Mobile element insertion detection in 89,874 clinical exomes.

PURPOSE: Exome sequencing (ES) is increasingly used for the diagnosis of rare genetic disease. However, some pathogenic sequence variants within the exome go undetected due to the technical difficulty of identifying them. Mobile element insertions (MEIs) are a known cause of genetic disease in humans but have been historically difficult to detect via ES and similar targeted sequencing methods. METHODS: We developed and applied a novel MEI detection method prospectively to samples received for clinical ES beginning in November 2017. Positive MEI findings were confirmed by an orthogonal method and reported back to the ordering provider. In this study, we examined 89,874 samples from 38,871 cases. RESULTS: Diagnostic MEIs were present in 0.03% (95% binomial test confidence interval: 0.02-0.06%) of all cases and account for 0.15% (95% binomial test confidence interval: 0.08-0.25%) of cases with a molecular diagnosis. One diagnostic MEI was a novel founder event. Most patients with pathogenic MEIs had prior genetic testing, three of whom had previous negative DNA sequencing analysis of the diagnostic gene. CONCLUSION: MEI detection from ES is a valuable diagnostic tool, reveals molecular findings that may be undetected by other sequencing assays, and increases diagnostic yield by 0.15%.

Targeted mutation screening panels expose systematic population bias in detection of cystic fibrosis risk.

PURPOSE: Carrier screening for mutations contributing to cystic fibrosis (CF) is typically accomplished with panels composed of variants that are clinically validated primarily in patients of European descent. This approach has created a static genetic and phenotypic profile for CF. An opportunity now exists to reevaluate the disease profile of CFTR at a global population level. METHODS: CFTR allele and genotype frequencies were obtained from a nonpatient cohort with more than 60,000 unrelated personal genomes collected by the Exome Aggregation Consortium. Likely disease-contributing mutations were identified with the use of public database annotations and computational tools. RESULTS: We identified 131 previously described and likely pathogenic variants and another 210 untested variants with a high probability of causing protein damage. None of the current genetic screening panels or existing CFTR mutation databases covered a majority of deleterious variants in any geographical population outside of Europe. CONCLUSIONS: Both clinical annotation and mutation coverage by commercially available targeted screening panels for CF are strongly biased toward detection of reproductive risk in persons of European descent. South and East Asian populations are severely underrepresented, in part because of a definition of disease that preferences the phenotype associated with European-typical CFTR alleles.

Downregulation of miR-23a and miR-27a following experimental traumatic brain injury induces neuronal cell death through activation of proapoptotic Bcl-2 proteins.

MicroRNAs (miRs) are small noncoding RNAs that negatively regulate gene expression at the post-transcriptional level. To identify miRs that may regulate neuronal cell death after experimental traumatic brain injury (TBI), we profiled miR expression changes during the first several days after controlled cortical impact (CCI) in mice. miR-23a and miR-27a were rapidly downregulated in the injured cortex in the first hour after TBI. These changes coincided with increased expression of the proapoptotic Bcl-2 family members Noxa, Puma, and Bax. In an etoposide-induced in vitro model of apoptosis in primary cortical neurons, miR-23a and miR-27a were markedly downregulated as early as 1 h after exposure, before the upregulation of proapoptotic Bcl-2 family molecules. Administration of miR-23a and miR-27a mimics attenuated etoposide-induced changes in Noxa, Puma, and Bax, reduced downstream markers of caspase-dependent (cytochrome c release and caspase activation) and caspase-independent (apoptosis-inducing factor release) pathways, and limited neuronal cell death. In contrast, miRs hairpin inhibitors enhanced etoposide-induced neuronal apoptosis and caspase activation. Importantly, administration of miR-23a and miR-27a mimics significantly reduced activation of Puma, Noxa, and Bax as well as attenuated markers of caspase-dependent and -independent apoptosis after TBI. Furthermore, miR-23a and miR-27a mimics significantly attenuated cortical lesion volume and neuronal cell loss in the hippocampus after TBI. These findings indicate that post-traumatic decreases in miR-23a and miR-27a contribute to neuronal cell death after TBI by upregulating proapoptotic Bcl-2 family members, thus providing a novel therapeutic target.

