents of premature infants. We feel that tt is possible to stop the current epidemic of preventable childhood blindness from ROP with concerted action by all stakeholders.

KBG syndrome involving a single-nucleotide duplication in ANKRD11

PubMed Central

Kleyner, Robert; Malcolmson, Janet; Tegay, David; Ward, Kenneth; Maughan, Annette; Maughan, Glenn; Nelson, Lesa; Wang, Kai; Robison, Reid; Lyon, Gholson J.

2016-01-01

KBG syndrome is a rare autosomal dominant genetic condition characterized by neurological involvement and distinct facial, hand, and skeletal features. More than 70 cases have been reported; however, it is likely that KBG syndrome is underdiagnosed because of lack of comprehensive characterization of the heterogeneous phenotypic features. We describe the clinical manifestations in a male currently 13 years of age, who exhibited symptoms including epilepsy, severe developmental delay, distinct facial features, and hand anomalies, without a positive genetic diagnosis. Subsequent exome sequencing identified a novel de novo heterozygous single base pair duplication (c.6015dupA) in ANKRD11, which was validated by Sanger sequencing. This single-nucleotide duplication is predicted to lead to a premature stop codon and loss of function in ANKRD11, thereby implicating it as contributing to the proband's symptoms and yielding a molecular diagnosis of KBG syndrome. Before molecular diagnosis, this syndrome was not recognized in the proband, as several key features of the disorder were mild and were not recognized by clinicians, further supporting the concept of variable expressivity in many disorders. Although a diagnosis of cerebral folate deficiency has also been given, its significance for the proband's condition remains uncertain. PMID:27900361

Rhnull syndrome: identification of a novel mutation in RHce.

PubMed

Rosa, K A; Reid, M E; Lomas-Francis, C; Powell, V I; Costa, F F; Stinghen, S T; Watanabe, A M; Carboni, E K; Baldon, J P; Jucksch, M M F; Castilho, L

2005-11-01

The deficiency of Rh proteins on red blood cells (RBCs) from individuals of the Rh(null) amorph type are the result of homozygosity for a silent RHCE in cis with a deleted RHD. A novel mutation in RHce was identified in two Caucasian Brazilian girls with the amorph type of Rh(null) who were born to parents who were first cousins. RBCs from the Rh(null) sisters and from family members were analyzed by serology and flow cytometry with specific antibodies. Genomic DNA and transcripts were tested by polymerase chain reaction and sequence analysis. Rh(null) RBCs were nonreactive with anti-Rh and anti-LW. Molecular analyses showed a deletion of RHD and of one nucleotide (960/963; GGGG-->GGG) in exon 7 of the RHce. This deletion introduced a frameshift after Gly321, a new C-terminal sequence, and a premature stop codon, resulting in a shorter predicted protein with 357 amino acids. The detection of a unique RHce transcript indicated that the two sisters were homozygous, whereas the other family members were heterozygous for the mutation. A novel mutation resulting in the amorph Rh(null) with loss of Rh antigen expression is described.

A novel SMAD3 mutation caused multiple aneurysms in a patient without osteoarthritis symptoms.

PubMed

Courtois, Audrey; Coppieters, Wouter; Bours, Vincent; Defraigne, Jean-Olivier; Colige, Alain; Sakalihasan, Natzi

2017-04-01

Heterozygous mutations in the SMAD3 gene were recently described as the cause of a form of non-syndromic familial aortic thoracic aneurysm and dissection (FTAAD) transmitted as an autosomal dominant disorder and often associated with early-onset osteoarthritis. This new clinical entity, called aneurysms-osteoarthritis syndrome (AOS) or Loeys-Dietz syndrome 3 (LDS3), is characterized by aggressive arterial damages such as aneurysms, dissections and tortuosity throughout the arterial tree. We report, here, the case of a 45 year-old man presenting multiple visceral arteries and abdominal aortic aneurysms but without dissection of the thoracic aorta and without any sign of osteoarthritis. Exome-sequencing revealed a new frameshift heterozygous c.455delC (p.Pro152Hisfs*34) mutation in the SMAD3 gene. This deletion is located in the exon 3 coding for the linker region of the protein and causes a premature stop codon at positions 556-558 in the exon 4. The same mutation was found in the proband's mother and sister who had open surgery for abdominal aortic aneurysm and in one of his children who was 5 year-old and did not present aneurysm yet. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Seventeen {alpha}-hydroxylase deficiency with one base pair deletion of the cytochrome P450c17 (CYP17) gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oshiro, Chikara; Takasu, Nobuyuki; Wakugami, Tamio

1995-08-01

Mutation of the cytochrome P450c17 (CYP17) gene causes 17{alpha}-hydroxylase deficiency (170HD). Recently, several researchers have elucidated the molecular basis of 170HD by gene analysis. We experienced a case of 170HD and intended to reveal the abnormality of the CYP17 gene in this Japanese female with 170HD. Leukocytes were obtained from the patient, her mother and sister, and normal control subjects. We amplified the CYP17 gene using polymerase chain reaction and performed the sequence analysis using the dideoxy terminator method and restriction enzyme analysis. We found that the patient had one base-pair deletion at the position of amino acid 438. Anmore » indentical result was obtained with restriction enzyme analysis. This G deletion altered the reading frame and resulted in a premature stop codon at position 443; the ligand of heme iron (Cys: cystine 442) was absent. This small mutation may account for the patient`s clinical manifestations of 170HD. This is the first case of 170HD with only one base pair deletion of the CYP17 gene. 18 refs., 3 figs.« less

[Mutation screening of MITF gene in patients with Waardenburg syndrome type 2].

PubMed

Chen, Jing; Yang, Shu-Zhi; Liu, Jun; Han, Bing; Wang, Guo-Jian; Zhang, Xin; Kang, Dong-Yang; Dai, Pu; Young, Wie-Yen; Yuan, Hui-Jun

2008-04-01

Warrgenburg syndrome type 2 (WS2) is the most common autosomal dominantly-inherited syndrome with hearing loss. MITF (microphthalmia associated transcription factor)is a basic-helix-loop-helix-luecine zipper (bHLHZip) factor which regulates expression of tyrosinase, and is involved in melanocyte differentiation. Mutations in MITF associated with WS2 have been identified in some but not all affected families. Here, we report a three-generation Chinese family with a point mutation in the MITF gene causing WS2. The proband exhibits congenital severe sensorineural hearing loss, heterochromia iridis and facial freckles. One of family members manifests sensorineural deafness, and the other patients show premature greying or/and freckles. This mutation, heterozygous deletion c.639delA, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking normal interaction with its target DNA motif. This mutation is a novel mutation and the third case identified in exon 7 of MITF in WS2. Though there is only one base pair distance between this novel mutation and the other two documented cases and similar amino acids change, significant difference is seen in clinical phenotype, which suggests genetic background may play an important role.

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.

Deletion in the EVC2 Gene Causes Chondrodysplastic Dwarfism in Tyrolean Grey Cattle

PubMed Central

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. PMID:24733244

New insights into the interplay between the translation machinery and nonsense-mediated mRNA decay factors.

PubMed

Raimondeau, Etienne; Bufton, Joshua C; Schaffitzel, Christiane

2018-06-19

Faulty mRNAs with a premature stop codon (PTC) are recognized and degraded by nonsense-mediated mRNA decay (NMD). Recognition of a nonsense mRNA depends on translation and on the presence of NMD-enhancing or the absence of NMD-inhibiting factors in the 3'-untranslated region. Our review summarizes our current understanding of the molecular function of the conserved NMD factors UPF3B and UPF1, and of the anti-NMD factor Poly(A)-binding protein, and their interactions with ribosomes translating PTC-containing mRNAs. Our recent discovery that UPF3B interferes with human translation termination and enhances ribosome dissociation in vitro , whereas UPF1 is inactive in these assays, suggests a re-interpretation of previous experiments and modification of prevalent NMD models. Moreover, we discuss recent work suggesting new functions of the key NMD factor UPF1 in ribosome recycling, inhibition of translation re-initiation and nascent chain ubiquitylation. These new findings suggest that the interplay of UPF proteins with the translation machinery is more intricate than previously appreciated, and that this interplay quality-controls the efficiency of termination, ribosome recycling and translation re-initiation. © 2018 The Author(s).

A patient with a unique frameshift mutation in GPC3, causing Simpson-Golabi-Behmel syndrome, presenting with craniosynostosis, penoscrotal hypospadias, and a large prostatic utricle.

PubMed

Villarreal, Diana D; Villarreal, Humberto; Paez, Ana Maria; Peppas, Dennis; Lynch, Jane; Roeder, Elizabeth; Powers, George C

2013-12-01

We present a Hispanic male with the clinical and molecular diagnosis of Simpson-Golabi-Behmel syndrome (SGBS). The patient was born with multiple anomalies not entirely typical of SGBS patients, including penoscrotal hypospadias, a large prostatic utricle, and left coronal craniosynostosis. In addition, he demonstrated endocrine anomalies including a low random cortisol level suspicious for adrenal insufficiency and low testosterone level. To our knowledge, this is the first report of a prostatic utricle in SGBS and the second report of craniosynostosis. The unique disease-causing mutation likely arose de novo in the mother. It is a deletion-insertion that leads to a frameshift at the p.p. S359 [corrected] residue of GPC3 and a premature stop codon after five more amino acids. p. S359 [corrected] is the same residue that is normally cleaved by the Furin convertase, although the significance of this novel mutation with respect to the patient's multiple anomalies is unknown. We present this case as the perinatal course of a patient with unique features of SGBS and a confirmed molecular diagnosis. © 2013 Wiley Periodicals, Inc.

Novel FAM20A mutation causes autosomal recessive amelogenesis imperfecta.

PubMed

Volodarsky, Michael; Zilberman, Uri; Birk, Ohad S

2015-06-01

To relate the peculiar phenotype of amelogenesis imperfecta in a large Bedouin family to the genotype determined by whole genome linkage analysis. Amelogenesis imperfecta (AI) is a broad group of inherited pathologies affecting enamel formation, characterized by variability in phenotypes, causing mutations and modes of inheritance. Autosomal recessive or compound heterozygous mutations in FAM20A, encoding sequence similarity 20, member A, have been shown to cause several AI phenotypes. Five members from a large consanguineous Bedouin family presented with hypoplastic amelogenesis imperfecta with unerupted and resorbed permanent molars. Following Soroka Medical Center IRB approval and informed consent, blood samples were obtained from six affected offspring, five obligatory carriers and two unaffected siblings. Whole genome linkage analysis was performed followed by Sanger sequencing of FAM20A. The sequencing unravelled a novel homozygous deletion mutation in exon 11 (c.1523delC), predicted to insert a premature stop codon (p.Thr508Lysfs*6). We provide an interesting case of novel mutation in this rare disorder, in which the affected kindred is unique in the large number of family members sharing a similar phenotype. Copyright © 2015 Elsevier Ltd. All rights reserved.

Highly Efficient Genome Editing via CRISPR/Cas9 to Create Clock Gene Knockout Cells.

PubMed

Korge, Sandra; Grudziecki, Astrid; Kramer, Achim

2015-10-01

Targeted genome editing using CRISPR/Cas9 is a relatively new, revolutionary technology allowing for efficient and directed alterations of the genome. It has been widely used for loss-of-function studies in animals and cell lines but has not yet been used to study circadian rhythms. Here, we describe the application of CRISPR/Cas9 genome editing for the generation of an F-box and leucine-rich repeat protein 3 (Fbxl3) knockout in a human cell line. Genomic alterations at the Fbxl3 locus occurred with very high efficiency (70%-100%) and specificity at both alleles, resulting in insertions and deletions that led to premature stop codons and hence FBXL3 knockout. Fbxl3 knockout cells displayed low amplitude and long period oscillations of Bmal1-luciferase reporter activity as well as increased CRY1 protein stability in line with previously published phenotypes for Fbxl3 knockout in mice. Thus, CRISPR/Cas9 genome editing should be highly valuable for studying circadian rhythms not only in human cells but also in classic model systems as well as nonmodel organisms. © 2015 The Author(s).

Inherited biallelic CSF3R mutations in severe congenital neutropenia.

PubMed

Triot, Alexa; Järvinen, Päivi M; Arostegui, Juan I; Murugan, Dhaarini; Kohistani, Naschla; Dapena Díaz, José Luis; Racek, Tomas; Puchałka, Jacek; Gertz, E Michael; Schäffer, Alejandro A; Kotlarz, Daniel; Pfeifer, Dietmar; Díaz de Heredia Rubio, Cristina; Ozdemir, Mehmet Akif; Patiroglu, Turkan; Karakukcu, Musa; Sánchez de Toledo Codina, José; Yagüe, Jordi; Touw, Ivo P; Unal, Ekrem; Klein, Christoph

2014-06-12

Severe congenital neutropenia (SCN) is characterized by low numbers of peripheral neutrophil granulocytes and a predisposition to life-threatening bacterial infections. We describe a novel genetic SCN type in 2 unrelated families associated with recessively inherited loss-of-function mutations in CSF3R, encoding the granulocyte colony-stimulating factor (G-CSF) receptor. Family A, with 3 affected children, carried a homozygous missense mutation (NM_000760.3:c.922C>T, NP_000751.1:p.Arg308Cys), which resulted in perturbed N-glycosylation and aberrant localization to the cell surface. Family B, with 1 affected infant, carried compound heterozygous deletions provoking frameshifts and premature stop codons (NM_000760.3:c.948_963del, NP_000751.1:p.Gly316fsTer322 and NM_000760.3:c.1245del, NP_000751.1:p.Gly415fsTer432). Despite peripheral SCN, all patients had morphologic evidence of full myeloid cell maturation in bone marrow. None of the patients responded to treatment with recombinant human G-CSF. Our study highlights the genetic and morphologic SCN variability and provides evidence both for functional importance and redundancy of G-CSF receptor-mediated signaling in human granulopoiesis. © 2014 by The American Society of Hematology.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ion, A.; Telvi, L.; Galacteros, F.

We describe a pedigree presenting X-linked severe mental retardation associated with multiple congenital abnormalities and 46,XY gonadal dysgenesis, leading in one family member to female gender assignment. Female carriers are unaffected. The dysmorphic features are similar to those described in the {alpha}-thalassemia and mental retardation (ATR-X) syndrome, although there is no clinical evidence of {alpha}-thalassemia in this family. In addition, the family had other clinical features not previously observed in the ATR-X syndrome, including partial optic-nerve atrophy and partial ocular albinism. Mutations in a putative DNA helicase, termed XH2, have been reported to give rise to the ATR-X syndrome. Wemore » screened the YCH2 gene for mutations in affected members of the family and identified a 4-bp deletion at an intron/exon boundary that removes an invariant 3{prime} splice-acceptor site. The mutation cosegregates with the syndrome. The genomic deletion causes missplicing of the pre-mRNA, which results in the loss of 8 bp of coding sequence, thereby generating a frameshift and a downstream premature stop codon. Our finding increases the range of clinical features associated with mutations in the XH2 gene. 17 refs., 4 figs., 2 tabs.« less

Inherited biallelic CSF3R mutations in severe congenital neutropenia

PubMed Central

Triot, Alexa; Järvinen, Päivi M.; Arostegui, Juan I.; Murugan, Dhaarini; Kohistani, Naschla; Dapena Díaz, José Luis; Racek, Tomas; Puchałka, Jacek; Gertz, E. Michael; Schäffer, Alejandro A.; Kotlarz, Daniel; Pfeifer, Dietmar; Díaz de Heredia Rubio, Cristina; Ozdemir, Mehmet Akif; Patiroglu, Turkan; Karakukcu, Musa; Sánchez de Toledo Codina, José; Yagüe, Jordi; Touw, Ivo P.; Unal, Ekrem

2014-01-01

Severe congenital neutropenia (SCN) is characterized by low numbers of peripheral neutrophil granulocytes and a predisposition to life-threatening bacterial infections. We describe a novel genetic SCN type in 2 unrelated families associated with recessively inherited loss-of-function mutations in CSF3R, encoding the granulocyte colony-stimulating factor (G-CSF) receptor. Family A, with 3 affected children, carried a homozygous missense mutation (NM_000760.3:c.922C>T, NP_000751.1:p.Arg308Cys), which resulted in perturbed N-glycosylation and aberrant localization to the cell surface. Family B, with 1 affected infant, carried compound heterozygous deletions provoking frameshifts and premature stop codons (NM_000760.3:c.948_963del, NP_000751.1:p.Gly316fsTer322 and NM_000760.3:c.1245del, NP_000751.1:p.Gly415fsTer432). Despite peripheral SCN, all patients had morphologic evidence of full myeloid cell maturation in bone marrow. None of the patients responded to treatment with recombinant human G-CSF. Our study highlights the genetic and morphologic SCN variability and provides evidence both for functional importance and redundancy of G-CSF receptor-mediated signaling in human granulopoiesis. PMID:24753537

Identification a nonsense mutation of APC gene in Chinese patients with familial adenomatous polyposis.

PubMed

Li, Haishan; Zhang, Lingling; Jiang, Quan; Shi, Zhenwang; Tong, Hanxing

2017-04-01

Familial adenomatous polyposis (FAP; Mendelian of Inherintance in Man ID, 175100) is a rare autosomal dominant disorder characterized by the development of numerous adenomatous polyps throughout the colon and rectum associated with an increased risk of colorectal cancer. FAP is at time accompanied with certain extraintestinal manifestations such as congenital hypertrophy of the retinal pigment epithelium, dental disorders and desmoid tumors. It is caused by mutations in the adenomatous polyposis coli ( APC ) gene. The present study reported on a Chinese family with FAP. Polymerase chain reaction and direct sequencing of the full coding sequence of the APC gene were performed to identify the mutation in this family. A nonsense mutation of the APC gene was identified in this pedigree. It is a heterozygous G>T substitution at position 2,971 in exon 15 of the APC gene, which formed a premature stop codon at amino acid residue 991 (p.Glu991*). The resulting truncated protein lacked 1,853 amino acids. The present study expanded the database on APC gene mutations in FAP and enriched the spectrum of known germline mutations of the APC gene. Prophylactic proctocolectomy may be considered as a possible treatment for carriers of the mutation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kreysing, J.; Bohne, W.; Boesenberg, C.

The authors identified a patient suffering from late-infantile metachromatic leukodystrophy (MLD) who has a residual arylsulfatase A (ARSA) activity of about 10%. Fibroblasts of the patient show significant sulfatide degradation activity exceeding that of adult MLD patients. Analysis of the ARSA gene in this patient revealed heterozygosity for two new mutant alleles: in one allele, deletion of C 447 in exon 2 leads to a frameshift and to a premature stop codon at amino acid position 105; in the second allele, a G[yields]A transition in exon 5 causes a Gly[sup 309][yields]Ser substitution. Transient expression of the mutant Ser[sup 309]-ARSA resultedmore » in only 13% enzyme activity of that observed in cells expressing normal ARSA. The mutant ARSA is correctly targeted to the lyosomes but is unstable. These findings are in contrast to previous results showing that the late-infantile type of MLD is always associated with the complete absence of ARSA activity. The expression of the mutant ARSA protein may be influenced by particular features of oligodendrocytes, such that the level of mutant enzymes is lower in these cells than in others. 23 refs., 4 figs., 2 tabs.« less

Mutation in the Auxiliary Calcium-Channel Subunit CACNA2D4 Causes Autosomal Recessive Cone Dystrophy

PubMed Central

Wycisk, Katharina Agnes; Zeitz, Christina; Feil, Silke; Wittmer, Mariana; Forster, Ursula; Neidhardt, John; Wissinger, Bernd; Zrenner, Eberhart; Wilke, Robert; Kohl, Susanne; Berger, Wolfgang

2006-01-01

Retinal signal transmission depends on the activity of high voltage–gated l-type calcium channels in photoreceptor ribbon synapses. We recently identified a truncating frameshift mutation in the Cacna2d4 gene in a spontaneous mouse mutant with profound loss of retinal signaling and an abnormal morphology of ribbon synapses in rods and cones. The Cacna2d4 gene encodes an l-type calcium-channel auxiliary subunit of the α2δ type. Mutations in its human orthologue, CACNA2D4, were not yet known to be associated with a disease. We performed mutation analyses of 34 patients who received an initial diagnosis of night blindness, and, in two affected siblings, we detected a homozygous nucleotide substitution (c.2406C→A) in CACNA2D4. The mutation introduces a premature stop codon that truncates one-third of the corresponding open reading frame. Both patients share symptoms of slowly progressing cone dystrophy. These findings represent the first report of a mutation in the human CACNA2D4 gene and define a novel gene defect that causes autosomal recessive cone dystrophy. PMID:17033974

Recurrent pulmonary embolism associated with deep venous thrombosis diagnosed as protein s deficiency owing to a novel mutation in PROS1: A case report.

PubMed

Huang, Xiaojie; Xu, Fangfang; Assa, Carmel Rebecca; Shen, Laigen; Chen, Bing; Liu, Zhenjie

2018-05-01

Protein S (PS) deficiency that can be inherited or acquired is an independent risk factor for venous thromboembolism (VTE). In this report, we present a case of recurrent pulmonary embolism (PE) and deep venous thrombosis (DVT) due to PS deficiency. A 32-year-old male patient with significant decrease in PS activity was detected by laboratory tests. Genetic examination of the PROS1 gene showed a transition of G to T in exon 14 (c.1792 G>T, p.E598X), which was a paternal inherited heterozygous G1792T substitution in the laminin G-type repeat domain, generating a premature stop codon at Glu598. We considered that the inherited PS deficiency due to a PROS1 gene mutation may associate with recurrent VTE. The patient was suggested to have an extended anticoagulant therapy to avoid a severe VTE event. The patient was discharged home with continued oral anticoagulants and was still seen in clinic for follow-up. It is necessary for the young patient with recurrent idiopathic thrombosis to perform an inherited PS deficiency test and receive anticoagulant therapy for an extended period.

Mutated cancer autoantigen implicated cause of paraneoplastic myasthenia gravis.

PubMed

Zekeridou, Anastasia; Griesmann, Guy E; Lennon, Vanda A

2018-05-09

Anti-tumor immune responses are postulated to initiate paraneoplastic neurological disorders when proteins normally restricted to neural cells are expressed as oncoproteins. Mutated oncopeptides could bypass self-tolerant T-cells to activate cytotoxic effector T-lymphocytes and requisite helper T-lymphocytes to stimulate autoantibody production by B-lymphocytes. We investigated muscle-type nicotinic acetylcholine receptor (AChR) antigen expression at transcriptional and protein levels in a small-cell lung cancer line (SCLC) established from a patient with AChR-IgG-positive myasthenia gravis. I We identified mRNA transcripts encoding the two AChR α1-subunit isoforms, and seven alternative-splicing products, three yielding premature stop codons. Despite detecting native muscle-type AChR pentamers in the tumor, we did not identify mutant α1-peptides. However, we found α1-subunit-derived peptides bound to tumor MHC1-protein. In a control SCLC from an ANNA-1(anti-Hu)-IgG-positive patient, we identified MHC1-complexed Hu protein-derived peptides, but not AChR peptides. Our findings support onconeural protein products as pertinent immunogens initiating paraneoplastic neurological autoimmunity. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Complete mitogenome of the semi-aquatic grasshopper Oxya intricate (Stål.) (Insecta: Orthoptera: Catantopidae).

PubMed

Dong, Jia-Jia; Guan, De-Long; Xu, Sheng-Quan

2016-09-01

The complete mitogenome of Oxya intricate (Stål.) has been reconstructed from whole-genome Illumina sequencing data with an average coverage of 294×. The circular genome is 15,466 bp in length, and consists of 22 transfer RNAs (tRNAs), 13 protein-coding genes (PCGs), 2 ribosomal RNAs (rRNAs) and 1 D-loop region. All PCGs are initiated with ATN codons, and are terminated with TAR codons except for ND5 with the incomplete stop codon T. The nucleotide composition is asymmetric (42.5%A, 14.6%C, 10.6%G, 32.3%T) with an overall GC content of 25.2%. These data would contribute to the design of novel molecular markers for population and evolutionary studies of this and related orthopteran species.

Family acholeplasmataceae (including phytoplasmas)

USDA-ARS?s Scientific Manuscript database

The family Acholeplasmataceae was originally established to accommodate the genus Acholeplasma, comprising the mollicutes that could be cultivated without the supplement of cholesterol and that use UGA as a stop codon instead of coding for tryptophan. It was later shown that the phytoplasmas, a larg...

The Enterococcus faecalis EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT

PubMed Central

Montealegre, Maria Camila; La Rosa, Sabina Leanti; Roh, Jung Hyeob; Harvey, Barrett R.

2015-01-01

ABSTRACT The endocarditis and biofilm-associated pili (Ebp) are important in Enterococcus faecalis pathogenesis, and the pilus tip, EbpA, has been shown to play a major role in pilus biogenesis, biofilm formation, and experimental infections. Based on in silico analyses, we previously predicted that ATT is the EbpA translational start codon, not the ATG codon, 120 bp downstream of ATT, which is annotated as the translational start. ATT is rarely used to initiate protein synthesis, leading to our hypothesis that this codon participates in translational regulation of Ebp production. To investigate this possibility, site-directed mutagenesis was used to introduce consecutive stop codons in place of two lysines at positions 5 and 6 from the ATT, to replace the ATT codon in situ with ATG, and then to revert this ATG to ATT; translational fusions of ebpA to lacZ were also constructed to investigate the effect of these start codons on translation. Our results showed that the annotated ATG does not start translation of EbpA, implicating ATT as the start codon; moreover, the presence of ATT, compared to the engineered ATG, resulted in significantly decreased EbpA surface display, attenuated biofilm, and reduced adherence to fibrinogen. Corroborating these findings, the translational fusion with the native ATT as the initiation codon showed significantly decreased expression of β-galactosidase compared to the construct with ATG in place of ATT. Thus, these results demonstrate that the rare initiation codon of EbpA negatively regulates EbpA surface display and negatively affects Ebp-associated functions, including biofilm and adherence to fibrinogen. PMID:26015496

Systematic asymmetric nucleotide exchanges produce human mitochondrial RNAs cryptically encoding for overlapping protein coding genes.

PubMed

Seligmann, Hervé

2013-05-07

GenBank's EST database includes RNAs matching exactly human mitochondrial sequences assuming systematic asymmetric nucleotide exchange-transcription along exchange rules: A→G→C→U/T→A (12 ESTs), A→U/T→C→G→A (4 ESTs), C→G→U/T→C (3 ESTs), and A→C→G→U/T→A (1 EST), no RNAs correspond to other potential asymmetric exchange rules. Hypothetical polypeptides translated from nucleotide-exchanged human mitochondrial protein coding genes align with numerous GenBank proteins, predicted secondary structures resemble their putative GenBank homologue's. Two independent methods designed to detect overlapping genes (one based on nucleotide contents analyses in relation to replicative deamination gradients at third codon positions, and circular code analyses of codon contents based on frame redundancy), confirm nucleotide-exchange-encrypted overlapping genes. Methods converge on which genes are most probably active, and which not, and this for the various exchange rules. Mean EST lengths produced by different nucleotide exchanges are proportional to (a) extents that various bioinformatics analyses confirm the protein coding status of putative overlapping genes; (b) known kinetic chemistry parameters of the corresponding nucleotide substitutions by the human mitochondrial DNA polymerase gamma (nucleotide DNA misinsertion rates); (c) stop codon densities in predicted overlapping genes (stop codon readthrough and exchanging polymerization regulate gene expression by counterbalancing each other). Numerous rarely expressed proteins seem encoded within regular mitochondrial genes through asymmetric nucleotide exchange, avoiding lengthening genomes. Intersecting evidence between several independent approaches confirms the working hypothesis status of gene encryption by systematic nucleotide exchanges. Copyright © 2013 Elsevier Ltd. All rights reserved.

Novel mutations responsible for α-thalassemia in Iranian families.

PubMed

Bayat, Nooshin; Farashi, Samaneh; Hafezi-Nejad, Nima; Faramarzi, Negin; Ashki, Mehri; Vakili, Shadi; Imanian, Hashem; Khosravi, Mohsen; Azar-Keivan, Azita; Najmabadi, Hossein

2013-01-01

α-Thalassemia (α-thal) is usually caused by deletions on the α-globin gene cluster and the role of point mutations is less well investigated. In the present study, a total of 1048 individuals with hypochromic microcytic anemia, who did not present the most common α-thal deletions, were referred for α-globin gene DNA sequencing. The nucleotide changes were studied and a total of five new mutations was identified, of which three were located on the α2 gene [codon7 (Lys→Stop), codon 34 (Leu→Pro) and codon 83 (Leu→Arg)] and two on the α1 gene [IVS-I-116 (A>G) and codon 44 (+C)]. These novel mutations not only explain new findings by molecular analysis of the α-globin gene but also have clinical importance due to their changes in α-globin production in means of decreased hemoglobin (Hb) related values. Moreover, considerations of its role in combination with other mutations, and the possibility of causing Hb H (β4) are yet to be studied.

Polymerization of non-complementary RNA: systematic symmetric nucleotide exchanges mainly involving uracil produce mitochondrial RNA transcripts coding for cryptic overlapping genes.

PubMed

Seligmann, Hervé

2013-03-01

Usual DNA→RNA transcription exchanges T→U. Assuming different systematic symmetric nucleotide exchanges during translation, some GenBank RNAs match exactly human mitochondrial sequences (exchange rules listed in decreasing transcript frequencies): C↔U, A↔U, A↔U+C↔G (two nucleotide pairs exchanged), G↔U, A↔G, C↔G, none for A↔C, A↔G+C↔U, and A↔C+G↔U. Most unusual transcripts involve exchanging uracil. Independent measures of rates of rare replicational enzymatic DNA nucleotide misinsertions predict frequencies of RNA transcripts systematically exchanging the corresponding misinserted nucleotides. Exchange transcripts self-hybridize less than other gene regions, self-hybridization increases with length, suggesting endoribonuclease-limited elongation. Blast detects stop codon depleted putative protein coding overlapping genes within exchange-transcribed mitochondrial genes. These align with existing GenBank proteins (mainly metazoan origins, prokaryotic and viral origins underrepresented). These GenBank proteins frequently interact with RNA/DNA, are membrane transporters, or are typical of mitochondrial metabolism. Nucleotide exchange transcript frequencies increase with overlapping gene densities and stop densities, indicating finely tuned counterbalancing regulation of expression of systematic symmetric nucleotide exchange-encrypted proteins. Such expression necessitates combined activities of suppressor tRNAs matching stops, and nucleotide exchange transcription. Two independent properties confirm predicted exchanged overlap coding genes: discrepancy of third codon nucleotide contents from replicational deamination gradients, and codon usage according to circular code predictions. Predictions from both properties converge, especially for frequent nucleotide exchange types. Nucleotide exchanging transcription apparently increases coding densities of protein coding genes without lengthening genomes, revealing unsuspected functional DNA coding potential. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

The complete mitochondrial genome of Gryllotalpa unispina Saussure, 1874 (Orthoptera: Gryllotalpoidea: Gryllotalpidae).

PubMed

Zhang, Yulong; Shao, Dandan; Cai, Miao; Yin, Hong; Zhang, Daochuan

2016-01-01

The complete mitochondrial genome of Gryllotalpa unispina was 15,513 bp in length and contained 70.9% AT. All G. unispina protein-coding sequences except for the nad2 started with a typical ATN codon. The usual termination codons (TAA) and incomplete stop codons (T) were found from 13 protein-coding genes. All tRNA genes were folded into the typical cloverleaf secondary structure, except trnS(AGN) lacking the dihydrouridine arm. The sizes of the large and small ribosomal RNA genes were 1245 and 725 bp, respectively. The A + T-rich region was 917 bp in length with 76.8%. The orientation and gene order of the G. unispina mitogenome were identical to the G. orientalis and G. pluvialis, there was no phenomenon of "DK rearrangement" which has been widely reported in Caelifera.

The complete mitochondrial genome of Hydra vulgaris (Hydroida: Hydridae).

PubMed

Pan, Hong-Chun; Fang, Hong-Yan; Li, Shi-Wei; Liu, Jun-Hong; Wang, Ying; Wang, An-Tai

2014-12-01

The complete mitochondrial genome of Hydra vulgaris (Hydroida: Hydridae) is composed of two linear DNA molecules. The mitochondrial DNA (mtDNA) molecule 1 is 8010 bp long and contains six protein-coding genes, large subunit rRNA, methionine and tryptophan tRNAs, two pseudogenes consisting respectively of a partial copy of COI, and terminal sequences at two ends of the linear mtDNA, while the mtDNA molecule 2 is 7576 bp long and contains seven protein-coding genes, small subunit rRNA, methionine tRNA, a pseudogene consisting of a partial copy of COI and terminal sequences at two ends of the linear mtDNA. COI gene begins with GTG as start codon, whereas other 12 protein-coding genes start with a typical ATG initiation codon. In addition, all protein-coding genes are terminated with TAA as stop codon.

The complete mitochondrial genome of Setaria digitata (Nematoda: Filarioidea): Mitochondrial gene content, arrangement and composition compared with other nematodes.

PubMed

Yatawara, Lalani; Wickramasinghe, Susiji; Rajapakse, R P V J; Agatsuma, Takeshi

2010-09-01

In the present study, we determined the complete mitochondrial (mt) genome sequence (13,839bp) of parasitic nematode Setaria digitata and its structure and organization compared with Onchocerca volvulus, Dirofilaria immitis and Brugia malayi. The mt genome of S. digitata is slightly larger than the mt genomes of other filarial nematodes. S. digitata mt genome contains 36 genes (12 protein-coding genes, 22 transfer RNAs and 2 ribosomal RNAs) that are typically found in metazoans. This genome contains a high A+T (75.1%) content and low G+C content (24.9%). The mt gene order for S. digitata is the same as those for O. volvulus, D. immitis and B. malayi but it is distinctly different from other nematodes compared. The start codons inferred in the mt genome of S. digitata are TTT, ATT, TTG, ATG, GTT and ATA. Interestingly, the initiation codon TTT is unique to S. digitata mt genome and four protein-coding genes use this codon as a translation initiation codon. Five protein-coding genes use TAG as a stop codon whereas three genes use TAA and four genes use T as a termination codon. Out of 64 possible codons, only 57 are used for mitochondrial protein-coding genes of S. digitata. T-rich codons such as TTT (18.9%), GTT (7.9%), TTG (7.8%), TAT (7%), ATT (5.7%), TCT (4.8%) and TTA (4.1%) are used more frequently. This pattern of codon usage reflects the strong bias for T in the mt genome of S. digitata. In conclusion, the present investigation provides new molecular data for future studies of the comparative mitochondrial genomics and systematic of parasitic nematodes of socio-economic importance. 2010 Elsevier B.V. All rights reserved.

Differential functional readthrough over homozygous nonsense mutations contributes to the bleeding phenotype in coagulation factor VII deficiency.

PubMed

Branchini, A; Ferrarese, M; Lombardi, S; Mari, R; Bernardi, F; Pinotti, M

2016-10-01

Essentials Potentially null homozygous Factor(F)7 nonsense mutations are associated to variable bleeding symptoms. Readthrough of p.Ser112X (life-threatening) and p.Cys132X (moderate) stop codons was investigated. Readthrough-mediated insertion of wild-type or tolerated residues produce functional proteins. Functional readthrough over homozygous F7 nonsense mutations contributes to the bleeding phenotype. Background Whereas the rare homozygous nonsense mutations causing factor (F)VII deficiency may predict null conditions that are almost completely incompatible with life, they are associated with appreciable differences in hemorrhagic symptoms. The misrecognition of premature stop codons (readthrough) may account for variable levels of functional full-length proteins. Objectives To experimentally evaluate the basal and drug-induced levels of FVII resulting from the homozygous p.Cys132X and p.Ser112X nonsense mutations that are associated with moderate (132X) or life-threatening (112X) symptoms, and that are predicted to undergo readthrough with (132X) or without (112X) production of wild-type FVII. Methods We transiently expressed recombinant FVII (rFVII) nonsense and missense variants in human embryonic kidney 293 cells, and evaluated secreted FVII protein and functional levels by ELISA, activated FX generation, and coagulation assays. Results The levels of functional FVII produced by p.Cys132X and p.Ser112X mutants (rFVII-132X, 1.1% ± 0.2% of wild-type rFVII; rFVII-112X, 0.5% ± 0.1% of wild-type rFVII) were compatible with the occurrence of spontaneous readthrough, which was magnified by the addition of G418 - up to 12% of the wild-type value for the rFVII-132X nonsense variant. The predicted missense variants arising from readthrough abolished (rFVII-132Trp/Arg) or reduced (rFVII-112Trp/Cys/Arg, 22-45% of wild-type levels) secretion and function. These data suggest that the appreciable rescue of p.Cys132X function was driven by reinsertion of the wild-type residue, whereas the minimal p.Ser112X function was explained by missense changes permitting FVII secretion and function. Conclusions The extent of functional readthrough might explain differences in the bleeding phenotype of patients homozygous for F7 nonsense mutations, and prevent null conditions even for the most readthrough-unfavorable mutations. © 2016 International Society on Thrombosis and Haemostasis.

Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria

PubMed Central

Jackson, Christopher J; Norman, John E; Schnare, Murray N; Gray, Michael W; Keeling, Patrick J; Waller, Ross F

2007-01-01

Background Dinoflagellates comprise an ecologically significant and diverse eukaryotic phylum that is sister to the phylum containing apicomplexan endoparasites. The mitochondrial genome of apicomplexans is uniquely reduced in gene content and size, encoding only three proteins and two ribosomal RNAs (rRNAs) within a highly compacted 6 kb DNA. Dinoflagellate mitochondrial genomes have been comparatively poorly studied: limited available data suggest some similarities with apicomplexan mitochondrial genomes but an even more radical type of genomic organization. Here, we investigate structure, content and expression of dinoflagellate mitochondrial genomes. Results From two dinoflagellates, Crypthecodinium cohnii and Karlodinium micrum, we generated over 42 kb of mitochondrial genomic data that indicate a reduced gene content paralleling that of mitochondrial genomes in apicomplexans, i.e., only three protein-encoding genes and at least eight conserved components of the highly fragmented large and small subunit rRNAs. Unlike in apicomplexans, dinoflagellate mitochondrial genes occur in multiple copies, often as gene fragments, and in numerous genomic contexts. Analysis of cDNAs suggests several novel aspects of dinoflagellate mitochondrial gene expression. Polycistronic transcripts were found, standard start codons are absent, and oligoadenylation occurs upstream of stop codons, resulting in the absence of termination codons. Transcripts of at least one gene, cox3, are apparently trans-spliced to generate full-length mRNAs. RNA substitutional editing, a process previously identified for mRNAs in dinoflagellate mitochondria, is also implicated in rRNA expression. Conclusion The dinoflagellate mitochondrial genome shares the same gene complement and fragmentation of rRNA genes with its apicomplexan counterpart. However, it also exhibits several unique characteristics. Most notable are the expansion of gene copy numbers and their arrangements within the genome, RNA editing, loss of stop codons, and use of trans-splicing. PMID:17897476

Haploinsufficiency of HDAC4 Causes Brachydactyly Mental Retardation Syndrome, with Brachydactyly Type E, Developmental Delays, and Behavioral Problems

PubMed Central

Williams, Stephen R.; Aldred, Micheala A.; Der Kaloustian, Vazken M.; Halal, Fahed; Gowans, Gordon; McLeod, D. Ross; Zondag, Sara; Toriello, Helga V.; Magenis, R. Ellen; Elsea, Sarah H.

2010-01-01

Brachydactyly mental retardation syndrome (BDMR) is associated with a deletion involving chromosome 2q37. BDMR presents with a range of features, including intellectual disabilities, developmental delays, behavioral abnormalities, sleep disturbance, craniofacial and skeletal abnormalities (including brachydactyly type E), and autism spectrum disorder. To date, only large deletions of 2q37 have been reported, making delineation of a critical region and subsequent identification of candidate genes difficult. We present clinical and molecular analysis of six individuals with overlapping deletions involving 2q37.3 that refine the critical region, reducing the candidate genes from >20 to a single gene, histone deacetylase 4 (HDAC4). Driven by the distinct hand and foot anomalies and similar cognitive features, we identified other cases with clinical findings consistent with BDMR but without a 2q37 deletion, and sequencing of HDAC4 identified de novo mutations, including one intragenic deletion probably disrupting normal splicing and one intragenic insertion that results in a frameshift and premature stop codon. HDAC4 is a histone deacetylase that regulates genes important in bone, muscle, neurological, and cardiac development. Reportedly, Hdac4−/− mice have severe bone malformations resulting from premature ossification of developing bones. Data presented here show that deletion or mutation of HDAC4 results in reduced expression of RAI1, which causes Smith-Magenis syndrome when haploinsufficient, providing a link to the overlapping findings in these disorders. Considering the known molecular function of HDAC4 and the mouse knockout phenotype, taken together with deletion or mutation of HDAC4 in multiple subjects with BDMR, we conclude that haploinsufficiency of HDAC4 results in brachydactyly mental retardation syndrome. PMID:20691407

Haploinsufficiency of HDAC4 causes brachydactyly mental retardation syndrome, with brachydactyly type E, developmental delays, and behavioral problems.

PubMed

Williams, Stephen R; Aldred, Micheala A; Der Kaloustian, Vazken M; Halal, Fahed; Gowans, Gordon; McLeod, D Ross; Zondag, Sara; Toriello, Helga V; Magenis, R Ellen; Elsea, Sarah H

2010-08-13

Brachydactyly mental retardation syndrome (BDMR) is associated with a deletion involving chromosome 2q37. BDMR presents with a range of features, including intellectual disabilities, developmental delays, behavioral abnormalities, sleep disturbance, craniofacial and skeletal abnormalities (including brachydactyly type E), and autism spectrum disorder. To date, only large deletions of 2q37 have been reported, making delineation of a critical region and subsequent identification of candidate genes difficult. We present clinical and molecular analysis of six individuals with overlapping deletions involving 2q37.3 that refine the critical region, reducing the candidate genes from >20 to a single gene, histone deacetylase 4 (HDAC4). Driven by the distinct hand and foot anomalies and similar cognitive features, we identified other cases with clinical findings consistent with BDMR but without a 2q37 deletion, and sequencing of HDAC4 identified de novo mutations, including one intragenic deletion probably disrupting normal splicing and one intragenic insertion that results in a frameshift and premature stop codon. HDAC4 is a histone deacetylase that regulates genes important in bone, muscle, neurological, and cardiac development. Reportedly, Hdac4(-/-) mice have severe bone malformations resulting from premature ossification of developing bones. Data presented here show that deletion or mutation of HDAC4 results in reduced expression of RAI1, which causes Smith-Magenis syndrome when haploinsufficient, providing a link to the overlapping findings in these disorders. Considering the known molecular function of HDAC4 and the mouse knockout phenotype, taken together with deletion or mutation of HDAC4 in multiple subjects with BDMR, we conclude that haploinsufficiency of HDAC4 results in brachydactyly mental retardation syndrome.

Mapping of herpes simplex virus-1 neurovirulence to. gamma. sub 1 34. 5, a gene nonessential for growth in culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chou, J.; Roizman, B.; Kern, E.R.

1990-11-30

The gene designated {gamma}{sub 1}34.5 maps in the inverted repeats flanking the long unique sequence of herpes simplex virus-1 (HSV-1) DNA, and therefore it is present in two copies per genome. This gene is not essential for viral growth in cell culture. Four recombinant viruses were genetically engineered to test the function of this gene. These were (i) a virus from which both copies of the gene were deleted, (ii) a virus containing a stop codon in both copies of the gene, (iii) a virus containing after the first codon an insert encoding a 16-amino acid epitope known to reactmore » with a specific monoclonal antibody, and (iv) a virus in which the deleted sequences were restored. The viruses from which the gene was deleted or which carried stop codons were avirulent on intracerebral inoculation of mice. The virus with the gene tagged by the sequence encoding the epitope was moderately virulent, whereas the restored virus reacquired the phenotype of the parent virus. Significant amounts of virus were recovered only from brains of animals inoculated with virulent viruses. Inasmuch as the product of the {gamma}{sub 1}34.5 gene extended the host range of the virus by enabling it to replicate and destroy brain cells, it is a viral neurovirulence factor.« less

Introduction of translation stop condons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity

USGS Publications Warehouse

Garver, Kyle A.; Conway, Carla M.; Kurath, Gael

2006-01-01

A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine.

Identification of a novel mutation in the human growth hormone receptor gene (GHR) in a patient with Laron syndrome.

PubMed

Gennero, Isabelle; Edouard, Thomas; Rashad, Mona; Bieth, Eric; Conte-Aurio, Françoise; Marin, Françoise; Tauber, Maithé; Salles, Jean Pierre; El Kholy, Mohamed

2007-07-01

Deletions and mutations in the growth hormone receptor (GHR) gene are the underlying etiology of Laron syndrome (LS) or growth hormone (GH) insensitivity syndrome (GHIS), an autosomal recessive disease. Most patients are distributed in or originate from Mediterranean and Middle-Eastern countries. Sixty mutations have been described so far. We report a novel mutation in the GHR gene in a patient with LS. Genomic DNA sequencing of exon 5 revealed a TT insertion at nucleotide 422 after codon 122. The insertion resulted in a frameshift introducing a premature termination codon that led to a truncated receptor. We present clinical, biochemical and molecular evidence of LS as the result of this homozygous insertion.

The highly conserved codon following the slippery sequence supports -1 frameshift efficiency at the HIV-1 frameshift site.

PubMed

Mathew, Suneeth F; Crowe-McAuliffe, Caillan; Graves, Ryan; Cardno, Tony S; McKinney, Cushla; Poole, Elizabeth S; Tate, Warren P

2015-01-01

HIV-1 utilises -1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating -1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the 'intercodon') contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules-eRF1 protein or a cognate suppressor tRNA-were able to access and decode the intercodon prior to -1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted) ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.

The complete mitochondrial genome of Chinese green hydra, Hydra sinensis (Hydroida: Hydridae).

PubMed

Pan, Hong-Chun; Qian, Xiao-Cheng; Li, Ping; Li, Xiao-Fei; Wang, An-Tai

2014-02-01

The complete mitochondrial genome of Chinese green hydra, Hydra sinensis (Hydroida: Hydridae) is a linear molecule of 16,189 bp in length, containing 13 protein-coding genes, small and large subunit ribosomal RNAs, methionine and tryptophan transfer RNAs, a pseudogene consisting of a partial copy of COI and terminal sequences at two ends of the linear mitochondrial DNA. The A + T content of the overall base composition of H-strand is 77.2% (T: 41.7%; C: 10.9%; A: 35.5%; and G: 11.9%). COI and ND1 genes begin with GTG as start codon, while other 11 protein-coding genes start with a typical ATG initiation codon. COII, ATP8, ATP6, COIII, ND5, ND6, ND3, ND1, ND4 and COI genes are terminated with TAA as stop codon, ND4L ends with TAG, ND2 ends with TA and Cyt b ends with T.

Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice

PubMed Central

Hogan, Chad H.; Oldenburg, Darby G.; Kara, Mehmet

2018-01-01

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host. PMID:29390024

The origin of ambling horses.

PubMed

Wutke, Saskia; Andersson, Leif; Benecke, Norbert; Sandoval-Castellanos, Edson; Gonzalez, Javier; Hallsson, Jón Hallsteinn; Lõugas, Lembi; Magnell, Ola; Morales-Muniz, Arturo; Orlando, Ludovic; Pálsdóttir, Albína Hulda; Reissmann, Monika; Muñoz-Rodríguez, Mariana B; Ruttkay, Matej; Trinks, Alexandra; Hofreiter, Michael; Ludwig, Arne

2016-08-08

Horseback riding is the most fundamental use of domestic horses and has had a huge influence on the development of human societies for millennia. Over time, riding techniques and the style of riding improved. Therefore, horses with the ability to perform comfortable gaits (e.g. ambling or pacing), so-called 'gaited' horses, have been highly valued by humans, especially for long distance travel. Recently, the causative mutation for gaitedness in horses has been linked to a substitution causing a premature stop codon in the DMRT3 gene (DMRT3_Ser301STOP) [1]. In mice, Dmrt3 is expressed in spinal cord interneurons and plays an important role in the development of limb movement coordination [1]. Genotyping the position in 4396 modern horses from 141 breeds revealed that nowadays the mutated allele is distributed worldwide with an especially high frequency in gaited horses and breeds used for harness racing [2]. Here, we examine historic horse remains for the DMRT3 SNP, tracking the origin of gaitedness to Medieval England between 850 and 900 AD. The presence of the corresponding allele in Icelandic horses (9(th)-11(th) century) strongly suggests that ambling horses were brought from the British Isles to Iceland by Norse people. Considering the high frequency of the ambling allele in early Icelandic horses, we believe that Norse settlers selected for this comfortable mode of horse riding soon after arrival. The absence of the allele in samples from continental Europe (including Scandinavia) at this time implies that ambling horses may have spread from Iceland and maybe also the British Isles across the continent at a later date. Copyright © 2016 Elsevier Ltd. All rights reserved.

Endocrine and molecular investigations in a cohort of 25 adolescent males with prominent/persistent pubertal gynecomastia.

PubMed

Paris, F; Gaspari, L; Mbou, F; Philibert, P; Audran, F; Morel, Y; Biason-Lauber, A; Sultan, C

2016-03-01

Pubertal gynecomastia is a common condition observed in up to 65% of adolescent males. It is usually idiopathic and tends to regress within 1-2 years. In this descriptive cross-sectional study, we investigated 25 adolescent males with prominent (>B3) and/or persistent (>2 years) pubertal gynecomastia (P/PPG) to determine whether a hormonal/genetic defect might underline this condition. Endocrine investigation revealed the absence of hormonal disturbance for 18 boys (72%). Three patients presented Klinefelter syndrome and three a partial androgen insensitivity syndrome (PAIS) as a result of p.Ala646Asp and p.Ala45Gly mutations of the androgen receptor gene. The last patient showed a 17α-hydroxylase/17,20-lyase deficiency as a result of a compound heterozygous mutation of the CYP17A1 gene leading to p.Pro35Thr(P35T) and p.Arg239Stop(R239X) in the P450c17 protein. Enzymatic activity was analyzed: the mutant protein bearing the premature stop codon R239X showed a complete loss of 17α-hydroxylase and 17,20-lyase activity. The mutant P35T seemed to retain 15-20% of 17α-hydroxylase and about 8-10% of 17,20-lyase activity. This work demonstrates that P/PPG had an endocrine/genetic cause in 28% of our cases. PAIS may be expressed only by isolated gynecomastia as well as by 17α-hydroxylase/17,20-lyase deficiency. Isolated P/PPG is not always a 'physiological' condition and should thus be investigated through adequate endocrine and genetic investigations, even though larger studies are needed to better determine the real prevalence of genetic defects in such patients. © 2016 American Society of Andrology and European Academy of Andrology.

Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice.

PubMed

Van Skike, Nick D; Minkah, Nana K; Hogan, Chad H; Wu, Gary; Benziger, Peter T; Oldenburg, Darby G; Kara, Mehmet; Kim-Holzapfel, Deborah M; White, Douglas W; Tibbetts, Scott A; French, Jarrod B; Krug, Laurie T

2018-02-01

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host.

Mitochondrial genetic codes evolve to match amino acid requirements of proteins.

PubMed

Swire, Jonathan; Judson, Olivia P; Burt, Austin

2005-01-01

Mitochondria often use genetic codes different from the standard genetic code. Now that many mitochondrial genomes have been sequenced, these variant codes provide the first opportunity to examine empirically the processes that produce new genetic codes. The key question is: Are codon reassignments the sole result of mutation and genetic drift? Or are they the result of natural selection? Here we present an analysis of 24 phylogenetically independent codon reassignments in mitochondria. Although the mutation-drift hypothesis can explain reassignments from stop to an amino acid, we found that it cannot explain reassignments from one amino acid to another. In particular--and contrary to the predictions of the mutation-drift hypothesis--the codon involved in such a reassignment was not rare in the ancestral genome. Instead, such reassignments appear to take place while the codon is in use at an appreciable frequency. Moreover, the comparison of inferred amino acid usage in the ancestral genome with the neutral expectation shows that the amino acid gaining the codon was selectively favored over the amino acid losing the codon. These results are consistent with a simple model of weak selection on the amino acid composition of proteins in which codon reassignments are selected because they compensate for multiple slightly deleterious mutations throughout the mitochondrial genome. We propose that the selection pressure is for reduced protein synthesis cost: most reassignments give amino acids that are less expensive to synthesize. Taken together, our results strongly suggest that mitochondrial genetic codes evolve to match the amino acid requirements of proteins.

Mutations in SMG9, Encoding an Essential Component of Nonsense-Mediated Decay Machinery, Cause a Multiple Congenital Anomaly Syndrome in Humans and Mice

PubMed Central

Shaheen, Ranad; Anazi, Shams; Ben-Omran, Tawfeg; Seidahmed, Mohammed Zain; Caddle, L. Brianna; Palmer, Kristina; Ali, Rehab; Alshidi, Tarfa; Hagos, Samya; Goodwin, Leslie; Hashem, Mais; Wakil, Salma M.; Abouelhoda, Mohamed; Colak, Dilek; Murray, Stephen A.; Alkuraya, Fowzan S.

2016-01-01

Nonsense-mediated decay (NMD) is an important process that is best known for degrading transcripts that contain premature stop codons (PTCs) to mitigate their potentially harmful consequences, although its regulatory role encompasses other classes of transcripts as well. Despite the critical role of NMD at the cellular level, our knowledge about the consequences of deficiency of its components at the organismal level is largely limited to model organisms. In this study, we report two consanguineous families in which a similar pattern of congenital anomalies was found to be most likely caused by homozygous loss-of-function mutations in SMG9, encoding an essential component of the SURF complex that generates phospho-UPF1, the single most important step in NMD. By knocking out Smg9 in mice via CRISPR/Cas9, we were able to recapitulate the major features of the SMG9-related multiple congenital anomaly syndrome we observed in humans. Surprisingly, human cells devoid of SMG9 do not appear to have reduction of PTC-containing transcripts but do display global transcriptional dysregulation. We conclude that SMG9 is required for normal human and murine development, most likely through a transcriptional regulatory role, the precise nature of which remains to be determined. PMID:27018474

Intronic splicing mutations in PTCH1 cause Gorlin syndrome.

