Clinical and genetic characterisation of dystrophin-deficient muscular dystrophy in a family of Miniature Poodle dogs

Four full-sibling intact male Miniature Poodles were evaluated at 4–19 months of age. One was clinically normal and three were affected. All affected dogs were reluctant to exercise and had generalised muscle atrophy, a stiff gait and a markedly elevated serum creatine kinase activity. Two affected dogs also showed poor development, learning difficulties and episodes of abnormal behaviour. In these two dogs, investigations into forebrain structural and metabolic diseases were unremarkable; electromyography demonstrated fibrillation potentials and complex repetitive discharges in the infraspinatus, supraspinatus and epaxial muscles. Histopathological, immunohistochemical and immunoblotting analyses of muscle biopsies were consistent with dystrophin-deficient muscular dystrophy. DNA samples were obtained from all four full-sibling male Poodles, a healthy female littermate and the dam, which was clinically normal. Whole genome sequencing of one affected dog revealed a >5 Mb deletion on the X chromosome, encompassing the entire DMD gene. The exact deletion breakpoints could not be experimentally ascertained, but we confirmed that this region was deleted in all affected males, but not in the unaffected dogs. Quantitative polymerase chain reaction confirmed all three affected males were hemizygous for the mutant X chromosome, while the wildtype chromosome was observed in the unaffected male littermate. The female littermate and the dam were both heterozygous for the mutant chromosome. Forty-four Miniature Poodles from the general population were screened for the mutation and were homozygous for the wildtype chromosome. The finding represents a naturally-occurring mutation causing dystrophin-deficient muscular dystrophy in the dog

Predispositional genome sequencing in healthy adults: design, participant characteristics, and early outcomes of the PeopleSeq Consortium

Background: Increasing numbers of healthy individuals are undergoing predispositional personal genome sequencing. Here we describe the design and early outcomes of the PeopleSeq Consortium, a multi-cohort collaboration of predispositional genome sequencing projects, which is examining the medical, behavioral, and economic outcomes of returning genomic sequencing information to healthy individuals. Methods: Apparently healthy adults who participated in four of the sequencing projects in the Consortium were included. Web-based surveys were administered before and after genomic results disclosure, or in some cases only after results disclosure. Surveys inquired about sociodemographic characteristics, motivations and concerns, behavioral and medical responses to sequencing results, and perceived utility. Results: Among 1395 eligible individuals, 658 enrolled in the Consortium when contacted and 543 have completed a survey after receiving their genomic results thus far (mean age 53.0 years, 61.4% male, 91.7% white, 95.5% college graduates). Most participants (98.1%) were motivated to undergo sequencing because of curiosity about their genetic make-up. The most commonly reported concerns prior to pursuing sequencing included how well the results would predict future risk (59.2%) and the complexity of genetic variant interpretation (56.8%), while 47.8% of participants were concerned about the privacy of their genetic information. Half of participants reported discussing their genomic results with a healthcare provider during a median of 8.0 months after receiving the results; 13.5% reported making an additional appointment with a healthcare provider specifically because of their results. Few participants (< 10%) reported making changes to their diet, exercise habits, or insurance coverage because of their results. Many participants (39.5%) reported learning something new to improve their health that they did not know before. Reporting regret or harm from the decision to undergo sequencing was rare (< 3.0%). Conclusions: Healthy individuals who underwent predispositional sequencing expressed some concern around privacy prior to pursuing sequencing, but were enthusiastic about their experience and not distressed by their results. While reporting value in their health-related results, few participants reported making medical or lifestyle changes

A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome

Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes

Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

Protein synthesis is catalyzed in the peptidyl transferase center (PTC), located in the large (50S) subunit of the ribosome. No high-resolution structure of the intact ribosome has contained a complete active site including both A- and P-site tRNAs. In addition, although past structures of the 50S subunit have found no ordered proteins at the PTC, biochemical evidence suggests that specific proteins are capable of interacting with the 3′ ends of tRNA ligands. Here we present structures, at 3.6-Å and 3.5-Å resolution respectively, of the 70S ribosome in complex with A- and P-site tRNAs that mimic pre- and post-peptidyl-transfer states. These structures demonstrate that the PTC is very similar between the 50S subunit and the intact ribosome. They also reveal interactions between the ribosomal proteins L16 and L27 and the tRNA substrates, helping to elucidate the role of these proteins in peptidyl transfer

Improving the iMM904 S. cerevisiae metabolic model using essentiality and synthetic lethality data