Validation of a high resolution NGS method for detecting spinal muscular atrophy carriers among phase 3 participants in the 1000 Genomes Project.

BACKGROUND: Spinal muscular atrophy (SMA) is the most common pan-ethnic cause of early childhood death due to mutations in a single gene, SMN1. Most chromosome 5 homologs have a functional gene and dysfunctional copy, SMN2, with a single synonymous base substitution that results in faulty RNA splicing. However, the copy number of SMN1 and SMN2 is highly variable, and one in 60 adults worldwide are SMA carriers. Although population-wide screening is recommended, current SMA carrier tests have not been incorporated into targeted gene panels. METHODS: Here we describe a novel computational protocol for determining SMA carrier status based solely on individual exome data. Our method utilizes a Bayesian hierarchical model to quantify an individual's carrier probability given only his or her SMN1 and SMN2 reads at six loci of interest. RESULTS: We find complete concordance with results obtained with the current qPCR-based testing standard in known SMA carriers and affecteds. We applied our protocol to the phase 3 cohort of the 1,000 Genomes Project and found carrier frequencies in multiple populations consistent with the present literature. CONCLUSION: Our process is a convenient, robust alternative to qPCR, which can easily be integrated into the analysis of large multi-gene NGS carrier screens.

Strong oviposition preference for Bt over non-Bt maize in Spodoptera frugiperda and its implications for the evolution of resistance.

BACKGROUND: Transgenic crops expressing Bt toxins have substantial benefits for growers in terms of reduced synthetic insecticide inputs, area-wide pest management and yield. This valuable technology depends upon delaying the evolution of resistance. The 'high dose/refuge strategy', in which a refuge of non-Bt plants is planted in close proximity to the Bt crop, is the foundation of most existing resistance management. Most theoretical analyses of the high dose/refuge strategy assume random oviposition across refugia and Bt crops. RESULTS: In this study we examined oviposition and survival of Spodoptera frugiperda across conventional and Bt maize and explored the impact of oviposition behavior on the evolution of resistance in simulation models. Over six growing seasons oviposition rates per plant were higher in Bt crops than in refugia. The Cry1F Bt maize variety retained largely undamaged leaves, and oviposition preference was correlated with the level of feeding damage in the refuge. In simulation models, damage-avoiding oviposition accelerated the evolution of resistance and either led to requirements for larger refugia or undermined resistance management altogether. Since larval densities affected oviposition preferences, pest population dynamics affected resistance evolution: larger refugia were weakly beneficial for resistance management if they increased pest population sizes and the concomitant degree of leaf damage. CONCLUSIONS: Damaged host plants have reduced attractiveness to many insect pests, and crops expressing Bt toxins are generally less damaged than conventional counterparts. Resistance management strategies should take account of this behavior, as it has the potential to undermine the effectiveness of existing practice, especially in the tropics where many pests are polyvoltinous. Efforts to bring down total pest population sizes and/or increase the attractiveness of damaged conventional plants will have substantial benefits for slowing the evolution of resistance.

Infarto cerebral embólico como complicación perioperatoria de un mixoma auricular.

Perioperative stroke is one of the complications that can occur during the surgical procedure and up to 30 days after it. A 52-year-old woman with no neurological symptoms and a diagnosis of intracardiac mass. She underwent cardiac surgery with resection of the tumor that was compatible with atrial myxoma. In the immediate postoperative, she presented neurological symptoms and was diagnosed with basal ganglia infarction of embolic etiology. Perioperative stroke appears most frequently in cardiovascular surgery but is rarely reported in heart tumor resection surgery.