PubMed

Bholah, Zaynab; Smith, Miriam J; Byers, Helen J; Miles, Emma K; Evans, D Gareth; Newman, William G

2014-09-01

Gorlin syndrome is an autosomal dominant disorder characterized by multiple early-onset basal cell carcinoma, odontogenic keratocysts and skeletal abnormalities. It is caused by heterozygous mutations in the tumour suppressor PTCH1. Routine clinical genetic testing, by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to confirm a clinical diagnosis of Gorlin syndrome, identifies a mutation in 60-90 % of cases. We undertook RNA analysis on lymphocytes from ten individuals diagnosed with Gorlin syndrome, but without known PTCH1 mutations by exonic sequencing or MLPA. Two altered PTCH1 transcripts were identified. Genomic DNA sequence analysis identified an intron 7 mutation c.1068-10T>A, which created a strong cryptic splice acceptor site, leading to an intronic insertion of eight bases; this is predicted to create a frameshift p.(His358Alafs*12). Secondly, a deep intronic mutation c.2561-2057A>G caused an inframe insertion of 78 intronic bases in the cDNA transcript, leading to a premature stop codon p.(Gly854fs*3). The mutations are predicted to cause loss of function of PTCH1, consistent with its tumour suppressor function. The findings indicate the importance of RNA analysis to detect intronic mutations in PTCH1 not identified by routine screening techniques.

Truncating Mutations of MAGEL2, a Gene within the Prader-Willi Locus, Are Responsible for Severe Arthrogryposis

PubMed Central

Mejlachowicz, Dan; Nolent, Flora; Maluenda, Jérome; Ranjatoelina-Randrianaivo, Hanitra; Giuliano, Fabienne; Gut, Ivo; Sternberg, Damien; Laquerrière, Annie; Melki, Judith

2015-01-01

Arthrogryposis multiplex congenita (AMC) is characterized by the presence of multiple joint contractures resulting from reduced or absent fetal movement. Here, we report two unrelated families affected by lethal AMC. By genetic mapping and whole-exome sequencing in a multiplex family, a heterozygous truncating MAGEL2 mutation leading to frameshift and a premature stop codon (c.1996delC, p.Gln666Serfs∗36) and inherited from the father was identified in the probands. In another family, a distinct heterozygous truncating mutation leading to frameshift (c.2118delT, p.Leu708Trpfs∗7) and occurring de novo on the paternal allele of MAGEL2 was identified in the affected individual. In both families, RNA analysis identified the mutated paternal MAGEL2 transcripts only in affected individuals. MAGEL2 is one of the paternally expressed genes within the Prader-Willi syndrome (PWS) locus. PWS is associated with, to varying extents, reduced fetal mobility, severe infantile hypotonia, childhood-onset obesity, hypogonadism, and intellectual disability. MAGEL2 mutations have been recently reported in affected individuals with features resembling PWS and called Schaaf-Yang syndrome. Here, we show that paternal MAGEL2 mutations are also responsible for lethal AMC, recapitulating the clinical spectrum of PWS and suggesting that MAGEL2 is a PWS-determining gene. PMID:26365340

Novel folliculin (FLCN) mutation and familial spontaneous pneumothorax.

PubMed

Zhu, J-F; Shen, X-Q; Zhu, F; Tian, L

2017-01-01

Familial spontaneous pneumothorax is one of the characteristics of Birt-Hogg-Dubé syndrome (BHDS), which is an autosomal dominant disease caused by the mutation of folliculin (FLCN). To investigate the mutation of FLCN gene in a familial spontaneous pneumothorax. Prospective case study. Clinical and genetic data of a Chinese family with four patients who presented spontaneous pneumothorax in the absence of skin lesions or renal tumors were collected. CT scan of patient's lung was applied for observation of pneumothorax. DNA sequencing of the coding exons (4-14 exons) of FLCN was performed for all 11 members of the family and 100 unrelated healthy controls. CT scan of patient's lung showed spontaneous pneumothorax. A mutation (c. 510C > G) that leads to a premature stop codon (p. Y170X) was found in the proband using DNA sequencing of coding exons (4-14 exons) of FLCN. This mutation was also observed in the other affected members of the family. A nonsense mutation of FLCN was found in a spontaneous pneumothorax family. Our results expand the mutational spectrum of FLCN in patients with BHDS. © The Author 2016. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Autism-Associated Insertion Mutation (InsG) of Shank3 Exon 21 Causes Impaired Synaptic Transmission and Behavioral Deficits

PubMed Central

Speed, Haley E.; Kouser, Mehreen; Xuan, Zhong; Reimers, Jeremy M.; Ochoa, Christine F.; Gupta, Natasha; Liu, Shunan

2015-01-01

SHANK3 (also known as PROSAP2) is a postsynaptic scaffolding protein at excitatory synapses in which mutations and deletions have been implicated in patients with idiopathic autism, Phelan–McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. In this study, we have created a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3G). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3G/G mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3G/G mice also exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. This work identifies clear alterations in synaptic function and behavior in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. Furthermore, these findings lay the foundation for future studies aimed to validate and study region-selective and temporally selective genetic reversal studies in the Shank3G/G mouse that was engineered with such future experiments in mind. PMID:26134648

WNT10A mutation results in severe tooth agenesis in a family of three sisters.

PubMed

Abid, M F; Simpson, M A; Barbosa, I A; Seppala, M; Irving, M; Sharpe, P T; Cobourne, M T

2018-06-21

To identify the genetic basis of severe tooth agenesis in a family of three affected sisters. A family of three sisters with severe tooth agenesis was recruited for whole-exome sequencing to identify potential genetic variation responsible for this penetrant phenotype. The unaffected father was tested for specific mutations using Sanger sequencing. Gene discovery was supplemented with in situ hybridization to localize gene expression during human tooth development. We report a nonsense heterozygous mutation in exon 2 of WNT10A c.321C>A[p.Cys107*] likely to be responsible for the severe tooth agenesis identified in this family through the creation of a premature stop codon, resulting in truncation of the amino acid sequence and therefore loss of protein function. In situ hybridization showed expression of WNT10A in odontogenic epithelium during the early and late stages of human primary tooth development. WNT10A has previously been associated with both syndromic and non-syndromic forms of tooth agenesis, and this report further expands our knowledge of genetic variation underlying non-syndromic forms of this condition. We also demonstrate expression of WNT10A in the epithelial compartment of human tooth germs during development. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thangavelu, Pulari U.; Gupta, Vipul; Dixit, Narendra M., E-mail: narendra@chemeng.iisc.ernet.in

The contest between the host factor APOBEC3G (A3G) and the HIV-1 protein Vif presents an attractive target of intervention. The extent to which the A3G–Vif interaction must be suppressed to tilt the balance in favor of A3G remains unknown. We employed stochastic simulations and mathematical modeling of the within-host dynamics and evolution of HIV-1 to estimate the fraction of progeny virions that must incorporate A3G to render productive infection unsustainable. Using three different approaches, we found consistently that a transition from sustained infection to suppression of productive infection occurred when the latter fraction exceeded ∼0.8. The transition was triggered bymore » A3G-induced hypermutations that led to premature stop codons compromising viral production and was consistent with driving the basic reproductive number, R{sub 0}, below unity. The fraction identified may serve as a quantitative guideline for strategies targeting the A3G–Vif axis. - Highlights: • We perform simulations and mathematical modeling of the role of APOBEC3G in suppressing HIV-1 infection. • In three distinct ways, we estimate that when over 80% of progeny virions carry APOBEC3G, productive HIV-1 infection would be suppressed. • Our estimate of this critical fraction presents quantitative guidelines for strategies targeting the APOBEC3G–Vif axis.« less

Generation of obese rat model by transcription activator-like effector nucleases targeting the leptin receptor gene.

PubMed

Chen, Yuting; Lu, Wenqing; Gao, Na; Long, Yi; Shao, Yanjiao; Liu, Meizhen; Chen, Huaqing; Ye, Shixin; Ma, Xueyun; Liu, Mingyao; Li, Dali

2017-02-01

The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases (TALENs) technology to generate Leptin receptor (Lepr) knockout rats on the Sprague Dawley (SD) genetic background. Through direct injection of in vitro transcribed mRNA of TALEN pairs into SD rat zygotes, somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.

A family of oculofaciocardiodental syndrome (OFCD) with a novel BCOR mutation and genomic rearrangements involving NHS.

PubMed

Kondo, Yukiko; Saitsu, Hirotomo; Miyamoto, Toshinobu; Nishiyama, Kiyomi; Tsurusaki, Yoshinori; Doi, Hiroshi; Miyake, Noriko; Ryoo, Na-Kyung; Kim, Jeong Hun; Yu, Young Suk; Matsumoto, Naomichi

2012-03-01

Oculofaciocardiodental syndrome (OFCD) is an X-linked dominant disorder associated with male lethality, presenting with congenital cataract, dysmorphic face, dental abnormalities and septal heart defects. Mutations in BCOR (encoding BCL-6-interacting corepressor) cause OFCD. Here, we report on a Korean family with common features of OFCD including bilateral 2nd-3rd toe syndactyly and septal heart defects in three affected females (mother and two daughters). Through the mutation screening and copy number analysis using genomic microarray, we identified a novel heterozygous mutation, c.888delG, in the BCOR gene and two interstitial microduplications at Xp22.2-22.13 and Xp21.3 in all the three affected females. The BCOR mutation may lead to a premature stop codon (p.N297IfsX80). The duplication at Xp22.2-22.13 involved the NHS gene causative for Nance-Horan syndrome, which is an X-linked disorder showing similar clinical features with OFCD in affected males, and in carrier females with milder presentation. Considering the presence of bilateral 2nd-3rd toe syndactyly and septal heart defects, which is unique to OFCD, the mutation in BCOR is likely to be the major determinant for the phenotypes in this family.

Analysis of a Spontaneous Non-Motile and Avirulent Mutant Shows That FliM Is Required for Full Endoflagella Assembly in Leptospira interrogans.

PubMed

Fontana, Célia; Lambert, Ambroise; Benaroudj, Nadia; Gasparini, David; Gorgette, Olivier; Cachet, Nathalie; Bomchil, Natalia; Picardeau, Mathieu

2016-01-01

Pathogenic Leptospira strains are responsible for leptospirosis, a worldwide emerging zoonotic disease. These spirochetes are unique amongst bacteria because of their corkscrew-like cell morphology and their periplasmic flagella. Motility is reported as an important virulence determinant, probably favoring entry and dissemination of pathogenic Leptospira in the host. However, proteins constituting the periplasmic flagella and their role in cell shape, motility and virulence remain poorly described. In this study, we characterized a spontaneous L. interrogans mutant strain lacking motility, correlated with the loss of the characteristic hook-shaped ends, and virulence in the animal model. Whole genome sequencing allowed the identification of one nucleotide deletion in the fliM gene resulting in a premature stop codon, thereby preventing the production of flagellar motor switch protein FliM. Genetic complementation restored cell morphology, motility and virulence comparable to those of wild type cells. Analyses of purified periplasmic flagella revealed a defect in flagella assembly, resulting in shortened flagella compared to the wild type strain. This also correlated with a lower amount of major filament proteins FlaA and FlaB. Altogether, these findings demonstrate that FliM is required for full and correct assembly of the flagella which is essential for motility and virulence.

Brooke-Spiegler syndrome: report of 10 patients from 8 families with novel germline mutations: evidence of diverse somatic mutations in the same patient regardless of tumor type.

PubMed

Sima, Radek; Vanecek, Tomas; Kacerovska, Denisa; Trubac, Pavel; Cribier, Bernard; Rutten, Arno; Vazmitel, Marina; Spagnolo, Dominic V; Litvik, Radek; Vantuchova, Yvetta; Weyers, Wolfgang; Pearce, Robert L; Pearn, John; Michal, Michal; Kazakov, Dmitry V

2010-06-01

Brooke-Spiegler syndrome (BSS) is an inherited autosomal dominant disease characterized by the development of multiple adnexal cutaneous neoplasms including spiradenoma, cylindroma, spiradenocylindroma, and trichoepithelioma (cribriform trichoblastoma). BSS patients have various mutations in the CYLD gene, a tumor suppressor gene located on chromosome 16q. Our search of the literature revealed 51 germline CYLD mutations reported to date. Somatic CYLD mutations have rarely been investigated. We studied 10 patients from 8 families with BSS. Analysis of germline mutations of the CYLD gene was performed using either peripheral blood or nontumorous tissue. In addition, 19 formalin-fixed paraffin-embedded tumor samples were analyzed for somatic mutations, including loss of heterozygosity studies. A total of 38 tumors were available for histopathologic review. We have identified 8 novel germline mutations, all of which consisted of substitutions, deletions, and insertions/duplications and all except one led to premature stop codons. The substitution mutation in a single case was also predicted to disrupt protein function and seems causally implicated in tumor formation. We demonstrate for the first time that somatic events, loss of heterozygosity, or sequence mutations may differ among multiple neoplasms even of the same histologic type, occurring in the same patient.

Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping.

PubMed

Gao, Quan Q; Wyatt, Eugene; Goldstein, Jeff A; LoPresti, Peter; Castillo, Lisa M; Gazda, Alec; Petrossian, Natalie; Earley, Judy U; Hadhazy, Michele; Barefield, David Y; Demonbreun, Alexis R; Bönnemann, Carsten; Wolf, Matthew; McNally, Elizabeth M

2015-11-02

Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations.

Mis-Spliced Lr34 Transcript Events in Winter Wheat.

PubMed

Fang, Tilin; Carver, Brett F; Hunger, Robert M; Yan, Liuling

2017-01-01

Lr34 in wheat is a non-race-specific gene that confers resistance against multiple fungal pathogens. The resistant allele Lr34 and the susceptible allele Lr34s can be distinguished by three polymorphisms that cause alternation of deduced amino acid sequences of Lr34 at the protein level. In seedlings of a cultivar carrying the resistant Lr34r allele, only a portion (35%) of its transcripts was correctly spliced and the majority (65%) of its transcripts were incorrectly spliced due to multiple mis-splicing events. Lr34 mis-splicing events were also observed at adult plant age when this gene exerts its function. All of the mis-spliced Lr34r cDNA transcripts observed in this study resulted in a premature stop codon due to a shift of the open reading frame; hence, the mis-spliced Lr34r cDNAs were deduced to encode incomplete proteins. Even if a cultivar has a functional Lr34 gene, its transcripts might not completely splice in a correct pattern. These findings suggested that the partial resistance conferred by a quantitative gene might be due to mis-splicing events in its transcripts; hence, the resistance of the gene could be increased by eliminating or mutating regulators that cause mis-splicing events in wheat.

PLEKHG5 deficiency leads to an intermediate form of autosomal-recessive Charcot–Marie–Tooth disease

PubMed Central

Azzedine, Hamid; Zavadakova, Petra; Planté-Bordeneuve, Violaine; Vaz Pato, Maria; Pinto, Nuno; Bartesaghi, Luca; Zenker, Jennifer; Poirot, Olivier; Bernard-Marissal, Nathalie; Arnaud Gouttenoire, Estelle; Cartoni, Romain; Title, Alexandra; Venturini, Giulia; Médard, Jean-Jacques; Makowski, Edward; Schöls, Ludger; Claeys, Kristl G.; Stendel, Claudia; Roos, Andreas; Weis, Joachim; Dubourg, Odile; Leal Loureiro, José; Stevanin, Giovanni; Said, Gérard; Amato, Anthony; Baraban, Jay; LeGuern, Eric; Senderek, Jan; Rivolta, Carlo; Chrast, Roman

2013-01-01

Charcot–Marie–Tooth disease (CMT) comprises a clinically and genetically heterogeneous group of peripheral neuropathies characterized by progressive distal muscle weakness and atrophy, foot deformities and distal sensory loss. Following the analysis of two consanguineous families affected by a medium to late-onset recessive form of intermediate CMT, we identified overlapping regions of homozygosity on chromosome 1p36 with a combined maximum LOD score of 5.4. Molecular investigation of the genes from this region allowed identification of two homozygous mutations in PLEKHG5 that produce premature stop codons and are predicted to result in functional null alleles. Analysis of Plekhg5 in the mouse revealed that this gene is expressed in neurons and glial cells of the peripheral nervous system, and that knockout mice display reduced nerve conduction velocities that are comparable with those of affected individuals from both families. Interestingly, a homozygous PLEKHG5 missense mutation was previously reported in a recessive form of severe childhood onset lower motor neuron disease (LMND) leading to loss of the ability to walk and need for respiratory assistance. Together, these observations indicate that different mutations in PLEKHG5 lead to clinically diverse outcomes (intermediate CMT or LMND) affecting the function of neurons and glial cells. PMID:23777631

A rare male patient with classic Rett syndrome caused by MeCP2_e1 mutation.

PubMed

Tokaji, Narumi; Ito, Hiromichi; Kohmoto, Tomohiro; Naruto, Takuya; Takahashi, Rizu; Goji, Aya; Mori, Tatsuo; Toda, Yoshihiro; Saito, Masako; Tange, Shoichiro; Masuda, Kiyoshi; Kagami, Shoji; Imoto, Issei

2018-03-01

Rett syndrome (RTT) is a severe neurodevelopmental disorder typically affecting females. It is mainly caused by loss-of-function mutations that affect the coding sequence of exon 3 or 4 of methyl-CpG-binding protein 2 (MECP2). Severe neonatal encephalopathy resulting in death before the age of 2 years is the most common phenotype observed in males affected by a pathogenic MECP2 variant. Mutations in MECP2 exon 1 affecting the MeCP2_e1 isoform are relatively rare causes of RTT in females, and only one case of a male patient with MECP2-related severe neonatal encephalopathy caused by a mutation in MECP2 exon 1 has been reported. This is the first reported case of a male with classic RTT caused by a 5-bp duplication in the open-reading frame of MECP2 exon 1 (NM_001110792.1:c.23_27dup) that introduced a premature stop codon [p.(Ser10Argfs*36)] in the MeCP2_e1 isoform, which has been reported in one female patient with classic RTT. Therefore, both males and females displaying at least some type of MeCP2_e1 mutation may exhibit the classic RTT phenotype. © 2018 Wiley Periodicals, Inc.

Mx1 and Mx2 key antiviral proteins are surprisingly lost in toothed whales

PubMed Central

Braun, Benjamin A.; Marcovitz, Amir; Camp, J. Gray; Jia, Robin; Bejerano, Gill

2015-01-01

Viral outbreaks in dolphins and other Delphinoidea family members warrant investigation into the integrity of the cetacean immune system. The dynamin-like GTPase genes Myxovirus 1 (Mx1) and Mx2 defend mammals against a broad range of viral infections. Loss of Mx1 function in human and mice enhances infectivity by multiple RNA and DNA viruses, including orthomyxoviruses (influenza A), paramyxoviruses (measles), and hepadnaviruses (hepatitis B), whereas loss of Mx2 function leads to decreased resistance to HIV-1 and other viruses. Here we show that both Mx1 and Mx2 have been rendered nonfunctional in Odontoceti cetaceans (toothed whales, including dolphins and orcas). We discovered multiple exon deletions, frameshift mutations, premature stop codons, and transcriptional evidence of decay in the coding sequence of both Mx1 and Mx2 in four species of Odontocetes. We trace the likely loss event for both proteins to soon after the divergence of Odontocetes and Mystocetes (baleen whales) ∼33–37 Mya. Our data raise intriguing questions as to what drove the loss of both Mx1 and Mx2 genes in the Odontoceti lineage, a double loss seen in none of 56 other mammalian genomes, and suggests a hitherto unappreciated fundamental genetic difference in the way these magnificent mammals respond to viral infections. PMID:26080416

A nonsense mutation of PEPD in four Amish children with prolidase deficiency.

PubMed

Wang, Heng; Kurien, Biji T; Lundgren, David; Patel, Nisha C; Kaufman, K M; Miller, David L; Porter, Andrew C; D'Souza, Anil; Nye, Leah; Tumbush, John; Hupertz, Vera; Kerr, Douglas S; Kurono, S; Matsumoto, H; Scofield, R Hal

2006-03-15

Encoded by the peptidase D (PEPD) gene located at 19q12-q13.11, prolidase is a ubiquitous cytosolic enzyme that catalyzes hydrolysis of oligopeptides with a C-terminal proline or hydroxyproline. We describe here four Amish children with a severe phenotype of prolidase deficiency in the Geauga settlements of Ohio as the first report of prolidase deficiency in the Amish population as well as in the United States. The patients presented with infection, hepatosplenomegaly, or thrombocytopenia, in contrast to most cases previously reported in the literature, presenting with skin ulcers. All four patients had typical facial features, classic skin ulcers, and multisystem involvement. Recurrent infections, asthma-like chronic reactive airway disease, hyperimmunoglobulins, hepatosplenomegaly with mildly elevated aspartate transaminase (AST), anemia, and thrombocytopenia were common and massive imidodipeptiduria was universal. Prolidase activity in our patients is nearly undetectable. Direct sequencing of PCR-amplified genomic DNA for all of the exons from the four patients revealed the same homozygous single nucleotide mutation c.793 T > C in exon 11, resulting in a premature stop-codon at amino acid residue 265 (p.R265X). It is speculated that the severe phenotype in these patients might be associated with the type of the PEPD gene mutation. 2006 Wiley-Liss, Inc.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing.

PubMed

Elaswad, Ahmed; Khalil, Karim; Cline, David; Page-McCaw, Patrick; Chen, Wenbiao; Michel, Maximilian; Cone, Roger; Dunham, Rex

2018-01-20

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Reduced Anthocyanins in Petioles codes for a GST anthocyanin transporter that is essential for the foliage and fruit coloration in strawberry.

PubMed

Luo, Huifeng; Dai, Cheng; Li, Yongping; Feng, Jia; Liu, Zhongchi; Kang, Chunying

2018-04-27

The red color of the foliage and fruit in strawberry comes from anthocyanins stored in the vacuole; however, how this anthocyanin accumulation is regulated remains unclear. A reduced anthocyanin in petioles (rap) mutant was identified in an N-ethyl-N-nitrosourea (ENU) mutagenized population of YW5AF7, a white-fruited variety of the wild strawberry Fragaria vesca. The causative mutation was identified to be a premature stop codon in a glutathione S-transferase (GST) gene. In addition to the foliage coloration, RAP also mediates fruit pigmentation and acts downstream of the fruit-specific transcription factor FvMYB10. Among all eight GST genes in the same subfamily, RAP is most abundantly expressed in the ripening fruit. Expression analysis and transient expression assays demonstrated that RAP is the principal transporter of anthocyanins among the paralogs. Moreover, domain-swap experiments showed that both the N- and C-terminals of RAP are essential for the binding capability of anthocyanins. In addition, transient knock-down of RAP resulted in reduced fruit coloration in cultivated strawberry. Collectively, our results demonstrate that RAP encodes the principal GST transporter of anthocyanins in the strawberry foliage and fruit, and it could be modified to alter the fruit color in strawberry.

Using Microarrays to Facilitate Positional Cloning: Identification of Tomosyn as an Inhibitor of Neurosecretion

PubMed Central

Dybbs, Michael; Ngai, John; Kaplan, Joshua M

2005-01-01

Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate positional cloning. This method relies on the fact that premature stop codons (i.e., nonsense mutations) constitute a frequent class of mutations isolated in screens and that nonsense mutant messenger RNAs are efficiently degraded by the conserved nonsense-mediated decay pathway. They validate this strategy by identifying two previously uncharacterized mutations: (1) tom-1, a mutation found in a forward genetic screen for enhanced acetylcholine secretion in Caenorhabditis elegans, and (2) an apparently spontaneous mutation in the hif-1 transcription factor gene. They further demonstrate the broad applicability of this strategy using other known mutants in C. elegans, Arabidopsis, and mouse. Characterization of tom-1 mutants suggests that TOM-1, the C. elegans ortholog of mammalian tomosyn, functions as an endogenous inhibitor of neurotransmitter secretion. These results also suggest that microarray hybridizations have the potential to significantly reduce the time and effort required for positional cloning. PMID:16103915

Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping

PubMed Central

Gao, Quan Q.; Wyatt, Eugene; Goldstein, Jeff A.; LoPresti, Peter; Castillo, Lisa M.; Gazda, Alec; Petrossian, Natalie; Earley, Judy U.; Hadhazy, Michele; Barefield, David Y.; Demonbreun, Alexis R.; Bönnemann, Carsten; Wolf, Matthew; McNally, Elizabeth M.

2015-01-01

Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations. PMID:26457733

Two novel compound heterozygous mutations in the BCKDHB gene that cause the intermittent form of maple syrup urine disease.

PubMed

Guo, Yi; Liming, Liu; Jiang, Li

2015-12-01

Intermittent maple syrup urine disease (MSUD) is a potentially life-threatening metabolic disorder caused by a deficiency of branched chain α-ketoacid dehydrogenase (BCKD) complex. In contrast to classic MSUD, children with the intermittent form usually have an atypical clinical manifestation. Here, we describe the presenting symptoms and clinical course of a Chinese boy with intermittent MSUD. Mutation analysis identified two previously unreported mutations in exon 7 of the BCKDHB gene: c.767A > G (p.Y256C) and c.768C > G (p.Y256X); the parents were each heterozygous for one of these mutations. In silico analysis predicted Y256C probably affects protein structure; Y256X leads to a premature stop codon. This case demonstrates intermittent MSUD should be suspected in cases with symptoms of recurrent encephalopathy, especially ataxia or marked drowsiness, which usually present after the neonatal period and in conjunction with infection. symmetrical basal ganglia damage but normal myelination in the posterior limb will assist differential diagnosis; alloisoleucine is a useful diagnostic marker and mutation analysis may be of prognostic value. These novel mutations Y256C and Y256X result in the clinical manifestation of a variant form of MSUD, expanding the mutation spectrum of this disease.

Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay

PubMed Central

Nguyen, Thi A.; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M.; Xu, Xialian; Miller, Bruce L.; Rigo, Frank; Ferguson, Shawn M.; Walther, Tobias C.; Farese, Robert V.

2018-01-01

Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized GrnR493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous GrnR493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in GrnR493X mice and cell lines and in fibroblasts from patients containing the GRNR493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. PMID:29511098

A Frameshift Mutation within LAMC2 Is Responsible for Herlitz Type Junctional Epidermolysis Bullosa (HJEB) in Black Headed Mutton Sheep

PubMed Central

Mömke, Stefanie; Kerkmann, Andrea; Wöhlke, Anne; Ostmeier, Miriam; Hewicker-Trautwein, Marion; Ganter, Martin; Kijas, James; Distl, Ottmar

2011-01-01

Junctional epidermolysis bullosa (JEB) is a hereditary mechanobullous skin disease in humans and animals. A Herlitz type JEB was identified in German Black Headed Mutton (BHM) sheep and affected lambs were reproduced in a breeding trial. Affected lambs showed skin and mucous membranes blistering and all affected lambs died within the first weeks of life. The pedigree data were consistent with a monogenic autosomal recessive inheritance. Immunofluorescence showed a reduced expression of laminin 5 protein which consists of 3 subunits encoded by the genes LAMA3, LAMB3 and LAMC2. We screened these genes for polymorphisms. Linkage and genome-wide association analyses identified LAMC2 as the most likely candidate for HJEB. A two base pair deletion within exon 18 of the LAMC2 gene (FM872310:c.2746delCA) causes a frameshift mutation resulting in a premature stop codon (p.A928*) 13 triplets downstream of this mutation and in addition, introduces an alternative splicing of exon 18 LAMC2. This deletion showed a perfect co-segregation with HJEB in all 740 analysed BHM sheep. Identification of the LAMC2 deletion means an animal model for HJEB is now available to develop therapeutic approaches of relevance to the human form of this disease. PMID:21573221

MC1R studies in dogs with melanistic mask or brindle patterns.

PubMed

Schmutz, S M; Berryere, T G; Ellinwood, N M; Kerns, J A; Barsh, G S

2003-01-01

Black mask is a characteristic pattern in which red, yellow, tan, fawn, or brindle dogs exhibit a melanistic muzzle which may extend up onto the ears. Melanistic mask is inherited in several breeds as an autosomal dominant trait, and appears to be a fixed trait in a few breeds of dogs. A MC1R nonsense mutation, R306ter, has been shown to cause a completely red or yellow coat color in certain breeds such as Irish setters, yellow Labrador retrievers, and golden retrievers. The amino acid sequence for the melanocortin receptor 1 gene (MC1R) was examined in 17 dogs with melanistic masks from seven breeds, 19 dogs without melanistic masks, and 7 dogs in which their coat color made the mask difficult to distinguish. We also examined nine brindle dogs of four breeds, including three dogs who also had a black mask. No consistent amino acid change was observed in the brindle dogs. All dogs with a melanistic mask had at least one copy of a valine substitution for methionine at amino acid 264 (M264V) and none were homozygous for the premature stop codon (R306ter). These results suggest that black mask, but not brindle, is caused by a specific MC1R allele.

Construction of "Toxin Complex" in a Mutant Serotype C Strain of Clostridium botulinum Harboring a Defective Neurotoxin Gene.

PubMed

Suzuki, Tomonori; Nagano, Thomas; Niwa, Koichi; Uchino, Masataka; Tomizawa, Motohiro; Sagane, Yoshimasa; Watanabe, Toshihiro

2017-01-01

A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the "toxin complex." Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the "toxin complex" produced by C-N71. The "toxin complex" was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the "toxin complex." Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic "toxin complex" of C-N71 may be valuable for the development of an oral drug delivery system.

Mx1 and Mx2 key antiviral proteins are surprisingly lost in toothed whales.

PubMed

Braun, Benjamin A; Marcovitz, Amir; Camp, J Gray; Jia, Robin; Bejerano, Gill

2015-06-30

Viral outbreaks in dolphins and other Delphinoidea family members warrant investigation into the integrity of the cetacean immune system. The dynamin-like GTPase genes Myxovirus 1 (Mx1) and Mx2 defend mammals against a broad range of viral infections. Loss of Mx1 function in human and mice enhances infectivity by multiple RNA and DNA viruses, including orthomyxoviruses (influenza A), paramyxoviruses (measles), and hepadnaviruses (hepatitis B), whereas loss of Mx2 function leads to decreased resistance to HIV-1 and other viruses. Here we show that both Mx1 and Mx2 have been rendered nonfunctional in Odontoceti cetaceans (toothed whales, including dolphins and orcas). We discovered multiple exon deletions, frameshift mutations, premature stop codons, and transcriptional evidence of decay in the coding sequence of both Mx1 and Mx2 in four species of Odontocetes. We trace the likely loss event for both proteins to soon after the divergence of Odontocetes and Mystocetes (baleen whales) ∼33-37 Mya. Our data raise intriguing questions as to what drove the loss of both Mx1 and Mx2 genes in the Odontoceti lineage, a double loss seen in none of 56 other mammalian genomes, and suggests a hitherto unappreciated fundamental genetic difference in the way these magnificent mammals respond to viral infections.

A novel frameshift variant in the CADASIL gene NOTCH3: pathogenic or not?

PubMed

Schubert, V; Bender, B; Kinzel, M; Peters, N; Freilinger, T

2018-06-01

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) represents the most common monogenic cause of adult-onset ischemic stroke and vascular dementia. It is caused by heterozygous missense mutations in the NOTCH3 gene, encoding a transmembrane receptor protein on vascular smooth muscle cells. Classical CADASIL mutations affect conserved cysteine residues of the Notch3 protein. By contrast, the role of non-canonical genetic variation in NOTCH3, in particular of variants causing a hypomorphic Notch3 protein, is subject to an ongoing scientific debate. In this context, we here report a novel NOTCH3 frameshift variant in exon 18 (NM_000435.2: c.2853_2857delTCCCG), causing a frameshift and introducing a premature stop codon, which was detected in a 43-year-old woman and her father. Both carriers of the variant were carefully evaluated, including serial follow-up in the index. Neither clinical nor imaging features provided convincing evidence for a classical CADASIL phenotype, thus reinforcing the concept of hypomorphic NOTCH3 variants most likely not being causative for CADASIL. Our finding, which is discussed in the light of the published literature, has practical implications for interpreting results of NOTCH3 molecular genetic testing as well as patient counseling.

The Potato leafroll virus structural proteins manipulate overlapping, yet distinct protein interaction networks during infection

USDA-ARS?s Scientific Manuscript database

Potato leafroll virus (PLRV) produces a readthrough protein (RTP) via translational readthrough of the coat protein amber stop codon. The RTP functions as a structural component of the virion and as a non-incorporated protein in concert with numerous insect and plant proteins to regulate virus movem...

The first complete mitochondrial genome of Dacus longicornis (Diptera: Tephritidae) using next-generation sequencing and mitochondrial genome phylogeny of Dacini tribe

PubMed Central

Jiang, Fan; Pan, Xubin; Li, Xuankun; Yu, Yanxue; Zhang, Junhua; Jiang, Hongshan; Dou, Liduo; Zhu, Shuifang

2016-01-01

The genus Dacus is one of the most economically important tephritid fruit flies. The first complete mitochondrial genome (mitogenome) of Dacus species – D. longicornis was sequenced by next-generation sequencing in order to develop the mitogenome data for this genus. The circular 16,253 bp mitogenome is the typical set and arrangement of 37 genes present in the ancestral insect. The mitogenome data of D. longicornis was compared to all the published homologous sequences of other tephritid species. We discovered the subgenera Bactrocera, Daculus and Tetradacus differed from the subgenus Zeugodacus, the genera Dacus, Ceratitis and Procecidochares in the possession of TA instead of TAA stop codon for COI gene. There is a possibility that the TA stop codon in COI is the synapomorphy in Bactrocera group in the genus Bactrocera comparing with other Tephritidae species. Phylogenetic analyses based on the mitogenome data from Tephritidae were inferred by Bayesian and Maximum-likelihood methods, strongly supported the sister relationship between Zeugodacus and Dacus. PMID:27812024

Unusual AIP mutation and phenocopy in the family of a young patient with acromegalic gigantism.

PubMed

Imran, Syed Ali; Aldahmani, Khaled A; Penney, Lynette; Croul, Sidney E; Clarke, David B; Collier, David M; Iacovazzo, Donato; Korbonits, Márta

2018-01-01

Early-onset acromegaly causing gigantism is often associated with aryl-hydrocarbon-interacting receptor protein ( AIP ) mutation, especially if there is a positive family history. A15y male presented with tiredness and visual problems. He was 201 cm tall with a span of 217 cm. He had typical facial features of acromegaly, elevated IGF-1, secondary hypogonadism and a large macroadenoma. His paternal aunt had a history of acromegaly presenting at the age of 35 years. Following transsphenoidal surgery, his IGF-1 normalized and clinical symptoms improved. He was found to have a novel AIP mutation destroying the stop codon c.991T>C; p.*331R. Unexpectedly, his father and paternal aunt were negative for this mutation while his mother and older sister were unaffected carriers, suggesting that his aunt represents a phenocopy. Typical presentation for a patient with AIP mutation with excess growth and eunuchoid proportions.Unusual, previously not described AIP variant with loss of the stop codon.Phenocopy may occur in families with a disease-causing germline mutation.

Unusual AIP mutation and phenocopy in the family of a young patient with acromegalic gigantism

PubMed Central

Aldahmani, Khaled A; Penney, Lynette; Croul, Sidney E; Clarke, David B; Collier, David M; Iacovazzo, Donato; Korbonits, Márta

2018-01-01

Summary Early-onset acromegaly causing gigantism is often associated with aryl-hydrocarbon-interacting receptor protein (AIP) mutation, especially if there is a positive family history. A15y male presented with tiredness and visual problems. He was 201 cm tall with a span of 217 cm. He had typical facial features of acromegaly, elevated IGF-1, secondary hypogonadism and a large macroadenoma. His paternal aunt had a history of acromegaly presenting at the age of 35 years. Following transsphenoidal surgery, his IGF-1 normalized and clinical symptoms improved. He was found to have a novel AIP mutation destroying the stop codon c.991T>C; p.*331R. Unexpectedly, his father and paternal aunt were negative for this mutation while his mother and older sister were unaffected carriers, suggesting that his aunt represents a phenocopy. Learning points: Typical presentation for a patient with AIP mutation with excess growth and eunuchoid proportions. Unusual, previously not described AIP variant with loss of the stop codon. Phenocopy may occur in families with a disease-causing germline mutation. PMID:29472986

Computational Analysis and Low-Scale Constitutive Expression of Laccases Synthetic Genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris

PubMed Central

Reyes-Guzmán, Edwin Alfredo; Poutou-Piñales, Raúl A.; Reyes-Montaño, Edgar Antonio; Pedroza-Rodríguez, Aura Marina; Rodríguez-Vázquez, Refugio; Cardozo-Bernal, Ángela M.

2015-01-01

Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications. PMID:25611746

Computational analysis and low-scale constitutive expression of laccases synthetic genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris.

PubMed

Rivera-Hoyos, Claudia M; Morales-Álvarez, Edwin David; Poveda-Cuevas, Sergio Alejandro; Reyes-Guzmán, Edwin Alfredo; Poutou-Piñales, Raúl A; Reyes-Montaño, Edgar Antonio; Pedroza-Rodríguez, Aura Marina; Rodríguez-Vázquez, Refugio; Cardozo-Bernal, Ángela M

2015-01-01

Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications.

Beyond the Triplet Code: Context Cues Transform Translation.

PubMed

Brar, Gloria A

2016-12-15

The elucidation of the genetic code remains among the most influential discoveries in biology. While innumerable studies have validated the general universality of the code and its value in predicting and analyzing protein coding sequences, established and emerging work has also suggested that full genome decryption may benefit from a greater consideration of a codon's neighborhood within an mRNA than has been broadly applied. This Review examines the evidence for context cues in translation, with a focus on several recent studies that reveal broad roles for mRNA context in programming translation start sites, the rate of translation elongation, and stop codon identity. Copyright © 2016 Elsevier Inc. All rights reserved.

Restored PB1-F2 in the 2009 Pandemic H1N1 Influenza Virus Has Minimal Effects in Swine

PubMed Central

Pena, Lindomar; Loving, Crystal L.; Henningson, Jamie N.; Lager, Kelly M.; Lorusso, Alessio

2012-01-01

PB1-F2 is an 87- to 90-amino-acid-long protein expressed by certain influenza A viruses. Previous studies have shown that PB1-F2 contributes to virulence in the mouse model; however, its role in natural hosts—pigs, humans, or birds—remains largely unknown. Outbreaks of domestic pigs infected with the 2009 pandemic H1N1 influenza virus (pH1N1) have been detected worldwide. Unlike previous pandemic strains, pH1N1 viruses do not encode a functional PB1-F2 due to the presence of three stop codons resulting in premature truncation after codon 11. However, pH1N1s have the potential to acquire the full-length form of PB1-F2 through mutation or reassortment. In this study, we assessed whether restoring the full-length PB1-F2 open reading frame (ORF) in the pH1N1 background would have an effect on virus replication and virulence in pigs. Restoring the PB1-F2 ORF resulted in upregulation of viral polymerase activity at early time points in vitro and enhanced virus yields in porcine respiratory explants and in the lungs of infected pigs. There was an increase in the severity of pneumonia in pigs infected with isogenic virus expressing PB1-F2 compared to the wild-type (WT) pH1N1. The extent of microscopic pneumonia correlated with increased pulmonary levels of alpha interferon and interleukin-1β in pigs infected with pH1N1 encoding a functional PB1-F2 but only early in the infection. Together, our results indicate that PB1-F2 in the context of pH1N1 moderately modulates viral replication, lung histopathology, and local cytokine response in pigs. PMID:22379102

Novel insertion mutation in a non-Jewish Caucasian type 1 Gaucher disease patient

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choy, F.Y.M.; Humphries, M.L.; Ferreira, P.

1997-01-20

Gaucher disease is the most prevalent lysosomal storage disorder. It is autosomal recessive, resulting in lysosomal glucocerebrosidase deficiency. Three clinical forms of Gaucher disease have been described: type 1 (nonneuronopathic), type 2 (acute neuronopathic), and type 3 (subacute neuronopathic). We performed PCR-thermal cycle sequence analysis of glucocerebrosidase genomic DNA and identified a novel mutation in a non-Jewish type 1 Gaucher disease patient. It is a C insertion in exon 3 at cDNA nucleotide position 122 and genomic nucleotide position 1626. This mutation causes a frameshift and, subsequently, four of the five codons immediately downstream of the insertion were changed whilemore » the sixth was converted to a stop codon, resulting in premature termination of protein translation. The 122CC insertion abolishes a Cac81 restriction endonuclease cleavage site, allowing a convenient and reliable method for detection using RFLP analysis of PCR-amplified glucocerebrosidase genomic DNA. The mutation in the other Gaucher allele was found to be an A{r_arrow}G substitution at glucocerebrosidase cDNA nucleotide position 1226 that so far has only been reported among type 1 Gaucher disease patients. Since mutation 122CC causes a frameshift and early termination of protein translation, it most likely results in a meaningless transcript and subsequently no residual glucocerebrosidase enzyme activity. We speculate that mutation 122CC may result in a worse prognosis than mutations associated with partial activity. When present in the homozygous form, it could be a lethal allele similar to what has been postulated for the other known insertion mutation, 84GG. Our patient, who is a compound heterozygote 122CC/1226G, has moderately severe type 1 Gaucher disease. Her clinical response to Ceredase{reg_sign} therapy that began 31 months ago has been favorable, though incomplete. 30 refs., 3 figs., 2 tabs.« less

Targeting Nonsense Mutations in Diseases with Translational Read-Through-Inducing Drugs (TRIDs).

PubMed

Nagel-Wolfrum, Kerstin; Möller, Fabian; Penner, Inessa; Baasov, Timor; Wolfrum, Uwe

2016-04-01

In recent years, remarkable advances in the ability to diagnose genetic disorders have been made. The identification of disease-causing genes allows the development of gene-specific therapies with the ultimate goal to develop personalized medicines for each patient according to their own specific genetic defect. In-depth genotyping of many different genes has revealed that ~12% of inherited genetic disorders are caused by in-frame nonsense mutations. Nonsense (non-coding) mutations are caused by point mutations, which generate premature termination codons (PTCs) that cause premature translational termination of the mRNA, and subsequently inhibit normal full-length protein expression. Recently, a gene-based therapeutic approach for genetic diseases caused by nonsense mutations has emerged, namely the so-called translational read-through (TR) therapy. Read-through therapy is based on the discovery that small molecules, known as TR-inducing drugs (TRIDs), allow the translation machinery to suppress a nonsense codon, elongate the nascent peptide chain, and consequently result in the synthesis of full-length protein. Several TRIDs are currently under investigation and research has been performed on several genetic disorders caused by nonsense mutations over the years. These findings have raised hope for the usage of TR therapy as a gene-based pharmacogenetic therapy for nonsense mutations in various genes responsible for a variety of genetic diseases.

Coinheritance of a novel mutation on the HBA1 gene: c.187delG (p.W62fsX66) [codon 62 (-G) (α1)] with the α212 patchwork allele and Hb S [β6(A3)Glu→Val, GAG>GTG; HBB: c.20A>T].

PubMed

Scheps, Karen G; De Paula, Silvia M; Bitsman, Alicia R; Freigeiro, Daniel H; Basack, F Nora; Pennesi, Sandra P; Varela, Viviana

2013-01-01

We describe a novel frameshift mutation on the HBA1 gene (c.187delG), causative of α-thalassemia (α-thal) in a Black Cuban family with multiple sequence variants in the HBA genes and the Hb S [β6(A3)Glu→Val, GAG>GTG; HBB: c.20A>T] mutation. The deletion of the first base of codon 62 resulted in a frameshift at amino acid 62 with a putative premature termination codon (PTC) at amino acid 66 on the same exon (p.W62fsX66), which most likely triggers nonsense mediated decay of the resulting mRNA. This study also presents the first report of the α212 patchwork allele in Latin America and the description of two new sequence variants in the HBA2 region (c.-614G>A in the promoter region and c.95+39 C>T on the first intron).

Negative and Translation Termination-Dependent Positive Control of FLI-1 Protein Synthesis by Conserved Overlapping 5′ Upstream Open Reading Frames in Fli-1 mRNA

PubMed Central

Sarrazin, Sandrine; Starck, Joëlle; Gonnet, Colette; Doubeikovski, Alexandre; Melet, Fabrice; Morle, François

2000-01-01

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5′ upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG −41 and GUG −37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5′ uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5′ uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5′ uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons. PMID:10757781

A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein.

PubMed

Dar, Saira; Shuja, Rukhsana N; Shakoori, Abdul Rauf

2013-02-01

Metallothioneins (MTs) are metal binding proteins that are rich in cysteine residues constituting 10-30 % of the total protein, and in which the thiol groups bind to the metal ions. The increasing amount of metal ions in the medium have shown increased production of MTs by different organisms such as bacteria, protozoa and mammals like humans. PMCd1 is the first gene ever discovered in Paramecium, a ciliated protozoan, that could produce this MT in response to cadmium. In this study the PMCd1syn gene has been cloned in pET41a expression vector and expressed in an Escherichia coli BL21-codonplus strain for the first time. Since the gene PMCd1 amplified from Paramecium contained 10 codons, which could act as stop codons during expression in E. coli, this gene of 612 bps was synthesized to substitute these (stop) codons for the Paramecium sp. specific amino acids. For stability of the expressed protein, glutathione-S-transferase gene was fused with PMCd1syn gene and coexpressed. The cells expressing PMCd1syn demonstrated increased accumulation of cadmium. This is the first report of cadmium MT protein expressed from Paramecium species, particularly from synthetic MT gene (PMCd1syn). This fusion protein, the molecular weight of which has been confirmed to be 53.03 kDa with MALDI analysis, is rich in cysteine residues, and has been shown for the first time in this ciliate to bind to and sequester Cd(2+)-ions.

Two novel mutations in the alpha-galactosidase gene in Japanese classical hemizygotes with Fabry disease.

PubMed

Okumiya, T; Takenaka, T; Ishii, S; Kase, R; Kamei, S; Sakuraba, H

1996-09-01

Four alpha-galactosidase gene mutations were identified in Japanese male patients with Fabry disease who had no detectable alpha-galactosidase activity. Two of them were novel mutations, an 11-bp deletion in exon 2 and a g-1 to t substitution at the 3' end of the splice acceptor site in intron 1. The former caused a frameshift and led to the creation of a new stop codon at codon 118. The latter was predicted to provoke aberrant mRNA splicing followed by accelerated degradation of the mRNA. A nonsense mutation, R301X, and a 2-bp deletion starting at nucleotide position 718, which were reported previously, were also identified in unrelated patients.

alpha-Tubulin of Histriculus cavicola (Ciliophora; Hypotrichea).

PubMed

Pérez-Romero, P; Villalobo, E; Díaz-Ramos, C; Calvo, P; Santos-Rosa, F; Torres, A

1997-03-01

An alpha-tubulin gene fragment amplified by PCR from the hypotrichous ciliate Histriculus cavicola has been sequenced. This fragment, 1,182 bp long, contains an in-frame "stop" codon (UAA), which in other hypotrichous species codes for a glutamine residue. The comparison of the alpha-tubulin genes from several ciliates classes have revealed amino acid positions which could serve to distinguish these taxonomic groups.

Ancient nature of alternative splicing and functions of introns

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Kemin; Salamov, Asaf; Kuo, Alan

Using four genomes: Chamydomonas reinhardtii, Agaricus bisporus, Aspergillus carbonarius, and Sporotricum thermophile with EST coverage of 2.9x, 8.9x, 29.5x, and 46.3x respectively, we identified 11 alternative splicing (AS) types that were dominated by intron retention (RI; biased toward short introns) and found 15, 35, 52, and 63percent AS of multiexon genes respectively. Genes with AS were more ancient, and number of AS correlated with number of exons, expression level, and maximum intron length of the gene. Introns with tendency to be retained had either stop codons or length of 3n+1 or 3n+2 presumably triggering nonsense-mediated mRNA decay (NMD), but intronsmore » retained in major isoforms (0.2-6percent of all introns) were biased toward 3n length and stop codon free. Stopless introns were biased toward phase 0, but 3n introns favored phase 1 that introduced more flexible and hydrophilic amino acids on both ends of introns which would be less disruptive to protein structure. We proposed a model in which minor RI intron could evolve into major RI that could facilitate intron loss through exonization.« less

Culture adaptation of malaria parasites selects for convergent loss-of-function mutants.

PubMed

Claessens, Antoine; Affara, Muna; Assefa, Samuel A; Kwiatkowski, Dominic P; Conway, David J

2017-01-24

Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured in vitro for up to three months. Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture.

A CRISPR-Cas9 Generated MDCK Cell Line Expressing Human MDR1 Without Endogenous Canine MDR1 (cABCB1): An Improved Tool for Drug Efflux Studies.

PubMed

Karlgren, Maria; Simoff, Ivailo; Backlund, Maria; Wegler, Christine; Keiser, Markus; Handin, Niklas; Müller, Janett; Lundquist, Patrik; Jareborg, Anne-Christine; Oswald, Stefan; Artursson, Per

2017-09-01

Madin-Darby canine kidney (MDCK) II cells stably transfected with transport proteins are commonly used models for drug transport studies. However, endogenous expression of especially canine MDR1 (cMDR1) confounds the interpretation of such studies. Here we have established an MDCK cell line stably overexpressing the human MDR1 transporter (hMDR1; P-glycoprotein), and used CRISPR-Cas9 gene editing to knockout the endogenous cMDR1. Genomic screening revealed the generation of a clonal cell line homozygous for a 4-nucleotide deletion in the canine ABCB1 gene leading to a frameshift and a premature stop codon. Knockout of cMDR1 expression was verified by quantitative protein analysis and functional studies showing retained activity of the human MDR1 transporter. Application of this cell line allowed unbiased reclassification of drugs previously defined as both substrates and non-substrates in different studies using commonly used MDCK-MDR1 clones. Our new MDCK-hMDR1 cell line, together with a previously developed control cell line, both with identical deletions in the canine ABCB1 gene and lack of cMDR1 expression represent excellent in vitro tools for use in drug discovery. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Two-Exon Skipping within MLPH Is Associated with Coat Color Dilution in Rabbits

PubMed Central

Lehner, Stefanie; Gähle, Marion; Dierks, Claudia; Stelter, Ricarda; Gerber, Jonathan; Brehm, Ralph; Distl, Ottmar

2013-01-01

Coat color dilution turns black coat color to blue and red color to cream and is a characteristic in many mammalian species. Matings among Netherland Dwarf, Loh, and Lionhead Dwarf rabbits over two generations gave evidence for a monogenic autosomal recessive inheritance of coat colour dilution. Histological analyses showed non-uniformly distributed, large, agglomerating melanin granules in the hair bulbs of coat color diluted rabbits. We sequenced the cDNA of MLPH in two dilute and one black rabbit for polymorphism detection. In both color diluted rabbits, skipping of exons 3 and 4 was present resulting in altered amino acids at p.QGL[37-39]QWA and a premature stop codon at p.K40*. Sequencing of genomic DNA revealed a c.111-5C>A splice acceptor mutation within the polypyrimidine tract of intron 2 within MLPH. This mutation presumably causes skipping of exons 3 and 4. In 14/15 dilute rabbits, the c.111-5C>A mutation was homozygous and in a further dilute rabbit, heterozygous and in combination with a homozygous frame shift mutation within exon 6 (c.585delG). In conclusion, our results demonstrated a colour dilution associated MLPH splice variant causing a strongly truncated protein (p.Q37QfsX4). An involvement of further MLPH-associated mutations needs further investigations. PMID:24376820

Both a frameshift and a missense mutation of the STRA6 gene observed in an infant with the Matthew-Wood syndrome.

PubMed

Sadowski, Samantha; Chassaing, Nicolas; Gaj, Zuzanna; Czichos, Ewa; Wilczynski, Jan; Nowakowska, Dorota

2017-03-01

The Matthew-Wood syndrome is associated with mutations of the STRA6 gene. It combines a pulmonary agenesis/hypoplasia; microphthalmia/anophthalmia; congenital cardiac, digestive, and urogenital malformations; and diaphragmatic defects. A 23-year-old nulliparous woman was referred to our center after a fetal ultrasound examination at 26 weeks of pregnancy revealed an abnormal head shape, a heart malformation, multiple cysts in both kidneys, and dilated ureters. A male baby (46, XY; 3600g; Apgar score 1) was delivered at 38 weeks of gestation and died 1 hr later due to respiratory failure. The diagnosis of Matthew-Wood syndrome was suspected given the association of bilateral anophthalmia, agenesis of the left lung, and heart and kidney defects. It was confirmed by the identification of two deleterious mutations of the STRA6 gene. The child was a compound heterozygote for two previously reported mutations, a paternally inherited missense mutation (c.878C>T [p.Pro293Leu] and a maternally inherited frameshift mutation (c.50_52delACTinsCC [p. Asp17Alafs*55]), producing a premature stop codon. The diagnosis of Matthew-Wood syndrome should be considered in all fetuses with microphthalmia/anophthalmia. It requires an extensive ultrasound/MRI examination of the lung, heart, and diaphragm. Birth Defects Research 109:251-253, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A novel LEMD3 mutation common to patients with osteopoikilosis with and without melorheostosis.

PubMed

Couto, Ana R; Bruges-Armas, Jacome; Peach, Chris A; Chapman, Kay; Brown, Matthew A; Wordsworth, B Paul; Zhang, Yun

2007-08-01

Recent studies have reported loss of function mutations in the LEMD3 gene, encoding an inner nuclear membrane protein that influences Smad signaling, as a cause of osteopoikilosis, Buschke-Ollendorff syndrome, and melorheostosis. We investigated LEMD3 in a three-generation family with osteopoikilosis from the Azores, an affected father and daughter from Ireland with osteopoikilosis (the daughter also had melorheostosis), and two other individuals from the UK with isolated melorheostosis. We found a novel C to T substitution at position 2032 bp (cDNA) in exon 8 of LEMD3, resulting in a premature stop codon at amino acid position 678. This mutation co-segregates with the osteopoikilosis phenotype in both the Azorean family and the Irish family. It was not detected in any of the six unaffected family members or in 342 healthy Caucasian individuals. No LEMD3 mutations were detected in the two patients with sporadic melorheostosis. The LEMD3 mutation reported was clearly the cause of osteopoikilosis in the two families but its relationship to melorheostosis in one of the family members is still unclear. Perhaps unsurprisingly in what is a segmental disease, we did not find LEMD3 mutations in peripheral-blood-derived DNA from the two other individuals with sporadic melorheostosis. The nature of the additional genetic and/or environmental influences required for the development of melorheostosis in those with osteopoikilosis requires further investigation.