<p>Abstract</p> <p>Background</p> <p><it>Saccharomyces cerevisiae </it>is the first eukaryotic organism for which a multi-compartment genome-scale metabolic model was constructed. Since then a sequence of improved metabolic reconstructions for yeast has been introduced. These metabolic models have been extensively used to elucidate the organizational principles of yeast metabolism and drive yeast strain engineering strategies for targeted overproductions. They have also served as a starting point and a benchmark for the reconstruction of genome-scale metabolic models for other eukaryotic organisms. In spite of the successive improvements in the details of the described metabolic processes, even the recent yeast model (i.e., <it>i</it>MM904) remains significantly less predictive than the latest <it>E. coli </it>model (i.e., <it>i</it>AF1260). This is manifested by its significantly lower specificity in predicting the outcome of grow/no grow experiments in comparison to the <it>E. coli </it>model.</p> <p>Results</p> <p>In this paper we make use of the automated GrowMatch procedure for restoring consistency with single gene deletion experiments in yeast and extend the procedure to make use of synthetic lethality data using the genome-scale model <it>i</it>MM904 as a basis. We identified and vetted using literature sources 120 distinct model modifications including various regulatory constraints for minimal and YP media. The incorporation of the suggested modifications led to a substantial increase in the fraction of correctly predicted lethal knockouts (i.e., specificity) from 38.84% (87 out of 224) to 53.57% (120 out of 224) for the minimal medium and from 24.73% (45 out of 182) to 40.11% (73 out of 182) for the YP medium. Synthetic lethality predictions improved from 12.03% (16 out of 133) to 23.31% (31 out of 133) for the minimal medium and from 6.96% (8 out of 115) to 13.04% (15 out of 115) for the YP medium.</p> <p>Conclusions</p> <p>Overall, this study provides a roadmap for the computationally driven correction of multi-compartment genome-scale metabolic models and demonstrates the value of synthetic lethals as curation agents.</p

Cytoplasmic CUG RNA Foci Are Insufficient to Elicit Key DM1 Features

The genetic basis of myotonic dystrophy type I (DM1) is the expansion of a CTG tract located in the 3′ untranslated region of DMPK. Expression of mutant RNAs encoding expanded CUG repeats plays a central role in the development of cardiac disease in DM1. Expanded CUG tracts form both nuclear and cytoplasmic aggregates, yet the relative significance of such aggregates in eliciting DM1 pathology is unclear. To test the pathophysiology of CUG repeat encoding RNAs, we developed and analyzed mice with cardiac-specific expression of a beta-galactosidase cassette in which a (CTG)400 repeat tract was positioned 3′ of the termination codon and 5′ of the bovine growth hormone polyadenylation signal. In these animals CUG aggregates form exclusively in the cytoplasm of cardiac cells. A key pathological consequence of expanded CUG repeat RNA expression in DM1 is aberrant RNA splicing. Abnormal splicing results from the functional inactivation of MBNL1, which is hypothesized to occur due to MBNL1 sequestration in CUG foci or from elevated levels of CUG-BP1. We therefore tested the ability of cytoplasmic CUG foci to elicit these changes. Aggregation of CUG RNAs within the cytoplasm results both in Mbnl1 sequestration and in approximately a two fold increase in both nuclear and cytoplasmic Cug-bp1 levels. Significantly, despite these changes RNA splice defects were not observed and functional analysis revealed only subtle cardiac dysfunction, characterized by conduction defects that primarily manifest under anesthesia. Using a human myoblast culture system we show that this transgene, when expressed at similar levels to a second transgene, which encodes expanded CTG tracts and facilitates both nuclear focus formation and aberrant splicing, does not elicit aberrant splicing. Thus the lack of toxicity of cytoplasmic CUG foci does not appear to be a consequence of low expression levels. Our results therefore demonstrate that the cellular location of CUG RNA aggregates is an important variable that influences toxicity and support the hypothesis that small molecules that increase the rate of transport of the mutant DMPK RNA from the nucleus into the cytoplasm may significantly improve DM1 pathology

Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT Ann Arbor)

HPRT Ann Arbor is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in two brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased K m for both substrates, a normal V max , a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric focusing conditions. We have cloned a full-length cDNA for HPRT Ann Arbor and determined its complete nucleotide sequence. A single nucleotide change (T→G) at nucleotide position 396 has been identified. This transversion predicts an amino acid substitution from isoleucine (ATT) to methionine (ATG) in codon 132, which is located within the putative 5′-phosphoribosyl-1-pyrophosphate (PRPP)-binding site of HPRT.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47622/1/439_2004_Article_BF00291707.pd