El accidente cerebrovascular perioperatorio es una de las complicaciones que pueden presentarse durante el proceder quirúrgico y hasta los 30 días posteriores al mismo. Se presenta el caso de una mujer de 52 años, sin síntomas neurológicos y con diagnóstico de masa intracardiaca. Se le realizó cirugía cardiaca con resección del tumor, que resultó compatible con mixoma auricular. En el posoperatorio inmediato presentó sintomatología neurológica y se diagnosticó infarto de ganglios basales de etiología embólica. El accidente cerebrovascular perioperatorio aparece con mayor frecuencia en las cirugías cardiovasculares, pero rara vez es reportado en cirugías de exéresis de tumores cardiacos.

NmDef02, a novel antimicrobial gene isolated from Nicotiana megalosiphon confers high-level pathogen resistance under greenhouse and field conditions.

Plant defensins are small cysteine-rich peptides that inhibit the growth of a broad range of microbes. In this article, we describe NmDef02, a novel cDNA encoding a putative defensin isolated from Nicotiana megalosiphon upon inoculation with the tobacco blue mould pathogen Peronospora hyoscyami f.sp. tabacina. NmDef02 was heterologously expressed in the yeast Pichia pastoris, and the purified recombinant protein was found to display antimicrobial activity in vitro against important plant pathogens. Constitutive expression of NmDef02 gene in transgenic tobacco and potato plants enhanced resistance against various plant microbial pathogens, including the oomycete Phytophthora infestans, causal agent of the economically important potato late blight disease, under greenhouse and field conditions.

El verdadero origen histórico del trauma cardíaco penetrante / The true historical origin of penetrating cardiac trauma

Introducción. Los traumatismos cardíacos son lesiones graves y con elevado índice de letalidad, aspecto que influye en el interés mostrado por los lectores cada vez que aparecen reportados en publicaciones científicas. En ocasiones existe cierto grado de incongruencia a la hora de establecer el origen histórico de sucesos o eventos ocurridos y relacionados con la historia de la medicina. En el caso del trauma cardíaco penetrante han sido descritos diversos orígenes en algunos de los artículos científicos publicados, lo cual puede generar un grado de duda en los lectores. Métodos. Se realizó una revisión de la literatura, médica y no médica, para buscar información que ayudara a esclarecer el verdadero origen histórico de esta entidad. Resultados. El trauma cardíaco penetrante fue descrito por primera vez en la obra griega titulada La Ilíada, escrita por Homero en el siglo VIII A.C., y no en El papiro quirúrgico de Edwin Smith, como varios autores mencionan. Conclusiones. De todos los eventos de trauma cardíaco penetrante descritos, el más irrefutable es el narrado en el canto XIII, donde se cuenta la muerte de Alcátoo, producto de una herida cardíaca ocasionada por una lanza arrojada por Idomeneo durante una batalla

Introduction. Cardiac traumatic injuries are serious injuries with a high lethality rate, an aspect that influences the interest shown by readers each time they appear reported in scientific publications. Sometimes there is a certain degree of inconsistency when it comes to establishing the historical origin of occurrences or events related to the history of medicine. In the case of penetrating cardiac trauma, different origins have been described in some of the published scientific articles, which may generate a degree of doubt in the readers. Methods. A review of the medical and non-medical literature was carried out to seek information that would help to clarify the true historical origin of this entity. Results. Penetrating cardiac trauma was first described in a Greek work entitled The Iliad, written by Homer in the 8th century B.C., and not in The Surgical Papyrus of Edwin Smith as several authors mention. Conclusions. Of all the events of penetrating cardiac trauma described, the most irrefutable is the one narrated in canto XIII, where it recounts the death of Alcathous product of a cardiac wound caused by a spear thrown by Idomeneo during a battle.

High-throughput SuperSAGE for gene expression analysis of Nicotiana tabacum-Rhizoctonia solani interaction.