Novel and recurrent germline LEMD3 mutations causing Buschke-Ollendorff syndrome and osteopoikilosis but not isolated melorheostosis.

PubMed

Zhang, Y; Castori, M; Ferranti, G; Paradisi, M; Wordsworth, B P

2009-06-01

Mutations in the LEMD3 gene were recently incriminated in Buschke-Ollendorff syndrome (BOS) and osteopoikilosis, with or without melorheostosis. The relationship of this gene with isolated sporadic melorheostosis is less clear. We investigated LEMD3 in a two-generation BOS family showing an extremely variable expression of the disease, in a sporadic patient with skin features of BOS, and in an additional subject with isolated melorheostosis. We identified two different mutations, both resulting in a premature stop codon, in the two cases of BOS. The mutation (c.2564G>A) reported in the familial case is novel, while that observed in the sporadic case (c.1963C>T) has been previously reported in an American woman with osteopoikilosis and melorheostosis who had a family history of isolated osteopoikilosis. The search for mutations in DNA extracted from the peripheral blood, as well as skin and bone biopsies of the patient with melorheostosis failed to identify any pathogenic change. Our results further expand the LEMD3 mutation repertoire, corroborate the extreme interfamilial and intrafamilial clinical variability of LEMD3 mutations, and underline the lack of a clear phenotype-genotype correlation in BOS. The present study supports the general conclusion that LEMD3 mutations do not contribute to isolated sporadic melorheostosis. The genetic or epigenetic influences that are responsible for the development of melorheostosis require further investigation.

Autism-Associated Insertion Mutation (InsG) of Shank3 Exon 21 Causes Impaired Synaptic Transmission and Behavioral Deficits.

PubMed

Speed, Haley E; Kouser, Mehreen; Xuan, Zhong; Reimers, Jeremy M; Ochoa, Christine F; Gupta, Natasha; Liu, Shunan; Powell, Craig M

2015-07-01

SHANK3 (also known as PROSAP2) is a postsynaptic scaffolding protein at excitatory synapses in which mutations and deletions have been implicated in patients with idiopathic autism, Phelan-McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. In this study, we have created a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3(G)). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3(G/G) mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3(G/G) mice also exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. This work identifies clear alterations in synaptic function and behavior in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. Furthermore, these findings lay the foundation for future studies aimed to validate and study region-selective and temporally selective genetic reversal studies in the Shank3(G/G) mouse that was engineered with such future experiments in mind. Copyright © 2015 the authors 0270-6474/15/359648-18$15.00/0.

A novel silent deletion, an insertion mutation and a nonsense mutation in the TCOF1 gene found in two Chinese cases of Treacher Collins syndrome.

PubMed

Wang, Yan; Yin, Xiao-Juan; Han, Tao; Peng, Wei; Wu, Hong-Lin; Liu, Xin; Feng, Zhi-Chun

2014-12-01

Treacher Collins syndrome (TCS) is the most common and well-known craniofacial disorder caused by mutations in the genes involved in pre-rRNA transcription, which include the TCOF1 gene. This study explored the role of TCOF1 mutations in Chinese patients with TCS. Mutational analysis of the TCOF1 gene was performed in three patients using polymerase chain reaction and direct sequencing. Among these three patients, two additional TCOF1 variations, a novel 18 bp deletion and a novel 1 bp insertion mutation, were found in patient 1, together with a novel nonsense mutation (p.Ser476X) and a previously reported 4 bp deletion (c.1872_1875delTGAG) in other patients. Pedigree analysis allowed for prediction of the character of the mutation, which was either pathological or not. The 18 bp deletion of six amino acids, Ser-Asp-Ser-Glu-Glu-Glu (798*803), which was located in the CKII phosphorylation site of treacle, seemed relatively benign for TCS. By contrast, another novel mutation of c.1072_1073insC (p.Gln358ProfsX23) was a frameshift mutation and expected to result in a premature stop codon. This study provides insights into the functional domain of treacle and illustrates the importance of clinical and family TCS screening for the interpretation of novel sequence alterations.

Genetic analysis of Chinese families reveals a novel truncation allele of the retinitis pigmentosa GTPase regulator gene

PubMed Central

Hu, Fang; Zeng, Xiang-Yun; Liu, Lin-Lin; Luo, Yao-Ling; Jiang, Yi-Ping; Wang, Hui; Xie, Jing; Hu, Cheng-Quan; Gan, Lin; Huang, Liang

2014-01-01

AIM To make comprehensive molecular diagnosis for retinitis pigmentosa (RP) patients in a consanguineous Han Chinese family using next generation sequencing based Capture-NGS screen technology. METHODS A five-generation Han Chinese family diagnosed as non-syndromic X-linked recessive RP (XLRP) was recruited, including four affected males, four obligate female carriers and eleven unaffected family members. Capture-NGS was performed using a custom designed capture panel covers 163 known retinal disease genes including 47 RP genes, followed by the validation of detected mutation using Sanger sequencing in all recruited family members. RESULTS Capture-NGS in one affected 47-year-old male reveals a novel mutation, c.2417_2418insG:p.E806fs, in exon ORF15 of RP GTPase regulator (RPGR) gene results in a frameshift change that results in a premature stop codon and a truncated protein product. The mutation was further validated in three of four affected males and two of four female carriers but not in the other unaffected family members. CONCLUSION We have identified a novel mutation, c.2417_2418insG:p.E806fs, in a Han Chinese family with XLRP. Our findings expand the mutation spectrum of RPGR and the phenotypic spectrum of XLRP in Han Chinese families, and confirms Capture-NGS could be an effective and economic approach for the comprehensive molecular diagnosis of RP. PMID:25349787

Nonsense variant in COL7A1 causes recessive dystrophic epidermolysis bullosa in Central Asian Shepherd dogs.

PubMed

Niskanen, Julia; Dillard, Kati; Arumilli, Meharji; Salmela, Elina; Anttila, Marjukka; Lohi, Hannes; Hytönen, Marjo K

2017-01-01

A rare hereditary mechanobullous disorder called epidermolysis bullosa (EB) causes blistering in the skin and the mucosal membranes. To date, nineteen EB-related genes have been discovered in human and other species. We describe here a novel EB variant in dogs. Two newborn littermates of Central Asian Shepherd dogs with severe signs of skin blistering were brought to a veterinary clinic and euthanized due to poor prognosis. In post-mortem examination, the puppies were shown to have findings in the skin and the mucosal membranes characteristic of EB. A whole-genome sequencing of one of the affected puppies was performed to identify the genetic cause. The resequencing data were filtered under a recessive model against variants from 31 other dog genomes, revealing a homozygous case-specific nonsense variant in one of the known EB-causing genes, COL7A1 (c.4579C>T, p.R1527*). The variant results in a premature stop codon and likely absence of the functional protein in the basement membrane of the skin in the affected dogs. This was confirmed by immunohistochemistry using a COL7A1 antibody. Additional screening of the variant indicated full penetrance and breed specificity at ~28% carrier frequency. In summary, this study reveals a novel COL7A1 variant causing recessive dystrophic EB and provides a genetic test for the eradication of the disease from the breed.

A novel mechanism of myostatin regulation by its alternative splicing variant during myogenesis in avian species.

PubMed

Shin, Sangsu; Song, Yan; Ahn, Jinsoo; Kim, Eunsoo; Chen, Paula; Yang, Shujin; Suh, Yeunsu; Lee, Kichoon

2015-11-15

Myostatin (MSTN) is a key negative regulator of muscle growth and development, and an increase of muscle mass is achieved by inhibiting MSTN signaling. In the current study, five alternative splicing isoforms of MSTN mRNAs in avian species were identified in various tissues. Among these five, three truncated forms of myostatin, MSTN-B, -C, and -E created premature stop codons and produced partial MSTN prodomains encoded from exon 1. MSTN-B is the second dominant isoform following full-length MSTN-A, and their expression was dynamically regulated during muscle development of chicken, turkey, and quail in vivo and in vitro. To clarify the function of MSTN-B, two stable cell lines of quail myoblasts (QM7) were generated to overexpress MSTN-A or MSTN-B. Interestingly, MSTN-B promoted both cell proliferation and differentiation similar to the function of the MSTN prodomain to counteract the negative role of MSTN on myogenesis. The coimmunoprecipitation assay revealed that MSTN-B binds to MSTN-A and reduces the generation of mature MSTN. Furthermore, the current study demonstrated that the partial prodomain encoded from exon 1 is critical for binding of MSTN-B to MSTN-A. Altogether, these data imply that alternative splicing isoforms of MSTN could negatively regulate pro-myostatin processing in muscle cells and prevent MSTN-mediated inhibition of myogenesis in avian species. Copyright © 2015 the American Physiological Society.

A Novel Nonsense Mutation in Exon 5 of KIND1 Gene in an Iranian Family with Kindler Syndrome.

PubMed

Heidari, Mohammad Mehdi; Khatami, Mehri; Kargar, Saeed; Azari, Mojdeh; Hoseinzadeh, Hassan; Fallah, Hamedeh

2016-06-01

Kindler syndrome (KS) is an autosomal recessive skin disease characterized by actual blistering, photosensitivity and a progressive poikiloderma. The disorder results from rare mutations in the KIND1 gene. This gene contains 15 exons and expresses two kindlin-1 isoforms. The aim of this investigation was to analyze mutations in the exons 1 to 15 of KIND1 gene in an Iranian family clinically affected with Kindler syndrome. The mutations analysis of 15 coding exons of KIND1 gene was performed with PCR-SSCP and direct sequencing in 14 subjects from one Iranian family clinically affected with Kindler syndrome. We identified eight new nucleotide changes in KIND1 in this family. These changes were found in g.3892delA, g.3951T>C, g.3962T>G, g.4190G>T, g.7497G>A, g.11076T>C, g.11102C>T and g.13177C>T positions. Among them, the g.13177C>T mutation resulting in the formation of a premature stop codon (Q226X) was detected only in seven affected family individuals as homozygous but was not present in 100 unrelated healthy controls. This study suggests that nonsense mutation may lead to incomplete and non-functional protein products and is pathogenic and has meaningful implications for the diagnosis of patients with Kindler syndrome.

Adrenocorticotropin-dependent precocious puberty of testicular origin in a boy with X-linked adrenal hypoplasia congenita due to a novel mutation in the DAX1 gene.

PubMed

Domenice, S; Latronico, A C; Brito, V N; Arnhold, I J; Kok, F; Mendonca, B B

2001-09-01

Primary adrenal insufficiency is a rare condition in pediatric age, and its association with precocious sexual development is very uncommon. We report a 2-yr-old Brazilian boy with DAX1 gene mutation whose first clinical manifestation was isosexual gonadotropin-independent precocious puberty. He presented with pubic hair, enlarged penis and testes, and advanced bone age. T levels were elevated, whereas basal and GnRH-stimulated LH levels were compatible with a prepubertal pattern. Chronic GnRH agonist therapy did not reduce T levels, supporting the diagnosis of gonadotropin-independent precocious puberty. Testotoxicosis was ruled out after normal sequencing of exon 11 of the LH receptor gene. At age 3 yr he developed clinical and hormonal features of severe primary adrenal insufficiency. The entire coding region of the DAX1 gene was analyzed through direct sequencing. A nucleotide G insertion between nucleotides 430 and 431 in exon 1, resulting in a novel frameshift mutation and a premature stop codon at position 71 of DAX-1, was identified. Surprisingly, steroid replacement therapy induced a clear decrease in testicular size and T levels to the prepubertal range. These findings suggest that chronic excessive ACTH levels resulting from adrenal insufficiency may stimulate Leydig cells and lead to gonadotropin-independent precocious puberty in some boys with DAX1 gene mutations.

Mutations in the calcium-related gene IL1RAPL1 are associated with autism.

PubMed

Piton, Amélie; Michaud, Jacques L; Peng, Huashan; Aradhya, Swaroop; Gauthier, Julie; Mottron, Laurent; Champagne, Nathalie; Lafrenière, Ronald G; Hamdan, Fadi F; Joober, Ridha; Fombonne, Eric; Marineau, Claude; Cossette, Patrick; Dubé, Marie-Pierre; Haghighi, Pejmun; Drapeau, Pierre; Barker, Philip A; Carbonetto, Salvatore; Rouleau, Guy A

2008-12-15

In a systematic sequencing screen of synaptic genes on the X chromosome, we have identified an autistic female without mental retardation (MR) who carries a de novo frameshift Ile367SerfsX6 mutation in Interleukin-1 Receptor Accessory Protein-Like 1 (IL1RAPL1), a gene implicated in calcium-regulated vesicle release and dendrite differentiation. We showed that the function of the resulting truncated IL1RAPL1 protein is severely altered in hippocampal neurons, by measuring its effect on neurite outgrowth activity. We also sequenced the coding region of the close related member IL1RAPL2 and of NCS-1/FREQ, which physically interacts with IL1RAPL1, in a cohort of subjects with autism. The screening failed to identify non-synonymous variant in IL1RAPL2, whereas a rare missense (R102Q) in NCS-1/FREQ was identified in one autistic patient. Furthermore, we identified by comparative genomic hybridization a large intragenic deletion of exons 3-7 of IL1RAPL1 in three brothers with autism and/or MR. This deletion causes a frameshift and the introduction of a premature stop codon, Ala28GlufsX15, at the very beginning of the protein. All together, our results indicate that mutations in IL1RAPL1 cause a spectrum of neurological impairments ranging from MR to high functioning autism.

Diversified clinical presentations associated with a novel sal-like 4 gene mutation in a Chinese pedigree with Duane retraction syndrome.

PubMed

Yang, Ming-ming; Ho, Mary; Lau, Henry H W; Tam, Pancy O S; Young, Alvin L; Pang, Chi Pui; Yip, Wilson W K; Chen, LiJia

2013-01-01

To determine the underlying genetic cause of Duane retraction syndrome (DRS) in a non-consanguineous Chinese Han family. Detailed ophthalmic and physical examinations were performed on all members from a pedigree with DRS. All exons and their adjacent splicing junctions of the sal-like 4 (SALL4) gene were amplified with polymerase chain reaction and analyzed with direct sequencing in all the recruited family members and 200 unrelated control subjects. Clinical examination revealed a broad spectrum of phenotypes in the DRS family. Mutation analysis of SALL4 identified a novel heterozygous duplication mutation, c.1919dupT, which was completely cosegregated with the disease in the family and absent in controls. This mutation was predicted to cause a frameshift, introducing a premature stop codon, when translated, resulting in a truncated SALL4 protein, i.e., p.Met640IlefsX25. Bioinformatics analysis showed that the affected region of SALL4 shared a highly conserved sequence across different species. Diversified clinical manifestations were observed in the c.1919dupT carriers of the family. We identified a novel truncating mutation in the SALL4 gene that leads to diversified clinical features of DRS in a Chinese family. This mutation is predicted to result in a truncated SALL4 protein affecting two functional domains and cause disease development due to haploinsufficiency through nonsense-mediated mRNA decay.

Recurrent elevated liver transaminases and acute liver failure in two siblings with novel bi-allelic mutations of NBAS.

PubMed

Regateiro, Frederico S; Belkaya, Serkan; Neves, Nélson; Ferreira, Sandra; Silvestre, Paula; Lemos, Sónia; Venâncio, Margarida; Casanova, Jean-Laurent; Gonçalves, Isabel; Jouanguy, Emmanuelle; Diogo, Luísa

2017-08-01

Acute liver failure (ALF) in children can be life-threatening. Although many causes are known, ALF remains unexplained in about half of the cases. Recently, bi-allelic mutations in NBAS were reported to underlie recurrent episodes of elevated liver transaminases (ELT) and ALF in the context of diverse extrahepatic phenotypes. We here describe two sisters, born to non-consanguineous Portuguese parents, who had short stature and presented with recurrent episodes of severe ELT triggered by febrile respiratory viral infections since early childhood. Patient 1 had mild facial dysmorphism and died during the second ELT crisis at 3-11/12 years of age. Patient 2, currently 9 years old, had multiple episodes of ELT (>30), twice with ALF, often accompanied by extensive urticaria and facial angioedema. Whole-exome and Sanger sequencing revealed that both patients carried previously undescribed compound heterozygous mutations of NBAS (NM_015909.3): c.680A > C (p.His227Pro), affecting an evolutionarily conserved residue, and c.1749G > A (p.Trp583*), causing a premature stop codon. Both mutations are predicted to be highly damaging. The parents and two younger siblings are healthy and heterozygous for one or another mutant allele. The multiplex kindred reported herein expands the genotypic and phenotypic spectrum of this recently described clinical syndrome due to autosomal recessive NBAS deficiency. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Respiratory chain complex I deficiency due to NDUFA12 mutations as a new cause of Leigh syndrome.

PubMed

Ostergaard, Elsebet; Rodenburg, Richard J; van den Brand, Mariël; Thomsen, Lise Lykke; Duno, Morten; Batbayli, Mustafa; Wibrand, Flemming; Nijtmans, Leo

2011-11-01

This study investigated a girl with Leigh syndrome born to first-cousin parents of Pakistani descent with an isolated respiratory chain complex I deficiency in muscle and fibroblasts. Her early development was delayed, and from age 2 years she started losing motor abilities. Cerebral MRI showed basal ganglia lesions typical of Leigh syndrome. A genome-wide search for homozygosity was performed with the Affymetrix GeneChip 50K Xba array. The analysis revealed several homozygous regions. Three candidate genes were identified, and in one of the genes, NDUFA12, a homozygous c.178C→T mutation leading to a premature stop codon (p.Arg60X) was found. Western blot analysis showed absence of NDUFA12 protein in patient fibroblasts and functional complementation by a baculovirus system showed restoration of complex I activity. NDUFA12 mutations are apparently not a frequent cause of complex I deficiency, since mutations were not found by screening altogether 122 complex I deficient patients in two different studies. NDUFA12 encodes an accessory subunit of complex I and is a paralogue of NDUFAF2. Despite the complete absence of NDUFA12 protein, a fully assembled and enzymatically active complex I could be found, albeit in reduced amounts. This suggests that NDUFA12 is required either at a late step in the assembly of complex I, or in the stability of complex I.

The Disruption of Celf6, a Gene Identified by Translational Profiling of Serotonergic Neurons, Results in Autism-Related Behaviors

PubMed Central

Dougherty, Joseph D.; Maloney, Susan E.; Wozniak, David F.; Rieger, Michael A.; Sonnenblick, Lisa; Coppola, Giovanni; Mahieu, Nathaniel G.; Zhang, Juliet; Cai, Jinlu; Patti, Gary J.; Abrahams, Brett S.; Geschwind, Daniel H.; Heintz, Nathaniel

2013-01-01

The immense molecular diversity of neurons challenges our ability to understand the genetic and cellular etiology of neuropsychiatric disorders. Leveraging knowledge from neurobiology may help parse the genetic complexity: identifying genes important for a circuit that mediates a particular symptom of a disease may help identify polymorphisms that contribute to risk for the disease as a whole. The serotonergic system has long been suspected in disorders that have symptoms of repetitive behaviors and resistance to change, including autism. We generated a bacTRAP mouse line to permit translational profiling of serotonergic neurons. From this, we identified several thousand serotonergic-cell expressed transcripts, of which 174 were highly enriched, including all known markers of these cells. Analysis of common variants near the corresponding genes in the AGRE collection implicated the RNA binding protein CELF6 in autism risk. Screening for rare variants in CELF6 identified an inherited premature stop codon in one of the probands. Subsequent disruption of Celf6 in mice resulted in animals exhibiting resistance to change and decreased ultrasonic vocalization as well as abnormal levels of serotonin in the brain. This work provides a reproducible and accurate method to profile serotonergic neurons under a variety of conditions and suggests a novel paradigm for gaining information on the etiology of psychiatric disorders. PMID:23407934

Lipoxygenase pathways in Homo neanderthalensis: functional comparison with Homo sapiens isoforms

PubMed Central

Chaitidis, Pavlos; Adel, Susan; Anton, Monika; Heydeck, Dagmar; Kuhn, Hartmut; Horn, Thomas

2013-01-01

Lipoxygenases (LOX) have been implicated in biosynthesis of pro- and anti-inflammatory mediators, and a previous report suggested compromised leukotriene signaling in H. neanderthalensis. To search for corresponding differences in leukotriene biosynthesis, we screened the Neandertal genome for LOX genes and found that, as modern humans, this archaic hominid contains six LOX genes (nALOX15, nALOX12, nALOX5, nALOX15B, nALOX12B, and nALOXE3) and one pseudogene. In the Neandertal genome, 60–75% of the amino acids of the different human LOX isoforms have been identified, and the degree of identity varies between 96 and 99%. Most functional amino acids (iron ligands, specificity determinants, calcium and ATP-binding sites, membrane-binding determinants, and phosphorylation sites) are well conserved in the Neandertal LOX isoforms, and expression of selected neandertalized human LOX mutants revealed no major functional defects. However, in nALOX12 and nALOXE3, two premature stop codons were found, leading to inactive enzyme species. These data suggest that ALOX15, ALOX5, ALOX15B, and ALOX12B should have been present as functional enzymes in H. neanderthalensis and that in contrast to lower nonhuman primates (M. mulatta) and other mammals (mice, rats), this ancient hominid expressed a 15-lipoxygenating ALOX15. Expression of ALOXE3 and ALOX12 was compromised, which might have caused problems in epidermal differentiation. PMID:23475662

Coffin-Siris syndrome with café-au-lait spots, obesity and hyperinsulinism caused by a mutation in the ARID1B gene

PubMed Central

Sonmez, Fatma Mujgan; Uctepe, Eyyup; Gunduz, Mehmet; Gormez, Zeliha; Erpolat, Seval; Oznur, Murat; Sagiroglu, Mahmut Samil; Demirci, Huseyin; Gunduz, Esra

2016-01-01

Summary Coffin-Siris syndrome (CSS) (MIM 135900) is characterized by developmental delay, severe speech impairment, distinctive facial features, hypertrichosis, aplasia or hypoplasia of the distal phalanx or nail of the fifth digit and agenesis of the corpus callosum. Recently, it was shown that mutations in the ARID1B gene are the main cause of CSS, accounting for 76% of identified mutations. Here, we report a 15 year-old female patient who was admitted to our clinic with seizures, speech problems, dysmorphic features, bilaterally big, large thumb, café-au-lait (CAL) spots, obesity and hyperinsulinism. First, the patient was thought to have an association of neurofibromatosis and Rubinstein Taybi syndrome. Because of the large size of the NF1 gene for neurofibromatosis and CREBBP gene for Rubinstein Taybi syndrome, whole exome sequence analysis (WES) was conducted and a novel ARID1B mutation was identified. The proband WES test identified a novel heterozygous frameshift mutation c.3394_3395insTA in exon 13 of ARID1B (NM_017519.2) predicting a premature stop codon p.(Tyr1132Leufs*67). Sanger sequencing confirmed the heterozygous c.3394_3395insTA mutation in the proband and that it was not present in her parents indicating de novo mutation. Further investigation and new cases will help to understand this phenomenon better. PMID:27672547

Coffin-Siris syndrome with café-au-lait spots, obesity and hyperinsulinism caused by a mutation in the ARID1B gene.

PubMed

Sonmez, Fatma Mujgan; Uctepe, Eyyup; Gunduz, Mehmet; Gormez, Zeliha; Erpolat, Seval; Oznur, Murat; Sagiroglu, Mahmut Samil; Demirci, Huseyin; Gunduz, Esra

2016-08-01

Coffin-Siris syndrome (CSS) (MIM 135900) is characterized by developmental delay, severe speech impairment, distinctive facial features, hypertrichosis, aplasia or hypoplasia of the distal phalanx or nail of the fifth digit and agenesis of the corpus callosum. Recently, it was shown that mutations in the ARID1B gene are the main cause of CSS, accounting for 76% of identified mutations. Here, we report a 15 year-old female patient who was admitted to our clinic with seizures, speech problems, dysmorphic features, bilaterally big, large thumb, café-au-lait (CAL) spots, obesity and hyperinsulinism. First, the patient was thought to have an association of neurofibromatosis and Rubinstein Taybi syndrome. Because of the large size of the NF1 gene for neurofibromatosis and CREBBP gene for Rubinstein Taybi syndrome, whole exome sequence analysis (WES) was conducted and a novel ARID1B mutation was identified. The proband WES test identified a novel heterozygous frameshift mutation c.3394_3395insTA in exon 13 of ARID1B (NM_017519.2) predicting a premature stop codon p.(Tyr1132Leufs*67). Sanger sequencing confirmed the heterozygous c.3394_3395insTA mutation in the proband and that it was not present in her parents indicating de novo mutation. Further investigation and new cases will help to understand this phenomenon better.

ACTN3 R577X polymorphism and performance phenotypes in young Chinese male soldiers.

PubMed

Shang, Xuya; Zhang, Fu; Zhang, Li; Huang, Changlin

2012-01-01

Alpha-actinin-3 (ACTN3) is absent in 18% of healthy Caucasian individuals owing to homozygosity for a premature stop codon (X) at amino acid 577 (rs1815739). Previous studies have shown a strong association between ACTN3 genotype and human athletic performance. In a study of 452 young Chinese male soldiers, we examined the distribution of ACTN3 genotypes and alleles and analysed the association between ACTN3 genotypes and athletic performance. We found that the frequencies of the ACTN3 R577X genotype (RR 39.8%, RX 43.4%, and XX 16.8%) and R577X allele (R 61.5%, X 38.5%) in young Chinese males were not significantly different from those in Caucasians. We only observed a significant association (P = 0.025) between ACTN3 R577X genotypes and grip strength. Participants with the XX genotype displayed significantly lower handgrip strength than individuals with the RR genotype (P = 0.021), but the difference between XX and RX means (P = 0.258) and that between RR and RX means (P = 0.42) was not significant. We did not observe a strong association between the ACTN3 R577X genotypes and sprint phenotypes or endurance phenotypes. In conclusion, our results indicate that the ACTN3 R577X polymorphism is most strongly associated with grip strength in young Chinese male soldiers.

A novel whole exon deletion in WWOX gene causes early epilepsy, intellectual disability and optic atrophy.

PubMed

Ben-Salem, Salma; Al-Shamsi, Aisha M; John, Anne; Ali, Bassam R; Al-Gazali, Lihadh

2015-05-01

Recent studies have implicated the WW domain-containing oxidoreductase encoding gene (WWOX) in a severe form of autosomal recessive neurological disorder. This condition showed an overlapping spectrum of clinical features including spinocerebellar ataxia associated with generalized seizures and delayed psychomotor development to growth retardation, spasticity, and microcephaly. We evaluated a child from a consanguineous Emirati family that presented at birth with growth retardation, microcephaly, epileptic seizures, and later developed spasticity and delayed psychomotor development. Screening for deletions and duplications using whole-chromosomal microarray analysis identified a novel homozygous microdeletion encompassing exon 5 of the WWOX gene. Analysis of parental DNA indicated that this deletion was inherited from both parents and lies within a large region of homozygosity. Sanger sequencing of the cDNA showed that the deletion resulted in exon 5 skipping leading to a frame-shift and creating a premature stop codon at amino acid position 212. Quantification of mRNA revealed striking low level of WWOX expression in the child and moderate level of expression in the mother compared to a healthy control. To the best of our knowledge, this is the first homozygous germline structural variation in WWOX gene resulting in truncated transcripts that were presumably subject to NMD pathway. Our findings extend the clinical and genetic spectrum of WWOX mutations and support a crucial role of this gene in neurological development.

Compound Heterozygous Desmoplakin Mutations Result in a Phenotype with a Combination of Myocardial, Skin, Hair, and Enamel Abnormalities

PubMed Central

Mahoney, Mỹ G.; Sadowski, Sara; Brennan, Donna; Pikander, Pekka; Saukko, Pekka; Wahl, James; Aho, Heikki; Heikinheimo, Kristiina; Bruckner-Tuderman, Leena; Fertala, Andrzej; Peltonen, Juha; Uitto, Jouni; Peltonen, Sirkku

2014-01-01

Desmoplakin (DP) anchors the intermediate filament cytoskeleton to the desmosomal cadherins and thereby confers structural stability to tissues. In this study, we present a patient with extensive mucocutaneous blisters, epidermolytic palmoplantar keratoderma, nail dystrophy, enamel dysplasia, and sparse woolly hair. The patient died at the age of 14 years from undiagnosed cardiomyopathy. The skin showed hyperplasia and acantholysis in the mid- and lower epidermal layers, whereas the heart showed extensive fibrosis and fibrofatty replacement in both ventricles. Immunofluorescence microscopy showed a reduction in the C-terminal domain of DP in the skin and oral mucosa. Sequencing of the DP gene showed undescribed mutations in the maternal and paternal alleles. Both mutations affected exon 24 encoding the C-terminal domain. The paternal mutation, c.6310delA, leads to a premature stop codon. The maternal mutation, c.7964 C to A, results in a substitution of an aspartic acid for a conserved alanine residue at amino acid 2655 (A2655D). Structural modeling indicated that this mutation changes the electrostatic potential of the mutated region of DP, possibly altering functions that depend on intermolecular interactions. To conclude, we describe a combination of DP mutation phenotypes affecting the skin, heart, hair, and teeth. This patient case emphasizes the importance of heart examination of patients with desmosomal genodermatoses. PMID:19924139

A Novel de novo CDH1 Germline Variant Aids in the Classification of C-terminal E-cadherin Alterations Predicted to Escape Nonsense-Mediated mRNA Decay.

PubMed

Krempely, Kate; Karam, Rachid

2018-05-24

Most truncating CDH1 pathogenic alterations confer an elevated lifetime risk of diffuse gastric cancer and lobular breast cancer. However, transcripts containing carboxyl-terminal (C-terminal) premature stop codons have been demonstrated to escape the nonsense-mediated mRNA decay (NMD) pathway, and gastric and breast cancer risks associated with these truncations should be carefully evaluated. A female patient underwent multigene panel testing due to a personal history of invasive lobular breast cancer diagnosed at age 54, which identified the germline CDH1 nonsense alteration, c.2506G>T (p.E836*), in the last exon of the gene. Subsequent parental testing for the alteration was negative and additional short tandem repeat analysis confirmed the familial relationships and the de novo occurrence in the proband. Based on the de novo occurrence, clinical history, and rarity in general population databases, this alteration was classified as a likely pathogenic variant. This is the most C-terminal pathogenic alteration reported to date. Additionally, this alteration contributed to the classification of six other upstream CDH1 C-terminal truncating variants as pathogenic or likely pathogenic. Identifying the most distal pathogenic alteration provides evidence to classify other C-terminal truncating variants as either pathogenic or benign, a fundamental step to offering pre-symptomatic screening and prophylactic procedures to the appropriate patients. Cold Spring Harbor Laboratory Press.

A novel RLBP1 gene geographical area-related mutation present in a young patient with retinitis punctata albescens.

PubMed

Scimone, Concetta; Donato, Luigi; Esposito, Teresa; Rinaldi, Carmela; D'Angelo, Rosalia; Sidoti, Antonina

2017-08-01

Autosomal recessive forms of retinitis punctata albescens (RPA) have been described. RPA is characterized by progressive retinal degeneration due to alteration in visual cycle and consequent deposit of photopigments in retinal pigment epithelium. Five loci have been linked to RPA onset. Among these, the retinaldehyde-binding protein 1 gene, RLBP1, is the most frequently involved and several founder mutations were reported. We report results of a genetic molecular investigation performed on a large Sicilian family in which appears a young woman with RPA. The proband is in homozygous condition for a novel RLBP1 single-pair deletion, and her healthy parents, both heterozygous, are not consanguineous. Thenovelc.398delC (p.P133Qfs*258) involves the exon 6 and leads to a premature stop codon, resulting in a truncated protein entirely missing of CRAL-TRIO lipid-binding domain. Pedigree analysis showed other non-consanguineous relatives heterozygous for the same mutation in the family. Extension of mutation research in the native town of the proband revealed its presence also in healthy subjects, in a heterozygous condition. A novel RLBP1 truncating mutation was detected in a young girl affected by RPA. Although her parents are not consanguineous, the mutation was observed in a homozygous condition. Being them native of the same small Sicilian town of Fiumedinisi, the hypothesis of a geographical area-related mutation was assessed and confirmed.

Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay.

PubMed

Nguyen, Andrew D; Nguyen, Thi A; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M; Xu, Xialian; Miller, Bruce L; Rigo, Frank; Ferguson, Shawn M; Huang, Eric J; Walther, Tobias C; Farese, Robert V

2018-03-20

Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized Grn R493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous Grn R493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in Grn R493X mice and cell lines and in fibroblasts from patients containing the GRN R493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. Copyright © 2018 the Author(s). Published by PNAS.

Genetic features of Mycobacterium tuberculosis modern Beijing sublineage

PubMed Central

Liu, Qingyun; Luo, Tao; Dong, Xinran; Sun, Gang; Liu, Zhu; Gan, Mingyun; Wu, Jie; Shen, Xin; Gao, Qian

2016-01-01

Mycobacterium tuberculosis (MTB) Beijing strains have caused a great concern because of their rapid emergence and increasing prevalence in worldwide regions. Great efforts have been made to investigate the pathogenic characteristics of Beijing strains such as hypervirulence, drug resistance and favoring transmission. Phylogenetically, MTB Beijing family was divided into modern and ancient sublineages. Modern Beijing strains displayed enhanced virulence and higher prevalence when compared with ancient Beijing strains, but the genetic basis for this difference remains unclear. In this study, by analyzing previously published sequencing data of 1082 MTB Beijing isolates, we determined the genetic changes that were commonly present in modern Beijing strains but absent in ancient Beijing strains. These changes include 44 single-nucleotide polymorphisms (SNPs) and two short genomic deletions. Through bioinformatics analysis, we demonstrated that these genetic changes had high probability of functional effects. For example, 4 genes were frameshifted due to premature stop mutation or genomic deletions, 19 nonsynonymous SNPs located in conservative codons, and there is a significant enrichment in regulatory network for all nonsynonymous mutations. Besides, three SNPs located in promoter regions were verified to alter downstream gene expressions. Our study precisely defined the genetic features of modern Beijing strains and provided interesting clues for future researches to elucidate the mechanisms that underlie this sublineage's successful expansion. These findings from the analysis of the modern Beijing sublineage could provide us a model to understand the dynamics of pathogenicity of MTB. PMID:26905026

RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay.

PubMed

Dean, Kimberly M; Grayhack, Elizabeth J

2012-12-01

We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.

Mucopolysaccharidosis IVA: Four new exonic mutations in patients with N-acetylgalactosamine-6-sulfate sulfatase deficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomatsu, Shunji; Fukuda, Seiji; Yamagishi, Atsushi

1996-05-01

We report four new mutations in Japanese patients with mucopolysaccharidosis IVA (MPSIVA) who were heterozygous for a common double gene deletion. A nonsense mutation of CAG to TAG at codon 148 in exon 4 was identified, resulting in a change of Q to a stop codon and three missense mutations: V (GTC) to A (GCC) at codon 138 in exon 4, P (CCC) to S (TCC) at codon 151 in exon 5, and P (CCC) to L (CTC) at codon 151 in exon 5. Introduction of these mutations into the normal GALNS cDNA and transient expression in cultured fibroblasts resultedmore » in a significant decrease in the enzyme activity. V138A and Q148X mutations result in changes of restriction site, which were analyzed by restriction-enzyme assay. P151S and P151L mutations that did not alter the restriction site were detected by direct sequencing or allele specific oligohybridization. Detection of the double gene deletion was initially done using Southern blots and was confirmed by PCR. Haplotypes were determined using seven polymorphisms to the GALNS locus in families with the double gene deletion. Haplotype analysis showed that the common double gene deletion occurred on a single haplotype, except for some variation in a VNTR-like polymorphism. This finding is consistent with a common founder for all individuals with this mutation. 48 refs., 5 figs., 1 tab.« less

Comparative mitochondrial genome analysis of Daphnis nerii and other lepidopteran insects reveals conserved mitochondrial genome organization and phylogenetic relationships

PubMed Central

Sun, Yu; Chen, Chen; Gao, Jin; Abbas, Muhammad Nadeem; Kausar, Saima; Qian, Cen; Wang, Lei; Wei, Guoqing; Zhu, Bao-Jian

2017-01-01

In the present study, the complete sequence of the mitochondrial genome (mitogenome) of Daphnis nerii (Lepidoptera: Sphingidae) is described. The mitogenome (15,247 bp) of D.nerii encodes13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and an adenine (A) + thymine (T)-rich region. Its gene complement and order is similar to that of other sequenced lepidopterans. The 12 PCGs initiated by ATN codons except for cytochrome c oxidase subunit 1 (cox1) gene that is seemingly initiated by the CGA codon as documented in other insect mitogenomes. Four of the 13 PCGs have the incomplete termination codon T, while the remainder terminated with the canonical stop codon. This mitogenome has six major intergenic spacers, with the exception of A+T-rich region, spanning at least 10 bp. The A+T-rich region is 351 bp long, and contains some conserved regions, including ‘ATAGA’ motif followed by a 17 bp poly-T stretch, a microsatellite-like element (AT)9 and also a poly-A element. Phylogenetic analyses based on 13 PCGs using maximum likelihood (ML) and Bayesian inference (BI) revealed that D. nerii resides in the Sphingidae family. PMID:28598968

Changes in mitochondrial genetic codes as phylogenetic characters: Two examples from the flatworms

PubMed Central

Telford, Maximilian J.; Herniou, Elisabeth A.; Russell, Robert B.; Littlewood, D. Timothy J.

2000-01-01

Shared molecular genetic characteristics other than DNA and protein sequences can provide excellent sources of phylogenetic information, particularly if they are complex and rare and are consequently unlikely to have arisen by chance convergence. We have used two such characters, arising from changes in mitochondrial genetic code, to define a clade within the Platyhelminthes (flatworms), the Rhabditophora. We have sampled 10 distinct classes within the Rhabditophora and find that all have the codon AAA coding for the amino acid Asn rather than the usual Lys and AUA for Ile rather than the usual Met. We find no evidence to support claims that the codon UAA codes for Tyr in the Platyhelminthes rather than the standard stop codon. The Rhabditophora are a very diverse group comprising the majority of the free-living turbellarian taxa and the parasitic Neodermata. In contrast, three other classes of turbellarian flatworm, the Acoela, Nemertodermatida, and Catenulida, have the standard invertebrate assignments for these codons and so are convincingly excluded from the rhabditophoran clade. We have developed a rapid computerized method for analyzing genetic codes and demonstrate the wide phylogenetic distribution of the standard invertebrate code as well as confirming already known metazoan deviations from it (ascidian, vertebrate, echinoderm/hemichordate). PMID:11027335

Cohort Study of the Success of Controlled Weight Loss Programs for Obese Dogs.

PubMed

German, A J; Titcomb, J M; Holden, S L; Queau, Y; Morris, P J; Biourge, V

2015-01-01

Most weight loss studies in obese dogs assess rate and percentage of weight loss in the first 2-3 months, rather than the likelihood of successfully reaching target weight. To determine outcome of controlled weight loss programs for obese dogs, and to determine the factors associated with successful completion. 143 obese dogs undergoing a controlled weight loss program. This was a cohort study of obese dogs attending a referral weight management clinic. Dogs were studied during their period of weight loss, and cases classified according to outcome as "completed" (reached target weight), "euthanized" (was euthanized before reaching target weight), or "stopped prematurely" (program stopped early for other reasons). Factors associated with successful completion were assessed using simple and multiple logistic regression. 87/143 dogs (61%) completed their weight loss program, 11 [8%] died or were euthanized, and the remaining 45 [32%] stopped prematurely. Reasons for dogs stopping prematurely included inability to contact owner, refusal to comply with weight management advice, or development of another illness. Successful weight loss was positively associated with a faster rate (P < .001), a longer duration (P < .001), and feeding a dried weight management diet (P = .010), but negatively associated with starting body fat (P < .001), and use of dirlotapide (P = .0046). Just over half of all obese dogs on a controlled weight loss program reach their target weight. Future studies should better clarify reasons for success in individual cases, and also the role of factors such as activity and behavioral modification. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons

PubMed Central

Pranke, Iwona; Bidou, Laure; Martin, Natacha; Blanchet, Sandra; Hatton, Aurélie; Karri, Sabrina; Cornu, David; Costes, Bruno; Chevalier, Benoit; Tondelier, Danielle; Coupet, Matthieu; Edelman, Aleksander; Fanen, Pascale; Namy, Olivier; Sermet-Gaudelus, Isabelle

2018-01-01

Premature termination codons (PTCs) are generally associated with severe forms of genetic diseases. Readthrough of in-frame PTCs using small molecules is a promising therapeutic approach. Nonetheless, the outcome of preclinical studies has been low and variable. Treatment efficacy depends on: 1) the level of drug-induced readthrough, 2) the amount of target transcripts, and 3) the activity of the recoded protein. The aim of the present study was to identify, in the cystic fibrosis transmembrane conductance regulator (CFTR) model, recoded channels from readthrough therapy that may be enhanced using CFTR modulators. First, drug-induced readthrough of 15 PTCs was measured using a dual reporter system under basal conditions and in response to gentamicin and negamycin. Secondly, exon skipping associated with these PTCs was evaluated with a minigene system. Finally, incorporated amino acids were identified by mass spectrometry and the function of the predicted recoded CFTR channels corresponding to these 15 PTCs was measured. Nonfunctional channels were subjected to CFTR-directed ivacaftor-lumacaftor treatments. The results demonstrated that CFTR modulators increased activity of recoded channels, which could also be confirmed in cells derived from a patient. In conclusion, this work will provide a framework to adapt treatments to the patient's genotype by identifying the most efficient molecule for each PTC and the recoded channels needing co-therapies to rescue channel function. PMID:29497617

Genetic hotels for the standard genetic code: evolutionary analysis based upon novel three-dimensional algebraic models.

PubMed

José, Marco V; Morgado, Eberto R; Govezensky, Tzipe

2011-07-01

Herein, we rigorously develop novel 3-dimensional algebraic models called Genetic Hotels of the Standard Genetic Code (SGC). We start by considering the primeval RNA genetic code which consists of the 16 codons of type RNY (purine-any base-pyrimidine). Using simple algebraic operations, we show how the RNA code could have evolved toward the current SGC via two different intermediate evolutionary stages called Extended RNA code type I and II. By rotations or translations of the subset RNY, we arrive at the SGC via the former (type I) or via the latter (type II), respectively. Biologically, the Extended RNA code type I, consists of all codons of the type RNY plus codons obtained by considering the RNA code but in the second (NYR type) and third (YRN type) reading frames. The Extended RNA code type II, comprises all codons of the type RNY plus codons that arise from transversions of the RNA code in the first (YNY type) and third (RNR) nucleotide bases. Since the dimensions of remarkable subsets of the Genetic Hotels are not necessarily integer numbers, we also introduce the concept of algebraic fractal dimension. A general decoding function which maps each codon to its corresponding amino acid or the stop signals is also derived. The Phenotypic Hotel of amino acids is also illustrated. The proposed evolutionary paths are discussed in terms of the existing theories of the evolution of the SGC. The adoption of 3-dimensional models of the Genetic and Phenotypic Hotels will facilitate the understanding of the biological properties of the SGC.

Novel mutations in the connexin 32 gene associated with X-linked Charcot-Marie-Tooth disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, C.; Ainsworth, P.

1994-09-01

Charcot-Marie-Tooth disease is a pathologically and genetically hetergenous group of disorders that cause a progressive neuropathy, defined pathologically by degeneration of the myelin (CMT 1) of the axon (CMT 2) of the peripheral nerves. An X-linked type of the demyelinating form of this disorder (CMT X) has recently been linked to mutations in the connexin 32 (Cx32) gene, which codes for a 284 amino acid gap junction protein found in myelinated peripheral nerve. To date some 7 different mutations in this gene have been identified as being responsible for CMT X. The majority of these predict nonconservative amino acid substitutions,more » while one is a frameshift mutation which predicts a premature stop at codon 21. We report the results of molecular studies on three further local CMT X kindreds. The Cx32 gene was amplified by PCR in three overlapping fragments 300-450 bp in length using leukocyte-derived DNA as template. These were either sequenced directly using a deaza dGTP sequencing protocol, or were cloned and sequenced using a TA vector. In two of the kindreds the affected members carried a point mutation which was predicted to effect a non-conservative amino acid change within the first transmembrane domain. Both of these mutations caused a restriction site alteration (the loss of an Nla III and the creation of a Pvu II, respectively), and the former mutation was observed to segregate with the clinicial phenotype in affected family members. Affected members of the third kindred, which was a very large multigenerational family that had been extensively studied previously, were shown to carry a point mutation predicted to cause a premature truncation of the Cx32 gene product in the intracellular carboxy terminus. This mutation obliterated an Rsa I site which allowed a rapid screen of several other family members.« less

Potential role for a B-catenin coactivator (high mobility group AT-hook 1 protein) during the latency-reactivation cycle of bovine herpesverus 1

USDA-ARS?s Scientific Manuscript database

The latency-related (LR)-RNA encoded by bovine herpes virus 1 (BoHV-1) is abundantly expressed in latently infected sensory neurons. Although the LR gene encodes several products, ORF2 appears to play a dominant role during the latency-reactivation cycle because a mutant virus containing stop codons...

Unit-length line-1 transcripts in human teratocarcinoma cells.

PubMed Central

Skowronski, J; Fanning, T G; Singer, M F

1988-01-01

We have characterized the approximately 6.5-kilobase cytoplasmic poly(A)+ Line-1 (L1) RNA present in a human teratocarcinoma cell line, NTera2D1, by primer extension and by analysis of cloned cDNAs. The bulk of the RNA begins (5' end) at the residue previously identified as the 5' terminus of the longest known primate genomic L1 elements, presumed to represent "unit" length. Several of the cDNA clones are close to 6 kilobase pairs, that is, close to full length. The partial sequences of 18 cDNA clones and full sequence of one (5,975 base pairs) indicate that many different genomic L1 elements contribute transcripts to the 6.5-kilobase cytoplasmic poly(A)+ RNA in NTera2D1 cells because no 2 of the 19 cDNAs analyzed had identical sequences. The transcribed elements appear to represent a subset of the total genomic L1s, a subset that has a characteristic consensus sequence in the 3' noncoding region and a high degree of sequence conservation throughout. Two open reading frames (ORFs) of 1,122 (ORF1) and 3,852 (ORF2) bases, flanked by about 800 and 200 bases of sequence at the 5' and 3' ends, respectively, can be identified in the cDNAs. Both ORFs are in the same frame, and they are separated by 33 bases bracketed by two conserved in-frame stop codons. ORF 2 is interrupted by at least one randomly positioned stop codon in the majority of the cDNAs. The data support proposals suggesting that the human L1 family includes one or more functional genes as well as an extraordinarily large number of pseudogenes whose ORFs are broken by stop codons. The cDNA structures suggest that both genes and pseudogenes are transcribed. At least one of the cDNAs (cD11), which was sequenced in its entirety, could, in principle, represent an mRNA for production of the ORF1 polypeptide. The similarity of mammalian L1s to several recently described invertebrate movable elements defines a new widely distributed class of elements which we term class II retrotransposons. Images PMID:2454389

Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code

PubMed Central

Rozov, Alexey; Demeshkina, Natalia; Khusainov, Iskander; Westhof, Eric; Yusupov, Marat; Yusupova, Gulnara

2016-01-01

Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNALysUUU with hypermodified 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine–pyrimidine mismatches. We show that mnm5s2U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism. PMID:26791911

Truncated variants of apolipoprotein B cause hypobetalipoproteinaemia.

PubMed Central

Collins, D R; Knott, T J; Pease, R J; Powell, L M; Wallis, S C; Robertson, S; Pullinger, C R; Milne, R W; Marcel, Y L; Humphries, S E

1988-01-01

Familial hypobetalipoproteinaemia is a rare autosomal dominant disorder in which levels of apo-B-containing plasma lipoproteins are approximately half-normal in heterozygotes and virtually absent in homozygotes. Here we describe mutations of the apo-B gene that cause two different truncated variants of apo-B in unrelated individuals with hypobetalipoproteinaemia. One variant, apo-B(His1795----Met-Trp-Leu-Val-Thr-Term) is predicted to be 1799 amino acids long and arises from deletion of a single nucleotide (G) from leucine codon 1794. This protein was found at low levels in very low density and low density lipoprotein fractions in the blood. The second, shorter variant, apo-B(Arg1306----Term), is caused by mutation of a CpG dinucleotide in arginine codon 1306 converting it to a stop codon and predicting a protein of 1305 residues. The product of this allele could not be detected in the circulation. The differences in size and behaviour of these two variants compared to apo-B100 or apo-B48 point to domains that may be important for the assembly, secretion or stability of apo-B-containing lipoproteins. Images PMID:2843815

The mitochondrial genome of the multicolored Asian lady beetle Harmonia axyridis (Pallas) and a phylogenetic analysis of the Polyphaga (Insecta: Coleoptera).

PubMed

Niu, Fang-Fang; Zhu, Liang; Wang, Su; Wei, Shu-Jun

2016-07-01

Here, we report the mitochondrial genome sequence of the multicolored Asian lady beetle Harmonia axyridis (Pallas, 1773) (Coleoptera: Coccinellidae) (GenBank accession No. KR108208). This is the first species with sequenced mitochondrial genome from the genus Harmonia. The current length with partitial A + T-rich region of this mitochondrial genome is 16,387 bp. All the typical genes were sequenced except the trnI and trnQ. As in most other sequenced mitochondrial genomes of Coleoptera, there is no re-arrangement in the sequenced region compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN codons. Five, five and three protein-coding genes stop with termination codon TAA, TA and T, respectively. Phylogenetic analysis using Bayesian method based on the first and second codon positions of the protein-coding genes supported that the Scirtidae is a basal lineage of Polyphaga. The Harmonia and the Coccinella form a sister lineage. The monophyly of Staphyliniformia, Scarabaeiformia and Cucujiformia was supported. The Buprestidae was found to be a sister group to the Bostrichiformia.

Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

PubMed Central

Ko, Jae-hyeong; Llopis, Paula Montero; Heinritz, Jennifer; Jacobs-Wagner, Christine; Söll, Dieter

2013-01-01

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNAHis have distinctive identity elements, we constructed E. coli tRNAHis CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNAHis CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNAHis CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNAHis CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair. PMID:24386240

Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code.

PubMed

Rozov, Alexey; Demeshkina, Natalia; Khusainov, Iskander; Westhof, Eric; Yusupov, Marat; Yusupova, Gulnara

2016-01-21

Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNA(Lys)(UUU) with hypermodified 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine-pyrimidine mismatches. We show that mnm(5)s(2)U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism.

Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code

NASA Astrophysics Data System (ADS)

Rozov, Alexey; Demeshkina, Natalia; Khusainov, Iskander; Westhof, Eric; Yusupov, Marat; Yusupova, Gulnara

2016-01-01

Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNALysUUU with hypermodified 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine-pyrimidine mismatches. We show that mnm5s2U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism.

Therapy for Duchenne muscular dystrophy: renewed optimism from genetic approaches.

PubMed

Fairclough, Rebecca J; Wood, Matthew J; Davies, Kay E

2013-06-01

Duchenne muscular dystrophy (DMD) is a devastating progressive disease for which there is currently no effective treatment except palliative therapy. There are several promising genetic approaches, including viral delivery of the missing dystrophin gene, read-through of translation stop codons, exon skipping to restore the reading frame and increased expression of the compensatory utrophin gene. The lessons learned from these approaches will be applicable to many other disorders.

Multiple conversion between the genes encoding bacterial class-I release factors

PubMed Central

Ishikawa, Sohta A.; Kamikawa, Ryoma; Inagaki, Yuji

2015-01-01

Bacteria require two class-I release factors, RF1 and RF2, that recognize stop codons and promote peptide release from the ribosome. RF1 and RF2 were most likely established through gene duplication followed by altering their stop codon specificities in the common ancestor of extant bacteria. This scenario expects that the two RF gene families have taken independent evolutionary trajectories after the ancestral gene duplication event. However, we here report two independent cases of conversion between RF1 and RF2 genes (RF1-RF2 gene conversion), which were severely examined by procedures incorporating the maximum-likelihood phylogenetic method. In both cases, RF1-RF2 gene conversion was predicted to occur in the region encoding nearly entire domain 3, of which functions are common between RF paralogues. Nevertheless, the ‘direction’ of gene conversion appeared to be opposite from one another—from RF2 gene to RF1 gene in one case, while from RF1 gene to RF2 gene in the other. The two cases of RF1-RF2 gene conversion prompt us to propose two novel aspects in the evolution of bacterial class-I release factors: (i) domain 3 is interchangeable between RF paralogues, and (ii) RF1-RF2 gene conversion have occurred frequently in bacterial genome evolution. PMID:26257102

Truncated ORF1 proteins can suppress LINE-1 retrotransposition in trans

PubMed Central

Sokolowski, Mark; Chynces, May; deHaro, Dawn; Christian, Claiborne M.

2017-01-01

Abstract Long interspersed element 1 (L1) is an autonomous non-LTR retroelement that is active in mammalian genomes. Although retrotranspositionally incompetent and functional L1 loci are present in the same genomes, it remains unknown whether non-functional L1s have any trans effect on mobilization of active elements. Using bioinformatic analysis, we identified over a thousand of human L1 loci containing at least one stop codon in their ORF1 sequence. RNAseq analysis confirmed that many of these loci are expressed. We demonstrate that introduction of equivalent stop codons in the full-length human L1 sequence leads to the expression of truncated ORF1 proteins. When supplied in trans some truncated human ORF1 proteins suppress human L1 retrotransposition. This effect requires the N-terminus and coiled-coil domain (C-C) as mutations within the ORF1p C-C domain abolish the suppressive effect of truncated proteins on L1 retrotransposition. We demonstrate that the expression levels and length of truncated ORF1 proteins influence their ability to suppress L1 retrotransposition. Taken together these findings suggest that L1 retrotransposition may be influenced by coexpression of defective L1 loci and that these L1 loci may reduce accumulation of de novo L1 integration events. PMID:28431148

The primary structure of the Saccharomyces cerevisiae gene for 3-phosphoglycerate kinase.

PubMed Central

Hitzeman, R A; Hagie, F E; Hayflick, J S; Chen, C Y; Seeburg, P H; Derynck, R

1982-01-01

The DNA sequence of the gene for the yeast glycolytic enzyme, 3-phosphoglycerate kinase (PGK), has been obtained by sequencing part of a 3.1 kbp HindIII fragment obtained from the yeast genome. The structural gene sequence corresponds to a reading frame of 1251 bp coding for 416 amino acids with no intervening DNA sequences. The amino acid sequence is approximately 65 percent homologous with human and horse PGK protein sequences and is in general agreement with the published protein sequence for yeast PGK. As for other highly expressed structural genes in yeast, the coding sequence is highly codon biased with 95 percent of the amino acids coded for by a select 25 codons (out of 61 possible). Besides structural DNA sequence, 291 bp of 5'-flanking sequence and 286 bp of 3'-flanking sequence were determined. Transcription starts 36 nucleotides upstream from the translational start and stops 86-93 nucleotides downstream from the translational stop. These results suggest a non-polyadenylated mRNA length of 1373 to 1380 nucleotides, which is consistent with the observed length of 1500 nucleotides for polyadenylated PGK mRNA. A sequence TATATATAAA is found at 145 nucleotides upstream from the translational start. This sequence resembles the TATAAA box that is possibly associated with RNA polymerase II binding. Images PMID:6296791

Massive programmed translational jumping in mitochondria

PubMed Central

Lang, B. Franz; Jakubkova, Michaela; Hegedusova, Eva; Daoud, Rachid; Forget, Lise; Brejova, Brona; Vinar, Tomas; Kosa, Peter; Fricova, Dominika; Nebohacova, Martina; Griac, Peter; Tomaska, Lubomir; Burger, Gertraud; Nosek, Jozef

2014-01-01

Programmed translational bypassing is a process whereby ribosomes “ignore” a substantial interval of mRNA sequence. Although discovered 25 y ago, the only experimentally confirmed example of this puzzling phenomenon is expression of the bacteriophage T4 gene 60. Bypassing requires translational blockage at a “takeoff codon” immediately upstream of a stop codon followed by a hairpin, which causes peptidyl-tRNA dissociation and reassociation with a matching “landing triplet” 50 nt downstream, where translation resumes. Here, we report 81 translational bypassing elements (byps) in mitochondria of the yeast Magnusiomyces capitatus and demonstrate in three cases, by transcript analysis and proteomics, that byps are retained in mitochondrial mRNAs but not translated. Although mitochondrial byps resemble the bypass sequence in the T4 gene 60, they utilize unused codons instead of stops for translational blockage and have relaxed matching rules for takeoff/landing sites. We detected byp-like sequences also in mtDNAs of several Saccharomycetales, indicating that byps are mobile genetic elements. These byp-like sequences lack bypassing activity and are tolerated when inserted in-frame in variable protein regions. We hypothesize that byp-like elements have the potential to contribute to evolutionary diversification of proteins by adding new domains that allow exploration of new structures and functions. PMID:24711422

[Novel CHST6 compound heterozygous mutations cause macular corneal dystrophy in a Chinese family].

PubMed

Qi, Yan-hua; Dang, Xiu-hong; Su, Hong; Zhou, Nan; Liang, Ting; Wang, Zheng; Huang, Shang-zhi

2010-02-01

The aim of this study was to identify mutations of CHST6 gene in a Chinese family with macular corneal dystrophy (MCD) and to investigate the histopathological changes of MCD. Corneal button of the proband was obtained from penetrating keratoplasty for the treatment of severe corneal dystrophy. The sections and ultrathin sections of this specimen were examined under light microscope and transmission electron microscope (TEM). Genomic DNA was extracted from leukocytes in peripheral blood from the family members. The coding region of CHST6 was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by direct sequencing and restriction enzyme digestion. Histochemical study revealed positive results of colloidal iron stain. TEM revealed enlargement of smooth endoplasmic reticulum and the presence of intracytoplasmic vacuoles. Two mutations, Q298X Y358H, were identified in exon 3 of CHST6. Three patients were compound heterozygotes of these two mutations. The C892T transversion occurred at codon 298 turned the codon of glutamine to a stop codon; the T1072C transversion occurred at codon 358 caused a missense mutation, tyrosine to histidine. All six unaffected family members were heterozygotes. These two mutations were not detected in any of the 100 control subjects. The novel compound heterozygous mutation results in loss of CHST6 function and causes the occurrence of MCD. This is the first report of this gene mutation.

Thiamine-responsive megaloblastic anemia: early diagnosis may be effective in preventing deafness.

PubMed

Onal, Hasan; Bariş, Safa; Ozdil, Mine; Yeşil, Gözde; Altun, Gürkan; Ozyilmaz, Isa; Aydin, Ahmet; Celkan, Tiraje

2009-01-01

Thiamine-responsive megaloblastic anemia syndrome is an autosomal recessive disorder characterized by diabetes mellitus, megaloblastic anemia and sensorineural hearing loss. Mutations in the SLC19A2 gene, encoding a high-affinity thiamine transporter protein, THTR-1, are responsible for the clinical features associated with thiamine-responsive megaloblastic anemia syndrome in which treatment with pharmacological doses of thiamine correct the megaloblastic anemia and diabetes mellitus. The anemia can recur when thiamine is withdrawn. Thiamine may be effective in preventing deafness if started before two months. Our patient was found homozygous for a mutation, 242insA, in the nucleic acid sequence of exon B, with insertion of an adenine introducing a stop codon at codon 52 in the high-affinity thiamine transporter gene, SLC19A2, on chromosome 1q23.3.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baserga, S.J.; Benz, E.J. Jr.

A number of premature translation termination mutations (nonsense mutations) have been described in the human {alpha}- and {beta}-globin genes. Studies on mRNA isolated from patients with {beta}{sup 0}-thalassemia have shown that for both the {beta}-17 and the {beta}-39 mutations less than normal levels of {beta}-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human {beta}-globin mRNA). In vitro studies using the cloned {beta}-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. Themore » authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human {beta}-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation.« less

Mutations in STRA6 Cause a Broad Spectrum of Malformations Including Anophthalmia, Congenital Heart Defects, Diaphragmatic Hernia, Alveolar Capillary Dysplasia, Lung Hypoplasia, and Mental Retardation

PubMed Central

Pasutto, Francesca; Sticht, Heinrich; Hammersen, Gerhard; Gillessen-Kaesbach, Gabriele; FitzPatrick, David R.; Nürnberg, Gudrun; Brasch, Frank; Schirmer-Zimmermann, Heidemarie; Tolmie, John L.; Chitayat, David; Houge, Gunnar; Fernández-Martínez, Lorena; Keating, Sarah; Mortier, Geert; Hennekam, Raoul C. M.; von der Wense, Axel; Slavotinek, Anne; Meinecke, Peter; Bitoun, Pierre; Becker, Christian; Nürnberg, Peter; Reis, André; Rauch, Anita

2007-01-01

We observed two unrelated consanguineous families with malformation syndromes sharing anophthalmia and distinct eyebrows as common signs, but differing for alveolar capillary dysplasia or complex congenital heart defect in one and diaphragmatic hernia in the other family. Homozygosity mapping revealed linkage to a common locus on chromosome 15, and pathogenic homozygous mutations were identified in STRA6, a member of a large group of “stimulated by retinoic acid” genes encoding novel transmembrane proteins, transcription factors, and secreted signaling molecules or proteins of largely unknown function. Subsequently, homozygous STRA6 mutations were also demonstrated in 3 of 13 patients chosen on the basis of significant phenotypic overlap to the original cases. While a homozygous deletion generating a premature stop codon (p.G50AfsX22) led to absence of the immunoreactive protein in patient’s fibroblast culture, structural analysis of three missense mutations (P90L, P293L, and T321P) suggested significant effects on the geometry of the loops connecting the transmembrane helices of STRA6. Two further variations in the C-terminus (T644M and R655C) alter specific functional sites, an SH2-binding motif and a phosphorylation site, respectively. STRA6 mutations thus define a pleiotropic malformation syndrome representing the first human phenotype associated with mutations in a gene from the “STRA” group. PMID:17273977

Improved fermentation performance of a lager yeast after repair of its AGT1 maltose and maltotriose transporter genes.

PubMed

Vidgren, Virve; Huuskonen, Anne; Virtanen, Hannele; Ruohonen, Laura; Londesborough, John

2009-04-01

The use of more concentrated, so-called high-gravity and very-high-gravity (VHG) brewer's worts for the manufacture of beer has economic and environmental advantages. However, many current strains of brewer's yeasts ferment VHG worts slowly and incompletely, leaving undesirably large amounts of maltose and especially maltotriose in the final beers. alpha-Glucosides are transported into Saccharomyces yeasts by several transporters, including Agt1, which is a good carrier of both maltose and maltotriose. The AGT1 genes of brewer's ale yeast strains encode functional transporters, but the AGT1 genes of the lager strains studied contain a premature stop codon and do not encode functional transporters. In the present work, one or more copies of the AGT1 gene of a lager strain were repaired with DNA sequence from an ale strain and put under the control of a constitutive promoter. Compared to the untransformed strain, the transformants with repaired AGT1 had higher maltose transport activity, especially after growth on glucose (which represses endogenous alpha-glucoside transporter genes) and higher ratios of maltotriose transport activity to maltose transport activity. They fermented VHG (24 degrees Plato) wort faster and more completely, producing beers containing more ethanol and less residual maltose and maltotriose. The growth and sedimentation behaviors of the transformants were similar to those of the untransformed strain, as were the profiles of yeast-derived volatile aroma compounds in the beers.

MCM5: a new actor in the link between DNA replication and Meier-Gorlin syndrome.

PubMed

Vetro, Annalisa; Savasta, Salvatore; Russo Raucci, Annalisa; Cerqua, Cristina; Sartori, Geppo; Limongelli, Ivan; Forlino, Antonella; Maruelli, Silvia; Perucca, Paola; Vergani, Debora; Mazzini, Giuliano; Mattevi, Andrea; Stivala, Lucia Anna; Salviati, Leonardo; Zuffardi, Orsetta

2017-05-01

Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5. This gene encodes a subunit of the replicative helicase complex, which represents a component of the pre-RC. Both variants, a missense substitution within a conserved domain critical for the helicase activity, and a single base deletion causing a frameshift and a premature stop codon, were predicted to be detrimental for the MCM5 function. Although variants of MCM5 have never been reported in specific human diseases, defect of this gene in zebrafish causes a phenotype of growth restriction overlapping the one associated with orc1 depletion. Complementation experiments in yeast showed that the plasmid carrying the missense variant was unable to rescue the lethal phenotype caused by mcm5 deletion. Moreover cell-cycle progression was delayed in patient's cells, as already shown for mutations in the ORC1 gene. Altogether our findings support the role of MCM5 as a novel gene involved in MGORS, further emphasizing that this condition is caused by impaired DNA replication.

New horizons for cystic fibrosis treatment.

PubMed

Fajac, Isabelle; De Boeck, Kris

2017-02-01

Cystic fibrosis is an inherited multi-system disease associated with chronic lung infection, malabsorption, salt loss syndromes, male infertility and leading to numerous comorbidities. The landscape in cystic fibrosis care has changed markedly with currently more adult patients than children in many countries. Over 2000 different mutations in the CFTR gene have been reported and the majority are extremely rare. Understanding how CFTR mutations translate to disturbed synthesis or function of the CFTR protein has opened the way to 'personalized' treatments to correct the basic defect. The first 2 drugs have reached the clinic: a CFTR potentiator to augment CFTR channel function, and the combination of this potentiator with a corrector to increase CFTR expression at the cell membrane. To obtain robust correction of CFTR expression at the cell membrane, combinations of correctors with additive efficacy are under investigation. Other mutation type-specific treatments under clinical investigation are premature stop codon-read through drugs and antisense oligonucleotides that correct the basic defect at the mRNA level. Restoring the defective gene by gene editing can already be achieved ex vivo. Mutation agnostic treatments are explored as well: stabilizing CFTR expression at the cell membrane, circumventing the CFTR channel by blocking or activating other ion channels, and gene therapy. Combinations of these therapies can be anticipated. The pipeline of corrective strategies under clinical investigation is increasing continuously and a rising number of pharmaceutical companies are entering the field. Copyright © 2016 Elsevier Inc. All rights reserved.

Phenotypic spectrum associated with mutations of the mitochondrial polymerase gamma gene.

PubMed

Horvath, Rita; Hudson, Gavin; Ferrari, Gianfrancesco; Fütterer, Nancy; Ahola, Sofia; Lamantea, Eleonora; Prokisch, Holger; Lochmüller, Hanns; McFarland, Robert; Ramesh, V; Klopstock, Thomas; Freisinger, Peter; Salvi, Fabrizio; Mayr, Johannes A; Santer, Rene; Tesarova, Marketa; Zeman, Jiri; Udd, Bjarne; Taylor, Robert W; Turnbull, Douglass; Hanna, Michael; Fialho, Doreen; Suomalainen, Anu; Zeviani, Massimo; Chinnery, Patrick F

2006-07-01

Mutations in the gene coding for the catalytic subunit of the mitochondrial DNA (mtDNA) polymerase gamma (POLG1) have recently been described in patients with diverse clinical presentations, revealing a complex relationship between genotype and phenotype in patients and their families. POLG1 was sequenced in patients from different European diagnostic and research centres to define the phenotypic spectrum and advance understanding of the recurrence risks. Mutations were identified in 38 cases, with the majority being sporadic compound heterozygotes. Eighty-nine DNA sequence changes were identified, including 2 predicted to alter a splice site, 1 predicted to cause a premature stop codon and 13 predicted to cause novel amino acid substitutions. The majority of children had a mutation in the linker region, often 1399G-->A (A467T), and a mutation affecting the polymerase domain. Others had mutations throughout the gene, and 11 had 3 or more substitutions. The clinical presentation ranged from the neonatal period to late adult life, with an overlapping phenotypic spectrum from severe encephalopathy and liver failure to late-onset external ophthalmoplegia, ataxia, myopathy and isolated muscle pain or epilepsy. There was a strong gender bias in children, with evidence of an environmental interaction with sodium valproate. POLG1 mutations cause an overlapping clinical spectrum of disease with both dominant and recessive modes of inheritance. 1399G-->A (A467T) is common in children, but complete POLG1 sequencing is required to identify multiple mutations that can have complex implications for genetic counselling.

Rett-like phenotypes: expanding the genetic heterogeneity to the KCNA2 gene and first familial case of CDKL5-related disease.

PubMed

Allou, L; Julia, S; Amsallem, D; El Chehadeh, S; Lambert, L; Thevenon, J; Duffourd, Y; Saunier, A; Bouquet, P; Pere, S; Moustaïne, A; Ruaud, L; Roth, V; Jonveaux, P; Philippe, C

2017-03-01

Several genes have been implicated in Rett syndrome (RTT) in its typical and variant forms. We applied next-generation sequencing (NGS) to evaluate for mutations in known or new candidate genes in patients with variant forms of Rett or Rett-like phenotypes of unknown molecular aetiology. In the first step, we used NGS with a custom panel including MECP2, CDKL5, FOXG1, MEF2C and IQSEC2. In addition to a FOXG1 mutation in a patient with all core features of the congenital variant of RTT, we identified a missense (p.Ser240Thr) in CDKL5 in a patient who appeared to be seizure free. This missense was maternally inherited with opposite allele expression ratios in the proband and her mother. In the asymptomatic mother, the mutated copy of the CDKL5 gene was inactivated in 90% of blood cells. We also identified a premature stop codon (p.Arg926*) in IQSEC2 in a patient with a Rett-like phenotype. Finally, exome sequencing enabled us to characterize a heterozygous de novo missense (p.Val408Ala) in KCNA2 encoding the potassium channel Kv 1.2 in a girl with infantile-onset seizures variant of RTT. Our study expands the genetic heterogeneity of RTT and RTT-like phenotypes. Moreover, we report the first familial case of CDKL5-related disease. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Tissue specific roles for the ribosome biogenesis factor Wdr43 in zebrafish development.

PubMed

Zhao, Chengtian; Andreeva, Viktoria; Gibert, Yann; LaBonty, Melissa; Lattanzi, Victoria; Prabhudesai, Shubhangi; Zhou, Yi; Zon, Leonard; McCann, Kathleen L; Baserga, Susan; Yelick, Pamela C

2014-01-01

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.

Dwarfism and increased adiposity in the gh1 mutant zebrafish vizzini.

PubMed

McMenamin, Sarah K; Minchin, James E N; Gordon, Tiffany N; Rawls, John F; Parichy, David M

2013-04-01

Somatic growth and adipogenesis are closely associated with the development of obesity in humans. In this study, we identify a zebrafish mutant, vizzini, that exhibits both a severe defect in somatic growth and increased accumulation of adipose tissue. Positional cloning of vizzini revealed a premature stop codon in gh1. Although the effects of GH are largely through igfs in mammals, we found no decrease in the expression of igf transcripts in gh1 mutants during larval development. As development progressed, however, we found overall growth to be progressively retarded and the attainment of specific developmental stages to occur at abnormally small body sizes relative to wild type. Moreover, both subcutaneous (sc) and visceral adipose tissues underwent precocious development in vizzini mutants, and at maturity, the sizes of different fat deposits were greatly expanded relative to wild type. In vivo confocal imaging of sc adipose tissue (SAT) expansion revealed that vizzini mutants exhibit extreme enlargement of adipocyte lipid droplets without a corresponding increase in lipid droplet number. These findings suggest that GH1 signaling restricts SAT hypertrophy in zebrafish. Finally, nutrient deprivation of vizzini mutants revealed that SAT mobilization was greatly diminished during caloric restriction, further implicating GH1 signaling in adipose tissue homeostasis. Overall, the zebrafish gh1 mutant, vizzini, exhibits decreased somatic growth, increased adipose tissue accumulation, and disrupted adipose plasticity after nutrient deprivation and represents a novel model to investigate the in vivo dynamics of vertebrate obesity.

Congenital analbuminemia caused by a novel aberrant splicing in the albumin gene.

PubMed

Caridi, Gianluca; Dagnino, Monica; Erdeve, Omer; Di Duca, Marco; Yildiz, Duran; Alan, Serdar; Atasay, Begum; Arsan, Saadet; Campagnoli, Monica; Galliano, Monica; Minchiotti, Lorenzo

2014-01-01

Congenital analbuminemia is a rare autosomal recessive disorder manifested by the presence of a very low amount of circulating serum albumin. It is an allelic heterogeneous defect, caused by variety of mutations within the albumin gene in homozygous or compound heterozygous state. Herein we report the clinical and molecular characterization of a new case of congenital analbuminemia diagnosed in a female newborn of consanguineous (first degree cousins) parents from Ankara, Turkey, who presented with a low albumin concentration (< 8 g/L) and severe clinical symptoms. The albumin gene of the index case was screened by single-strand conformation polymorphism, heteroduplex analysis, and direct DNA sequencing. The effect of the splicing mutation was evaluated by examining the cDNA obtained by reverse transcriptase - polymerase chain reaction (RT-PCR) from the albumin mRNA extracted from proband's leukocytes. DNA sequencing revealed that the proband is homozygous, and both parents are heterozygous, for a novel G>A transition at position c.1652+1, the first base of intron 12, which inactivates the strongly conserved GT dinucleotide at the 5' splice site consensus sequence of this intron. The splicing defect results in the complete skipping of the preceding exon (exon 12) and in a frame-shift within exon 13 with a premature stop codon after the translation of three mutant amino acid residues. Our results confirm the clinical diagnosis of congenital analbuminemia in the proband and the inheritance of the trait and contribute to shed light on the molecular genetics of analbuminemia.

Gentamicin treatment in exercised mdx mice: Identification of dystrophin-sensitive pathways and evaluation of efficacy in work-loaded dystrophic muscle.

PubMed

De Luca, Annamaria; Nico, Beatrice; Rolland, Jean-François; Cozzoli, Anna; Burdi, Rosa; Mangieri, Domenica; Giannuzzi, Viviana; Liantonio, Antonella; Cippone, Valentina; De Bellis, Michela; Nicchia, Grazia Paola; Camerino, Giulia Maria; Frigeri, Antonio; Svelto, Maria; Camerino, Diana Conte

2008-11-01

Aminoglycosides force read through of premature stop codon mutations and introduce new mutation-specific gene-corrective strategies in Duchenne muscular dystrophy. A chronic treatment with gentamicin (32 mg/kg/daily i.p., 8-12 weeks) was performed in exercised mdx mice with the dual aim to clarify the dependence on dystrophin of the functional, biochemical and histological alterations present in dystrophic muscle and to verify the long term efficiency of small molecule gene-corrective strategies in work-loaded dystrophic muscle. The treatment counteracted the exercise-induced impairment of in vivo forelimb strength after 6-8 weeks. We observed an increase in dystrophin expression level in all the fibers, although lower than that observed in normal fibers, and found a concomitant recovery of aquaporin-4 at sarcolemma. A significant reduction in centronucleated fibers, in the area of necrosis and in the percentage of nuclear factor-kB-positive nuclei was observed in gastrocnemious muscle of treated animals. Plasma creatine kinase was reduced by 70%. Ex vivo, gentamicin restored membrane ionic conductance in mdx diaphragm and limb muscle fibers. No effects were observed on the altered calcium homeostasis and sarcolemmal calcium permeability, detected by electrophysiological and microspectrofluorimetric approaches. Thus, the maintenance of a partial level of dystrophin is sufficient to reinforce sarcolemmal stability, reducing leakiness, inflammation and fiber damage, while correction of altered calcium homeostasis needs greater expression of dystrophin or direct interventions on the channels involved.

An event of alternative splicing affects the expression of the NTRC gene, encoding NADPH-thioredoxin reductase C, in seed plants.

PubMed

Nájera, Victoria A; González, María Cruz; Pérez-Ruiz, Juan Manuel; Cejudo, Francisco Javier

2017-05-01

The NTRC gene encodes a NADPH-dependent thioredoxin reductase with a joint thioredoxin domain, exclusive of photosynthetic organisms. An updated search shows that although most species harbor a single copy of the NTRC gene, two copies were identified in different species of the genus Solanum, Glycine max and the moss Physcomitrella patens. The phylogenetic analysis of NTRCs from different sources produced a tree with the major groups of photosynthetic organisms: cyanobacteria, algae and land plants, indicating the evolutionary success of the NTRC gene among photosynthetic eukaryotes. An event of alternative splicing affecting the expression of the NTRC gene was identified, which is conserved in seed plants but not in algae, bryophytes and lycophytes. The alternative splicing event results in a transcript with premature stop codon, which would produce a truncated form of the enzyme. The standard splicing/alternative splicing (SS/AS) transcripts ratio was higher in photosynthetic tissues from Arabidopsis, Brachypodium and tomato, in line with the higher content of the NTRC polypeptide in these tissues. Moreover, environmental stresses such as cold or high salt affected the SS/AS ratio of the NTRC gene transcripts in Brachypodium seedlings. These results suggest that the alternative splicing of the NTRC gene might be an additional mechanism for modulating the content of NTRC in photosynthetic and non-photosynthetic tissues of seed plants. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular basis and clinical presentation of classic galactosemia in a Croatian population.

PubMed

Ramadža, Danijela Petković; Sarnavka, Vladimir; Vuković, Jurica; Fumić, Ksenija; Krželj, Vjekoslav; Lozić, Bernarda; Pušeljić, Silvija; Pereira, Hana; Silva, Maria João; Tavares de Almeida, Isabel; Barić, Ivo; Rivera, Isabel

2018-01-26

Classic galactosemia is an autosomal recessive disorder of galactose metabolism caused by severely decreased activity of galactose-1-phosphate uridylyltransferase (GALT) due to pathogenic mutations in the GALT gene. To date more than 330 mutations have been described, with p.Q188R and p.K285N being the most common in Caucasian populations. Although acute manifestations can be fully avoided by a galactose-restricted diet, chronic complications, such as neurological ones, cannot be prevented in a significant number of patients despite compliance with the dietary treatment. A cohort of 16 galactosemic Croatian patients, including one pair of siblings, was studied. Molecular characterization was performed by direct sequence analysis of the GALT gene. Sixteen patients were analyzed and only four different mutations were detected. As expected, p.Q188R and p.K285N were common, accounting for 40% and 37% of unrelated alleles, respectively. The third mutation accounting for 20% of mutant alleles was p.R123X causing a premature stop codon, is thus considered to be severe, which is in accordance with the phenotype presented by the homozygous patient described here. The fourth mutation p.E271D was found in a single allele. More than half of our patients manifested some chronic neurological complications. This is the first report on mutational and phenotypic spectra of classic galactosemia in Croatia that expands the knowledge on the mutational map of the GALT gene across Europe and reveals the genetic homogeneity of the Croatian population.

A new mutation within the porphobilinogen deaminase gene leading to a truncated protein as a cause of acute intermittent porphyria in an extended Indian family.

PubMed

Flachsová, E; Verma, I C; Ulbrichová, D; Saxena, R; Zeman, J; Saudek, V; Raman, C S; Martásek, P

2007-01-01

Based on Internet search, we were contacted by a 50-year-old man suffering from severe abdominal pain. Acute hepatic porphyria was considered from positive Watson-Schwartz test. He, not being a health professional, searched for centres with ability to do molecular diagnosis and for information about therapeutic possibilities. He asked his physician for haem-arginate (Normosang, Orphan Europe, Paris) treatment, arranged sending his blood to our laboratory and mediated genetic counselling for him and his family. Molecular analyses of the PBGD gene revealed a novel mutation in exon 15, the 973insG. Subsequently, genetic analysis was performed in 18 members of the proband's extensive family. In 12 members of the family, the same mutation was found. The mutation, which consisted of one nucleotide insertion, resulted in addition of four different amino acids leading to a protein that is prematurely truncated by the stop codon. The effect of this mutation was investigated by expression of the wildtype and mutated PBGD in a prokaryotic expression system. The mutation resulted in instability of the protein and loss of enzymatic function. The increasing access to a number of disease- and symptom-oriented web pages presents a new and unusual venue for gaining knowledge and enabling self-diagnosis and self-help. It is, therefore, important that diseaseoriented Internet pages for public use should be designed with clarity and accurate current knowledge based background.

[Molecular genetic analysis for a pedigree with severe hereditary coagulation factor VII deficiency].

PubMed

Ding, Qiu-lan; Wang, Hong-li; Wang, Xue-feng; Wang, Ming-shan; Fu, Qi-hua; Wu, Wen-man; Hu, Yi-qun; Wang, Zhen-yi

2003-10-01

To identify the genetic mutations of a severe inherited coagulation factor VII (FVII) deficiency pedigree. The diagnosis was validated by coagulant and haemostatic parameters. FVII gene mutations were screened in the propositus and his family members by DNA direct sequencing and confirmed by digestions of the restriction enzymes of the PCR production. Two heterozygous missense mutations were found in the propositus of the pedigree: a G to T transversion at position 9482 in exon 6 and a C to T mutation at position 11348 in exon 8 resulting in the amino acid substitution of Arg152 with Leu and Arg304 with Trp, respectively. A heterozygous single nucleotide deletion (C) at position 11487-11489(CCC) within exon 8 was identified, which predicted the frameshift mutation at position His351 followed by the changes of six corresponding amino acids and appearance of a premature protein caused by stop codon. The heterozygous mutations identified in the proband were derived from his father (Arg152 to Leu) and his mother (Arg304 to Trp mutation) and a heterozygous deletion (C) at position 11487-9(CCC). By tracing the other pedigree members, it was found that his grandmother had a heterozygous mutation of Arg304Trp and a heterozygous polymorphism of Arg353Gln and his grandfather had a heterozygous Arg152Leu mutation. Three heterozygous mutations were found in a pedigree with hereditary coagulation factor VII deficiency. Arg152Leu and deletion C at position 11487-9(CCC) were novel mutations.

Dwarfism and Increased Adiposity in the gh1 Mutant Zebrafish vizzini

PubMed Central

McMenamin, Sarah K.; Minchin, James E.N.; Gordon, Tiffany N.

2013-01-01

Somatic growth and adipogenesis are closely associated with the development of obesity in humans. In this study, we identify a zebrafish mutant, vizzini, that exhibits both a severe defect in somatic growth and increased accumulation of adipose tissue. Positional cloning of vizzini revealed a premature stop codon in gh1. Although the effects of GH are largely through igfs in mammals, we found no decrease in the expression of igf transcripts in gh1 mutants during larval development. As development progressed, however, we found overall growth to be progressively retarded and the attainment of specific developmental stages to occur at abnormally small body sizes relative to wild type. Moreover, both subcutaneous (sc) and visceral adipose tissues underwent precocious development in vizzini mutants, and at maturity, the sizes of different fat deposits were greatly expanded relative to wild type. In vivo confocal imaging of sc adipose tissue (SAT) expansion revealed that vizzini mutants exhibit extreme enlargement of adipocyte lipid droplets without a corresponding increase in lipid droplet number. These findings suggest that GH1 signaling restricts SAT hypertrophy in zebrafish. Finally, nutrient deprivation of vizzini mutants revealed that SAT mobilization was greatly diminished during caloric restriction, further implicating GH1 signaling in adipose tissue homeostasis. Overall, the zebrafish gh1 mutant, vizzini, exhibits decreased somatic growth, increased adipose tissue accumulation, and disrupted adipose plasticity after nutrient deprivation and represents a novel model to investigate the in vivo dynamics of vertebrate obesity. PMID:23456361

Single-Exome sequencing identified a novel RP2 mutation in a child with X-linked retinitis pigmentosa.

PubMed

Lim, Hassol; Park, Young-Mi; Lee, Jong-Keuk; Taek Lim, Hyun

2016-10-01

To present an efficient and successful application of a single-exome sequencing study in a family clinically diagnosed with X-linked retinitis pigmentosa. Exome sequencing study based on clinical examination data. An 8-year-old proband and his family. The proband and his family members underwent comprehensive ophthalmologic examinations. Exome sequencing was undertaken in the proband using Agilent SureSelect Human All Exon Kit and Illumina HiSeq 2000 platform. Bioinformatic analysis used Illumina pipeline with Burrows-Wheeler Aligner-Genome Analysis Toolkit (BWA-GATK), followed by ANNOVAR to perform variant functional annotation. All variants passing filter criteria were validated by Sanger sequencing to confirm familial segregation. Analysis of exome sequence data identified a novel frameshift mutation in RP2 gene resulting in a premature stop codon (c.665delC, p.Pro222fsTer237). Sanger sequencing revealed this mutation co-segregated with the disease phenotype in the child's family. We identified a novel causative mutation in RP2 from a single proband's exome sequence data analysis. This study highlights the effectiveness of the whole-exome sequencing in the genetic diagnosis of X-linked retinitis pigmentosa, over the conventional sequencing methods. Even using a single exome, exome sequencing technology would be able to pinpoint pathogenic variant(s) for X-linked retinitis pigmentosa, when properly applied with aid of adequate variant filtering strategy. Copyright © 2016 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.

Several Classical Mouse Inbred Strains, Including DBA/2, NOD/Lt, FVB/N, and SJL/J, Carry a Putative Loss-of-Function Allele of Gpr84

PubMed Central

2013-01-01

G protein–coupled receptor 84 (GPR84) is a 7-transmembrane protein expressed on myeloid cells that can bind to medium-chain free fatty acids in vitro. Here, we report the discovery of a 2-bp frameshift deletion in the second exon of the Gpr84 gene in several classical mouse inbred strains. This deletion generates a premature stop codon predicted to result in a truncated protein lacking the transmembrane domains 4-7. We sequenced Gpr84 exon 2 from 58 strains representing different groups in the mouse family tree and found that 14 strains are homozygous for the deletion. Some of these strains are DBA/1J, DBA/2J, FVB/NJ, LG/J, MRL/MpJ, NOD/LtJ, and SJL/J. However, the deletion was not found in any of the wild-derived inbred strains analyzed. Haplotype analysis suggested that the deletion originates from a unique mutation event that occurred more than 100 years ago, preceding the development of the first inbred strain (DBA), from a Mus musculus domesticus source. As GPR84 ostensibly plays a role in the biology of myeloid cells, it could be relevant 1) to consider the existence of this Gpr84 nonsense mutation in several mouse strains when choosing a mouse model to study immune processes and 2) to consider reevaluating data obtained using such strains. PMID:23616478

Several classical mouse inbred strains, including DBA/2, NOD/Lt, FVB/N, and SJL/J, carry a putative loss-of-function allele of Gpr84.

PubMed

Perez, Carlos J; Dumas, Aline; Vallières, Luc; Guénet, Jean-Louis; Benavides, Fernando

2013-01-01

G protein-coupled receptor 84 (GPR84) is a 7-transmembrane protein expressed on myeloid cells that can bind to medium-chain free fatty acids in vitro. Here, we report the discovery of a 2-bp frameshift deletion in the second exon of the Gpr84 gene in several classical mouse inbred strains. This deletion generates a premature stop codon predicted to result in a truncated protein lacking the transmembrane domains 4-7. We sequenced Gpr84 exon 2 from 58 strains representing different groups in the mouse family tree and found that 14 strains are homozygous for the deletion. Some of these strains are DBA/1J, DBA/2J, FVB/NJ, LG/J, MRL/MpJ, NOD/LtJ, and SJL/J. However, the deletion was not found in any of the wild-derived inbred strains analyzed. Haplotype analysis suggested that the deletion originates from a unique mutation event that occurred more than 100 years ago, preceding the development of the first inbred strain (DBA), from a Mus musculus domesticus source. As GPR84 ostensibly plays a role in the biology of myeloid cells, it could be relevant 1) to consider the existence of this Gpr84 nonsense mutation in several mouse strains when choosing a mouse model to study immune processes and 2) to consider reevaluating data obtained using such strains.

X-linked primary immunodeficiency associated with hemizygous mutations in the moesin (MSN) gene.

PubMed

Lagresle-Peyrou, Chantal; Luce, Sonia; Ouchani, Farid; Soheili, Tayebeh Shabi; Sadek, Hanem; Chouteau, Myriam; Durand, Amandine; Pic, Isabelle; Majewski, Jacek; Brouzes, Chantal; Lambert, Nathalie; Bohineust, Armelle; Verhoeyen, Els; Cosset, François-Loïc; Magerus-Chatinet, Aude; Rieux-Laucat, Frédéric; Gandemer, Virginie; Monnier, Delphine; Heijmans, Catherine; van Gijn, Marielle; Dalm, Virgil A; Mahlaoui, Nizar; Stephan, Jean-Louis; Picard, Capucine; Durandy, Anne; Kracker, Sven; Hivroz, Claire; Jabado, Nada; de Saint Basile, Geneviève; Fischer, Alain; Cavazzana, Marina; André-Schmutz, Isabelle

2016-12-01

We investigated 7 male patients (from 5 different families) presenting with profound lymphopenia, hypogammaglobulinemia, fluctuating monocytopenia and neutropenia, a poor immune response to vaccine antigens, and increased susceptibility to bacterial and varicella zoster virus infections. We sought to characterize the genetic defect involved in a new form of X-linked immunodeficiency. We performed genetic analyses and an exhaustive phenotypic and functional characterization of the lymphocyte compartment. We observed hemizygous mutations in the moesin (MSN) gene (located on the X chromosome and coding for MSN) in all 7 patients. Six of the latter had the same missense mutation, which led to an amino acid substitution (R171W) in the MSN four-point-one, ezrin, radixin, moesin domain. The seventh patient had a nonsense mutation leading to a premature stop codon mutation (R533X). The naive T-cell counts were particularly low for age, and most CD8 + T cells expressed the senescence marker CD57. This phenotype was associated with impaired T-cell proliferation, which was rescued by expression of wild-type MSN. MSN-deficient T cells also displayed poor chemokine receptor expression, increased adhesion molecule expression, and altered migration and adhesion capacities. Our observations establish a causal link between an ezrin-radixin-moesin protein mutation and a primary immunodeficiency that could be referred to as X-linked moesin-associated immunodeficiency. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Type I Glanzmann thrombasthenia caused by an apparently silent beta3 mutation that results in aberrant splicing and reduced beta3 mRNA.

PubMed

Xie, Jingli; Pabón, Dina; Jayo, Asier; Butta, Nora; González-Manchón, Consuelo

2005-05-01

We report a novel genetic defect in a patient with type I Glanzmann thrombasthenia. Flow cytometry analysis revealed undetectable levels of platelet glycoproteins alphaIIb and beta3, although residual amounts of both proteins were detectable in immunoblotting analysis. Sequence analysis of reversely transcribed platelet beta3 mRNA showed a 100-base pair deletion in the 3'-boundary of exon 11, that results in a frame shift and appearance of a premature STOP codon. Analysis of the corresponding genomic DNA fragment revealed the presence of a homozygous C1815T transition in exon 11. The mutation does not change the amino acid residue but it creates an ectopic consensus splice donor site that is used preferentially, causing splicing out of part of exon 11. The parents of the proband, heterozygous for this mutation, were asymptomatic and had reduced platelet content of alphaIIbbeta3. PCR-based relative quantification of beta3 mRNA failed to detect the mutant transcript in the parents and showed a marked reduction in the patient. The results suggest that the thrombasthenic phenotype is, mainly, the result of the reduced availability of beta3-mRNA, most probably due to activation of the nonsense-mediated mRNA decay mechanism. They also show the convenience of analyzing both genomic DNA and mRNA, in order to ascertain the functional consequences of single nucleotide substitutions.

The ACTN3 R577X Polymorphism Is Associated with Cardiometabolic Fitness in Healthy Young Adults

PubMed Central

Deschamps, Chelsea L.; Connors, Kimberly E.; Klein, Matthias S.; Johnsen, Virginia L.; Shearer, Jane; Vogel, Hans J.; Devaney, Joseph M.; Gordish-Dressman, Heather; Many, Gina M.; Barfield, Whitney; Hoffman, Eric P.; Kraus, William E.; Hittel, Dustin S.

2015-01-01

Homozygosity for a premature stop codon (X) in the ACTN3 “sprinter” gene is common in humans despite the fact that it reduces muscle size, strength and power. Because of the close relationship between skeletal muscle function and cardiometabolic health we examined the influence of ACTN3 R577X polymorphism over cardiovascular and metabolic characteristics of young adults (n = 98 males, n = 102 females; 23 ± 4.2 years) from our Assessing Inherent Markers for Metabolic syndrome in the Young (AIMMY) study. Both males and females with the RR vs XX genotype achieved higher mean VO2 peak scores (47.8 ± 1.5 vs 43.2 ±1.8 ml/O2/min, p = 0.002) and exhibited higher resting systolic (115 ± 2 vs 105 ± mmHg, p = 0.027) and diastolic (69 ± 3 vs 59 ± 3 mmHg, p = 0.005) blood pressure suggesting a role for ACTN3 in the maintenance of vascular tone. We subsequently identified the expression of alpha-actinin 3 protein in pulmonary artery smooth muscle, which may explain the genotype-specific differences in cardiovascular adaptation to acute exercise. In addition, we utilized targeted serum metabolomics to distinguish between RR and XX genotypes, suggesting an additional role for the ACTN3 R577X polymorphism in human metabolism. Taken together, these results identify significant cardiometabolic effects associated with possessing one or more functional copies of the ACTN3 gene. PMID:26107372

Novel variants in PAX6 gene caused congenital aniridia in two Chinese families.

PubMed

Zhang, R; Linpeng, S; Wei, X; Li, H; Huang, Y; Guo, J; Wu, Q; Liang, D; Wu, L

2017-06-01

PurposeTo reveal the underlying genetic defect in two four-generation Chinese families with aniridia and explore the pathologic mechanism.MethodsFull ophthalmic examinations were performed in two families with aniridia. The PAX6 gene was directly sequenced in patients of two families, and the detected variants were screened in unaffected family members and two hundred unrelated healthy controls. Real-time quantitative PCR was used to explore pathologic mechanisms of the two variants.ResultsAniridia, cataract, and oscillatory nystagmus were observed in patients of the two families. In addition, we observed corneal opacity and microphthalmus in family 1, and strabismus, left ectopia lentis, microphthalmus, and microcornea in family 2. Sanger sequencing detected a novel 1-bp duplication (c.50dupA) in family 1 and a novel 2-bp splice site deletion (c.765+1_765+2delGT) in family 2. Sequencing of cDNA indicated skipping of exon 9 caused by the splice site deletion, being predicted to cause a premature stop codon, as well as the duplication. The PAX6 mRNA significantly lower in patients with aniridia than in unaffected family members in both families, suggesting that the duplication and splice site deletion caused nonsense-mediated mRNA decay.ConclusionsOur study identified two novel PAX6 variants in two families with aniridia and revealed the pathogenicity of the variants; this would expand the variant spectrum of PAX6 and help us better understand the molecular basis of aniridia, thus facilitating genetic counseling.

A new mutation in the AFP gene responsible for a total absence of alpha feto-protein on second trimester maternal serum screening for Down syndrome

PubMed Central

Petit, François M; Hébert, Marylise; Picone, Olivier; Brisset, Sophie; Maurin, Marie-Laure; Parisot, Frédéric; Capel, Liliane; Benattar, Clarisse; Sénat, Marie-Victoire; Tachdjian, Gérard; Labrune, Philippe

2009-01-01

Alpha feto-protein (AFP) is a major plasma protein produced by the yolk sac and the liver during the fetal period. During the second trimester of pregnancy, APF and βhCG serum concentrations are commonly used for screening Down syndrome. AFP deficiency is rare (estimated to be 1/105 000 newborns) and only one sequence alteration has previously been reported in the AFP gene. We report a new mutation in exon 5 of the AFP gene, leading to a total absence of AFP on 2nd-trimester maternal serum screening for Down syndrome, confirmed on the amniotic fluid. Despite this, fetal development and birth were normal. After PCR-amplification, the whole AFP gene was sequenced. The new mutation was a guanine to adenine transition in position 543 creating a premature stop codon in position 181. In order to search for eventual modifications of the amniotic fluid profile, proteins were separated by electrophoresis and compared with 10 normal amniotic fluids sampled at the same developmental age (18 weeks). In the amniotic fluid of our patient albumin rate was reduced whereas alpha1 and beta protein fractions were increased, suggesting that AFP deficiency may modify the distribution of protein fractions. This observation emphasizes the complex molecular mechanisms of compensation of serum protein deficiency. Studies on other families with AFP deficiency are necessary to confirm this observation. PMID:18854864

Characterisation of InlA truncation in Listeria monocytogenes isolates from farm animals and human cases in the province of Quebec

PubMed Central

Fravalo, Philippe; Cherifi, Tamazight; Neira Feliciano, Kersti Dina; Letellier, Ann; Fairbrother, Julie-Hélène; Bekal, Sadjia

2017-01-01

The introduction of Listeria monocytogenes into the food production chain is a concern, with numerous grouped cases of listeriosis associated with milk-derived or pork-derived products have been documented. Management of this zoonotic pathogen considers all strains as an equal risk. Recently, a new perspective for characterisation of strain virulence was introduced with the discovery of the unaltered sequence of InlA as a determinant of strain virulence; this has also been reported as an infrequent finding among so-called environmental strains, that is, strains isolated from food or from surfaces in food industries. The aim of this study was to differentiate L monocytogenes strains isolated from animal cases versus those from human cases and to differentiate clinical strains from environmental ones using a Caenorhabditis elegans virulence testing model. In Quebec in 2013/2014, the surveillance of L monocytogenes clinical isolates registered a total of 20 strains of animal origin and 16 pulsed-field gel electrophoresis types isolated from human cases. The mixed PCR multiplex agglutination protocol used for geno-serotyping clearly discriminated genogroup IVB strains from bovine and human origins. The presence of a premature stop codon single nucleotide polymorphism in the inlA gene sequence in clinical strains and the identical behaviour of particular strains in the C elegans model are discussed in this paper from the perspective of industrial management of L monocytogenes risk. PMID:28761668

Genetic dissection of the Gpnmb network in the eye.

PubMed

Lu, Hong; Wang, Xusheng; Pullen, Matthew; Guan, Huaijin; Chen, Hui; Sahu, Shwetapadma; Zhang, Bing; Chen, Hao; Williams, Robert W; Geisert, Eldon E; Lu, Lu; Jablonski, Monica M

2011-06-13

To use a systematic genetics approach to investigate the regulation of Gpnmb, a gene that contributes to pigmentary dispersion syndrome (PDS) and pigmentary glaucoma (PG) in the DBA/2J (D2) mouse. Global patterns of gene expression were studied in whole eyes of a large family of BXD mouse strains (n = 67) generated by crossing the PDS- and PG-prone parent (DBA/2J) with a resistant strain (C57BL/6J). Quantitative trait locus (eQTL) mapping methods and gene set analysis were used to evaluate Gpnmb coexpression networks in wild-type and mutant cohorts. The level of Gpnmb expression was associated with a highly significant cis-eQTL at the location of the gene itself. This autocontrol of Gpnmb is likely to be a direct consequence of the known premature stop codon in exon 4. Both gene ontology and coexpression network analyses demonstrated that the mutation in Gpnmb radically modified the set of genes with which Gpnmb expression is correlated. The covariates of wild-type Gpnmb are involved in biological processes including melanin synthesis and cell migration, whereas the covariates of mutant Gpnmb are involved in the biological processes of posttranslational modification, stress activation, and sensory processing. These results demonstrated that a systematic genetics approach provides a powerful tool for constructing coexpression networks that define the biological process categories within which similarly regulated genes function. The authors showed that the R150X mutation in Gpnmb dramatically modified its list of genetic covariates, which may explain the associated ocular pathology.

Functions of the APC tumor suppressor protein dependent and independent of canonical WNT signaling: implications for therapeutic targeting.

PubMed

Hankey, William; Frankel, Wendy L; Groden, Joanna

2018-03-01

The acquisition of biallelic mutations in the APC gene is a rate-limiting step in the development of most colorectal cancers and occurs in the earliest lesions. APC encodes a 312-kDa protein that localizes to multiple subcellular compartments and performs diverse functions. APC participates in a cytoplasmic complex that promotes the destruction of the transcriptional licensing factor β-catenin; APC mutations that abolish this function trigger constitutive activation of the canonical WNT signaling pathway, a characteristic found in almost all colorectal cancers. By negatively regulating canonical WNT signaling, APC counteracts proliferation, promotes differentiation, facilitates apoptosis, and suppresses invasion and tumor progression. APC further antagonizes canonical WNT signaling by interacting with and counteracting β-catenin in the nucleus. APC also suppresses tumor initiation and progression in the colorectal epithelium through functions that are independent of canonical WNT signaling. APC regulates the mitotic spindle to facilitate proper chromosome segregation, localizes to the cell periphery and cell protrusions to establish cell polarity and appropriate directional migration, and inhibits DNA replication by interacting directly with DNA. Mutations in APC are often frameshifts, insertions, or deletions that introduce premature stop codons and lead to the production of truncated APC proteins that lack its normal functions and possess tumorigenic properties. Therapeutic approaches in development for the treatment of APC-deficient tumors are focused on the inhibition of canonical WNT signaling, especially through targets downstream of APC in the pathway, or on the restoration of wild-type APC expression.

Quantifying rare, deleterious variation in 12 human cytochrome P450 drug-metabolism genes in a large-scale exome dataset.

PubMed

Gordon, Adam S; Tabor, Holly K; Johnson, Andrew D; Snively, Beverly M; Assimes, Themistocles L; Auer, Paul L; Ioannidis, John P A; Peters, Ulrike; Robinson, Jennifer G; Sucheston, Lara E; Wang, Danxin; Sotoodehnia, Nona; Rotter, Jerome I; Psaty, Bruce M; Jackson, Rebecca D; Herrington, David M; O'Donnell, Christopher J; Reiner, Alexander P; Rich, Stephen S; Rieder, Mark J; Bamshad, Michael J; Nickerson, Deborah A

2014-04-15

The study of genetic influences on drug response and efficacy ('pharmacogenetics') has existed for over 50 years. Yet, we still lack a complete picture of how genetic variation, both common and rare, affects each individual's responses to medications. Exome sequencing is a promising alternative method for pharmacogenetic discovery as it provides information on both common and rare variation in large numbers of individuals. Using exome data from 2203 AA and 4300 Caucasian individuals through the NHLBI Exome Sequencing Project, we conducted a survey of coding variation within 12 Cytochrome P450 (CYP) genes that are collectively responsible for catalyzing nearly 75% of all known Phase I drug oxidation reactions. In addition to identifying many polymorphisms with known pharmacogenetic effects, we discovered over 730 novel nonsynonymous alleles across the 12 CYP genes of interest. These alleles include many with diverse functional effects such as premature stop codons, aberrant splicesites and mutations at conserved active site residues. Our analysis considering both novel, predicted functional alleles as well as known, actionable CYP alleles reveals that rare, deleterious variation contributes markedly to the overall burden of pharmacogenetic alleles within the populations considered, and that the contribution of rare variation to this burden is over three times greater in AA individuals as compared with Caucasians. While most of these impactful alleles are individually rare, 7.6-11.7% of individuals interrogated in the study carry at least one newly described potentially deleterious alleles in a major drug-metabolizing CYP.

Type 1 papillary renal cell carcinoma in a patient with schwannomatosis: Mosaic versus loss of SMARCB1 expression in respectively schwannoma and renal tumor cells.

PubMed

Hulsebos, Theo J M; Kenter, Susan; Baas, Frank; Nannenberg, Eline A; Bleeker, Fonnet E; van Minkelen, Rick; van den Ouweland, Ans M W; Wesseling, Pieter; Flucke, Uta

2016-04-01

In schwannomatosis, germline SMARCB1 or LZTR1 mutations predispose to the development of multiple benign schwannomas. Besides these, other tumors may occur in schwannomatosis patients. We present a 45-year-old male patient who developed multiple schwannomas and in addition a malignant type 1 papillary renal cell carcinoma (pRCC1). We identified a duplication of exon 7 of SMARCB1 on chromosome 22 in the constitutional DNA of the patient (c.796-2246_986 + 5250dup7686), resulting in the generation of a premature stop codon in the second exon 7 copy (p.Glu330*). The mutant SMARCB1 allele proved to be retained in three schwannomas and in the pRCC1 of the patient. Loss of heterozygosity analysis demonstrated partial loss of the wild-type SMARCB1 allele containing chromosome 22, suggesting loss of that chromosome in only a subset of tumor cells, in all four tumors. Immunohistochemical staining with a SMARCB1 antibody revealed a mosaic SMARCB1 expression pattern in the three benign schwannomas, but absence of expression in the malignant tumor cells of the pRCC1. To our knowledge, this difference in SMARCB1 protein expression has not been reported before. We conclude that a germline SMARCB1 mutation may predispose to the development of pRCC1, thereby further widening the spectrum of tumors that can develop in the context of schwannomatosis. © 2016 Wiley Periodicals, Inc.

In vitro antibacterial activity of rifampicin in combination with imipenem, meropenem and doripenem against multidrug-resistant clinical isolates of Pseudomonas aeruginosa.

PubMed

Hu, Yi-Fan; Liu, Chang-Pan; Wang, Nai-Yu; Shih, Shou-Chuan

2016-08-24

Multidrug-resistant Pseudomonas aeruginosa has emerged as one of the most important healthcare-associated pathogens. Colistin is regarded as the last-resort antibiotic for multidrug-resistant Gram-negative bacteria, but is associated with high rates of acute kidney injury. The aim of this in vitro study is to search for an alternative treatment to colistin for multidrug-resistant P. aeruginosa infections. Multidrug and carbapenem-resistant P. aeruginosa isolates were collected between January 2009 and December 2012 at MacKay Memorial Hospital. Minimal inhibitory concentrations (MICs) were determined for various antibiotic combinations. Carbapenemase-producing genes including bla VIM, other β-lactamase genes and porin mutations were screened by PCR and sequencing. The efficacy of carbapenems (imipenem, meropenem, doripenem) with or without rifampicin was correlated with the type of porin mutation (frameshift mutation, premature stop codon mutation) in multidrug-resistant P. aeruginosa isolates without carbapenemase-producing genes. Of the 71 multidrug-resistant clinical P. aeruginosa isolates, only six harboured the bla VIM gene. Imipenem, meropenem and doripenem were significantly more effective (reduced fold-change of MICs) when combined with rifampicin in bla VIM-negative isolates, especially in isolates with porin frameshift mutation. Imipenem + rifampicin combination has a low MIC against multidrug-resistant P. aeruginosa, especially in isolates with porin frameshift mutation. The imipenem + rifampicin combination may provide an alternative treatment to colistin for multidrug -resistant P. aeruginosa infections, especially for patients with renal insufficiency.

A High-Definition View of Functional Genetic Variation from Natural Yeast Genomes

PubMed Central

Bergström, Anders; Simpson, Jared T.; Salinas, Francisco; Barré, Benjamin; Parts, Leopold; Zia, Amin; Nguyen Ba, Alex N.; Moses, Alan M.; Louis, Edward J.; Mustonen, Ville; Warringer, Jonas; Durbin, Richard; Liti, Gianni

2014-01-01

The question of how genetic variation in a population influences phenotypic variation and evolution is of major importance in modern biology. Yet much is still unknown about the relative functional importance of different forms of genome variation and how they are shaped by evolutionary processes. Here we address these questions by population level sequencing of 42 strains from the budding yeast Saccharomyces cerevisiae and its closest relative S. paradoxus. We find that genome content variation, in the form of presence or absence as well as copy number of genetic material, is higher within S. cerevisiae than within S. paradoxus, despite genetic distances as measured in single-nucleotide polymorphisms being vastly smaller within the former species. This genome content variation, as well as loss-of-function variation in the form of premature stop codons and frameshifting indels, is heavily enriched in the subtelomeres, strongly reinforcing the relevance of these regions to functional evolution. Genes affected by these likely functional forms of variation are enriched for functions mediating interaction with the external environment (sugar transport and metabolism, flocculation, metal transport, and metabolism). Our results and analyses provide a comprehensive view of genomic diversity in budding yeast and expose surprising and pronounced differences between the variation within S. cerevisiae and that within S. paradoxus. We also believe that the sequence data and de novo assemblies will constitute a useful resource for further evolutionary and population genomics studies. PMID:24425782

Excess of genomic defects in a woolly mammoth on Wrangel island

PubMed Central

Slatkin, Montgomery

2017-01-01

Woolly mammoths (Mammuthus primigenius) populated Siberia, Beringia, and North America during the Pleistocene and early Holocene. Recent breakthroughs in ancient DNA sequencing have allowed for complete genome sequencing for two specimens of woolly mammoths (Palkopoulou et al. 2015). One mammoth specimen is from a mainland population 45,000 years ago when mammoths were plentiful. The second, a 4300 yr old specimen, is derived from an isolated population on Wrangel island where mammoths subsisted with small effective population size more than 43-fold lower than previous populations. These extreme differences in effective population size offer a rare opportunity to test nearly neutral models of genome architecture evolution within a single species. Using these previously published mammoth sequences, we identify deletions, retrogenes, and non-functionalizing point mutations. In the Wrangel island mammoth, we identify a greater number of deletions, a larger proportion of deletions affecting gene sequences, a greater number of candidate retrogenes, and an increased number of premature stop codons. This accumulation of detrimental mutations is consistent with genomic meltdown in response to low effective population sizes in the dwindling mammoth population on Wrangel island. In addition, we observe high rates of loss of olfactory receptors and urinary proteins, either because these loci are non-essential or because they were favored by divergent selective pressures in island environments. Finally, at the locus of FOXQ1 we observe two independent loss-of-function mutations, which would confer a satin coat phenotype in this island woolly mammoth. PMID:28253255

The scanning model for translation: an update

PubMed Central

1989-01-01

The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained. PMID:2645293

tRNAs as Therapeutic Agents of Breast Cancer

DTIC Science & Technology

2013-07-01

their anticodon sequence. Wild-type tRNA reads codon for serine, Suppressor (Sup) tRNA for amber stop, and killer tRNA for isoleucine . Figure 6...endoplasmic reticulum (ER) is a eukaryotic organelle that performs the major functions of synthesizing and packaging pro- teins. Overloading of...anticodons tested in HeLa, tRNASer with the AAU anticodon (tRNASer(AAU)) leads to the substitution of isoleucine with serine within the proteome and

Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type 1a

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chou, J.Y.; Lei, K.J.; Shelly, L.L.