OBJECTIVE: The ubiquitous soil pathogen Rhizoctonia solani causes serious diseases in different plant species. Despite the importance of this disease, little is known regarding the molecular basis of susceptibility. SuperSAGE technology and next-generation sequencing were used to generate transcript libraries during the compatible Nicotiana tabacum-R. solani interaction. Also, we used the post-transcriptional silencing to evaluate the function of a group of important genes. RESULTS: A total of 8960 and 8221 unique Tag sequences identified as differentially up- and down-regulated were obtained. Based on gene ontology classification, several annotated UniTags corresponded to defense response, metabolism and signal transduction. Analysis of the N. tabacum transcriptome during infection identified regulatory genes implicated in a number of hormone pathways. Silencing of an mRNA induced by salicylic acid reduced the susceptibility of N. tabacum to R. solani. We provide evidence that the salicylic acid pathway was involved in disease development. This is important for further development of disease management strategies caused by this pathogen.

EIL2 transcription factor and glutathione synthetase are required for defense of tobacco against tobacco blue mold.

In order to identify tobacco (Nicotiana megalosiphon) genes involved in broad-spectrum resistance to tobacco blue mold (Peronospora hyoscyami f. sp. tabacina), suppression subtractive hybridization was used to generate cDNA from transcripts that are differentially expressed during an incompatible interaction. After differential screening by membrane-based hybridization, clones corresponding to 182 differentially expressed genes were selected, sequenced, and analyzed. The cDNA collection comprised a broad repertoire of genes associated with various processes. Northern blot analysis of a subset of these genes confirmed the differential expression patterns between the compatible and incompatible interaction. Subsequent virus-induced gene silencing (VIGS) of four genes that were found to be differentially induced was pursued. While VIGS of a lipid transfer protein gene or a glutamate decarboxylase gene in Nicotiana megalosiphon did not affect blue mold resistance, silencing of an EIL2 transcription factor gene and a glutathione synthetase gene was found to compromise the resistance of Nicotiana megalosiphon to P. hyoscyami f. sp. tabacina. Potentially, these genes can be used to engineer resistance in blue mold-susceptible tobacco cultivars.

Identification of genes induced upon water-deficit stress in a drought-tolerant rice cultivar.

Among the abiotic stresses, the availability of water is the most important factor that limits the productive potential of higher plants. The identification of novel genes, determination of their expression patterns, and the understanding of their functions in stress adaptation is essential to improve stress tolerance. Amplified fragment length polymorphism analysis of cDNA was used to identify rice genes differentially expressed in a tolerant rice variety upon water-deficit stress. In total, 103 transcript-derived fragments corresponding to differentially induced genes were identified. The results of the sequence comparison in BLAST database revealed that several differentially expressed TDFs were significantly homologous to stress regulated genes/proteins isolated from rice or other plant species. Most of the transcripts identified here were genes related to metabolism, energy, protein biosynthesis, cell defence, signal transduction, and transport. New genes involved in the response to water-deficit stress in a tolerant rice variety are reported here.

Carrier Screening is a Deficient Strategy for Determining Sperm Donor Eligibility and Reducing Risk of Disease in Recipient Children.

AIMS: DNA-based carrier screening is a standard component of donor eligibility protocols practiced by U.S. sperm banks. Applicants who test positive for carrying a recessive disease mutation are typically disqualified. The aim of our study was to examine the utility of a range of screening panels adopted by the industry and the effectiveness of the screening paradigm in reducing a future child's risk of inheriting disease. METHODS: A cohort of 27 donor applicants, who tested negative on an initial cystic fibrosis carrier test, was further screened with three expanded commercial carrier testing panels. These results were then compared to a systematic analysis of the applicants' DNA using next-generation sequencing (NGS) data. RESULTS: The carrier panels detected serious pediatric disease mutations in one, four, or six donor applicants. Because each panel screens distinct regions of the genome, no single donor was uniformly identified as carrier positive by all three panels. In contrast, systematic NGS analysis identified all donors as carriers of one or more mutations associated with severe monogenic pediatric disease. These included 30 variants classified as "pathogenic" based on clinical observation and 66 with a high likelihood of causing gene dysfunction. CONCLUSION: Despite tremendous advances in variant identification, understanding, and analysis, the vast majority of disease-causing mutation combinations remain undetected by commercial carrier screening panels, which cover a narrow, and often distinct, subset of genes and mutations. The biological reality is that all donors and recipients carry serious recessive disease mutations. This challenges the utility of any screening protocol that anchors donor eligibility to carrier status. A more effective approach to reducing recessive disease risk would consider joint comprehensive analysis of both donor and recipient disease mutations. This type of high-resolution recessive disease risk analysis is now available and affordable, but industry practice must be modified to incorporate its use.