1994-09-01

Glycogen storage disease (GSD) type la (von Gierke disease) is caused by the deficiency of glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. The disease presents with clinical manifestations of severe hypoglycemia, hepatomegaly, growth retardation, lactic acidemia, hyperlipidemia, and hyperuricemia. We have succeeded in isolating a murine G6Pase cDNA from a normal mouse liver cDNA library by differentially screening method. We then isolated the human G6Pase cDNA and gene. To date, we have characterized the G6Pase genes of twelve GSD type la patients and uncovered a total of six different mutations. The mutations are comprised of R83C (an Arg atmore » codon 83 to a Cys), Q347X (a Gly at codon 347 to a stop codon), 459insTA (a two basepair insertion at nucleotide 459 yielding a truncated G6Pase of 129 residues), R295C (an Arg at codon 295 to a Cys), G222R (a Gly at codon 222 to an Arg) and {delta}F327 (a codon deletion for Phe-327 at nucleotides 1058 to 1060). The relative incidences of these mutations are 37.5% (R83C), 33.3% (Q347X), 16.6% (459insTA), 4.2% (G222R), 4.2% (R295C) and 4.2% ({delta}F327). Site-directed mutagenesis and transient expression assays demonstrated that the R83C, Q347X, R295C, and {delta}F327 mutations abolished whereas the G222R mutation greatly reduced G6Pase activity. We further characterized the structure-function requirements of amino acids 83, 222, and 295 in G6Pase catalysis. The identification of mutations in GSD type la patients has unequivocally established the molecular basis of the type la disorder. Knowledge of the mutations may be applied to prenatal diagnosis and opens the way for developing and evaluating new therapeutic approaches.« less

Mitochondrial genome of Pteronotus personatus (Chiroptera: Mormoopidae): comparison with selected bats and phylogenetic considerations.

PubMed

López-Wilchis, Ricardo; Del Río-Portilla, Miguel Ángel; Guevara-Chumacero, Luis Manuel

2017-02-01

We described the complete mitochondrial genome (mitogenome) of the Wagner's mustached bat, Pteronotus personatus, a species belonging to the family Mormoopidae, and compared it with other published mitogenomes of bats (Chiroptera). The mitogenome of P. personatus was 16,570 bp long and contained a typically conserved structure including 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one control region (D-loop). Most of the genes were encoded on the H-strand, except for eight tRNA and the ND6 genes. The order of protein-coding and rRNA genes was highly conserved in all mitogenomes. All protein-coding genes started with an ATG codon, except for ND2, ND3, and ND5, which initiated with ATA, and terminated with the typical stop codon TAA/TAG or the codon AGA. Phylogenetic trees constructed using Maximum Parsimony, Maximum Likelihood, and Bayesian inference methods showed an identical topology and indicated the monophyly of different families of bats (Mormoopidae, Phyllostomidae, Vespertilionidae, Rhinolophidae, and Pteropopidae) and the existence of two major clades corresponding to the suborders Yangochiroptera and Yinpterochiroptera. The mitogenome sequence provided here will be useful for further phylogenetic analyses and population genetic studies in mormoopid bats.

The mitochondrial genome of Polistes jokahamae and a phylogenetic analysis of the Vespoidea (Insecta: Hymenoptera).

PubMed

Song, Sheng-Nan; Chen, Peng-Yan; Wei, Shu-Jun; Chen, Xue-Xin

2016-07-01

The mitochondrial genome sequence of Polistes jokahamae (Radoszkowski, 1887) (Hymenoptera: Vespidae) (GenBank accession no. KR052468) was sequenced. The current length with partial A + T-rich region of this mitochondrial genome is 16,616 bp. All the typical mitochondrial genes were sequenced except for three tRNAs (trnI, trnQ, and trnY) located between the A + T-rich region and nad2. At least three rearrangement events occurred in the sequenced region compared with the pupative ancestral arrangement of insects, corresponding to the shuffling of trnK and trnD, translocation or remote inversion of tnnY and translocation of trnL1. All protein-coding genes start with ATN codons. Eleven, one, and another one protein-coding genes stop with termination codon TAA, TA, and T, respectively. Phylogenetic analysis using the Bayesian method based on all codon positions of the 13 protein-coding genes supports the monophyly of Vespidae and Formicidae. Within the Formicidae, the Myrmicinae and Formicinae form a sister lineage and then sister to the Dolichoderinae, while within the Vespidae, the Eumeninae is sister to the lineage of Vespinae + Polistinae.

Optimizing doped libraries by using genetic algorithms

NASA Astrophysics Data System (ADS)

Tomandl, Dirk; Schober, Andreas; Schwienhorst, Andreas

1997-01-01

The insertion of random sequences into protein-encoding genes in combination with biologicalselection techniques has become a valuable tool in the design of molecules that have usefuland possibly novel properties. By employing highly effective screening protocols, a functionaland unique structure that had not been anticipated can be distinguished among a hugecollection of inactive molecules that together represent all possible amino acid combinations.This technique is severely limited by its restriction to a library of manageable size. Oneapproach for limiting the size of a mutant library relies on `doping schemes', where subsetsof amino acids are generated that reveal only certain combinations of amino acids in a proteinsequence. Three mononucleotide mixtures for each codon concerned must be designed, suchthat the resulting codons that are assembled during chemical gene synthesis represent thedesired amino acid mixture on the level of the translated protein. In this paper we present adoping algorithm that `reverse translates' a desired mixture of certain amino acids into threemixtures of mononucleotides. The algorithm is designed to optimally bias these mixturestowards the codons of choice. This approach combines a genetic algorithm with localoptimization strategies based on the downhill simplex method. Disparate relativerepresentations of all amino acids (and stop codons) within a target set can be generated.Optional weighing factors are employed to emphasize the frequencies of certain amino acidsand their codon usage, and to compensate for reaction rates of different mononucleotidebuilding blocks (synthons) during chemical DNA synthesis. The effect of statistical errors thataccompany an experimental realization of calculated nucleotide mixtures on the generatedmixtures of amino acids is simulated. These simulations show that the robustness of differentoptima with respect to small deviations from calculated values depends on their concomitantfitness. Furthermore, the calculations probe the fitness landscape locally and allow apreliminary assessment of its structure.

Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames

PubMed Central

1996-01-01

An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules. PMID:8879204

Determining if an mRNA is a Substrate of Nonsense-Mediated mRNA Decay in Saccharomyces cerevisiae.

PubMed

Johansson, Marcus J O

2017-01-01

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic quality control mechanism which triggers decay of mRNAs harboring premature translation termination codons. In this chapter, I describe methods for monitoring the influence of NMD on mRNA abundance and decay rates in Saccharomyces cerevisiae. The descriptions include detailed methods for growing yeast cells, total RNA isolation, and Northern blotting. Although the chapter focuses on NMD, the methods can be easily adapted to assess the effect of other mRNA decay pathways.

Congenital deficiency of alpha feto-protein.

PubMed

Sharony, Reuven; Zadik, Idit; Parvari, Ruti

2004-10-01

Alpha-fetoprotein (AFP) is the main fetus serum glycoprotein with a very low concentration in the adult. AFP deficiency is a rare phenomenon. We studied two families with congenital AFP deficiency and searched for mutations in the AFP gene. We identified one mutation of 2 base deletion in exon 8, in both families, that leads to the congenital deficiency of AFP. The mutation nt930-931delCT (T294fs25X) creates a frameshift after codon 294 that leads to a stop codon after 24 amino acids, thus truncating the normal length of AFP of 609 amino acids. All the affected children were found to be homozygous for the mutation as was one of the fathers. The affected individuals were asymptomatic and presented normal development. This first identification of a mutation in the AFP gene demonstrates for the first time that deficiency of AFP is compatible with human normal fetal development and further reproduction in males.

Complete mitochondrial genome of the Freshwater Whipray Himantura dalyensis.

PubMed

Feutry, Pierre; Kyne, Peter M; Peng, Zaiqing; Pan, Lianghao; Chen, Xiao

2016-05-01

The complete mitochondrial genome of the Freshwater Whipray Himantura dalyensis is presented in this study. It is 17,693 bp in length and contains 37 genes in typical gene order and transcriptional orientation observed in vertebrates. There were a total of 86 bp short intergenic spacers and 22 bp overlaps in the genome. The overall base composition was 31.4% A, 25.5% C, 13.2% G and 29.9% T. Two start codons (GTG and ATG) and two stop codons (TAG and TAA/T) were found in 13 protein-coding genes. The length of 22 tRNA genes ranged from 68 (tRNA-Cys and tRNA-Ser2) to 75 bp (tRNA-Leu1). The origin of L-strand replication (OL) was found between the tRNA-Asn and tRNA-Cys genes. The base composition of the control region (1940 bp) was similar to the whole mitogenome.

Inhibition of Exotoxin Production by Mobile Genetic Element SCCmec-Encoded psm-mec RNA Is Conserved in Staphylococcal Species

PubMed Central

Saito, Yuki; Mao, Han; Sekimizu, Kazuhisa; Kaito, Chikara

2014-01-01

Staphylococcal species acquire antibiotic resistance by incorporating the mobile-genetic element SCCmec. We previously found that SCCmec-encoded psm-mec RNA suppresses exotoxin production as a regulatory RNA, and the psm-mec translation product increases biofilm formation in Staphylococcus aureus. Here, we examined whether the regulatory role of psm-mec on host bacterial virulence properties is conserved among other staphylococcal species, S. epidermidis and S. haemolyticus, both of which are important causes of nosocomial infections. In S. epidermidis, introduction of psm-mec decreased the production of cytolytic toxins called phenol-soluble modulins (PSMs) and increased biofilm formation. Introduction of psm-mec with a stop-codon mutation that did not express PSM-mec protein but did express psm-mec RNA also decreased PSM production, but did not increase biofilm formation. Thus, the psm-mec RNA inhibits PSM production, whereas the PSM-mec protein increases biofilm formation in S. epidermidis. In S. haemolyticus, introduction of psm-mec decreased PSM production, but did not affect biofilm formation. The mutated psm-mec with a stop-codon also caused the same effect. Thus, the psm-mec RNA also inhibits PSM production in S. haemolyticus. These findings suggest that the inhibitory role of psm-mec RNA on exotoxin production is conserved among staphylococcal species, although the stimulating effect of the psm-mec gene on biofilm formation is not conserved. PMID:24926994

Major histocompatibility complex variation in the endangered Przewalski's horse.

PubMed Central

Hedrick, P W; Parker, K M; Miller, E L; Miller, P S

1999-01-01

The major histocompatibility complex (MHC) is a fundamental part of the vertebrate immune system, and the high variability in many MHC genes is thought to play an essential role in recognition of parasites. The Przewalski's horse is extinct in the wild and all the living individuals descend from 13 founders, most of whom were captured around the turn of the century. One of the primary genetic concerns in endangered species is whether they have ample adaptive variation to respond to novel selective factors. In examining 14 Przewalski's horses that are broadly representative of the living animals, we found six different class II DRB major histocompatibility sequences. The sequences showed extensive nonsynonymous variation, concentrated in the putative antigen-binding sites, and little synonymous variation. Individuals had from two to four sequences as determined by single-stranded conformation polymorphism (SSCP) analysis. On the basis of the SSCP data, phylogenetic analysis of the nucleotide sequences, and segregation in a family group, we conclude that four of these sequences are from one gene (although one sequence codes for a nonfunctional allele because it contains a stop codon) and two other sequences are from another gene. The position of the stop codon is at the same amino-acid position as in a closely related sequence from the domestic horse. Because other organisms have extensive variation at homologous loci, the Przewalski's horse may have quite low variation in this important adaptive region. PMID:10430594

Pseudogenization of the tooth gene enamelysin (MMP20) in the common ancestor of extant baleen whales

PubMed Central

Meredith, Robert W.; Gatesy, John; Cheng, Joyce; Springer, Mark S.

2011-01-01

Whales in the suborder Mysticeti are filter feeders that use baleen to sift zooplankton and small fish from ocean waters. Adult mysticetes lack teeth, although tooth buds are present in foetal stages. Cladistic analyses suggest that functional teeth were lost in the common ancestor of crown-group Mysticeti. DNA sequences for the tooth-specific genes, ameloblastin (AMBN), enamelin (ENAM) and amelogenin (AMEL), have frameshift mutations and/or stop codons in this taxon, but none of these molecular cavities are shared by all extant mysticetes. Here, we provide the first evidence for pseudogenization of a tooth gene, enamelysin (MMP20), in the common ancestor of living baleen whales. Specifically, pseudogenization resulted from the insertion of a CHR-2 SINE retroposon in exon 2 of MMP20. Genomic and palaeontological data now provide congruent support for the loss of enamel-capped teeth on the common ancestral branch of crown-group mysticetes. The new data for MMP20 also document a polymorphic stop codon in exon 2 of the pygmy sperm whale (Kogia breviceps), which has enamel-less teeth. These results, in conjunction with the evidence for pseudogenization of MMP20 in Hoffmann's two-toed sloth (Choloepus hoffmanni), another enamel-less species, support the hypothesis that the only unique, non-overlapping function of the MMP20 gene is in enamel formation. PMID:20861053

Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein.

PubMed

Herrera, Victoria L M; Steffen, Martin; Moran, Ann Marie; Tan, Glaiza A; Pasion, Khristine A; Rivera, Keith; Pappin, Darryl J; Ruiz-Opazo, Nelson

2016-06-14

In contrast to rat and mouse databases, the NCBI gene database lists the human dual-endothelin1/VEGFsp receptor (DEspR, formerly Dear) as a unitary transcribed pseudogene due to a stop [TGA]-codon at codon#14 in automated DNA and RNA sequences. However, re-analysis is needed given prior single gene studies detected a tryptophan [TGG]-codon#14 by manual Sanger sequencing, demonstrated DEspR translatability and functionality, and since the demonstration of actual non-translatability through expression studies, the standard-of-excellence for pseudogene designation, has not been performed. Re-analysis must meet UNIPROT criteria for demonstration of a protein's existence at the highest (protein) level, which a priori, would override DNA- or RNA-based deductions. To dissect the nucleotide sequence discrepancy, we performed Maxam-Gilbert sequencing and reviewed 727 RNA-seq entries. To comply with the highest level multiple UNIPROT criteria for determining DEspR's existence, we performed various experiments using multiple anti-DEspR monoclonal antibodies (mAbs) targeting distinct DEspR epitopes with one spanning the contested tryptophan [TGG]-codon#14, assessing: (a) DEspR protein expression, (b) predicted full-length protein size, (c) sequence-predicted protein-specific properties beyond codon#14: receptor glycosylation and internalization, (d) protein-partner interactions, and (e) DEspR functionality via DEspR-inhibition effects. Maxam-Gilbert sequencing and some RNA-seq entries demonstrate two guanines, hence a tryptophan [TGG]-codon#14 within a compression site spanning an error-prone compression sequence motif. Western blot analysis using anti-DEspR mAbs targeting distinct DEspR epitopes detect the identical glycosylated 17.5 kDa pull-down protein. Decrease in DEspR-protein size after PNGase-F digest demonstrates post-translational glycosylation, concordant with the consensus-glycosylation site beyond codon#14. Like other small single-transmembrane proteins, mass spectrometry analysis of anti-DEspR mAb pull-down proteins do not detect DEspR, but detect DEspR-protein interactions with proteins implicated in intracellular trafficking and cancer. FACS analyses also detect DEspR-protein in different human cancer stem-like cells (CSCs). DEspR-inhibition studies identify DEspR-roles in CSC survival and growth. Live cell imaging detects fluorescently-labeled anti-DEspR mAb targeted-receptor internalization, concordant with the single internalization-recognition sequence also located beyond codon#14. Data confirm translatability of DEspR, the full-length DEspR protein beyond codon#14, and elucidate DEspR-specific functionality. Along with detection of the tryptophan [TGG]-codon#14 within an error-prone compression site, cumulative data demonstrating DEspR protein existence fulfill multiple UNIPROT criteria, thus refuting its pseudogene designation.

No Evidence for Natural Selection on Endogenous Borna-Like Nucleoprotein Elements after the Divergence of Old World and New World Monkeys

PubMed Central

Kobayashi, Yuki; Horie, Masayuki; Tomonaga, Keizo; Suzuki, Yoshiyuki

2011-01-01

Endogenous Borna-like nucleoprotein (EBLNs) elements were recently discovered as non-retroviral RNA virus elements derived from bornavirus in the genomes of various animals. Most of EBLNs appeared to be defective, but some of primate EBLN-1 to -4, which appeared to be originated from four independent integrations of bornavirus nucleoprotein (N) gene, have retained an open reading frame (ORF) for more than 40 million years. It was therefore possible that primate EBLNs have encoded functional proteins during evolution. To examine this possibility, natural selection operating on all ORFs of primate EBLN-1 to -4 was examined by comparing the rates of synonymous and nonsynonymous substitutions. The expected number of premature termination codons in EBLN-1 generated after the divergence of Old World and New World monkeys under the selective neutrality was also examined by the Monte Carlo simulation. As a result, natural selection was not identified for the entire region as well as parts of ORFs in the pairwise analysis of primate EBLN-1 to -4 and for any branch of the phylogenetic trees for EBLN-1 to -4 after the divergence of Old World and New World monkeys. Computer simulation also indicated that the absence of premature termination codon in the present-day EBLN-1 does not necessarily support the maintenance of function after the divergence of Old World and New World monkeys. These results suggest that EBLNs have not generally encoded functional proteins after the divergence of Old World and New World monkeys. PMID:21912690

No evidence for natural selection on endogenous borna-like nucleoprotein elements after the divergence of Old World and New World monkeys.

PubMed

Kobayashi, Yuki; Horie, Masayuki; Tomonaga, Keizo; Suzuki, Yoshiyuki

2011-01-01

Endogenous Borna-like nucleoprotein (EBLNs) elements were recently discovered as non-retroviral RNA virus elements derived from bornavirus in the genomes of various animals. Most of EBLNs appeared to be defective, but some of primate EBLN-1 to -4, which appeared to be originated from four independent integrations of bornavirus nucleoprotein (N) gene, have retained an open reading frame (ORF) for more than 40 million years. It was therefore possible that primate EBLNs have encoded functional proteins during evolution. To examine this possibility, natural selection operating on all ORFs of primate EBLN-1 to -4 was examined by comparing the rates of synonymous and nonsynonymous substitutions. The expected number of premature termination codons in EBLN-1 generated after the divergence of Old World and New World monkeys under the selective neutrality was also examined by the Monte Carlo simulation. As a result, natural selection was not identified for the entire region as well as parts of ORFs in the pairwise analysis of primate EBLN-1 to -4 and for any branch of the phylogenetic trees for EBLN-1 to -4 after the divergence of Old World and New World monkeys. Computer simulation also indicated that the absence of premature termination codon in the present-day EBLN-1 does not necessarily support the maintenance of function after the divergence of Old World and New World monkeys. These results suggest that EBLNs have not generally encoded functional proteins after the divergence of Old World and New World monkeys.

Assessment of possible allergenicity of hypothetical ORFs in common food crops using current bioinformatic guidelines and its implications for the safety assessment of GM crops.

PubMed

Young, Gregory J; Zhang, Shiping; Mirsky, Henry P; Cressman, Robert F; Cong, Bin; Ladics, Gregory S; Zhong, Cathy X

2012-10-01

Before a genetically modified (GM) crop can be commercialized it must pass through a rigorous regulatory process to verify that it is safe for human and animal consumption, and to the environment. One particular area of focus is the potential introduction of a known or cross-reactive allergen not previously present within the crop. The assessment of possible allergenicity uses the guidelines outlined by the Food and Agriculture Organization (FAO) and World Health Organization's (WHO) Codex Alimentarius Commission (Codex) to evaluate all newly expressed proteins. Some regulatory authorities have broadened the scope of the assessment to include all DNA reading frames between stop codons across the insert and spanning the insert/genomic DNA junctions. To investigate the utility of this bioinformatic assessment, all naturally occurring stop-to-stop frames in the non-transgenic genomes of maize, rice, and soybean, as well as the human genome, were compared against the AllergenOnline (www.allergenonline.org) database using the Codex criteria. We discovered thousands of frames that exceeded the Codex defined threshold for potential cross-reactivity suggesting that evaluating hypothetical ORFs (stop-to-stop frames) has questionable value for making decisions on the safety of GM crops. Copyright © 2012 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leighton, J.K.; Joyner, J.; Zamarripa, J.

Two different molecular weight forms of apoB are produced from a common initial transcript via editing of a Gln codon (CAA) to a stop codon (UAA), leading to a truncated translation product (apo BS) that consists of the amino terminal half of the larger form (apoBL). Previous studies have shown that fasting coordinately decreases lipogenesis and the secretion of very low density lipoprotein (VLDL) lipids and apoBS. Secretion of the apoBL is unaffected by fasting. We studied whether editing of apoB RNA is repressed by fasting, thus accounting for the selective decreased secretion of apoBS. Column chromatography of (35S)methionine-labeled lipoproteinsmore » secreted by hepatocytes from fed rats showed that essentially all of apoBL is secreted in the VLDL fraction, whereas a significant amount (15%) of apoBS is secreted associated as lipoproteins eluting in the HDL fractions. Fasting decreased the relative amount of apoBS that eluted in the VLDL fractions and increased the amount secreted in the HDL fractions. Consistent with previous results, hepatocytes from fasted rats show a selective twofold decrease in apoBS secretion. Fasting did not affect the relative abundance of apoB RNA, determined by slot blot hybridization assays using two different 32P-labeled cDNA probes coding either for both molecular weight forms or for only the large molecular weight form. However, quantitative of the editing of apoB RNA showed that fasting caused a 60% decrease in the amount of apoB RNA possessing the stop codon. These data show that the editing of apoB RNA is sensitive to metabolic state (i.e., fasting) resulting in a selective decrease in the secretion of apoBS. However, since the total secretion of apoB was decreased by fasting, while apoB mRNA levels remained constant, additional (post-transcriptional) mechanisms play a role in regulating apoB secretion.« less

Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera)

PubMed Central

Snell, Terry W; Shearer, Tonya L; Smith, Hilary A; Kubanek, Julia; Gribble, Kristin E; Welch, David B Mark

2009-01-01

Background Mate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating. Results A 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions, suggests that the repeats are kept nearly identical through a process of concerted evolution. Information-rich molecules like surface glycoproteins are well adapted for chemical communication and aquatic animals may have evolved signaling systems based on these compounds, whereas insects use cuticular hydrocarbons. Conclusion Owing to its critical role in mating, the mate recognition pheromone gene will be a useful molecular marker for exploring the mechanisms and rates of selection and the evolution of reproductive isolation and speciation using rotifers as a model system. The phylogenetic variation in the mate recognition pheromone gene can now be studied in conjunction with the large amount of ecological and population genetic data being gathered for the Brachionus plicatilis species complex to understand better the evolutionary drivers of cryptic speciation. PMID:19740420

Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera).

PubMed

Snell, Terry W; Shearer, Tonya L; Smith, Hilary A; Kubanek, Julia; Gribble, Kristin E; Welch, David B Mark

2009-09-09

Mate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating. A 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions, suggests that the repeats are kept nearly identical through a process of concerted evolution. Information-rich molecules like surface glycoproteins are well adapted for chemical communication and aquatic animals may have evolved signaling systems based on these compounds, whereas insects use cuticular hydrocarbons. Owing to its critical role in mating, the mate recognition pheromone gene will be a useful molecular marker for exploring the mechanisms and rates of selection and the evolution of reproductive isolation and speciation using rotifers as a model system. The phylogenetic variation in the mate recognition pheromone gene can now be studied in conjunction with the large amount of ecological and population genetic data being gathered for the Brachionus plicatilis species complex to understand better the evolutionary drivers of cryptic speciation.

Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice

PubMed Central

Kamal, Ram P.; Kumar, Amrita; Davis, Charles T.; Tzeng, Wen-Pin; Nguyen, Tung; Donis, Ruben O.; Katz, Jacqueline M.

2015-01-01

ABSTRACT Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses. PMID:25787281

Premature Ejaculation

MedlinePlus

... feel like you are about to have an orgasm. Then the stimulation stops for about 30 seconds. Once you regain control of your response, stimulation begins again. This process is repeated 3 or 4 times before you allow yourself to have an orgasm. The squeeze method works in a similar way. ...

An RNA decay factor wears a new coat: UPF3B modulates translation termination

PubMed Central

Gao, Zhaofeng; Wilkinson, Miles

2017-01-01

Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA turnover pathway that has been subject to intense scrutiny. NMD identifies and degrades subsets of normal RNAs, as well as abnormal mRNAs containing premature termination codons. A core factor in this pathway—UPF3B—is an adaptor protein that serves as an NMD amplifier and an NMD branch-specific factor. UPF3B is encoded by an X-linked gene that when mutated causes intellectual disability and is associated with neurodevelopmental disorders, including schizophrenia and autism. Neu-Yilik et al. now report a new function for UPF3B: it modulates translation termination. Using a fully reconstituted in vitro translation system, they find that UPF3B has two roles in translation termination. First, UPF3B delays translation termination under conditions that mimic premature translation termination. This could drive more efficient RNA decay by allowing more time for the formation of RNA decay-stimulating complexes. Second, UPF3B promotes the dissociation of post-termination ribosomal complexes that lack nascent peptide. This implies that UPF3B could promote ribosome recycling. Importantly, the authors found that UPF3B directly interacts with both RNA and the factors that recognize stop codons—eukaryotic release factors (eRFs)—suggesting that UPF3B serves as a direct regulator of translation termination. In contrast, a NMD factor previously thought to have a central regulatory role in translation termination—the RNA helicase UPF1—was found to indirectly interact with eRFs and appears to act exclusively in post-translation termination events, such as RNA decay, at least in vitro. The finding that an RNA decay-promoting factor, UFP3B, modulates translation termination has many implications. For example, the ability of UPF3B to influence the development and function of the central nervous system may be not only through its ability to degrade specific RNAs but also through its impact on translation termination and subsequent events, such as ribosome recycling. PMID:29333258

Role of the Integrin-Linked Kinase, ILK, in Mammary Carcinogensis

DTIC Science & Technology

2000-08-01

have been implicated in environmental stress clonei 6-10 responses in yeasts, plants and mammals, as well as regulating abscisic acid signal transduction...phosphatase 2C involved in abscisic acid signal transduction in higher plants. Proc. Natl Acad. Sci. USA, 95, 975-980. Strovel,E.T., Wu,D. and Sussman,D.J...contain a 450bp open reading frame, coding for 149 amino acids and a poly A tail 245bp downstream of the stop codon, although no polyadenylation site

DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli.

PubMed

Lalaouna, David; Morissette, Audrey; Carrier, Marie-Claude; Massé, Eric

2015-10-01

The 87 nucleotide long DsrA sRNA has been mostly studied for its translational activation of the transcriptional regulator RpoS. However, it also represses hns mRNA, which encodes H-NS, a major regulator that affects expression of nearly 5% of Escherichia coli genes. A speculative model previously suggested that DsrA would block hns mRNA translation by binding simultaneously to start and stop codon regions of hns mRNA (coaxial model). Here, we show that DsrA efficiently blocked translation of hns mRNA by base-pairing immediately downstream of the start codon. In addition, DsrA induced hns mRNA degradation by actively recruiting the RNA degradosome complex. Data presented here led to a model of DsrA action on hns mRNA, which supports a canonical mechanism of sRNA-induced mRNA degradation by binding to the translation initiation region. Furthermore, using MS2-affinity purification coupled with RNA sequencing technology (MAPS), we also demonstrated that DsrA targets rbsD mRNA, involved in ribose utilization. Surprisingly, DsrA base pairs far downstream of rbsD start codon and induces rapid degradation of the transcript. Thus, our study enables us to draw an extended DsrA targetome. © 2015 John Wiley & Sons Ltd.

Translation regulation of mammalian selenoproteins.

PubMed

Vindry, Caroline; Ohlmann, Théophile; Chavatte, Laurent

2018-05-09

Interest in selenium research has considerably grown over the last decades owing to the association of selenium deficiencies with an increased risk of several human diseases, including cancers, cardiovascular disorders and infectious diseases. The discovery of a genetically encoded 21 st amino acid, selenocysteine, is a fascinating breakthrough in molecular biology as it is the first addition to the genetic code deciphered in the 1960s. Selenocysteine is a structural and functional analog of cysteine, where selenium replaces sulfur, and its presence is critical for the catalytic activity of selenoproteins. The insertion of selenocysteine is a non-canonical translational event, based on the recoding of a UGA codon in selenoprotein mRNAs, normally used as a stop codon in other cellular mRNAs. Two RNA molecules and associated partners are crucial components of the selenocysteine insertion machinery, the Sec-tRNA [Ser]Sec devoted to UGA codon recognition and the SECIS elements located in the 3'UTR of selenoprotein mRNAs. The translational UGA recoding event is a limiting stage of selenoprotein expression and its efficiency is regulated by several factors. The control of selenoproteome expression is crucial for redox homeostasis and antioxidant defense of mammalian organisms. In this review, we summarize current knowledge on the co-translational insertion of selenocysteine into selenoproteins, and its layers of regulation. Copyright © 2018. Published by Elsevier B.V.

Selenium. Role of the Essential Metalloid in Health

PubMed Central

Kurokawa, Suguru; Berry, Marla J.

2015-01-01

Selenium is an essential micronutrient in mammals, but is also recognized as toxic in excess. It is a non-metal with properties that are intermediate between the chalcogen elements sulfur and tellurium. Selenium exerts its biological functions through selenoproteins. Selenoproteins contain selenium in the form of the 21st amino acid, selenocysteine (Sec), which is an analog of cysteine with the sulfur-containing side chain replaced by a Se-containing side chain. Sec is encoded by the codon UGA, which is one of three termination codons for mRNA translation in non-selenoprotein genes. Recognition of the UGA codon as a Sec insertion site instead of stop requires a Sec insertion sequence (SECIS) element in selenoprotein mRNAs and a unique selenocysteyl-tRNA, both of which are recognized by specialized protein factors. Unlike the 20 standard amino acids, Sec is biosynthesized from serine on its tRNA. Twenty-five selenoproteins are encoded in the human genome. Most of the selenoprotein genes were discovered by bioinformatics approaches, searching for SECIS elements downstream of in-frame UGA codons. Sec has been described as having stronger nucleophilic and electrophilic properties than cysteine, and Sec is present in the catalytic site of all selenoenzymes. Most selenoproteins, whose functions are known, are involved in redox systems and signaling pathways. However, several selenoproteins are not well characterized in terms of their function. The selenium field has grown dramatically in the last few decades, and research on selenium biology is providing extensive new information regarding its importance for human health. PMID:24470102

Evolution of Nucleotide Punctuation Marks: From Structural to Linear Signals.

PubMed

El Houmami, Nawal; Seligmann, Hervé

2017-01-01

We present an evolutionary hypothesis assuming that signals marking nucleotide synthesis (DNA replication and RNA transcription) evolved from multi- to unidimensional structures, and were carried over from transcription to translation. This evolutionary scenario presumes that signals combining secondary and primary nucleotide structures are evolutionary transitions. Mitochondrial replication initiation fits this scenario. Some observations reported in the literature corroborate that several signals for nucleotide synthesis function in translation, and vice versa. (a) Polymerase-induced frameshift mutations occur preferentially at translational termination signals (nucleotide deletion is interpreted as termination of nucleotide polymerization, paralleling the role of stop codons in translation). (b) Stem-loop hairpin presence/absence modulates codon-amino acid assignments, showing that translational signals sometimes combine primary and secondary nucleotide structures (here codon and stem-loop). (c) Homopolymer nucleotide triplets (AAA, CCC, GGG, TTT) cause transcriptional and ribosomal frameshifts. Here we find in recently described human mitochondrial RNAs that systematically lack mono-, dinucleotides after each trinucleotide (delRNAs) that delRNA triplets include 2x more homopolymers than mitogenome regions not covered by delRNA. Further analyses of delRNAs show that the natural circular code X (a little-known group of 20 translational signals enabling ribosomal frame retrieval consisting of 20 codons {AAC, AAT, ACC, ATC, ATT, CAG, CTC, CTG, GAA, GAC, GAG, GAT, GCC, GGC, GGT, GTA, GTC, GTT, TAC, TTC} universally overrepresented in coding versus other frames of gene sequences), regulates frameshift in transcription and translation. This dual transcription and translation role confirms for X the hypothesis that translational signals were carried over from transcriptional signals.

Massive Losses of Taste Receptor Genes in Toothed and Baleen Whales

PubMed Central

Feng, Ping; Zheng, Jinsong; Rossiter, Stephen J.; Wang, Ding; Zhao, Huabin

2014-01-01

Taste receptor genes are functionally important in animals, with a surprising exception in the bottlenose dolphin, which shows extensive losses of sweet, umami, and bitter taste receptor genes. To examine the generality of taste gene loss, we examined seven toothed whales and five baleen whales and sequenced the complete repertoire of three sweet/umami (T1Rs) and ten bitter (T2Rs) taste receptor genes. We found all amplified T1Rs and T2Rs to be pseudogenes in all 12 whales, with a shared premature stop codon in 10 of the 13 genes, which demonstrated massive losses of taste receptor genes in the common ancestor of whales. Furthermore, we analyzed three genome sequences from two toothed whales and one baleen whale and found that the sour taste marker gene Pkd2l1 is a pseudogene, whereas the candidate salty taste receptor genes are intact and putatively functional. Additionally, we examined three genes that are responsible for taste signal transduction and found the relaxation of functional constraints on taste signaling pathways along the ancestral branch leading to whales. Together, our results strongly suggest extensive losses of sweet, umami, bitter, and sour tastes in whales, and the relaxation of taste function most likely arose in the common ancestor of whales between 36 and 53 Ma. Therefore, whales represent the first animal group to lack four of five primary tastes, probably driven by the marine environment with high concentration of sodium, the feeding behavior of swallowing prey whole, and the dietary switch from plants to meat in the whale ancestor. PMID:24803572

Presence of tannins in sorghum grains is conditioned by different natural alleles of Tannin1

PubMed Central

Wu, Yuye; Li, Xianran; Xiang, Wenwen; Zhu, Chengsong; Lin, Zhongwei; Wu, Yun; Li, Jiarui; Pandravada, Satchidanand; Ridder, Dustan D.; Bai, Guihua; Wang, Ming L.; Trick, Harold N.; Bean, Scott R.; Tuinstra, Mitchell R.; Tesso, Tesfaye T.; Yu, Jianming

2012-01-01

Sorghum, an ancient old-world cereal grass, is the dietary staple of over 500 million people in more than 30 countries in the tropics and semitropics. Its C4 photosynthesis, drought resistance, wide adaptation, and high nutritional value hold the promise to alleviate hunger in Africa. Not present in other major cereals, such as rice, wheat, and maize, condensed tannins (proanthocyanidins) in the pigmented testa of some sorghum cultivars have been implicated in reducing protein digestibility but recently have been shown to promote human health because of their high antioxidant capacity and ability to fight obesity through reduced digestion. Combining quantitative trait locus mapping, meta-quantitative trait locus fine-mapping, and association mapping, we showed that the nucleotide polymorphisms in the Tan1 gene, coding a WD40 protein, control the tannin biosynthesis in sorghum. A 1-bp G deletion in the coding region, causing a frame shift and a premature stop codon, led to a nonfunctional allele, tan1-a. Likewise, a different 10-bp insertion resulted in a second nonfunctional allele, tan1-b. Transforming the sorghum Tan1 ORF into a nontannin Arabidopsis mutant restored the tannin phenotype. In addition, reduction in nucleotide diversity from wild sorghum accessions to landraces and cultivars was found at the region that codes the highly conserved WD40 repeat domains and the C-terminal region of the protein. Genetic research in crops, coupled with nutritional and medical research, could open the possibility of producing different levels and combinations of phenolic compounds to promote human health. PMID:22699509

A methods review on use of nonsense suppression to study 3′ end formation and other aspects of tRNA biogenesis

PubMed Central

Rijal, Keshab; Maraia, Richard J.; Arimbasseri, Aneeshkumar G.

2014-01-01

Suppressor tRNAs bear anticodon mutations that allow them to decode premature stop codons in metabolic marker gene mRNAs, that can be used as in vivo reporters of functional tRNA biogenesis. Here, we review key components of a suppressor tRNA system specific to S. pombe and its adaptations for use to study specific steps in tRNA biogenesis. Eukaryotic tRNA biogenesis begins with transcription initiation by RNA polymerase (pol) III. The nascent pre-tRNAs must undergo folding, 5′ and 3′ processing to remove the leader and trailer, nuclear export, and splicing if applicable, while multiple complex chemical modifications occur throughout the process. We review evidence that precursor-tRNA processing begins with transcription termination at the oligo(T) terminator element, which forms a 3′ oligo(U) tract on the nascent RNA, a sequence-specific binding site for the RNA chaperone, La protein. The processing pathway bifurcates depending on a poorly understood property of pol III termination that determines the 3′ oligo(U) length and therefore the affinity for La. We thus review the pol III termination process and the factors involved including advances using gene-specific random mutagenesis by dNTP analogs that identify key residues important for transcription termination in certain pol III subunits. The review ends with a ‘technical approaches’ section that includes a parts lists of suppressor-tRNA alleles, strains and plasmids, and graphic examples of its diverse uses. PMID:25447915

A small indel mutation in an anthocyanin transporter causes variegated colouration of peach flowers.

PubMed

Cheng, Jun; Liao, Liao; Zhou, Hui; Gu, Chao; Wang, Lu; Han, Yuepeng

2015-12-01

The ornamental peach cultivar 'Hongbaihuatao (HBH)' can simultaneously bear pink, red, and variegated flowers on a single tree. Anthocyanin content in pink flowers is extremely low, being only 10% that of a red flower. Surprisingly, the expression of anthocyanin structural and potential regulatory genes in white flowers was not significantly lower than that in both pink and red flowers. However, proteomic analysis revealed a GST encoded by a gene-regulator involved in anthocyanin transport (Riant)-which is expressed in the red flower, but almost undetectable in the variegated flower. The Riant gene contains an insertion-deletion (indel) polymorphism in exon 3. In white flowers, the Riant gene is interrupted by a 2-bp insertion in the last exon, which causes a frameshift and a premature stop codon. In contrast, both pink and red flowers that arise from bud sports are heterozygous for the Riant locus, with one functional allele due to the 2-bp deletion or a novel 1-bp insertion. Southern blot analysis indicated that the Riant gene occurs in a single copy in the peach genome and it is not interrupted by a transposon. The function of the Riant gene was confirmed by its ectopic expression in the Arabidopsis tt19 mutant, where it complements the anthocyanin phenotype, but not the proanthocyanidin pigmentation in seed coat. Collectively,these results indicate that a small indel mutation in the Riant gene, which is not the result of a transposon insertion or excision, causes variegated colouration of peach flowers. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Tissue Specific Roles for the Ribosome Biogenesis Factor Wdr43 in Zebrafish Development

PubMed Central

Zhao, Chengtian; Andreeva, Viktoria; Gibert, Yann; LaBonty, Melissa; Lattanzi, Victoria; Prabhudesai, Shubhangi; Zhou, Yi; Zon, Leonard; McCann, Kathleen L.; Baserga, Susan; Yelick, Pamela C.

2014-01-01

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome. PMID:24497835

Sequence Polymorphisms and Structural Variations among Four Grapevine (Vitis vinifera L.) Cultivars Representing Sardinian Agriculture

PubMed Central

Mercenaro, Luca; Nieddu, Giovanni; Porceddu, Andrea; Pezzotti, Mario; Camiolo, Salvatore

2017-01-01

The genetic diversity among grapevine (Vitis vinifera L.) cultivars that underlies differences in agronomic performance and wine quality reflects the accumulation of single nucleotide polymorphisms (SNPs) and small indels as well as larger genomic variations. A combination of high throughput sequencing and mapping against the grapevine reference genome allows the creation of comprehensive sequence variation maps. We used next generation sequencing and bioinformatics to generate an inventory of SNPs and small indels in four widely cultivated Sardinian grape cultivars (Bovale sardo, Cannonau, Carignano and Vermentino). More than 3,200,000 SNPs were identified with high statistical confidence. Some of the SNPs caused the appearance of premature stop codons and thus identified putative pseudogenes. The analysis of SNP distribution along chromosomes led to the identification of large genomic regions with uninterrupted series of homozygous SNPs. We used a digital comparative genomic hybridization approach to identify 6526 genomic regions with significant differences in copy number among the four cultivars compared to the reference sequence, including 81 regions shared between all four cultivars and 4953 specific to single cultivars (representing 1.2 and 75.9% of total copy number variation, respectively). Reads mapping at a distance that was not compatible with the insert size were used to identify a dataset of putative large deletions with cultivar Cannonau revealing the highest number. The analysis of genes mapping to these regions provided a list of candidates that may explain some of the phenotypic differences among the Bovale sardo, Cannonau, Carignano and Vermentino cultivars. PMID:28775732

Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes.

PubMed

Maury, Mylène M; Chenal-Francisque, Viviane; Bracq-Dieye, Hélène; Han, Lei; Leclercq, Alexandre; Vales, Guillaume; Moura, Alexandra; Gouin, Edith; Scortti, Mariela; Disson, Olivier; Vázquez-Boland, José A; Lecuit, Marc

2017-11-01

The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene ( hly ) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA - /LLO - ) mutants belonged to phylogenetically diverse clades of L. monocytogenes , and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA - /LLO - mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen. Copyright © 2017 Maury et al.

Common founder mutation in the LDL receptor gene causing familial hypercholesterolaemia in the Icelandic population.

PubMed

Gudnason, V; Sigurdsson, G; Nissen, H; Humphries, S E

1997-01-01

Haplotype analysis in 18 apparently unrelated families with familial hypercholesterolaemia (FH) in Iceland has identified at least five different chromosomes cosegregating with hypercholesterolaemia. The most common haplotype was identified in 11 of the 18 families, indicating a responsible for FH in the Icelandic population. By using single-strand conformation polymorphism (SSCP) and direct sequencing of amplified DNA, we identified a novel mutation (a T to a C) in the second nucleotide in the 5' part of intron 4 in the LDL receptor gene. This mutation was present in approximately 60% of the FH families (10/18), supporting the prediction of a common founder. These families could be traced to a common ancestor in half of the cases by going back no further than the eighteenth century. The mutation was predicted to affect correct splicing of exon 4, and analysis at the cellular level demonstrated an abnormal mRNA containing intron 4 sequence in lymphoblastoid cells from a patient carrying this mutation. Translation of the mRNA would lead to a premature stop codon and a truncated nonfunctional protein of 285 amino acids. The novel sequence change created a new restriction site for the restriction endonuclease NlaIII, and using this assay, 29 unrelated individuals with possible FH attending a lipid clinic for treatment were examined for this mutation. Two individuals in this group of patients were found to be carriers of this mutation, supporting the suggestion of a founder mutation. Using this assay for the detection of FH in the Icelandic population should identify > 60% of these individuals.

A novel Dutch mutation in UNC13D reveals an essential role of the C2B domain in munc13-4 function.

PubMed

Elstak, Edo D; te Loo, Maroeska; Tesselaar, Kiki; van Kerkhof, Peter; Loeffen, Jan; Grivas, Dimitris; Hennekam, Eric; Boelens, Jaap Jan; Hoogerbrugge, Peter M; van der Sluijs, Peter; van Gijn, Marielle E; van de Corput, Lisette

2012-04-01

UNC13D, encoding the protein munc13-4, is essential in intracellular trafficking and exocytosis of lytic granules. Mutations in this gene are associated with familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a genetically heterogeneous, rare autosomal recessive immune disorder. How mutations affect function of munc13-4 is poorly understood. Since 2006 we genetically identified seven FHL patients with mutations in UNC13D. Here, we report for the first time a c.2695C>T (p.Arg899X) mutation in exon 28 of UNC13D in three young unrelated Dutch patients. The mutation causes a premature stop codon and encodes munc13-4(1-899), which lacks the C-terminal C2 domain. Genealogical research and haplotyping of the patient families demonstrated that a single ancestral founder introduced the mutation in the Netherlands. We then characterized the mutant protein phenotypically in cell biological and immunological assays. Munc13-4(1-899) was correctly targeted to CD63-positive secretory lysosomes, although its stability was reduced and dynamic turnover on the granule membrane became uncoupled from receptor signaling. In accord, and in contrast to wild-type munc13-4, ectopically expressed mutant failed to rescue degranulation in cells with silenced endogenous munc13-4. The functional and clinical data showed that this novel Dutch founder mutation leads to severe early onset of FHL3 due to misfolding and degradation of munc13-4(1-899). Copyright © 2011 Wiley Periodicals, Inc.

The secreted esterase of Propionibacterium freudenreichii has a major role in cheese lipolysis.

PubMed

Abeijón Mukdsi, María Claudia; Falentin, Hélène; Maillard, Marie-Bernadette; Chuat, Victoria; Medina, Roxana Beatriz; Parayre, Sandrine; Thierry, Anne

2014-01-01

Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1(T) for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis.

The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis

PubMed Central

Abeijón Mukdsi, María Claudia; Falentin, Hélène; Maillard, Marie-Bernadette; Chuat, Victoria; Medina, Roxana Beatriz; Parayre, Sandrine

2014-01-01

Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1T for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis. PMID:24242250

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing.

PubMed

Margaglione, M; Santacroce, R; Colaizzo, D; Seripa, D; Vecchione, G; Lupone, M R; De Lucia, D; Fortina, P; Grandone, E; Perricone, C; Di Minno, G

2000-10-01

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the gamma-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated gamma-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

Congenital analbuminemia caused by a novel aberrant splicing in the albumin gene

PubMed Central

Caridi, Gianluca; Dagnino, Monica; Erdeve, Omer; Di Duca, Marco; Yildiz, Duran; Alan, Serdar; Atasay, Begum; Arsan, Saadet; Campagnoli, Monica; Galliano, Monica; Minchiotti, Lorenzo

2014-01-01

Introduction: Congenital analbuminemia is a rare autosomal recessive disorder manifested by the presence of a very low amount of circulating serum albumin. It is an allelic heterogeneous defect, caused by variety of mutations within the albumin gene in homozygous or compound heterozygous state. Herein we report the clinical and molecular characterization of a new case of congenital analbuminemia diagnosed in a female newborn of consanguineous (first degree cousins) parents from Ankara, Turkey, who presented with a low albumin concentration (< 8 g/L) and severe clinical symptoms. Materials and methods: The albumin gene of the index case was screened by single-strand conformation polymorphism, heteroduplex analysis, and direct DNA sequencing. The effect of the splicing mutation was evaluated by examining the cDNA obtained by reverse transcriptase - polymerase chain reaction (RT-PCR) from the albumin mRNA extracted from proband’s leukocytes. Results: DNA sequencing revealed that the proband is homozygous, and both parents are heterozygous, for a novel G>A transition at position c.1652+1, the first base of intron 12, which inactivates the strongly conserved GT dinucleotide at the 5′ splice site consensus sequence of this intron. The splicing defect results in the complete skipping of the preceding exon (exon 12) and in a frame-shift within exon 13 with a premature stop codon after the translation of three mutant amino acid residues. Conclusions: Our results confirm the clinical diagnosis of congenital analbuminemia in the proband and the inheritance of the trait and contribute to shed light on the molecular genetics of analbuminemia. PMID:24627724

HSJ1-related hereditary neuropathies: novel mutations and extended clinical spectrum.

PubMed

Gess, Burkhard; Auer-Grumbach, Michaela; Schirmacher, Anja; Strom, Tim; Zitzelsberger, Manuela; Rudnik-Schöneborn, Sabine; Röhr, Dominik; Halfter, Hartmut; Young, Peter; Senderek, Jan

2014-11-04

To determine the nature and frequency of HSJ1 mutations in patients with hereditary motor and hereditary motor and sensory neuropathies. Patients were screened for mutations by genome-wide or targeted linkage and homozygosity studies, whole-exome sequencing, and Sanger sequencing. RNA and protein studies of skin fibroblasts were used for functional characterization. We describe 2 additional mutations in the HSJ1 gene in a cohort of 90 patients with autosomal recessive distal hereditary motor neuropathy (dHMN) and Charcot-Marie-Tooth disease type 2 (CMT2). One family with a dHMN phenotype showed the homozygous splice-site mutation c.229+1G>A, which leads to retention of intron 4 in the HSJ1 messenger RNA with a premature stop codon and loss of protein expression. Another family, presenting with a CMT2 phenotype, carried the homozygous missense mutation c.14A>G (p.Tyr5Cys). This mutation was classified as likely disease-related by several automatic algorithms for prediction of possible impact of an amino acid substitution on the structure and function of proteins. Both mutations cosegregated with autosomal recessive inheritance of the disease and were absent from the general population. Taken together, in our cohort of 90 probands, we confirm that HSJ1 mutations are a rare but detectable cause of autosomal recessive dHMN and CMT2. We provide clinical and functional information on an HSJ1 splice-site mutation and report the detailed phenotype of 2 patients with CMT2, broadening the phenotypic spectrum of HSJ1-related neuropathies. © 2014 American Academy of Neurology.

rhIGF-1 Treatment Increases Bone Mineral Density and Trabecular Bone Structure in Children with PAPP-A2 Deficiency.

PubMed

Hawkins-Carranza, Federico G; Muñoz-Calvo, María T; Martos-Moreno, Gabriel Á; Allo-Miguel, Gonzalo; Del Río, Luis; Pozo, Jesús; Chowen, Julie A; Pérez-Jurado, Luis A; Argente, Jesús

2018-01-01

Our objective was to determine changes in bone mineral density (BMD), trabecular bone score (TBS), and body composition after 2 years of therapy with recombinant human insulin-like growth factor-1 (rhIGF-1) in 2 prepubertal children with a complete lack of circulating PAPP-A2 due to a homozygous mutation in PAPP-A2 (p.D643fs25*) resulting in a premature stop codon. Body composition, BMD, and bone structure were determined by dual-energy X-ray absorptiometry at baseline and after 1 and 2 years of rhIGF-1 treatment. Height increased from 132 to 145.5 cm (patient 1) and from 111.5 to 124.5 cm (patient 2). Bone mineral content increased from 933.40 to 1,057.97 and 1,152.77 g in patient 1, and from 696.12 to 773.26 and 911.51 g in patient 2, after 1 and 2 years, respectively. Whole-body BMD also increased after 2 years of rhIGF-1 from baseline 0.788 to 0.869 g/cm2 in patient 1 and from 0.763 to 0.829 g/cm2 in patient 2. After 2 years of treatment, both children had an improvement in TBS. During therapy, a slight increase in body fat mass was seen, with a concomitant increase in lean mass. No adverse effects were reported. Two years of rhIGF-1 improved growth, with a tendency to improve bone mass and bone microstructure and to modulate body composition. © 2018 S. Karger AG, Basel.

Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A

PubMed Central

PORADA, C. D.; SANADA, C.; LONG, C. R.; WOOD, J. A.; DESAI, J.; FREDERICK, N.; MILLSAP, L.; BORMANN, C.; MENGES, S. L.; HANNA, C.; FLORES-FOXWORTH, G.; SHIN, T.; WESTHUSIN, M. E.; LIU, W.; GLIMP, H.; ZANJANI, E. D.; LOZIER, J. N.; PLISKA, V.; STRANZINGER, G.; JOERG, H.; KRAEMER, D. C.; ALMEIDA-PORADA, G.

2010-01-01

Summary Background Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. Objectives To re-establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. Patients/methods Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. Results and conclusions The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non-existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame-shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients. PMID:19943872

Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes

PubMed Central

Maury, Mylène M.; Chenal-Francisque, Viviane; Bracq-Dieye, Hélène; Han, Lei; Leclercq, Alexandre; Vales, Guillaume; Moura, Alexandra; Gouin, Edith; Scortti, Mariela; Disson, Olivier

2017-01-01

ABSTRACT The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene (hly) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes. Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA. Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA−/LLO−) mutants belonged to phylogenetically diverse clades of L. monocytogenes, and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA−/LLO− mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen. PMID:28827366

A deletion in the VLDLR gene in Eurasier dogs with cerebellar hypoplasia resembling a Dandy-Walker-like malformation (DWLM).

PubMed

Gerber, Martina; Fischer, Andrea; Jagannathan, Vidhya; Drögemüller, Michaela; Drögemüller, Cord; Schmidt, Martin J; Bernardino, Filipa; Manz, Eberhard; Matiasek, Kaspar; Rentmeister, Kai; Leeb, Tosso

2015-01-01

Dandy-Walker-like malformation (DWLM) is the result of aberrant brain development and mainly characterized by cerebellar hypoplasia. DWLM affected dogs display a non-progressive cerebellar ataxia. Several DWLM cases were recently observed in the Eurasier dog breed, which strongly suggested a monogenic autosomal recessive inheritance in this breed. We performed a genome-wide association study (GWAS) with 9 cases and 11 controls and found the best association of DWLM with markers on chromosome 1. Subsequent homozygosity mapping confirmed that all 9 cases were homozygous for a shared haplotype in this region, which delineated a critical interval of 3.35 Mb. We sequenced the genome of an affected Eurasier and compared it with the Boxer reference genome and 47 control genomes of dogs from other breeds. This analysis revealed 4 private non-synonymous variants in the critical interval of the affected Eurasier. We genotyped these variants in additional dogs and found perfect association for only one of these variants, a single base deletion in the VLDLR gene encoding the very low density lipoprotein receptor. This variant, VLDLR:c.1713delC is predicted to cause a frameshift and premature stop codon (p.W572Gfs*10). Variants in the VLDLR gene have been shown to cause congenital cerebellar ataxia and mental retardation in human patients and Vldlr knockout mice also display an ataxia phenotype. Our combined genetic data together with the functional knowledge on the VLDLR gene from other species thus strongly suggest that VLDLR:c.1713delC is indeed causing DWLM in Eurasier dogs.

Intronic deletions in the SLC34A3 gene: A cautionary tale for mutation analysis of hereditary hypophosphatemic rickets with hypercalciuria

PubMed Central

Ichikawa, Shoji; Tuchman, Shamir; Padgett, Leah R.; Gray, Amie K.; Baluarte, H. Jorge; Econs, Michael J.

2013-01-01

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder, characterized by hypophosphatemia, variable degrees of rickets/osteomalacia, and hypercalciuria secondary to increased serum 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. HHRH is caused by mutations in the SLC34A3 gene, which encodes sodium-phosphate co-transporter type IIc. A 6 ½-year-old female presented with a history of nephrolithiasis. Her metabolic evaluation revealed increased 24- hour urine calcium excretion with high serum calcium, low intact parathyroid hormone (PTH) levels, and elevated 1,25(OH)2D level. In addition, the patient had low to low-normal serum phosphorus with high urine phosphorus. The patient had normal stature; without rachitic or boney deformities or a history of fractures. Genetic analysis of SLC34A3 revealed the patient to be a compound heterozygote for a novel single base pair deletion in exon 12 (c.1304delG) and 30-base pair deletion in intron 6 (g.1440–1469del). The single-base pair mutation causes a frameshift, which results in premature stop codon. The intronic deletion is likely caused by misalignment of the 4-basepair homologous repeats and results in the truncation of an already small intron to 63 bp, which would impair proper RNA splicing of the intron. This is the fourth unique intronic deletion identified in patients with HHRH, suggesting the frequent occurrence of sequence misalignments in SLC34A3 and the importance of screening introns in patients with HHRH. PMID:24176905

Intronic deletions in the SLC34A3 gene: a cautionary tale for mutation analysis of hereditary hypophosphatemic rickets with hypercalciuria.

PubMed

Ichikawa, Shoji; Tuchman, Shamir; Padgett, Leah R; Gray, Amie K; Baluarte, H Jorge; Econs, Michael J

2014-02-01

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder, characterized by hypophosphatemia, variable degrees of rickets/osteomalacia, and hypercalciuria secondary to increased serum 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. HHRH is caused by mutations in the SLC34A3 gene, which encodes sodium-phosphate co-transporter type IIc. A 6-1/2-year-old female presented with a history of nephrolithiasis. Her metabolic evaluation revealed increased 24-hour urine calcium excretion with high serum calcium, low intact parathyroid hormone (PTH), and elevated 1,25(OH)2D. In addition, the patient had low to low-normal serum phosphorus with high urine phosphorus. The patient had normal stature; without rachitic or boney deformities or a history of fractures. Genetic analysis of SLC34A3 revealed the patient to be a compound heterozygote for a novel single base pair deletion in exon 12 (c.1304delG) and 30-base pair deletion in intron 6 (g.1440-1469del). The single-base pair mutation causes a frameshift, which results in premature stop codon. The intronic deletion is likely caused by misalignment of the 4-basepair homologous repeats and results in the truncation of an already small intron to 63bp, which would impair proper RNA splicing of the intron. This is the fourth unique intronic deletion identified in patients with HHRH, suggesting the frequent occurrence of sequence misalignments in SLC34A3 and the importance of screening introns in patients with HHRH. © 2013.

A small indel mutation in an anthocyanin transporter causes variegated colouration of peach flowers

PubMed Central

Cheng, Jun; Liao, Liao; Zhou, Hui; Gu, Chao; Wang, Lu; Han, Yuepeng

2015-01-01

The ornamental peach cultivar ‘Hongbaihuatao (HBH)’ can simultaneously bear pink, red, and variegated flowers on a single tree. Anthocyanin content in pink flowers is extremely low, being only 10% that of a red flower. Surprisingly, the expression of anthocyanin structural and potential regulatory genes in white flowers was not significantly lower than that in both pink and red flowers. However, proteomic analysis revealed a GST encoded by a gene—regulator involved in anthocyanin transport (Riant)—which is expressed in the red flower, but almost undetectable in the variegated flower. The Riant gene contains an insertion-deletion (indel) polymorphism in exon 3. In white flowers, the Riant gene is interrupted by a 2-bp insertion in the last exon, which causes a frameshift and a premature stop codon. In contrast, both pink and red flowers that arise from bud sports are heterozygous for the Riant locus, with one functional allele due to the 2-bp deletion or a novel 1-bp insertion. Southern blot analysis indicated that the Riant gene occurs in a single copy in the peach genome and it is not interrupted by a transposon. The function of the Riant gene was confirmed by its ectopic expression in the Arabidopsis tt19 mutant, where it complements the anthocyanin phenotype, but not the proanthocyanidin pigmentation in seed coat. Collectively,these results indicate that a small indel mutation in the Riant gene, which is not the result of a transposon insertion or excision, causes variegated colouration of peach flowers. PMID:26357885

Disorders with similar clinical phenotypes reveal underlying genetic interaction: SATB2 acts as an activator of the UPF3B gene

PubMed Central

Leoyklang, Petcharat; Suphapeetiporn, Kanya; Srichomthong, Chalurmpon; Tongkobpetch, Siraprapa; Fietze, Stefanie; Dorward, Heidi; Cullinane, Andrew R.; Gahl, William A.; Huizing, Marjan; Shotelersuk, Vorasuk

2014-01-01

Two syndromic cognitive impairment disorders have very similar craniofacial dysmorphisms. One is caused by mutations of SATB2, a transcription regulator, and the other by heterozygous mutations leading to premature stop codons in UPF3B, encoding a member of the nonsense-mediated mRNA decay complex. Here we demonstrate that the products of these two causative genes function in the same pathway. We show that the SATB2 nonsense mutation in our patient leads to a truncated protein that localizes to the nucleus, forms a dimer with wild-type SATB2 and interferes with its normal activity. This suggests that the SATB2 nonsense mutation has a dominant negative effect. The patient’s leukocytes had significantly decreased UPF3B mRNA compared to controls. This effect was replicated both in vitro, where siRNA knockdown of SATB2 in HEK293 cells resulted in decreased UPF3B expression, and in vivo, where embryonic tissue of Satb2 knock-out mice showed significantly decreased Upf3b expression. Furthermore, chromatin immunoprecipitation demonstrates that SATB2 binds to the UPF3B promoter, and a luciferase reporter assay confirmed that SATB2 expression significantly activates gene transcription using the UPF3B promoter. These findings indicate that SATB2 acts as an activator UPF3B expression through binding to its promoter. This study emphasizes the value of recognizing disorders with similar clinical phenotypes to explore underlying mechanisms of genetic interaction. PMID:23925499

A Homozygous Mutation in a Novel Zinc-Finger Protein, ERIS, Is Responsible for Wolfram Syndrome 2

PubMed Central

Amr, Sami ; Heisey, Cindy ; Zhang, Min ; Xia, Xia-Juan ; Shows, Kathryn H. ; Ajlouni, Kamel ; Pandya, Arti ; Satin, Leslie S. ; El-Shanti, Hatem ; Shiang, Rita 

2007-01-01

A single missense mutation was identified in a novel, highly conserved zinc-finger gene, ZCD2, in three consanguineous families of Jordanian descent with Wolfram syndrome (WFS). It had been shown that these families did not have mutations in the WFS1 gene (WFS1) but were mapped to the WFS2 locus at 4q22-25. A G→C transversion at nucleotide 109 predicts an amino acid change from glutamic acid to glutamine (E37Q). Although the amino acid is conserved and the mutation is nonsynonymous, the pathogenesis for the disorder is because the mutation also causes aberrant splicing. The mutation was found to disrupt messenger RNA splicing by eliminating exon 2, and it results in the introduction of a premature stop codon. Mutations in WFS1 have also been found to cause low-frequency nonsyndromic hearing loss, progressive hearing loss, and isolated optic atrophy associated with hearing loss. Screening of 377 probands with hearing loss did not identify mutations in the WFS2 gene. The WFS1-encoded protein, Wolframin, is known to localize to the endoplasmic reticulum and plays a role in calcium homeostasis. The ZCD2-encoded protein, ERIS (endoplasmic reticulum intermembrane small protein), is also shown to localize to the endoplasmic reticulum but does not interact directly with Wolframin. Lymphoblastoid cells from affected individuals show a significantly greater rise in intracellular calcium when stimulated with thapsigargin, compared with controls, although no difference was observed in resting concentrations of intracellular calcium. PMID:17846994

Permanent neonatal diabetes caused by a homozygous nonsense mutation in the glucokinase gene.

PubMed

Rubio-Cabezas, O; Díaz González, F; Aragonés, A; Argente, J; Campos-Barros, A

2008-06-01

Glucokinase deficiency is an unfrequent cause of permanent neonatal diabetes (PND), as only seven patients have been reported, either homozygous for a missense or frameshift mutation or compound heterozygous for both of them. We report here the first known case caused by a homozygous nonsense mutation (Y61X) in the glucokinase gene (GCK) that introduces a premature stop codon, generating a truncated protein that is predicted to be completely inactive as it lacks both the glucose- and the adenosine triphosphate-binding sites. The proband, born to consanguineous parents, was a full-term, intra-uterine growth-retarded male newborn who presented with a glycaemia of 129 mg/dL (7.16 mmol/L) on his second day of life, increasing thereafter up to 288 mg/dL (15.98 mmol/L) and 530 mg/dL (29.41 mmol/L) over the next 24 h, in the face of low serum insulin (<3 muIU/mL; <20.83 pmol/L). He was put on insulin on the third day of life. Insulin has never been discontinued since then. The patient was tested negative for anti-insulin and islet cell antibodies at age 5 months. His father had non-progressive, impaired fasting glucose for several years. The mother was found to be mildly hyperglycaemic only when her glucose was checked after the child was diagnosed. In conclusion, biallelic GCK loss should be considered as a potential cause of PND in children born to consanguineous parents, even if they are not known to be diabetic at the time of PND presentation.

A novel mutation in the AVPR2 gene (222delA) associated with X-linked nephrogenic diabetes insipidus in a boy with growth failure.

PubMed

Abaci, Ayhan; Wood, Kent; Demir, Korcan; Büyükgebiz, Atilla; Böber, Ece; Kopp, Peter

2010-01-01

To study the case of a 2 10/12-year-old boy who had growth failure and delayed bone maturation. We reviewed the history, which revealed that he had had polyuria, polydipsia, lack of weight gain, and frequent vomiting since the age of 5 months. On physical examination, his height was 86 cm (-1.93 standard deviation [SD]), his weight 10.5 kg (-2.67 SD), and he had motor and mental retardation. His maternal great-grandfather also had polyuria and polydipsia (but not diabetes mellitus), suggesting X-linked nephrogenic diabetes insipidus as the underlying cause. The patient underwent a water deprivation-desmopressin test. The coding region of the AVPR2 gene was amplified by polymerase chain reaction and submitted to direct sequence analysis. The water deprivation test confirmed the diagnosis of diabetes insipidus, and administration of desmopressin did not diminish his water secretion. Direct sequencing of the AVPR2 gene revealed a novel deletion of adenine at position 222 (222delA) in exon 2. This mutation is predicted to lead to a frameshift beginning at amino acid 75 and a premature stop codon at position 115 (FS75>115X). His height and weight, as well as his motor skills, improved after initiation of therapy with hydrochlorothiazide and amiloride. Growth delay can be associated with diabetes insipidus. The X-linked nephrogenic diabetes insipidus in this boy is caused by a novel mutation in the AVPR2 gene that is predicted to truncate the receptor protein.

Eight previously unidentified mutations found in the OA1 ocular albinism gene

PubMed Central

Mayeur, Hélène; Roche, Olivier; Vêtu, Christelle; Jaliffa, Carolina; Marchant, Dominique; Dollfus, Hélène; Bonneau, Dominique; Munier, Francis L; Schorderet, Daniel F; Levin, Alex V; Héon, Elise; Sutherland, Joanne; Lacombe, Didier; Said, Edith; Mezer, Eedy; Kaplan, Josseline; Dufier, Jean-Louis; Marsac, Cécile; Menasche, Maurice; Abitbol, Marc

2006-01-01

Background Ocular albinism type 1 (OA1) is an X-linked ocular disorder characterized by a severe reduction in visual acuity, nystagmus, hypopigmentation of the retinal pigmented epithelium, foveal hypoplasia, macromelanosomes in pigmented skin and eye cells, and misrouting of the optical tracts. This disease is primarily caused by mutations in the OA1 gene. Methods The ophthalmologic phenotype of the patients and their family members was characterized. We screened for mutations in the OA1 gene by direct sequencing of the nine PCR-amplified exons, and for genomic deletions by PCR-amplification of large DNA fragments. Results We sequenced the nine exons of the OA1 gene in 72 individuals and found ten different mutations in seven unrelated families and three sporadic cases. The ten mutations include an amino acid substitution and a premature stop codon previously reported by our team, and eight previously unidentified mutations: three amino acid substitutions, a duplication, a deletion, an insertion and two splice-site mutations. The use of a novel Taq polymerase enabled us to amplify large genomic fragments covering the OA1 gene. and to detect very likely six distinct large deletions. Furthermore, we were able to confirm that there was no deletion in twenty one patients where no mutation had been found. Conclusion The identified mutations affect highly conserved amino acids, cause frameshifts or alternative splicing, thus affecting folding of the OA1 G protein coupled receptor, interactions of OA1 with its G protein and/or binding with its ligand. PMID:16646960

Mechanisms of Comorbidities Associated With the Metabolic Syndrome: Insights from the JCR:LA-cp Corpulent Rat Strain.

PubMed

Diane, Abdoulaye; Pierce, W David; Kelly, Sandra E; Sokolik, Sharon; Borthwick, Faye; Jacome-Sosa, Miriam; Mangat, Rabban; Pradillo, Jesus Miguel; Allan, Stuart McRae; Ruth, Megan R; Field, Catherine J; Hutcheson, Rebecca; Rocic, Petra; Russell, James C; Vine, Donna F; Proctor, Spencer D

2016-01-01

Obesity and its metabolic complications have emerged as the epidemic of the new millennia. The use of obese rodent models continues to be a productive component of efforts to understand the concomitant metabolic complications of this disease. In 1978, the JCR:LA-cp rat model was developed with an autosomal recessive corpulent ( cp ) trait resulting from a premature stop codon in the extracellular domain of the leptin receptor. Rats that are heterozygous for the cp trait are lean-prone, while those that are homozygous ( cp/cp ) spontaneously display the pathophysiology of obesity as well as a metabolic syndrome (MetS)-like phenotype. Over the years, there have been formidable scientific contributions that have originated from this rat model, much of which has been reviewed extensively up to 2008. The premise of these earlier studies focused on characterizing the pathophysiology of MetS-like phenotype that was spontaneously apparent in this model. The purpose of this review is to highlight areas of recent advancement made possible by this model including; emerging appreciation of the "thrifty gene" hypothesis in the context of obesity, the concept of how chronic inflammation may drive obesogenesis, the impact of acute forms of inflammation to the brain and periphery during chronic obesity, the role of dysfunctional insulin metabolism on lipid metabolism and vascular damage, and the mechanistic basis for altered vascular function as well as novel parallels between the human condition and the female JCR:LA-cp rat as a model for polycystic ovary disease (PCOS).

Molecular basis of a shattering resistance boosting global dissemination of soybean

PubMed Central

Funatsuki, Hideyuki; Suzuki, Masaya; Hirose, Aya; Inaba, Hiroki; Yamada, Tetsuya; Hajika, Makita; Komatsu, Kunihiko; Katayama, Takeshi; Sayama, Takashi; Ishimoto, Masao; Fujino, Kaien

2014-01-01

Pod dehiscence (shattering) is essential for the propagation of wild plant species bearing seeds in pods but is a major cause of yield loss in legume and crucifer crops. Although natural genetic variation in pod dehiscence has been, and will be, useful for plant breeding, little is known about the molecular genetic basis of shattering resistance in crops. Therefore, we performed map-based cloning to unveil a major quantitative trait locus (QTL) controlling pod dehiscence in soybean. Fine mapping and complementation testing revealed that the QTL encodes a dirigent-like protein, designated as Pdh1. The gene for the shattering-resistant genotype, pdh1, was defective, having a premature stop codon. The functional gene, Pdh1, was highly expressed in the lignin-rich inner sclerenchyma of pod walls, especially at the stage of initiation in lignin deposition. Comparisons of near-isogenic lines indicated that Pdh1 promotes pod dehiscence by increasing the torsion of dried pod walls, which serves as a driving force for pod dehiscence under low humidity. A survey of soybean germplasm revealed that pdh1 was frequently detected in landraces from semiarid regions and has been extensively used for breeding in North America, the world’s leading soybean producer. These findings point to a new mechanism for pod dehiscence involving the dirigent protein family and suggest that pdh1 has played a crucial role in the global expansion of soybean cultivation. Furthermore, the orthologs of pdh1, or genes with the same role, will possibly be useful for crop improvement. PMID:25468966

Mutation in TOR1AIP1 encoding LAP1B in a form of muscular dystrophy: a novel gene related to nuclear envelopathies.

PubMed

Kayman-Kurekci, Gulsum; Talim, Beril; Korkusuz, Petek; Sayar, Nilufer; Sarioglu, Turkan; Oncel, Ibrahim; Sharafi, Parisa; Gundesli, Hulya; Balci-Hayta, Burcu; Purali, Nuhan; Serdaroglu-Oflazer, Piraye; Topaloglu, Haluk; Dincer, Pervin

2014-07-01

We performed genome-wide homozygosity mapping and mapped a novel myopathic phenotype to chromosomal region 1q25 in a consanguineous family with three affected individuals manifesting proximal and distal weakness and atrophy, rigid spine and contractures of the proximal and distal interphalangeal hand joints. Additionally, cardiomyopathy and respiratory involvement were noted. DNA sequencing of torsinA-interacting protein 1 (TOR1AIP1) gene encoding lamina-associated polypeptide 1B (LAP1B), showed a homozygous c.186delG mutation that causes a frameshift resulting in a premature stop codon (p.E62fsTer25). We observed that expression of LAP1B was absent in the patient skeletal muscle fibres. Ultrastructural examination showed intact sarcomeric organization but alterations of the nuclear envelope including nuclear fragmentation, chromatin bleb formation and naked chromatin. LAP1B is a type-2 integral membrane protein localized in the inner nuclear membrane that binds to both A- and B-type lamins, and is involved in the regulation of torsinA ATPase. Interestingly, luminal domain-like LAP1 (LULL1)-an endoplasmic reticulum-localized partner of torsinA-was overexpressed in the patient's muscle in the absence of LAP1B. Therefore, the findings suggest that LAP1 and LULL1 might have a compensatory effect on each other. This study expands the spectrum of genes associated with nuclear envelopathies and highlights the critical function for LAP1B in striated muscle. Copyright © 2014 Elsevier B.V. All rights reserved.

Correction of nonsense BMPR2 and SMAD9 mutations by ataluren in pulmonary arterial hypertension.

PubMed

Drake, Kylie M; Dunmore, Benjamin J; McNelly, Lauren N; Morrell, Nicholas W; Aldred, Micheala A

2013-09-01

Heritable pulmonary arterial hypertension (HPAH) is a serious lung vascular disease caused by heterozygous mutations in the bone morphogenetic protein (BMP) pathway genes, BMPR2 and SMAD9. One noncanonical function of BMP signaling regulates biogenesis of a subset of microRNAs. We have previously shown that this function is abrogated in patients with HPAH, making it a highly sensitive readout of BMP pathway integrity. Ataluren (PTC124) is an investigational drug that permits ribosomal readthrough of premature stop codons, resulting in a full-length protein. It exhibits oral bioavailability and limited toxicity in human trials. Here, we tested ataluren in lung- or blood-derived cells from patients with HPAH with nonsense mutations in BMPR2 (n = 6) or SMAD9 (n = 1). Ataluren significantly increased BMP-mediated microRNA processing in six of the seven cases. Moreover, rescue was achieved even for mutations exhibiting significant nonsense-mediated mRNA decay. Response to ataluren was dose dependent, and complete correction was achieved at therapeutic doses currently used in clinical trials for cystic fibrosis. BMP receptor (BMPR)-II protein levels were normalized and ligand-dependent phosphorylation of downstream target Smads was increased. Furthermore, the usually hyperproliferative phenotype of pulmonary artery endothelial and smooth muscle cells was reversed by ataluren. These results indicate that ataluren can effectively suppress a high proportion of BMPR2 and SMAD9 nonsense mutations and correct BMP signaling in vitro. Approximately 29% of all HPAH mutations are nonsense point mutations. In light of this, we propose ataluren as a potential new personalized therapy for this significant subgroup of patients with PAH.

Functions of the APC tumor suppressor protein dependent and independent of canonical WNT signaling: Implications for therapeutic targeting

PubMed Central

Hankey, William; Frankel, Wendy L.

2018-01-01

The acquisition of biallelic mutations in the APC gene is a rate-limiting step in the development of most colorectal cancers and occurs in the earliest lesions. APC encodes a 312-kDa protein that localizes to multiple subcellular compartments and performs diverse functions. APC participates in a cytoplasmic complex that promotes the destruction of the transcriptional licensing factor β-catenin; APC mutations that abolish this function trigger constitutive activation of the canonical WNT signaling pathway, a characteristic found in almost all colorectal cancers. By negatively regulating canonical WNT signaling, APC counteracts proliferation, promotes differentiation, facilitates apoptosis and suppresses invasion and tumor progression. APC further antagonizes canonical WNT signaling by interacting with and counteracting β-catenin in the nucleus. APC also suppresses tumor initiation and progression in the colorectal epithelium through functions that are independent of canonical WNT signaling. APC regulates the mitotic spindle to facilitate proper chromosome segregation, localizes to the cell periphery and cell protrusions to establish cell polarity and appropriate directional migration, and inhibits DNA replication by interacting directly with DNA. Mutations in APC are often frameshifts, insertions or deletions that introduce premature stop codons and lead to the production of truncated APC proteins that lack its normal functions and possess tumorigenic properties. Therapeutic approaches in development for the treatment of APC-deficient tumors are focused on the inhibition of canonical WNT signaling, especially through targets downstream of APC in the pathway, or on the restoration of wild-type APC expression. PMID:29318445

The A31P missense mutation in cardiac myosin binding protein C alters protein structure but does not cause haploinsufficiency.

PubMed

van Dijk, Sabine J; Bezold Kooiker, Kristina; Mazzalupo, Stacy; Yang, Yuanzhang; Kostyukova, Alla S; Mustacich, Debbie J; Hoye, Elaine R; Stern, Joshua A; Kittleson, Mark D; Harris, Samantha P

2016-07-01

Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function. Copyright © 2016 Elsevier Inc. All rights reserved.

Mutation profile of all 49 exons of the human myosin VIIA gene, and haplotype analysis, in Usher 1B families from diverse origins.

PubMed

Adato, A; Weil, D; Kalinski, H; Pel-Or, Y; Ayadi, H; Petit, C; Korostishevsky, M; Bonne-Tamir, B

1997-10-01

Usher syndrome types I (USH1A-USH1E) are a group of autosomal recessive diseases characterized by profound congenital hearing loss, vestibular areflexia, and progressive visual loss due to retinitis pigmentosa. The human myosin VIIA gene, located on 11q14, has been shown to be responsible for Usher syndrome type 1B (USH1B). Haplotypes were constructed in 28 USH1 families by use of the following polymorphic markers spanning the USH1B locus: D11S787, D11S527, D11S1789, D11S906, D11S4186, and OMP. Affected individuals and members of their families from 12 different ethnic origins were screened for the presence of mutations in all 49 exons of the myosin VIIA gene. In 15 families myosin VIIA mutations were detected, verifying their classification as USH1B. All these mutations are novel, including three missense mutations, one premature stop codon, two splicing mutations, one frameshift, and one deletion of >2 kb comprising exons 47 and 48, a part of exon 49, and the introns between them. Three mutations were shared by more than one family, consistent with haplotype similarities. Altogether, 16 USH1B haplotypes were observed in the 15 families; most haplotypes were population specific. Several exonic and intronic polymorphisms were also detected. None of the 20 known USH1B mutations reported so far in other world populations were identified in our families.

Overexpression of the DNA mismatch repair factor, PMS2, confers hypermutability and DNA damage tolerance.

PubMed

Gibson, Shannon L; Narayanan, Latha; Hegan, Denise Campisi; Buermeyer, Andrew B; Liskay, R Michael; Glazer, Peter M

2006-12-08

Inherited defects in genes associated with DNA mismatch repair (MMR) have been linked to familial colorectal cancer. Cells deficient in MMR are genetically unstable and demonstrate a tolerance phenotype in response to certain classes of DNA damage. Some sporadic human cancers also show abnormalities in MMR gene function, typically due to diminished expression of one of the MutL homologs, MLH1. Here, we report that overexpression of the MutL homolog, human PMS2, can also cause a disruption of the MMR pathway in mammalian cells, resulting in hypermutability and DNA damage tolerance. A mouse fibroblast cell line carrying a recoverable lambda phage shuttle vector for mutation detection was transfected with either a vector designed to express hPMS2 or with an empty vector control. Cells overexpressing hPMS2 were found to have elevated spontaneous mutation frequencies at the cII reporter gene locus. They also showed an increase in the level of mutations induced by the alkylating agent, methynitrosourea (MNU). Clonogenic survival assays demonstrated increased survival of the PMS2-overexpressing cells following exposure to MNU, consistent with the induction of a damage tolerance phenotype. Similar results were seen in cells expressing a mutant PMS2 gene, containing a premature stop codon at position 134 and representing a variant found in an individual with familial colon cancer. These results show that dysregulation of PMS2 gene expression can disrupt MMR function in mammalian cells and establish an additional carcinogenic mechanism by which cells can develop genetic instability and acquire resistance to cytotoxic cancer therapies.

Cryptic tRNAs in chaetognath mitochondrial genomes.

PubMed

Barthélémy, Roxane-Marie; Seligmann, Hervé

2016-06-01

The chaetognaths constitute a small and enigmatic phylum of little marine invertebrates. Both nuclear and mitochondrial genomes have numerous originalities, some phylum-specific. Until recently, their mitogenomes seemed containing only one tRNA gene (trnMet), but a recent study found in two chaetognath mitogenomes two and four tRNA genes. Moreover, apparently two conspecific mitogenomes have different tRNA gene numbers (one and two). Reanalyses by tRNAscan-SE and ARWEN softwares of the five available complete chaetognath mitogenomes suggest numerous additional tRNA genes from different types. Their total number never reaches the 22 found in most other invertebrates using that genetic code. Predicted error compensation between codon-anticodon mismatch and tRNA misacylation suggests translational activity by tRNAs predicted solely according to secondary structure for tRNAs predicted by tRNAscan-SE, not ARWEN. Numbers of predicted stop-suppressor (antitermination) tRNAs coevolve with predicted overlapping, frameshifted protein coding genes including stop codons. Sequence alignments in secondary structure prediction with non-chaetognath tRNAs suggest that the most likely functional tRNAs are in intergenic regions, as regular mt-tRNAs. Due to usually short intergenic regions, generally tRNA sequences partially overlap with flanking genes. Some tRNA pairs seem templated by sense-antisense strands. Moreover, 16S rRNA genes, but not 12S rRNAs, appear as tRNA nurseries, as previously suggested for multifunctional ribosomal-like protogenomes. Copyright © 2016 Elsevier Ltd. All rights reserved.

A free VP3 C-terminus is essential for the replication of infectious bursal disease virus.

PubMed

Mosley, Yung-Yi C; Wu, Ching Ching; Lin, Tsang Long

2017-03-15

Green fluorescent protein (GFP) has been successfully incorporated into the viral-like particles of infectious bursal disease virus (IBDV) with a linker at the C-terminus of VP3 in a baculovirus system. However, when the same locus in segment A was used to express GFP by a reverse genetic (RG) system, no viable GFP-expressing IBDV was recovered. To elucidate the underlying mechanism, cDNA construct of segment A with only the linker sequence (9 amino acids) was applied to generate RG IBDV virus (rIBDV). Similarly, no rIBDV was recovered. Moreover, when the incubation after transfection was extended, wildtype rIBDV without the linker was recovered suggesting a free C-terminus of VP3 might be necessary for IBDV replication. On the other hand, rIBDV could be recovered when additional sequence (up to 40 nucleotides) were inserted at the 3' noncoding region (NCR) adjacent to the stop codon of VP3, suggesting that the burden of the linker sequence was not in the stretched genome size but the disruption of the VP3 function. Finally, when the stop codon of VP3 was deleted in segment A to extend the translation into the 3' NCR without introducing additional genomic sequence, no rIBDV was recovered. Our data suggest that a free VP3 C-terminus is essential for IBDV replication. Copyright © 2017 Elsevier B.V. All rights reserved.

Epidermolysis bullosa with congenital pyloric atresia: novel mutations in the beta 4 integrin gene (ITGB4) and genotype/phenotype correlations.

PubMed

Nakano, A; Pulkkinen, L; Murrell, D; Rico, J; Lucky, A W; Garzon, M; Stevens, C A; Robertson, S; Pfendner, E; Uitto, J

2001-05-01

Epidermolysis bullosa with pyloric atresia (EB-PA: OMIM 226730), also known as Carmi syndrome, is a rare autosomal recessive genodermatosis that manifests with neonatal mucocutaneous fragility associated with congenital pyloric atresia. The disease is frequently lethal within the first year, but nonlethal cases have been reported. Mutations in the genes encoding subunit polypeptides of the alpha 6 beta 4 integrin (ITGA6 and ITGB4) have been demonstrated in EB-PA patients. To extend the repertoire of mutations and to identify genotype-phenotype correlations, we examined seven new EB-PA families, four with lethal and three with nonlethal disease variants. DNA from patients was screened for mutations using heteroduplex analysis followed by nucleotide sequencing of PCR products spanning all beta 4 integrin-coding sequences. Mutation analysis disclosed 12 distinct mutations, 11 of them novel. Four mutations predicted a premature termination codon as a result of nonsense mutations or small out-of-frame insertions or deletions, whereas seven were missense mutations. This brings the total number of distinct ITGB4 mutations to 33. The mutation database indicates that premature termination codons are associated predominantly with the lethal EB-PA variants, whereas missense mutations are more prevalent in nonlethal forms. However, the consequences of the missense mutations are position dependent, and substitutions of highly conserved amino acids may have lethal consequences. In general, indirect immunofluorescence studies of affected skin revealed negative staining for beta 4 integrin in lethal cases and positive, but attenuated, staining in nonlethal cases and correlated with clinical phenotype. The data on specific mutations in EB-PA patients allows prenatal testing and preimplantation genetic diagnosis in families at risk.

Clinical and mutation-type analysis from an international series of 198 probands with a pathogenic FBN1 exons 24–32 mutation

PubMed Central

Faivre, L; Collod-Beroud, G; Callewaert, B; Child, A; Binquet, C; Gautier, E; Loeys, B L; Arbustini, E; Mayer, K; Arslan-Kirchner, M; Stheneur, C; Kiotsekoglou, A; Comeglio, P; Marziliano, N; Wolf, J E; Bouchot, O; Khau-Van-Kien, P; Beroud, C; Claustres, M; Bonithon-Kopp, C; Robinson, P N; Adès, L; De Backer, J; Coucke, P; Francke, U; De Paepe, A; Jondeau, G; Boileau, C

2009-01-01

Mutations in the FBN1 gene cause Marfan syndrome (MFS) and a wide range of overlapping phenotypes. The severe end of the spectrum is represented by neonatal MFS, the vast majority of probands carrying a mutation within exons 24–32. We previously showed that a mutation in exons 24–32 is predictive of a severe cardiovascular phenotype even in non-neonatal cases, and that mutations leading to premature truncation codons are under-represented in this region. To describe patients carrying a mutation in this so-called ‘neonatal' region, we studied the clinical and molecular characteristics of 198 probands with a mutation in exons 24–32 from a series of 1013 probands with a FBN1 mutation (20%). When comparing patients with mutations leading to a premature termination codon (PTC) within exons 24–32 to patients with an in-frame mutation within the same region, a significantly higher probability of developing ectopia lentis and mitral insufficiency were found in the second group. Patients with a PTC within exons 24–32 rarely displayed a neonatal or severe MFS presentation. We also found a higher probability of neonatal presentations associated with exon 25 mutations, as well as a higher probability of cardiovascular manifestations. A high phenotypic heterogeneity could be described for recurrent mutations, ranging from neonatal to classical MFS phenotype. In conclusion, even if the exons 24–32 location appears as a major cause of the severity of the phenotype in patients with a mutation in this region, other factors such as the type of mutation or modifier genes might also be relevant. PMID:19002209

The complete mitochondrial genome of Pomacea canaliculata (Gastropoda: Ampullariidae).

PubMed

Zhou, Xuming; Chen, Yu; Zhu, Shanliang; Xu, Haigen; Liu, Yan; Chen, Lian

2016-01-01

The mitochondrial genome of Pomacea canaliculata (Gastropoda: Ampullariidae) is the first complete mtDNA sequence reported in the genus Pomacea. The total length of mtDNA is 15,707 bp, which containing 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a 359 bp non-coding region. The A + T content of the overall base composition of H-strand is 71.7% (T: 41%, C: 12.7%, A: 30.7%, G: 15.6%). ATP6, ATP8, CO1, CO2, ND1-3, ND5, ND6, ND4L and Cyt b genes begin with ATG as start codon, CO3 and ND4 begin with ATA. ATP8, CO2-3, ND4L, ND2-6 and Cyt b genes are terminated with TAA as stop codon, ATP6, ND1, and CO1 end with TAG. A long non-coding region is found and a 23 bp repeat unit repeat 11 times in this region.

Characterization of six mutations in Exon 37 of neurofibromatosis type 1 gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Upadhyaya, M.; Osborn, M.; Maynard, J.

Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders, with an incidence of 1 in 3,000. We screened a total of 320 unrelated NF1 patients for mutations in exon 37 of the NF1 gene. Six independent mutations were identified, of which three are novel, and these include a recurrent nonsense mutation identified in 2 unrelated patients at codon 2281 (G2281X), a 1-bp insertion (6791 ins A) resulting in a change of TAG (tyrosine) to a TAA (stop codon), and a 3-bp deletion (6839 del TAC) which generated a frameshift. Another recurrent nonsense mutation, Y2264X, which was detectedmore » in 2 unrelated patients in this study, was also previously reported in 2 NF1 individuals. All the mutations were identified within a contiguous 49-bp sequence. Further studies are warranted to support the notion that this region of the gene contains highly mutable sequences. 17 refs., 2 figs., 1 tab.« less

The complete mitochondrial genome of the Aluterus monoceros.

PubMed

Li, Wenshen; Zhang, Guoqing; Wen, Xin; Wang, Qian; Chen, Guohua

2016-07-01

The complete mitochondrial genome of Aluterus monoceros (A. monoceros) has been sequenced. The mitochondrial genome of A. monoceros is 16,429 bp in length, consisting of 22 tRNA genes, 2 rRNA genes, 13 protein-coding genes and a D-loop region (Gen Bank accession number KP637022). The base A + T of the mitochondrial genome is 63.25%, including 33.16% of A, 30.09% of T and 20.74% of C. Twelve protein-coding genes start with a standard ATG as the initiation codon, expect for the COXI, which begins with GTG. Some of the termination codons are incomplete T or TA, except for the ND1, COXI, ATP8, ND4L1, ND5 and ND6, which stop with TAA. Construction of phylogenetic trees based on the entire mitochondrial genome sequence of 14 Tetrodontiformes species constructed has suggested that A. monoceros has closer relationship with Acreichthys tomentosus and Monacanthus chinensis, and they constitute a sister group.

Complete mitochondrial genome of the Yellow-spotted skate Okamejei hollandi (Rajiformes: Rajidae).

PubMed

Li, Weidong; Chen, Xiao; Liu, Wenai; Sun, Renjie; Zhou, Haolang

2016-07-01

The complete mitochondrial genome of the Yellow-spotted skate Okamejei hollandi was determined in this study. It is 16,974 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one putative control region. The overall base composition is 30.5% A, 27.8% C, 14.0% G, and 27.8% T. There are 28 bp short intergenic spaces located in 12 gene junctions and 31 bp overlaps located in nine gene junctions in the whole mitogenome. Two start codons (ATG and GTG) and two stop codons (TAG and TAA/T) were used in the protein-coding genes. The lengths of 22 tRNA genes range from 68 (tRNA-Ser2) to 75 (tRNA-Leu1) bp. The origin of L-strand replication (OL) sequence (37 bp) was identified between the tRNA-Asn and tRNA-Cys genes. The control region is 1311 bp in length with high A + T and poor G content.

A homolog of the variola virus B22 membrane protein contributes to ectromelia virus pathogenicity in the mouse footpad model.

PubMed

Reynolds, Sara E; Earl, Patricia L; Minai, Mahnaz; Moore, Ian; Moss, Bernard

2017-01-15

Most poxviruses encode a homolog of a ~200,000-kDa membrane protein originally identified in variola virus. We investigated the importance of the ectromelia virus (ECTV) homolog C15 in a natural infection model. In cultured mouse cells, the replication of a mutant virus with stop codons near the N-terminus (ECTV-C15Stop) was indistinguishable from a control virus (ECTV-C15Rev). However, for a range of doses injected into the footpads of BALB/c mice there was less mortality with the mutant. Similar virus loads were present at the site of infection with mutant or control virus whereas there was less ECTV-C15Stop in popliteal and inguinal lymph nodes, spleen and liver indicating decreased virus spread and replication. The latter results were supported by immunohistochemical analyses. Decreased spread was evidently due to immune modulatory activity of C15, rather than to an intrinsic viral function, as the survival of infected mice depended on CD4+ and CD8+ T cells. Published by Elsevier Inc.

mRNA translation and protein synthesis: an analysis of different modelling methodologies and a new PBN based approach

PubMed Central

2014-01-01

Background mRNA translation involves simultaneous movement of multiple ribosomes on the mRNA and is also subject to regulatory mechanisms at different stages. Translation can be described by various codon-based models, including ODE, TASEP, and Petri net models. Although such models have been extensively used, the overlap and differences between these models and the implications of the assumptions of each model has not been systematically elucidated. The selection of the most appropriate modelling framework, and the most appropriate way to develop coarse-grained/fine-grained models in different contexts is not clear. Results We systematically analyze and compare how different modelling methodologies can be used to describe translation. We define various statistically equivalent codon-based simulation algorithms and analyze the importance of the update rule in determining the steady state, an aspect often neglected. Then a novel probabilistic Boolean network (PBN) model is proposed for modelling translation, which enjoys an exact numerical solution. This solution matches those of numerical simulation from other methods and acts as a complementary tool to analytical approximations and simulations. The advantages and limitations of various codon-based models are compared, and illustrated by examples with real biological complexities such as slow codons, premature termination and feedback regulation. Our studies reveal that while different models gives broadly similiar trends in many cases, important differences also arise and can be clearly seen, in the dependence of the translation rate on different parameters. Furthermore, the update rule affects the steady state solution. Conclusions The codon-based models are based on different levels of abstraction. Our analysis suggests that a multiple model approach to understanding translation allows one to ascertain which aspects of the conclusions are robust with respect to the choice of modelling methodology, and when (and why) important differences may arise. This approach also allows for an optimal use of analysis tools, which is especially important when additional complexities or regulatory mechanisms are included. This approach can provide a robust platform for dissecting translation, and results in an improved predictive framework for applications in systems and synthetic biology. PMID:24576337

Outcome of Isolated Premature Menarche: A Retrospective and Follow-Up Study.

PubMed

Ejaz, Sehar; Lane, Andrew; Wilson, Thomas

2015-01-01

Isolated premature menarche is isolated or recurrent vaginal bleeding in a female in the absence of appropriate secondary sexual characters. Retrospective chart review of patients with premature menarche, followed by a telephone questionnaire. Charts of patients evaluated in the Pediatric Endocrine Clinic for premature vaginal bleeding from 1982-2013 were reviewed. Of 21 patients identified, 17 could be contacted. Five newly diagnosed patients were recruited during the course of the study. The median age at initial presentation was 7 years. Most patients presented with Tanner I sexual development. Some had more advanced breast staging, which regressed later. All had prepubertal baseline and stimulated gonadotropins. Most of the patients reported a single or few episodes of menses. Two reported continuation of irregular bleeding into adulthood. All reported an adult height within the midparental target height. Premature menarche in the absence of other appropriate secondary sexual characteristics is a benign entity. Most patients have a few isolated episodes of menses that stop spontaneously; some may continue to have periods into adulthood. Unlike true puberty, these patients do not demonstrate advanced skeletal maturation, and the adult height is normal. Fertility appears to be normal. © 2015 S. Karger AG, Basel.

Two alternative ways of start site selection in human norovirus reinitiation of translation.

PubMed

Luttermann, Christine; Meyers, Gregor

2014-04-25

The calicivirus minor capsid protein VP2 is expressed via termination/reinitiation. This process depends on an upstream sequence element denoted termination upstream ribosomal binding site (TURBS). We have shown for feline calicivirus and rabbit hemorrhagic disease virus that the TURBS contains three sequence motifs essential for reinitiation. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18 S rRNA leading to the model that hybridization between motif 1 and 18 S rRNA tethers the post-termination ribosome to the mRNA. Motif 2 and motif 2* are proposed to establish a secondary structure positioning the ribosome relative to the start site of the terminal ORF. Here, we analyzed human norovirus (huNV) sequences for the presence and importance of these motifs. The three motifs were identified by sequence analyses in the region upstream of the VP2 start site, and we showed that these motifs are essential for reinitiation of huNV VP2 translation. More detailed analyses revealed that the site of reinitiation is not fixed to a single codon and does not need to be an AUG, even though this codon is clearly preferred. Interestingly, we were able to show that reinitiation can occur at AUG codons downstream of the canonical start/stop site in huNV and feline calicivirus but not in rabbit hemorrhagic disease virus. Although reinitiation at the original start site is independent of the Kozak context, downstream initiation exhibits requirements for start site sequence context known for linear scanning. These analyses on start codon recognition give a more detailed insight into this fascinating mechanism of gene expression.

Purification and DNA binding properties of the blaI gene product, repressor for the beta-lactamase gene, blaP, of Bacillus licheniformis.

PubMed Central

Grossman, M J; Lampen, J O

1987-01-01

The location of the repressor gene, blaI, for the beta-lactamase gene blaP of Bacillus licheniformis 749, on the 5' side of blaP, was confirmed by sequencing the bla region of the constitutive mutant 749/C. An amber stop codon, likely to result in a nonfunctional truncated repressor, was found at codon 32 of the 128 codon blaI open reading frame (ORF) located 5' to blaP. In order to study the DNA binding activity of the repressor, the structural gene for blaI, from strain 749, with its ribosome binding site was expressed using a two plasmid T7 RNA polymerase/promotor system (S. Tabor and C. C. Richardson. Proc. Natl. Acad. Sci. 82, 1074-1078 (1985). Heat induction of this system in Escherichia coli K38 resulted in the production of BlaI as 5-10% of the soluble cell protein. Repressor protein was then purified by ammonium sulfate fractionation and cation exchange chromatography. The sequence of the N-terminal 28 amino acid residues was determined and was as predicted from the DNA. Binding of BlaI to DNA was detected by the slower migration of protein DNA complexes during polyacrylamide gel electrophoresis. BlaI was shown to selectively bind DNA fragments carrying the promoter regions of blaI and blaP. Images PMID:3498148

Novel SIL1 nonstop mutation in a Chinese consanguineous family with Marinesco-Sjögren syndrome and Dandy-Walker syndrome.

PubMed

Gai, Nan; Jiang, Chen; Zou, Yong-Yi; Zheng, Yu; Liang, De-Sheng; Wu, Ling-Qian

2016-07-01

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessive disorder, which is characterized by congenital cataracts, cerebellar ataxia, progressive muscle weakness, and delayed psychomotor development. SIL1, which is located at 5q31.2, is the only gene known to cause MSS. Dandy-Walker syndrome (DWS) is defined by hypoplasia, upward rotation of the cerebellar vermis, and cystic dilation of the fourth ventricle; however, its genetic pathogeny remains unclear. Here, we report a Chinese consanguineous family with MSS and DWS. Whole exome sequencing identified a novel nonstop mutation in SIL1. Sanger sequencing revealed that the mutation was segregated in this family according to a recessive mode of inheritance. We found that the mutation changed a stop codon (TGA) to an arginine codon (CGA), and no in-frame termination codon in the 3' untranslated region (UTR) of SIL1 could be found. The mRNA levels of SIL1 were decreased by 56.6% and 37.5% in immortalized lymphoblasts of the patients respectively; the protein levels of SIL1 were substantially decreased. This case study is the first report on Chinese MSS patients, MSS complicated by DWS, and a nonstop mutation in SIL1. Our findings imply the pathogenetic association between DWS and MSS. Copyright © 2016 Elsevier B.V. All rights reserved.

Ocular phenotypes associated with two mutations (R121W, C126X) in the Norrie disease gene.

PubMed

Kellner, U; Fuchs, S; Bornfeld, N; Foerster, M H; Gal, A

1996-06-01

To describe the ocular phenotypes associated with 2 mutations in the Norrie disease gene including a manifesting carrier. Ophthalmological examinations were performed in 2 affected males and one manifesting carrier. Genomic DNA was analyzed by direct sequencing of the Norrie disease gene. Family I: A 29-year-old male had the right eye enucleated at the age of 3 years. His left eye showed severe temporal dragging of the retina and central scars. Visual acuity was 20/300. DNA analysis revealed a C-to-T transition of the first nucleotide in codon 121 predicting the replacement of arginine-121 by tryptophan (R121W). Both the mother and maternal grandmother carry the same mutation in heterozygous form. Family 2: A 3-month-old boy presented with severe temporal dragging of the retina on both eyes and subsequently developed retinal detachment. Visual acuity was limited to light perception. His mother's left eye was amaurotic and phthitic. Her right eye showed severe retinal dragging, visual acuity was reduced to 20/60. DNA analysis revealed a T-to-A transversion of the third nucleotide in codon 126 creating a stop codon (C126X). The mother and maternal grandmother were carriers. Mutations in the Norrie disease gene can lead to retinal malformations of variable severity both in hemizygous males and manifesting carriers.

BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity.

PubMed

Jiang, Wen; Feng, Songjie; Huang, Shisheng; Yu, Wenxia; Li, Guanglei; Yang, Guang; Liu, Yajing; Zhang, Yu; Zhang, Lei; Hou, Yu; Chen, Jia; Chen, Jieping; Huang, Xingxu

2018-06-06

Base editor (BE), containing a cytidine deaminase and catalytically defective Cas9, has been widely used to perform base editing. However, the narrow editing window of BE limits its utility. Here, we developed a new editing technology named as base editor for programming larger C to U (T) scope (BE-PLUS) by fusing 10 copies of GCN4 peptide to nCas9(D10A) for recruiting scFv-APOBEC-UGI-GB1 to the target sites. The new system achieves base editing with a broadened window, resulting in an increased genome-targeting scope. Interestingly, the new system yielded much fewer unwanted indels and non-C-to-T conversions. We also demonstrated its potential use in gene disruption across the whole genome through induction of stop codons (iSTOP). Taken together, the BE-PLUS system offers a new editing tool with increased editing window and enhanced fidelity.

Novel APC gene mutations associated with protein alteration in diffuse type gastric cancer.

PubMed

Ghatak, Souvik; Chakraborty, Payel; Sarkar, Sandeep Roy; Chowdhury, Biswajit; Bhaumik, Arup; Kumar, Nachimuthu Senthil

2017-06-02

The role of adenomatous polyposis coli (APC) gene in mitosis might be critical for regulation of genomic stability and chromosome segregation. APC gene mutations have been associated to have a role in colon cancer and since gastric and colon tumors share some common genetic lesions, it is relevant to investigate the role of APC tumor suppressor gene in gastric cancer. We investigated for somatic mutations in the Exons 14 and 15 of APC gene from 40 diffuse type gastric cancersamples. Rabbit polyclonal anti-APC antibody was used, which detects the wild-type APC protein and was recommended for detection of the respective protein in human tissues. Cell cycle analysis was done from tumor and adjacent normal tissue. APC immunoreactivity showed positive expression of the protein in stages I, II, III and negative expression in Stages III and IV. Two novel deleterious variations (g.127576C > A, g.127583C > T) in exon 14 sequence were found to generate stop codon (Y622* and Q625*)in the tumor samples. Due to the generation of stop codon, the APC protein might be truncated and all the regulatory features could be lost which has led to the down-regulation of protein expression. Our results indicate that aneuploidy might occurdue to the codon 622 and 625 APC-driven gastric tumorigenesis, in agreement with our cell cycle analysis. The APC gene function in mitosis and chromosomal stability might be lost and G1 might be arrested with high quantity of DNA in the S phase. Six missense somatic mutations in tumor samples were detected in exon 15 A-B, twoof which showed pathological and disease causing effects based on SIFT, Polyphen2 and SNPs & GO score and were not previously reported in the literature or the public mutation databases. The two novel pathological somatic mutations (g.127576C > A, g.127583C > T) in exon 14 might be altering the protein expression leading to development of gastric cancer in the study population. Our study showed that mutations in the APC gene alter the protein expression and cell cycle regulation in diffuse type gastric adenocarcinoma.

'They have to show that they can make it': vitality as a criterion for the prognosis of premature infants.

PubMed

Brinchmann, B S

2000-03-01

In this article, the vitality of premature infants will be described and discussed. Vitality was one of the main factors in a grounded theory study in which the aim was to generate knowledge concerning the ethical decision-making processes with which nurses and physicians are faced in a neonatal unit. Which assessments underlie decisions about whether to start, continue or stop medical treatment of very sick premature babies? A descriptive study design, including 120 hours of field observations and 22 qualitative in-depth interviews with doctors and nurses, was chosen. Strauss and Glaser's comparative method was used to analyse the field observations and interviews. The findings indicate that life-and-death decisions are somewhat ambivalent; experience does not always make them easier. In situations of ambiguity, decisions also seem to be based upon the vitality of the babies concerned.

Definition of the mutation responsible for maple syrup urine disease in Poll Shorthorns and genotyping Poll Shorthorns and Poll Herefords for maple syrup urine disease alleles.

PubMed

Dennis, J A; Healy, P J

1999-08-01

The organisation of the E1alpha subunit of bovine branched-chain alpha-keto acid dehydrogenase gene was established. c DNA was cloned from Poll Shorthorn x Poll Hereford calves affected with Maple Syrup Urine Disease to identify the mutation responsible for the disease in Poll Shorthorns. Clones containing the c DNA sequences inherited from the Poll Shorthorn sire of the affected calves were identified. Paternal clones were sequenced and a cytidine to thymidine transition was found at nucleotide 1380. The mutation is predicted to substitute leucine in place of a highly conserved proline at codon 372. A polymerase chain reaction procedure was developed for detection of the 1380C-->T mutation in genomic DNA. Three Poll Shorthorn parents of affected calves and three affected Poll Shorthorn x Poll Hereford calves were heterozygous and an affected Poll Shorthorn calf was homozygous for this mutation. An improved polymerase chain reaction procedure was also devised to genotype Poll Herefords for the 248C-->T mutation. The procedures will facilitate disease prevention programs and assist in differential diagnosis of conditions in new-born calves that present with a rapid onset of progressive neurological disease and are characterised histologically by 'status spongiosus'. Maple Syrup Urine Disease (MSUD) is an autosomal recessive defect reported in humans (Danner and Elsas 1989), and in Poll Hereford (PH) and Poll Shorthorn (PS) calves (Harper et al 1986, Healy et al 1992). The clinical, biochemical and pathological manifestations of the disease are identical in the two breeds of cattle, and are characterised by the rapid onset of progressive neurological disease, leading to death within a few days of birth. The disease is caused by a deficiency of activity of the mitochondrial enzyme branched-chain alpha-keto acid dehydrogenase (BCKADH). This deficiency leads to elevated concentrations, in blood and tissues, of branched chain alpha-keto acids and their precursors, the branched chain amino acids, valine, leucine and isoleucine. BCKADH consists of four subunits E1alpha, E1beta, E2 and E3 that are encoded by separate genes, and MSUD may result from deficiency of any of the subunits. In PH s, the disease in caused by premature termination of translation, of the E1alpha subunit, that is induced by a cytidine to thymidine transition exon 2 (248C-->T), that converts the glutamine codon -6 to a stop codon (Q-6ST; Zhang et al 1990). We have shown that MSUD -affected PSxPH calves are heterozygous at the PH locus, illustrating molecular heterogeneity exists for bovine MSUD (Healy and Dennis 1994a). The fact that these crossbred calves are affected, indicates the PS, like the PH mutation, resides in the E1alpha subunit. Copyright 1999 Harcourt Publishers Ltd.

Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry.

PubMed

Taylor, Ethan Will; Ruzicka, Jan A; Premadasa, Lakmini; Zhao, Lijun

2016-01-01

Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3' end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation.

Maple Syrup Urine Disease in Cypriot Families: Identification of Three Novel Mutations and Biochemical Characterization of the p.Thr211Met Mutation in the E1α Subunit

PubMed Central

Georgiou, Theodoros; Chuang, Jacinta L.; Wynn, R. Max; Stylianidou, Goula; Korson, Mark; Chuang, David T.