Expression of a Nicotiana tabacum pathogen-induced gene is involved in the susceptibility to black shank.

Many host genes induced during compatible plant-pathogen interactions constitute targets of pathogen virulence factors that act to suppress host defenses. In order to identify Nicotiana tabacum L. genes for pathogen-induced proteins involved in susceptibility to the oomycete Phytophthora parasitica var. nicotianae, we used SuperSAGE technology combined with next-generation sequencing to identify transcripts that were differentially upregulated during a compatible interaction. We identified a pathogen-induced gene (NtPIP) that was rapidly induced only during the compatible interaction. Virus-induced gene silencing of NtPIP reduced the susceptibility of N. tabacum to P. parasitica var. nicotianae. Additionally, transient expression of NtPIP in the resistant species Nicotiana megalosiphon Van Heurck & Mull. Arg. compromised the resistance to P. parasitica var. nicotianae. This pathogen-induced protein is therefore a positive regulator of the susceptibility response against an oomycete pathogen in tobacco.

'Candidatus Liberibacter asiaticus', Causal Agent of Citrus Huanglongbing, Is Reduced by Treatment with Brassinosteroids.

Huanglongbing (HLB) constitutes the most destructive disease of citrus worldwide, yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, 'Candidatus Liberibacter asiaticus'. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced in leaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified. The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB.

Identification of sugarcane genes induced in disease-resistant somaclones upon inoculation with Ustilago scitaminea or Bipolaris sacchari.

To understand the molecular basis of a specific plant-pathogen interaction, it is important to identify plant genes that respond to the pathogen attack. Amplified fragment length polymorphism (AFLP) analysis of cDNA was used to identify sugarcane genes differentially expressed in disease-resistant but not in susceptible sugarcane somaclones in response to inoculation with either Ustilago scitaminea or Bipolaris sacchari (also known as Helminthosporium sacchari or Drechslera sacchari), causal agents of smut and eyespot respectively. In total 62 differentially regulated genes were identified, of which 10 were down-regulated and 52 were induced. Of these 52, 19 transcript derived fragments showed homology to known plant gene sequences, most of them related to defense or signaling. The total set of differentially expressed sugarcane genes can be an important resource for further studies aimed at understanding sugarcane pathogen defense.

High efficacy of a 20 amino acid peptide of the acidic ribosomal protein P0 against the cattle tick, Rhipicephalus microplus.

Current strategies to control cattle ticks use integrated control programs (ICP) that include vaccination. Reduction in the use of chemicals and in the cost of tick control, the delay or elimination of acaricide resistance and the decreasing of environmental pollution are the advantages of using these programs. This integrated program is potentially applicable to all genotypes of chemical resistant ticks. However, the problem here is to improve the efficacy of anti-tick vaccines. The P0 protein is a structural component of the ribosome of all organisms. We have identified an immunogenic region of ribosomal protein P0 from Rhipicephalus spp. ticks that is not very conserved compared to the orthologous protein in their hosts. A synthetic 20 amino acid peptide from this sequence was effective as a vaccine against Rhipicephalus sanguineus infestations in an immunization and challenge experiment using rabbits. In this paper, the same peptide used as vaccine against the cattle tick Rhipicephalus Boophilus microplus shows a significant diminution in the number of engorged females recovered, in the weight of females and the weight of egg masses. The number of eggs hatched was also significantly reduced for the vaccinated group, with an overall effectivity for the antigen pP0 of 96%. These results, together with the conserved sequence of the P0 peptide among ticks, suggest that this antigen could be a good broad spectrum vaccine candidate. It would be expected to be active against many species of ticks and thus has promise in an ICP for effective control of ticks and thereby to improve the efficiency and productivity of the livestock industry.