2009-01-01

We report five mutations, three of them novel, responsible for maple syrup urine disease in four unrelated Cypriot families. The five children studied are the first cases of classic maple syrup urine disease to be reported among Cypriots. The first novel mutation identified is a single-base deletion in exon 6 of the Elα gene (c.718delG), which leads to a frameshift after Ala240 and to a stop codon 89 residues further downstream. The other two novel mutations identified are in the Elβ subunit: a two-base deletion in exon 6, c.662_663delCC, which leads to a frameshift after Ala221 and creates a stop codon 17 residues further downstream, as well as a splice mutation, IVS3[+3]delA, which results in the skipping of exon 3. The two known mutations identified are in the Elα gene: the G > C transversion at the 3′-splice acceptor site, (IVS5-1G > C), which results in the deletion of the entire exon 6, and the missense mutation in exon 5 (c.632C > T), which corresponds to a p.Thr211Met substitution. The p.Thr211Met substitution is located in a potassium-ion pocket in the E1 component required for stability of the bound cofactor thiamine diphosphate. The mutant E1 protein harboring the p.Thr211Met substitution was shown unable to bind thiamine diphosphate, leading to undetectable E1 activity. PMID:19715473

SLC3A1 and SLC7A9 mutations in autosomal recessive or dominant canine cystinuria: a new classification system.

PubMed

Brons, A-K; Henthorn, P S; Raj, K; Fitzgerald, C A; Liu, J; Sewell, A C; Giger, U

2013-01-01

Cystinuria, one of the first recognized inborn errors of metabolism, has been reported in many dog breeds. To determine urinary cystine concentrations, inheritance, and mutations in the SLC3A1 and SLC7A9 genes associated with cystinuria in 3 breeds. Mixed and purebred Labrador Retrievers (n = 6), Australian Cattle Dogs (6), Miniature Pinschers (4), and 1 mixed breed dog with cystine urolithiasis, relatives and control dogs. Urinary cystinuria and aminoaciduria was assessed and exons of the SLC3A1 and SLC7A9 genes were sequenced from genomic DNA. In each breed, male and female dogs, independent of neuter status, were found to form calculi. A frameshift mutation in SLC3A1 (c.350delG) resulting in a premature stop codon was identified in autosomal-recessive (AR) cystinuria in Labrador Retrievers and mixed breed dogs. A 6 bp deletion (c.1095_1100del) removing 2 threonines in SLC3A1 was found in autosomal-dominant (AD) cystinuria with a more severe phenotype in homozygous than in heterozygous Australian Cattle Dogs. A missense mutation in SLC7A9 (c.964G>A) was discovered in AD cystinuria in Miniature Pinschers with only heterozygous affected dogs observed to date. Breed-specific DNA tests were developed, but the prevalence of each mutation remains unknown. These studies describe the first AD inheritance and the first putative SLC7A9 mutation to cause cystinuria in dogs and expand our understanding of this phenotypically and genetically heterogeneous disease, leading to a new classification system for canine cystinuria and better therapeutic management and genetic control in these breeds. Copyright © 2013 by the American College of Veterinary Internal Medicine.

Massive losses of taste receptor genes in toothed and baleen whales.

PubMed

Feng, Ping; Zheng, Jinsong; Rossiter, Stephen J; Wang, Ding; Zhao, Huabin

2014-05-06

Taste receptor genes are functionally important in animals, with a surprising exception in the bottlenose dolphin, which shows extensive losses of sweet, umami, and bitter taste receptor genes. To examine the generality of taste gene loss, we examined seven toothed whales and five baleen whales and sequenced the complete repertoire of three sweet/umami (T1Rs) and ten bitter (T2Rs) taste receptor genes. We found all amplified T1Rs and T2Rs to be pseudogenes in all 12 whales, with a shared premature stop codon in 10 of the 13 genes, which demonstrated massive losses of taste receptor genes in the common ancestor of whales. Furthermore, we analyzed three genome sequences from two toothed whales and one baleen whale and found that the sour taste marker gene Pkd2l1 is a pseudogene, whereas the candidate salty taste receptor genes are intact and putatively functional. Additionally, we examined three genes that are responsible for taste signal transduction and found the relaxation of functional constraints on taste signaling pathways along the ancestral branch leading to whales. Together, our results strongly suggest extensive losses of sweet, umami, bitter, and sour tastes in whales, and the relaxation of taste function most likely arose in the common ancestor of whales between 36 and 53 Ma. Therefore, whales represent the first animal group to lack four of five primary tastes, probably driven by the marine environment with high concentration of sodium, the feeding behavior of swallowing prey whole, and the dietary switch from plants to meat in the whale ancestor. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Automated sequence analysis and editing software for HIV drug resistance testing.

PubMed

Struck, Daniel; Wallis, Carole L; Denisov, Gennady; Lambert, Christine; Servais, Jean-Yves; Viana, Raquel V; Letsoalo, Esrom; Bronze, Michelle; Aitken, Sue C; Schuurman, Rob; Stevens, Wendy; Schmit, Jean Claude; Rinke de Wit, Tobias; Perez Bercoff, Danielle

2012-05-01

Access to antiretroviral treatment in resource-limited-settings is inevitably paralleled by the emergence of HIV drug resistance. Monitoring treatment efficacy and HIV drugs resistance testing are therefore of increasing importance in resource-limited settings. Yet low-cost technologies and procedures suited to the particular context and constraints of such settings are still lacking. The ART-A (Affordable Resistance Testing for Africa) consortium brought together public and private partners to address this issue. To develop an automated sequence analysis and editing software to support high throughput automated sequencing. The ART-A Software was designed to automatically process and edit ABI chromatograms or FASTA files from HIV-1 isolates. The ART-A Software performs the basecalling, assigns quality values, aligns query sequences against a set reference, infers a consensus sequence, identifies the HIV type and subtype, translates the nucleotide sequence to amino acids and reports insertions/deletions, premature stop codons, ambiguities and mixed calls. The results can be automatically exported to Excel to identify mutations. Automated analysis was compared to manual analysis using a panel of 1624 PR-RT sequences generated in 3 different laboratories. Discrepancies between manual and automated sequence analysis were 0.69% at the nucleotide level and 0.57% at the amino acid level (668,047 AA analyzed), and discordances at major resistance mutations were recorded in 62 cases (4.83% of differences, 0.04% of all AA) for PR and 171 (6.18% of differences, 0.03% of all AA) cases for RT. The ART-A Software is a time-sparing tool for pre-analyzing HIV and viral quasispecies sequences in high throughput laboratories and highlighting positions requiring attention. Copyright © 2012 Elsevier B.V. All rights reserved.

An ARHGEF10 Deletion Is Highly Associated with a Juvenile-Onset Inherited Polyneuropathy in Leonberger and Saint Bernard Dogs

PubMed Central

Minor, Katie M.; Shelton, G. Diane; Patterson, Edward E.; Bley, Tim; Oevermann, Anna; Bilzer, Thomas; Leeb, Tosso

2014-01-01

An inherited polyneuropathy (PN) observed in Leonberger dogs has clinical similarities to a genetically heterogeneous group of peripheral neuropathies termed Charcot-Marie-Tooth (CMT) disease in humans. The Leonberger disorder is a severe, juvenile-onset, chronic, progressive, and mixed PN, characterized by exercise intolerance, gait abnormalities and muscle atrophy of the pelvic limbs, as well as inspiratory stridor and dyspnea. We mapped a PN locus in Leonbergers to a 250 kb region on canine chromosome 16 (Praw = 1.16×10−10, Pgenome, corrected = 0.006) utilizing a high-density SNP array. Within this interval is the ARHGEF10 gene, a member of the rho family of GTPases known to be involved in neuronal growth and axonal migration, and implicated in human hypomyelination. ARHGEF10 sequencing identified a 10 bp deletion in affected dogs that removes four nucleotides from the 3′-end of exon 17 and six nucleotides from the 5′-end of intron 17 (c.1955_1958+6delCACGGTGAGC). This eliminates the 3′-splice junction of exon 17, creates an alternate splice site immediately downstream in which the processed mRNA contains a frame shift, and generates a premature stop codon predicted to truncate approximately 50% of the protein. Homozygosity for the deletion was highly associated with the severe juvenile-onset PN phenotype in both Leonberger and Saint Bernard dogs. The overall clinical picture of PN in these breeds, and the effects of sex and heterozygosity of the ARHGEF10 deletion, are less clear due to the likely presence of other forms of PN with variable ages of onset and severity of clinical signs. This is the first documented severe polyneuropathy associated with a mutation in ARHGEF10 in any species. PMID:25275565

Listeria monocytogenes Isolates Carrying Virulence-Attenuating Mutations in Internalin A Are Commonly Isolated from Ready-to-Eat Food Processing Plant and Retail Environments.

PubMed

VAN Stelten, A; Roberts, A R; Manuel, C S; Nightingale, K K

2016-10-01

Listeria monocytogenes is a human foodborne pathogen that may cause an invasive disease known as listeriosis in susceptible individuals. Internalin A (InlA; encoded by inlA) is a virulence factor that facilitates crossing of host cell barriers by L. monocytogenes . At least 19 single nucleotide polymorphisms (SNPs) in inlA that result in a premature stop codon (PMSC) have been described worldwide. SNPs leading to a PMSC in inlA have been shown to be causally associated with attenuated virulence. L. monocytogenes pathogens carrying virulence-attenuating (VA) mutations in inlA have been commonly isolated from ready-to-eat (RTE) foods but rarely have been associated with human disease. This study was conducted to determine the prevalence of VA SNPs in inlA among L. monocytogenes from environments associated with RTE food production and handling. More than 700 L. monocytogenes isolates from RTE food processing plant (n = 409) and retail (n = 319) environments were screened for the presence of VA SNPs in inlA. Overall, 26.4% of isolates from RTE food processing plant and 32.6% of isolates from retail environments carried a VA mutation in inlA. Food contact surfaces sampled at retail establishments were significantly (P < 0.0001) more likely to be contaminated by a L. monocytogenes isolate carrying a VA mutation in inlA (56% of 55 isolates) compared with nonfood contact surfaces (28% of 264 isolates). Overall, a significant proportion of L. monocytogenes isolated from RTE food production and handling environments have reduced virulence. These data will be useful in the revision of current and the development of future risk assessments that incorporate strain-specific virulence parameters.

A LINE-1 Insertion in DLX6 Is Responsible for Cleft Palate and Mandibular Abnormalities in a Canine Model of Pierre Robin Sequence

PubMed Central

Wolf, Zena T.; Leslie, Elizabeth J.; Arzi, Boaz; Jayashankar, Kartika; Karmi, Nili; Jia, Zhonglin; Rowland, Douglas J.; Young, Amy; Safra, Noa; Sliskovic, Saundra; Murray, Jeffrey C.; Wade, Claire M.; Bannasch, Danika L.

2014-01-01

Cleft palate (CP) is one of the most commonly occurring craniofacial birth defects in humans. In order to study cleft palate in a naturally occurring model system, we utilized the Nova Scotia Duck Tolling Retriever (NSDTR) dog breed. Micro-computed tomography analysis of CP NSDTR craniofacial structures revealed that these dogs exhibit defects similar to those observed in a recognizable subgroup of humans with CP: Pierre Robin Sequence (PRS). We refer to this phenotype in NSDTRs as CP1. Individuals with PRS have a triad of birth defects: shortened mandible, posteriorly placed tongue, and cleft palate. A genome-wide association study in 14 CP NSDTRs and 72 unaffected NSDTRs identified a significantly associated region on canine chromosome 14 (24.2 Mb–29.3 Mb; praw = 4.64×10−15). Sequencing of two regional candidate homeobox genes in NSDTRs, distal-less homeobox 5 (DLX5) and distal-less homeobox 6 (DLX6), identified a 2.1 kb LINE-1 insertion within DLX6 in CP1 NSDTRs. The LINE-1 insertion is predicted to insert a premature stop codon within the homeodomain of DLX6. This prompted the sequencing of DLX5 and DLX6 in a human cohort with CP, where a missense mutation within the highly conserved DLX5 homeobox of a patient with PRS was identified. This suggests the involvement of DLX5 in the development of PRS. These results demonstrate the power of the canine animal model as a genetically tractable approach to understanding naturally occurring craniofacial birth defects in humans. PMID:24699068

A single-nucleotide polymorphism that accounts for allelic variation in the Lr34 gene and leaf rust reaction in hard winter wheat.

PubMed

Cao, Shuanghe; Carver, Brett F; Zhu, Xinkai; Fang, Tilin; Chen, Yihua; Hunger, Robert M; Yan, Liuling

2010-07-01

Leaf rust, caused by Puccinia triticina Eriks, is one of the most common and persistent wheat diseases in the US Great Plains. We report that the Lr34 gene was mapped in the center of a QTL for leaf rust reaction and explained 18-35% of the total phenotypic variation in disease severity of adult plants in a Jagger x 2174 population of recombinant inbred lines (RILs) field-tested for 3 years. The sequence of the complete Lr34 gene was determined for the susceptible Jagger allele and for the resistant 2174 allele. The two alleles had exactly the same sequence as the resistant allele reported previously in Chinese Spring at three polymorphic sites in intron 4, exon 11, and exon 12. A G/T polymorphism was found in exon 22, where a premature stop codon was found in the susceptible Jagger allele (Lr34E22s), confirming a previous report, due to a point mutation compared with the resistant 2174 allele (Lr34E22r). We have experimentally demonstrated a tight association between the point mutation at exon 22 of Lr34 and leaf rust susceptibility in a segregating biparental population. A PCR marker was developed to distinguish between the Lr34E22r and Lr34E22s alleles. A survey of 33 local hard winter wheat cultivars indicated that 7 cultivars carry the Lr34E22s allele and 26 cultivars carry the Lr34E22r allele. This study significantly improves our genetic understanding of allelic variation in the Lr34 gene and provides a functional molecular tool to improve leaf rust resistance in a major US wheat gene pool.

Significance of the evolutionary α1,3-galactosyltransferase (GGTA1) gene inactivation in preventing extinction of apes and old world monkeys.

PubMed

Galili, Uri

2015-01-01

The α1,3-galactosyltransferase (α1,3GT or GGTA1) gene displays unique evolutionary characteristics. This gene appeared early in mammalian evolution and is absent in other vertebrates. The α1,3GT gene is active in marsupials, nonprimate placental mammals, lemurs (prosimians) and New World monkeys, encoding the α1,3GT enzyme that synthesizes a carbohydrate antigen called "α-gal epitope." The α-gal epitope is present in large numbers on cell membrane glycolipids and glycoproteins. The α1,3GT gene was inactivated in ancestral Old World monkeys and apes by frameshift single-base deletions forming premature stop codons. Because of this gene inactivation, humans, apes, and Old World monkeys lack α-gal epitopes and naturally produce an antibody called the "anti-Gal antibody" which binds specifically to α-gal epitopes and which is the most abundant antibody in humans. The evolutionary event that resulted in the inactivation of the α1,3GT gene in ancestral Old World primates could have been mediated by a pathogen endemic to Eurasia-Africa landmass that exerted pressure for selection of primate populations lacking the α-gal epitope. Once the α-gal epitope was eliminated, primates could produce the anti-Gal antibody, possibly as means of defense against pathogens expressing this epitope. This assumption is supported by the fossil record demonstrating an almost complete extinction of apes in the late Miocene and failure of Old World monkeys to radiate into multiple species before that period. A present outcome of this evolutionary event is the anti-Gal-mediated rejection of mammalian xenografts expressing α-gal epitopes in humans, apes, and Old World monkeys.

Virulence factors and resistance to antimicrobials in Listeria monocytogenes serotype 1/2c isolated from food.

PubMed

Gelbíčová, T; Pantůček, R; Karpíšková, R

2016-08-01

The aim of this study was to assess the potential risk posed to the human population by the presence of Listeria monocytogenes serotype 1/2c in food based on the characterization of virulence factors of Listeria involved in the invasion of host cells and sensitivity to antimicrobial agents. In addition to sequencing of the inlA and inlB genes, the presence of genes lapB, aut, fbpA, ami, vip and llsX was tested. A premature stop codon (PMSC) in the inlA gene was detected in all tested strains of serotype 1/2c and, concurrently, two novel PMSC mutation types were identified. However, neither PMSC in the inlB gene nor deletion of the lapB, aut, fbpA, ami and vip genes were found in any of the strains. The presence of the llsX gene was not confirmed. Even though all L. monocytogenes strains showed sensitivity to the tested antimicrobials on the basis of their phenotype, sequencing revealed the presence of IS1542 insertion in the inlA gene, indicating the possibility of sharing of mobile genetic elements associated with antimicrobial resistance among strains. Other than the presence of PMSCs in the inlA gene, no PMSC in inlB or deletion of other factors linked to the invasiveness of listeria were detected. Tested strains showed sensitivity to antibiotics used in the therapy of listeriosis. Strains of L. monocytogenes serotype 1/2c typically carry a PMSC in the inlA gene, but these strains still represent a potential threat to public health. The possibility of transfer of IS1542, associated with resistance to vancomycin, between enterococci and Listeria spp. was revealed. © 2016 The Society for Applied Microbiology.

Molecular analysis of congenital goitres with hypothyroidism caused by defective thyroglobulin synthesis. Identification of a novel c.7006C>T [p.R2317X] mutation and expression of minigenes containing nonsense mutations in exon 7.

PubMed

Machiavelli, Gloria A; Caputo, Mariela; Rivolta, Carina M; Olcese, María C; Gruñeiro-Papendieck, Laura; Chiesa, Ana; González-Sarmiento, Rogelio; Targovnik, Héctor M

2010-01-01

Thyroglobulin (TG) deficiency is an autosomal-recessive disorder that results in thyroid dyshormonogenesis. A number of distinct mutations have been identified as causing human hypothyroid goitre. The purpose of this study was to identify and characterize new mutations in the TG gene in an attempt to increase the understanding of the genetic mechanism responsible for this disorder. A total of six patients from four nonconsanguineous families with marked impairment of TG synthesis were studied. Single-strand conformation polymorphism (SSCP) analysis, sequencing of DNA, genotyping, expression of chimeric minigenes and bioinformatic analysis were performed. Four different inactivating TG mutations were identified: one novel mutation (c.7006C>T [p.R2317X]) and three previously reported (c.886C>T [p.R277X], c.6701C>A [p.A2215D] and c.6725G>A [p.R2223H]). Consequently, one patient carried a compound heterozygous for p.R2223H/p.R2317X mutations; two brothers showed a homozygous p.A2215D substitution and the remaining three patients, from two families with typical phenotype, had a single p.R277X mutated allele. We also showed functional evidences that premature stop codons inserted at different positions in exon 7, which disrupt exonic splicing enhancer (ESE) sequences, do not interfere with exon definition and processing. In this study, we have identified a novel nonsense mutation p.R2317X in the acetylcholinesterase homology domain of TG. We have also observed that nonsense mutations do not interfere with the pre-mRNA splicing of exon 7. The results are in accordance with previous observations confirming the genetic heterogeneity of TG defects.

An Unusual Splice Defect in the Mitofusin 2 Gene (MFN2) Is Associated with Degenerative Axonopathy in Tyrolean Grey Cattle

PubMed Central

Drögemüller, Cord; Reichart, Ursula; Seuberlich, Torsten; Oevermann, Anna; Baumgartner, Martin; Kühni Boghenbor, Kathrin; Stoffel, Michael H.; Syring, Claudia; Meylan, Mireille; Müller, Simone; Müller, Mathias; Gredler, Birgit

2011-01-01

Tyrolean Grey cattle represent a local breed with a population size of ∼5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome) in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS) and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2), is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE) and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle. PMID:21526202

An unusual splice defect in the mitofusin 2 gene (MFN2) is associated with degenerative axonopathy in Tyrolean Grey cattle.

PubMed

Drögemüller, Cord; Reichart, Ursula; Seuberlich, Torsten; Oevermann, Anna; Baumgartner, Martin; Kühni Boghenbor, Kathrin; Stoffel, Michael H; Syring, Claudia; Meylan, Mireille; Müller, Simone; Müller, Mathias; Gredler, Birgit; Sölkner, Johann; Leeb, Tosso

2011-04-15

Tyrolean Grey cattle represent a local breed with a population size of ∼5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome) in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS) and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2), is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE) and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle.

Recurrent loss of sex is associated with accumulation of deleterious mutations in Oenothera.

PubMed

Hollister, Jesse D; Greiner, Stephan; Wang, Wei; Wang, Jun; Zhang, Yong; Wong, Gane Ka-Shu; Wright, Stephen I; Johnson, Marc T J

2015-04-01

Sexual reproduction is nearly universal among eukaryotes. Theory predicts that the rarity of asexual eukaryotic species is in part caused by accumulation of deleterious mutations and heightened extinction risk associated with suppressed recombination and segregation in asexual species. We tested this prediction with a large data set of 62 transcriptomes from 29 species in the plant genus Oenothera, spanning ten independent transitions between sexual and a functionally asexual genetic system called permanent translocation heterozygosity. Illumina short-read sequencing and de novo transcript assembly yielded an average of 16.4 Mb of sequence per individual. Here, we show that functionally asexual species accumulate more deleterious mutations than sexual species using both population genomic and phylogenetic analysis. At an individual level, asexual species exhibited 1.8 × higher heterozygosity than sexual species. Within species, we detected a higher proportion of nonsynonymous polymorphism relative to synonymous variation within asexual compared with sexual species, indicating reduced efficacy of purifying selection. Asexual species also exhibited a greater proportion of transcripts with premature stop codons. The increased proportion of nonsynonymous mutations was also positively correlated with divergence time between sexual and asexual species, consistent with Muller's ratchet. Between species, we detected repeated increases in the ratio of nonsynonymous to synonymous divergence in asexual species compared with sexually reproducing sister taxa, indicating increased accumulation of deleterious mutations. These results confirm that an important advantage of sex is that it facilitates selection against deleterious alleles, which might help to explain the dearth of extant asexual species. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Truncation of LEAFY COTYLEDON1 Protein Is Required for Asexual Reproduction in Kalanchoë daigremontiana1[OPEN

PubMed Central

Garcês, Helena M.P.; Koenig, Daniel; Townsley, Brad T.; Kim, Minsung; Sinha, Neelima R.

2014-01-01

Kalanchoë daigremontiana reproduces asexually by generating numerous plantlets on its leaf margins. The formation of plantlets requires the somatic initiation of organogenic and embryogenic developmental programs in the leaves. However, unlike normal embryogenesis in seeds, leaf somatic embryogenesis bypasses seed dormancy to form viable plantlets. In Arabidopsis (Arabidopsis thaliana), seed dormancy and embryogenesis are initiated by the transcription factor LEAFY COTYLEDON1 (LEC1). The K. daigremontiana ortholog of LEC1 is expressed during leaf somatic embryo development. However, KdLEC1 encodes for a LEC1-type protein that has a unique B domain, with 11 unique amino acids and a premature stop codon. Moreover, the truncated KdLEC1 protein is not functional in Arabidopsis. Here, we show that K. daigremontiana transgenic plants expressing a functional, chimeric KdLEC1 gene under the control of Arabidopsis LEC1 promoter caused several developmental defects to leaf somatic embryos, including seed dormancy characteristics. The dormant plantlets also behaved as typical dormant seeds. Transgenic plantlets accumulated oil bodies and responded to the abscisic acid biosynthesis inhibitor fluridone, which broke somatic-embryo dormancy and promoted their normal development. Our results indicate that having a mutated form of LEC1 gene in K. daigremontiana is essential to bypass dormancy in the leaf embryos and generate viable plantlets, suggesting that the loss of a functional LEC1 promotes viviparous leaf somatic embryos and thus enhances vegetative propagation in K. daigremontiana. Mutations resulting in truncated LEC1 proteins may have been of a selective advantage in creating somatic propagules, because such mutations occurred independently in several Kalanchoë species, which form plantlets constitutively. PMID:24664206

Truncation of LEAFY COTYLEDON1 protein is required for asexual reproduction in Kalanchoë daigremontiana.

PubMed

Garcês, Helena M P; Koenig, Daniel; Townsley, Brad T; Kim, Minsung; Sinha, Neelima R

2014-05-01

Kalanchoë daigremontiana reproduces asexually by generating numerous plantlets on its leaf margins. The formation of plantlets requires the somatic initiation of organogenic and embryogenic developmental programs in the leaves. However, unlike normal embryogenesis in seeds, leaf somatic embryogenesis bypasses seed dormancy to form viable plantlets. In Arabidopsis (Arabidopsis thaliana), seed dormancy and embryogenesis are initiated by the transcription factor LEAFY COTYLEDON1 (LEC1). The K. daigremontiana ortholog of LEC1 is expressed during leaf somatic embryo development. However, KdLEC1 encodes for a LEC1-type protein that has a unique B domain, with 11 unique amino acids and a premature stop codon. Moreover, the truncated KdLEC1 protein is not functional in Arabidopsis. Here, we show that K. daigremontiana transgenic plants expressing a functional, chimeric KdLEC1 gene under the control of Arabidopsis LEC1 promoter caused several developmental defects to leaf somatic embryos, including seed dormancy characteristics. The dormant plantlets also behaved as typical dormant seeds. Transgenic plantlets accumulated oil bodies and responded to the abscisic acid biosynthesis inhibitor fluridone, which broke somatic-embryo dormancy and promoted their normal development. Our results indicate that having a mutated form of LEC1 gene in K. daigremontiana is essential to bypass dormancy in the leaf embryos and generate viable plantlets, suggesting that the loss of a functional LEC1 promotes viviparous leaf somatic embryos and thus enhances vegetative propagation in K. daigremontiana. Mutations resulting in truncated LEC1 proteins may have been of a selective advantage in creating somatic propagules, because such mutations occurred independently in several Kalanchoë species, which form plantlets constitutively.

A novel CYP27B1 mutation causes a feline vitamin D-dependent rickets type IA.

PubMed

Grahn, Robert A; Ellis, Melanie R; Grahn, Jennifer C; Lyons, Leslie A

2012-08-01

A 12-week-old domestic cat presented at a local veterinary clinic with hypocalcemia and skeletal abnormalities suggestive of rickets. Osteomalacia (rickets) is a disease caused by impaired bone mineralization leading to an increased prevalence of fractures and deformity. Described in a variety of species, rickets is most commonly caused by vitamin D or calcium deficiencies owing to both environmental and or genetic abnormalities. Vitamin D-dependent rickets type 1A (VDDR-1A) is a result of the enzymatic pathway defect caused by mutations in the 25-hydroxyvitamin D(3)-1-alpha-hydroxylase gene [cytochrome P27 B1 (CYP27B1)]. Calcitriol, the active form of vitamin D(3), regulates calcium homeostasis, which requires sufficient dietary calcium availability and correct hormonal function for proper bone growth and maintenance. Patient calcitriol concentrations were low while calcidiol levels were normal suggestive of VDDR-1A. The entire DNA coding sequencing of CYP27B1 was evaluated. The affected cat was wild type for previously identified VDDR-1A causative mutations. However, six novel mutations were identified, one of which was a nonsense mutation at G637T in exon 4. The exon 4 G637T nonsense mutation results in a premature protein truncation, changing a glutamic acid to a stop codon, E213X, likely causing the clinical presentation of rickets. The previously documented genetic mutation resulting in feline VDDR-1A rickets, as well as the case presented in this research, result from novel exon 4 CYP27B1 mutations, thus exon 4 should be the initial focus of future sequencing efforts.

No evidence for involvement of SDHD in neuroblastoma pathogenesis

PubMed Central

De Preter, Katleen; Vandesompele, Jo; Hoebeeck, Jasmien; Vandenbroecke, Caroline; Smet, Jöel; Nuyts, Annick; Laureys, Geneviève; Combaret, Valérie; Van Roy, Nadine; Roels, Frank; Van Coster, Rudy; Praet, Marleen; De Paepe, Anne; Speleman, Frank

2004-01-01

Background Deletions in the long arm of chromosome 11 are observed in a subgroup of advanced stage neuroblastomas with poor outcome. The deleted region harbours the tumour suppressor gene SDHD that is frequently mutated in paraganglioma and pheochromocytoma, which are, like neuroblastoma, tumours originating from the neural crest. In this study, we sought for evidence for involvement of SDHD in neuroblastoma. Methods SDHD was investigated on the genome, transcriptome and proteome level using mutation screening, methylation specific PCR, real-time quantitative PCR based homozygous deletion screening and mRNA expression profiling, immunoblotting, functional protein analysis and ultrastructural imaging of the mitochondria. Results Analysis at the genomic level of 67 tumour samples and 37 cell lines revealed at least 2 bona-fide mutations in cell lines without allelic loss at 11q23: a 4bp-deletion causing skip of exon 3 resulting in a premature stop codon in cell line N206, and a Y93C mutation in cell line NMB located in a region affected by germline SDHD mutations causing hereditary paraganglioma. No evidence for hypermethylation of the SDHD promotor region was observed, nor could we detect homozygous deletions. Interestingly, SDHD mRNA expression was significantly reduced in SDHD mutated cell lines and cell lines with 11q allelic loss as compared to both cell lines without 11q allelic loss and normal foetal neuroblast cells. However, protein analyses and assessment of mitochondrial morphology presently do not provide clues as to the possible effect of reduced SDHD expression on the neuroblastoma tumour phenotype. Conclusions Our study provides no indications for 2-hit involvement of SDHD in the pathogenesis of neuroblastoma. Also, although a haplo-insufficient mechanism for SDHD involvement in advanced stage neuroblastoma could be considered, the present data do not provide consistent evidence for this hypothesis. PMID:15331017

Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer

PubMed Central

Bortolussi, Giulia; Zentilin, Lorena; Baj, Gabriele; Giraudi, Pablo; Bellarosa, Cristina; Giacca, Mauro; Tiribelli, Claudio; Muro, Andrés F.

2012-01-01

Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.—Bortolussi, G., Zentilin, L., Baj, G., Giraudi, P., Bellarosa, C., Giacca, M., Tiribelli, C., Muro, A. F. Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer. PMID:22094718

Genetic Characterization of Simian Foamy Viruses Infecting Humans

PubMed Central

Rua, Réjane; Betsem, Edouard; Calattini, Sara; Saib, Ali

2012-01-01

Simian foamy viruses (SFVs) are retroviruses that are widespread among nonhuman primates (NHPs). SFVs actively replicate in their oral cavity and can be transmitted to humans after NHP bites, giving rise to a persistent infection even decades after primary infection. Very few data on the genetic structure of such SFVs found in humans are available. In the framework of ongoing studies searching for SFV-infected humans in south Cameroon rainforest villages, we studied 38 SFV-infected hunters whose times of infection had presumably been determined. By long-term cocultures of peripheral blood mononuclear cells with BHK-21 cells, we isolated five new SFV strains and obtained complete genomes of SFV strains from chimpanzee (Pan troglodytes troglodytes; strains BAD327 and AG15), monkey (Cercopithecus nictitans; strain AG16), and gorilla (Gorilla gorilla; strains BAK74 and BAD468). These zoonotic strains share a very high degree of similarity with their NHP counterparts and have a high degree of conservation of the genetic elements important for viral replication. Interestingly, analysis of FV DNA sequences obtained before cultivation revealed variants with deletions in both the U3 region and tas that may correlate with in vivo chronicity in humans. Genomic changes in bet (a premature stop codon) and gag were also observed. To determine if such changes were specific to zoonotic strains, we studied local SFV-infected chimpanzees and found the same genomic changes. Our study reveals that natural polymorphism of SFV strains does exist at both the intersubspecies level (gag, bet) and the intrasubspecies (U3, tas) levels but does not seem to reflect a viral adaptation specific to zoonotic SFV strains. PMID:23015714

Multiplex screening for RB1 germline mutations in 106 patients with hereditary retinoblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lohmann, D.R.; Brandt, B.; Passarge, E.

1994-09-01

The identification of germline mutations in the retinoblastoma susceptibility gene (RB1) is important for genetic counseling in hereditary retinoblastoma. Due to the complex genomic organization of this gene and the heterogeneity of mutations, efficient screening procedures are important for rapid mutation detection. We have developed methods based on simultaneous analysis of multiple regions of this gene in an ABI automated DNA fragment analyzer to examine 106 patients with hereditary retinoblastoma in which no alteration was identified by Southern blot hybridization. Primers for the amplification of all 27 exons of the RB1 gene as well as the promoter and poly(A) signalmore » sequences were labelled with distinct fluorescent dyes (FAM, HEX, TAMRA) to enable simultaneous electrophoretic analysis of PCR products with similar mobility. PCR fragments distinguishable by size or color were co-amplified by multiplex PCR and analyzed for length by GENESCAN analysis. Using this approach, small deletions ranging from 1 bp to 22 bp were identified in 24 patients (23%). Short sequence repeats or polypyrimidine runs were present in the vicinity of most of these deletions. In 4 patients (4%), insertions from 1 bp to 4 bp were found. The majority of length mutations resulted in a truncated gene product due to frameshift and premature termination. No mutation was identified in exons 25 to 27 possibly indicating that the encoded protein domains have minor functional importance. In order to screen for base substitutions that are not detectable by fragment length analysis, we adapted heteroduplex analysis for the use in the DNA fragment analyzer. During the optimization of this method we detected 10 single base substitutions most of which generated stop codons. Intriguingly, two identical missense mutations were identified in two unrelated families with a low-penetrance phenotype.« less

Ferrochelatase gene mutation in Singapore and a novel frame-shift mutation in an Asian boy with erythropoietic protoporphyria.

PubMed

Alagappan, Uma; Pramono, Zacharias A D; Chong, Wei-Sheng

2017-03-01

Erythropoietic protoporphyria (EPP) is a rare inherited disorder of heme biosynthesis caused by decreased activity of the enzyme ferrochelatase (FECH ). The frequency of the hypomorphic c.333-48C allele in a population directly contributes to the prevalence of EPP in the same population. This study sought to identify the molecular basis of EPP in a Chinese patient from Singapore and the c.333-48C allele frequency among the Chinese population in Singapore. FECH gene was screened for mutation in the patient's DNA sample by polymerase chain reaction amplification and DNA sequencing. To validate the identified mutation, the FECH region harboring the mutation was screened in DNA samples from all healthy controls. One patient and 46 ethnically matched healthy controls were included in the study. A novel c.474dupC which leads to a frameshift and premature stop codon was identified in one allele, while the other allele showed to carry c.333-48C and c.337C>T variants in the patient's FECH. The frequency of the c.333-48C hypomorphic allele is 27% among Chinese population in Singapore. c.474dupC in one allele trans to hypomorphic c.333-48C and c.337C>T allele in FECH gene may be the underlying cause of the clinical EPP of the studied patient. The FECH hypomorphic c.333-48C allele frequency in Singapore is lower than the Han Chinese (41.3%) and Japanese (43%) populations but nearly the same as the Southeast Asian (31%) population and higher than the European (2.7-11%) population. © 2016 The International Society of Dermatology.

PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features

PubMed Central

Low, Karen J; Ansari, Morad; Abou Jamra, Rami; Clarke, Angus; El Chehadeh, Salima; FitzPatrick, David R; Greenslade, Mark; Henderson, Alex; Hurst, Jane; Keller, Kory; Kuentz, Paul; Prescott, Trine; Roessler, Franziska; Selmer, Kaja K; Schneider, Michael C; Stewart, Fiona; Tatton-Brown, Katrina; Thevenon, Julien; Vigeland, Magnus D; Vogt, Julie; Willems, Marjolaine; Zonana, Jonathan; Study, D D D; Smithson, Sarah F

2017-01-01

PUF60 encodes a nucleic acid-binding protein, a component of multimeric complexes regulating RNA splicing and transcription. In 2013, patients with microdeletions of chromosome 8q24.3 including PUF60 were found to have developmental delay, microcephaly, craniofacial, renal and cardiac defects. Very similar phenotypes have been described in six patients with variants in PUF60, suggesting that it underlies the syndrome. We report 12 additional patients with PUF60 variants who were ascertained using exome sequencing: six through the Deciphering Developmental Disorders Study and six through similar projects. Detailed phenotypic analysis of all patients was undertaken. All 12 patients had de novo heterozygous PUF60 variants on exome analysis, each confirmed by Sanger sequencing: four frameshift variants resulting in premature stop codons, three missense variants that clustered within the RNA recognition motif of PUF60 and five essential splice-site (ESS) variant. Analysis of cDNA from a fibroblast cell line derived from one of the patients with an ESS variants revealed aberrant splicing. The consistent feature was developmental delay and most patients had short stature. The phenotypic variability was striking; however, we observed similarities including spinal segmentation anomalies, congenital heart disease, ocular colobomata, hand anomalies and (in two patients) unilateral renal agenesis/horseshoe kidney. Characteristic facial features included micrognathia, a thin upper lip and long philtrum, narrow almond-shaped palpebral fissures, synophrys, flared eyebrows and facial hypertrichosis. Heterozygote loss-of-function variants in PUF60 cause a phenotype comprising growth/developmental delay and craniofacial, cardiac, renal, ocular and spinal anomalies, adding to disorders of human development resulting from aberrant RNA processing/spliceosomal function. PMID:28327570

Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia

PubMed Central

Alaa el Din, Ferdos; Patri, Sylvie; Thoreau, Vincent; Rodriguez-Ballesteros, Montserrat; Hamade, Eva; Bailly, Sabine; Gilbert-Dussardier, Brigitte; Abou Merhi, Raghida; Kitzis, Alain

2015-01-01

Hereditary Hemorrhagic Telangiectasia syndrome (HHT) or Rendu-Osler-Weber (ROW) syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1or ALK1) genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1), the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val) affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7) was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations. PMID:26176610

Phenotypic Variation in Patients with Homozygous c.1678G>T Mutation in EVC Gene: Report of Two Mexican Families with Ellis-van Creveld Syndrome

PubMed Central

Ibarra-Ramirez, Marisol; Campos-Acevedo, Luis Daniel; Lugo-Trampe, Jose; Martínez-Garza, Laura E.; Martinez-Glez, Víctor; Valencia-Benitez, María; Lapunzina, Pablo; Ruiz-Peréz, Víctor

2017-01-01

Case series Patient: — Final Diagnosis: Ellis van Creveld syndrome Symptoms: Conical teeth • polydactyly • short stature Medication: — Clinical Procedure: — Specialty: Pediatrics and Neonatology Objective: Rare disease Background: Ellis-van Creveld syndrome is an autosomal recessive chondro-ectodermal dysplasia characterized by disproportionate short stature, limb shortening, narrow chest, postaxial polydactyly and dysplastic nails and teeth. In addition, 60% of cases present congenital heart defects. Ellis-van Creveld syndrome is predominantly caused by mutations in the EVC or EVC2 (4p16) genes, with only a few cases caused by mutations in WDR35. Case Report: Here, we report on two Mexican families with patients diagnosed with Ellis-van Creveld syndrome. Family 1 includes four patients: three females of 15, 18, and 23 years of age and a 7-year old male. Family 2 has only one affected newborn male. All patients exhibited multiple features including hypodontia, dysplastic teeth, extra frenula, mild short stature, distal limb shortening, postaxial polydactyly of hands and feet, nail dystrophy, and knee joint abnormalities. Only two patients had an atrial septal defect. In all cases, molecular analysis by Sanger sequencing identified the same homozygous mutation in exon 12 of EVC, c.1678G>T, which leads to a premature stop codon. Conclusions: The mutation c.1678G>T has been previously reported in another Mexican patient and it appears to be a recurrent mutation in Mexico which could represent a founder mutation. The large number of patients in this case allows the clinical variability and spectrum of manifestations present in individuals with Ellis-van Creveld syndrome even if they carry the same homozygous mutation in a same family. PMID:29229899

Two novel partial deletions of LDL-receptor gene in Italian patients with familial hypercholesterolemia (FH Siracusa and FH Reggio Emilia).

PubMed

Garuti, R; Lelli, N; Barozzini, M; Tiozzo, R; Ghisellini, M; Simone, M L; Li Volti, S; Garozzo, R; Mollica, F; Vergoni, W; Bertolini, S; Calandra, S

1996-03-01

In the present study we report two novel partial deletions of the LDL-R gene. The first (FH Siracusa), found in an FH-heterozygote, consists of a 20 kb deletion spanning from the 5' flanking region to the intron 2 of the LDL-receptor gene. The elimination of the promoter and the first two exons prevents the transcription of the deleted allele, as shown by Northern blot analysis of LDL-R mRNA isolated from the proband's fibroblasts. The second deletion (FH Reggio Emilia), which eliminates 11 nucleotides of exon 10, was also found in an FH heterozygote. The characterization of this deletion was made possible by a combination of techniques such as single strand conformation polymorphism (SSCP) analysis, direct sequence of exon 10 and cloning of the normal and deleted exon 10 from the proband's DNA. The 11 nt deletion occurs in a region of exon 10 which contains three triplets (CTG) and two four-nucleotides (CTGG) direct repeats. This structural feature might render this region more susceptible to a slipped mispairing during DNA duplication. Since this deletion causes a shift of the BamHI site at the 5' end of exon 10, a method has been devised for its rapid screening which is based on the PCR amplification of exon 10 followed by BamHI digestion. FH Reggio Emilia deletion produces a shift in the reading frame downstream from Lys458, leading to a sequence of 51 novel amino acids before the occurrence of a premature stop codon (truncated receptor). However, since RT-PCR failed to demonstrate the presence of the mutant LDL-R mRNA in proband fibroblasts, it is likely that the amount of truncated receptor produced in these cells is negligible.

Whole-Genome SNP Association in the Horse: Identification of a Deletion in Myosin Va Responsible for Lavender Foal Syndrome

PubMed Central

Brooks, Samantha A.; Gabreski, Nicole; Miller, Donald; Brisbin, Abra; Brown, Helen E.; Streeter, Cassandra; Mezey, Jason; Cook, Deborah; Antczak, Douglas F.

2010-01-01

Lavender Foal Syndrome (LFS) is a lethal inherited disease of horses with a suspected autosomal recessive mode of inheritance. LFS has been primarily diagnosed in a subgroup of the Arabian breed, the Egyptian Arabian horse. The condition is characterized by multiple neurological abnormalities and a dilute coat color. Candidate genes based on comparative phenotypes in mice and humans include the ras-associated protein RAB27a (RAB27A) and myosin Va (MYO5A). Here we report mapping of the locus responsible for LFS using a small set of 36 horses segregating for LFS. These horses were genotyped using a newly available single nucleotide polymorphism (SNP) chip containing 56,402 discriminatory elements. The whole genome scan identified an associated region containing these two functional candidate genes. Exon sequencing of the MYO5A gene from an affected foal revealed a single base deletion in exon 30 that changes the reading frame and introduces a premature stop codon. A PCR–based Restriction Fragment Length Polymorphism (PCR–RFLP) assay was designed and used to investigate the frequency of the mutant gene. All affected horses tested were homozygous for this mutation. Heterozygous carriers were detected in high frequency in families segregating for this trait, and the frequency of carriers in unrelated Egyptian Arabians was 10.3%. The mapping and discovery of the LFS mutation represents the first successful use of whole-genome SNP scanning in the horse for any trait. The RFLP assay can be used to assist breeders in avoiding carrier-to-carrier matings and thus in preventing the birth of affected foals. PMID:20419149

Molecular and phenotypic characterization of Listeria monocytogenes from U.S. Department of Agriculture Food Safety and Inspection Service surveillance of ready-to-eat foods and processing facilities.

PubMed

Ward, Todd J; Evans, Peter; Wiedmann, Martin; Usgaard, Thomas; Roof, Sherry E; Stroika, Steven G; Hise, Kelley

2010-05-01

A panel of 501 Listeria monocytogenes isolates obtained from the U.S. Department of Agriculture Food Safety and Inspection Service monitoring programs for ready-to-eat (RTE) foods were subtyped by multilocus genotyping (MLGT) and by sequencing the virulence gene inlA, which codes for internalin. MLGT analyses confirmed that clonal lineages associated with previous epidemic outbreaks were rare (7.6%) contaminants of RTE meat and poultry products and their production environments. Conversely, sequence analyses revealed mutations leading to 11 different premature stop codons (PMSCs) in inlA, including three novel PMSC mutations, and revealed that the frequency of these virulence-attenuating mutations among RTE isolates (48.5%) was substantially higher than previously appreciated. Significant differences (P < 0.001) in the frequency of inlA PMSCs were observed between lineages and between major serogroups, which could partially explain differences in association of these subtypes with human listeriosis. Interrogation of single-nucleotide polymorphisms responsible for PMSCs in inlA improved strain resolution among isolates with the 10 most common pulsed-field gel electrophoresis (PFGE) patterns, 8 of which included isolates with a PMSC in inlA. The presence or absence of PMSCs in inlA accounted for significant differences (P < 0.05) in Caco-2 invasion efficiencies among isolates with identical PFGE patterns, and the proportion of PulseNet entries from clinical sources was significantly higher (P < 0.001) for PFGE patterns exclusively from isolates with full-length inlA. These results indicated that integration of PFGE and DNA sequence-based subtyping provides an improved framework for prediction of relative risk associated with L. monocytogenes strains from RTE foods.

Unusual presentation of hepatitis B serological markers in an Amerindian community of Venezuela with a majority of occult cases

PubMed Central

2011-01-01

Background Occult hepatitis B infection (OBI) is characterized by the presence of hepatitis B virus (HBV) DNA in the absence of HBsAg in the serum of patients. The aim of this study was to characterize HBV infection among a Piaroa community, an Amerindian group which exhibits significant evidence of exposure to HBV but relatively low presence of HBsAg, and to explore the presence of OBI in this population. Results Of 150 sera, with 17% anti-HBc and 1.3% HBsAg prevalence, 70 were tested for the presence of HBV DNA. From these, 25 (36%) were found positive for HBV DNA by PCR in the core region. Two of these 25 sera were HBsAg positive, indicating an overt infection. Of the remaining 68 sera tested, 23 exhibited OBI. Of these, 13 were HBV DNA out of 25 anti-HBc positive (52%) and 10 HBV DNA positive, out of 43 anti-HBc negative (23%), with a statistical significance of p = 0.03. Viral DNA and HBsAg were present intermittently in follow up sera of 13 individuals. Sequence analysis in the core region of the amplified DNA products showed that all the strains belonged to HBV genotype F3. The OBI isolates displayed 96-100% nucleotide identity between them. One isolate exhibited the co-circulation of a wild type variant with a variant with a premature stop codon at the core protein, and a variant exhibiting a deletion of 28 amino acids. Conclusions The frequency of OBI found in this Amerindian group warrants further studies in other communities exhibiting different degrees of HBV exposure. PMID:22152023

Diverse Geno- and Phenotypes of Persistent Listeria monocytogenes Isolates from Fermented Meat Sausage Production Facilities in Portugal ▿

PubMed Central

Ferreira, V.; Barbosa, J.; Stasiewicz, M.; Vongkamjan, K.; Moreno Switt, A.; Hogg, T.; Gibbs, P.; Teixeira, P.; Wiedmann, M.

2011-01-01

The persistence of Listeria monocytogenes in food-associated environments represents a key factor in transmission of this pathogen. To identify persistent and transient strains associated with production of fermented meat sausages in northern Portugal, 1,723 L. monocytogenes isolates from raw material and finished products from 11 processors were initially characterized by random amplification of polymorphic DNA (RAPD), PCR-based molecular serotyping, and epidemic clone characterization, as well as cadmium, arsenic, and tetracycline resistance typing. Pulsed-field gel electrophoresis (PFGE) typing of 240 representative isolates provided evidence for persistence of L. monocytogenes for periods of time ranging from 10 to 32 months for all seven processors for which isolates from different production dates were available. Among 50 L. monocytogenes isolates that included one representative for each PFGE pattern obtained from a given sample, 12 isolates showed reduced invasion efficiency in Caco-2 cells, including 8 isolates with premature stop codons in inlA. Among 41 isolates representing sporadic and persistent PFGE types, 22 isolates represented lysogens. Neither strains with reduced invasion nor lysogens were overrepresented among persistent isolates. While the susceptibility of isolates to lysogenic phages also did not correlate with persistence, it appeared to be associated with molecular serotype. Our data show the following. (i) RAPD may not be suitable for analysis of large sets of L. monocytogenes isolates. (ii) While a large diversity of L. monocytogenes subtypes is found in Portuguese fermented meat sausages, persistence of L. monocytogenes in this food chain is common. (iii) Persistent L. monocytogenes strains are diverse and do not appear to be characterized by unique genetic or phenotypic characteristics. PMID:21378045

Identification of a novel dentin matrix protein-1 (DMP-1) mutation and dental anomalies in a kindred with autosomal recessive hypophosphatemia.

PubMed

Turan, Serap; Aydin, Cumhur; Bereket, Abdullah; Akcay, Teoman; Güran, Tülay; Yaralioglu, Betul Akmen; Bastepe, Murat; Jüppner, Harald

2010-02-01

An autosomal recessive form of hypophosphatemia (ARHP) was recently shown to be caused by homozygous mutations in DMP1, the gene encoding dentin matrix protein-1 (DMP-1), a non-collagenous bone matrix protein with an important role in the development and mineralization of bone and teeth. Here, we describe a previously not reported consanguineous ARHP kindred in which the three affected individuals carry a novel homozygous DMP-1 mutation. The index case presented at the age of 3 years with bowing of his legs and showed hypophosphatemia due to insufficient renal phosphate retention. Serum alkaline phosphatase activity was elevated, with initially normal PTH. FGF23 was inappropriately normal at an older age while being treated with oral phosphate and 1,25(OH)(2)D. Similar clinical and biochemical findings, except for elevated FGF23 levels, were present in his 16-month-old brother and his 12.5-year-old female cousin; the parents of the three affected children are first-degree cousins. Nucleotide sequence analysis was performed on PCR-amplified exons encoding DMP-1 and flanking intronic regions. A novel homozygous frame-shift mutation (c.485Tdel; p.Glu163ArgfsX53) in exon 6 resulting in a premature stop codon was identified in all effected individuals. The parents and available unaffected siblings were heterozygous for c.485Tdel. Tooth growth and shape were normal for the index case, his affected brother and cousin, but their permanent and deciduous teeth displayed enlarged pulp chambers. The identified genetic mutation underscores the importance of DMP-1 mutations in the pathogenesis of ARHP. Furthermore, DMP-1 mutations appear to contribute, through yet unknown mechanisms, to tooth development. (c) 2009 Elsevier Inc. All rights reserved.

Genetic variation of six desaturase genes in flax and their impact on fatty acid composition.

PubMed

Thambugala, Dinushika; Duguid, Scott; Loewen, Evelyn; Rowland, Gordon; Booker, Helen; You, Frank M; Cloutier, Sylvie

2013-10-01

Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability of genes encoding stearoyl-ACP desaturase (SAD), and fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6 and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variations. While most of the SNPs and all the indels were silent mutations, both genes carried nonsense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant effects of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles.

High-resolution genetic mapping of allelic variants associated with cell wall chemistry in Populus

DOE PAGES

Muchero, Wellington; Guo, Jianjun; Difazio, Stephen P.; ...

2015-01-23

We report the identification of six genetic loci and the allelic-variants associated with Populus cell wall phenotypes determined independently using pyrolysis Molecular Beam Mass Spectrometry (pyMBMS), saccharification assay and wet chemistry in two partially overlapping populations of P. trichocarpa genotypes sampled from multiple environments in the Pacific Northwest of North America. All 6 variants co-located with a quantitative trait locus (QTL) hotspot on chromosome XIV for lignin content, syringyl to guaiacyl (S/G) ratio, 5- and 6- carbon sugars identified in an interspecific P. trichocarpa x P. deltoides pseudo-backcross mapping pedigree. Genomic intervals containing an amino acid transporter, a MYB transcriptionmore » factor, an angustifolia CtBP transcription factor, a copper transport protein ATOX1-related, a Ca 2+ transporting ATPase and a protein kinase were identified within 5 QTL regions. Each interval contained single nucleotide polymorphisms (SNPs) that were significantly associated to cell-wall phenotypes, with associations exceeding the chromosome-wise Bonferroni-adjusted p-values in at least one environment. cDNA sequencing for allelic variants of 3 of the 6 genes identified polymorphisms leading to premature stop codons in the MYB transcription factor and protein kinase. On the other hand, variants of the Angustifolia CtBP transcription factor exhibited a polyglutamine (PolyQ) length polymorphism. Results from transient protoplast assays suggested that each of the polymorphisms conferred allelic differences in activation of cellulose, hemicelluloses and lignin pathway marker genes, with truncated and short PolyQ alleles exhibiting significantly reduced marker gene activation. Genes identified in this study represent novel targets for reducing cell wall recalcitrance for lignocellulosic biofuels production using plant biomass.« less

A comparison of the carotenoid accumulation in Capsicum varieties that show different ripening colours: deletion of the capsanthin-capsorubin synthase gene is not a prerequisite for the formation of a yellow pepper.

PubMed

Ha, Sun-Hwa; Kim, Jung-Bong; Park, Jong-Sug; Lee, Shin-Woo; Cho, Kang-Jin

2007-01-01

Ripe pepper (Capsicum sp.) fruits can display a range of colours from white to deep red. To understand better the regulatory mechanisms of the carotenoid biosynthetic pathways that underlie these ripening colours, Capsicum varieties that show seven different fully ripe colour types were analysed. The levels and composition of the carotenoid accumulation in these samples at different stages of ripening were measured, and the resulting data were analysed in conjunction with the expression patterns of the carotenoid biosynthetic genes. It was found that red peppers accumulate increasing levels of total carotenoids during ripening, whereas non-red peppers accumulate lower levels of total carotenoids of varying composition. The expression levels of the phytoene synthase, phytoene desaturase, and capsanthin-capsorubin synthase (Ccs) genes are high in peppers with high levels of total carotenoid, whereas one or two of these genes are not expressed in peppers with lower levels of total carotenoid. Surprisingly, it was found that the Ccs gene is present in two Capsicum varieties whose ripe colour is yellow. This gene has never previously been shown to be present in yellow peppers. Sequence analyses of the Ccs gene further revealed two structural mutations in yellow peppers that may result in either a premature stop-codon or a frame-shift. Taken together with the fact that the Ccs transcript is not detectable in yellow peppers, our current results suggest that nonsense-mediated transcriptional gene silencing of Ccs and not the deletion of this gene is responsible for yellow ripening in Capsicum.

A rare IL33 loss-of-function mutation reduces blood eosinophil counts and protects from asthma

PubMed Central

Sulem, Patrick; Bjornsdottir, Unnur Steina; Lim, Ai Ching; Sveinbjornsson, Gardar; Brown, Michael; Garrett, Logan; Jonasdottir, Adalbjorg; Jonasdottir, Aslaug; Sigurdsson, Asgeir; Magnusson, Olafur T.; Eyjolfsson, Gudmundur I.; Olafsson, Isleifur; Onundarson, Pall Torfi; Sigurdardottir, Olof; Gislason, David; Gislason, Thorarinn; Ludviksson, Bjorn Runar; Ludviksdottir, Dora; Boezen, H. Marike; Heinzmann, Andrea; Porsbjerg, Celeste; Waage, Johannes; Backer, Vibeke; Deichmann, Klaus A.; Bønnelykke, Klaus; Bisgaard, Hans; Gudbjartsson, Daniel F.; Johnston, James A.; Jonsdottir, Ingileif; Stefansson, Kari

2017-01-01

IL-33 is a tissue-derived cytokine that induces and amplifies eosinophilic inflammation and has emerged as a promising new drug target for asthma and allergic disease. Common variants at IL33 and IL1RL1, encoding the IL-33 receptor ST2, associate with eosinophil counts and asthma. Through whole-genome sequencing and imputation into the Icelandic population, we found a rare variant in IL33 (NM_001199640:exon7:c.487-1G>C (rs146597587-C), allele frequency = 0.65%) that disrupts a canonical splice acceptor site before the last coding exon. It is also found at low frequency in European populations. rs146597587-C associates with lower eosinophil counts (β = -0.21 SD, P = 2.5×10–16, N = 103,104), and reduced risk of asthma in Europeans (OR = 0.47; 95%CI: 0.32, 0.70, P = 1.8×10–4, N cases = 6,465, N controls = 302,977). Heterozygotes have about 40% lower total IL33 mRNA expression than non-carriers and allele-specific analysis based on RNA sequencing and phased genotypes shows that only 20% of the total expression is from the mutated chromosome. In half of those transcripts the mutation causes retention of the last intron, predicted to result in a premature stop codon that leads to truncation of 66 amino acids. The truncated IL-33 has normal intracellular localization but neither binds IL-33R/ST2 nor activates ST2-expressing cells. Together these data demonstrate that rs146597587-C is a loss of function mutation and support the hypothesis that IL-33 haploinsufficiency protects against asthma. PMID:28273074

Impaired Heme Binding and Aggregation of Mutant Cystathionine β-Synthase Subunits in Homocystinuria

PubMed Central

Janošík, Miroslav; Oliveriusová, Jana; Janošíková, Bohumila; Sokolová, Jitka; Kraus, Eva; Kraus, Jan P.; Kožich, Viktor

2001-01-01

During the past 20 years, cystathionine β-synthase (CBS) deficiency has been detected in the former Czechoslovakia with a calculated frequency of 1:349,000. The clinical manifestation was typical of homocystinuria, and about half of the 21 patients were not responsive to pyridoxine. Twelve distinct mutations were detected in 30 independent homocystinuric alleles. One half of the alleles carried either the c.833 T→C or the IVS11−2A→C mutation; the remaining alleles contained private mutations. The abundance of five mutant mRNAs with premature stop codons was analyzed by PCR-RFLP. Two mRNAs, c.828_931ins104 (IVS7+1G→A) and c.1226 G→A, were severely reduced in the cytoplasm as a result of nonsense-mediated decay. In contrast, the other three mRNAs—c.19_20insC, c.28_29delG, and c.210_235del26 (IVS1−1G→C)—were stable. Native western blot analysis of 14 mutant fibroblast lines showed a paucity of CBS antigen, which was detectable only in aggregates. Five mutations—A114V (c.341C→T), A155T (c.463G→A), E176K (c.526G→A), I278T (c.833T→C), and W409_G453del (IVS11−2A→C)—were expressed in Escherichia coli. All five mutant proteins formed substantially more aggregates than did the wild-type CBS, and no aggregates contained heme. These data suggest that abnormal folding, impaired heme binding, and aggregation of mutant CBS polypeptides may be common pathogenic mechanisms in CBS deficiency. PMID:11359213

A novel pathogenic variant in an Iranian Ataxia telangiectasia family revealed by next-generation sequencing followed by in silico analysis.

PubMed

Tabatabaiefar, Mohammad Amin; Alipour, Paria; Pourahmadiyan, Azam; Fattahi, Najmeh; Shariati, Laleh; Golchin, Neda; Mohammadi-Asl, Javad

2017-08-15

Ataxia telangiectasia (A-T) is a neurodegenerative autosomal recessive disorder with the main characteristics of progressive cerebellar degeneration, sensitivity to ionizing radiation, immunodeficiency, telangiectasia, premature aging, recurrent sinopulmonary infections, and increased risk of malignancy, especially of lymphoid origin. Ataxia Telangiectasia Mutated gene, ATM, as a causative gene for the A-T disorder, encodes the ATM protein, which plays an important role in the activation of cell-cycle checkpoints and initiation of DNA repair in response to DNA damage. Targeted next-generation sequencing (NGS) was performed on an Iranian 5-year-old boy presented with truncal and limb ataxia, telangiectasia of the eye, Hodgkin lymphoma, hyper pigmentation, total alopecia, hepatomegaly, and dysarthria. Sanger sequencing was used to confirm the candidate pathogenic variants. Computational docking was done using the HEX software to examine how this change affects the interactions of ATM with the upstream and downstream proteins. Three different variants were identified comprising two homozygous SNPs and one novel homozygous frameshift variant (c.80468047delTA, p.Thr2682ThrfsX5), which creates a stop codon in exon 57 leaving the protein truncated at its C-terminal portion. Therefore, the activation and phosphorylation of target proteins are lost. Moreover, the HEX software confirmed that the mutated protein lost its interaction with upstream and downstream proteins. The variant was classified as pathogenic based on the American College of Medical Genetics and Genomics guideline. This study expands the spectrum of ATM pathogenic variants in Iran and demonstrates the utility of targeted NGS in genetic diagnostics. Copyright © 2017. Published by Elsevier B.V.

Prevalence of SHANK3 variants in patients with different subtypes of autism spectrum disorders.

PubMed

Boccuto, Luigi; Lauri, Maria; Sarasua, Sara M; Skinner, Cindy D; Buccella, Daniela; Dwivedi, Alka; Orteschi, Daniela; Collins, Julianne S; Zollino, Marcella; Visconti, Paola; Dupont, Barb; Tiziano, Danilo; Schroer, Richard J; Neri, Giovanni; Stevenson, Roger E; Gurrieri, Fiorella; Schwartz, Charles E

2013-03-01

Autism spectrum disorders (ASDs) include three main conditions: autistic disorder (AD), pervasive developmental disorder, not otherwise specified (PDD-NOS), and Asperger syndrome. It has been shown that many genes associated with ASDs are involved in the neuroligin-neurexin interaction at the glutamate synapse: NLGN3, NLGN4, NRXN1, CNTNAP2, and SHANK3. We screened this last gene in two cohorts of ASD patients (133 patients from US and 88 from Italy). We found 5/221 (2.3%) cases with pathogenic alterations: a 106 kb deletion encompassing the SHANK3 gene, two frameshift mutations leading to premature stop codons, a missense mutation (p.Pro141Ala), and a splicing mutation (c.1820-4 G>A). Additionally, in 17 patients (7.7%) we detected a c.1304+48C>T transition affecting a methylated cytosine in a CpG island. This variant is reported as SNP rs76224556 and was found in both US and Italian controls, but it results significantly more frequent in our cases than in the control cohorts. The variant is also significantly more common among PDD-NOS cases than in AD cases. We also screened this gene in an independent replication cohort of 104 US patients with ASDs, in which we found a missense mutation (p.Ala1468Ser) in 1 patient (0.9%), and in 8 patients (7.7%) we detected the c.1304+48C>T transition. While SHANK3 variants are present in any ASD subtype, the SNP rs76224556 appears to be significantly associated with PDD-NOS cases. This represents the first evidence of a genotype-phenotype correlation in ASDs and highlights the importance of a detailed clinical-neuropsychiatric evaluation for the effective genetic screening of ASD patients.

Prevalence of SHANK3 variants in patients with different subtypes of autism spectrum disorders

PubMed Central

Boccuto, Luigi; Lauri, Maria; Sarasua, Sara M; Skinner, Cindy D; Buccella, Daniela; Dwivedi, Alka; Orteschi, Daniela; Collins, Julianne S; Zollino, Marcella; Visconti, Paola; DuPont, Barb; Tiziano, Danilo; Schroer, Richard J; Neri, Giovanni; Stevenson, Roger E; Gurrieri, Fiorella; Schwartz, Charles E

2013-01-01

Autism spectrum disorders (ASDs) include three main conditions: autistic disorder (AD), pervasive developmental disorder, not otherwise specified (PDD-NOS), and Asperger syndrome. It has been shown that many genes associated with ASDs are involved in the neuroligin–neurexin interaction at the glutamate synapse: NLGN3, NLGN4, NRXN1, CNTNAP2, and SHANK3. We screened this last gene in two cohorts of ASD patients (133 patients from US and 88 from Italy). We found 5/221 (2.3%) cases with pathogenic alterations: a 106 kb deletion encompassing the SHANK3 gene, two frameshift mutations leading to premature stop codons, a missense mutation (p.Pro141Ala), and a splicing mutation (c.1820-4 G>A). Additionally, in 17 patients (7.7%) we detected a c.1304+48C>T transition affecting a methylated cytosine in a CpG island. This variant is reported as SNP rs76224556 and was found in both US and Italian controls, but it results significantly more frequent in our cases than in the control cohorts. The variant is also significantly more common among PDD-NOS cases than in AD cases. We also screened this gene in an independent replication cohort of 104 US patients with ASDs, in which we found a missense mutation (p.Ala1468Ser) in 1 patient (0.9%), and in 8 patients (7.7%) we detected the c.1304+48C>T transition. While SHANK3 variants are present in any ASD subtype, the SNP rs76224556 appears to be significantly associated with PDD-NOS cases. This represents the first evidence of a genotype–phenotype correlation in ASDs and highlights the importance of a detailed clinical-neuropsychiatric evaluation for the effective genetic screening of ASD patients. PMID:22892527

Comparative analyses of plastid genomes from fourteen Cornales species: inferences for phylogenetic relationships and genome evolution.

PubMed

Fu, Chao-Nan; Li, Hong-Tao; Milne, Richard; Zhang, Ting; Ma, Peng-Fei; Yang, Jing; Li, De-Zhu; Gao, Lian-Ming

2017-12-08

The Cornales is the basal lineage of the asterids, the largest angiosperm clade. Phylogenetic relationships within the order were previously not fully resolved. Fifteen plastid genomes representing 14 species, ten genera and seven families of Cornales were newly sequenced for comparative analyses of genome features, evolution, and phylogenomics based on different partitioning schemes and filtering strategies. All plastomes of the 14 Cornales species had the typical quadripartite structure with a genome size ranging from 156,567 bp to 158,715 bp, which included two inverted repeats (25,859-26,451 bp) separated by a large single-copy region (86,089-87,835 bp) and a small single-copy region (18,250-18,856 bp) region. These plastomes encoded the same set of 114 unique genes including 31 transfer RNA, 4 ribosomal RNA and 79 coding genes, with an identical gene order across all examined Cornales species. Two genes (rpl22 and ycf15) contained premature stop codons in seven and five species respectively. The phylogenetic relationships among all sampled species were fully resolved with maximum support. Different filtering strategies (none, light and strict) of sequence alignment did not have an effect on these relationships. The topology recovered from coding and noncoding data sets was the same as for the whole plastome, regardless of filtering strategy. Moreover, mutational hotspots and highly informative regions were identified. Phylogenetic relationships among families and intergeneric relationships within family of Cornales were well resolved. Different filtering strategies and partitioning schemes do not influence the relationships. Plastid genomes have great potential to resolve deep phylogenetic relationships of plants.

Whole exome sequencing identified 1 base pair novel deletion in BCL2-associated athanogene 3 (BAG3) gene associated with severe dilated cardiomyopathy (DCM) requiring heart transplant in multiple family members.

PubMed

Rafiq, Muhammad Arshad; Chaudhry, Ayeshah; Care, Melanie; Spears, Danna A; Morel, Chantal F; Hamilton, Robert M

2017-03-01

Dilated cardiomyopathy (DCM) is characterized by dilation and impaired contraction of the left ventricle or both ventricles. Among hereditary DCM, the genetic causes are heterogeneous, and include mutations encoding cytoskeletal, nucleoskeletal, mitochondrial, and calcium-handling proteins. We report three severely affected males, in a four-generation pedigree, with DCM phenotype who underwent cardiac transplant. Cardiomegaly with marked biventricular dilation and fibrosis were noticeable histopathological findings. The affected males had tested negative on a 46-gene pancardiomyopathy panel. Whole Exome Sequencing (WES) was performed to reveal mutation in the gene responsible in generation of DCM phenotypes. The 1-bp (Chr10:121435979delC; c.913delC) novel heterozygous deletion in exon 4 of BAG3, was identified in three affected males, resulted in frame-shift and a premature termination codon (p.Met306-Stop) producing a truncated BAG3 protein lacking functionally important PXXP and BAG domains. WES data were further utilized to map 10 SNP markers around the discovered mutation to generate shared disease haplotype in all affected individuals encompassing 11 Mb on 10q25.3-26.2 harboring BAG3. Finally genotypes were inferred for the unavailable/deceased individuals in the pedigrees. Here we propose that Chr10:121435979delC in BAG3 is a causal mutation in these subjects. Our and earlier studies indicate that BAG3 mutations are associated with DCM phenotypes. BAG3 should be added to cardiomyopathy gene panels for screening of DCM patients, and patients previously considered gene elusive should undergo sequencing of the BAG3 gene. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Succinate Dehydrogenase (SDH) D Subunit (SDHD) Inactivation in a Growth-Hormone-Producing Pituitary Tumor: A New Association for SDH?

PubMed Central

Xekouki, Paraskevi; Pacak, Karel; Almeida, Madson; Wassif, Christopher A.; Rustin, Pierre; Nesterova, Maria; de la Luz Sierra, Maria; Matro, Joey; Ball, Evan; Azevedo, Monalisa; Horvath, Anelia; Lyssikatos, Charalampos; Quezado, Martha; Patronas, Nicholas; Ferrando, Barbara; Pasini, Barbara; Lytras, Aristides; Tolis, George

2012-01-01

Background: Mutations in the subunits B, C, and D of succinate dehydrogenase (SDH) mitochondrial complex II have been associated with the development of paragangliomas (PGL), gastrointestinal stromal tumors, papillary thyroid and renal carcinoma (SDHB), and testicular seminoma (SDHD). Aim: Our aim was to examine the possible causative link between SDHD inactivation and somatotropinoma. Patients and Methods: A 37-yr-old male presented with acromegaly and hypertension. Other family members were found with PGL. Elevated plasma and urinary levels of catecholamines led to the identification of multiple PGL in the proband in the neck, thorax, and abdomen. Adrenalectomy was performed for bilateral pheochromocytomas (PHEO). A GH-secreting macroadenoma was also found and partially removed via transsphenoidal surgery (TTS). Genetic analysis revealed a novel SDHD mutation (c.298_301delACTC), leading to a frame shift and a premature stop codon at position 133 of the protein. Loss of heterozygosity for the SDHD genetic locus was shown in the GH-secreting adenoma. Down-regulation of SDHD protein in the GH-secreting adenoma by immunoblotting and immunohistochemistry was found. A literature search identified other cases of multiple PGL and/or PHEO in association with pituitary tumors. Conclusion: We describe the first kindred with a germline SDHD pathogenic mutation, inherited PGL, and acromegaly due to a GH-producing pituitary adenoma. SDHD loss of heterozygosity, down-regulation of protein in the GH-secreting adenoma, and decreased SDH enzymatic activity supports SDHD's involvement in the pituitary tumor formation in this patient. Older cases of multiple PGL and PHEO and pituitary tumors in the literature support a possible association between SDH defects and pituitary tumorigenesis. PMID:22170724

Precise gene editing of chicken Na+/H+ exchange type 1 (chNHE1) confers resistance to avian leukosis virus subgroup J (ALV-J).

PubMed

Lee, Hong Jo; Lee, Kyung Youn; Jung, Kyung Min; Park, Kyung Je; Lee, Ko On; Suh, Jeong-Yong; Yao, Yongxiu; Nair, Venugopal; Han, Jae Yong

2017-12-01

Avian leukosis virus subgroup J (ALV-J), first isolated in the late 1980s, has caused economic losses to the poultry industry in many countries. As all chicken lines studied to date are susceptible to ALV infection, there is enormous interest in developing resistant chicken lines. The ALV-J receptor, chicken Na + /H + exchange 1 (chNHE1) and the critical amino acid sequences involved in viral attachment and entry have already been characterized. However, there are no reported attempts to induce resistance to the virus by targeted genome modification of the receptor sequences. In an attempt to induce resistance to ALV-J infection, we used clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR/Cas9)-based genome editing approaches to modify critical residues of the chNHE1 receptor in chicken cells. The susceptibility of the modified cell lines to ALV-J infection was examined using enhanced green fluorescent protein (EGFP)-expressing marker viruses. We showed that modifying the chNHE1 receptor by artificially generating a premature stop codon induced absolute resistance to viral infection, with mutations of the tryptophan residue at position 38 (Trp38) being very critical. Single-stranded oligodeoxynucleotide (ssODN)-mediated targeted recombination of the Trp38 region revealed that deletions involving the Trp38 residue were most effective in conferring resistance to ALV-J. Moreover, protein structure analysis of the chNHE1 receptor sequence suggested that its intrinsically disordered region undergoes local conformational changes through genetic alteration. Collectively, these results demonstrate that targeted mutations on chNHE1 alter the susceptibility to ALV-J and the technique is expected to contribute to develop disease-resistant chicken lines. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome Sequence and Analysis of Escherichia coli MRE600, a Colicinogenic, Nonmotile Strain that Lacks RNase I and the Type I Methyltransferase, EcoKI

PubMed Central

Kurylo, Chad M.; Alexander, Noah; Dass, Randall A.; Parks, Matthew M.; Altman, Roger A.; Vincent, C. Theresa; Mason, Christopher E.; Blanchard, Scott C.

2016-01-01

Escherichia coli strain MRE600 was originally identified for its low RNase I activity and has therefore been widely adopted by the biomedical research community as a preferred source for the expression and purification of transfer RNAs and ribosomes. Despite its widespread use, surprisingly little information about its genome or genetic content exists. Here, we present the first de novo assembly and description of the MRE600 genome and epigenome. To provide context to these studies of MRE600, we include comparative analyses with E. coli K-12 MG1655 (K12). Pacific Biosciences Single Molecule, Real-Time sequencing reads were assembled into one large chromosome (4.83 Mb) and three smaller plasmids (89.1, 56.9, and 7.1 kb). Interestingly, the 7.1-kb plasmid possesses genes encoding a colicin E1 protein and its associated immunity protein. The MRE600 genome has a G + C content of 50.8% and contains a total of 5,181 genes, including 4,913 protein-encoding genes and 268 RNA genes. We identified 41,469 modified DNA bases (0.83% of total) and found that MRE600 lacks the gene for type I methyltransferase, EcoKI. Phylogenetic, taxonomic, and genetic analyses demonstrate that MRE600 is a divergent E. coli strain that displays features of the closely related genus, Shigella. Nevertheless, comparative analyses between MRE600 and E. coli K12 show that these two strains exhibit nearly identical ribosomal proteins, ribosomal RNAs, and highly homologous tRNA species. Substantiating prior suggestions that MRE600 lacks RNase I activity, the RNase I-encoding gene, rna, contains a single premature stop codon early in its open-reading frame. PMID:26802429

Protective role of alpha-actinin-3 in the response to an acute eccentric exercise bout.

PubMed

Vincent, Barbara; Windelinckx, An; Nielens, Henri; Ramaekers, Monique; Van Leemputte, Marc; Hespel, Peter; Thomis, Martine A

2010-08-01

The ACTN3 gene encodes for the alpha-actinin-3 protein, which has an important structural function in the Z line of the sarcomere in fast muscle fibers. A premature stop codon (R577X) polymorphism in the ACTN3 gene causes a complete loss of the protein in XX homozygotes. This study investigates a possible role for the alpha-actinin-3 protein in protecting the fast fiber from eccentric damage and studies repair mechanisms after a single eccentric exercise bout. Nineteen healthy young men (10 XX, 9 RR) performed 4 series of 20 maximal eccentric knee extensions with both legs. Blood (creatine kinase; CK) and muscle biopsy samples were taken to study differential expression of several anabolic (MyoD1, myogenin, MRF4, Myf5, IGF-1), catabolic (myostatin, MAFbx, and MURF-1), and contraction-induced muscle damage marker genes [cysteine- and glycine-rich protein 3 (CSRP3), CARP, HSP70, and IL-6] as well as a calcineurin signaling pathway marker (RCAN1). Baseline mRNA content of CSRP3 and MyoD1 was 49 + or - 12 and 67 + or - 25% higher in the XX compared with the RR group (P = 0.01-0.045). However, satellite cell number was not different between XX and RR individuals. After eccentric exercise, XX individuals tended to have higher serum CK activity (P = 0.10) and had higher pain scores than RR individuals. However, CSRP3 (P = 0.058) and MyoD1 (P = 0.08) mRNA expression tended to be higher after training in RR individuals compared with XX alpha-actinin-3-deficient subjects. This study suggests a protective role of alpha-actinin-3 protein in muscle damage after eccentric training and an improved stress-sensor signaling, although effects are small.

A Frameshift Mutation in KIT is Associated with  White Spotting in the Arabian Camel.

PubMed

Holl, Heather; Isaza, Ramiro; Mohamoud, Yasmin; Ahmed, Ayeda; Almathen, Faisal; Youcef, Cherifi; Gaouar, Semir; Antczak, Douglas F; Brooks, Samantha

2017-03-09

While the typical Arabian camel is characterized by a single colored coat, there are rare populations with white spotting patterns. White spotting coat patterns are found in virtually all domesticated species, but are rare in wild species. Theories suggest that white spotting is linked to the domestication process, and is occasionally associated with health disorders. Though mutations have been found in a diverse array of species, fewer than 30 genes have been associated with spotting patterns, thus providing a key set of candidate genes for the Arabian camel. We obtained 26 spotted camels and 24 solid controls for candidate gene analysis. One spotted and eight solid camels were whole genome sequenced as part of a separate project. The spotted camel was heterozygous for a frameshift deletion in KIT (c.1842delG, named KITW1 for White spotting 1), whereas all other camels were wild-type (KIT+/KIT+). No additional mutations unique to the spotted camel were detected in the EDNRB, EDN3, SOX10, KITLG, PDGFRA, MITF, and PAX3 candidate white spotting genes. Sanger sequencing of the study population identified an additional five kITW1/KIT+ spotted camels. The frameshift results in a premature stop codon five amino acids downstream, thus terminating KIT at the tyrosine kinase domain. An additional 13 spotted camels tested KIT+/KIT+, but due to phenotypic differences when compared to the KITW1/KIT+ camels, they likely represent an independent mutation. Our study suggests that there are at least two causes of white spotting in the Arabian camel, the newly described KITW1 allele and an uncharacterized mutation.

Comprehensive approach to study complement C4 in systemic lupus erythematosus: Gene polymorphisms, protein levels and functional activity.

PubMed

Tsang-A-Sjoe, M W P; Bultink, I E M; Korswagen, L A; van der Horst, A; Rensink, I; de Boer, M; Hamann, D; Voskuyl, A E; Wouters, D

2017-12-01

Genetic variation of the genes encoding complement component C4 is strongly associated with systemic lupus erythematosus (SLE), a chronic multi-organ auto-immune disease. This study examined C4 and its isotypes on a genetic, protein, and functional level in 140 SLE patients and 104 healthy controls. Gene copy number (GCN) variation, silencing CT-insertion, and the retroviral HERV-K(C4) insertion) were analyzed with multiplex ligation-dependent probe amplification. Increased susceptibility to SLE was found for low GCN (≪2) of C4A. Serositis was the only clinical manifestation associated with low C4A GCN. One additional novel silencing mutation in the C4A gene was found by Sanger sequencing. This mutation causes a premature stop codon in exon 11. Protein concentrations of C4 isoforms C4A and C4B were determined with ELISA and were significantly lower in SLE patients compared to healthy controls. To study C4 isotypes on a functional level, a new C4 assay was developed, which distinguishes C4A from C4B by its binding capacity to amino or hydroxyl groups, respectively. This assay showed high correlation with ELISA and detected crossing over of Rodgers and Chido antigens in 3.2% (8/244) of individuals. The binding capacity of available C4 to its substrates was unaffected in SLE. Our study provides, for the first time, a complete overview of C4 in SLE from genetic variation to binding capacity using a novel test. As this test detects crossing over of Rodgers and Chido antigens, it will allow for more accurate measurement of C4 in future studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of the equine 2'-5' oligoadenylate synthetase 1 (OAS1) and ribonuclease L (RNASEL) innate immunity genes

PubMed Central

Rios, Jonathan J; Perelygin, Andrey A; Long, Maureen T; Lear, Teri L; Zharkikh, Andrey A; Brinton, Margo A; Adelson, David L

2007-01-01

Background The mammalian OAS/RNASEL pathway plays an important role in antiviral host defense. A premature stop-codon within the murine Oas1b gene results in the increased susceptibility of mice to a number of flaviviruses, including West Nile virus (WNV). Mutations in either the OAS1 or RNASEL genes may also modulate the outcome of WNV-induced disease or other viral infections in horses. Polymorphisms in the human OAS gene cluster have been previously utilized for case-control analysis of virus-induced disease in humans. No polymorphisms have yet been identified in either the equine OAS1 or RNASEL genes for use in similar case-control studies. Results Genomic sequence for equine OAS1 was obtained from a contig assembly generated from a shotgun subclone library of CHORI-241 BAC 100I10. Specific amplification of regions of the OAS1 gene from 13 horses of various breeds identified 33 single nucleotide polymorphisms (SNP) and two microsatellites. RNASEL cDNA sequences were determined for 8 mammals and utilized in a phylogenetic analysis. The chromosomal location of the RNASEL gene was assigned by FISH to ECA5p17-p16 using two selected CHORI-241 BAC clones. The horse genomic RNASEL sequence was assembled. Specific amplification of regions of the RNASEL gene from 13 horses identified 31 SNPs. Conclusion In this report, two dinucleotide microsatellites and 64 single nucleotide polymorphisms within the equine OAS1 and RNASEL genes were identified. These polymorphisms are the first to be reported for these genes and will facilitate future case-control studies of horse susceptibility to infectious diseases. PMID:17822564

Identification of a Novel Dentin Matrix Protein-1 (DMP-1) Mutation and Dental Anomalies in a Kindred with Autosomal Recessive Hypophosphatemia

PubMed Central

Turan, Serap; Aydin, Cumhur; Bereket, Abdullah; Akcay, Teoman; Güran, Tülay; Yaralioglu, Betul Akmen; Bastepe, Murat; Jüppner, Harald

2009-01-01

An autosomal recessive form of hypophosphatemia (ARHP) was recently shown to be caused by homozygous mutations in DMP1, the gene encoding dentin matrix protein-1 (DMP-1), a non-collagenous bone matrix protein with an important role in the development and mineralization of bone and teeth. Here, we report a previously not reported consanguineous ARHP kindred in which the three affected individuals carry a novel homozygous DMP-1 mutation. The index case presented at the age of 3 years with bowing of his legs, and showed hypophosphatemia due to insufficient renal phosphate retention. Serum alkaline phosphatase activity was elevated, with initially normal PTH. FGF23 was inappropriately normal at an older age while being treated with oral phosphate and 1,25(OH)2D. Similar clinical and biochemical findings, except for elevated FGF23 levels, were present in his 16 month-old brother and his 12.5 year-old female cousin; the parents of the three affected children are first-degree cousins. Nucleotide sequence analysis was performed on PCR-amplified exons encoding DMP-1 and flanking intronic regions. A novel homozygous frame-shift mutation (c.485Tdel; p.Glu163ArgfsX53) in exon 6 resulting in a premature stop codon was identified in all effected individuals. The parents and available unaffected siblings were heterozygous for c.485Tdel. Tooth growth and shape were normal for the index case, his affected brother and cousin, but their permanent and deciduous teeth displayed enlarged pulp chambers. The identified genetic mutation underscores the importance of DMP-1 mutations in the pathogenesis of ARHP. Furthermore, DMP-1 mutations appear to contribute, through yet unknown mechanisms, to tooth development. PMID:19796717

Whole genome sequencing for typing and characterisation of Listeria monocytogenes isolated in a rabbit meat processing plant.

PubMed

Palma, Federica; Pasquali, Frédérique; Lucchi, Alex; Cesare, Alessandra De; Manfreda, Gerardo

2017-08-16

Listeria monocytogenes is a food-borne pathogen able to survive and grow in different environments including food processing plants where it can persist for month or years. In the present study the discriminatory power of Whole Genome Sequencing (WGS)-based analysis (cgMLST) was compared to that of molecular typing methods on 34 L. monocytogenes isolates collected over one year in the same rabbit meat processing plant and belonging to three genotypes (ST14, ST121, ST224). Each genotype included isolates indistinguishable by standard molecular typing methods. The virulence potential of all isolates was assessed by Multi Virulence-Locus Sequence Typing (MVLST) and the investigation of a representative database of virulence determinant genes. The whole genome of each isolate was sequenced on a MiSeq platform. The cgMLST, MVLST, and in silico identification of virulence genes were performed using publicly available tools. Draft genomes included a number of contigs ranging from 13 to 28 and N50 ranging from 456298 to 580604. The coverage ranged from 41 to 187X. The cgMLST showed a significantly superior discriminatory power only in comparison to ribotyping, nevertheless it allows the detection of two singletons belonging to ST14 that were not observed by other molecular methods. All ST14 isolates belonged to VT107, which 7-loci concatenated sequence differs for only 4 nucleotides to VT1 (Epidemic clone III). Analysis of virulence genes showed the presence of a fulllength inlA version in all ST14 isolates and of a mutated version including a premature stop codon (PMSC) associated to attenuated virulence in all ST121 isolates.

Functional characterization of the novel intronic nucleotide change c.288+9C>T within the BCKDHA gene: understanding a variant presentation of maple syrup urine disease.

PubMed

Fernández-Guerra, Paula; Navarrete, Rosa; Weisiger, Kara; Desviat, Lourdes R; Packman, Seymour; Ugarte, Magdalena; Rodríguez-Pombo, Pilar

2010-12-01

Mutations in any of the three different genes--BCKDHA, BCKDHB, and DBT--encoding for the E1α, E1β, and E2 catalytic components of the branched-chain α-ketoacid dehydrogenase complex can cause maple syrup urine disease (MSUD). Disease severity ranges from the classic to the mildest variant types and precise genotypes, mostly based on missense mutations, have been associated to the less severe presentations of the disease. Herein, we examine the consequences at the messenger RNA (mRNA) level of the novel intronic alteration c.288+9C>T found in heterozygous fashion in a BCKDHA variant MSUD patient who also carries the nucleotide change c.745G>A (p.Gly249Ser), previously described as a severe change. Direct analysis of the processed transcripts from the patient showed--in addition to a low but measurable level of normal mRNA product--an aberrantly spliced mRNA containing a 7-bp fragment of intron 2, which could be rescued when the patient's cells were treated with emetine. This aberrant transcript with a premature stop codon would be unstable, supporting the possible activation of nonsense-mediated mRNA decay pathway. Consistent with this finding, minigene splicing assays demonstrated that the point mutation c.288+9C>T is sufficient to create a cryptic splice site and cause the observed 7-bp insertion. Furthermore, our results strongly suggest that the c.288+9C>T allele in the patient generates both normal and aberrant transcripts that could sustain the variant presentation of the disease, highlighting the importance of correct genotyping to establish genotype-phenotype correlations and as basis for the development of therapeutic interventions.

In silico prediction of the pathogenic effect of a novel variant of BCKDHA leading to classical maple syrup urine disease identified using clinical exome sequencing.

PubMed

Fernández-Lainez, Cynthia; Aláez-Verson, Carmen; Ibarra-González, Isabel; Enríquez-Flores, Sergio; Carrillo-Sanchez, Karol; Flores-Lagunes, Leonardo; Guillén-López, Sara; Belmont-Martínez, Leticia; Vela-Amieva, Marcela

2018-04-16

Maple syrup urine disease (MSUD) is a metabolic disorder caused by mutations in three of the branched-chain α-keto acid dehydrogenase complex (BCKDC) genes. Classical MSUD symptom can be observed immediately after birth and include ketoacidosis, irritability, lethargy, and coma, which can lead to death or irreversible neurodevelopmental delay in survivors. The molecular diagnosis of MSUD can be time-consuming and difficult to establish using conventional Sanger sequencing because it could be due to pathogenic variants of any of the BCKDC genes. Next-generation sequencing-based methodologies have revolutionized the molecular diagnosis of inborn errors in metabolism and offer a superior approach for genotyping these patients. Here, we report an MSUD case whose molecular diagnosis was performed by clinical exome sequencing (CES), and the possible structural pathogenic effect of a novel E1α subunit pathogenic variant was analyzed using in silico analysis of α and β subunit crystallographic structure. Molecular analysis revealed a new homozygous non-sense c.1267C>T or p.Gln423Ter variant of BCKDHA. The novel BCKDHA variant is considered pathogenic because it caused a premature stop codon that probably led to the loss of the last 22 amino acid residues of the E1α subunit C-terminal end. In silico analysis of this region showed that it is in contact with several residues of the E1β subunit mainly through polar contacts, hydrogen bonds, and hydrophobic interactions. CES strategy could benefit the patients and families by offering precise and prompt diagnosis and better genetic counseling. Copyright © 2018 Elsevier B.V. All rights reserved.

Porin Deficiency in Carbapenem-Resistant Enterobacter aerogenes Strains.

PubMed

Hao, Min; Ye, Meiping; Shen, Zhen; Hu, Fupin; Yang, Yang; Wu, Shi; Xu, Xiaogang; Zhu, Sihui; Qin, Xiaohua; Wang, Minggui

2018-03-13

The more frequent reports of carbapenem-resistant Enterobacteriaceae have raised the alarm for public health. Apart from the production of carbapenemases, deficiency (decreased or loss of expression) of outer membrane proteins (OMPs) has been proposed as a potentially important mechanism of carbapenem resistance. The aim of the present study was to evaluate the contribution of the major OMPs to carbapenem resistance in Enterobacter aerogenes (CREA) isolates and also investigate the role of small RNAs (sRNAs) in inducing porin-associated permeability defects. The differential expression of OMPs was analyzed in four clinical CREA isolates. omp35 and omp36 genes were further investigated by whole-genome sequencing, induction of meropenem resistance, sRNA overexpression, OMP complementation assays, and reverse transcription-quantitative PCR. All four isolates examined were deficient in omp35 and omp36. Functional restoration of these two genes confirmed their contribution to carbapenem resistance. The meropenem induction assay further revealed that porin deficiency plays a role in carbapenem resistance under antibiotic selection pressure. Single-point mutations in omp36 leading to premature stop codons were detected in two of the isolates. Elevated expression levels of the sRNAs micF and micC were detected in the other two porin-deficient isolates, which were predicted to be potential porin regulators from whole-genome sequencing. Overexpression of micF and micC downregulated the expression of Omp35 and Omp36, respectively. Porin deficiency plays an important role in carbapenem resistance among clinical E. aerogenes isolates under regulation of the sRNAs micC and micF. Furthermore, overexpression of micC and micF had a minor to no impact on carbapenem minimum inhibitory concentrations, and thus, the regulatory mechanism is likely to be complex.

Erythrocytic Pyruvate Kinase Mutations Causing Hemolytic Anemia, Osteosclerosis, and Secondary Hemochromatosis in Dogs

PubMed Central

Gultekin, G. Inal; Raj, K.; Foureman, P.; Lehman, S.; Manhart, K.; Abdulmalik, O.; Giger, U.

2013-01-01

Background Erythrocytic pyruvate kinase (PK) deficiency, first documented in Basenjis, is the most common inherited erythroenzymopathy in dogs. Objectives To report 3 new breed-specific PK-LR gene mutations and a retrospective survey of PK mutations in a small and selected group of Beagles and West Highland White Terriers (WHWT). Animals Labrador Retrievers (2 siblings, 5 unrelated), Pugs (2 siblings, 1 unrelated), Beagles (39 anemic, 29 other), WHWTs (22 anemic, 226 nonanemic), Cairn Terrier (n = 1). Methods Exons of the PK-LR gene were sequenced from genomic DNA of young dogs (<2 years) with persistent highly regenerative hemolytic anemia. Results A nonsense mutation (c.799C>T) resulting in a premature stop codon was identified in anemic Labrador Retriever siblings that had osteosclerosis, high serum ferritin concentrations, and severe hepatic secondary hemochromatosis. Anemic Pug and Beagle revealed 2 different missense mutations (c.848T>C, c.994G>A, respectively) resulting in intolerable amino acid changes to protein structure and enzyme function. Breed-specific mutation tests were developed. Among the biased group of 248 WHWTs, 9% and 35% were homozygous (affected) and heterozygous, respectively, for the previously described mutation (mutant allele frequency 0.26). A PK-deficient Cairn Terrier had the same insertion mutation as the affected WHWTs. Of the selected group of 68 Beagles, 35% were PK-deficient and 3% were carriers (0.37). Conclusions and Clinical Importance Erythrocytic PK deficiency is caused by different mutations in different dog breeds and causes chronic severe hemolytic anemia, hemosiderosis, and secondary hemochromatosis because of chronic hemolysis and, an as yet unexplained osteosclerosis. The newly developed breed-specific mutation assays simplify the diagnosis of PK deficiency. PMID:22805166

A Frameshift Mutation in KIT is Associated with White Spotting in the Arabian Camel

PubMed Central

Holl, Heather; Isaza, Ramiro; Mohamoud, Yasmin; Ahmed, Ayeda; Almathen, Faisal; Youcef, Cherifi; Gaouar, Semir; Antczak, Douglas F.; Brooks, Samantha

2017-01-01

While the typical Arabian camel is characterized by a single colored coat, there are rare populations with white spotting patterns. White spotting coat patterns are found in virtually all domesticated species, but are rare in wild species. Theories suggest that white spotting is linked to the domestication process, and is occasionally associated with health disorders. Though mutations have been found in a diverse array of species, fewer than 30 genes have been associated with spotting patterns, thus providing a key set of candidate genes for the Arabian camel. We obtained 26 spotted camels and 24 solid controls for candidate gene analysis. One spotted and eight solid camels were whole genome sequenced as part of a separate project. The spotted camel was heterozygous for a frameshift deletion in KIT (c.1842delG, named KITW1 for White spotting 1), whereas all other camels were wild-type (KIT+/KIT+). No additional mutations unique to the spotted camel were detected in the EDNRB, EDN3, SOX10, KITLG, PDGFRA, MITF, and PAX3 candidate white spotting genes. Sanger sequencing of the study population identified an additional five KITW1/KIT+ spotted camels. The frameshift results in a premature stop codon five amino acids downstream, thus terminating KIT at the tyrosine kinase domain. An additional 13 spotted camels tested KIT+/KIT+, but due to phenotypic differences when compared to the KITW1/KIT+ camels, they likely represent an independent mutation. Our study suggests that there are at least two causes of white spotting in the Arabian camel, the newly described KITW1 allele and an uncharacterized mutation. PMID:28282952

Within-host whole genome analysis of an antibiotic resistant Pseudomonas aeruginosa strain sub-type in cystic fibrosis.

PubMed

Sherrard, Laura J; Tai, Anna S; Wee, Bryan A; Ramsay, Kay A; Kidd, Timothy J; Ben Zakour, Nouri L; Whiley, David M; Beatson, Scott A; Bell, Scott C

2017-01-01

A Pseudomonas aeruginosa AUST-02 strain sub-type (M3L7) has been identified in Australia, infects the lungs of some people with cystic fibrosis and is associated with antibiotic resistance. Multiple clonal lineages may emerge during treatment with mutations in chromosomally encoded antibiotic resistance genes commonly observed. Here we describe the within-host diversity and antibiotic resistance of M3L7 during and after antibiotic treatment of an acute pulmonary exacerbation using whole genome sequencing and show both variation and shared mutations in important genes. Eleven isolates from an M3L7 population (n = 134) isolated over 3 months from an individual with cystic fibrosis underwent whole genome sequencing. A phylogeny based on core genome SNPs identified three distinct phylogenetic groups comprising two groups with higher rates of mutation (hypermutators) and one non-hypermutator group. Genomes were screened for acquired antibiotic resistance genes with the result suggesting that M3L7 resistance is principally driven by chromosomal mutations as no acquired mechanisms were detected. Small genetic variations, shared by all 11 isolates, were found in 49 genes associated with antibiotic resistance including frame-shift mutations (mexA, mexT), premature stop codons (oprD, mexB) and mutations in quinolone-resistance determining regions (gyrA, parE). However, whole genome sequencing also revealed mutations in 21 genes that were acquired following divergence of groups, which may also impact the activity of antibiotics and multi-drug efflux pumps. Comparison of mutations with minimum inhibitory concentrations of anti-pseudomonal antibiotics could not easily explain all resistance profiles observed. These data further demonstrate the complexity of chronic and antibiotic resistant P. aeruginosa infection where a multitude of co-existing genotypically diverse sub-lineages might co-exist during and after intravenous antibiotic treatment.

Splicing mutation in Sbf1 causes nonsyndromic male infertility in the rat.

PubMed

Liška, František; Chylíková, Blanka; Janků, Michaela; Šeda, Ondřej; Vernerová, Zdeňka; Pravenec, Michal; Křen, Vladimír

2016-09-01

In the inbred SHR/OlaIpcv rat colony, we identified males with small testicles and inability to reproduce. By selectively breeding their parents, we revealed the infertility to segregate as an autosomal recessive Mendelian character. No other phenotype was observed in males, and females were completely normal. By linkage using a backcross with Brown Norway strain, we mapped the locus to a 1.2Mbp segment on chromosome 7, harboring 35 genes. Sequencing of candidate genes revealed a G to A substitution in a canonical 'AG' splice site of intron 37 in Sbf1 (SET binding factor 1, alias myotubularin-related protein 5). This leads to either skipping exon 38 or shifting splicing one base downstream, invariantly resulting in frameshift, premature stop codon and truncation of the protein. Western blotting using two anti-Sbf1 antibodies revealed absence of the full-length protein in the mutant testis. Testicles of the mutant males were significantly smaller compared with SHR from 4weeks, peaked at 84% wild-type weight at 6weeks and declined afterward to 28%, reflecting massive germ cell loss. Histological examination revealed lower germ cell number; latest observed germ cell stage were round spermatids, resulting in the absence of sperm in the epididymis (azoospermia). SBF1 is a member of a phosphatase family lacking the catalytical activity. It probably modulates the activity of a phosphoinositol phosphatase MTMR2. Human homozygotes or compound heterozygotes for missense SBF1 mutations exhibit Charcot-Marie-Tooth disease (manifested mainly as progressive neuropathy), while a single mouse knockout reported in the literature identified male infertility as the only phenotype manifestation. © 2016 Society for Reproduction and Fertility.

Overproduction of active efflux pump and variations of OprD dominate in imipenem-resistant Pseudomonas aeruginosa isolated from patients with bloodstream infections in Taiwan.

PubMed

Kao, Cheng-Yen; Chen, Shu-Sheng; Hung, Kuei-Hsiang; Wu, Hsiu-Mei; Hsueh, Po-Ren; Yan, Jing-Jou; Wu, Jiunn-Jong

2016-06-13

The emergence of imipenem-resistant Pseudomonas aeruginosa (IRPA) has become a great concern worldwide. The aim of this study was to investigate resistance mechanisms associated with bloodstream isolated IRPA strains in Taiwan. A total of 78 non-duplicated IRPA isolates were isolated from patients with bloodstream infection. The average prevalence of imipenem-resistance in those isolates was 5.9 % during a 10-year longitudinal surveillance in Taiwan. PFGE results showed high clonal diversity among the 78 isolates. VIM-2, VIM-3, OXA-10, and OXA-17 β-lactamases were identified in 2 (2.6 %), 3 (3.8 %), 2 (2.6 %), and 1 (1.3 %) isolates, respectively. Active efflux pumps, AmpC β-lactamase overproduction, and extended-spectrum AmpC cephalosporinases (ESACs) were found in 58 (74.4 %), 25 (32.1 %) and 15 (19.2 %) of IRPA isolates, respectively. oprD mutations with amino acid substitution, shortened putative loop L7, premature stop codon caused by point mutation, frameshift by nucleotide insertion or deletion, and interruption by insertion sequence were found in 19 (24.4 %), 18 (23.1 %), 15 (19.2 %), 14 (17.9 %), and 10 (12.8 %) of isolates, respectively. This study suggests that alterations in the OprD protein and having an active efflux pump are the main mechanisms associated with bloodstream isolated IRPA. Overproduction of AmpC, ESACs, and the presence of VIM- and OXA-type β-lactamases play additional roles in reduced susceptibility to imipenem in P. aeruginosa isolates in Taiwan.

Multidrug Resistant Pseudomonas aeruginosa Causing Prosthetic Valve Endocarditis: A Genetic-Based Chronicle of Evolving Antibiotic Resistance.

PubMed

Domitrovic, T Nicholas; Hujer, Andrea M; Perez, Federico; Marshall, Steven H; Hujer, Kristine M; Woc-Colburn, Laila E; Parta, Mark; Bonomo, Robert A

2016-10-01

Background.  Successful treatment of infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa is thwarted by the emergence of antibiotic resistance and biofilm formation on prosthetic devices. Our aims were to decipher the molecular basis of resistance in a unique case of prosthetic valve endocarditis (PVE) caused by MDR P. aeruginosa . Methods.  Five sequential MDR P. aeruginosa blood isolates collected during a 7-month period were recovered from a patient suffering from PVE previously exposed to β-lactam antibiotics. Minimum inhibitory concentrations (MICs) of several classes of antibiotics were used to indicate clinical resistance characteristics; relatedness of the isolates was determined using multilocus sequence typing and repetitive sequence-based polymerase chain reaction. Amplification and sequencing of regulatory and resistance genes was performed. Results.  All isolates belonged to ST 298, possessed bla PDC-16 , and were resistant to fluoroquinolones and carbapenems. In the course of therapy, we observed a >2-fold increase in cephalosporin resistance (4 µg/mL to >16 µg/mL). Sequencing of the AmpC regulator, amp R, revealed a D135N point mutation in cephalosporin-resistant isolates. Common carbapenemase genes were not identified. All isolates demonstrated a premature stop codon at amino acid 79 of the outer membrane protein OprD and mutations in the quinolone resistance-determining regions of gyr A and par C. Point mutations in nal C, an efflux pump regulator, were also observed. Conclusions.  In this analysis, we chart the molecular evolution of β-lactam resistance in a case of PVE. We show that mutations in regulatory genes controlling efflux and cephalosporinase production contributed to the MDR phenotype.

Stickler syndrome caused by COL2A1 mutations: genotype–phenotype correlation in a series of 100 patients

PubMed Central

Hoornaert, Kristien P; Vereecke, Inge; Dewinter, Chantal; Rosenberg, Thomas; Beemer, Frits A; Leroy, Jules G; Bendix, Laila; Björck, Erik; Bonduelle, Maryse; Boute, Odile; Cormier-Daire, Valerie; De Die-Smulders, Christine; Dieux-Coeslier, Anne; Dollfus, Hélène; Elting, Mariet; Green, Andrew; Guerci, Veronica I; Hennekam, Raoul C M; Hilhorts-Hofstee, Yvonne; Holder, Muriel; Hoyng, Carel; Jones, Kristi J; Josifova, Dragana; Kaitila, Ilkka; Kjaergaard, Suzanne; Kroes, Yolande H; Lagerstedt, Kristina; Lees, Melissa; LeMerrer, Martine; Magnani, Cinzia; Marcelis, Carlo; Martorell, Loreto; Mathieu, Michèle; McEntagart, Meriel; Mendicino, Angela; Morton, Jenny; Orazio, Gabrielli; Paquis, Véronique; Reish, Orit; Simola, Kalle O J; Smithson, Sarah F; Temple, Karen I; Van Aken, Elisabeth; Van Bever, Yolande; van den Ende, Jenneke; Van Hagen, Johanna M; Zelante, Leopoldo; Zordania, Riina; De Paepe, Anne; Leroy, Bart P; De Buyzere, Marc; Coucke, Paul J; Mortier, Geert R

2010-01-01

Stickler syndrome is an autosomal dominant connective tissue disorder caused by mutations in different collagen genes. The aim of our study was to define more precisely the phenotype and genotype of Stickler syndrome type 1 by investigating a large series of patients with a heterozygous mutation in COL2A1. In 188 probands with the clinical diagnosis of Stickler syndrome, the COL2A1 gene was analyzed by either a mutation scanning technique or bidirectional fluorescent DNA sequencing. The effect of splice site alterations was investigated by analyzing mRNA. Multiplex ligation-dependent amplification analysis was used for the detection of intragenic deletions. We identified 77 different COL2A1 mutations in 100 affected individuals. Analysis of the splice site mutations showed unusual RNA isoforms, most of which contained a premature stop codon. Vitreous anomalies and retinal detachments were found more frequently in patients with a COL2A1 mutation compared with the mutation-negative group (P<0.01). Overall, 20 of 23 sporadic patients with a COL2A1 mutation had either a cleft palate or retinal detachment with vitreous anomalies. The presence of vitreous anomalies, retinal tears or detachments, cleft palate and a positive family history were shown to be good indicators for a COL2A1 defect. In conclusion, we confirm that Stickler syndrome type 1 is predominantly caused by loss-of-function mutations in the COL2A1 gene as >90% of the mutations were predicted to result in nonsense-mediated decay. On the basis of binary regression analysis, we developed a scoring system that may be useful when evaluating patients with Stickler syndrome. PMID:20179744

Characterization of the RpoS Status of Clinical Isolates of Salmonella enterica

PubMed Central

Robbe-Saule, Véronique; Algorta, Gabriela; Rouilhac, Isabelle; Norel, Françoise

2003-01-01

The stationary-phase-inducible sigma factor, σS (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella. We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the σS protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of σS, showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease. PMID:12902215

Growth hormone receptor gene mutations in two Italian patients with Laron Syndrome.

PubMed

Fassone, L; Corneli, G; Bellone, S; Camacho-Hübner, C; Aimaretti, G; Cappa, M; Ubertini, G; Bona, G

2007-05-01

Laron Syndrome (LS) represents a condition characterized by GH insensitivity caused by molecular defects in the GH receptor (GHR) gene or in the post-receptor signalling pathway. We report the molecular characterization of two unrelated Italian girls from Sicily diagnosed with LS. The DNA sequencing of the GHR gene revealed the presence of different nonsense mutations, occurring in the same background haplotype. The molecular defects occurred in the extracellular domain of the GHR leading to a premature termination signal and to a truncated non-functional receptor. In one patient, a homozygous G to T transversion, in exon 6, led to the mutation GAA to TAA at codon 180 (E180X), while in the second patient a homozygous C to T transition in exon 7 was detected, causing the CGA to TAA substitution at codon 217 (R217X). Both probands presented the polymorphisms Gly168Gly and Ile544Leu in a homozygous state in exons 6 and 10, respectively. The E180X represents a novel defect of the GHR gene, while the R217X mutation has been previously reported in several patients from different ethnic backgrounds but all from countries located in the Mediterranean and Middle Eastern region.

A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes.

PubMed

Liu, H X; Cartegni, L; Zhang, M Q; Krainer, A R

2001-01-01

Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.

Premature chain termination is a unifying mechanism for COL1A1 null alleles in osteogenesis imperfecta type I cell strains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willing, M.C.; Deschenes, S.P.; Roberts, E.J.

Nonsense and frameshift mutations, which predict premature termination of translation, often cause a dramatic reduction in the amount of transcript from the mutant allele (nonsense-mediated mRNA decay). In some genes, these mutations also influence RNA splicing and induce skipping of the exon that contains the nonsense codon. To begin to dissect how premature termination alters the metabolism of RNA from the COL1A1 gene, we studied nonsense and frameshift mutations distributed over exons 11-49 of the gene. These mutations were originally identified in 10 unrelated families with osteogenesis imperfecta (OI) type I. We observed marked reduction in steady-state amounts of mRNAmore » from the mutant allele in both total cellular and nuclear RNA extracts of cells from affected individuals, suggesting that nonsense-mediated decay of COL1A1 RNA is a nuclear phenomenon. Position of the mutation within the gene did not influence this observation. None of the mutations induced skipping of either the exon containing the mutation or, for the frameshifts, the downstream exons with the new termination sites. Our data suggest that nonsense and frameshift mutations throughout most of the COL1A1 gene result in a null allele, which is associated with the predictable mild clinical phenotype, OI type I. 42 refs., 6 figs., 1 tab.« less

Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis.

PubMed

Legnini, Ivano; Di Timoteo, Gaia; Rossi, Francesca; Morlando, Mariangela; Briganti, Francesca; Sthandier, Olga; Fatica, Alessandro; Santini, Tiziana; Andronache, Adrian; Wade, Mark; Laneve, Pietro; Rajewsky, Nikolaus; Bozzoni, Irene

2017-04-06

Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Dynamic Modeling of GAIT System Reveals Transcriptome Expansion and Translational Trickle Control Device

PubMed Central

Yao, Peng; Potdar, Alka A.; Arif, Abul; Ray, Partho Sarothi; Mukhopadhyay, Rupak; Willard, Belinda; Xu, Yichi; Yan, Jun; Saidel, Gerald M.; Fox, Paul L.

2012-01-01

SUMMARY Post-transcriptional regulatory mechanisms superimpose “fine-tuning” control upon “on-off” switches characteristic of gene transcription. We have exploited computational modeling with experimental validation to resolve an anomalous relationship between mRNA expression and protein synthesis. Differential GAIT (Gamma-interferon Activated Inhibitor of Translation) complex activation repressed VEGF-A synthesis to a low, constant rate despite high, variable VEGFA mRNA expression. Dynamic model simulations indicated the presence of an unidentified, inhibitory GAIT element-interacting factor. We discovered a truncated form of glutamyl-prolyl tRNA synthetase (EPRS), the GAIT constituent that binds the 3’-UTR GAIT element in target transcripts. The truncated protein, EPRSN1, prevents binding of functional GAIT complex. EPRSN1 mRNA is generated by a remarkable polyadenylation-directed conversion of a Tyr codon in the EPRS coding sequence to a stop codon (PAY*). By low-level protection of GAIT element-bearing transcripts, EPRSN1 imposes a robust “translational trickle” of target protein expression. Genome-wide analysis shows PAY* generates multiple truncated transcripts thereby contributing to transcriptome expansion. PMID:22386318

DOE Office of Scientific and Technical Information (OSTI.GOV)

Galloway, Chad A.; Smith, Harold C., E-mail: harold.smith@rochester.edu

Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is {approx}80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expressionmore » of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However, transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.« less