First Report of a Closterovirus-Like Particle Associated with Pineapple Plants (Ananas comosus cv. Smooth Cayenne) Affected with Pineapple Mealybug Wilt in Cuba.

Pineapple mealybug wilt (PMW) was first described in Hawaii in 1910 and has since spread wherever pineapple is cultivated. PMW is currently one of the most important field diseases of pineapple in Cuba, causing up to 40% crop loss. Leaves of plants affected with PMW turn bronze-red and lose turgidity, and the tips curve down. Root system size is decreased and fruits are unmarketable because the flesh is fibrous and sour. Leaves from wilt-affected pineapple plants were selected from the Experimental Station "Tomás Roig" at Ciego de Avila, Cuba. Asymptomatic plants derived from meristem tissue and cultivated under greenhouse conditions were also collected. Both samples were analyzed by several virus purification procedures and finally purified with a protocol modified from Gunashinge and German (1). Partially purified samples from symptomatic and asymptomatic plants were negatively stained and examined by a JEOL (JEM-2000 EX) transmission electron microscope at a magnification of ×30,000. Sixteen, long, flexuous, rod-shaped, viruslike particles (estimated length 1,200 to 1,450 nm; width about 12 nm) were observed in symptomatic leaves, using electron microscope micrographs. These particles were similar to those of the pineapple clostero-like virus found in Hawaii (1) and Australia (2). Particles were observed from symptomatic plants but not from plants propagated by primary meristem culture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of viral proteins indicated that the coat protein has a molecular mass of approximately 23 kDa. This is the first report of closterovirus-like particles associated with pineapple plants (Ananas comosus cv. Smooth Cayenne) affected with PMW in Cuba. References: (1) U. B. Gunasinge and T. L. German. Phytopathology 79:1337, 1989. (2) W. Wakman et al. Aust. J. Agric. Res. 46:947, 1995.

Microtubules underlie dysfunction in duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal X-linked degenerative muscle disease caused by the absence of the microtubule-associated protein dystrophin, which results in a disorganized and denser microtubule cytoskeleton. In addition, mechanotransduction-dependent activation of calcium (Ca(2+)) and reactive oxygen species (ROS) signaling underpins muscle degeneration in DMD. We show that in muscle from adult mdx mice, a model of DMD, a brief physiologic stretch elicited microtubule-dependent activation of NADPH (reduced-form nicotinamide adenine dinucleotide phosphate) oxidase-dependent production of ROS, termed X-ROS. Further, X-ROS amplified Ca(2+) influx through stretch-activated channels in mdx muscle. Consistent with the importance of the microtubules to the dysfunction in mdx muscle, muscle cells with dense microtubule structure, such as those from adult mdx mice or from young wild-type mice treated with Taxol, showed increased X-ROS production and Ca(2+) influx, whereas cells with a less dense microtubule network, such as young mdx or adult mdx muscle treated with colchicine or nocodazole, showed little ROS production or Ca(2+) influx. In vivo treatments that disrupted the microtubule network or inhibited NADPH oxidase 2 reduced contraction-induced injury in adult mdx mice. Furthermore, transcriptome analysis identified increased expression of X-ROS-related genes in human DMD skeletal muscle. Together, these data show that microtubules are the proximate element responsible for the dysfunction in Ca(2+) and ROS signaling in DMD and could be effective therapeutic targets for intervention.